Discovery of CNS-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00611

Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress response. To investigate the therapeutic value of modulating this pathway in neurological disease, we have completed medicinal chemistry studie

Full text of DOI:10.1021/acsmedchemlett.9b00611

Subscriber access provided by GRIFFITH UNIVERSITY
Letter
Discovery of CNS-Penetrant Apoptosis
Signal-Regulating Kinase 1 (ASK1) Inhibitors
Zhili Xin, Martin K. Himmelbauer, J. Howard Jones, Istvan Enyedy, Rab Gilfillan, Thomas Hesson, Kristopher
King, Douglas J Marcotte, Paramasivam Murugan, Joseph C. Santoro, and Felix Gonzalez-Lopez de Turiso
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00611 • Publication Date (Web): 12 Feb 2020
Downloaded from pubs.acs.org on February 16, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 6
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Discovery of CNS-Penetrant Apoptosis Signal-Regulating Kinase 1
(ASK1) Inhibitors
Zhili Xin,a Martin K. Himmelbauer,a J. Howard Jones,a Istvan Enyedy,a Rab Gilfillan,a Thomas
Hesson,b Kristopher King,c Douglas J. Marcotte,d Paramasivam Murugan,b Joseph C. Santoro,b and
Felix Gonzalez-Lopez de Turisoa,*
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aMedicinal Chemistry; bBioassays; cDrug Metabolism and Pharmacokinetics; dPhysical Biochemistry and Molecular
Design. Biotherapeutic and Medicinal Science. Biogen. 225 Binney Street, Cambridge, MA 02142. United States.
KEYWORDS. Apoptosis signal-regulating kinase 1 (ASK1), rational design, CNS penetration, neurodegenerative diseases.
ABSTRACT: Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress
response. To investigate the therapeutic value of modulating this pathway in neurological disease we have completed
medicinal chemistry studies to identify novel CNS-penetrant ASK1 inhibitors starting from peripherally restricted compounds
reported in the literature. This effort led to the discovery of 21, a novel ASK1 inhibitor with good potency (cell IC50 = 138 nM),
low clearance (rat Cl/Clu = 0.36/6.7 L/hr/kg) and good CNS penetration (rat Kp,uu = 0.38).
Apoptosis signal-regulating kinase (ASK1) is a member of
the mitogen-activated protein kinase kinase kinase
(MAP3K) family that activates downstream MAP kinases
(MAPKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs
in response to stress.1 This pathway plays an essential role
in the regulation of both apoptosis and inflammation2 and
genetic animal models of neurodegeneration have
implicated ASK1 in a number of diseases including
amyotrophic lateral sclerosis (ALS)3 and multiple sclerosis.4
Specifically, genetic deletion of ASK1 in Cu/Zn-superoxide
dismutase (SOD1)mut mice extended the life span of these
animals5 and this event was found to be concurrent with
reduced motor neuron loss in the spinal cord. These
findings were partially recapitulated in SOD1 mice that had
been administered with the ASK1 inhibitors K811 (1, Figure
1) or K812 (2, Figure 1)6 at disease onset (28 weeks), thus
linking modulation of the kinase activity of ASK1 with
disease progression in ALS. Despite the potential benefit
associated with targeting this pathway in neurological
disease,7 the only ASK1 inhibitor in the clinic is selonsertib
(3, GS-4997, Figure 1), which is in Phase II clinical trials for
the treatment of liver fibrosis in combination with other
alternative approach to gain access to potent and brain
penetrant ASK1 inhibitors that relied on the optimization of
the phenyl group in the initially deconstructed analog 5
(Figure 2, bottom).
