Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.12.026

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.

Full text of DOI:10.1016/j.bmcl.2017.12.026

Accepted Manuscript
Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors
Andrii Monastyrskyi, Simon Bayle, Victor Quereda, Wayne Grant, Michael
Cameron, Derek Duckett, William Roush
PII:
S0960-894X(17)31186-1
https://doi.org/10.1016/j.bmcl.2017.12.026
BMCL 25482
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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19 October 2017
9 December 2017
13 December 2017
Please cite this article as: Monastyrskyi, A., Bayle, S., Quereda, V., Grant, W., Cameron, M., Duckett, D., Roush,
W., Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.026
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors
Andrii Monastyrskyia , Simon Bayleb , Victor Queredab , Wayne Grantb , Michael Cameronb , Derek
Duckettb , and William Rousha, *
aDepartment of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
bDepartment of Molecular Medicine, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is
described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high
throughput screening, we used tools of structure-based drug design to develop a series of potent
kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar
inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline
scaffold ASK1 inhibitors described herein are ATP competitive.
Revised
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
Apoptosis signal-regulating kinase (ASK1)
High throughput screening
Structure-based drug design
Kinase
Inhibitor
The apoptosis signal-regulating kinase 1 (ASK1) is a
serine/threonine protein kinase in the triple mitogen activated
protein kinase (MAP3K) family that plays a critical role in the
cellular response to a wide variety of environmental and
biological stressors. Specifically, ASK1 is activated in response
to increases in reactive oxygen species (ROS), endoplasmic
reticulum (ER) stress, cytotoxic cytokines, and the activation of
certain G protein-coupled receptor (GPCR) agonists.1 ASK1
activates both the p38 MAP and the c-jun-N-terminal (JNK)
kinase pathways, through activation of the MAP2Ks,
MKK3/MKK6 and MKK4/MKK7 respectively.1 The MAPK
signaling pathway is involved in a variety of fundamental cellular
processes such as cell survival, growth, proliferation,
differentiation, stress response, and apoptosis.2 As a result, ASK1
is activated in a number of human pathological conditions
including cancer, autoimmune disease, diabetes, cardiac,
inflammatory and several neurodegenerative diseases.3
Consequently, inhibition of ASK1 represents an attractive
strategy to provide significant benefit in multiple stress-induced
diseased states.
there are only a few reports of in vivo studies of these analogues.8
Specifically, Gilead has progressed a small molecule ASK1
inhibitor (GS-4997, selonsertib, compound 2, Fig.1) into the
clinic with mixed results. While the Phase II studies in
pulmonary arterial hypertension and diabetic kidney disease
failed to reach primary end points, the Phase II studies for
GS4779 in nonalchololic steatoheaptitis (NASH) demonstrated
improvement in primary endpoints, including improvements in
liver fibrosis and reduction in progression to cirrhosis, prompting
further Phase III studies. An earlier compound (GS-9679), also
from Gilead, demonstrated pre-clinical efficacy in models of
myocardial ischemia-reperfusion injury,9 acetaminophen (APAP)
induced liver injury10 and diabetic nephropathy.11
Several structural classes of ASK1 inhibitors, mostly from
industry4 but also from academia5, have been identified over the
last decade. In 2012, Terao et al (Takeda) reported imidazo[1,2-
α]pyridine 1 (Fig. 1) as a potent ASK1 inhibitor derived from
structure-based drug design.4b GSK,6 Merck7 and Gilead8
revealed some of their efforts on ASK1 a few years later.
Figure 1. Structures of Takeda’s imidazo[1,2-α]pyridine (1) ASK1
inhibitor and clinical candidate GS-4997 (2, Gilead)
Herein, we present our efforts using high-throughput
screening (HTS) and structure-based design that led to the
identification and development of 2-arylquinazolines as a new
ASK1 inhibitor scaffolds.
A—lt—hou—gh several series of ASK1 inhibitors have been identified,
* Corresponding author. Tel.: +1-561-228-2450; fax: +1-561-228-3092; e-mail: roush@scripps.edu

2-aminoquinazolines
2-aminopyrimidines
4-aminopurines
Molecule
Scaffold to scaffold similarity based
on Tanimoto score > 0.9
ASK1 % inh
<50%
>80%
Figure 2. Chemical structure similarity network graph and ASK1 potencies of the 114 primary screen hits. Topologically similar hits cluster together in organic
fashion. Hits were abstracted to scaffolds using Murcko algorithm in order to construct the graph. Each of the screening hits is represented as a node, and
nodes are connected via edges according to scaffold similarity (Tanimoto coefficient > 0.9). Molecular nodes are coded to reflect potency against ASK1 (small,
red - inhibition < 50%; large, green - inhibition > 80%).
In order to identify new small molecules to inhibit ASK1, we
developed and performed a HTS campaign of a proprietary
primary assay was used to determine the IC50’s of these
compounds. In parallel, we analyzed structural features of all 114
initial hits and clustered them into different scaffolds using the
Bemis-Murcko algorithm.13 This method dissects molecules into
ring systems, linker, framework, and side chain atoms. The
chemical structure similarity network graph and the ASK1
potencies were visualized in Cytoscape (v 3.4.0) using the Y-files
organic layout method (Fig. 2).14 Many of the 114 hits were
classified into several large clusters with unique scaffolds while
others are present as singlets. We identified a series of new
ASK1 inhibitors from six different structural classes (purine,
pyridine, pyrimidine, triazine, benzo-fused pyrimidine, and
quinazoline) with biochemical IC50 activities against ASK1
ranging from 0.3 to 10 µM. Out of the six structural classes,
purines, as well as 2-aminoquinazolines and the related 2-
aminopyrimidines formed the largest clusters (Fig. 2).
focused kinase inhibitor library using
a novel amplified
luminescent proximity homogenous (Alphascreen) assay.12 HTS
was carried out at the Scripps Florida HTS Facility on a subset of
the Scripps Drug Discovery Library (SDDL) containing 16,000
drug-like compounds based on known kinase inhibitor scaffolds.
The compounds were screened in a 384-well format through an
in vitro assay using the purified stress-activated ASK1
signalosome complex, and monitoring the phosphorylation of its
full-length native substrate, MKK6, as previously described.12
Compounds were analyzed at a single concentration of 7.5 µM
(0.6% DMSO) using 7.5 µM staurosporine, a promiscuous ATP-
competitive inhibitor, as a positive control. The ASK1 HTS
campaign demonstrated an average fourfold signal to background
(S/B) and Z’ factor (0.6 0.07) for the entire primary screen
indicative of a robust asasay.12
In order to understand the structural basis of observed ASK1
activity, we performed molecular docking studies using
coordinates of the ASK1•1 (PDB ID:4BHN4b) crystal complex.
Receptor grids were generated using Glide 7.415 in standard
precision (SP) mode, without any constraints and validated by
A total of 114 confirmed hits showed more than 40%
inhibition of ASK1 (average of two runs) at 7.5 µM. The 27
analogues, that displayed more than 90% inhibition, were
validated (in triplicate) using an orthogonal assay and the

