6-Amino[1,2,5]oxadiazolo[3,4- b]pyrazin-5-ol Derivatives as Efficacious Mitochondrial Uncouplers in STAM Mouse Model of Nonalcoholic Steatohepatitis

Article abstract of DOI:10.1021/acs.jmedchem.0c00542

Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating nonalcoholic steatohepatitis (NASH). In this study, we report the structure-activity relationship profiling of a 6-amino[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrate that a wide array of substituents is tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a readout. In particular, compound SHS4121705 (12i) displayed an EC50 of 4.3 μM in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the STAM mouse model of NASH, administration of 12i at 25 mg kg-1 day-1 lowered liver triglyceride levels and improved liver markers such as alanine aminotransferase, NAFLD activity score, and fibrosis. Importantly, no changes in body temperature or food intake were observed. As potential treatment of NASH, mitochondrial uncouplers show promise for future development.

Full text of DOI:10.1021/acs.jmedchem.0c00542

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Article
6-Amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol Derivatives
as Efficacious Mitochondrial Uncouplers in STAM
Mouse Model of Non-alcoholic Steatohepatitis
Joseph Salamoun, Christopher J Garcia, Stefan Hargett, Jacob Murray, Sing-Young Chen, Martina Baretta,
Stephanie Alexopoulos, Divya Shah, Ellen Olzomer, Simon Tucker, Kyle Hoehn, and Webster L. Santos
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00542 • Publication Date (Web): 11 May 2020
Downloaded from pubs.acs.org on May 12, 2020
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-Amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol Derivatives as Efficacious
Mitochondrial Uncouplers in STAM Mouse Model of Non-alcoholic
Steatohepatitis
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Joseph M. Salamoun,1 Christopher J. Garcia,1,‡ Stefan R. Hargett, Jacob H. Murray, Sing-
,‡
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Young Chen, Martina Beretta, Stephanie J. Alexopoulos, Divya P. Shah, Ellen M. Olzomer,
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2,3,*
and Webster L. Santos1,*
Simon P. Tucker, Kyle L. Hoehn,
1Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech,
Blacksburg, Virginia 24061, United States.
2Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia
22908, United States.
3School of Biotechnology and Biomolecular Sciences, University of New South Wales,
Kensington, NSW 2033, Australia.
4Continuum Biosciences, Pty Ltd Sydney, Australia 2035 and Continuum Biosciences Inc.,
Boston, Massachusetts 02116, United States.
Corresponding Author Email: santosw@vt.edu; k.hoehn@unsw.edu.au
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Abstract
Small molecule mitochondrial uncouplers have recently garnered great interest for their potential
in treating nonalcoholic steatohepatitis (NASH). In this study, we report the structure-activity
relationship profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the
hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We
demonstrate that a wide array of substituents is tolerated with this novel scaffold that increased
cellular metabolic rates in vitro using changes in oxygen consumption rate as a read-out. In
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particular, compound SHS4121705 (12i) displayed an EC of 4.3 M in L6 myoblast cells and
50
excellent oral bioavailability and liver exposure in mice. In the STAM mouse model of NASH,
administration of 12i at 25 mg/kg/day lowered liver triglyceride levels and improved liver
markers such as alanine aminotransferase, NAFLD activity score, and fibrosis. Importantly, no
changes in body temperature or food intake was observed. As potential treatment of NASH,
mitochondrial uncouplers show promise for future development.
Keywords
Oxidative phosphorylation, small molecule mitochondrial uncoupler, protonophore, BAM15,
NASH.
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Introduction
Cellular respiration occurs in mitochondria wherein nutrient oxidation leads to the
production of adenosine triphosphate (ATP), an energy rich molecule that fuels cellular function
and signaling pathways.1, 2 This process, also known as oxidative phosphorylation, occurs by the
generation of a proton motive force (pmf) driven by the efflux of protons from the matrix to the
mitochondrial intermembrane space. The resulting electrochemical gradient drives ATP
production as protons re-enter the mitochondrial matrix via the enzyme ATP synthase.
Alternatively, protons may re-enter the mitochondrial matrix independently of ATP synthase,
thus bypassing the production of ATP, through a process known as mitochondrial uncoupling.3, 4
This proton leak is a natural process in mammals that occurs as both basal and protein-induced
leaks.5,6 As proton leak occurs, cells compensate by increasing the metabolic rate to regenerate
the proton motive force and sustain an adequate supply of ATP. Inducible proton leak is a
process mediated by activation of uncoupling proteins (UCPs) among others and activated by
various stimuli such as oxidative stress or cold exposure.7-11
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In addition to naturally occurring mitochondrial uncoupling, proton leaks can also be
induced with small molecules that transport protons, by a protonation/deprotonation mechanism,
from the mitochondrial intermembrane space into the mitochondrial matrix.4, 12-14 Lipophilic
weak acids comprise a major category of protonophores (Figure 1). Their ability to modulate
cellular respiration has been explored for therapeutic use, especially for obesity and adult onset
diabetes (T2D).7, 15-22 Additionally, mitochondrial uncouplers may be useful in the treatment of
non-alcoholic fatty liver disease by slowing down or even preventing hepatic steatosis and
nonalcoholic steatohepatitis (NASH) phenotypes.19-21, 23, 24, 26 These encouraging results are
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particularly relevant to today’s clinical challenges as non-alcoholic fatty liver disease (NAFLD)
is becoming an epidemic affecting more than 100 million adults and children in the United States
alone.27-29 The lack of non-invasive diagnostic tools for NASH makes it difficult to confirm the
number of people affected; however, the incidence of NASH is increasing and will soon become
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a significant contributor to the number of liver transplantation. NASH, an advanced form of
NAFLD, is associated with hepatocyte injury with ballooning, lobular inflammation, and often
fibrosis and cirrhosis. Due to the lack of FDA-approved therapeutics for NASH, drug discovery
in this field is highly active and was the subject of a recent review.31
N
N
OH
N
O
O
N
NH
Cl
NO2
NO2
O
NO2
N
S
Cl
N
H
H
NO2
OH
OCF3
O
2,4-Dinitrophenol
FCCP (2)
Niclosamide (3)
Nitazoxanide (4)
(DNP, 1)
H C
3
Cl
N
S
Cl
N
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F
F F F F F O
NH2
CF3
HN
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N
Cl
CN
CF3
O
H
CF3
F
F F F F
F
Cl
F
OPC-163493 (5)
Perfluorooctanesulfonamide (6)
4,5,6,7-Tetrachloro-2-trifluoromethylbenzimidazole
TTFB, 7)
(
F
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H
N
H
N
N
N
N
N
N
N
N
N
O
O
NH
NH
F
F
BAM15 (8)
9
Figure 1. Structures of select protonophore mitochondrial uncouplers to showcase chemical diversity of the weak
acids.
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In addition to increasing energy expenditure, protonophores reduce the production of
reactive oxygen species32-34 and have been explored for their potential use as anti-
3
5
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anti-ageing,40-42 and antibacterial agents. Despite the great
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neurodegenerative, anti-cancer,
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preclinical promise of mitochondrial uncouplers for treatment of numerous serious medical
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conditions, clinical translation has been challenging due to the promiscuous mechanisms of
action of many uncouplers. In fact, any molecule that meets a certain lipophilicity and acidity
threshold is potentially an uncoupler, albeit unselectively through both mitochondrial and non-
mitochondrial membranes.45 Therefore, there is a need for the rational design of novel
uncouplers with drug-like properties suitable for translation into the clinic.