At the outset the potency of 5 was determined using both a
biochemical11 and an internally developed cellular
,
,
1
assay12 1
and this analog was found to be a weak ASK1
inhibitor (IC50 = 607 nM, cell IC50 > 20 M, Table 1). The poor
potency of this compound was rationalized by docking
experiments that predicted a binding mode on which the
lipophilic phenyl group in 5 would be placed in a solvent-
exposed area (Figure 2). This insight led us to pursue the
synthesis of compounds on which the phenyl group would
be replaced with more polar 5-member heterocyclic groups
,
(Table 1).15 1 The first analog synthesized following this
strategy was pyrazole 6, and it was found to be 20-fold more
potent in the biochemical assay relative to 5 (6, IC50 = 29 nM
vs 5, IC50 = 607 nM, Table 1). To evaluate the importance of
the attachment point of the pyrazole to the amino-pyridine
fragment, the regioisomeric 4-substituted pyrazole 7 was
then synthesized and this analog was found to have
improved cellular potency relative to 6 (7, cell IC50 = 6.8 M
vs 6, cell IC50 > 20 M). To mask the H-bond of the amide in
7, substituents with the ability to form an intramolecular H-
bond were then introduced in the pyrazole. Among these,
the methoxy group had the biggest impact in potency and
the resulting analog 8 (cell IC50 = 90 nM) was also found to
have low in vivo clearance (Cl/Clu = 1.6/4.0 L/hr/kg) in a
rat PK experiment.1 An initial attempt to further improve
the profile of this inhibitor by implementing the previously
described macrocyclization strategy10 led to the
identification of analog 9 (cell IC50 = 32 nM)1 but this
compound had high in vivo clearance in a rat PK experiment
(Cl/Clu = 4.7/21 L/hr/kg) and also suffered from high efflux
ratio (ER) (9, Table 1, ER = 26).1 Given the shortcomings of
this macrocyclization strategy, the effort shifted towards
the synthesis of analogs with reduced polar surface area
N
O
O
O
O
NH
O
N
N
N
N
O
N
H
N
N
F
N
X
3 (GS-4997)
Selonsertib
1, X=N, K811
2, X=C, K812
agents.9
Figure 1: ASK1 inhibitors reported in the literature
To gain a better understanding of the impact of modulating
the ASK1 pathway in neurological disease we have recently
disclosed the identification of macrocyclic inhibitors with
good potency, pharmacokinetic (PK) profile and moderate
CNS penetration following a deconstruction-cyclization
strategy (analog 4, Figure 2, top).10 Herein we describe an
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 6
O
O
1
2
3
4
5
6
7
N
N
N
N
Cyclization
Ref 9
H
N
Val 757
F
4
Val 757
O
cell IC50 = 95 nM
Kpuu = 0.17
Deconstruct
solvent-exposed
Lys 709
N
Lys 709
N
N
N
H
N
substituents
8
9
5
Solvent
exposed
region
O
N
O
This paper:
Phenyl group
optimization
N
N
N
N
H
N
N
N
N
R
Solvent
exposed
region
N
N
H
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
N
3 (GS-4997)
Solvent
exposed
region
(PSA) in an attempt to reduce their high ER. Among these,
analog
Figure 2. Binding mode of compound 3 bound to ASK1 (PDB code: 6OYT) and optimization strategy. Compound 3 occupies the ATP-
binding pocket of ASK1 and interacts with the hinge binder residue (Val 757) and with the catalytic lysine residue (Lys 709).
Compound 5 was predicted to retain these two key interactions and place the phenyl group in a solvent exposed region.
Table 1. In vitro and in vivo data for 1 and analogs 5-11
Bioc.
IC50
Cell
IC50
MDCK-
MDR1
Rat
Cl/Clu
RLM, HLM
(mL/min/kg)
RPPB/
d
Analog
3
Structure
MW/PSA/MPO
445/90/4.3
HPPB (fu)
(nM)a
(nM)b
Papp (ER)c
(L/hr/kg)
N
O
N
N
N
H
N
N
5.5
20
0.7/63
1.1/4.9
60, 13
26, <5
1.2/5.2
30/-
-
N
F
O
N
N
N
N
N
N
607
>20000
-/-
307/73/5.6
5
H
N
O
N
N
29
108
3.9
>20000
6797
90
2.8/14
0.3/56
1.7/15
1.8/26
0.3/104
10/2.2
18, 10
<11, <5
<11, 14
36, <5
<11, <5
72, 9
58/-
-/-
-/-
311/90/5.8
311/90/5.8
341/100/5.5
340/100/5.8
340/87/5.8
340/87/5.8
6
7
H
N
N
N
O
N
N
N
H
N
N
N
N
57/70
39/40
23/29
59/17
8/13
N
O
N
H
N
N
1.6/4.0e
4.7/21f
-/-
N
N
N
N
N
N
8
N
N
N
N
OMe
O
N
N
N
H
6.8
32
9
N
N
N
O
O
N
H
N
6164
>20000
10
OMe
O
N
H
N
2.4
93
3.4/42g
N
N
11
OMe
aBiochemical assay. bInhibition of ASK1 autophosphorylation. IC50 results are the geometric mean of a minimum of two
determination. cMDCK-MDR1 human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6 cm/s); ER =
B-A/A-B efflux ratio. dClu = Cl/fu. eDMSO:PG (1:1) used as vehicle; Vdss: 1.8 L/kg; t1/2: 0.9 hr. fDMSO:PG (1:1) used as vehicle; Vdss: 2.7
L/kg; t1/2: 0.7 hr. gDMA:EtOH:PG:water (1:1:3:5) used as vehicle; Vdss: 3.5 L/kg; t1/2: 2.0 hr.
