Towards the development of an in vivo chemical probe for cyclin G associated kinase (GAK)

Article abstract of DOI:10.3390/molecules24224016

SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC₅₀ = 1.4 μM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

Full text of DOI:10.3390/molecules24224016

molecules
Article
Towards the Development of an In vivo Chemical
Probe for Cyclin G Associated Kinase (GAK)
Christopher R. M. Asquith 1,2,
*
, James M. Bennett 3, Lianyong Su 4, Tuomo Laitinen 5,
Jonathan M. Elkins 3,6, Julie E. Pickett 1, Carrow I. Wells 1, Zengbiao Li 4, Timothy M. Willson 1
and William J. Zuercher 1,7,
*
1
Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA; julie.pickett@unc.edu (J.E.P.); carrow.wells@unc.edu (C.I.W.);
tim.willson@unc.edu (T.M.W.)
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA
Structural Genomics Consortium and Target Discovery Institute, Nuffield Department of Clinical Medicine,
University of Oxford, Oxford OX3 7DQ, UK; james.bennett@ndm.ox.ac.uk (J.M.B.);
jon.elkins@sgc.ox.ac.uk (J.M.E.)
Drumetix Laboratories, Greensboro, NC 27409, USA; lsu@drumetix.com (L.S.); zli@drumetix.com (Z.L.)
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland;
tuomo.laitinen@uef.fi
2
3
4
5
6
7
Structural Genomics Consortium, Universidade Estadual de Campinas-UNICAMP, Campinas,
São Paulo 13083-886, Brazil
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, USA
*
Correspondence: chris.asquith@unc.edu (C.R.M.A); william.zuercher@unc.edu (W.J.Z.); Tel.: +1919-962-5349
(C.R.M.A. & W.J.Z.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Diego Muñoz-Torrero, F. Javier Luque and Marçal Pastor-Anglada
Received: 14 October 2019; Accepted: 2 November 2019; Published: 6 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: SGC-GAK-1 (
kinase (GAK). However,
P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which
limited its utility in in vivo studies. Chemical modifications of that improved metabolic stability,
generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells
was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 M), showing improved stability
1) is a potent, selective, cell-active chemical probe for cyclin G-associated
1
was rapidly metabolized in mouse liver microsomes by cytochrome
1
µ
in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an
alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome
P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK
inhibitors. Taken together, these approaches point towards the development of an in vivo chemical
probe for the dark kinase GAK.
Keywords: cyclin G associated kinase (GAK); kinase inhibitor design; in vivo kinase design;
1-aminobenzotriazole (ABT)
1. Introduction
Cyclin G-associated kinase (GAK) is a 160 kDa member of the numb-associated kinase (NAK)
family of serine/threonine kinases [
of cyclin G, and is ubiquitously expressed across tissues [
Golgi complex, cytoplasm, and nucleus [ ]. GAK has been genetically associated with a diverse
range of biological processes. Genome-wide association studies have identified single nucleotide
1]. GAK was originally identified as a directly associated partner
2
]. Within the cell, GAK localizes to the
3
Molecules 2019, 24, 4016; doi:10.3390/molecules24224016
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4016
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polymorphisms in GAK that are associated with susceptibility to Parkinson’s disease [
4]. Knock down
of GAK has shown that it is required for the maintenance of centrosome maturation and progression
through mitosis [ ]. Conversely, GAK was found to be overexpressed in osteosarcoma cell lines and
5
tissues, where it contributes to proliferation and survival [6].
The biological implications of GAK depletion or over-expression prompt the question of whether
there is therapeutic utility in targeting its kinase domain with small molecules. GAK is a collateral
target of several clinical kinase inhibitors, including the epidermal growth factor receptor (EGFR)
inhibitors erlotinib and gefitinib (Figure 1), but until recently potent selective cell-active inhibitors of
GAK had not been identified [
remained untested. As part of an ongoing effort to generate chemical probes for dark kinases, we
recently described SGC-GAK-1 ( ) as a high-quality cell-active chemical probe for GAK (Figure 1) [ ].
SGC-GAK-1 ( ) showed IC50 = 48 nM in a GAK live cell target engagement assay and proved to be
7] and thus the biological consequences of selective GAK inhibition
1
8,9
1
more than 50-fold selective for GAK across a panel of over 400 human kinases.
OMe
F
Cl
MeO
NH
N
NH
N
O
MeO
NH
O
O
O
N
N
OMe
OMe
N
Br
OMe
N
Erlotinib
GAK - KD = 3.1 nM
EGFR - KD = 0.7 nM
Gefitinib
GAK - KD = 13 nM
EGFR - KD = 1.0 nM
SGC-GAK-1 - (1)
GAK DSF
Tm = 7.7 °C
GAK Ki = 3.1 nM
GAK nanoBRET IC50 = 48 nM
NAK binding selectivity > 16000
>50-fold selectivity over nearest kinase
Figure 1. Examples of reported GAK inhibitors.
GAK is a potential therapeutic target for the treatment of androgen-independent prostate cancer.
Expression of GAK is known to increase during prostate cancer progression to androgen independence
and has been positively correlated with Gleason scores in resections from prostate cancer patients [10].
We have demonstrated that
constitutively-active splice variants of the androgen receptor [
1
potently inhibits the viability of prostate cancer cells that contain
]. These results prompted us to identify
9
a chemical probe of GAK that is suitable for use in rodent models of prostate cancer. We now describe
our efforts in the optimization of 4-aminoquinolines as potential in vivo chemical probes for GAK.
2. Results
2.1. Initial Investigation of P450 Metabolism of 1
The metabolic stability of
1 was evaluated in mouse liver microsomes (MLMs) to assess the
potential for use as an in vivo chemical probe. SGC-GAK-1 (1) was incubated with MLMs in the
presence or absence of added NADPH, and the compound was quantified at various time points
over an hour. No degradation of 1 was observed in the absence of NADPH. However, 1 was rapidly
consumed (T1/2 = 5.7 min; Clint = 990 mL/min/kg) in the presence of NADPH. This result suggests that
1 would be prone to rapid first pass metabolism when dosed in vivo.
The necessity of NADPH for the metabolism of
oxidation. To further understand the metabolic fate of
experiment. 10 M of 1 was incubated for 5 min (ratio of 0.5 with respect to propranolol, n = 4) in MLMs
1
is consistent with cytochrome P450-mediated
1
, we conducted a metabolite identification
µ
at 37 ◦C in the presence of an excess of NADPH before quenching with methanol. After centrifugation,
the supernatant was subjected to analysis by LCMS, and structures of metabolites were assigned on the
basis of their molecular weight and fragmentation pattern. The primary metabolite
as a loss of CH2 from the 3,4,5-trimethoxyaniline (Figure 2 and Figure S1). Minor metabolites were
identified as an alternate loss of CH2 from the 3,4,5-trimethoxyaniline ( ), the loss of 2 CH2 ( ), and the
2 was identified
3
×
4

Molecules 2019, 24, 4016
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formation of a cyclic acetal (
5). Formation of
5
likely occurs via the product of a mono-demethylation
intercepting an intermediate oxocarbenium ion formed during a second oxidative demethylation.
Finally, two minor species with quinoline ring oxidation ( and ) were observed at low abundance.
The liver microsomal stability and the metabolite identification experiments identified cytochrome
6
7
P450-mediated oxidation as the primary route of metabolism of
major metabolic liability.
1, with the trimethoxyaniline as the
OH
OMe
OH
HO
HO
MeO
MeO
MeO
NH
MeO
NH
N
NH
N
Br
Br
Br
N
2
3
4
Exact demethylation
position not determined
32.4%
Exact demethylation
position not determined
2.6%
Exact demethylation
position not determined
1.2%
OMe
OMe
O
O
MeO
MeO
MeO
MeO
NH
N
OH
NH
N
OH
MeO
NH
N
Br
Br
Br
6
7
5
Exact quinoline hydroxylation Exact quinoline hydroxylation
6.3%
position not determined
0.1%
position not determined
0.7%
Figure 2. Metabolites of 1 identified (2–7) along with percentage abundance in respect to the parent
compound. Compounds are ordered by HPLC retention time (see Supplementary Materials).
2.2. Synthesis of Analogs of 1
In an attempt to address the poor metabolic stability of
analogs in which the trimethoxyaniline was replaced by various bioisosteres (
13 31, and 33 43 were synthesized through nucleophilic aromatic displacement of the corresponding
4-chloroquinolines (Scheme 1) to furnish the products with good to excellent yields (54–89%) [ 11 14].
Additional analogs (23 24, and 27) were synthesized by the same route with modest yields (12–38%)
1
, we prepared a focused array of
8–
43). Compounds 11
8
–
,
–
–
8
,
9,
–
,
due to the reduced nucleophilicity of their respective anilines. An alternative route employing a
Buchwald–Hartwig cross-coupling with 4-chloro-6-trifluoromethylquinoline enabled access to the
6-trifluoromethyl analogs (12) and (32) in 21% and 17% yields, respectively (Scheme 2) [8,9,12–14].
Scheme 1. General displacement synthetic procedure.
Scheme 2. General Buchwald-Hartwig cross-coupling procedure.