Perhaps the most pertinent example of a mitochondrial uncoupler used in humans, 2,4-
dinitrophenol (DNP, 1) promoted upwards of 50% increase in metabolism and had weight-loss-
inducing effects.46-49 Although widely used as a diet aid in the early 1930’s, DNP was quickly
banned by the FDA due to adverse effects including increased body temperature,47, 50-52
cataracts,53-56 blindness, and death in some patients. Mechanistic studies now indicate that, in
addition to the mitochondria, DNP also depolarizes the plasma membrane resulting in a narrow
therapeutic window.57-60 FCCP (2), which is highly toxic in animals, has a limited use as an in
vitro control in biochemical assays.61-65 In an effort to develop effective strategies to address
safety concerns, uncoupling activity was identified in FDA-approved drugs such as niclosamide
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(3), a well-known anthelmintic drug widely investigated in a number of disease models, most
notably cancer for its ability to act on other targets including STAT3, Wnt, and PKA signaling.66,
67
Likewise, an anti-parasitic drug nitazoxanide (4) was recently shown to exhibit uncoupling
activity68 and is currently in phase 2 clinical trial for NASH-associated fibrosis
(ClinicalTrials.gov Identifier: NCT03656068).
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Given the broad definition of a protonophore and the lack of a defined biological target,
the chemical space is quite vast. As shown in Figure 1, phenols (e.g. DNP), dicyano hydrazones
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(e.g. FCCP), aryl amides (e.g. niclosamide and nitazoxanide), triazoles (e.g. OPC-163493),
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70, 71
and carboranes72, 73 are among the diverse
perfluoro alkyl sulfonamides, benzimidazoles,
class of protonophores studied. Recently, through a high throughput screen, we reported a novel
chemical scaffold in BAM15 (8), which elicits selective depolarization of the mitochondrial
inner membrane.74 The mitochondrial-specificity of 8 is a unique departure from other
protonophores and offers a potential breakthrough to overcome the toxicity of protonophores.
Detailed structure-activity relationship (SAR) profiling of 8 identified the oxadiazolo-pyrazine
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5
core decorated with aniline as essential for uncoupling properties. Replacement of the aniline
moiety with phenol or alkylation of the aniline N-H resulted in a total loss of activity. This result
was an indication that the aniline N-H in 8 was the source of the acidic proton.76 The
[1,2,5]oxadiazolo[3,4-b]pyrazine core is strongly electron withdrawing and is likely the main
driver of 8’s acidity. Replacing the bicyclic core with pyrazine or triazine, while maintaining two
7
7
aniline moieties, generally led to lower potency and efficacy. Interestingly, a subtle change to 8
such as regioisomers of the fluoro aniline provided the unsymmetrical analog 9 that was
efficacious in a streptozotocin (STZ)-induced mouse model of NASH. Compound 9 lowered in
liver triglyceride levels and reduced inflammation and fibrosis scores.78
Overall, the SAR with the [1,2,5]oxadiazolo[3,4-b]pyrazine core and two amine moieties
showed a narrow set of tolerated substituents.75, 78 The limitations include the solubility and
pharmacokinetic properties of these compounds. To improve the physicochemical properties of
new derivatives, we replaced the acidic N-H moiety in 9 with a hydroxyl functional group
(Figure 2).79 Since O-H is more acidic than N-H, the hydroxyl group may obviate the need for
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two anilines and widen the scope of tolerated aryl and alkyl substituents. In this study, we
performed an SAR investigation of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol derivatives as
mitochondrial uncouplers. Our studies identified mitochondrial uncoupler 12i that possessed
favorable pharmacokinetic properties and desired liver exposure. In a streptozotocin (STZ)-
induced mouse model of NASH, not only did 12i demonstrated improvement in NAFLD activity
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score and fibrosis, but it also decreased liver triglyceride and ALT levels.
F
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delocalized
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Figure 2. Design of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol scaffold as mitochondrial uncoupler.
Results and Discussion
We hypothesized that the acidic proton of the anilines can be replaced by a hydroxyl
group since the pKa of the hydroxyl group is expected to decrease because of the electron
withdrawing capacity of the furazano pyrazine ring (Figure 2). An important intrinsic property of
these protonophores is their ability to cycle between the intermembrane space and mitochondrial
matrix with concomitant transport of protons; hence, the molecule must be able to penetrate the
inner mitochondrial membrane both as a neutral and charged species. Efficient penetration
through the membrane is likely facilitated by ‘hiding the negative charge’ rather than anion
transport (e.g. via the adenine nucleotide transporter (ANT)) as activity of the parent molecule
74
BAM15 is not affected by the ANT inhibitor carboxyatractyloside; in this case, the oxygen
anion is stabilized by delocalization of electrons in the furazan and pyrazine rings through
various resonance forms. Furthermore, maintaining one amino group provides a convenient
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synthetic handle to modulate the lipophilicity of the molecule, another important parameter for
membrane crossing. To test this hypothesis, we synthesized and developed the structure-activity
relationship profile of a series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol derivatives.
The synthesis of the pyrazinol derivatives initiated with the common intermediate 5,6-
dichloro-[1,2,5]oxadiazolo[3,4-b]pyrazine (11), synthesized as described before (Scheme 1).80-82,
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Then in a one-pot reaction, intermediate 11 was directly substituted with a sub-stoichiometric
equivalent of the requisite alkyl or aryl amines followed by hydroxylation with aqueous
potassium hydroxide and then quenching with dilute hydrochloric acid to afford the desired
neutral derivatives 12. Similarly, a methyl ether derivative 13 was synthesized to test the
importance of the hydroxyl proton by the one-pot amination of 11 followed by reaction with
sodium methoxide in methanol.
H
NH2
N
N
N
Cl
Cl
N
N
N
a, b
N
N
c
N
N
R
O
O
O
NH2
N
OH
10
11
12
d
H
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N
O
O
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13
Scheme 1. Reagents and conditions: (a) oxalic acid, 10% HCl, reflux, 4 h, 72%; (b) PCl
c) one pot reaction: i) amine, Et N or K CO , THF or acetone, rt or 75 °C; ii) KOH(aq) at rt for 2 h; (d) one pot
reaction: 4-trifluoromethoxyaniline, Et N, THF, rt; then NaOMe in MeOH at rt for 1 h.
5 3
, POCl , 95 °C, 2 h, 77%;
(
3
2
3
3
To investigate the effect of the strongly electron withdrawing furazan ring, pyrazine
derivatives were synthesized (Scheme 2). Mono-substitution of 2,3-dichloropyrazine (14) with
benzyl alcohol afforded the desired intermediate 15. The aniline moiety was introduced via a
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Buchwald-Hartwig cross-coupling reaction, followed by catalytic hydrogenation to cleave the
benzyl group and generate the desired hydroxy pyrazine analogs 16.