10, on which the central pyridine ring had been replaced
with a phenyl group, was found to be a very weak ASK1
inhibitor in the biochemical assay (IC50 = 6.2 M) and,
despite its reduced PSA, it was still found to be a P-gp
substrate (ER = 104). In contrast, replacement of the
triazole ring in 8 with an imidazole led to the identification
of inhibitor 11, which had good potency (cell IC50 = 93 nM)
and low ER (2.2) but it was found to have high clearance in
ACS Paragon Plus Environment

Page 3 of 6
ACS Medicinal Chemistry Letters
a rat PK experiment (Cl/Clu = 3.4/42 L/hr/kg).
1
Table 2. In vitro and in vivo data for pyrazole analogs 8 and 12-21
2
3
O
N
N
H
N
R
4
5
N
N
N
N
OMe
6
7
8
9
Rat
Cl/Clu
(L/hr/kg)
Bioc. IC50
(nM)a
Cell IC50
(nM)b
MDCK-MDR1c
Papp/ER
RLM, HLM
(mL/min/kg)
RPPB/
HPPB (fu)
d
Analog
8
R
3.9
4.1
6.9
7.3
17
90
22
1.7/15
1.5/17
1.7/16
0.6/47
4.5/3.5
1.9/9.3
1.8/16
<11, 14
<11, 21
62, <5
31, 5
39/40
10/16
1.6/4.0e
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
-
12
12
3.3/18.7
20/42
-
13
35
1.6/8.1f
14
O
N
299
1710
11
32, 7
3.0/2.3
1.2/1.9
7.3/9.7
-
-
-
15
N
134
5.3
120, 16
77, 8
16
N
17
N
5.6
28
0.8/16
75, 24
1.0/1.1
-
18
N
1.9
16
21
21
74
2.5/4.5
1.6/19
5.0/5.0
136, 81
22, <5
<11, 6
0.7/0.5
14/27
-
19
20
21
Me2N
N
-
N
N
138
5.3/5.4
0.36/6.7g
N
aBiochemical assay. bInhibition of ASK1 autophosphorylation. IC50 results are the geometric mean of a minimum of two
determination. cMDCK-MDR1 human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6 cm/s); ER =
B-A/A-B efflux ratio. dClu = Cl/fu. eDMSO:PG (1:1) used as vehicle; Vdss: 1.8 L/kg; t1/2: 0.9 hr. fDMA:EtOH:PG:water (1:1:3:5) used as
vehicle; Vdss: 1.3 L/kg; t1/2: 1.8 hr. gDMA:EtOH:PG:water (1:1:3:5) used as vehicle. Vdss: 2.3 L/kg, t1/2: 5.2 hr.
Given the limited success of this optimization strategy it
was decided to focus the synthetic effort towards
modification of the N-methyl substituent in the pyrazole
analog 8. In an attempt to increase the structural diversity
of these inhibitors a small library of N-alkylated and N-
arylated pyrazoles was synthesized (Table 2). These studies
revealed that introduction of small alkyl groups in the
pyrazole N (analogs 12-14) resulted in compounds with
improved cellular potency. Specifically, analog 13 on which
a cyclopropylmethyl group had been introduced was over
7-fold more potent relative to the parent methyl substituted
analog 8 (13, cell IC50 = 12 nM vs 8, cell IC50 = 90 nM) but all
the analogs synthesized with an alkyl group were found to
have high ER (Table 2, analogs 12-14). In contrast,
introduction of a 2-pyridyl group (analog 15) was found to
have significantly lower ER (3.5) as well as an acceptable
level of cellular potency (IC50 = 299 nM). Systematic
synthesis of the other pyridyl rings was then carried out
(analogs 16 and 17) and it was found that the analog
containing a 3-pyridyl ring (17) had dramatically improved
cellular potency relative to 15 (a 25-fold improvement in
potency was observed) although this analog was a P-gp
substrate (17, ER = 16). Subsequent introduction of a
cyclopropyl substituent in 17 led to analog 18 which was
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 6
Figure 4: Mouse PK experiment with analog 21 (10 mg/kg, PO)
and projected dose to cover the cellular IC50 in the brain for 12
hours
found to have good potency but similarly high ER (16). In
contrast, introduction of a dimethylamino group led to a
compound with moderate ER (4.5) but its microsomal
stability was poor (19, RLM, HLM = 136, 81 mL/min/kg).