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2.3. Metabolite Invesitigation Focused 4-Anilinoquinolines Analogs 8–13
The metabolic stability of the 4-anilinoquinolines (8–13) were evaluated in MLMs (Table 1).
Half-lives for metabolic clearance were normalized to propranolol in each experimental run to control
for variations in the MLM preparations. Changing the substitution of the core quinoline heterocycle
to 6,7-dimethoxy, as in
maintained an aniline O-demethylation species as the primary metabolite. Modification of the aniline
portion had a wide range effects on metabolic stability. The 2,3-methylenedioxy analog showed similar
stability to , with an aniline ring oxidation as the primary metabolite. Surprisingly, modification of
8, marginally increased stability relative to the 6-bromo substitution and
9
8
the trimethoxy to the difluoro (10 and 11) yielded compounds with equal or less metabolic stability
than 1. Metabolite identification experiments with the fluorinated analogs continued to show aniline
oxidation as the primary site of metabolism: direct ring oxidation was observed with 10 and hydroxyl
replacement of a fluorine with 11. However, replacement of the trimethoxyaniline with a perfluorinated
aniline resulted in a significantly more stable analog 12. Finally, the similar stability of 6-Br quinoline
and 6-CF3 quinoline 13 showed that switching of the 6-substituents had little effect on metabolism.
1
Table 1. Initial microsomal stability assessment (see Supplementary Materials for details).
Primary
Metabolism Site
Primary
Metabolite Mass
T1/2 Ratio b
Cmpd
R
X
T1/2 (min) a
8
9
10
11
12
13
3,4,5-(OMe)3
2,3-O-CH2-O-
2,5-F
2,4-F
2,3,4,5,6-F5
3,4,5-(OMe)3
6,7-(OMe)2
6-Br
6-Br
6-Br
6-CF3
6-CF3
12.2
8.9
1.7
3.2
45.6
6.4
1.6
1.9
0.17
0.47
7.0
Aniline
Aniline
Aniline
Aniline
nt
Parent − CH2
Parent + O
Parent + O
Parent − F + OH
nt
0.78
Aniline
Parent + O
nt: not tested; a mean average, n = 2; b T1/2 ratio is the normalization with respect to internal control (propranolol).
The analogs (
transfer (TR-FRET) ligand-binding displacement assay and a live cell NanoBRET target engagement
assay (Table 2) [ 12 15]. The 6,7-dimethoxyquinoline displayed a high GAK potency biochemically
and in cells (IC50 = 25 nM), as did the methylenedioxy compound (IC50 = 22 nM). The two
8–13) were also assessed for GAK activity in a Time-resolved fluorescence energy
8,
9
,
–
8
9
difluoroaniline isomers 10 and 11 had nanomolar Ki values in the biochemical assay but showed
a larger drop-off in potency in the live cell NanoBRET assay, suggesting that they had poorer cell
penetration. The metabolically stable pentafluoro aniline analog 12 had only weak GAK affinity in
the biochemical assay (Ki = 880 nM), and did not progress to cellular evaluation. Finally, 13 showed
a 4-fold loss in cellular GAK potency relative to 1. Considering both the MLM stability and cellular
GAK activity of these initial analogs, we selected the methylenedioxy compound
of optimization.
9 for further rounds
Table 2. GAK affinity measurements and microsomal clearance of initial quinoline series.
Biochemical a
GAK Ki (nM)
Live Cell b
Cmpd
R
X
GAK IC50 (nM) c
0.54 d
1.9
15
25 d
22
8
9
10
11
12
13
3,4,5-(OMe)3
2,3-O-CH2-O-
2,5-F
2,4-F
2,3,4,5,6-F5
6,7-(OMe)2
6-Br
6-Br
6-Br
6-CF3
6-CF3
870
2700
nt
35
880
3.9 d
180 d
3,4,5-(OMe)3
nt: not tested; a TR-FRET assay; b nanoBRET; c mean average, n = 2; d reference [12].

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2.4. Design and Tesing of 4-Anilinoquinolines Analogs 14–43
Docking of
1 and 9 into a model of GAK derived from the co-crystal structure with gefitinib
(Figure 3) was used to define key structural features for favorable binding using Schrödinger
Maestro [16]. The compounds were minimized using LigPrep before docking into the ATP-binding
site prepared from PDB structure 5Y7Z [17]. As expected, the most favorable docking pose placed
the quinoline nitrogen in a position to accept a hydrogen bond from the backbone NH of the hinge
region residue C126. The 5-membered ring of the methylenedioxy in
it could form a weak hydrophobic interaction with T123 [18]. Based on these results, we designed and
prepared a series of isosteric replacements of the substituted aniline (14 43) that would maintain a
9 adopted a conformation where
–
favorable interaction with GAK and be less prone to oxidative metabolism. The analogs were assessed
for GAK activity in the biochemical and live cell assays and for metabolic stability in MLM (Figure 4
and Table 3).
Figure 3. Docking of
1 (A) and 9 (B) into the ATP binding site of GAK (PDB: 5Y7Z). The protein is
shown as a ribbon view with key amino acids as sticks with carbon atoms in grey, nitrogen in blue,
oxygen in red, and sulphur in yellow. The GAK inhibitor is shown as sticks with carbon atoms in green,
nitrogen in blue, and oxygen in red. The ATP-binding pocket is shown in light grey. Hydrogen bonds
from the hinge residues to the inhibitor are highlighted as yellow dashed lines.
Figure 4. Aniline fragments incorporated into compounds 9 and 14–43 in Table 3.

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Results of the initial compound evaluation underscored the difficulty in the concurrent optimization
of GAK potency and metabolic stability (Table 4). Consistent with the docking model supporting a
favorable fit for bicyclic aniline fragments, a majority of the analogs (14–43) demonstrated potent GAK
activity in a biochemical assay (Ki < 100 nM). Importantly, all GAK active analogs (14–43) maintained
good NAK family selectivity in biochemical assays (Table S11). Unfortunately, potent biochemical
GAK activity did not necessarily translate to potent GAK activity in cells. Most of the heterocycles
showed lower GAK activity in the live cell NanoBRET assay compared to the methylenedioxy analog
9
, although six compounds demonstrated submicromolar IC50 values in cells: benzofurans 19 and 20,
benzoxazole 23, benzothiazole 24, benzothiadiazole 29, and indazole 32. Interestingly, we observed
that a switch of the 2,3-substitution in the aniline heterocycle to the corresponding 3,4-substitution often
led to greatly increased metabolic stability, with six compounds demonstrating T1/2 ratios ≥ 2-fold
higher than 9: indazoles 34 and 35, benzimidazolone 39 and 42, and benzoxazolidinones 40 and 41.
The benzimidazolone 39 was the most stable heterocycle in MLM with a T1/2 of >200 min and T1/2 ratio
> 25. Unfortunately, the matched pair of indazoles 34 and 35 were the only compounds to maintain
any measurable activity in the live cell GAK NanoBRET assay, with IC50 = 1.4 and 2.6
µM, respectively.
However, this level of potency was 30- to 60-fold lower than SGC-GAK-1 (
minimum criteria for use as an in vivo chemical probe for GAK.
1) and did not meet our
Table 3. GAK affinity measurements and microsomal clearance of isosteric replacements of 9.
Live Cell c
Biochemical b
GAK Ki (nM)
Cmpd a
X
T1/2 Ratio e
T1/2 (Min) d
GAK IC50 (nM) d
9
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
CF3
CF3
Br
Br
Br
Br
Br
Br
Br
Br
Br
1.9
1.4
740
460
5.2
29
1.9
4.3
37
19
19
11
26
23
4.1
41
5.3
2.8
3.6
14
720
34
20
nt
47
62
22
120
>5000
>5000
>5000
2500
180
570
1400
>5000
340
220
>5000
3300
>5000
2500
400
>5000
>5000
760
>5000
2600
8.9
1.8
1.0
2.0
1.7
0.82
1.7
0.9
5.2
1.4
1.7
5.3
2.6
4.1
8.1
1.2
1.6
1.9
0.27
0.12
0.24
0.16
0.10
0.16
0.08
0.64
0.17
0.21
0.68
0.32
0.50
1.0
0.15
0.20
0.09
0.10
0.32
0.90
9.8
9.8
2.2
0.92
1.0
>25
6.9
3.4
4.2
1.9
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
0.73
0.80
2.5
5.9
46.4
45.6
14.8
7.5
1400
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
8.1
360
680
110
1600
1700
>200
55.9
27.8
33.6
15.6
nt: not tested; a R groups are shown in Figure 4; b TR-FRET assay; c nanoBRET; d mean average, n = 2; e T1/2 ratio is
the normalization with respect to internal control (propranolol).

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2.5. Metabolite ID Profiling and Kinome Scan of 35
Despite the limited utility of indazole 35 as an in vivo chemical probe for GAK we decided
to perform a metabolite identification experiment to elaborate the origin of its improved stability.
Following incubation in MLM, metabolites were identified that were nucleated around the aniline ring.
Oxidation of the phenyl ring of the indazole yielded the major metabolites (44–45). Minor metabolites
were identified as hydroxylation on an indazole nitrogen (46) (Figure 5). Notably, the quinoline core
remained less metabolically labile than the indazole heterocycle.
Figure 5. Compound 35 and corresponding metabolites (44–46) identified along with percentage
abundance ordered by retention time (see Supplementary Materials).
Compound 35 was also submitted to a KinomeScan assay to determine whether the indazole
moiety had changed the selectivity profile across > 400 human kinases. The results (Figure 6) showed
activity on only four kinases (GAK, MEK5, TGFBR2 & RIPK2) with a 1
µM concentration of 35. So,
although activity on GAK was reduced compared to
spectrum kinase inhibition profile in vitro.
1, the indazole 35 still maintained a narrow
Figure 6. Representation of the human kinome based on sequence identity-displaying KinomeScan
results for 35 screened at 1 M. Red dots represent hits differentiated by size; kinases inhibited at 1
GAK, 90%; MEK5, 89%; TGFBR2, 82%; RIPK2, 71% (see Supplementary Materials).
µ
µM