H
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Cl
Cl
N
N
Cl
N
N
N
a
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Scheme 2. Reagents and conditions: (a) NaH, BnOH, THF, rt, 2 h, 87%; (b) i. Pd
mol%), arylamine (1.3 equiv.), K CO
MeOH, rt, 1.5 h, 12-37% over two steps.
16
2
dba
3
(10 mol%), Xantphos (10
2
3
(2.3 equiv.), dioxane, 110 °C, 16 h; ii. 10% Pd/C (5 mol %), H2 (g) (1 atm),
With [1,2,5] oxadiazolo[3,4-b]pyrazin-5-ol and pyrazine derivatives in hand, we tested
their activity as mitochondrial uncoupler using an Agilent Seahorse XF analyzer, which
measures oxygen consumption rate (OCR) in L6 rat myoblast cells.83 In this assay, cells were
monitored over a 90 minute period after treatment with increasing concentrations of compound.
OCR increases as mitochondrial uncouplers cycle protons through the mitochondrial inner
membrane. BAM15 was used as a positive control because it induces maximal cellular
78
respiration. To benchmark the activity of new analogs, the activities are reported as integrated
areas under the curve relative to BAM15 to normalize the data and take into account inter-assay
variabilities across the large number of compounds studied. Our ideal mitochondrial uncouplers
are expected to have low EC values and OCR activity close to BAM15.
5
0
Inspired by previous work on 9,78 fluorinated analogs in the ortho, meta and para
positions were synthesized. The unsymmetrical 9 contains a 2-fluoroaniline and 3-fluoroaniline
that stimulates respiration in L6 rat myoblast cells with improved EC value (190 nM) relative
5
0
to BAM15. The first analog 12a, a direct derivative of 9 containing a 2-fluoroaniline, retained
4% of BAM15 uncoupling capability, albeit at a 40x loss of potency (Table 1). The
7
corresponding meta (12b) and para (12c) derivatives were likewise effective although with
9
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
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decreased activity. Nonetheless, the activities were sufficiently compelling from this chemical
scaffold. From the handling of these compounds during synthesis and purification, it was
apparent that the physical properties (polarity and solubility) were significantly different from 9.
Taken together, an extensive SAR profiling of novel derivatives were pursued (Tables 1-5).
The first series of analogs incorporated a diverse array of electron withdrawing groups
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Table 1). A variety of halogenated moieties were well-tolerated including trifluoromethyl (12d–
1
2f), trifluoromethoxy (12g and 12i), OCF CHF (12m), chloro (12j and 12k) and
2
2
difluorobenzodioxole (12o), which all had >60% of BAM15 OCR and an EC ≤ 13 µM. The
5
0
OCF analogs (12g–12i) were the most promising with activity in the single digit micromolar
3
range and the para-substituted derivative 12i having the best potency EC at 4.3 µM. Anilines
5
0
with multiple halogenated groups (12q–12ab) were less tolerated except for 12v; however, this
compound is > 2 fold less potent than 12i. Interestingly, both the aniline N-H and O-H were
required for activity. To determine the effect of acidic protons in N-H and O-H in the scaffold of
1
2a, the corresponding methylated versions were synthesized. N-methylated analog 12ae was
completely devoid of uncoupling activity. Likewise, methylation of the phenolic oxygen (13)
significantly abrogated the compound’s activity suggesting the role of the hydroxyl group as a
protonophore.
1
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Table 1. Oxygen consumption rates of halogenated aniline derivatives in L6 rat myoblast cells.a
N
N
R
N
N
O
OH
%
of BAM15
OCR
% of BAM15
Entry
R
EC50, µM
Entry
R
EC50, µM
b
b
OCR
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
BAM15
-
100%
71%
0.31 ± 0.06
12p
12q
OH
29%
45%
34.0 ± 8.0
CF3
CF3
F
H
N
F
9
-
0.19 ± 0.01
9.7 ± 1.8
F
F
H
H
N
N
12a
74%
67%
12.5 ± 0.8
31.2 ± 2.0
12r
12s
NA
--
F
H
N
H
N
F
F
F
1
2b
46%
23.4 ± 4.2
F
F
H
N
H
N
1
2c
51%
38.6 ± 4.8
12t
47%
5.1 ± 0.3
CF3
CF3
F
H
H
N
N
12d
2e
2f
88%
66%
68%
10.6 ± 0.7
13.3
12u
12v
12w
30%
76%
32%
2.6 ± 0.2
11.6 ± 1.2
4.9 ± 1.3
CF3
F
H
N
H
N
CF3
1
CF3
CF3
H
N
F
H
N
1
10.4 ± 1.4
CF3
H
N
OCF3
CF3
H
N
1
2g
67%
47%
7.1 ± 0.5
12x
10%
4.5 ± 1.2
CF3
F
H
H
N
OCF3
N
12h
2i
5.1 ± 0.4
4.3 ± 0.7
6.3 ± 0.7
11.7 ± 1.3
12y
12z
27%
32%
26%
8%
2.3 ± 0.7
2.9 ± 0.3
3.0 ± 0.2
1.4 ± 0.4
OCF3
F
H
N
H
N
1
6
4%
(
SHS4121705)
OCF3
OCF3
Cl
Cl
H
N
H
N
1
2j
2k
2l
84%
72%
46%
12aa
12ab
OCF3
Cl
H
H
N
N
1
Cl
F
OCF3
OCH3
H
H
N
N
1
20.6 ± 2.1
7.0 ± 1.3
12ac
12ad
50%
27%
3.8 ± 0.7
O
F
F
OCF3
H
N
F
H
N
NO2
F
O
1
2m
86%
53.5 ± 6.4
F
OCF3
H
F
N
N
F
F
1
2n
2o
55%
70%
1.9 ± 0.3
7.6 ± 0.9
12ae
13
NA
NA
--
--
O
Cl
H
N
O
F
F
1
-
O
a
Highest tested concentration is 200 µM.
Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15. NA = No Activity.
b
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Oxygen consumption rates of alkoxy, cyano, and thio- aniline derivatives in L6 rat myoblast cells.a
N
R
N
O
N
N
OH
%
of BAM15
OCR
% of BAM15
Entry
2af
12ag
R
EC50, µM Entry
R
EC50, µM
1.8 ± 0.1
46.9 ± 4.8
b
b
OCR
H
H
N
CF3
O
N
OCH3
1
41%
55%
75.4 ± 9.3 12ao
60%
27%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
H
N
O
O
2.3 ± 0.2
3.7 ± 1.4
12ap
12aq
O
H
H
N
N
1
2ah
56%
50%
44%
66%
2.4 ± 0.4
3.0 ± 0.3
O
O
CF3
H
N
H
N
O
1
2ai
42.3 ± 8.3
12ar
O
CF3
H
N
H
CH3
N
1
2aj
57%
60%
43%
7.0 ± 0.7
10.4 ± 1.2
4.0 ± 0.3
12as
12at
12au
NA
29%
46%
--
O
F
CF3
CF3
CF3
SO Me
2
H
N
H
N
SF5
1
2ak
2al
3.7 ± 0.2
3.9 ± 0.6
O
H
N
H
N
CF3
1
O
SF5
H
N
F
H
N
1
2am
24%
46%
4.2 ± 0.2
2.1 ± 0.5
12av
24%
NA
1.4 ± 0.2
--
O
CF3
SCF3
CN
H
N
CF3
O
H
N
1
2an
12aw
CF3
a
Highest tested concentration is 200 µM.
Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15. NA = No Activity.
b
After the encouraging result from the OCF analog 12i, a series of alkoxy analogs was
3
generated (Table 2). Whereas the methoxy analog 12af was moderately efficacious, the butoxy
analogs (12ag–12ah) were significantly more potent than the methoxy derivative (12af),
presumably due to the increased lipophilicity favoring permeation of the mitochondrial
8
4
membrane. Because phenylalkylether groups are known to be a metabolic liability, fluorinated
analogs were introduced (12ai–12an) to potentially enhance metabolic stability.85 However,
8
6
many of these compounds showed diminished efficacy or potency. The cyclopropyl methoxy
analog 12ao, a four carbon derivative of the butoxy chain in 12ah and 12an, had a promising
result with 60% of BAM15 OCR and an EC of 1.8 µM. Additionally, the benzyl derivative
5
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2ar had good activity and potency. The pentafluorosulfanyl group (12at and 12au), which is a
8
7
proposed isostere for the trifluoromethyl group, and 12av, the sulfur derivative of 12i, had low
efficacy. The electron withdrawing sulfonyl moiety (12as) and a cyano group (12aw) were not
tolerated.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We next investigated the effect of other rings as well as alkyl groups on the aniline ring
(Table 3). A sharp decrease in potency was observed with aniline derivative 12ax and N-
7
8
methylation (12ay). In stark contrast to our prior SAR studies, the alkyl anilines were well-
tolerated in the absence of any halogens or strongly electron withdrawing groups. More
specifically, anilines containing isomers of butane were among the most efficacious and potent,
e.g. tert-butyl (12bg and 12bh) and n-butyl (12bj). Unfortunately, the half-life of these analogs
in mice is short (< 1 h) as alkyl moieties are susceptible to ω-oxidation (Table 6).88 To
circumvent this issue, fluorine groups were added to potentially enhance the metabolic
8
9
stability. Interestingly, with the exception of 12bm and 12bu, the combination of halogens and
alkyl groups (12bn–12br, and 12bt) led to poor activity similar to the fluorinated alkoxy
analogs. The incorporation of additional polar groups such as a primary alcohol (12bs), a cyano
group (12bv) or a ketone (12bw) were not tolerated.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Oxygen consumption rates (OCR) of alkyl substituted aniline derivatives in L6 rat myoblast cells.a
N
N
R
N
N
O
OH
%
of BAM15
OCR
% of BAM15
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Entry
R
EC50, µM
66.7 ± 22.7
--
Entry
R
EC50, µM
2.4 ± 0.1
4.7 ± 3.0
6.7 ± 1.6
1.1 ± 0.2
1.5 ± 1.0
3.3 ± 0.2
b
b
OCR
H
N
H
N
12ax
12ay
12az
52%
NA
12bk
12bl
51%
33%
65%
14%
5%
H
N
N
H
N
F
H
N
69%
66%
64%
14%
4.0 ± 0.5
20.4 ± 3.9
51.9 ± 10.2
>100
12bm
12bn
12bo
12bp
F
H
H
N
N
1
2ba
12bb
2bc
2bd
2be
12bf
2bg
H
N
F
H
N
F
H
H
N
N
1
5%
H
OCF3
OCF3
H
N
N
1
26%
74%
63%
3.0 ± 0.2
25.2 ± 9.1
10.7 ± 1.9
12bq
12br
12bs
NA
NA
NA
--
--
--
H
N
H
N
1
H
N
H
N
OH
CH3
CF3
H
N
H
N
1
73%
2.2 ± 0.3
12bt
86%
32.8 ± 8.0
H
CH3
N
H
N
1
2bh
81%
18%
3.9 ± 1.0
12bu
12bv
69%
23%
4.6 ± 0.9
OCF3
H
H
N
N
1
2bi
112.8 ± 30.4
35.7 ± 5.5
CN
H
H
N
N
12bj
76%
2.1 ± 0.3
12bw
42%
93.7 ± 16.9
O
a
Highest tested concentration is 200 µM.
Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15. NA = No Activity.
b
We next investigated whether alkyl amines were effective as mitochondrial uncouplers
Table 4). When alkyl and cycloalkyl groups were xchanged for the aniline, greatly diminished
(
efficacy and potency were observed with the exception of 1-adamantyl amine (12ce) with 60%
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of BAM15 OCR and an EC50 value of 7.0 µM. The analog 12ce was selected for
pharmacokinetic studies as a representative of alkyl amines (Table 6).
Table 4. Oxygen consumption rates (OCR) of alkyl amine derivatives in L6 rat myoblast cells.a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R
N
O
N
N
OH
%
of BAM15
OCR
% of BAM15
Entry
12bx
R
EC50, µM
Entry
R
EC50, µM
b
b
OCR
H
H
N
N
NA
--
12cd
63%
38.3 ± 5.0
H
N
H
N
12by
19%
16%
8%
3.4 ± 0.3
65.5 ± 10.2
109.8 ± 45.8
12ce
12cf
12cg
60%
42%
23%
7.0 ± 1.0
22.8 ± 2.5
2.0 ± 0.3
H
H
N
N
1
2bz
2ca
12cb
2cc
H
N
H
N
1
CF3
H
N
H
N
20%
32%
53.2 ± 11.3
13.4 ± 4.1
12ch
12ci
NA
--
HO
OCF3
H
N
N
1
H
43%
4.9 ± 0.5
a
Highest tested concentration is 200 µM.
Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15. NA = No Activity.
b
Finally, we determined the effect of the furazan ring (Table 5). Replacing the
[1,2,5]oxadiazolo[3,4-b]pyrazine core with a pyrazine resulted in complete loss of activity.
Unfortunately, all compounds synthesized bearing fluoro (16a), alkyl (16b), electron
withdrawing groups (16c–16f), and biphenyls (16g and 16h) were inactive. These results
highlight the significance of the electron withdrawing effect of the bicyclic core.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Table 5. Oxygen consumption rates (OCR) of pyrazine derivatives in L6 rat myoblast cells.
N
R
N
OH
%
of BAM15
OCR
% of BAM15
Entry
6a
6b
6c
R
EC50, µM Entry
R
EC50, µM
b
b
OCR
F
H
N
H
N
1
NA
NA
--
--
16e
16f
NA
NA
--
--
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CF3
CF3
H
N
H
N
1
C10H21
H
N
H
N
1
NA
NA
--
--
16g
16h
O
NA
NA
--
--
OCF3
CF3
H
N
H
N
1
6d
CF3
F
a
Highest tested concentration is 200 µM.
b
Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15. NA = No Activity.
Table 6. Pharmacokinetic profile of select analogs in mice.a
Compound
2i
SHS4121705)
Structure
Cmax (µM)
t1/2 (h)
1
H
N
N
N
N
(
O
80.9
5.7
N
OH
OCF3
H
N
N
N
N
N
1
2ah
O
3.4
0.3
0.4
OH
O
H
N
N
N
N
1
2az
O
12.5
N
OH
H
N
N
N
N
O
1
2bh
2bj
2ce
22.3
12
0.6
0.7
1.1
N
OH
H
N
N
N
1
O
N
N
OH
H
N
N
N
1
18
O
N
N
OH
a
Compounds were administered at 10 mg/kg body weight by oral
gavage to male mice. Plasma samples collected over 8-24 h were
analyzed by LC-MS/MS. Cmax = maximal plasma concentration. t1/2
=
half-life.