Introduction of a pyrimidine substituent led to analog 20
which had good microsomal stability (RLM, HLM = 22, <5
mL/min/kg) and cellular potency (74 nM) but high ER (19).
Interestingly, analog 21 on which a pyrazine ring had been
introduced, was found to have a good balance of potency
(cell IC50 = 138 nM) and ER (5.0) and its good microsomal
stability (RLM, HLM = <11, 6 mL/min/kg) was found to
correlate well with a low in vivo clearance in a rat PK
experiment (Cl/Clu = 0.36/6.7 L/hr/kg).
To confirm the binding mode of compound 21 an X-ray
cocrystal structure with human ASK1 was elucidated
(Figure 3). As expected, the same H-bond interactions of the
carbonyl group of the amide to the backbone NH of Val 757
and the triazole N to the catalytic Lys observed in 3 were
also present in the cocrystal structure of analog 21 with
ASK1. As predicted, an intramolecular H-bond between the
methoxy group and the amide was also observed.2
1
2
3
4
5
6
7
8
To predict the target coverage upon oral dosing of 21, a
mouse PK experiment on which this compound was
administered at 10 mg/kg (PO) was also carried out (Figure
4). This experiment revealed that the projected dose to
cover the cellular IC50/IC70/IC90 at trough in the brain for 12
hours were 50/115/435 mg/kg respectively making this
compound a suitable candidate for further in vivo studies.23
In summary, systematic optimization of the solvent-
exposed substituent of a peripherally restricted ASK1
inhibitor led to the identification of the CNS penetrant
analog 21. This compound was found to have good
biochemical and cellular potency, low clearance and good
brain penetration in rodents making it a suitable candidate
for more advanced in vivo pharmacological studies.
9
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
(i) Experimental procedures for the synthesis of all the
compounds described in this manuscript, (ii) experimental
conditions for cocrystallization, collection and refinement
statistics for compound 21 and (iii) kinase selectivity
profile for compound 21. The Supporting Information is
available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*Email: turiso@gmail.com. Telephone: 617-914-1295.
Author Contributions
Figure 3: Overlay of the cocrystal structure of analogs 3 and 21
in the kinase domain (659-951) of human ASK1. Key
interactions of the ligands with Val 757 and Lys 709 are
represented with yellow dashes (PDB code: 6VRE).
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Compounds 11 and 21 displayed good potency and low ER
and were selected for further in vivo characterization.
Specifically, rat Kp,uu experiments (Table 3) revealed that
both analogs had satisfactory CNS penetration (compound
11, Kp,uu = 0.6, compound 21, Kp,uu = 0.38).21 Further in vitro
characterization showed that inhibitor 21 had superior CYP
inhibition profile (relative to 11) and it was also inactive in
a hERG assay (Table 3). Based on this data, analog 21 was
selected for a kinase selectivity profile against a panel of
468 kinases and it was found to be moderately selective (S-
score (10) = 0.07).22
Notes
The authors declare no competing financial interest
ACKNOWLEDGMENTS
We thank Kevin Barry for chiral separation support,
Michael Dechantsreiter for parallel synthesis support, Sam
Cziria for compound handling, Josh Johnson for support
with collection of analytical data and Jay Chodaparambil for
X-ray support. We also thank Xiaoqing Wu, Xiaoping Wang
and Wenkui Zhao for efforts coordinating the synthesis of
some of the compounds included in this manuscript and
Janaky Coomaraswamy and Eric Tien for helpful
discussions.