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2.6. Electronic Fuikui Modelling Aniline of 4-Anilinoquinolines
In order to rationalize the observed structure activity relationships within the MLM results and
gage propensity for reactive oxidation events to occur, we attempted to model the compound electronics
using frontier molecular orbital theory and Fukui indices [19] (Figure 7). The negative values for f
−
correspond to regions that lose electron density when the molecule undergoes an electrophilic attack
or when the molecule itself acts as a nucleophile [19]. Unfortunately, the results did not align with the
structure activity relationships observed in the MLM.
Figure 7. Electronic mapping of the scaffolds showing Fuikui minus visualized with two isovalues,
0.002 (white) and 0.004 (green): (A) 9, (B) 17, (C) 19, (D) 20, (E) 14, (F) 32, (G) 35, (H) 37, (I) 39, (J) 40.
2.7. Modelling of Lead Compounds in XenoSite
We also analyzed the five lead compounds that retained GAK activity (
the P450 modelling software XenoSite (https://swami.wustl.edu/xenosite/) [20]. The XenoSite models
were trained on a large set of publicly available P450 metabolism data [20 22] to predict the sites of
1, 8, 9, 14, and 35) using
–
metabolism by CYP1A2, -2A6, -2B6, -2C19, -2C8, -2C9, -2D6, -2E1 and -3A4 (Figures 8–13) [20].
Several functional groups were predicted by XenoSite to be metabolically labile, including
the trimethoxy functionality of
1
and
8
and the benzo[d][1,3]dioxol-4-yl of
9
and 14. The bromine
, but this did
substitution of appeared to deactivate the core quinoline structure compared with
1
8
not translate to a large increase in stability (Figure 8; Figure 9). This analysis highlights that the main
metabolism is likely to occur on the aniline ring system. The methylenes of the benzo[d][1,3]dioxol-4-yl
of
9 and 14 were also predicted to be a hot spot for metabolism (Figure 10; Figure 11). In contrast,
the indazole of 35 showed less propensity for metabolism, a prediction that was consistent with the
observed MLM experimental data (Figure 12).
Figure 8. Compound 1 in XenoSite’s P450 Metabolism P450 1.0 algorithm.

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Figure 9. Compound 8 in XenoSite’s P450 Metabolism P450 1.0 algorithm.
Figure 10. Compound 9 in XenoSite’s P450 Metabolism P450 1.0 algorithm.
Figure 11. Compound 14 in XenoSite’s P450 Metabolism P450 1.0 algorithm.
Figure 12. Compound 35 in XenoSite’s P450 Metabolism P450 1.0 algorithm.

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Figure 13. Summary image highlighting the predicted metabolic profiles of 1, 8, 9, 14, and 35 in
XenoSite’s P450 Metabolism P450 1.0 algorithm.
2.8. 4-Anilinoquinolines Stablisation with ABT
Having modelled the P450 metabolism, we measured the stability of analogs 8, 9, 14, and 35
in MLM in comparison to 1. The experiments were run with and without the broad spectrum P450
inhibitor, 1-aminobenzotriazole (ABT) [23] (Table 4). In the absence of ABT, we observed increased
stability of
However, the benzo[d][1,3]dioxol-4-yl analogs
half-lives. In the presence of ABT, , and 35 showed greatly increased stability. Surprisingly, ABT
did not, however, increase the stability of analogs and 14. ABT is reported to inhibit 100% of CYP2A6
8
and 35, with a 5- to 20-fold increase in the half-life in MLM compared to
1 (Table 5).
9
and 14 showed higher intrinsic clearance and shorter
1, 8
9
and -3A4 activity but residual activity (up to 20%) can be measured with CYP1A2, -2B6, -2C8, -2C9,
-2C19, and -2D6 [24]. CYP2C9 was particularly resistant to inhibition by ABT, with 60% of the activity
remaining. A separate study indicated that ABT caused a concentration-dependent inhibition of
CYP1A2, -2B6, -2C9, -2C19, -2D6, and -3A4 [25]. Taken together, our results suggest that CYP2C9
or -2C8 may be responsible for the metabolism of the benzo[d][1,3]dioxol-4-yl group of
9 and 14 in
MLM. XenoSite predicts a metabolism hotspot for these analogs with CYP2C9 or -2C8 that ABT may
be unable to block in MLM.
Table 4. Pharmacokinetic profile of 1 and 8 with and without pre-administration of ABT.
T1/2 (min) a
(−) ABT
Clint
(mL/min/kg)
T1/2 (min) a
(+) ABT
Clint
(mL/min/kg)
T1/2 Ratio b
T1/2 Ratio
Cmpd
1
8
9
14
35
a
5.7
18
0.74
0.74
46
0.58
1.3
0.05
0.05
9.8
990
320
7610
7550
120
130
>200
1.8
2.5
170
13.2
14.7
0.13
0.18
17.5
40
<28
3140
2290
35
mean average, n = 2; b T1/2 ratio following normalization with respect to an internal control (propranolol).
2.9. In Vivo Stabilisation of 1 and 8 with ABT
Having been unable to identify a potent (sub-µM) cell-active GAK inhibitor with improved
metabolic stability in MLM, we decided to test an alternative strategy to enable dosing of a GAK
chemical probe in vivo. Since in vitro inhibition of P450 enzymes by cotreatment with ABT in MLM
had greatly increased the metabolic stability of
A pharmacokinetic experiment was performed in which C57BL/6 mice were dosed orally with 10 mg/kg
of or with or without 2 h pre-treatment with 50 mg/kg ABT (Table 5). As predicted by the microsomal
stability experiment, orally dosed or were rapidly cleared in mice with a T1/2 < 1 h and a low Cmax
(<100 ng/mL). In contrast, pretreatment with ABT resulted in a dramatically extended T1/2 with a
1 and 8, we explored the effect of co-dosing in vivo.
1
8
1
8

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>20-fold higher exposure for
1
and 8-fold higher exposure for
8. In the absence of ABT, the plasma
concentration
pretreatment with ABT resulted in a sustained plasma concentration of
IC50 for 6 h with a 10 mg/kg dose (Figure 14A,B). This shift was even more pronounced for
s
fell below the cellular IC50 after approximately 1 h for
1
and after 2 h for
at 2-fold above the cellular
with
8. In contrast,
1
8
2-fold above the cellular IC50 maintained for >8 h with a 10 mg/kg dose (Figure 14C,D). These results
demonstrate that the decreased metabolism observed upon co-dosing of ABT in MLM can translate to
increased exposure of 4-anilinoquinoline GAK inhibitors in vivo.
Table 5. Pharmacokinetic profile of 1 and 8 with and without pre-administration of ABT.
Experiment
Tmax (h)
Cmax (ng/mL)
AUCinf (h·ng/mL)
10 mpk 1
2 h pretreat 50 mpk ABT 10 mpk 1
10 mpk 8
0.5
0.5
1.0
2.0
97
650
19
98
1900
71
2 h pretreat 50 mpk ABT 10 mpk 8
107
569
Figure 14. Plasma concentration profiles of 1 (A,B) and 8 (C,D) in mice (n = 18) following a 10 mg/kg
po dose with (green) or without (red) 2 h pretreatment with 50 mg/kg ABT.
3. Discussion
Chemical probes are potent and selective modulators of protein function that are useful as
reagents to elucidate the biology of their targets [26 27]. The use of chemical probes in phenotypic
,
models of disease can also provide powerful evidence to support target validation for drug discovery.
Positive results in cellular experiments naturally lead to interest in using the chemical probe for target
validation in animal models. However, many chemical probes are not amenable to use in vivo because
they are unable to attain sufficient exposure to their target protein to support valid experiments.
The pharmacokinetic profiles of probes can be addressed through careful chemical optimization of the
series but at the cost of significant investments of time and resources.
A common pharmacokinetic liability of compounds is that they are rapidly metabolized by
cytochrome P450 enzymes in the liver and cleared from the animal, a process known as first pass
metabolism. As a result, compounds are routinely evaluated for stability in MLM prior to progression

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to in vivo pharmacokinetic studies [28]. It has been well established that molecules with low MLM
stability are prone to rapid first pass metabolism in vivo. The challenge of optimizing compounds to
improve in vivo pharmacokinetic properties while maintaining their target potency and selectivity has
vexed many medicinal chemistry programs, including our own exploration of the dark kinase GAK.
The application of the machine learning algorithms such as XenoSite to develop predictive models
of P450 metabolism has greatly improved the design of molecules with fewer metabolic liabilities.
The XenoSite model accurately predicted the reduced metabolic liability of indazole 35, which showed
improved stability in liver microsomes compared to
1. However, the modest cellular GAK activity of 35
(IC50 = 1.4 M) meant that it was unsuitable for use as a chemical probe. Unfortunately, all of the other
µ
4-aminoquinolines analogs with improved metabolic stability had even poorer GAK activity. We found
that replacement of the 3,4,5-trimethoxy substitution with 5-membered heterocycles annulated at the
aniline 2,3-positions yielded several compounds that maintained high GAK activity in biochemical
assays, but without improvement in microsomal stability. On the other hand, compounds with
3,4-annulation of heterocycles in general proved to be significantly more metabolically stable, but at the
cost of weakened GAK activity. The identification of 3,4,5-trimethoxyphenyl heterocyclic replacement
groups with increased microsomal stability is a valuable learning tool. The 3,4,5-trimethoxyphenyl
group is a commonly encountered substructure in ATP-competitive inhibitors of protein kinases
and in a variety of other molecules with biological activity, such as the antibiotic trimethoprim, the
antihypertensive reserpine, the anti-inflammatory colchicine, and the DHFR-inhibitor trimetrexate for
the treatment of leiomyosarcoma [29]. Thus, other medicinal chemistry campaigns requiring isosteres
of the 3,4,5-trimethoxyphenyl group may consider incorporation of select heterocyclic replacements
identified here.
Although our original goal of the chemical optimization of 1 to decrease intrinsic metabolism while
retaining potent GAK activity remained unrealized, we were able to use co-dosing with ABT to reduce
P450 metabolism and increase in vivo exposure. ABT is a potent inhibitor of CYP3A4, CYP2D6, and
several other P450 enzymes, and has been successfully employed to enhance the pharmacokinetics of
metabolically labile compounds [24,30]. Although not an approved drug for administration to patients,
co-dosing with ABT to block oxidative metabolism in animal models is significantly less demanding of
time and resources than chemical optimization, and may be broadly applicable for the development of
in vivo chemical probes [31]. For example, co-dosing of ABT with the antifungal APX001 increased
exposure of the compound and enhanced its efficacy in a murine model of invasive candidiasis [32].
Long-term dosing of ABT is also possible. ABT has displayed a well-behaved pharmacokinetic profile
over five days when dosing with an osmotic pump [25]. Minimal side effects were observed for chronic
ABT dosing, the most prominent of which was delayed gastric emptying [33]. ABT can also be utilized
in species other than mice for in vivo studies. ABT is a competent cytochrome P450 inhibitor in rats,
guinea pigs, and other species, and has been used to enhance pharmacokinetic exposure in these
animals [30,34]. Our results further extend the utility of ABT by demonstrating an increase of in vivo
exposure for certain 4-anilinoquinoline GAK inhibitors in mice.
Not all 4-anilinoquinolines showed reduced MLM metabolism in the presence of ABT.
The 4-anilinoquinolines 9 and 14 containing the benzo[d][1,3]dioxol-4-yl group had shorter half-lives
than
1 in MLM and were still rapidly metabolized in the presence of ABT. Notably, the XenoSite
model predicted that the benzo[d][1,3]dioxol-4-yl group was a hotspot for metabolism by CYP2C9
and CYP2C8, two P450s that are not strongly inhibited by ABT. For compounds such as these, it
may be possible to use the co-dosing of other P450 inhibitors such as atipamezole to block CYP2C8/9
activity [35]. The development of a range of broad spectrum P450 inhibitors with overlapping profiles
that can be used safely in animal models to block P450 metabolism would be a powerful addition to
the medicinal chemist’s toolbox.

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4. Materials and Methods
4.1. Computational Methods
Small molecules were prepared using the ligprep module of Schrödinger (Schrödinger Release
2018-4: LigPrep, Schrödinger, LLC, New York, NY, USA, 2018.) Structures were superimposed using
flexible ligand alignment functionality (Schrödinger Release 2018-4: Maestro, Schrödinger, LLC,
New York, NY, USA, 2018) using compound
for common substructure recognition [14].
8 as a template and employing the Bemis–Murcko method
4.2. Biology Methods
4.2.1. NAK Family TR-FRET Assays
Assays performed as previously described [12].
4.2.2. GAK NanoBRET Assay
Assays performed as previously described [9].
4.2.3. Metabolite Identification
Compounds were incubated at a concentration of 10 µM with mouse liver microsomes in the
presence of an excess of NADPH. The duplicate incubations were performed in 0.5 mL 96-well plates
in a shaking water bath maintained at 37 ◦C for 30 min and quenched by the addition of an equal
volume of methanol (see Supplementary Materials for full details and reports).
4.2.4. Pharmacokinetic Profiling
Pharmacokinetic profiling was performed by SAI Life Sciences (Hyderabad, India). All procedures
of the present study were in accordance with the guidelines provided by the Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA) as published in the Gazette
of India, 15 December 1998. Prior approval of the Institutional Animal Ethics Committee (IAEC)
was obtained before initiation of the study. Healthy male C57BL/6 mice (8–12 weeks old) weighing
between 20 and 35 g were procured from (Envigo Research Private Ltd., City, Hyderabad, India).
Three mice were housed in each cage. Temperature and humidity were maintained at 22
±
3 ◦C and
30–70%, respectively, and illumination was controlled to give a sequence of a 12 h light and 12 h dark
cycle. Temperature and humidity were recorded by an automatically controlled data logger system.
All animals were provided a laboratory rodent diet (Envigo Research Private Ltd., Hyderabad, India).
Reverse osmosis water treated with ultraviolet light was provided ad libitum. Eighteen male mice
were weighed and divided in to two groups (Group 1 and Group 2), with nine mice in each group.
Animals in Group 1 were administered orally with a solution formulation of
Animals in Group 2 were administered orally with a solution formulation of ABT (2 h before dosing
of or ) at 50 mg/kg dose. After 2 h of ABT administration, animals were administered orally with
solution formulation of or at 10 mg/kg dose. Solution formulation of or was prepared in 5%
NMP, 5% solutol HS-15 in normal saline. The dosing volume administered was 10 mL/kg for the oral
route. Blood (~60 L) samples were collected from a set of three mice at each time point in a labeled
1 or 8 at 10 mg/kg dose.
1
8
1
8
1
8
µ
micro centrifuge tube containing K2EDTA solution as anticoagulant at pre-dose, 0.5, 1, 2, 4, 6, 8, 12,
and 24 h. Plasma samples were separated by centrifugation at 4000 rpm for 10 min and stored below
◦
−
70 C until bioanalysis. Concentrations of
1
or
8
in mice plasma samples were determined by a
or was provided for data analysis
LC-MS/MS method. The plasma concentration-time data for
1
8
by the bioanalytical group (Sai Life Sciences Ltd., Pune, India). The plasma concentration-time data
were then used for the pharmacokinetic analysis. A non-compartmental analysis module in Phoenix
WinNonlin (Version 6.3, Certara USA Inc., Raleigh, NC, USA) was used to assess the pharmacokinetic
parameters. Maximum concentration (Cmax) and time to reach the maximum concentration (Tmax
)

Molecules 2019, 24, 4016
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were the observed values. The areas under the concentration time curve (AUClast and AUCinf) were
calculated by linear trapezoidal rule.
4.2.5. KinomeScan Assay
KinomeScan Assay on 35 was performed as previously described at EuroFins DiscoverX (Fremont,
CA, USA) [7].
4.3. Chemistry Methods and Compound Characterization
4.3.1. General Information
All reactions were performed using flame-dried round-bottomed flasks or reaction vessels.
Where appropriate, reactions were carried out under an inert atmosphere of nitrogen with dry
solvents, unless otherwise stated. Yields refer to chromatographically and spectroscopically pure
isolated yields. Reagents were purchased at the highest commercial quality and used without further
purification. Reactions were monitored by thin-layer chromatography carried out on 0.25-mm E.
Merck silica gel plates (60F-254) using ultraviolet light as a visualizing agent. NMR spectra were
recorded on a Varian Inova 400 (Varian, Palo Alto, CA, USA) or Inova 500 spectrometer (Varian, Palo
Alto, CA, USA) and calibrated using residual undeuterated solvent as an internal reference (CDCl3:
1H NMR = 7.26 13C NMR = 77.16). The following abbreviations or combinations thereof were used to
,
explain the multiplicities observed: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad. Liquid chromatography (LC) and high resolution mass spectra (HRMS) were recorded
on a ThermoFisher hybrid LTQ FT (ICR 7T) (ThermoFisher, Waltham, MA USA). The University
of Southampton (Southampton, UK) small molecule x-ray facility collected and analyzed all X-ray
diffraction data.
Compounds
1, 8, and 13 were synthesized as previously reported [8,9,12]. Compound spectra of
9
–12
,
14
–
43 are included in the Supplementary Materials. All compounds were >98% pure by 1H/13C
NMR and LCMS.
4.3.2. General Procedure for the Synthesis of 4-anilinoquinolines (Scheme 1)
6-Bromo-4-chloroquinoline (1.0 equiv.) and 3,4,5-trimethoxyaniline (1.1 equiv.) were suspended in
ethanol (10 mL) and refluxed for 18 h. The crude mixture was purified by flash chromatography using
EtOAc:hexane followed by 1–5% methanol in EtOAc, solvent was removed under reduced pressure to
afford the desired product (1, 8–11, 13–31, and 33–43).
4.3.3. General Procedure for the Synthesis of 4-anilinoquinolines (Scheme 2)
4-Chloro-6-(trifluoromethyl)quinoline (1.0 equiv.), aniline (1.1 equiv.), bis(dibenzylideneacetone)
palladium(0) (Pd(dba)2) (0.1 equiv.), 2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl (XPhos)
(0.15 equiv.), and cesium carbonate (2.0 equiv.) were all suspended in dimethylformamide 15 mL and
degassed for 5 min. The mixture was heated to 140 ◦C for 18 h. The crude mixture was passed through
a plug of Celite 545 before been purified by flash chromatography 20–100% ethyl acetate/hexane
followed by 1–5% methanol/ethyl acetate and solvent removed under reduced pressure to afford the
desired product (12 and 32).
4.3.4. Compound Characterization
N-(Benzo[d][1,3]dioxol-4-yl)-6-bromoquinolin-4-amine (
9
) was obtained as a yellow solid (157 mg,
1
0.458 mmol, 74%). m.p. > 270 ◦C decomp.; H NMR (400 MHz, DMSO-d6)
δ
11.21 (s, 1H), 9.24 (d,
J = 1.9 Hz, 1H), 8.57 (d, J = 6.9 Hz, 1H), 8.25–8.02 (m, 2H), 7.16–6.81 (m, 3H), 6.65 (d, J = 6.9 Hz, 1H),
6.11 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 153.1, 148.8, 142.9, 141.5, 137.2, 136.5, 126.3, 122.6, 122.5,
120.0, 119.5, 119.1, 118.4, 107.9, 101.84, 101.80. HRMS m/z [M + H]+ calculated for C16H12N2O2Br:
343.0082, found 343.0072, LC tR = 3.43 min, >98% purity.

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6-Bromo-N-(2,5-difluorophenyl)quinolin-4-amine (10) was obtained as a colorless solid (125 mg, 0.373 mmol,
60%). m.p. 187–189 ◦C; 1H NMR (400 MHz, DMSO-d6)
11.11 (s, 1H), 9.21 (d, J = 2.0 Hz, 1H), 8.59 (d,
J = 6.9 Hz, 1H), 8.20 (dd, J = 9.1, 1.9 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.78–7.46 (m, 2H), 7.38–7.25 (m,
1H), 6.56 (dd, J = 6.9, 2.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) 161.5 (dd, J = 247.9, 11.7 Hz), 157.1
δ
δ
(dd, J = 251.6, 13.2 Hz), 154.5, 143.4, 137.3, 136.7, 130.2 (dd, J = 10.2, 2.0 Hz), 126.2, 122.7, 121.0 (dd,
J = 12.6, 3.9 Hz), 120.2, 118.4, 113.0 (dd, J = 22.6, 3.7 Hz), 105.8 (dd, J = 27.1, 24.0 Hz), 100.9 (d, J = 1.8
Hz). HRMS m/z [M + H]+ calculated for C15H10N2F2Br: 334.9995, found 334.9985, LC tR = 3.65 min,
>98% purity.
6-Bromo-N-(2,4-difluorophenyl)quinolin-4-amine (11) was obtained as a colorless solid (94 mg, 0.281 mmol,
45%). m.p. 301–303 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 11.18 (s, 1H), 9.20 (d, J = 2.0 Hz, 1H), 8.62 (d,
J = 6.8 Hz, 1H), 8.20 (dd, J = 9.0, 2.0 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.65–7.48 (m, 2H), 7.40 (ddt, J = 9.2
,
,
8.0, 3.5 Hz, 1H), 6.68 (dd, J = 6.8, 2.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) 158.2 (dd, J = 242.3
δ
2.3 Hz), 153.2 (dd, J = 245.2, 2.9 Hz), 153.9, 143.6, 137.4, 136.7, 126.2, 125.6 (dd, J = 14.7, 11.0 Hz),
122.8, 120.3, 118.5, 118.4 (dd, J = 22.5, 9.7 Hz), 116.3 (dd, J = 23.9, 8.2 Hz), 115.5, 115.2, 101.5 (d, J = 2.3
Hz). HRMS m/z [M + H]+ calculated for C15H10N2F2Br: 334.9995, found 334.9985, LC tR = 3.63 min
>98% purity.
,
6-Bromo-N-(perfluorophenyl)quinolin-4-amine (12) was obtained as a brown solid (51.4 mg, 0.136 mmol,
21%) m.p. > 300 ◦C; 1H NMR (400 MHz, Methanol-d4)
8.91 (d, J = 4.8 Hz, 1H), 8.56 (dp, J = 1.9,
0.9 Hz, 1H), 8.25 (dp, J = 8.8, 0.8 Hz, 1H), 8.05 (dd, J = 8.8, 2.1 Hz, 1H), 7.79 (d, J = 4.8 Hz, 1H).
13C NMR (100 MHz, Methanol-d4) 153.7, 150.9 (d, J = 1.3 Hz), 145.0, 140.7–140.3 (m, 1C), 139.1–138.8,
δ
δ
138.2–137.2, 136.7–136.5, 134.6–134.3, 131.9, 130.7(q, J = 32.9 Hz), 127.4 (q, J = 3.1 Hz), 127.0, 126.57,
124.1, 123.9, 123.2 (q, J = 4.6 Hz). HRMS m/z [M + H]+ calculated for C16H7N2F8: 379.0481, found
379.0473, LC tR = 4.38 min, >98% purity.
6-Bromo-N-(5-fluorobenzo[d][1,3]dioxol-4-yl)quinolin-4-amine (14) was obtained as a beige solid (174 mg,
0.483 mmol, 78%). m.p. 224–227 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.08 (s, 1H), 9.26 (d, J = 1.7 Hz
1H), 8.63 (d, J = 6.8 Hz, 1H), 8.28–8.06 (m, 2H), 7.04 (dd, J = 8.7, 4.1 Hz, 1H), 6.94 (dd, J = 10.8, 8.6 Hz,
1H), 6.67 (dd, J = 6.8, 1.2 Hz, 1H), 6.17 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
153.8, 152.4 (d,
,
δ
J = 240.7 Hz), 144.8 (d, J = 1.8 Hz), 143.9 (d, J = 6.3 Hz), 143.4, 137.3, 136.8, 126.1, 122.7, 120.4, 118.4,
108.7 (d, J = 20.5 Hz), 107.9 (d, J = 22.4 Hz), 107.5 (d, J = 9.2 Hz), 103.2, 101.7. HRMS m/z [M + H]+
calculated for C16H10N2O2FBr: 359.9910, found 360.9987, LC tR = 3.53 min, >98% purity.
6-Bromo-N-(2,3-dimethoxyphenyl)quinolin-4-amine (15) was obtained as a colorless solid (187 mg,
◦
1
0.520 mmol, 84%). m.p. 150–153 C; H NMR (400 MHz, DMSO-d6)
δ 11.03–10.85 (br s, 1H),
9.19 (d, J = 2.0 Hz, 1H), 8.50 (d, J = 6.9 Hz, 1H), 8.18 (dd, J = 9.0, 2.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.26
(dd, J = 8.4, 7.7 Hz, 1H), 7.20 (dd, J = 8.4, 1.7 Hz, 1H), 7.00 (dd, J = 7.7, 1.7 Hz, 1H), 6.39 (d, J = 6.9 Hz,
1H), 3.89 (s, 3H), 3.67 (s, 3H). 13C NMR (101 MHz, DMSO- d6)
δ 155.1, 154.0, 144.8, 143.2, 137.7, 137.0,
130.5, 126.6, 125.2, 123.0, 120.3, 119.9, 118.6, 113.5, 101.6, 61.2, 56.5. HRMS m/z [M + H]+ calculated for
C17H16N2O2Br: 359.0395, found 359.0284, LC tR = 3.62 min, >98% purity.
6-Bromo-N-(2,2-difluorobenzo[d][1,3]dioxol-4-yl)quinolin-4-amine (16) was obtained as a colorless solid
(190 mg, 0.501 mmol, 81%). m.p. 272–274 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.36 (s, 1H), 9.22 (d,
J = 2.0 Hz, 1H), 8.68 (d, J = 6.8 Hz, 1H), 8.21 (dd, J = 9.0, 2.0 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.50 (dd,
J = 7.5, 1.7 Hz, 1H), 7.44–7.21 (m, 2H), 6.82 (d, J = 6.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
153.2,
δ
144.1, 143.6, 137.4, 137.1, 136.8, 131.0 (t, J = 254.8 Hz), 126.3, 125.5, 122.7, 122.2, 120.4 (d, J = 3.0 Hz, 2C),
118.7, 109.2, 101.9. HRMS m/z [M + H]+ calculated for C16H10N2O2F2Br: 378.9894, found 378.9880,
LC tR = 4.13 min, >98% purity.
6-Bromo-N-(2,3-dihydrobenzofuran-4-yl)quinolin-4-amine (17) was obtained as a yellow solid (133 mg,
0.390 mmol, 64%). m.p. 291–293 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.09 (s, 1H), 9.19 (d, J = 2.0 Hz,
1H), 8.52 (d, J = 6.9 Hz, 1H), 8.17 (dd, J = 9.0, 2.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H),
6.89 (ddd, J = 10.4, 8.0, 0.8 Hz, 2H), 6.56 (d, J = 6.9 Hz, 1H), 4.59 (t, J = 8.7 Hz, 2H), 3.10 (t, J = 8.7 Hz
,

Molecules 2019, 24, 4016
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2H). 13C NMR (100 MHz, DMSO-d6)
δ
161.4, 153.6, 143.1, 137.3, 136.6, 133.7, 129.5, 126.3, 124.2, 122.5,
119.9, 118.5, 117.8, 108.6, 100.9, 71.2, 27.9. HRMS m/z [M + H]+ calculated for C17H14N2OBr: 341.0289,
found 341.0283, LC tR = 3.59 min, >98% purity.
6-Bromo-N-(2,3-dihydrobenzofuran-7-yl)quinolin-4-amine (18) was obtained as a yellow solid (173 mg,
◦
1
0.507 mmol, 82%). m.p. 301–304 C; H NMR (400 MHz, DMSO-d6)
1H), 8.52 (d, J = 6.9 Hz, 1H), 8.22–8.04 (m, 2H), 7.32 (dq, J = 7.3, 1.1 Hz, 1H), 7.23–7.15 (m, 1H), 6.99
(dd, J = 7.9, 7.3 Hz, 1H), 6.44 (d, J = 6.9 Hz, 1H), 4.59 (t, J = 8.7 Hz, 2H), 3.30 (t, J = 8.7 Hz, 2H). 13
δ 11.08 (s, 1H), 9.25–9.17 (m,
C
NMR (101 MHz, DMSO-d6)
δ 153.9, 153.5, 142.5, 137.1, 136.4, 129.8, 126.2, 125.5, 124.6, 122.4, 121.3,
119.8, 119.1, 118.2, 101.5, 71.9, 29.3. HRMS m/z [M + H]+ calculated for C17H14N2OBr: 341.0289, found
341.0285 LC tR = 3.70 min, >98% purity.
N-(Benzofuran-4-yl)-6-bromoquinolin-4-amine (19) was obtained as a yellow solid (162 mg, 0.476 mmol,
77%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 11.36 (s, 1H), 9.27 (d, J = 2.0 Hz, 1H), 8.50 (d,
J = 6.9 Hz, 1H), 8.20 (dd, J = 9.0, 2.0 Hz, 1H), 8.16–7.90 (m, 2H), 7.73 (dt, J = 8.3, 0.9 Hz, 1H), 7.51 (t,
J = 8.0 Hz, 1H), 7.38 (dd, J = 7.7, 0.8 Hz, 1H), 6.91 (dd, J = 2.2, 1.0 Hz, 1H), 6.52 (d, J = 6.9 Hz, 1H).
13C NMR (100 MHz, DMSO- d6)
δ 155.4, 154.0, 146.7, 143.0, 137.4, 136.5, 130.0, 129.7, 126.5, 125.3,
123.7, 122.5, 120.2, 119.9, 118.7, 111.1, 105.2, 101.0. HRMS m/z [M + H]+ calculated for C17H12N2OBr:
339.0133, found 339.0129, LC tR = 3.82 min, >98% purity.
N-(Benzofuran-7-yl)-6-bromoquinolin-4-amine (20) was obtained as a yellow solid (157 mg, 0.464 mmol,
75%). m.p. 256–259 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 11.37 (s, 1H), 9.27 (d, J = 2.0 Hz, 1H), 8.53
(d, J = 6.9 Hz, 1H), 8.21 (dd, J = 9.1, 2.0 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.79
(dd, J = 6.0, 2.9 Hz, 1H), 7.55–7.26 (m, 2H), 7.13 (d, J = 2.1 Hz, 1H), 6.46 (d, J = 6.9 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6)
δ 154.0, 148.3, 146.7, 143.1, 137.3, 136.7, 129.5, 126.2, 124.0, 122.6, 122.0, 121.3,
121.2, 120.4, 118.5, 107.5, 101.4. HRMS m/z [M + H]+ calculated for C17H12N2OBr: 339.0133, found
339.0129, LC tR = 4.36 min, >98% purity.
N-(6-Bromoquinolin-4-yl)benzo[d]is◦oxazol-7-amine (21) was obtained as a colorless solid (141 mg,
1
0.414 mmol, 67%). m.p. > 300 C; H NMR (400 MHz, DMSO-d6)
δ
11.49 (s, 1H), 9.45 (s, 1H),
9.29 (d, J = 2.0 Hz, 1H), 8.45 (d, J = 6.9 Hz, 1H), 8.31 (dd, J = 6.8, 2.4 Hz, 1H), 8.21 (dd, J = 9.0, 2.0 Hz,
1H), 8.11 (d, J = 9.0 Hz, 1H), 7.75–7.62 (m, 2H), 6.41 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ
157.6, 154.5, 148.3, 142.7, 137.3, 136.6, 135.8, 130.7, 126.4, 126.2, 123.9, 122.6, 122.5, 120.0, 118.4,
101.9. HRMS m/z [M + H]+ calculated for C16H11N3OBr: 340.0085, found 340.0074, LC tR = 3.18 min
,
>98% purity.
N-(6-Bromoquinolin-4-yl)benzo[d]isoxazol-4-amine (22) was obtained as a dark green solid (122 mg,
0.359 mmol, 58%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
11.37 (s, 1H), 9.31 (s, 1H), 9.24 (d,
J = 2.0 Hz, 1H), 8.58 (d, J = 6.8 Hz, 1H), 8.22 (dd, J = 9.0, 1.9 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.93–7.70
(m, 2H), 7.53 (dd, J = 4.6, 3.7 Hz, 1H), 6.85 (d, J = 6.8 Hz, 1H). 13C NMR (101 MHz, DMSO- d6)
162.7,
δ
δ
153.9, 145.7, 143.7, 137.6, 136.7, 131.8, 131.4, 126.5, 122.8, 120.2, 120.1, 119.2, 117.4, 108.6, 101.6. HRMS
m/z [M + H]+ calculated for C16H11N3OBr: 340.0085, found 340.0084, LC tR = 3.38 min, >98% purity.
N-(6-Bromoquinolin-4-yl)benzo[d]oxazol-7-amine (23) was obtained as a dark yellow solid (65 mg,
◦
1
0.192 mmol, 31%). m.p. 305–308 C; H NMR (400 MHz, DMSO-d6)
δ 10.80 (s, 1H), 10.16 (s,
1H), 9.10 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 6.9 Hz, 1H), 8.18 (dd, J = 9.0, 2.0 Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H),
7.49 (d, J = 7.9 Hz, 1H), 7.21 (t, J = 7.9 Hz, 1H), 6.50 (d, J = 6.9 Hz, 1H). 13C NMR (176 MHz, DMSO-d6)
δ
160.9, 154.9, 149.2, 143.9, 143.3, 138.2, 136.8, 128.5, 127.0, 126.9, 126.3, 123.5, 121.1, 120.0, 119.1,
101.5. HRMS m/z [M + H]+ calculated for C16H11N3OBr: 340.0085, found 340.0084, LC tR = 3.67 min
,
>98% purity.
N-(6-Bromoquinolin-4-yl)benzo[d]thiazol-7-amine (24) was obtained as a dark yellow solid (84 mg,
0.235 mmol, 38%). m.p. 240–243 ◦C; 1H NMR (400 MHz, DMSO-d6)
9.54 (s, 1H), 9.39 (d, J = 0.4 Hz,
1H), 8.73 (t, J = 1.4 Hz, 1H), 8.44 (d, J = 5.2 Hz, 1H), 8.05 (dd, J = 8.2, 1.0 Hz, 1H), 7.96–7.75 (m, 2H), 7.66 (t,
δ

Molecules 2019, 24, 4016
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J = 7.9 Hz, 1H), 7.55–7.46 (m, 1H), 6.29 (d, J = 5.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ
156.1, 154.9,
151.1, 147.5, 145.9, 134.7, 132.4, 131.5, 129.2, 127.3, 124.7, 121.8, 120.9, 120.4, 117.9, 103.6. HRMS m/z
[M + H]+ calculated for C16H11N3F: 355.9857, found 355.9846, LC tR = 3.20 min, >98% purity.
N-(6-Bromoquinolin-4-yl)benzo[d]thiaz◦ol-4-amine (25) was obtained as a colorless solid (150 mg,
1
0.421 mmol, 68%). m.p. 281–283 C; H NMR (400 MHz, DMSO- d6)
δ
11.46 (s, 1H), 9.45 (s,
1H), 9.27 (d, J = 2.0 Hz, 1H), 8.45 (d, J = 6.9 Hz, 1H), 8.37–8.25 (m, 1H), 8.21 (dd, J = 9.0, 2.0 Hz, 1H), 8.10
(d, J = 9.0 Hz, 1H), 7.75–7.59 (m, 2H), 6.42 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ
157.6,
154.4, 148.3, 142.7, 137.4, 136.6, 135.8 130.7, 126.4, 126.2, 123.9, 122.7, 122.5, 120.0, 118.4, 101.9. HRMS m/z
[M + H]+ calculated for C16H11N3SBr: 355.9857, found 355.9846, LC tR = 3.30 min, >98% purity.
N-(Benzo[b]thiophen-4-yl)-6-bromoquinolin-4-amine (26) was obtained as a dark yellow solid (147 mg,
0.414 mmol, 67%). m.p. 308–311 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.40 (s, 1H), 9.28 (d, J = 2.0 Hz
,
,
,
1H), 8.46 (d, J = 6.9 Hz, 1H), 8.20 (dd, J = 9.0, 2.0 Hz, 1H), 8.16 (dt, J = 8.1, 0.9 Hz, 1H), 8.11 (d, J = 9.0 Hz
1H), 7.88 (dd, J = 5.5, 0.4 Hz, 1H), 7.60–7.52 (m, 1H), 7.49 (dd, J = 7.5, 1.0 Hz, 1H), 7.37 (dd, J = 5.6
0.8 Hz, 1H), 6.37 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 154.6, 143.0, 141.1, 137.4, 136.6,
135.8, 131.4, 129.1, 126.5, 125.3, 122.8, 122.5, 122.4, 121.3, 119.8, 118.6, 100.9. HRMS m/z [M + H]+
calculated for C17H12N2SBr: 354.9905, found 354.9900, LC tR = 4.64 min, >98% purity.
N-(1H-Benzo[d]imidazol-7-yl)-6-bromoquinolin-4-amine (27) was obtained as a dark yellow solid (25 mg,
0.074 mmol, 12%). m.p. 270–273 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
8.85 (d, J = 1.4 Hz, 1H), 8.37 (d,
J = 5.3 Hz, 1H), 8.15 (s, 1H), 7.80 (d, J = 1.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.24 (t, J = 7.7 Hz, 1H), 7.18
(d, J = 7.5 Hz, 1H), 6.49 (dd, J = 5.3, 1.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 175.3, 151.0, 151.0,
147.9, 147.5, 141.9, 141.6, 139.8, 132.0, 131.3, 129.2, 125.1, 122.0, 121.0, 117.3, 117.0. HRMS m/z [M + H]+
calculated for C16H12N4Br: 339.0245, found 339.0232, LC tR = 2.46 min, >98% purity.
N-(6-Bromoquinolin-4-yl)benzo[c][1,2,5]oxadiazol-4-amine (28) was obtained as a black solid (152 mg,
◦
1
0.445 mmol, 72%). m.p. > 300 C; H NMR (400 MHz, DMSO-d6)
δ 11.65 (s, 1H), 9.29 (d, J = 2.0
Hz, 1H), 8.63 (d, J = 6.8 Hz, 1H), 8.23 (dd, J = 9.0, 1.9 Hz, 1H), 8.18–8.06 (m, 2H), 7.83–7.70 (m, 2H),
6.94 (d, J = 6.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 153.6, 150.3, 145.9, 143.3, 137.4, 136.8, 133.2,
128.0, 126.4, 125.7, 122.8, 120.5, 118.9, 115.3, 103.5. HRMS m/z [M + H]+ calculated for C15H10N4OBr:
341.0038, found 341.0027, LC tR = 3.39 min, >98% purity.
N-(6-Bromoquinolin-4-yl)benzo[c][1,2,5]thiadiazol-4-amine (29) was obtained as a yellow solid (164 mg,
0.458 mmol, 74%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.54 (s, 1H), 9.28 (d, J = 2.0 Hz,
1H), 8.48 (d, J = 6.8 Hz, 1H), 8.19 (ddd, J = 8.5, 6.4, 1.6 Hz, 2H), 8.12 (d, J = 9.0 Hz, 1H), 7.95–7.78
(m, 2H), 6.61 (d, J = 6.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 155.6, 153.8, 149.9, 143.3, 137.9,
136.5, 130.3, 129.3, 126.2, 126.1, 123.2, 120.8, 120.1, 118.8, 103.0. HRMS m/z [M + H]+ calculated for
C15H10N4SBr: 356.9810, found 356.9798, LC tR = 3.34 min, >98% purity.
6-Bromo-N-(1-methyl-1H-benzo[d][1,2,3]triazol-4-yl)quinolin-4-amine (30) was obtained as a colorless solid
(118 mg, 0.334 mmol, 54%). m.p. 202–205 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 9.54 (s, 1H), 8.82 (dd,
J = 1.9, 0.8 Hz, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.98–7.75 (m, 2H), 7.64 (dd, J = 8.3, 0.9 Hz, 1H), 7.57
(dd, J = 8.3, 7.3 Hz, 1H), 7.31 (dd, J = 7.3, 1.0 Hz, 1H), 6.73 (d, J = 5.3 Hz, 1H), 4.33 (s, 3H). 13C NMR
(100 MHz, DMSO-d6)
δ 151.0, 147.5, 146.9, 139.6, 135.0, 132.2, 131.7, 131.4, 128.0, 124.9, 121.4, 117.9,
116.2, 106.2, 104.9, 34.3. HRMS m/z [M + H]+ calculated for C16H13N5Br: 354.0354, found 354.0343,
LC tR = 3.04 min, >98% purity.
6-Bromo-N-(1-methyl-1H-indazol-4-yl)quinolin-4-amine (31) was obtained as a dark gray solid (131 mg,
0.371 mmol, 60%). m.p. 206–209 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 11.38 (s, 1H), 9.29 (d, J = 2.0 Hz,
1H), 8.49 (d, J = 6.9 Hz, 1H), 8.19 (dd, J = 9.0, 2.0 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 0.9 Hz,
1H), 7.75 (dt, J = 8.5, 0.8 Hz, 1H), 7.56 (dd, J = 8.5, 7.3 Hz, 1H), 7.26 (dd, J = 7.3, 0.7 Hz, 1H), 6.59 (d,
J = 6.9 Hz, 1H), 4.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 154.1, 142.9, 141.0, 137.4, 136.5, 130.9,

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129.3, 126.8, 126.5, 122.5, 119.9, 119.2, 118.8, 117.2, 109.6, 101.3, 35.7. HRMS m/z [M + H]+ calculated for
C17H14N4Br: 353.0402, found 353.0340, LC tR = 3.48 min, >98% purity.
6-Bromo-N-(1H-indazol-4-yl)quinolin-4-amine (32) was obtained as a bright yellow solid (174 mg,
◦
1
0.513 mmol, 83%). m.p. > 300 C; H NMR (400 MHz, DMSO-d6)
δ 13.51 (s, 1H), 11.34 (s, 1H),
9.27 (d, J = 2.0 Hz, 1H), 8.50 (d, J = 6.9 Hz, 1H), 8.20 (dd, J = 9.0, 2.0 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H),
8.02 (d, J = 1.1 Hz, 1H), 7.65 (dt, J = 8.4, 0.9 Hz, 1H), 7.52 (dd, J = 8.4, 7.2 Hz, 1H), 7.23 (dd, J = 7.3,
0.7 Hz, 1H), 6.60 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 154.1, 143.0, 141.4, 137.4, 136.5,
131.9, 129.2, 126.8, 126.4, 122.5, 119.9, 118.8, 118.6, 117.1, 110.1, 101.2. HRMS m/z [M + H]+ calculated
for C16H12N4Br: 339.0245, found 339.0234, LC tR = 3.08 min, >98% purity.
N-(1H-Indazol-4-yl)-6-(trifluoromethyl)quinolin-4-amine (33) was obtained as a dark yellow solid (36 mg,
0.110 mmol, 17%). m.p. 106–109 ◦C; 1H NMR (700 MHz, DMSO-d6)
δ
9.27 (d, J = 4.6 Hz, 1H), 8.89 (s,
1H), 8.82 (d, J = 2.0 Hz, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.15 (dd, J = 8.9, 2.1 Hz, 1H), 8.02 (d, J = 4.6 Hz
1H), 6.99 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 8.3, 7.1 Hz, 1H), 6.40 (d, J = 7.0 Hz, 1H), 5.51 (s, 1H), 3.50 (s,
,
1H). 13C NMR (176 MHz, DMSO-d6)
δ 154.0, 150.6, 144.4, 138.8, 131.7, 128.1 (q, J = 32.1 Hz), 126.7, 126.6,
126.1, 125.2, 123.6, 123.4, 121.7, 118.4, 107.4, 104.8, 40.3. HRMS m/z [M + H]+ calculated for C17H12N4F3:
329.1014, found 353.0340, LC tR = 5.66 min, >98% purity.
N-(1H-Indazol-7-yl)-6-(trifluoromethyl)quinolin-4-amine (34) was obtained as a bright yellow solid (189 mg,
0.576 mmol, 89%). m.p. 251–254 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
13.43 (s, 1H), 11.89–11.60 (m, 1H),
9.55–9.41 (m, 1H), 8.56 (d, J = 7.0 Hz, 1H), 8.47–8.21 (m, 2H), 8.15 (d, J = 1.0 Hz, 1H), 7.93 (dd, J = 8.5
0.7 Hz, 1H), 7.70 (dt, J = 1.9, 0.9 Hz, 1H), 7.21 (dd, J = 8.5, 1.8 Hz, 1H), 6.89 (d, J = 7.0 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) 156.2, 144.4, 140.6, 135.2, 134.1 (1C, br s), 129.7 (1C, d, J = 3.1 Hz), 128.3, 127.2
,
δ
(q, J = 32.9 Hz), 125.6, 123.3 (d, J = 4.2 Hz), 122.8, 122.5, 122.3 (d, J = 5.9 Hz), 118.6, 117.2, 107.4 (1C, br
s), 101.6. HRMS m/z [M + H]+ calculated for C17H12N4F3: 329.1014, found 329.1006, LC tR = 3.42 min
>98% purity.
,
6-Bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) was obtained as an orange solid (180 mg, 0.532 mmol,
86%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 13.34 (s, 1H), 11.19 (s, 1H), 9.18 (d, J = 2.0 Hz,
1H), 8.51 (d, J = 6.9 Hz, 1H), 8.47–8.09 (m, 2H), 8.07 (d, J = 9.0 Hz, 1H), 7.95 (dd, J = 8.5, 0.7 Hz,
1H), 7.66 (dt, J = 1.9, 0.9 Hz, 1H), 7.20 (dd, J = 8.5, 1.8 Hz, 1H), 6.88 (d, J = 6.9 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6)
δ 154.3, 143.1, 140.2, 137.4, 136.5, 135.0, 133.8, 126.2, 122.6, 122.1, 121.9, 119.8,
118.7, 118.2, 106.7, 100.6. HRMS m/z [M + H]+ calculated for C16H12N4Br: 339.0245, found 339.0243,
LC tR = 3.33 min, >98% purity.
6-Bromo-N-(5-fluoro-1H-indazol-6-◦yl)quinolin-4-amine (36) was obtained as a beige solid (188 mg,
1
0.526 mmol, 85%). m.p. > 300 C; H NMR (400 MHz, DMSO-d6)
δ
13.51 (s, 1H), 11.24 (s, 1H),
9.23 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 6.9 Hz, 1H), 8.49–8.13 (m, 2H), 8.11 (d, J = 9.0 Hz, 1H), 7.88 (d,
J = 10.3 Hz, 1H), 7.80 (dd, J = 6.3, 1.1 Hz, 1H), 6.61 (dd, J = 6.9, 2.5 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6)
δ 154.8, 151.9 (d, J = 240.3 Hz), 143.3, 137.3, 136.7, 136.5, 134.4–133.0 (m, 1C), 126.2, 123.9 (d,
J = 17.7 Hz), 122.6, 121.6 (d, J = 9.5 Hz), 120.1, 118.4, 109.8, 106.5 (d, J = 22.1 Hz), 101.2. HRMS m/z
[M + H]+ calculated for C16H11N4FBr: 357.0151, found 357.0150, LC tR = 3.43 min, >98% purity.
6-Bromo-N-(1H-indazol-5-yl)quinolin-4-amine (37) was obtained as a mustard solid (172 mg, 0.507 mmol,
82%). m.p. 260–263 ◦C; 1H NMR (400 MHz, Methanol-d4)
δ
8.89 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 7.0 Hz
,
1H), 8.28–8.00 (m, 2H), 8.00–7.81 (m, 2H), 7.76 (dt, J = 8.8, 0.9 Hz, 1H), 7.46 (dd, J = 8.8, 1.9 Hz, 1H),
6.82 (d, J = 7.0 Hz, 1H). 13C NMR (100 MHz, Methanol-d4)
δ 157.2, 143.8, 140.8, 138.8, 138.3, 135.4,
131.1, 126.9, 126.1, 124.9, 123.3, 122.0, 120.0, 119.3, 113.1, 101.6. HRMS m/z [M + H]+ calculated for
C16H12N4Br: 339.0245, found 339.0243, LC tR = 3.18 min, >98% purity.
6-[(6-Bromoquinolin-4-yl)amino]-2,3-dihydro-1H-isoindol-1-one (38) was obtained as a yellow solid (160 mg,
0.452 mmol, 73%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.19 (s, 1H), 9.18 (d, J = 2.0 Hz, 1H),
8.77 (s, 1H), 8.54 (d, J = 6.9 Hz, 1H), 8.16 (dd, J = 9.0, 2.0 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.95–7.72 (m,