Based on the OCR assay, we selected representative compounds to determine their
suitability for in vivo studies in a mouse model of NASH. Thus, mice were dosed with 10 mg/kg
of compound per oral and pharmacokinetic parameters were determined. As shown in Table 6,
the majority of the compounds had poor oral bioavailability (< 22 µM) and short half-life (1.1 h
or less). Fortunately, 12i (SHS4121705) resulted in a peak plasma concentration of ~81 µM and
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a half-life of 5.7 h after administering by oral gavage (Figure 3A). We then evaluated the effect
of SHS4121705 on core body temperature as a function of dose at 5, 20, 50 mg/kg along with
vehicle control. As shown in Figure 3B, there is no effect on core body temperature detected by
rectal probe thermometer at up to 4 hours after an acute dose. This is an important safety
parameter as increased body temperature is a characteristic of some toxic protonophores such as
1
1
1
1
1
1
1
1
1
1
2
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DNP. In addition, tissue distribution of SHS4121705 in the liver is crucial as complications of
NASH are manifested in the liver. After dosing animals with 10 mg/kg of SHS4121705 by oral
gavage, mice were sacrificed 1 hour after exposure and levels of compound were determined by
liquid chromatography tandem mass spectrometry (LC/MS-MS). As illustrated in Figure 3C,
SHS4121705 exposure was highest in the liver (15.8 ± 1.3 µg/g) and kidney (9.2 ± 1.2 µg/g).
The favorable PK properties of SHS4121705 is supported by the improvement of
physicochemical properties (solubility in PBS, pH 7.4: >500 µM; cLogP = 5.2) when compared
to starting lead compound 9 (solubility in PBS, pH 7.4: <10 µM; cLogP = 6.4). We also found
that SHS4121705 has no activity on ion channels such as hERG (see SI for details). Taken
together, the favorable physicochemical properties, excellent oral bioavailability and liver
exposure suggest that SHS4121705 was a suitable candidate for in vivo efficacy studies against
NASH.
The in vivo efficacy was determined using the Stelic animal model (STAM) of NASH
with Telmisartan as a positive control, as described before.78,90 Over the course of 21 days,
SHS4121705 was administered with food at a dose of 25 mg/kg/d. As a control, Telmisartan was
dosed 10 mg/kg daily by oral gavage. During the treatment period, food intake was monitored
and SHS4121705 showed no effect on food consumption (331 ± 27 g/cage) as compared to
vehicle (318 ± 13 g/cage). The Telmisartan control group showed a 9% decrease in food intake
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(
246 ± 2 g/cage). As shown in Figure 4A-B, mice treated with SHS4121705 had a lower fibrosis
score measured by Picrosirius Red stained area (0.62 ± 0.07%) compared to untreated vehicle
control (0.91 ± 0.09%). SHS4121705 anti-fibrosis efficacy was similar to the positive control
telmisartan (0.60 ± 0.07%). Mice treated with SHS4121705 had a 2-point improvement in NAS
score compared to vehicle control mice (Figure 4C, 2.63 ± 0.18 compared to 4.63 ± 0.26 in
vehicle), resulting from a lower score in all three liver NAS markers: steatosis (0 ± 0),
inflammation (2.38 ± 0.18) and ballooning (0.25 ± 0.16) when compared to vehicle (steatosis 1.0
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±
0), inflammation (2.75 ± 0.16), ballooning (0.88 ± 0.23) (Figure 4D-F). Importantly, a
complete reduction in steatosis score was observed in SHS4121705 treated mice. Ballooning
score was also decreased by SHS4121705, which is notable as early studies have indicated that
ballooning is one of the few histological features associated with risk of cirrhosis development in
9
1
NAFLD. Additionally, ballooning is linked to the attraction of inflammatory cells, suggesting
that longer in vivo studies, that allow for greater accumulation of inflammatory cells in the
vehicle group, could show a further reduction in inflammation score.92
The impact of SHS4121705 on whole body physiology was investigated. Unlike
telmisartan-treated animals, there was no change in body or liver weight following SHS4121705
treatment (Figure 5A-C). Both SHS4121705 and telmisartan decreased liver triglyceride content
(Figure 5D). SHS4121705 did not alter plasma triglyceride, cholesterol or glucose levels, while
telmisartan raised plasma cholesterol and glucose (Figure 5E-G). Both SHS4121705 and
telmisartan decreased alanine aminotransferase (ALT), which is a marker of liver damage,
(Figure 5H) without altering levels of aspartate aminotransferase (AST) (Figure 5I). Overall, the
physiological data indicate that mitochondrial uncoupler SHS4121705 decreased biochemical
markers of liver damage and decreased steatosis, inflammation and ballooning resulting in a 2-
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point improvement in NAS score. These results suggest that mitochondrial uncouplers have
potential as NASH therapeutics.
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Figure 3. Acute dose studies of SHS4121705. To measure pharmacokinetics (A) SHS4121705 was administered at
1
0 mg/kg body weight by oral gavage and plasma samples were collected at the time points shown and analyzed by
LC-MS/MS. Body temperature (B) was measured using a rectal temperature probe following an acute dose of 5-50
mg/kg SHS4121705, or vehicle. No significant changes were detected in body temperature, as assessed by Two-
Way Repeated Measures ANOVA. Tissue distribution (C) of SHS4121705 was measured at 1 hour after an acute
oral gavage of at 10 mg/kg in C57BL/6 male mice. Compound concentration was measured in tissues (liver, gonadal
white adipose tissue (WAT), mixed quadriceps muscle (Quad), kidney, heart and brain) by LC-MS/MS. n=3
C57BL/6 male mice for all experiments.
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Figure 4. Histological results from liver tissue of STAM mice. Representative images of hematoxylin & eosin and
Sirius Red staining of fixed tissue (A). Fibrosis was measured by quantification of Sirius Red-positive area (B).
NAFLD Activity Scores (C) were calculated from pathologist scoring of steatosis (D), inflammation (E) and
ballooning (F). * indicates p<0.05. Statistical significance was assessed by Kruskal-Wallis test. n=8 male mice per
group.
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Figure 5. Physiological parameters of STAM mice. Telmisartan, but not SHS4121705, decreased body weight (A),
liver weight (B) and liver-to-body weight ratio (C). Both drug treatments decreased liver triglyceride (D).
SHS4121705 did not change plasma triglyceride (E) or cholesterol (F) levels, nor whole blood glucose (G).
SHS4121705 and Telmisartan treatment decreased plasma ALT (H) but had no effect on plasma AST (I). * indicates
p<0.05. Statistical significance assessed by One-Way ANOVA for normally distributed data (D, G, I) and Kruskal-
Wallis test for non-parametric data (A-C, E-F, H). n=8 male mice per group.