10 mg/kg PO in Mouse
1000
Plasma (Measured)
ABBREVIATIONS
Brain (Predicted)
100
Cellular IC50
ALS, amyotrophic lateral sclerosis; ASK1, apoptosis signal-
regulating kinase; Cl, clearance; CNS, central nervous
system; ER, efflux ratio; HLM, human liver microsomes;
HPPB, human plasma protein binding; JNK, c-Jun N-terminal
kinase; MAPK, mitogen-activated kinase; MAP3K, mitogen-
activated kinases kinase kinase; MDCK, Madin-Darby canine
kidney; MDR1, multidrug resistance 1; MS, multiple
sclerosis; NASH, nonalcoholic steatohepatitis; PD,
Parkinson’s disease; PG: propylene glycol; P-gp, P-
glycoprotein; PK, pharmacokinetics; RLM, rat liver
10
1
0
6
12
18
24
Time (hr)
ACS Paragon Plus Environment

Page 5 of 6
ACS Medicinal Chemistry Letters
microsomes; RPPB, rat plasma protein binding; SOD1,
superoxide dismutase 1.
1
2
3
4
Table 3.
r
r
d
A ME
r
r
r r
r
d
CYP
MDCK-
MDR1c
Papp/ER
hERG IC50
(M)
Bioc. IC50
(nM)a
Cell IC50
(nM)b
RLM, HLM
(mL/min/kg)
RPPB/
HPPB (fu)
Cl/Clu
Rat
Kp,uu
3A4/2C9,f
IC50 ( M)
Analog
(L/hr/kg)d
e
5
6
7
8
9
3.9
2.4
21
90
93
<11, 14
72, 9
1.7/15
10/2.2
5.0/5.0
39/40
8/13
1.6/4.0
3.4/42
-
>10/10
5.9/0.7
8.8/>10
30
15
8
0.61
11
138
<11, 6
5.3/5.4
0.36/6.7
0.38
>30
21
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aBiochemical assay. bInhibition of ASK1 autophosphorylation. IC50 results are the geometric mean of a minimum of two
determinations. cMDCK-MDR1 human P-gp transfected cell line. Papp–Apparent permeability Apical-to-Basolateral (10-6 cm/s); ER =
B-A/A-B efflux ratio. dClu=Cl/fu. eDMA:EtOH:PG:water (1:1:3:5) was used as the vehicle; fmidazolam and tolbutamide were used as
substrates
O
N
N
N
H
N
N
N
N
N
N
OMe
Val 757
21
Lys 709
ASK1 IC50 = 21 nM, cell IC50 = 138 nM
Rat Cl/Clu = 0.36/6.7 L/hr/kg. Rat Kp,uu = 0.38
Selectivity S-score (10) @100 nM = 0.07
REFERENCES
1
Kawarazaki, Y.; Ichijo, H.; Naguro, I. Apoptosis Signal-Regulating Kinase 1 as a Therapeutic Target. Expert Opin. Ther. Targets. 2014, 18,
651-664.
2 Matsukawa, J.; Matsuzawa, A.; Takeda, K.; Ichijo, H. The ASK1-MAP Kinase Cascades in Mammalian Stress Response. J. Biochem. 2004, 136,
261-265.
3 Nishitoh, H.; Kadowaki, H.; Nagai, A.; Maruyama, T.; Yokota, T.; Fukutomi, H.; Noguchi, T.; Matsuzawa, A.; Takeda, K.; Ichijo, H. ALS-Linked
Mutant SOD1 Induces ER Stress- and ASK1-Dependent Motor Neuron Death by Targeting Derlin-1. Genes Dev. 2008, 22, 1451-1464.
4
Guo, X.; Harada, C.; Namekata, K.; Matsuzawa, A.; Camps, M.; Ji, H.; Swinnen, D.; Jorand-Lebrun, C.; Muzerelle, M.; Vitte, P.-A.; Rückle, T.;
Kimura, A.; Kohyama, K.; Matsumoto, Y.; Ichijo, H.; Harada, T. Regulation of the Severity of Neuroinflammation and Demyelination by TLR-
ASK1-p38 pathways. EMBO Mol. Med. 2010, 2, 504-515.
5 Relative to their ASK1 wild type littermates.
6 Fujisawa, T.; Takahashi, M.; Tsukamoto, Y.; Yamaguchi, N.; Nakoji, M.; Endo, M.; Kodaira, H.; Hayashi, Y.; Nishitoh, H.; Naguro, I.; Homma, K.;
Ichijo, H. The ASK1-Specific Inhibitors K811 and K812 Prolong Survival in a Mouse Model of Amyotrophic Lateral Sclerosis. Human Molecular
Genetics. 2016, 25(2), 245-253.