docking 1 and 2 into the kinase ATP binding site. The docked
model for 1 was in good agreement with the reported crystal
structures coordinates. The binding poses and scores of the top
27 screening hits (>90% inhibition) were subsequently analyzed
in comparison to the known ASK1 inhibitors 1 and 2. Of these,
HTS hit 3 adopted a binding pose featuring hydrogen bond
interactions with the hinge residue Val757 that closely matched
those of the Takeda inhibitor (Fig. 3). The hinge binding regions
of 1 and 3 are highlighted with purple circles in Fig. 3.
small number of monosubstituted 2-arylquinazolines 10-13 with
logD values from 2.7 to 4.2 were synthesized and tested.
Molecular docking studies suggested the possibility of picking up
additional hinge binding interactions with the side chain of
Gln756 upon the introduction of an appropriate hydrogen bond
donor-acceptor unit. Gratifyingly, 3-acetamide analogue 12
showed a >10 fold improvement in potency (IC50 = 0.9 µM) in
comparison to the initial quinazoline 9. We then extended the
scaffold towards the DFG loop with the intent to gain favorable
hydrogen bond interactions with Lys709 and Asp822. However,
none of the 2-aryl-6-bromo-qunazolines 14-18 synthesized were
active against ASK1. Installation of 3-aminophenyl (19), 1H-
pyrazol-4-yl (20) and 1-methyl-1H-pyrazol-4-yl (21) at the C6
position of the unsubstituted 2-arylquinazoline also resulted in no
potency improvement (IC50 > 10 µM). At the same time,
incorporating the 3-acetamide unit (12) into the C6 1H-pyrazol-
4-yl (20) led to identification of the most active compound in this
series, 23 (IC50 = 0.2 µM). The predicted binding mode of 23
compared to Takeda 1 is presented in Fig. 4. As can be seen
from this image, 23 is predicted to maintain the favorable
interactions with Val-757 in the hinge binding region, but also to
pick up favorable interactions with Lys-709. 3-N-
phenylacetamide showed superior activity in comparison to other
substituents such as anilines (25-27) and aryl halogens used for
aromatic substitution reaction. Interestingly, the N-
phenylpivalamide analogues (28, 29), that were predicted by
molecular docking software to achieve favorable interactions in
the outer, solvent-exposed pocket, did not show improvement in
ASK1 activity. At the same time, on the phenyl side of the
quinazoline, the presence of hydrogen-bond donor (i.e. 1H-
pyrozole) is absolutely required to maintain kinase activity.
Figure 3. Hinge binding regions of 1 and 3 highlighted with purple
circles (upper panel). Comparative docking of 1 (orange) and 3 (cyan).
H-bond interactions with Val757, Lys709 and Asp822 are shown (lower
panel).
Table 1. Summary of ASK1 activity and physico-chemical
properties for selected number of purine series of inhibitors
Because 3 is approximately 250-fold less active as an ASK1
inhibitor compared to 1, additional inhibitors were designed with
the intent to (i) maintain the hinge-binding interactions with
Val757 highlighted in Fig. 3 and (ii) to gain interactions with
Lys709 and Asp822 which are achievable with compound 1 but
are predicted not to occur with 3. A set of inhibitors were
synthesized, by incorporating structural units present in other hits
from the HTS campaign. These inhibitors fall into two top
“enriched” scaffold groups from the HTS (Fig. 2): compounds
with pyrimidine or quinazoline cores; and compounds with
purine cores.
ASK1b
IC50 (µM)
Cmpd
R
logDc
4
5
6
7
8
H
> 10
> 10
> 10
> 10
> 10
1.74
1.88
2.34
0.97
1.84
4-F
3-Cl
Guided by the molecular modeling results, we initially
focused on the purine series of ASK1 inhibitors that showed
superior docking and Glide scores. A selected number of
examples from this exercise with logD values from 1.0 to 2.3 are
shown in Table 1. The general procedure that was utilized for
their synthesis relies on nucleophilic aromatic substitution (SNAr)
reactions of the indicated, readily commercially available
heterocycles.16 Unfortunately, none of the synthesized
compounds presented in Table 1 showed activity in the ASK1
biochemical assay.
3-NHAc
3-NMe2
aReagents and conditions: DIPEA, 2-propanol, 90 ºC, 18h, 35-
80% yield.
bMeasured in triplicates using developed Alphascreen assay12
cCalculated with Pipeline Pilot workflow application
(Accelrys) at pH 7.4.
Next, a set of 2-arylquinazolines analogues was synthesized
via nucleophilic aromatic substitution (SNAr) followed by Pd-
mediated cross-coupling (Suzuki)17 reactions starting from
commercially available 2-choloro-6-bromo-qunazoline. The
summary of the quinazoline scaffold structure-activity
relationship (SAR) studies is summarized in Table 2. First, a