CONCLUSION
Mitochondrial uncouplers have been shown to increase cellular respiration, uncoupling oxidative
phosphorylation and nutrient metabolism. Therapeutic application of uncouplers has been
supported in myriad disease models such as obesity and fatty liver disease. Promising drug
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candidates include the FDA-approved drug niclosamide, which is currently being repurposed and
has efficacy in fatty liver models, although off-target effects may persist. Our discovery of
[1,2,5]oxadiazolo[3,4-b]pyrazine derivatives point to a new class of mitochondria-selective
uncouplers with in vivo efficacy in a mouse model of NASH. Our investigation focused on the
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[1,2,5]oxadiazolo[3,4-b]pyrazine core modified with a hydroxyl group to expand the tolerated
chemical space and enable modulation of physicochemical properties. Our investigations
revealed that this new modification preserved the protonophoric activity with both electron
withdrawing and electron donating groups tolerated. Electron donating alkyl groups were among
the most potent and efficacious; however, electron withdrawing groups approached the same
potency and efficacy with greatly improved pharmacokinetic properties. In particular, we
discovered that SHS4121705 (12i) had excellent oral bioavailability and exposure in liver that
encouraged the advancement into in vivo efficacy studies. In the STAM mouse model of NASH,
SHS4121705 improved NAFLD activity score as expected of an anti-NASH agent, lowered liver
triglyceride levels, and decreased liver ALT and fibrosis without affecting body temperature or
food intake. Taken together, these results support the continued development of this novel
scaffold of mitochondrial uncouplers as potential agents for the treatment of NASH.
Experimental Section
General Material and Synthetic Procedures
All solvents used were either dried with a PureSolv solvent purification system prior to
use or purchased in anhydrous form. All chemical reagents were purchased from commercial
sources and used without further purification. Thin layer chromatography (TLC) was performed
on aluminum-backed silica gel, 200 µm, F254. Column chromatography was performed on flash
grade silica gel, 40-63 µm, with a Teledyne ISCO Combiflash Rf purification system.
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NMR spectra were recorded using an Agilent 400-MR 400 MHz, a Varian Inova 400 MHz
1
or Bruker Avance II 500. H NMR chemical shifts are reported in ppm with the solvent
resonance as an internal standard ((CD ) SO: 2.50 ppm; (CD ) CO: 2.05 ppm; CDCl : 7.26
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3 2
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ppm). C NMR chemical shifts are reported in ppm with the solvent resonance as the internal
standard ((CD ) SO: 39.52 ppm; (CD ) CO: 29.84/206.26 ppm; CDCl : 77.16 ppm). Data are
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reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
br = broad, m = multiplet), coupling constants (Hz), and integration. HPLC and high resolution
mass spectroscopy (HRMS) was performed on Thermo Electron TSQ triple quadrupole mass
spectrometer equipped with an ESI source. HPLC condition 1: Phenomenex LUNA column (150
mm x 2.0 mm, 5 µm, C18) using a solvent system of 1% formic acid in H O (mobile phase A)
2
and 1% formic acid in acetonitrile (mobile phase B) on an Agilent 1100 binary pump with a
gradient of 50-95% mobile phase AB at a flow rate of 0.2 mL/min. HPLC condition 2:
Phenomenex Luna (150 mm x 4.5 mm, 5 µm, C18) using a solvent system of 0.1% TFA in
water (mobile phase A) and 0.1% TFA in acetonitrile (mobile phase B) on an Agilent 1200
binary pump with a gradient of 5-95% mobile phase AB at a flow rate of 1.5 mL/min. Unless
otherwise noted, HPLC condition 1 was used. All compounds tested in biological assays are
>
95% pure by HPLC analyses unless noted otherwise.
General Procedure A. In a screw-cap vial or round-bottom flask, the requisite amine (0.70 –
0.98 mmol) was added to a stirring mixture of dichloro 11 (0.200 g, 1.05 mmol) in anhydrous
THF (or acetone where indicated) (0.1 – 0.2 M). Then, Et N (1.1 mmol or 2.2 mmol when an
3
amine salt is used) was added and the resulting dark mixture was stirred at room temperature
(unless otherwise indicated) for 2 – 20 h. The reaction mixture was diluted with an aqueous
solution of KOH (6 equiv.) and stirring at room temperature was continued for 0.5 – 2 h. The
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mixture was acidified with 1 M aqueous HCl and extracted with EtOAc. The organic layer was
washed with brine, dried (Na SO ), and concentrated to a residue. The residue was purified by
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chromatography on SiO using a MeOH/CH Cl or EtOAc/hexanes solvent system to yield the
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desired product. When additional purification was needed, the solid was dissolved in a minimal
amount of hot acetone, allowed to cool to room temperature, and precipitated by the addition of
hexanes. The precipitate was filtered, rinsed with hexanes, and collected to yield the desired
product 12.
General Procedure B. To a round-bottom flask containing a stir bar was added dichloro 11
(1.05 mmol) which was diluted with anhydrous THF (4 mL), followed by the amine (0.838
mmol, 0.8 equiv.). The resulting solution was stirred at 50 °C for 16 h. KOH (6.28 mmol, 6.0
equiv.) in H O (8 mL) and THF (2 mL) was added and stirring was continued for an additional 1
2
h. The mixture was then acidified with 1 M aqueous HCl and extracted with EtOAc. The organic
layer was washed with brine, dried (Na SO ), and concentrated to a solid. The solid was purified
2
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by chromatography on SiO using a MeOH/CH Cl solvent system to yield the desired compound
2
2
2
1
2.
General Procedure C. To anhydrous THF (25 mL) was added sodium hydride (1.5 equiv., 60%
dispersion) and allowed to stir for 5 min. Benzyl alcohol (1.0 equiv.) in anhydrous THF (15 mL)
was added dropwise over a minute and allowed to stir for 30 min. 2,3-Dichloropyrazine (14) (1.0
equiv.) in THF (10 mL) was added and the reaction was allowed to stir at room temperature for 2
h (monitored by TLC). Upon completion, the reaction was quenched slowly with isopropanol.
The solvent was evaporated under reduced pressure and the crude product was purified by
chromatography on SiO using EtOAc/hexanes to produce intermediate 15.
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A vial was charged with Pd dba (0.05 equiv.), Xantphos (0.10 equiv.), 2-(benzyloxy)-3-
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chloropyrazine (15) (1.00 equiv.), and K CO (2.00 equiv.). The vial was sealed with a septum
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and then evacuated and backfilled with argon (3x). Deoxygenated anhydrous dioxane (4 mL)
was added through the septum, followed by the requisite aryl amine (1.05 equiv.). The resulting
mixture was stirred at 110 °C for 16 h and then allowed to cool to room temperature. The solid
material was filtered through Celite and washed with EtOAc. The filtrate was concentrated under
reduced pressure in a round-bottom flask and diluted with methanol (4 mL). The flask was
flushed with nitrogen for 5 min. 10% Pd/C (0.05 equiv.) was added and the mixture was stirred
under an atmosphere of hydrogen gas (1 atm, balloon) for 1.5 h at room temperature. The
reaction was then filtered through a Celite plug washing with EtOAc, concentrated under
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reduced pressure, and the crude product was purified by chromatography on SiO using
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EtOAc/hexanes to yield the desired compound 16.