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2H), 7.70 (dd, J = 8.0, 2.0 Hz, 1H), 6.85 (d, J = 6.9 Hz, 1H), 4.45 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 169.0, 154.0, 143.3, 142.9, 137.5, 136.9, 136.5, 134.3, 128.4, 126.2, 125.4, 122.7, 119.9, 119.4, 118.8, 100.5,
45.0. HRMS m/z [M + H]+ calculated for C17H13N3OBr: 354.0242, found 354.0240, LC tR = 3.46 min,
>98% purity.
5-[(6-Bromoquinolin-4-yl)amino]-2,3-dihydro-1H-1,3-benzodiazol-2-one (39) was obtained as a yellow solid
(180 mg, 0.507 mmol, 82%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 10.88 (p, J = 2.4 Hz, 3H),
9.07 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 7.0 Hz, 1H), 8.15 (dd, J = 9.0, 2.0 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H),
7.08 (d, J = 8.7 Hz, 1H), 7.04–6.96 (m, 2H), 6.73 (d, J = 7.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
δ
155.4, 154.5, 143.0, 137.5, 136.4, 130. 7, 129.7, 129.1, 126.0, 122.7, 119.6, 118.4, 117.9, 109.2, 106.1,
100.3. HRMS m/z [M + H]+ calculated for C16H12N4OBr: 355.0194, found 355.0195, LC tR = 3.43 min,
>98% purity.
6-[(6-Bromoquinolin-4-yl)amino]-2,3-dihydro-1,3-benzoxazol-2-one (40) was obtained as a yellow solid
(165 mg, 0.464 mmol, 75%). m.p. > 300 ◦C; 1H NMR (400 MHz, Methanol-d4)
δ
8.84 (d, J = 1.9 Hz, 1H),
8.40 (d, J = 6.9 Hz, 1H), 8.14 (dd, J = 9.0, 2.0 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.40 (dd, J = 1.7, 0.8 Hz,
1H), 7.35–7.18 (m, 2H), 6.89 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, Methanol-d4)
δ 156.9, 156.5, 146.1,
144.6, 138.1, 132.8, 131.6, 126.8, 123.9, 123.8, 122.9, 122.0, 120.2, 111.7, 109.0, 101.9. HRMS m/z [M + H]+
calculated for C16H11N3O2Br: 356.0035, found 356.0032, LC tR = 3.66 min, >98% purity.
5-[(6-Bromoquinolin-4-yl)amino]-2,3-dihydro-1,3-benzoxazol-2-one (41) was obtained as a yellow solid
(163 mg, 0.458 mmol, 74%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
12.01 (s, 1H), 11.07 (s, 1H),
9.14 (d, J = 2.0 Hz, 1H), 8.51 (d, J = 6.9 Hz, 1H), 8.16 (dd, J = 9.0, 2.0 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 7.46
(d, J = 8.4 Hz, 1H), 7.30–6.99 (m, 2H), 6.79 (d, J = 6.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
154.5,
δ
154.4, 143.1, 142.3, 137.4, 136.5, 132.8, 131.6, 126.2, 122.5, 119.8, 119.2, 118.5, 110.5, 107.5, 100.5. HRMS
m/z [M + H]+ calculated for C16H11N3O2Br: 356.0035, found 356.0032, LC tR = 3.71 min, >98% purity.
5-[(6-Bromoquinolin-4-yl)amino]-1-methyl-2,3-dihydro-1H-1,3-benzodiazol-2-one (42) was obtained as a
yellow solid (162 mg, 0.439 mmol, 71%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ
11.16 (s, 1H),
11.10 (s, 1H), 9.17 (d, J = 2.0 Hz, 1H), 8.46 (d, J = 7.0 Hz, 1H), 8.13 (dd, J = 9.0, 1.9 Hz, 1H), 8.05 (d,
J = 9.0 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.17–6.98 (m, 2H), 6.71 (d, J = 7.0 Hz, 1H), 3.33 (s, 3H). 13
C
NMR (100 MHz, DMSO-d6)
δ 154.6, 154.5, 142.7, 137.3, 136.4, 130.3, 130.1, 129.1, 126.2, 122.4, 119.6,
118.4, 118.0, 108.3, 106.3, 100.2, 26.6. HRMS m/z [M + H]+ calculated for C17H14N4OBr: 369.0351, found
369.0350 LC tR = 3.70 min, >98% purity.
5-((6-Bromoquinolin-4-yl)amino)-1,3-dihydrobenzo[c]thiophene 2,2-dioxide (43) was obtained as an orange
solid (205 mg, 0.526 mmol, 85%). m.p. > 300 ◦C; 1H NMR (400 MHz, DMSO-d6)
δ 11.19 (s, 1H), 9.19
(d, J = 2.0 Hz, 1H), 8.55 (d, J = 6.9 Hz, 1H), 8.17 (dd, J = 9.0, 1.9 Hz, 1H), 8.09 (d, J = 9.0 Hz, 1H),
7.58 (d, J = 8.2 Hz, 1H), 7.54–7.36 (m, 2H), 6.88 (d, J = 6.9 Hz, 1H), 4.59 (d, J = 7.1 Hz, 4H). 13C NMR
(100 MHz, DMSO-d6)
δ 153.9, 143.2, 137.4, 136.9 136.6, 134.1, 131.5, 127.5, 126.3, 125.0, 122.5, 120.0,
118.7, 100.5, 56.1, 55.8. HRMS m/z [M + H]+ calculated for C17H14N2O2SBr: 388.9959, found 388.9955,
LC tR = 3.65 min, >98% purity.
4.3.5. Mass Spectrometry Method
Samples were analyzed with a ThermoFisher Q Exactive HF-X (ThermoFisher, Bremen, Germany)
mass spectrometer coupled with a Waters Acquity H-class liquid chromatograph system (Waters
Corporation, Milford, MA, USA). Samples were introduced via a heated electrospray source (HESI)
at a flow rate of 0.6 mL/min. Electrospray source conditions were set as: spray voltage 3.0 kV,
sheath gas (nitrogen) 60 arb, auxiliary gas (nitrogen) 20 arb, sweep gas (nitrogen) 0 arb, nebulizer
temperature 375 ◦C, capillary temperature 380 ◦C, RF funnel 45 V. The mass range was set to 150–2000
m/z. All measurements were recorded at a resolution setting of 120,000.
Separations were conducted on a Waters Acquity UPLC BEH C18 column (2.1
particle size). LC conditions were set at 100% water with 0.1% formic acid (A) ramped linearly over 9.8
×
50 mM, 1.7 µM

Molecules 2019, 24, 4016
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min to 95% acetonitrile with 0.1% formic acid (B) and held until 10.2 min. At 10.21 min the gradient
was switched back to 100% A and allowed to re-equilibrate until 11.25 min. Injection volume for all
samples was 3 µL.
Xcalibur (ThermoFisher) was used to analyze the data. Solutions were analyzed at 0.1 mg/mL or
less based on responsiveness to the ESI mechanism. Molecular formula assignments were determined
with Molecular Formula Calculator. All observed species were singly charged, as verified by unit
m/z separation between mass spectral peaks corresponding to the 12C and 13C12Cc-1 isotope for each
elemental composition.
4.4. Abbreviations Used
cyclin G-associated kinase (GAK)
mouse liver microsomes (MLM)
nicotinamide adenine dinucleotide phosphate (NADPH)
liquid chromatography mass spectra (LCMS)
mitogen-activated protein kinase kinase 5 (MEK5/MAP2K5)
transforming growth factor beta receptor 2 (TGFBR2)
receptor-interacting serine/threonine-protein kinase 2 (RIPK2)
1-aminobenzotriazole(ABT)
milligrams per kilogram (MPK)
N-methyl-2-pyrrolidoneolidone (NMP)
Bis(dibenzylideneacetone)palladium(0) (Pd(dba)2)
2-Dicyclohexylphosphino-20,40,60-triisopropylbiphenyl (XPhos)
5. Conclusions
We found that optimization of a series of 4-anilinoquinolines GAK inhibitors to decrease liver
metabolism was not compatible with maintaining potent enzyme activity. Nonetheless, several
heterocyclic replacements of the trimethoxyaniline moiety were identified that had greatly improved
metabolic stability and may be useful as bioisosteres in other lead optimization projects. Co-dosing with
the P450 inhibitor ABT proved to be a more general approach to blocking the metabolism of several
4-anilinoquinolines GAK inhibitors in MLM and in vivo. The availability of additional non-toxic
broad spectrum P450 inhibitors could further broaden the implementation of this approach to other
chemical probes.
Supplementary Materials: The following are available online at ‘http://www.mdpi.com/1420-3049/24/22/4016/s1,
HRMS, 1H, and 13C NMR spectra of all new compounds. Metabolite identification profiling and Kinomescan data
on compound 35.
Author Contributions: C.R.M.A. and W.J.Z. conceived and designed the study; C.R.M.A., J.M.B., L.S., T.L., J.E.P.
and Z.L. performed the experiments; C.R.M.A., J.M.B., L.S., T.L., J.M.E., J.E.P., C.I.W. and Z.L. analyzed the data;
C.R.M.A., J.E.P., T.M.W. and W.J.Z. edited the paper; C.R.M.A., T.M.W. and W.J.Z. wrote the paper.
Funding: The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG,
Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada,
Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt
Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São
Paulo Research Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z].
Acknowledgments: We thank Brandie M. Ehrmann for LC-MS/HRMS support provided by the Mass Spectrometry
Core Laboratory at the University of North Carolina at Chapel Hill. We would also like to thank the Biocenter
Finland/DDCB for financial support towards the goals of our work and the CSC-IT Center for Science Ltd. (Finland)
for the allocation of computational resources.
Conflicts of Interest: The authors declare no conflict of interest.

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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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