Table 2. Summary of ASK1 activity and physico-chemical
properties for selected number of purine series of inhibitors
ASK1c
IC50 (µM)
Cmpd
R1
R2
logDd
9
H
3-Cl
H
H
> 10
> 10
> 10
0.9
3.54
4.15
3.69
2.78
2.71
4.31
4.46
3.55
3.48
5.35
10
11
12
13
14
15
16
17
18
4-F
H
3-NHAc
3-NH2
H
H
H
> 10
> 10
> 10
> 10
> 10
> 10
Br
Br
Br
Br
Br
4-F
Figure 4. Comparative docking of 1 (orange) and 23 (green). H-bond
3-NHAc
3-NH2
3-NHPiv
interactions with Val757, Lys709 and Asp822 are shown.
In order to confirm the ATP competitive nature of the 2-
arylquinazolines, we have performed detailed enzyme kinetic
studies with compound 12. The results of the experiment were
visualized using a Lineweaver–Burk plot based on six different
concentrations of ATP, with or without compound 12. As with
any competitive inhibition where Vmax stays unchanged with
increased Km values when the inhibitor concentration was raised,
12-mediated inhibition of ASK1 activity is an ATP competitive
(Fig. 5, also supporting information). Inhibition of ASK1 activity
by compound 12 in ATP competitive manner in combination
with the molecular modeling studies of the 2-arylquinazolines
binding in the active site of ASK1, strongly suggest that other
analogues, including 23, bind to the kinase in a similar fashion.
19
20
H
H
> 10
> 10
4.36
3.50
21
22
23
24
25
26
27
28
29
H
> 10
> 10
0.2
3.62
3.60
2.73
2.86
3.53
2.67
2.79
4.53
4.66
3-NHAc
3-NHAc
3-NHAc
3-NH2
> 10
> 10
0.6
3-NH2
3-NH2
> 10
> 10
> 10
3-NHPiv
3-NHPiv
aReagents and conditions: (a)DIPEA, 2-propanol, 90 ºC, 18h,
15-65% yields. (b) R2B(OH)2, Pd(Ph3P)4, sodium carbonate aq,
dioxane:water (2:1), MW 110 ºC, 30 min, 65-85% yields.
cMeasured in triplicates using developed Alphascreen assay12
dCalculated with Pipeline Pilot workflow application
(Accelrys) at pH 7.4
Figure 5. ASK1 inhibition by 2-arylquinazoline 12 is ATP competitive.
To determine the cell-based activity of our new analogues, we
monitored the endogenous levels of the ASK1 downstream
substrate, MKK6, under stress-induced conditions. This assay is
designed to specifically measure the phosphorylation of
endogenous MKK6 at Ser207 (MKK6ser207). Notably, ASK1-/-
mice provide significant protection from injury in models of type
1 diabetes induced by streptozotocin and in β-cells from the
pancreas by tumor necrosis factor α (TNFα).4b Using the rat INS-
1e cell line we demonstrated that compound 12 protects against

TNFα-induced MKK6ser207 phosphorylation (Fig. 6). These
results confirm ASK1 activation in response to TNFα treatment.
Supplementary data associated with this article can be found,
in the online version, at
Acknowledgments
This work was supported by NIH NIGMS grant R01GM122109
(D.D. and W.R.).
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In addition to testing inhibitory activity against ASK1 and the
cell-based substrate (MKK6) phosphorylation, three of our best
compounds from the 2-arylquinazoline series (12, 23 and 26)
were taken forward to a core set of ADME assays (solubility,
microsome stability, inhibition of cytochrome P450 1A2, 2C9,
2D6, and 3A4) to assess the drug-like properties of the
increasingly optimized candidates. The ADME data is
summarized in Table 3.
Table 3. ADME properties of selected ASK1 inhibitors series of
inhibitors
aSolubility in pH 7.4 phosphate buffered saline.
bMicrosome stability using human, mouse and rat liver
Microsome
stability (min)
(H/M/R)b
CYP inhibition (%)c
Pharmacol, 2012;60:276; (b) Toldo S, Breckenridge
Mezzaroma E, et al. J Am Heart Assoc, 2012;1:e002360.
D G,
Solubility
(µM)a
Cmpd
1A2 2C9 2D6 3A4
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Pharmacol, 2015;286:1.
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2014;12:229.
12
23
100
10
6 / 2 / 4
42 / 2 / 4
29 / 2 / 3
46 /13 30
94
49
67
20
98
93
13
52
56
-13
88
26
17
52
sunitinib
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While all three inhibitors showed moderate to excellent
aqueous solubility (10-100 µM), compounds were particularly
unstable in the hepatic microsomal stability assay (especially in
rodent liver microsomes, 2-4 min). The CYP inhibition profile of
23 (>85% inhibition of CYPs 2C9 and 3A4 at 10 µM) also
indicates that further improvements are required. We suspect that
the pyrazole unit of 23 is responsible for the CYP inhibition, as
12 exhibits <20% inhibition of 2C9 and 3A4 at 10 µM.
Additional molecular modeling, design and structure refinement
are underway to address these ADME liabilities.
17. Miyaura N, Suzuki A. Chem. Rev., 1995;95:2457.
In summary, we have developed a new series of ASK1
inhibitors by optimization of a HTS-derived set of hits. These
efforts led to identification of 2-arylquinalzolines 12 and 23 with
submicromolar inhibitory activities against ASK1. A detailed
enzyme kinetic study and molecular modeling both confirm the
ATP competitive nature of inhibition. This new series of ASK1
inhibitors displays a promising potency profile for further
development as in vivo probes.
Supplementary data

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Discovery of 2-arylquinazoline derivatives as
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Andrii Monastyrskyi, Simon Bayle, Victor Quereda, Wayne Grant, Michael Cameron, Derek Duckett, and
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