Characterizations:
6
-((2-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12a). Synthesized by
1
procedure A to yield 12a in 8% as a pale yellow solid: H NMR ((CD ) CO 500 MHz) δ 12.18
3
2
,
1
9
(
br s, 1 H), 9.23 (s, 1 H), 8.51 (t, J = 8.2 Hz, 1 H), 7.35-7.27 (m, 3 H); F NMR ((CD ) CO, 376
3 2
1
3
MHz) δ -129.33 to -129.40 (m, 1 F); C NMR ((CD ) CO, 126 MHz) δ 155.0 (d, J = 245.7
3
2
CF
Hz), 153.5, 151.4, 150.2, 145.1, 127.2 (d, J = 7.7 Hz), 126.3 (d, J = 10.4 Hz), 125.6 (d, J =
CF
CF
CF
-
-
3
.9 Hz), 124.1, 116.2 (d, JCF = 19.5 Hz); HRMS (ESI ) m/z calc’d. for C H FN O (M-H)
10 5 5 2
246.0433, found 246.0438.
6
-((3-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12b). Synthesized by
1
procedure A to yield 12b in 10% as a pale yellow solid: H NMR ((CD ) CO 500 MHz) δ 12.09
3
2
,
(br s, 1 H), 9.64 (s, 1 H), 8.13 (dt, J = 11.6, 2.4 Hz, 1 H), 7.90 (dd, J = 8.2, 2.0 Hz, 1 H), 7.48 (q,
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J = 8.0 Hz, 1 H), 6.99 (dt, J = 8.4, 2.5 Hz, 1 H); F NMR ((CD ) CO, 376 MHz) δ -113.15 to -
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1
13.22 (m, 1 F); C NMR ((CD ) CO, 126 MHz) δ 163.6 (d, J = 242.4 Hz), 153.4, 151.4,
3 2 CF
150.2, 145.1, 140.4 (d, JCF = 11.1 Hz), 131.3 (d, JCF = 9.4 Hz), 117.9 (d, JCF = 3.1 Hz), 112.3 (d,
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-
JCF = 21.3 Hz), 109.0 (d, JCF = 26.9 Hz); HRMS (ESI ) m/z calc’d. for C H FN O (M-H)
10
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46.0433, found 246.0440.
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-((4-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12c). Synthesized by
1
procedure B to yield 12c in 44% as a yellow solid (HPLC condition 2): H NMR ((CD ) CO, 400
3
2
MHz) δ 11.69 (s, 1H), 9.58 (s, 1H), 8.19 – 8.11 (m, 2H), 7.28 – 7.19 (m, 2H); 19F NMR
1
3
(
(CD ) CO, 376 MHz) δ -118.44 – -118.56 (m, 1F); C NMR (101 MHz, Acetone-d ) δ 160.6
3
2
6
(d, J = 243.0 Hz), 153.6, 151.2, 150.4, 145.1, 135.0 (d, J = 2.7 Hz), 124.2 (d, J = 8.0 Hz), 116.3
+
+
(
d, J = 22.8 Hz); HRMS (ESI ) m/z calc’d for C H FN O (M+H) 248.0578, found 248.0591.
10 7 5 2
6
-((2-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12d).
1
Synthesized by procedure B to yield 12d in 5% as a yellow solid: H NMR (400 MHz, Acetone-
d6) δ 12.1 (s, 1H), 9.36 (s, 1H), 8.55 (d, J = 8.1 Hz, 1H), 7.88 – 7.79 (m, 2H), 7.50 (tp, J = 7.7,
1
9
+
1
.0 Hz, 1H); F NMR ((CD ) CO, 376 MHz) δ rotamers -61.19 (s), -61.21 (s); HRMS (ESI )
3 2
+
m/z calc’d for C H F N O (M+H) 298.0546, found 298.0538.
11
7
3
5
2
6
-((3-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12e).
Synthesized by procedure B to yield 12e in 79% as a yellow solid (HPLC condition 2): H NMR
(CD ) CO, 400 MHz) δ 10.41 (s, 1H), 9.78 (s, 1H), 8.58 (s, 1H), 8.44 – 8.38 (m, 1H), 7.69 (t,
1
(
3 2
1
9
13
8.0 Hz, 1H), 7.57 – 7.52 (m, 1H); F NMR ((CD ) CO, 376 MHz) δ -63.23 (s, 3F); C NMR
3
2
(
(CD ) CO, 126 MHz) δ 153.5, 151.6, 150.1, 145.2, 139.5, 131.4 (q, J = 32.1 Hz), 130.8,
3
2
C
F
1
25.6, 125.1 (q, JCF = 271.6 Hz), 122.1 (q, JCF = 3.8 Hz), 118.6 (q, JCF = 4.1 Hz); HRMS (ESI-)
-
m/z Calcd. for C H F N O (M-H) 296.0401, found 296.0425.
1
1
5
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5
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-((4-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12f).
1
Synthesized by procedure A to yield 12f in 66% as a yellow solid: H NMR ((CD ) CO, 500
3
2
1
9
MHz) δ 12.11 (br s, 1 H), 9.77 (s, 1 H), 8.37 (d, J = 8.3 Hz, 2 H), 7.80 (d, J = 8.3 Hz, 2 H); F
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
3
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3
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4
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5
5
5
5
5
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0
1
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8
9
0
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8
9
0
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7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
NMR ((CD ) CO, 376 MHz) δ -62.59 (s, 3 F); C NMR ((CD ) CO, 126 MHz) δ 153.4, 151.6,
3
2
3 2
1
1
6
50.2, 145.1, 142.1, 127.0 (q, JCF = 3.8 Hz), 126.7 (q, JCF = 32.6 Hz), 125.3 (q, JCF = 270.8 Hz),
-
-
22.2; HRMS (ESI ) m/z calc’d. for C H F N O (M-H) 296.0401, found 296.0419.
11
5
3
5
2
-((2-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12g).
1
Synthesized by procedure A to yield 12g in 27% as a yellow solid: H NMR ((CD ) CO, 500
3
2
MHz) δ 12.25 (br s, 1H), 9.34 (s, 1H), 8.75 (d, J = 8.1, 1H), 7.57 – 7.50 (m, 2H), 7.38 (t, J = 7.5
Hz, 1H); 19F NMR ((CD ) CO, 376 MHz) δ rotamers -58.516 (s), -58.522 (s); 13C NMR
3
2
((CD ) CO, 126 MHz) δ 153.7, 151.3, 150.2, 145.2, 140.5 (q, J = 1.2 Hz), 130.8, 128.9, 126.8,
3 2 CF
+
+
1
23.7, 121.9, 121.6 (q, JCF = 258.0 Hz); HRMS (ES ) m/z calc’d. for C H F N O (M+H)
11 7 3 5 3
314.0496, found 314.0474.
6
-((3-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12h).
1
Synthesized by procedure A to yield 12h in 64% as a yellow solid: H NMR ((CD ) CO, 500
3
2
MHz) δ 12.01 (br s, 1H), 9.73 (s, 1 H), 8.29 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 8.3 Hz,
1
9
13
1
H), 7.18 (d, J = 8.3 Hz, 1H); F NMR ((CD ) CO, 376 MHz) δ -58.5 (s, 3F); C NMR
3 2
((CD ) CO, 126 MHz) δ 153.4, 151.5, 150.2, 150.1 (q, J = 3.8 Hz), 145.1, 140.3, 131.3, 121.5
3 2 CF
-
-
(q, JCF = 255.9 Hz), 120.7, 117.8, 114.6; HRMS (ESI ) m/z calc’d. for C H F N O (M-H)
11
5
3
5
3
312.0350, found 312.0372.
6
1
-((4-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (SHS4121705,
1
2i). Synthesized by procedure A to yield SHS4121705 (12i) in 75% as a yellow solid: H NMR
((CD ) CO 500 MHz) δ 12.07 (br s, 1H), 9.67 (s, 1H), 8.27 – 8.24 (m, 2H), 7.43 (d, J = 9.1 Hz,
3
2
,
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19
13
2
1
H); F NMR ((CD ) CO, 376 MHz) δ -58.79 (s, 3F); C NMR ((CD ) CO, 126 MHz) δ 153.5,
3
2
3 2
51.3, 150.3, 146.4 (q, JCF = 2.0 Hz), 145.1, 137.7, 123.7, 122.5, 121.5 (q, JCF = 255.3 Hz);
-
-
HRMS (ESI ) m/z calc’d for C H F N O (M-H) 312.0350, found 312.0369.
1
1
5
3
5
3
0
1
2
3
4
5
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8
9
0
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0
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0
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0
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-((2-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12j). Synthesized by
1
procedure A to yield 12j in 24% as a yellow solid: H NMR ((CD ) CO, 400 MHz) δ 12.26 (br s,
3
2
1
H), 9.51 (br s, 1H), 8.78 – 8.75 (m, 1H), 7.62 – 7.59 (m, 1H), 7.54 – 7.50 (m, 1H), 7.30 – 7.26
1
3
(
m, 1H); C NMR ((CD ) CO, 126 MHz) δ *rotamers 153.7, 151.2, 150.2, 145.1, 134.8, 130.4,
3 2
-
-
1
29.0, 127.0, 126.9*, 125.3, 123.1, 123.0*; HRMS (ESI ) m/z calc’d for C H ClN O (M-H)
10 5 5 2
2
62.0137, found 262.0160.
6-((4-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12k). Synthesized by
1
procedure A to yield 12k in 59% as a yellow solid: H NMR ((CD ) CO, 500 MHz) δ 12.06 (br
3
2
1
3
s, 1 H), 9.61 (br s, 1H), 8.18 – 8.15 (m, 2H), 7.50 – 7.47 (m, 2H); C NMR ((CD ) CO, 126
3
2
+
MHz) δ 153.5, 151.3, 150.3, 145.1, 137.6, 130.4, 129.7, 123.7; HRMS (ES ) m/z calc’d for
+
C H ClN O (M+H) 264.0283, found 264.0289.
1
0
7
5
2
6
-((4-(Difluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12l).
1
Synthesized by procedure A to yield 12l in 72% as a pale yellow solid: H NMR ((CD ) CO, 500
3 2
MHz) δ 12.05 (br s, 1H), 9.60 (s, 1H), 8.19 – 8.15 (m, 2H), 7.31 – 7.27 (m, 2H), 7.01 (t, JHF
=
7
4.3 Hz, 1H); 19F NMR ((CD ) CO, 376 MHz) δ -82.64 (d, J = 74.1 Hz, 2F); 13C NMR
3
2
(
(CD ) CO, 126 MHz) δ 153.5, 151.2, 150.4, 149.1 (t, J = 3.2 Hz), 145.1, 136.0, 123.8, 120.5,
3 2 CF
+
+
117.5 (t, JCF = 257.4 Hz); HRMS (ES ) m/z calc’d for C H F N O (M+H) 296.0590, found
11
8
2
5
3
2
96.0594.
6
-((4-(1,1,2,2-Tetrafluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12m).
1
Synthesized by procedure A to yield 12m in 70% as a yellow solid: H NMR ((CD ) CO, 500
3
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MHz) δ 12.07 (br s, 1H), 9.66 (s, 1H), 8.25 – 8.21 (m, 2H), 7.40 – 7.38 (m, 2H), 6.53 (tt, JHF
=
1
9
5
2.5, 3.0 Hz, 1H); F NMR ((CD ) CO, 376 MHz) δ -89.10 (td, J = 5.5, 2.7 Hz, 2F), -138.63
3 2
1
3
(dt, J = 52.4, 5.8 Hz, 2F); C NMR ((CD ) CO, 126 MHz) δ 153.5, 151.3, 150.3, 146.1 (t, J =
CF
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
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5
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5
5
5
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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7
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9
0
1
2
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8
9
0
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8
9
0
2
.1 Hz), 145.1, 137.3, 123.6, 123.1, 117.6 (tt, JCF = 270.0, 28.6 Hz), 109.1 (tt, JCF = 249.0, 40.7
+
+
Hz); HRMS (ES ) m/z calc’d. for C H F N O (M+H) 346.0558, found 346.0562.
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2
8
4
5
3
6
-((4-(Chlorodifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol
(12n).
1
Synthesized by procedure A to yield 12n in 65% as a pale yellow solid: H NMR ((CD ) CO,
3
2
1
9
4
00 MHz) δ 12.08 (br s, 1H), 9.69 (s, 1H), 8.29 – 8.25 (m, 2H), 7.46 – 7.43 (m, 2H); F NMR
1
3
(
(CD ) CO, 376 MHz) δ -26.23 (s, 2F); C NMR ((CD ) CO, 126 MHz) δ 153.5, 151.4, 150.3,
3 2 3 2
+
147.5, 145.1, 137.9, 126.3 (t, JCF = 286.8 Hz), 123.6, 122.9; HRMS (ES ) m/z calc’d. for
+
C H ClF N O (M+H) 330.0200, found 330.0202.
1
1
7
2
5
3
6
-((2,2-Difluorobenzo[d][1,3]dioxol-5-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12o).
1
Synthesized by procedure A to yield 12o in 59% as a yellow solid: H NMR ((CD ) CO, 500
3
2
MHz) δ 12.08 (br s, 1H), 9.71 (s, 1H), 8.23 (d, J = 2.2 Hz, 1 H), 7.91 (dd, J = 8.8, 2.2 Hz, 1H),
1
9
13
7
1
1
6
.38 (d, J = 8.8 Hz, 1H); F NMR ((CD ) CO, 376 MHz) δ -51.12 (s, 2F); C NMR ((CD ) CO,
3 2 3 2
25 MHz) δ 153.5, 151.4, 150.2, 145.1, 144.2, 141.1, 135.3, 132.7 (t, JCF = 252.9 Hz), 118.1,
+
+
10.7, 104.8; HRMS (ES ) m/z calc’d. for C H F N O (M+H) 310.0382, found 310.0386.
1
1
6
2
5
4
-((4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-
b]pyrazin-5-ol (12p). In a screw-cap vial, a mixture of 11 (0.200 g, 1.05 mmol), 2-(4-
aminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (0.125 g, 0.482 mmol), and NaHCO (0.100 g,
3
1
.19 mmol) in acetone/H O (9:1, 4 mL) was stirred at room temperature for 18 h. The mixture
2
was diluted with an aqueous solution of KOH (0.320 g in 4 mL) and stirring was continued for 2
h. The mixture was acidified with 1 M HCl and extracted with EtOAc. The organic layer was
2
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