7 Fujisawa, T. Therapeutic Application of Apoptosis Signal-Regulating Kinase 1 Inhibitors. Adv. Biol. Regul. 2017, 66, 85-90 and references
therein.
ClinicalTrials.gov. Search result page. https://clinicaltrials.gov/ct2/results?cond=&term=NCT02781584+&cntry=&state=&city=&dist=
(accessed 2/11/2020).
9 Bansal, R.; Nagorniewicz, B.; Prakash, J. Clinical Advancements in the Targeted Therapies Against Liver Fibrosis. Mediators. Inflamm. 2016,
1-26.
10 Himmelbauer, M. K; Xin, Z.; Jones, J. H.; Enyedy, I.; King, K.; Marcotte, D. J.; Murugan, P.; Santoro, J. C.; Hesson, T.; Spilker, K.; Johnson, J. L.;
Luzzio, M. J.; Gilfillan, R.; Gonzalez-Lopez de Turiso, F. Rational Design and Optimization of a Novel Class of Macrocyclic Apoptosis Signal-
Regulating Kinase 1 Inhibitors. J. Med. Chem. 2019, 62, 10740-10756 and references therein.
11
The biochemical potency was determined at Reaction Biology corporation using the assay conditions described in the supporting
information of reference 10.
12 This assay quantified the level of ASK1 phosphorylation at residue T848 (see also the supporting information of reference 10).
13 Prediction of the ability of these analogs to enter the CNS was performed using an in vitro MDCK-MDR1 assay as previously described. In
these experiments, an MDCK NIH cell line transfected with MDR1 was used as an in vitro predictor of CNS penetration. Good correlation
between the ER in this assay and brain exposure has been reported when using this cell line (see reference 14).
14 Kikuchi, R.; de Morais, S. M.; Kalvass, J. C. In Vitro P-Glycoprotein Efflux Ratio Can Predict the In Vivo Brain Penetration Regardless of
Biopharmaceutics Drug Disposition Classification System Class. Drug Metab. Dispos. 2013, 12, 2012-2017.
15 Pyrazole-containing ASK1 inhibitors have been recently reported in the literature although these compounds were not optimized for CNS-
penetration (see reference 16).
1
Gibson, T. S.; Johnson, B.; Fanjul, A.; Halkowycz, P.; Dougan, D. R.; Cole, D.; Swann, S. Structure-Based Drug Design of Novel ASK1
Inhibitors Using an Integrated Lead Optimization Strategy. Bioorg. Med. Chem. Lett. 2017, 27, 1709-1713.
17 All in vivo PK studies were conducted in accordance with Biogen institutional animal care and use committee (IACUC) guidelines
1
This compound was synthesized as the racemate and the enantiomers were separated using chiral SFC. See Supporting Information.
19 This finding, in combination with the difficulty associated with synthesizing these analogs, led us to the deprioritization of this approach.
2
In addition to the highlighted interactions the isopropyl group of 3 also interacts with the side chain of Val 694. The pyrazine ring forms
a carbonyl- interaction with the carbonyl of Gly 759 and the pyrazole ring stacks against Gly 760. A non-conventional CH-C=O hydrogen
bond of the pyrazole with the hinge amino acid residue: Val 757 can also be invoked.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 6
1
2
3
4
5
6
7
8
9
21 In these experiments the compounds were administered via an IV infusion (using DMA:EtOH:PG:water in a 1:1:3:5 ratio as the vehicle) in
the carotid artery for a period of four hours (1 mg/kg, 0.1 mg/mL) to reach steady state. After this time the plasma and brain concentration
levels were quantified, and the values were adjusted by the measured protein binding in plasma and brain homogenate to calculate the Kp,uu
.
22 See supporting information.
23
This dose compares favorably to that projected for compound 2 (internal in vitro and in vivo data suggests that the estimated dose to
cover the cellular IC50 at trough in the brain for 12 hours for compound 2 would be >100 fold higher relative to compound 21). Additional
in vivo experiments with 21 and other ASK1 inhibitors will be reported in the future.
10
11
12
13
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
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment