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

Joseph M Salamoun; Christopher J Garcia; Stefan R Hargett; Jacob H Murray; Sing-Young Chen; Martina Beretta; Stephanie J Alexopoulos; Divya P Shah; Ellen M Olzomer; Simon P Tucker; Kyle L Hoehn; Webster L Santos

doi:10.1021/acs.jmedchem.0c00542

. Author manuscript; available in PMC: 2024 Apr 24.

Published in final edited form as: J Med Chem. 2020 May 27;63(11):6203–6224. doi: 10.1021/acs.jmedchem.0c00542

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

Joseph M Salamoun ^1,^#, Christopher J Garcia ^2,^#, Stefan R Hargett ³, Jacob H Murray ⁴, Sing-Young Chen ⁵, Martina Beretta ⁶, Stephanie J Alexopoulos ⁷, Divya P Shah ⁸, Ellen M Olzomer ⁹, Simon P Tucker ¹⁰, Kyle L Hoehn ¹¹, Webster L Santos ¹²

¹Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

²Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

³Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia 22908, United States

⁴Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

⁵School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

⁶School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

⁷School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

⁸School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

⁹School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

¹⁰Continuum Biosciences, Pty Ltd., Sydney 2035, Australia; Continuum Biosciences Inc., Boston, Massachusetts 02116, United States

¹¹Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia 22908, United States; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

¹²Departments of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

Author Contributions

J.M.S. and C.J.G. contributed equally.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

^✉

Corresponding Authors Kyle L. Hoehn – Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia 22908, United States; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia; k.hoehn@unsw.edu.au, Webster L. Santos – Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States; santosw@vt.edu

PMCID: PMC11042500 NIHMSID: NIHMS1984181 PMID: 32392051

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 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.

Graphical Abstract

graphic file with name nihms-1984181-f0008.jpg

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

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 nonalcoholic fatty liver disease by slowing down or even preventing hepatic steatosis and nonalcoholic steatohepatitis (NASH) phenotypes.^{19–21,23–26} These encouraging results are particularly relevant to today’s clinical challenges as nonalcoholic 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 noninvasive 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 a significant contributor to the number of liver transplantations.³⁰ 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

Figure 1. — Structures of select protonophore mitochondrial uncouplers to showcase chemical diversity of the weak acids.

In addition to increasing energy expenditure, protonophores reduce the production of reactive oxygen species^32–34 and have been explored for their potential use as antineurodegenerative,³⁵ anticancer,^36–39 antiaging,^40–42 and antibacterial agents.⁴³ Despite the great preclinical promise of mitochondrial uncouplers for treatment of numerous serious medical 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 nonmitochondrial membranes.⁴⁴ 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 upward of 50% increase in metabolism and had weight-loss-inducing effects.^45–48 Although widely used as a diet aid in the early 1930s, DNP was quickly banned by the FDA due to adverse effects including increased body temperature,^47,49–51 cataracts,^52–55 blindness,⁵⁴ and death in some patients.⁴⁸ Mechanistic studies now indicate that, in addition to the mitochondria, DNP also depolarizes the plasma membrane resulting in a narrow therapeutic window.^56–59 FCCP (2), which is highly toxic in animals, has a limited use as an in vitro control in biochemical assays.^60–64 In an effort to develop effective strategies to address safety concerns, uncoupling activity was identified in FDA-approved drugs such as niclosamide (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.^65,66 Likewise, an antiparasitic drug nitazoxanide (4) was recently shown to exhibit uncoupling activity⁶⁷ and is currently in phase 2 clinical trial for NASH-associated fibrosis (ClinicalTrials.gov identifier: NCT03656068).

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), dicyanohydrazones (e.g., FCCP), arylamides (e.g., niclosamide and nitazoxanide),⁶⁷ triazoles (e.g., OPC-163493),²² perfluoroalkyl-sulfonamides,⁶⁸ benzimidazoles,^69,70 and carboranes^71,72 are among the diverse 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.^73,74 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 oxadiazolopyrazine core decorated with aniline as essential for uncoupling properties.⁷⁵ 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 aniline moieties generally led to lower potency and efficacy.⁷⁷ Interestingly, a subtle change to 8 such as regioisomers of the fluoroaniline provided the unsymmetrical analog 9 that was efficacious in a streptozotocin (STZ)-induced mouse model of NASH. Compound 9 lowered 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 two anilines and widen the scope of tolerated aryl and alkyl substituents. In this study, we performed a 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 demonstrate improvement in NAFLD activity score and fibrosis but it also decreased liver triglyceride and ALT levels.

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 pK_a 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 as both 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 BAM15 is not affected by the ANT inhibitor carboxyatractyloside;⁷³ 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 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 was initiated with the common intermediate 5,6-dichloro[1,2,5]oxadiazolo[3,4-b]pyrazine (11), synthesized as described before (Scheme 1).^80–82,78 Then in a one-pot reaction, intermediate 11 was directly substituted with a substoichiometric equivalent of the requisite alkyl- or arylamines 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.

Scheme 1a — ^aReagents and conditions: (a) oxalic acid, 10% HCl (aq), reflux, 4 h, 72%; (b) PCl₅, POCl₃, 95 °C, 2 h, 77%; (c) one pot reaction: (i) amine, Et₃N or K₂CO₃, THF or acetone, rt or 75 °C; (ii) KOH_(aq) at rt for 2 h; (d) one pot reaction: 4-trifluoromethoxyaniline, Et₃N, THF, rt; then NaOMe in MeOH at rt for 1 h.

To investigate the effect of the strongly electron withdrawing furazan ring, pyrazine derivatives were synthesized (Scheme 2). Monosubstitution of 2,3-dichloropyrazine (14) with benzyl alcohol afforded the desired intermediate 15. The aniline moiety was introduced via a Buchwald–Hartwig cross-coupling reaction,⁷⁷ followed by catalytic hydrogenation to cleave the benzyl group and generate the desired hydroxypyrazine analogs 16.

Scheme 2a — ^aReagents and conditions: (a) NaH, BnOH, THF, rt, 2 h, 87%; (b) (i) Pd₂dba₃ (10 mol %), Xantphos (10 mol %), arylamine (1.3 equiv), K₂CO₃ (2.3 equiv), dioxane, 110 °C, 16 h; (ii) 10% Pd/C (5 mol %), H_{2 (g)} (1 atm), MeOH, rt, 1.5 h, 12–37% over two steps.

With [1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol and pyrazine derivatives in hand, we tested their activity as mitochondrial uncouplers 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 min 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 respiration.⁷⁸ 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 interassay variabilities across the large number of compounds studied. Our ideal mitochondrial uncouplers are expected to have low EC₅₀ values and OCR activity close to BAM15.

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 stimulate respiration in L6 rat myoblast cells with an improved EC₅₀ value (190 nM) relative to BAM15. The first analog 12a, a direct derivative of 9 containing a 2-fluoroaniline, retained 74% of BAM15 uncoupling capability, albeit at a 40× loss of potency (Table 1). The corresponding meta (12b) and para (12c) derivatives were likewise effective although with 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 was pursued (Tables 1–5).

Table 1.

Oxygen Consumption Rates of Halogenated Aniline Derivatives in L6 Rat Myoblast Cells^a



Entry	R	% of BAM15 OCR^b	EC₅₀, μM	Entry	R	% of BAM15 OCR^b	EC₅₀, μM

BAM15	-	100%	0.31 ± 0.06	12p		29%	34.0 ± 8.0
9	-	71%	0.19 ± 0.01	12q		45%	9.7 ± 1.8
12a		74%	12.5 ± 0.8	12r		NA	--
12b		67%	31.2 ± 2.0	12s		46%	23.4 ± 4.2
12c		51%	38.6 ± 4.8	12t		47%	5.1 ± 0.3
12d		88%	10.6 ± 0.7	12u		30%	2.6 ± 0.2
12e		66%	13.3	12v		76%	11.6 ± 1.2
12f		68%	10.4 ± 1.4	12w		32%	4.9 ± 1.3
12g		67%	7.1 ± 0.5	12x		10%	4.5 ± 1.2
12h		47%	5.1 ± 0.4	12y		27%	2.3 ± 0.7
12i^(SHS4121705)		64%	4.3 ± 0.7	12z		32%	2.9 ± 0.3
12j		84%	6.3 ± 0.7	12aa		26%	3.0 ± 0.2
12k		72%	11.7 ± 1.3	12ab		8%	1.4 ± 0.4
12l		46%	20.6 ± 2.1	12ac		50%	3.8 ± 0.7
12m		86%	7.0 ± 1.3	12ad		27%	53.5 ± 6.4
12n		55%	1.9 ± 0.3	12ae		NA	--
12o		70%	7.6 ± 0.9	13	-	NA	--

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Highest tested concentration is 200 μM.

Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15.

NA = no activity.

Table 5.

Oxygen Consumption Rates (OCR) of Pyrazine Derivatives in L6 Rat Myoblast Cells^a



Entry	R	% of BAM15 OCR^b	EC₅₀, μM	Entry	R	% of BAM15 OCR^b	EC₅₀, μM

16a		NA	--	16e		NA	--
16b		NA	--	16f		NA	--
16c		NA	--	16g		NA	--
16d		NA	--	16h		NA	--

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Highest tested concentration is 200 μM.

Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15.

NA = no activity.

The first series of analogs incorporated a diverse array of electron withdrawing groups (Table 1). A variety of halogenated moieties were well-tolerated including trifluoromethyl (12d–12f), trifluoromethoxy (12g and 12i), OCF₂CHF₂ (12m), chloro (12j and 12k), and difluorobenzodioxole (12o), which all had >60% of BAM15 OCR and an EC₅₀ ≤ 13 μM. The OCF₃ analogs (12g–12i) were the most promising with activity in the single digit micromolar range and with the para-substituted derivative 12i having the best potency EC₅₀ at 4.3 μM. Anilines 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 12a, 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.

After the encouraging result from the OCF₃ analog 12i, a series of alkoxy analogs was 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 membrane. Because phenylalkyl ether groups are known to be a metabolic liability,⁸⁴ fluorinated analogs were introduced (12ai–12an) to potentially enhance metabolic stability.⁸⁵ However, many of these compounds showed diminished efficacy or potency.⁸⁶ The cyclopropylmethoxy 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 EC₅₀ of 1.8 μM. Additionally, the benzyl derivative 12ar had good activity and potency. The pentafluorosulfanyl group (12at and 12au), which is a proposed isostere for the trifluoromethyl group,⁸⁷ and 12av, the sulfur derivative of 12i, had low efficacy. The electron withdrawing sulfonyl moiety (12as) and a cyano group (12aw) were not tolerated.

Table 2.

Oxygen Consumption Rates of Alkoxy, Cyano, and Thioaniline Derivatives in L6 Rat Myoblast Cells^a



Entry	R	% of BAM15 OCR^b	EC₅₀, μM	Entry	R	% of BAM15 OCR^b	EC₅₀, μM

12af		41%	75.4 ± 9.3	12ao		60%	1.8 ± 0.1
12ag		55%	2.3 ± 0.2	12ap		27%	46.9 ± 4.8
12ah		56%	3.7 ± 1.4	12aq		44%	2.4 ± 0.4
12ai		50%	42.3 ± 8.3	12ar		66%	3.0 ± 0.3
12aj		57%	7.0 ± 0.7	12as		NA	--
12ak		60%	10.4 ± 1.2	12at		29%	3.7 ± 0.2
12al		43%	4.0 ± 0.3	12au		46%	3.9 ± 0.6
12am		24%	4.2 ± 0.2	12av		24%	1.4 ± 0.2
12an		46%	2.1 ± 0.5	12aw		NA	--

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Highest tested concentration is 200 μM.

Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15.

NA = no activity.

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-methylation (12ay). In stark contrast to our prior SAR studies,⁷⁸ the alkylanilines 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 stability.⁸⁹ 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) was not tolerated.

Table 3.

Oxygen Consumption Rates (OCR) of Alkyl Substituted Aniline Derivatives in L6 Rat Myoblast Cells^a



Entry	R	% of BAM15 OCR^b	EC₅₀, μM	Entry	R	% of BAM15 OCR^b	EC₅₀, μM

12ax		52%	66.7 ± 22.7	12bk		51%	2.4 ± 0.1
12ay		NA	--	12bl		33%	4.7 ± 3.0
12az		69%	4.0 ± 0.5	12bm		65%	6.7 ± 1.6
12ba		66%	20.4 ± 3.9	12bn		14%	1.1 ± 0.2
12bb		64%	51.9 ± 10.2	12bo		5%	1.5 ± 1.0
12bc		14%	>100	12bp		5%	3.3 ± 0.2
12bd		26%	3.0 ± 0.2	12bq		NA	--
12be		74%	25.2 ± 9.1	12br		NA	--
12bf		63%	10.7 ± 1.9	12bs		NA	--
12bg		73%	2.2 ± 0.3	12bt		86%	32.8 ± 8.0
12bh		81%	3.9 ± 1.0	12bu		69%	4.6 ± 0.9
12bi		18%	112.8 ± 30.4	12bv		23%	35.7 ± 5.5
12bj		76%	2.1 ± 0.3	12bw		42%	93.7 ± 16.9

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Highest tested concentration is 200 μM.

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	C_max (μM)	t_1/2 (h)

12i (SHS4121705)	80.9	5.7
12ah	3.4	0.3
12az	12.5	0.4
12bh	22.3	0.6
12bj	12	0.7
12ce	18	1.1

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Compounds were administered at 10 mg/kg body weight by oral gavage to male mice. Plasma samples were analyzed by LC–MS/MS. C_max = maximal plasma concentration. t_1/2 = half-life.

We next investigated whether alkylamines were effective as mitochondrial uncouplers (Table 4). When alkyl and cycloalkyl groups replaced the phenyl ring, greatly diminished efficacy and potency were observed with the exception of 1-adamantylamine (12ce) with 60% of BAM15 OCR and an EC₅₀ value of 7.0 μM. The analog 12ce was selected for pharmacokinetic studies as a representative of alkylamines (Table 6).

Table 4.

Oxygen Consumption Rates (OCR) of Alkylamine Derivatives in L6 Rat Myoblast Cells^a



Entry	R	% of BAM15 OCR^b	EC₅₀, μM	Entry	R	% of BAM15 OCR^b	EC₅₀, μM

12bx		NA	--	12cd		63%	38.3 ± 5.0
12by		19%	3.4 ± 0.3	12ce		60%	7.0 ± 1.0
12bz		16%	65.5 ± 10.2	12cf		42%	22.8 ± 2.5
12ca		8%	109.8 ± 45.8	12cg		23%	2.0 ± 0.3
12cb		20%	53.2 ± 11.3	12ch		NA	--
12cc		32%	13.4 ± 4.1	12ci		43%	4.9 ± 0.5

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Highest tested concentration is 200 μM.

Ratio of integrated area under OCR dose curve above baseline relative to that of BAM15.

NA = no activity.

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.

On the basis of 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 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 h after an acute dose. This is an important safety parameter as increased body temperature is a characteristic of some toxic protonophores such as DNP.⁴⁵ 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 h 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 are 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 Supporting Information 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.

Figure 3. — Acute dose studies of **SHS4121705**. To measure pharmacokinetics, (A) **SHS4121705** was administered at 10 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 h 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.

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⁻¹ d⁻¹. As a control, telmisartan was dosed at 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 (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 antifibrosis 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 ± 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 NAFLD.⁹¹ Additionally, ballooning is linked to the attraction of inflammatory cells, suggesting that longer in vivo studies, which allow for greater accumulation of inflammatory cells in the vehicle group, could show a further reduction in inflammation score.⁹²

Figure 4. — Histological results from liver tissue of STAM mice. Representative images of hematoxylin and 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.

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-point improvement in NAS score. These results suggest that mitochondrial uncouplers have potential as NASH therapeutics.

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 or whole blood glucose (G). **SHS412170**5 and telmisartan treatment decreased plasma ALT (H) but had no effect on plasma AST (I). * indicates p < 0.05. Statistical significance was assessed by one-way ANOVA for normally distributed data (D, G, I) and Kruskal–Wallis test for nonparametric 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 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 [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.

NMR spectra were recorded using an Agilent 400-MR 400 MHz, a Varian Inova 400 MHz, or Bruker Avance II 500. ¹H NMR chemical shifts are reported in ppm with the solvent resonance as an internal standard ((CD₃)₂SO, 2.50 ppm; (CD₃)₂CO, 2.05 ppm; CDCl₃, 7.26 ppm). ¹³C NMR chemical shifts are reported in ppm with the solvent resonance as the internal standard ((CD₃)₂SO, 39.52 ppm; (CD₃)₂CO, 29.84/206.26 ppm; CDCl₃, 77.16 ppm). Data are 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) were performed on a Thermo Electron TSQ triple quadrupole mass spectrometer equipped with an ESI source. HPLC condition 1: Phenomenex LUNA column (150 mm × 2.0 mm, 5 μm, C18) using a solvent system of 1% formic acid in H₂O (mobile phase A) 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 × 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, Et₃N (1.1 or 2.2 mmol when an 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 mixture was acidified with 1 M aqueous HCl and extracted with EtOAc. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated to a residue. The residue was purified by chromatography on SiO₂ using a MeOH/CH₂Cl₂ or EtOAc/hexanes solvent system to yield the 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 H₂O (8 mL) and THF (2 mL) was added, and stirring was continued for an additional 1 h. The mixture was then acidified with 1 M aqueous HCl and extracted with EtOAc. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated to a solid. The solid was purified by chromatography on SiO₂ using a MeOH/CH₂Cl₂ solvent system to yield the desired compound 12.

General Procedure C.

To anhydrous THF (25 mL) was added sodium hydride (1.5 equiv, 60% dispersion), and the mixture was 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 SiO₂ using EtOAc/hexanes to produce intermediate 15.

A vial was charged with Pd₂dba₃ (0.05 equiv), Xantphos (0.10 equiv), 2-(benzyloxy)-3-chloropyrazine (15) (1.00 equiv), and K₂CO₃ (2.00 equiv). The vial was sealed with a septum and then evacuated and backfilled with argon (3×). Deoxygenated anhydrous dioxane (4 mL) was added through the septum, followed by the requisite arylamine (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 reduced pressure, and the crude product was purified by chromatography on SiO₂ using 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 procedure A to yield 12a in 8% as a pale yellow solid: ¹H NMR ((CD₃)₂CO_, 500 MHz) δ 12.18 (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); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −129.33 to −129.40 (m, 1 F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 155.0 (d, J_CF = 245.7 Hz), 153.5, 151.4, 150.2, 145.1, 127.2 (d, J_CF = 7.7 Hz), 126.3 (d, J_CF = 10.4 Hz), 125.6 (d, J_CF = 3.9 Hz), 124.1, 116.2 (d, J_CF = 19.5 Hz); HRMS (ESI⁻) m/z calcd for C₁₀H₅FN₅O₂ (M − H)⁻ 246.0433, found 246.0438.

6-((3-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12b).

Synthesized by procedure A to yield 12b in 10% as a pale yellow solid: ¹H NMR ((CD₃)₂CO_, 500 MHz) δ 12.09 (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, J = 8.0 Hz, 1 H), 6.99 (dt, J = 8.4, 2.5 Hz, 1 H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −113.15 to −113.22 (m, 1 F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 163.6 (d, J_CF = 242.4 Hz), 153.4, 151.4, 150.2, 145.1, 140.4 (d, J_CF = 11.1 Hz), 131.3 (d, J_CF = 9.4 Hz), 117.9 (d, J_CF = 3.1 Hz), 112.3 (d, J_CF = 21.3 Hz), 109.0 (d, J_CF = 26.9 Hz); HRMS (ESI⁻) m/z calcd for C₁₀H₅FN₅O₂ (M − H)⁻ 246.0433, found 246.0440.

6-((4-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12c).

Synthesized by procedure B to yield 12c in 44% as a yellow solid (HPLC condition 2): ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.69 (s, 1H), 9.58 (s, 1H), 8.19–8.11 (m, 2H), 7.28–7.19 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −118.44 to −118.56 (m, 1F); ¹³C NMR (101 MHz, acetone-d₆) δ 160.6 (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 calcd for C₁₀H₇FN₅O₂ (M + H)⁺ 248.0578, found 248.0591.

6-((2-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12d).

Synthesized by procedure B to yield 12d in 5% as a yellow solid: ¹H 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, 1.0 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −61.19 (s), −61.21 (s); HRMS (ESI⁺) m/z calcd for C₁₁H₇F₃N₅O₂ (M + H)⁺ 298.0546, found 298.0538.

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): ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.41 (s, 1H), 9.78 (s, 1H), 8.58 (s, 1H), 8.44–8.38 (m, 1H), 7.69 (t, 8.0 Hz, 1H), 7.57–7.52 (m, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −63.23 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.6, 150.1, 145.2, 139.5, 131.4 (q, J_CF = 32.1 Hz), 130.8, 125.6, 125.1 (q, J_CF = 271.6 Hz), 122.1 (q, J_CF = 3.8 Hz), 118.6 (q, J_CF = 4.1 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₅F₃N₅O₂ (M − H)⁻ 296.0401, found 296.0425.

6-((4-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12f).

Synthesized by procedure A to yield 12f in 66% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 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); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.59 (s, 3 F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.4, 151.6, 150.2, 145.1, 142.1, 127.0 (q, J_CF = 3.8 Hz), 126.7 (q, J_CF = 32.6 Hz), 125.3 (q, J_CF = 270.8 Hz), 122.2; HRMS (ESI⁻) m/z calcd for C₁₁H₅F₃N₅O₂ (M − H)⁻ 296.0401, found 296.0419.

6-((2-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12g).

Synthesized by procedure A to yield 12g in 27% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 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 ((CD₃)₂CO, 376 MHz) δ rotamers −58.516 (s), −58.522 (s); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.7, 151.3, 150.2, 145.2, 140.5 (q, J_CF = 1.2 Hz), 130.8, 128.9, 126.8, 123.7, 121.9, 121.6 (q, J_CF = 258.0 Hz); HRMS (ES⁺) m/z calcd for C₁₁H₇F₃N₅O₃ (M + H)⁺ 314.0496, found 314.0474.

6-((3-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12h).

Synthesized by procedure A to yield 12h in 64% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 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 H), 7.18 (d, J = 8.3 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.5 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.4, 151.5, 150.2, 150.1 (q, J_CF = 3.8 Hz), 145.1, 140.3, 131.3, 121.5 (q, J_CF = 255.9 Hz), 120.7, 117.8, 114.6; HRMS (ESI⁻) m/z calcd for C₁₁H₅F₃N₅O₃ (M − H)⁻ 312.0350, found 312.0372.

6-((4-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (SHS4121705, 12i).

Synthesized by procedure A to yield SHS4121705 (12i) in 75% as a yellow solid: ¹H NMR ((CD₃)₂CO_, 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, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.79 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.3, 150.3, 146.4 (q, J_CF = 2.0 Hz), 145.1, 137.7, 123.7, 122.5, 121.5 (q, J_CF = 255.3 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₅F₃N₅O₃ (M − H)⁻ 312.0350, found 312.0369.

6-((2-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12j).

Synthesized by procedure A to yield 12j in 24% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.26 (br s, 1H), 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 (m, 1H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ *rotamers 153.7, 151.2, 150.2, 145.1, 134.8, 130.4, 129.0, 127.0, 126.9*, 125.3, 123.1, 123.0*; HRMS (ESI⁻) m/z calcd for C₁₀H₅ClN₅O₂ (M − H)⁻ 262.0137, found 262.0160.

6-((4-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12k).

Synthesized by procedure A to yield 12k in 59% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.06 (br s, 1 H), 9.61 (br s, 1H), 8.18–8.15 (m, 2H), 7.50–7.47 (m, 2H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.3, 150.3, 145.1, 137.6, 130.4, 129.7, 123.7; HRMS (ES⁺) m/z calcd for C₁₀H₇ClN₅O₂ (M + H)⁺ 264.0283, found 264.0289.

6-((4-(Difluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12l).

Synthesized by procedure A to yield 12l in 72% as a pale yellow solid: ¹H NMR ((CD₃)₂CO, 500 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, J_HF = 74.3 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −82.64 (d, J = 74.1 Hz, 2F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.2, 150.4, 149.1 (t, J_CF = 3.2 Hz), 145.1, 136.0, 123.8, 120.5, 117.5 (t, J_CF = 257.4 Hz); HRMS (ES⁺) m/z calcd for C₁₁H₈F₂N₅O₃ (M + H)⁺ 296.0590, found 296.0594.

6-((4-(1,1,2,2-Tetrafluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12m).

Synthesized by procedure A to yield 12m in 70% as a yellow solid:¹H NMR ((CD₃)₂CO, 500 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, J_HF = 52.5, 3.0 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −89.10 (td, J = 5.5, 2.7 Hz, 2F), −138.63 (dt, J = 52.4, 5.8 Hz, 2F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.3, 150.3, 146.1 (t, J_CF = 2.1 Hz), 145.1, 137.3, 123.6, 123.1, 117.6 (tt, J_CF = 270.0, 28.6 Hz), 109.1 (tt, J_CF = 249.0, 40.7 Hz); HRMS (ES⁺) m/z calcd for C₁₂H₈F₄N₅O₃ (M + H)⁺ 346.0558, found 346.0562.

6-((4-(Chlorodifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12n).

Synthesized by procedure A to yield 12n in 65% as a pale yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.08 (br s, 1H), 9.69 (s, 1H), 8.29–8.25 (m, 2H), 7.46–7.43 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −26.23 (s, 2F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.5, 151.4, 150.3, 147.5, 145.1, 137.9, 126.3 (t, J_CF = 286.8 Hz), 123.6, 122.9; HRMS (ES⁺) m/z calcd for C₁₁H₇ClF₂N₅O₃ (M + H)⁺ 330.0200, found 330.0202.

6-((2,2-Difluorobenzo[d][1,3]dioxol-5-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12o).

Synthesized by procedure A to yield 12o in 59% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 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), 7.38 (d, J = 8.8 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −51.12 (s, 2F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.5, 151.4, 150.2, 145.1, 144.2, 141.1, 135.3, 132.7 (t, J_CF = 252.9 Hz), 118.1, 110.7, 104.8; HRMS (ES⁺) m/z calcd for C₁₁H₆F₂N₅O₄ (M + H)⁺ 310.0382, found 310.0386.

6-((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 NaHCO₃ (0.100 g, 1.19 mmol) in acetone/H₂O (9:1, 4 mL) was stirred at room temperature for 18 h. The mixture 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 washed with brine, dried (Na₂SO₄), and concentrated to an orange residue. The residue was purified by chromatography on SiO₂ (gradient, 0–3% MeOH/CH₂Cl₂) to yield a sticky yellow solid (0.196 g). The solid was dissolved in a minimal amount of acetone and then precipitated by the addition of hexanes. The precipitate was filtered, rinsed with hexanes, and collected to yield 12p (0.155 g, 81%) as an off-white solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.08 (br s, 1H), 9.69 (s, 1H), 8.30–8.28 (m, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.52 (br s, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −75.64 (s, 6F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.5, 151.5, 150.3, 145.1, 140.4, 128.5, 127.7, 124.1 (q, J_CF = 288.1 Hz), 122.0, 78.1 (p, J_CF = 29.7 Hz); HRMS (ES⁺) m/z calcd for C₁₃H₈F₆N₅O₃ (M + H)⁺ 396.0526, found 396.0530.

6-((2,3-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12q).

Synthesized by procedure B to yield 12q in 29% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.09 (s, 1H), 9.35 (s, 1H), 8.24–8.17 (m, 1H), 7.38–7.30 (m, 1H), 7.29–7.20 (m, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −139.53 to −139.84 (m, 1F), −152.07 to −152.34 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.4, 151.8, 151.3 (dd, J_CF = 245.6, 11.0 Hz), 150.2, 145.3, 143.4 (dd, J_CF = 247.2, 14.6 Hz), 128.2 (dd, J_CF = 7.8, 1.9 Hz), 125.4 (dd, J_CF = 8.0, 5.0 Hz), 120.1 (d, J_CF = 3.5 Hz), 114.7 (d, J_CF = 16.9 Hz); HRMS (ESI⁺) m/z calcd for C₁₀H₆F₂N₅O₂ (M + H)⁺ 266.0484, found 266.0479.

6-((2,6-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12r).

Synthesized by procedure B to yield 12r in 23% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.67 (s, 1H), 9.41 (s, 1H), 7.54–7.45 (m, 1H), 7.22–7.15 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −117.87 to −117.95 (m, 2F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 159.4 (dd, J_CF = 250.9, 4.7 Hz), 153.4, 153.0, 150.3, 145.4, 130.2 (t, J_CF = 9.9 Hz), 114.7 (t, J_CF = 16.6 Hz), 112.9–112.5 (m); HRMS (ESI⁺) m/z calcd for C₁₀H₆F₂N₅O₂ (M + H)⁺ 266.0484, found 266.0480.

6-((3,5-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12s).

Synthesized by procedure B to yield 12s in 19% as a yellow solid. ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.11 (s, 1H), 9.76 (s, 1H), 7.97–7.88 (m, 2H), 6.87 (tt, J = 9.1, 2.3 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −110.23 to −110.33 (m, 2F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 163.84 (dd, J_CF = 244.1, 14.8 Hz), 153.2, 151.5, 149.9, 145.0, 141.1 (t, J_CF = 13.9 Hz), 105.0 (dd, J_CF = 21.1, 9.1 Hz), 100.6 (t, J_CF = 26.2 Hz); HRMS (ESI⁺) m/z calcd for C₁₀H₆F₂N₅O₂ (M + H)⁺ 266.0484, found 266.0488.

6-((2-Fluoro-5-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12t).

Synthesized by procedure B to yield 12t in 26% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.99 (s, 1H), 9.40 (s, 1H), 8.90 (dd, J = 7.4, 1.9 Hz, 1H), 7.72–7.66 (m, 1H), 7.63–7.56 (m, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.66 (s, 3F), −122.44 to −122.53 (m, 1F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 157.0 (d, J_CF = 252.7 Hz), 153.5, 151.9, 150.1, 145.4, 127.6 (d, J_CF = 4.0 Hz), 127.4 (d, J_CF = 11.6 Hz), 124.9 (q, J_CF = 271.4 Hz), 124.5 (dd, J_CF = 9.5, 4.3 Hz), 121.5–121.4, (m), 117.5 (d, J_CF = 21.2 Hz); HRMS (ESI⁺) m/z calcd for C₁₁H₆F₄N₅O₂ (M + H)⁺ 316.0452, found 316.0476.

6-((2-Fluoro-4-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12u).

Synthesized by procedure B to yield 12u in 14% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.92 (s, 1H), 9.37 (s, 1H), 8.81 (t, J = 8.3 Hz, 1H), 7.79–7.67 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.82 (s, 3F), −127.05 (t, J = 10.5 Hz, 1F); ¹³C NMR (126 MHz, acetone-d₆) δ 154.2 (d, J = 248.5 Hz), 153.4, 151.6, 150.0, 145.2, 130.1 (d, J = 11.3 Hz), 127.7 (dd, J = 35.9, 7.6 Hz), 124.4 (dq, J = 271.1, 3.1 Hz), 124.1, 123.0 (p, J = 4.3 Hz), 113.7 (dq, J = 23.1, 4.2 Hz); HRMS (ESI⁺) m/z calcd for C₁₁H₆F₄N₅O₂ (M + H)⁺ 316.0452, found 316.0453.

6-((3-Fluoro-4-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12v).

Synthesized by procedure B to yield 12v in 11% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.65 (s, 1H), 9.92 (s, 1H), 8.37 (ddd, J = 13.4, 2.0, 0.9 Hz, 1H), 8.18–8.12 (m, 1H), 7.82 (d, J = 8.5 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −61.27 (d, J = 12.3 Hz, 3F), −114.54 to −114.73 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ *rotamers 160.6 (dq, J_CF = 251.6, 2.5 Hz), 153.3*, 153.2, 151.8*, 151.7, 149.9, 145.2, 144.4–144.0 (m), 128.8–128.5 (m), 123.8 (dd, J_CF = 270.0, 2.3 Hz), 117.6 (d, J_CF = 3.5 Hz)*, 117.5 (d, J_CF = 3.6 Hz), 113.9 (dd, J_CF = 33.0, 12.8 Hz), 109.8 (d, J_CF = 26.3 Hz)*, 109.7 (d, J_CF = 26.4 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₄F₄N₅O₂ (M − H)⁻ 314.0306, found 314.0352.

6-((2-Fluoro-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12w).

Synthesized by procedure B to yield 12w in 21% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.21 (s, 1H), 9.41 (s, 1H), 8.75–8.65 (m, 1H), 7.69–7.51 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −61.75 (d, J = 13.0 Hz, 3F), −129.74 to −129.93 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ *rotamers 152.4, 152.3*, 151.5 (dd, J_CF = 256.3, 2.4 Hz), 151.0, 150.9*, 149.1, 149.2*, 144.3, 128.2 (d, J_CF = 18.9 Hz), 126.8, 125.4 (q, J_CF = 271.6 Hz), 124.9 (dd, J_CF = 5.1, 2.1 Hz), 123.0 (dd, J_CF = 4.7, 1.2 Hz), 117.9 (dd, J_CF = 33.1, 10.8 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₄F₄N₅O₂ (M − H)⁻ 314.0306, found 314.0306.

6-((3,5-Bis(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12x).

Synthesized by procedure B to yield 12x in 6% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.20 (s, 1H), 10.07 (s, 1H), 8.89 (d, J = 1.8 Hz, 2H), 7.87 (s, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −63.57 (s, 6F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.2, 151.9, 149.9, 145.2, 140.6, 132.7 (q, J_CF = 33.3 Hz), 124.4 (q, J_CF = 272.0 Hz), 122.1 (q, J_CF = 4.1 Hz), 118.6–118.5 (m); HRMS (ESI−) m/z calcd for C₁₀H₄BrFN₅O₂ (M − H)⁻ 364.0274, found 364.0261.

6-((2-Fluoro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12y).

Synthesized by procedure B to yield 12y in 23% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.53 (s, 1H), 9.30 (s, 1H), 8.56 (t, J = 8.9 Hz, 1H), 7.61–7.28 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −59.02 (s, 3F), −123.51 to −123.59 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 155.1 (d, J_CF = 249.9 Hz), 153.4, 151.7, 150.1, 146.6 (dd, J_CF = 10.7, 2.3 Hz), 145.3, 125.7 (d, J_CF = 10.7 Hz), 125.5 (d, J_CF = 2.3 Hz), 121.3 (q, J_CF = 256.6 Hz), 118.3 (dd, J_CF = 4.2, 1.2 Hz), 110.4 (dd, J_CF = 23.6, 1.3 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₄F₄N₅O₃ (M − H)⁻ 330.0255, found 330.0257.

6-((3-Fluoro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12z).

Synthesized by procedure A to yield 12z in 51% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.10 (br s, 1H), 9.82 (br s, 1H), 8.40–8.36 (m, 1H), 8.08–8.04 (m, 1H), 7.62–7.58 (m, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −59.87 to −59.88 (m, 3F), −128.86 to −128.96 (m, 1F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 154.9 (d, J_CF = 248.7 Hz), 153.3, 151.5, 150.1, 145.1, 139.2 (d, J_CF = 10.0 Hz), 133.1 (d, J_CF = 12.5 Hz), 125.2, 121.5 (q, J_CF = 257.2 Hz), 118.6 (d, J_CF = 3.7 Hz), 110.9 (d, J_CF = 24.4 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₄F₄N₅O₃ (M − H)⁻ 330.0256, found 330.0289.

6-((3-Chloro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aa).

Synthesized by procedure A to yield 12aa in 51% as a yellow-orange solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.05 (br s, 1H), 9.78 (s, 1H), 8.53 (d, J = 2.7 Hz, 1H), 8.21 (dd, J = 9.1, 2.6 Hz, 1H), 7.60 (dq, J = 9.0, 1.4 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.87 to −58.88 (m, 3 F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.3, 151.5, 150.1, 145.2, 141.9 (q, J_CF = 2.0 Hz), 138.7, 127.8, 124.3, 123.8, 122.1, 121.5 (q, J_CF = 257.3 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₄ClF₃N₅O₃ (M − H)⁻ 345.9960, found 345.9949.

6-((2-Chloro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ab).

Synthesized by procedure B to yield 12ab in 9% as a yellow solid (HPLC condition 2): ¹H NMR ((CD₃)₂CO, 400 MHz) δ 13.73 (s, 1H), 9.51 (s, 1H), 8.84 (d, J = 9.3 Hz, 1H), 7.66 (d, J = 3.5 Hz, 1H), 7.58–7.50 (m, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.99 (s, 3F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.7, 151.4, 150.1, 146.2 (q, J_CF = 2.4 Hz), 145.3, 134.3, 126.4, 124.4, 123.6 (q, J_CF = 1.3 Hz), 121.9 (q, J_CF = 1.1 Hz), 121.4 (q, J_CF = 256.4 Hz); HRMS (ESI⁺) m/z calcd for C₁₁H₆ClF₃N₅O₃ (M + H)⁺ 348.0105, found 348.0089.

6-((2-Methoxy-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ac).

Synthesized by procedure A to yield 12ac in 68% as an orange solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.20 (br s, 1H), 9.46 (s, 1H), 8.86 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 2.6 Hz, 1H), 7.09 (dt, J = 9.0, 2.6 Hz, 1H), 4.10 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.70 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.8, 151.1, 150.6, 150.3, 146.5 (q, J_CF = 2.1 Hz), 145.1, 126.7, 121.5 (q, J_CF = 255.6 Hz), 121.3, 113.7, 105.7, 57.3; HRMS (ESI⁻) m/z calcd for C₁₂H₇F₃N₅O₄ (M − H)⁻ 342.0456, found 342.0448.

6-((3-Nitro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ad).

Synthesized by procedure B to yield 12ad in 2% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.06 (s, 1H), 9.05 (d, J = 2.7 Hz, 1H), 8.61 (dd, J = 9.1, 2.7 Hz, 1H), 7.80 (dq, J = 9.1, 1.3 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.76 9 (s, 3F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.4, 151.8, 149.9, 145.4, 143.7, 138.7, 137.3, 127.7, 125.3 (q, J_CF = 1.2 Hz), 121.3 (q, J_CF = 258.5 Hz), 118.9; HRMS (ESI⁻) m/z calcd for C₁₁H₄F₃N₆O₅ (M − H)⁻ 357.0200, found 357.0198.

6-((2-Fluorophenyl)(methyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ae).

Synthesized by procedure A to yield 12ae in 42% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.63 (br s, 1H), 7.47–7.42 (m, 1H), 7.40–7.34 (m, 1H), 7.26–7.20 (m, 2H), 3.54 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −125.48 to −125.68 (m, 1 F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 158.0 (d, J_CF = 245.9 Hz), 154.6, 152.3, 150.4, 145.7, 134.6 (d, J_CF = 12.8 Hz), 129.7 (d, J_CF = 7.8 Hz), 128.7, 125.6 (d, J_CF = 3.9 Hz), 116.7 (d, J_CF = 20.2 Hz), 42.6; HRMS (ESI⁻) m/z calcd for C₁₁H₇FN₅O₂ (M − H)⁻ 260.0589, found 260.0593.

6-((3-Methoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12af).

Synthesized by procedure B to yield 12af in 36% as a yellow solid: ¹H NMR ((CD₃)₂SO, 400 MHz) δ 13.27 (s, 1H), 10.16 (s, 1H), 7.74–7.66 (m, 2H), 7.32 (t, J = 8.2 Hz, 1H), 6.77 (dd, J = 8.0, 2.0 Hz, 1H), 3.77 (s, 3H); ¹³C NMR ((CD₃)₂SO, 126 MHz) δ 159.34, 153.1, 150.7, 149.6, 144.5, 138.9, 129.5, 113.9, 110.0, 107.7, 55.1; HRMS (ESI⁺) m/z calcd for C₁₁H₁₀N₅O₃ (M + H)⁺ 260.0778, found 260.0793.

6-((3-Butoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ag).

In a vial, 1-iodobutane (0.20 mL, 1.8 mmol) was added to a stirring mixture of N-(3-hydroxyphenyl)acetamide (0.150 g, 0.992 mmol) and K₂CO₃ (0.200 g, 1.45 mmol) in acetone (1.5 mL). The resulting mixture was stirred for 2 d. Additional 1-iodobutane (0.20 mL, 1.8 mmol) was added, and the mixture was heated to 40 °C for 9 h. The mixture was diluted with EtOAc, washed with brine, dried (Na₂SO₄), and concentrated to a clear oil (0.177 g). The oil was heated in a mixture of 6 M aq HCl (5 mL) and dioxane (3 mL) at 90 °C for 6 h. The mixture was allowed to cool to rt, basified with 1 M NaOH, and extracted with EtOAc (2×). The combined organic layers were washed with brine, dried (Na₂SO₄), and concentrated to yield 3-butoxyaniline as a crude orange oil (0.123 g).

Product 12ag was synthesized using the crude aniline by procedure A in 37% as an off-white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.04 (br s, 1H), 9.42 (s, 1H), 7.83 (t, J = 2.2 Hz, 1H), 7.68 (ddd, J =8.2, 2.1, 0.9 Hz, 1H), 7.33 (t, J = 8.2 Hz, 1 H), 6.79 (ddd, J = 8.2, 2.5, 0.9 Hz, 1H), 4.04 (t, J = 6.5 Hz, 2H), 1.82–1.75 (m, 2H), 1.56–1.47 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 160.5, 153.6, 151.0, 150.4, 145.0, 139.7, 130.5, 114.1, 111.9, 108.4, 68.4, 32.0, 19.9, 14.1; HRMS (ES⁺) m/z calcd for C₁₄H₁₆N₅O₃ (M + H)⁺ 302.1248, found 302.1251.

6-((4-Butoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ah).

Synthesized by procedure B to yield 12ah in 75% as tan solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.97 (s, 1H), 9.40 (s, 1H), 8.16–7.87 (m, 2H), 7.08–6.89 (m, 2H), 4.02 (t, J = 6.5 Hz, 2H), 1.87–1.69 (m, 2H), 1.63–1.42 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 156.6, 152.8, 149.7, 149.6, 144.1, 130.6, 122.7, 114.4, 67.6, 31.2, 18.9, 13.2; HRMS (ESI⁺) m/z calcd for C₁₄H₁₆N₅O₃ (M + H)⁺ 302.1247, found 302.1253.

6-((4-(2,2,2-Trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ai).

In a round-bottom flask, a mixture of 12ai-int (see Supporting Information) and iron (325 mesh, 0.200 g, 3.58 mmol) in AcOH/MeOH (1:1, 4 mL) was stirred at 50 °C for 2 h. The mixture was filtered through Celite, rinsing with EtOAc, and concentrated to a dark oil. The oil was quenched with sat. aq NaHCO₃, diluted with brine, and extracted with EtOAc. The organic layer was dried (Na₂SO₄) and concentrated to yield 4-(2,2,2-trifluoroethoxy)aniline as a crude dark oil (0.539 g).

Compound 12ai was synthesized using the crude aniline by procedure A in 60% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.00 (br s, 1H), 9.50 (s, 1H), 8.11–8.08 (m, 2H), 7.17–7.15 (m, 2H), 4.71 (q, J_HF = 8.6 Hz, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −74.73 (t, J = 8.6 Hz, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 155.6, 153.7, 151.0, 150.5, 145.1, 133.3, 124.9 (q, J_CF = 289.2 Hz), 123.8, 116.1, 66.4 (q, J_CF = 35.1 Hz); HRMS (ESI⁻) m/z calcd for C₁₂H₇F₃N₅O₃ (M − H)⁻ 326.0506, found 326.0508.

6-((3-Methyl-4-(2,2,2-trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aj).

In a round-bottom flask, a mixture of 12aj-int (see Supporting Information) and iron (325 mesh, 0.200 g, 3.58 mmol) in AcOH/MeOH (1:1, 4 mL) was stirred at 50 °C for 2 h. The mixture was filtered through Celite, rinsing with EtOAc, and concentrated to a dark oil. The oil was quenched with sat. aq NaHCO₃, diluted with brine, and extracted with EtOAc. The organic layer was dried (Na₂SO₄) and concentrated to yield 3-methyl-4-(2,2,2-trifluoroethoxy)aniline as a crude dark oil (0.143 g).

Product 12aj was synthesized using the crude aniline by procedure A in 66% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.98 (s, 1H), 9.40 (s, 1H), 8.00 (dd, J = 8.9, 2.7 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 4.70 (q, J_HF = 8.5 Hz, 2H), 2.28 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −74.92 (t, J = 8.5 Hz, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.8, 153.7, 150.9, 150.5, 145.1, 133.0, 128.3, 124.99 (q, J_CF = 277.3 Hz), 124.98, 121.0, 113.4, 66.7 (q, J_CF = 34.8 Hz), 16.2; HRMS (ESI⁻) m/z calcd for C₁₃H₉F₃N₅O₃ (M − H)⁻ 340.0663, found 340.0667.

6-((3-Fluoro-4-(2,2,2-trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ak).

Synthesized by procedure B using 12ak-int (see Supporting Information) to yield 12ak in 53% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 9.62 (s, 1H), 8.18 (dd, J = 13.5, 3.0 Hz, 1H), 7.94–7.86 (m, 1H), 7.37 (t, J_HF = 9.6 Hz, 1H), 4.79 (q, J_HF = 8.9 Hz, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −74.94 (t, J = 8.9 Hz, 3F), −132.89 to −133.04 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ *rotamers 153.5, 153.4*, 152.9 (d, J_CF = 244.5 Hz), 151.2, 151.1*, 150.3, 145.1, 143.2 (dd, J_CF = 11.1, 1.3 Hz), 134.2 (d, J_CF = 9.6 Hz), 124.7 (q, J_CF = 276.7 Hz), 118.4 (d, J_CF = 3.8 Hz), 118.3 (d, J_CF = 3.8 Hz)*, 117.7 (d, J_CF = 2.7 Hz), 110.9 (d, J_CF = 23.7 Hz), 110.8 (d, J_CF = 23.8 Hz)*, 67.7 (q, J_CF = 35.6 Hz). HRMS (ESI⁺) m/z calcd for C₁₂H₈F₄N₅O₃ (M + H)⁺ 346.0557, found 346.0574.

6-((4-(2,2,2-Trifluoroethoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12al).

In a round-bottom flask, a mixture of 12al-int (0.200 g) (see Supporting Information) and iron (325 mesh, 0.200 g, 3.58 mmol) in AcOH/MeOH (1:1, 4 mL) was stirred at 50 °C for 2 h. The mixture was filtered through Celite, rinsing with EtOAc, and concentrated to a dark oil. The oil was quenched with sat. aq NaHCO₃, diluted with brine, and extracted with EtOAc. The organic layer was dried (Na₂SO₄) and concentrated to yield 4-(2,2,2-trifluoroethoxy)-3-(trifluoromethyl)aniline as a clear oil (0.152 g): ¹H NMR ((CD₃)₂CO, 400 MHz) δ 7.09 (d, J = 8.8 Hz, 1H), 6.97–6.96 (m, 1H), 6.92–6.89 (m, 1H), 4.83 (br s, 2H), 4.60 (q, J_HF = 8.6 Hz, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.38 (s, 3F), −75.03 (t, J = 8.6 Hz, 3F).

Product 12al was synthesized using the crude aniline by procedure A in 46% as an orange solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.03 (br s, 1H), 9.73 (s, 1H), 8.48 (d, J = 2.8 Hz, 1H), 8.44 (dd, J = 9.0, 2.8 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 4.88 (q, J_HF = 8.4 Hz, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.78 (m, 3F), −74.84 (t, J = 8.5 Hz, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.5, 152.8, 151.4, 150.3, 145.2, 133.2, 127.6, 124.6 (q, J_CF = 276.8 Hz), 124.2 (q, J_CF = 271.5 Hz), 121.4 (q, J_CF = 5.6 Hz), 119.8 (q, J_CF = 31.4 Hz), 115.7, 67.0 (q, J_CF = 35.7 Hz); HRMS (ESI⁻) m/z calcd for C₁₃H₆F₆N₅O₃ (M − H)⁻ 394.0380, found 394.0384.

6-((3-Fluoro-4-(4,4,4-trifluorobutoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12am).

In a sealed microwave vial, a mixture of 1,2-difluoro-4-nitrobenzene (0.350 g, 2.20 mmol), 4,4,4-trifluorobutan-1-ol (0.35 mL, 3.3 mmol), and K₂CO₃ (0.700 g, 5.07 mmol) in DMF (2 mL) was stirred at 80 °C for 22 h. The mixture was allowed to cool to rt, diluted with water and brine, and extracted with EtOAc (2×). The combined organic layers were dried (Na₂SO₄) and concentrated to a crude yellow liquid (0.657 g). In a 6 dram vial, a mixture of the liquid (0.657 g) and iron (325 mesh, 0.650 g, 11.6 mmol) in AcOH/MeOH (1:1, 5 mL) was stirred at 50 °C for 2 h under an atmosphere of N₂. The mixture was filtered through Celite (EtOAc), quenched with sat. aq NaHCO₃, and extracted with EtOAc (2×). The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by chromatography on SiO₂ (gradient 20–30% EtOAc/hexanes) to yield 3-fluoro-4-(4,4,4-trifluorobutoxy)aniline (0.421 g) as a crude orange liquid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 6.86 (dd, J = 9.5, 8.7 Hz, 1H), 6.48 (dd, J = 13.4, 2.6 Hz, 1H), 6.39 (ddd, J = 8.7, 2.7, 1.3 Hz, 1H), 4.57 (s, 2H), 4.01 (t, J = 6.1 Hz, 2H), 2.49–2.36 (m, 2H), 2.00–1.93 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −67.05 (t, J = 11.2 Hz, 3F), −134.85 to −134.91 (m, 1F).

Product 12am was synthesized using the crude aniline by procedure A in 60% as a yellow solid: ¹H NMR ((CD₃)₂SO, 500 MHz) δ 13.28 (br s, 1H), 10.34 (s, 1H), 8.01 (dd, J = 13.7, 2.6 Hz, 1H), 7.85 (ddd, J = 9.0, 2.5, 1.3 Hz, 1H), 7.23 (t, J = 9.3 Hz, 1H), 4.13 (t, J = 6.2 Hz, 2H), 2.51–2.38 (m, 2H), 1.99–1.93 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −67.01 (t, J = 11.3 Hz, 3F), −133.97 to −134.035 (m, 1F); ¹³C NMR ((CD₃)₂SO, 125 MHz) δ 152.9, 150.9 (d, J_CF = 242.1 Hz), 150.5, 149.6, 144.5, 142.9 (d, J_CF = 10.8 Hz), 131.5 (d, J_CF = 9.6 Hz), 127.6 (q, J_CF = 276.1 Hz), 117.9 (d, J_CF = 3.4 Hz), 115.2 (d, J_CF = 2.5 Hz), 109.9 (d, J_CF = 23.1 Hz), 67.4, 29.4 (q, J_CF = 28.0 Hz), 21.6 (q, J_CF = 3.1 Hz); HRMS (ES⁻) m/z calcd for C₁₄H₁₀F₄N₅O₃ (M − H)⁻ 372.0725, found 372.0726.

6-((4-(4,4,4-Trifluorobutoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12an).

In a sealed microwave vial, a mixture of 1-fluoro-4-nitro-2-(trifluoromethyl)-benzene (0.400 g, 1.91 mmol), 4,4,4-trifluorobutan-1-ol (0.35 mL, 3.3 mmol), and K₂CO₃ (0.700 g, 5.07 mmol) in DMF (2 mL) was stirred at 80 °C for 22 h. The mixture was allowed to cool to rt, diluted with water and brine, and extracted with EtOAc (2×). The combined organic layers were dried (Na₂SO₄) and concentrated to a crude orange liquid (0.913 g). In a 6 dram vial, a mixture of the liquid (0.913 g) and iron (325 mesh, 0.650 g, 11.6 mmol) in AcOH/MeOH (1:1, 5 mL) was stirred at 50 °C for 2 h under an atmosphere of N₂. The mixture was filtered through Celite (EtOAc), quenched with sat. aq NaHCO₃, and extracted with EtOAc (2×). The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by chromatography on SiO₂ (gradient, 20–30% EtOAc/hexanes) to yield 4-(4,4,4-trifluorobutoxy)-3-(trifluoromethyl)aniline (0.452 g) as a crude orange liquid that solidified to an off-white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 6.99–6.96 (m, 1H), 6.95–6.94 (m, 1H), 6.90–6.86 (m, 1H), 4.65 (br s, 2H), 4.10–4.07 (m, 2H), 2.49–2.36 (m, 2H), 2.04–1.98 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.81 (d, J = 4.0 Hz, 3F), −67.13 (dt, J = 11.0, 3.5 Hz, 3F).

Product 12an was synthesized using the crude aniline by procedure A in 53% as a yellow solid: ¹H NMR ((CD₃)₂SO, 500 MHz) δ 13.28 (br s, 1H), 10.45 (s, 1H), 8.37 (d, J = 2.6 Hz, 1H), 8.28 (dd, J = 9.1, 2.7 Hz, 1H), 7.34 (d, J = 9.1 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 2.47–2.37 (m, 2H), 2.00–1.94 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.81 (s, 3F), −67.09 (t, J_HF = 11.5 Hz, 3F); ¹³C NMR ((CD₃)₂SO, 125 MHz) δ 153.0, 152.8, 150.8, 149.6, 144.6, 130.8, 127.6 (q, J_CF = 276.0 Hz), 127.4, 123.5 (q, J_CF = 272.1 Hz), 120.5 (q, J_CF = 5.9 Hz), 116.8 (q, J_CF = 30.4 Hz), 114.0, 66.8, 29.3 (q, J_CF = 28.3 Hz), 21.6 (q, J_CF = 3.2 Hz); HRMS (ES⁻) m/z calcd for C₁₅H₁₀F₆N₅O₃ (M − H)⁻ 422.0693, found 422.0696.

6-((4-(Cyclopropylmethoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol) (12ao).

In a sealed vial, a mixture of 1-fluoro-4-nitro-2-(trifluoromethyl)benzene (0.250 g, 1.20 mmol), potassium carbonate (0.400 g, 2.89 mmol), and cyclopropylmethanol (0.25 mL, 3.1 mmol) in DMF (2 mL) was stirred at 80 °C for 22 h. The mixture was allowed to cool to rt, diluted with water and brine, and extracted with EtOAc (2×). The combined organic layers were washed with brine (2×), dried (Na₂SO₄), and concentrated to a crude oil. A mixture of the oil and iron (325 mesh, 0.300 g, 5.37 mmol) in AcOH/MeOH (1:1, 3 mL) was stirred at 50 °C for 2 h under an atmosphere of N₂. The mixture was quenched with sat. aq NaHCO₃ and extracted with EtOAc (2×). The combined organic layers were washed with sat. aq NaHCO₃ (2×) and then brine, dried (Na₂SO₄), and concentrated to yield 4-(cyclopropylmethoxy)-3-(trifluoromethyl)aniline as a yellow oil (0.240 g). The oil was used crude in the next reaction. ¹H NMR ((CD₃)₂CO, 400 MHz) δ 6.95–6.92 (m, 2H), 6.87–6.83 (m, 1H), 4.59 (br s, 2H), 3.84 (d, J = 6.5 Hz, 2H), 1.25–1.15 (m, 1H), 0.57–0.52 (m, 2H), 0.36–0.31 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.20 (s, 3F).

Product 12ao was synthesized using the crude aniline by procedure A in 40% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.02 (br s, 1H), 9.65 (s, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.34 (dd, J = 9.0, 2.8 Hz, 1H), 7.31 (d, J = 9.0 Hz, 1H), 4.07 (d, J = 6.6 Hz, 2H), 1.34–1.28 (m, 1H), 0.63–0.60 (m, 2H), 0.43–0.40 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.71 (s, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 155.0 (q, J_CF = 1.9 Hz), 153.5, 151.3, 150.4, 145.1, 131.4, 127.6, 124.6 (q, J_CF = 271.9 Hz), 121.3 (q, J_CF = 5.5 Hz), 119.2 (q, J_CF = 30.7 Hz), 115.1, 74.1, 10.7, 3.3; HRMS (ES⁺) m/z calcd for C₁₅H₁₃F₃N₅O₃ (M + H)⁺ 368.0965, found 368.0966.

6-(Benzo[d][1,3]dioxol-5-ylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ap).

Synthesized by procedure A to yield 12ap in 60% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.00 (br s, 1H), 9.44 (s, 1H), 7.77 (d, J = 2.3 Hz, 1H), 7.56 (dd, J = 8.5, 2.2 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.05 (s, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 150.8, 150.5, 148.7, 145.8, 145.0, 132.9, 115.6, 108.8, 103.9, 102.5; HRMS (ESI⁻) m/z calcd for C₁₁H₆N₅O₄ (M − H)⁻ 272.0425, found 272.0430.

6-((4-(Benzyloxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aq).

Synthesized by procedure A at 75 °C to yield 12aq in 30% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.99 (br s, 1H), 9.45 (s, 1H), 8.05–8.02 (m, 2H), 7.52–7.50 (m, 2H), 7.42–7.39 (m, 2H), 7.35–7.32 (m, 1H), 7.13–7.09 (m, 2H), 5.17 (s, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 157.1, 153.7, 150.8, 150.6, 145.1, 138.3, 131.9, 129.3, 128.7, 128.5, 123.7, 115.8, 70.7; HRMS (ESI⁺) m/z calcd for C₁₇H₁₄N₅O₃ (M + H)⁺ 336.1091, found 336.1088.

6-((3-((4-(Trifluoromethyl)benzyl)oxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ar).

Synthesized by procedure A using 12ar-int (see Supporting Information) to yield 12ar in 60% as an off-white solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.06 (br s, 1H), 9.45 (s, 1H), 7.98 (t, J = 2.3 Hz, 1H), 7.76 (app s, 4H), 7.70 (dd, J = 8.2, 2.0 Hz, 1H), 7.36 (t, J = 8.2 Hz, 1H), 6.90 (dd, J = 8.3, 2.4 Hz, 1H), 5.29 (s, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.9 (s, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 159.8, 153.5, 151.1, 150.3, 145.0, 142.9, 139.8, 130.6, 130.2 (q, J_CF = 32.1 Hz), 128.9, 126.2 (q, J_CF = 3.9 Hz), 125.3 (q, J_CF = 271.3 Hz), 114.7, 112.3, 108.7, 69.7; HRMS (ES⁺) m/z calcd for C₁₈H₁₃F₃N₅O₃ (M + H)⁺ 404.0965, found 404.0961.

6-((4-(Methylsulfonyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12as).

Synthesized by procedure A to yield 12as (94% purity using HPLC condition 1) in 40% as a light yellow solid: ¹H NMR ((CD₃)₂SO, 500 MHz) δ 13.37 (br s, 1H), 10.60 (s, 1H), 8.31 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 3.21 (s, 3H); ¹³C NMR ((CD₃)₂SO, 125 MHz) δ 152.9, 151.3, 149.5, 144.7, 142.3, 136.0, 127.9, 121.7, 43.7; HRMS (ESI⁻) m/z calcd for C₁₁H₈N₅O₄S (M − H)⁻ 306.0302, found 306.0307.

6-((3-(Pentafluoro-l6-sulfaneyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12at).

Synthesized by procedure A to yield 12at in 37% as a colorless solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.05 (br s, 1H), 9.88 (s, 1H), 8.77–8.76 (m, 1H), 8.46–8.42 (m, 1H), 7.74–7.69 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −120.78 to −121.70 (m, 5F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 154.6 (p, J_CF = 17.1 Hz), 153.4, 151.7, 150.1, 145.2, 139.4, 130.5, 125.6, 122.9 (p, J_CF = 4.3 Hz), 119.7 (p, J_CF = 4.9 Hz); HRMS (ESI⁺) m/z calcd for C₁₀H₇F₅N₅O₂S (M + H)⁺ 356.0235, found 356.0232.

6-((4-(Pentafluoro-l6-sulfaneyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12au).

Synthesized by procedure A to yield 12au in 38% as a colorless cloth-like solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.10 (br s, 1H), 9.83 (s, 1H), 8.38 (d, J = 8.9 Hz, 2H), 7.99–7.96 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −120.74 to −121.67 (m, 5F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.4, 151.6, 150.1 (p, J_CF = 17.5 Hz), 145.2, 141.8, 127.8 (p, J_CF = 4.8 Hz), 121.9; HRMS (ESI⁻) m/z calcd for C₁₀H₅F₅N₅O₂S (M − H)⁻ 354.0090, found 354.0080.

6-((4-((Trifluoromethyl)thio)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12av).

Synthesized by procedure A to yield 12av in 23% as a pale yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.11 (br s, 1H), 9.74 (s, 1H), 8.33–8.31 (m, 2H), 7.82–7.80 (m, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −44.23 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.4, 151.5, 150.2, 145.1, 141.6, 138.1, 130.8 (q, J_CF = 307.1 Hz), 123.0, 119.7 (q, J_CF = 2.2 Hz); HRMS (ESI⁻) m/z calcd for C₁₁H₅F₃N₅O₂S (M − H)⁻ 328.0122, found 328.0113.

3-((6-Hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-yl)amino)-benzonitrile (12aw).

Synthesized by procedure B to yield 12aw in 17% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.09 (s, 1H), 9.78 (s, 1H), 8.63–8.59 (m, 1H), 8.42 (dd, J = 8.3, 1.1 Hz, 1H), 7.69 (dd, J = 7.9, 0.5 Hz, 1H), 7.62 (dd, J = 7.9, 1.3 Hz, 1H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.4, 151.7, 150.1, 145.2, 139.7, 131.1, 129.1, 126.6, 125.0, 119.1, 113.6; HRMS (ESI⁺) m/z calcd for C₁₁H₇N₆O₂ (M + H)⁺ 255.0625, found 255.0639.

6-(Phenylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ax).

Synthesized by procedure B to yield 12ax in 39% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.04 (s, 1H), 9.50 (s, 1H), 8.12 (dd, J = 9.0, 1.2 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.23 (t, J = 7.7 Hz, 1H); ¹³C NMR ((CD₃)₂SO, 126 MHz) δ 153.0, 150.8, 149.7, 144.5, 137.8, 128.6, 124.8, 121.8; HRMS (ESI⁺) m/z calcd for C₁₀H₈N₅O₂ (M + H)⁺ 230.0672, found 230.0671.

6-(Methyl(phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ay).

Synthesized by procedure A to yield 12ay in 93% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.50 (br s, 1H), 7.42–7.37 (m, 2H), 7.34–7.29 (m, 3H), 3.54 (s, 3H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 154.6, 152.3, 150.6, 147.4, 145.8, 129.8, 127.7, 126.5, 43.5; HRMS (ESI⁻) m/z calcd for C₁₁H₈N₅O₂ (M − H)⁻ 242.0683, found 242.0694.

6-(Naphthalen-2-ylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12az).

Synthesized by procedure A to yield 12az in 17% as an orange solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.00 (br s, 1H), 9.66 (s, 1H), 8.86 (d, J = 2.3 Hz, 1H), 8.07 (dd, J = 8.9, 2.3 Hz, 1H), 8.09–7.90 (m, 3H), 7.54 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.7, 151.3, 150.5, 145.2, 136.2, 134.7, 132.0, 129.5, 128.8, 128.5, 127.6, 126.4, 122.0, 119.1; HRMS (ESI⁺) m/z calcd for C₁₄H₁₀N₅O₂ (M + H)⁺ 280.0829, found 280.0835.

6-(Naphthalen-1-ylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ba).

Synthesized by procedure A to yield 12ba in 46% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.10 (br s, 1H), 9.77 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 8.09–8.07 (m, 1 H), 8.02–8.00 (m, 1H), 7.90 (d, J = 8.2, 1H), 7.64–7.57 (m, 3H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.9, 152.9, 150.7, 145.3, 135.2, 133.5, 129.5, 129.0, 127.7, 127.4, 127.2, 126.5, 123.0, 122.7; HRMS (ESI⁻) m/z calcd for C₁₄H₈N₅O₂ (M − H)⁻ 278.0683, found 278.0689.

6-(p-Tolylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bb).

Synthesized by procedure B to yield 12bb in 39% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.96 (s, 1H), 9.42 (s, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 2.34 (s, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.7, 150.9, 150.5, 145.0, 136.1, 135.5, 130.2, 122.0, 20.9; HRMS (ESI⁺) m/z calcd for C₁₁H₁₀N₅O₂ (M + H)⁺ 244.0829, found 244.0812.

6-((2,6-Dimethylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bc).

Synthesized by procedure B to yield 12bc in 39% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.97 (s, 1H), 9.25 (s, 1H), 7.21–7.13 (m, 3H), 2.26 (s, 6H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.5, 152.9, 150.9, 145.4, 136.7, 135.2, 128.9, 128.5, 18.4; HRMS (ESI⁺) m/z calcd for C₁₂H₁₂N₅O₂ (M + H)⁺ 258.0985, found 258.0993.

6-((3,4-Dimethylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bd).

Synthesized by procedure B to yield 12bd in 48% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.93 (s, 1H), 9.32 (s, 1H), 7.86 (dd, J = 8.2, 2.4 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 2.28 (s, 3H), 2.25 (s, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.7, 150.9, 150.6, 145.1, 137.9, 136.4, 134.4, 130.7, 123.2, 119.6, 20.1, 19.3. HRMS (ESI⁻) m/z calcd for C₁₂H₁₀N₅O₂ (M − H)⁻ 256.0840, found 256.0847.

6-((3-Ethylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12be).

Synthesized by procedure A to yield 12be in 86% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.04 (br s, 1H), 9.42 (br s, 1H), 7.98 (dd, J = 8.3, 2.3 Hz, 1H), 7.93 (t, J = 2.0 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 2.69 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ *rotamers 153.64, 153.60*, 151.1, 151.0*, 150.5, 145.9, 145.0, 138.64, 138.55*, 129.7, 125.5, 121.5, 121.4*, 119.5, 119.4*, 29.5, 15.9; HRMS (ESI⁻) m/z calcd for C₁₂H₁₀N₅O₂ (M − H)⁻ 256.0840, found 256.0856.

6-((4-Ethylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bf).

Synthesized by procedure B to yield 12bf in 56% as a yellow solid. ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.92 (s, 1H), 9.44 (s, 1H), 8.07–7.94 (m, 2H), 7.33–7.26 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H); ¹³C NMR ((CD₃)₂CO 101 MHz) δ *rotamers 153.6, 153.6*, 150.9, 150.8*, 150.5, 145.0, 142.0, 141.9*, 136.3, 136.2, 129.0, 122.1, 122.1, 28.9, 16.0; HRMS (ESI⁺) m/z calcd for C₁₂H₁₂N₅O₂ (M + H)⁺ 258.0985, found 258.0984.

6-((3-(tert-Butyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bg).

Synthesized by procedure B to yield 12bg in 47% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.93 (s, 1H), 9.53–9.41 (m, 1H), 8.11–8.05 (m, 2H), 7.41–7.35 (m, 1H), 7.31–7.25 (m, 1H), 1.35 (s, 9H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.6, 152.8, 151.0, 150.5, 145.0, 138.4, 129.4, 122.9, 119.4, 119.2, 35.4, 31.6; HRMS (ESI⁻) m/z calcd for C₁₄H₁₄N₅O₂ (M − H)⁻ 284.1153, found 284.1152.

6-((4-(tert-Butyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bh).

Synthesized by procedure B to yield 12bh in 64% as a tan solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.98 (s, 1H), 9.44 (s, 1H), 8.24–7.79 (m, 2H), 7.71–7.27 (m, 2H), 1.34 (s, 9H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.7, 150.1, 149.6, 147.9, 144.1, 135.1, 125.6, 120.9, 34.1, 30.7; HRMS (ESI⁺) m/z calcd for C₁₄H₁₆N₅O₂ (M + H)⁺ 286.12985, found 286.1289.

6-(Mesitylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bi).

Synthesized by procedure B to yield 12bi in 25% as a colorless solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.83 (s, 1H), 9.17 (s, 1H), 6.96 (s, 2H), 2.28 (s, 3H), 2.21 (s, 6H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.6, 153.0, 151.1, 145.5, 138.1, 136.4, 132.7, 129.6, 21.1, 18.4; HRMS (ESI⁻) m/z calcd for C₁₃H₁₂N₅O₂ (M − H)⁻ 270.0996, found 270.0992.

6-((4-Butylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bj).

Synthesized by procedure B to yield 12bj in 33% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.04 (s, 1H), 9.42 (s, 1H), 8.25–7.74 (m, 2H), 7.50–7.01 (m, 2H), 2.67–2.58 (m, 2H), 1.66–1.55 (m, 2H), 1.43–1.30 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ *rotamers 153.6, 153.5*, 150.8, 150.7*, 150.4, 144.9, 140.6, 136.2, 136.1*, 129.5, 122.0, 121.9*, 35.6, 34.4, 22.9, 14.2; HRMS (ESI⁺) m/z calcd for C₁₄H₁₆N₅O₂ (M + H)⁺ 286.1298, found 286.1307.

6-((4-Pentylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bk).

Synthesized by procedure B to yield 12bk in 55% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.95 (s, 1H), 9.43 (s, 1H), 8.05–7.97 (m, 2H), 7.31–7.24 (m, 2H), 2.63 (t, J = 7.7 Hz, 2H), 1.69–1.58 (m, 2H), 1.42–1.28 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.7, 150.9, 150.5, 145.0, 140.7, 136.3, 129.6, 122.1, 35.9, 32.2, 32.0, 23.2, 14.3; HRMS (ESI⁺) m/z calcd for C₁₅H₁₈N₅O₂ (M + H)⁺ 300.1455, found 300.1443.

6-((4-Hexylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bl).

Synthesized by procedure B to yield 12bl in 40% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.98 (s, 1H), 9.43 (s, 1H), 8.04–7.97 (m, 2H), 7.31–7.25 (m, 2H), 2.63 (t, J = 7.5 Hz, 2H), 1.69–1.57 (m, 2H), 1.41–1.25 (m, 6H), 0.88 (t, J = 6.9 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.7, 150.9, 150.5, 145.1, 140.7, 136.3, 129.6, 122.1, 36.0, 32.5, 32.3, 29.7, 23.3, 14.4; HRMS (ESI⁺) m/z calcd for C₁₆H₂₀N₅O₂ (M + H)⁺ 314.1611, found 314.1602.

6-((4-Ethyl-2-fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bm).

Synthesized by procedure B to yield 12bm in 33% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.54 (s, 1H), 9.17 (s, 1H), 8.35 (t, J = 8.3 Hz, 1H), 7.23–7.13 (m, 2H), 2.69 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −129.38 to −129.48 (m), −129.63 to −129.71 (m); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 155.0 (d, J_CF = 245.5 Hz), 153.6, 151.4, 150.4, 145.2, 144.4 (d, J_CF = 6.9 Hz), 124.8 (d, J_CF = 3.4 Hz), 124.1 (d, J_CF = 16.1 Hz), 123.8 (d, J_CF = 10.7 Hz), 115.5 (d, J_CF = 19.0 Hz), 28.9 (d, J_CF = 1.6 Hz), 15.7; HRMS (ESI⁺) m/z calcd for C₁₂H₁₁FN₅O₂ (M + H)⁺ 276.0891, found 276.0893.

6-((4-Butyl-2-fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bn).

Synthesized by procedure B to yield 12bn in 21% as a yellow solid (HPLC condition 2): ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.13 (s, 1H), 9.19 (s, 1H), 8.41–8.33 (m, 1H), 7.20–7.14 (m, 2H), 2.72–2.62 (t, J = 7.6 Hz, 2H), 1.69–1.58 (m, 2H), 1.44–1.31 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −129.46 to −129.54 (m); HRMS (ESI⁺) m/z calcd for C₁₄H₁₅FN₅O₂ (M + H)⁺ 304.1204, found 304.1207.

6-((2-Fluoro-4-pentylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bo).

Synthesized by procedure B with 12bo-int2 (see Supporting Information) to yield 12bo in 37% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.96 (s, 1H), 9.17 (s, 1H), 8.37 (q, J = 8.1 Hz, 1H), 7.20–7.13 (m, 2H), 2.66 (t, J = 7.7 Hz, 2H), 1.72–1.60 (m, 2H), 1.40–1.30 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −129.56 to −129.65 (m), −129.80 to −129.89 (m); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 154.9 (d, J_CF = 245.6 Hz), 153.6, 151.3, 150.4, 145.2, 143.0 (d, J_CF = 7.1 Hz), 125.4 (d, J_CF = 3.2 Hz), 124.0, 123.8 (d, J_CF = 10.8 Hz), 115.9 (d, J_CF = 18.9 Hz), 35.9 (d, J_CF = 1.5 Hz), 32.1, 31.7, 23.2, 14.3; HRMS (ESI⁺) m/z calcd for C₁₅H₁₇FN₅O₂ (M + H)⁺ 318.1360, found 318.1365.

6-((2-Fluoro-4-hexylphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bp).

Synthesized by procedure B with 12bp-int2 (see Supporting Information) to yield 12bp in 50% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.91 (s, 1H), 9.16 (s, 1H), 8.35 (t, J = 8.3 Hz, 1H), 7.19–7.13 (m, 2H), 2.66 (t, J = 7.6 Hz, 2H), 1.70–1.60 (m, 2H), 1.41–1.26 (m, 6H), 0.89 (t, J = 7.0 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −129.58 to −129.71 (m), −129.83 to −129.92 (m); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 154.9 (d, J_CF = 245.6 Hz), 153.6, 151.3, 150.3, 145.1, 142.9 (d, J_CF = 7.0 Hz), 125.4 (d, J_CF = 3.1 Hz), 123.9, 123.8 (d, J_CF = 10.7 Hz), 115.9 (d, J_CF = 18.7 Hz), 35.9 (d, J_CF = 1.4 Hz), 32.4, 31.9, 29.6, 23.3, 14.4; HRMS (ESI⁺) m/z calcd for C₁₆H₁₉FN₅O₂ (M + H)⁺ 332.1517, found 332.1515.

6-((4-Pentyl-2-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bq).

Synthesized by procedure B with 12bq-int2 (see Supporting Information) to yield 12bq in 47% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.19 (s, 1H), 9.28 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 7.42–7.34 (m, 2H), 2.71 (t, J = 7.9 Hz, 2H), 1.72–1.62 (m, 2H), 1.40–1.30 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −58.42 (d, J = 1.3 Hz), −58.44 (d, J = 1.3 Hz); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.7, 150.2, 149.3, 144.2, 141.5, 139.4, 127.8 (q, J_CF = 1.5 Hz), 127.4, 122.5, 120.8 (q, J_CF = 259.2 Hz), 120.8, 34.9, 31.2, 30.8, 22.2, 13.4; HRMS (ESI⁺) m/z calcd for C₁₆H₁₇F₃N₅O₃ (M + H)⁺ 384.1278, found 384.1296.

6-((4-Hexyl-2-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12br).

Synthesized by procedure B with 12br-int2 (see Supporting Information) to yield 12br in 14% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.14 (s, 1H), 9.27 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 7.42–7.34 (m, 2H), 2.71 (t, J = 7.4 Hz, 2H), 1.72–1.60 (m, 2H), 1.39–1.28 (m, 6H), 0.87 (t, J = 7.0 Hz, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ rotamers −58.31 (d, J = 1.9 Hz), −58.39 (d, J = 1.8 Hz); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.8, 151.3, 150.3, 145.3, 142.5, 140.5 (q, J_CF = 2.4 Hz), 128.8, 128.5, 123.7, 121.8, 121.7 (q, J_CF = 258.0 Hz), 35.9, 32.4, 32.1, 29.6, 23.3, 14.4; HRMS (ESI⁺) m/z calcd for C₁₇H₁₉F₃N₅O₃ (M + H)⁺ 398.1434, found 398.1441.

6-((4-(2-Hydroxyethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bs).

A round-bottom flask containing 11 (0.200 g, 1.05 mmol) was evacuated and refilled with N₂ (3×). The flask was cooled in an ice bath and the solid was sequentially diluted with anhydrous THF (2 mL), a mixture of 2-(4-aminophenyl)ethan-1-ol (0.130 g, 0.948 mmol) in THF (2 mL), and Et₃N (0.15 mL, 1.1 mmol). The mixture, cooled in an ice bath, was stirred for 1.5 h, diluted with EtOAc, filtered through a SiO₂ plug (EtOAc) to remove the salts, and concentrated to a residue. The residue was diluted with a solution of KOH (320 mg, 5.70 mmol) in H₂O (4 mL) and THF (2 mL). The resulting solution was stirred at rt for 1 h, acidified with 1 M aq HCl, and extracted with EtOAc. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated to a pale yellow residue (0.150 g). The residue was purified by chromatography on SiO₂ (gradient, 0–5% MeOH/CH₂Cl₂) to yield product (0.135 g) as an off-white solid at ~90% purity by NMR. The solid was diluted with EtOAc and then hexanes for a final mix of ~70% EtOAc/hexanes. The solid was filtered, rinsed with hexanes, and collected to afford 12bs (0.106 g, at 95% purity by NMR, 39% yield) as a pale yellow solid: ¹H NMR ((CD₃)₂SO, 400 MHz) δ 13.3 (br s, 1H), 10.2 (s, 1H), 7.91–7.88 (m, 2H), 7.26–7.23 (m, 2H), 4.66 (br s, 1H), 3.60 (t, J = 7.1 Hz, 2H), 2.72 (t, J = 7.1 Hz, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.7, 151.0, 150.5, 145.1, 137.7, 136.6, 130.3, 122.1, 63.8, 39.8.; HRMS (ESI⁻) m/z calcd for C₁₂H₁₀N₅O₃ (M − H) 272.0789, found 272.0796.

6-((2-Methyl-5-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bt).

Synthesized by procedure B to yield 12bt in 17% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.98 (s, 1H), 9.34 (s, 1H), 8.46–8.36 (m, 1H), 7.62–7.50 (m, 2H), 2.49 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.87 (s, 3F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.7, 151.3, 149.5, 144.3, 136.4, 136.3, 131.5, 128.3 (q, J_CF = 32.4 Hz), 124.3 (q, J_CF = 271.2 Hz), 122.5 (q, J_CF = 3.9 Hz), 120.7 (q, J_CF = 4.1 Hz), 17.0; HRMS (ESI⁺) m/z calcd for C₁₂H₉F₃N₅O₂ (M + H)⁺ 312.0702, found 312.0700.

6-((2-Methyl-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bu).

Synthesized by procedure A to yield 12bu in 42% as a pale yellow crystalline solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.08 (br s, 1H), 9.23 (s, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 2.8 Hz, 1H), 7.29 (dd, J = 8.7, 2.8 Hz, 1H), 2.44 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.62 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.7, 152.1, 150.5, 147.4 (q, J_CF = 1.7 Hz), 145.2, 135.5, 135.4, 126.7, 123.9, 121.5 (q, J_CF = 255.5 Hz), 119.8, 17.9; HRMS (ESI⁺) m/z calcd for C₁₂H₉F₃N₅O₃ (M + H)⁺ 328.0652, found 328.0650.

2-(4-((6-Hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-yl)amino)phenyl)-2-methylpropanenitrile (12bv).

Synthesized by procedure A to yield 12bv in 78% as an off-white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 12.06 (s, 1H), 9.61 (s, 1H), 8.20–8.15 (m, 2H), 7.66–7.61 (m, 2H), 1.76 (s, 6H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 153.6, 151.3, 150.4, 145.1, 139.4, 138.1, 126.6, 125.1, 122.5, 37.6, 29.2; HRMS (ES⁺) m/z calcd for C₁₄H₁₃N₆O₂ (M + H)⁺ 297.1095, found 297.1092.

1-(4-((6-Hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-yl)amino)phenyl)ethan-1-one (12bw).

A round-bottom flask containing 11 (0.200 g, 1.05 mmol), 4-aminoacetophenone (0.130 g, 0.962 mmol), and K₂CO₃ (0.200 g, 1.45 mmol) was diluted with acetone/H₂O (9:1, 3 mL), and the resulting dark mixture was stirred at room temperature. After 2 h, the mixture was diluted with a solution of KOH in H₂O (0.320 g in 4 mL) and stirring was continued for an additional 30 min. The mixture was acidified with 1 M HCl and extracted with EtOAc. The organic layer was washed with brine, dried (MgSO₄), and concentrated to a yellow residue. The residue was purified by chromatography on SiO₂ (gradient: 0–2% MeOH/CH₂Cl₂) to afford 12bw (93% purity using HPLC condition 1) in 19% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 12.13 (br s, 1H), 9.73 (s, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.5 Hz, 2H), 2.60 (s, 3H); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 196.7, 153.5, 151.4, 150.2, 145.2, 142.7, 134.5, 130.2, 121.5, 26.6; HRMS (ES⁺) m/z calcd for C₁₂H₁₀N₅O₃ (M + H)⁺ 272.0778, found 272.0793.

6-(Isopropylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bx).

Synthesized by procedure B to yield 12bx in 60% as a tan solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.44 (s, 1H), 7.59 (s, 1H), 4.56–4.11 (m, 1H), 1.31 (d, J = 6.6 Hz, 6H); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.3, 152.5, 150.9, 144.8, 44.0, 21.7; HRMS (ESI⁺) m/z calcd for C₇H₁₀N₅O₂ (M + H)⁺ 196.0829, found 196.0826.

6-(Octylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12by).

A round-bottom flask containing 11 (0.200 g, 1.05 mmol) and K₂CO₃ (0.200 g, 1.45 mmol) in acetone/H₂O (9:1, 3 mL) was slowly diluted with a solution of n-octylamine (0.125 g, 0.967 mmol) in acetone/H₂O (9:1, 1 mL), and the resulting dark mixture was stirred at rt. After 2 h, the mixture was diluted with a solution of KOH in H₂O (0.320 g in 4 mL) and stirring was continued for an additional 30 min. The mixture was acidified with 1 M HCl and extracted with EtOAc. The organic layer was washed with brine, dried (MgSO₄), and concentrated to a brown residue. The residue was purified by chromatography on SiO₂ (gradient, 0.5–2% MeOH/CH₂Cl₂) to yield a crude brown solid. The solid was dissolved in EtOAc and extracted with 2–3 M NaOH. The aqueous layer was acidified with aq HCl and extracted with EtOAc (3×). The organic layer was washed with brine, dried (MgSO₄), and concentrated to yield 12by (9%) as an off-white solid: ¹H NMR ((CD₃)₂SO, 500 MHz) δ 13.05 (s, 1H), 8.73 (t, J = 6.1 Hz, 1H), 3.36 (q, J = 6.8 Hz, 2H), 1.58 (p, J = 7.2 Hz, 2H), 1.33–1.20 (m, 10H), 0.85 (t, J = 6.7 Hz, 3H); ¹³C NMR ((CD₃)₂SO, 125 MHz) δ 152.8, 152.7, 150.2, 144.4, 40.7, 31.2, 28.7, 28.6, 27.8, 26.4, 22.1, 13.9; HRMS (ES⁺) m/z calcd for C₁₂H₂₀N₅O₂ (M + H)⁺ 266.1612, found 266.1628.

6-(Phenethylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12bz).

Synthesized by procedure A to yield 12bz in 20% as a colorless solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.80 (br s, 1H), 8.01 (s, 1H), 7.33–7.17 (m, 5H), 3.85–3.77 (m, 2H), 3.07–2.99 (m, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 153.3, 150.9, 145.0, 140.0, 129.7, 129.3, 127.2, 43.4, 35.0; HRMS (ESI⁻) m/z calcd for C₁₂H₁₀N₅O₂ (M − H)⁻ 256.0840, found 256.0845.

6-((3,3,3-Trifluoropropyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ca).

Synthesized by procedure A to yield 12ca (94% purity using HPLC condition 1) in 66% yield as a light yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.82 (br s, 1H), 8.20 (s, 1H), 3.86 (q, J = 6.7 Hz, 2H), 2.72 (qt, J = 11.1, 7.0 Hz, 2H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −65.98 to −66.05 (m, 3 F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.8, 153.2, 150.7, 145.1, 127.6 (q, J_CF = 276.1 Hz), 35.4 (q, J_CF = 4.0 Hz), 32.7 (q, J_CF = 27.8 Hz); HRMS (ESI⁻) m/z calcd for C₇H₅F₃N₅O₂ (M − H)⁻ 248.0401, found 248.0413.

6-((4-Methylbenzyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12cb).

A round-bottom flask containing 11 (0.200 g, 1.05 mmol) and K₂CO₃ (0.200 g, 1.45 mmol) in acetone/H₂O (9:1, 3 mL) was slowly diluted with a solution of p-tolylmethanamine (0.120 g, 0.990 mmol) in acetone/H₂O (9:1, 1 mL), and the resulting dark mixture was stirred at rt. After 1.5 h, the mixture was diluted with a solution of KOH in H₂O (0.320 g in 4 mL) and stirring was continued for an additional 30 min. The mixture was acidified with 1 M HCl and extracted with EtOAc. The organic layer was washed with brine, dried (MgSO₄), and concentrated to a brown residue. The residue was purified by chromatography on SiO₂ (0.7% MeOH/CH₂Cl₂) to yield 12cb in 29% as an off-white solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.76 (br s, 1H), 8.39 (s, 1H), 7.33 (d, J = 7.7 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 4.73 (d, J = 6.4 Hz, 2H), 2.29 (s, 3H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 153.5, 150.9, 145.1, 137.7, 135.8, 129.9, 128.8, 45.0, 21.1; HRMS (ESI⁺) m/z calcd for C₁₂H₁₂N₅O₂ (M + H)⁺ 258.0986, found 258.0993.

6-((4-(Trifluoromethoxy)benzyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12cc).

Synthesized by procedure A to yield 12cc in 36% as a colorless solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.77 (br s, 1H), 8.59 (s, 1H), 7.59 (app d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 4.83 (d, J = 6.5 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.65 (s, 3F); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.9, 153.5, 150.9, 149.1 (q, J_CF = 1.6 Hz), 145.2, 138.4, 130.6, 121.9, 121.4 (q, J_CF = 255.2 Hz), 44.5; HRMS (ESI⁻) m/z calcd for C₁₂H₇F₃N₅O₃ (M − H)⁻ 326.0506, found 326.0492.

6-(Benzhydrylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12 cd).

Synthesized by procedure A to yield 12 cd in 63% as a colorless solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.92 (br s, 1H), 8.30 (d, J = 8.7 Hz, 1H), 7.48–7.45 (m, 4H), 7.40–7.35 (m, 4H), 7.33–7.28 (m, 2H), 6.58 (d, J = 8.6 Hz, 1H); ¹³C NMR ((CD₃)₂CO, 100 MHz) δ 153.4, 152.9, 150.8, 145.2, 141.8, 129.5, 128.6, 128.4, 59.4; HRMS (ESI⁻) m/z calcd for C₁₇H₁₂N₅O₂ (M − H)⁻ 318.0996, found 318.1003.

6-(Adamantan-1-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ce).

Synthesized by procedure A at 75 °C to yield 12ce in 55% as an off-white solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.83 (br s, 1H), 6.97 (s, 1H), 2.30–2.24 (m, 6H), 2.16–2.12 (m, 3H), 1.80–1.72 (m, 6H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 152.0, 150.4, 144.7, 54.2, 41.0, 36.9, 30.3; HRMS (ESI⁻) m/z calcd for C₁₄H₁₆N₅O₂ (M − H)⁻ 286.1309, found 286.1311.

6-(Adamantan-2-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12cf).

Synthesized by procedure A with a reverse-phase HPLC purification using 0.1% TFA MeCN/H₂O solvent system to yield 12cf in 5% as a colorless solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.57 (br s, 1H), 7.38 (s, 1H), 4.27–4.24 (m, 1H), 2.18–2.16 (m, 2 H), 2.00–1.88 (m, 8H), 1.83–1.81 (m, 2H), 1.74–1.70 (m, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.9, 152.6, 151.0, 145.3, 56.2, 38.0, 37.6, 32.4, 31.9, 28.1; HRMS (ESI⁺) m/z calcd for C₁₄H₁₈N₅O₂ (M + H)⁺ 288.1455, found 288.1458.

6-(3,5-Dimethyladamantan-1-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12cg).

Synthesized by procedure A at 75 °C to yield 12cg in 38% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.84 (br s, 1H), 6.98 (s, 1H), 2.22 (hept, J = 3.2 Hz, 1H), 2.13–2.08 (m, 2H), 1.93–1.85 (m, 4H), 1.47 (dt, J = 12.2, 2.7 Hz, 2H), 1.37 (dt, J = 12.3, 2.8 Hz, 2H), 1.23 (qt, J = 12.4, 2.1 Hz, 2H), 0.90 (s, 6H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 152.0, 150.4, 144.7, 55.8, 51.1, 46.9, 43.2, 39.5, 31.0, 30.5; HRMS (ESI⁻) m/z calcd for C₁₆H₂₀N₅O₂ (M − H)⁻ 314.1622, found 314.1616.

6-(3-Hydroxyadamantan-1-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ch).

Synthesized by procedure A at 75 °C to yield 12ch in 30% as an off-white solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.84 (br s, 1H), 7.04 (s, 1H), 3.81 (br s, 1H), 2.31–2.28 (m, 2H), 2.22–2.11 (m, 6H), 1.77–1.68 (m, 4H), 1.67–1.57 (m, 2H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 152.0, 150.4, 144.7, 68.7, 56.6, 48.6, 45.0, 40.0, 35.7, 31.5; HRMS (ESI⁺) m/z calcd for C₁₄H₁₈N₅O₃ (M + H)⁺ 304.1404, found 304.1399.

6-(Adamantan-1-yl)ethyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ci).

Synthesized by procedure A at 75 °C to yield 12ci in 8% as a beige solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 11.84 (s, 1H), 7.27 (d, J = 9.7 Hz, 1H), 4.05 (dq, J = 10.0, 6.9 Hz, 1H), 1.99 (p, J = 3.2 Hz, 3H), 1.76–1.62 (m, 12H), 1.20 (d, J = 6.9 Hz, 3H); ¹³C NMR ((CD₃)₂CO, 125 MHz) δ 153.6, 153.3, 151.0, 144.9, 55.9, 39.1, 37.7, 37.3, 29.3, 13.9; HRMS (ESI⁻) m/z calcd for C₁₆H₂₀N₅O₂ (M − H)⁻ 314.1622, found 314.1618.

6-Methoxy-N-(4-(trifluoromethoxy)phenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-amine (13).

Synthesized by procedure A to yield 13 in 60% as a yellow solid: ¹H NMR ((CD₃)₂CO, 500 MHz) δ 9.70 (s, 1H), 8.19–8.10 (m, 2H), 7.45–7.36 (m, 2H), 4.23 (s, 3H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.78 (s, 3F); ¹³C NMR ((CD₃)₂CO, 126 MHz) δ 156.4, 151.7, 150.6, 147.7, 146.5 (q, J_CF = 2.3 Hz), 137.8, 124.1, 122.4, 121.5 (q, J_CF = 255.2 Hz), 56.5; HRMS (ESI⁺) m/z calcd for C₁₂H₉F₃N₅O₃⁺ (M + H)⁺ 328.0637, found 328.0652.

3-((2-Fluorophenyl)amino)pyrazin-2-ol (16a).

Synthesized by procedure C to yield 16a in 32% as an off white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.98 (s, 1H), 8.79–8.68 (m, 1H), 8.42 (s, 1H), 7.27–7.14 (m, 2H), 7.08–7.00 (m, 1H), 6.97 (d, J = 4.5 Hz, 1H), 6.91 (d, J = 4.5 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −134.06 to −134.15 (m, 1F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 153.3 (d, J_CF = 241.9 Hz), 149.7, 139.1, 136.5, 125.3 (d, J_CF = 3.9 Hz), 123.4 (d, J_CF = 7.7 Hz), 121.5, 120.7 (d, J_CF = 1.6 Hz), 116.9, 115.4 (d, J_CF = 19.1 Hz); HRMS (ESI⁺) calcd for C₁₀H₉FN₃O (M + H)⁺ 206.0724, found 206.0716.

3-((4-Decylphenyl)amino)pyrazin-2-ol (16b).

Synthesized by procedure C to yield 16b in 33% as an off white solid: ¹H NMR (CDCl₃, 400 MHz) δ 12.16 (s, 1H), 8.04 (s, 1H), 7.68 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 4.4 Hz, 1H), 6.67 (d, J = 4.4 Hz, 1H), 2.58 (t, J = 7.7 Hz, 2H), 1.60 (p, J = 7.2 Hz, 2H), 1.40–1.19 (m, 14H), 0.88 (t, J = 6.6 Hz, 3H); ¹³C NMR (CDCl₃, 101 MHz) δ 153.1, 148.9, 138.0, 136.5, 129.0, 123.4, 119.4, 114.0, 35.5, 32.0, 31.7, 29.8, 29.8, 29.7, 29.5, 29.4, 22.8, 14.3; HRMS (ESI⁺) calcd for C₂₀H₃₀N₃O (M + H)⁺ 328.2383, found 328.2383.

3-((4-(Trifluoromethoxy)phenyl)amino)pyrazin-2-ol (16c).

Synthesized by procedure C to yield 16c to yield in 12% as an off white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.90 (s, 1H), 8.77 (s, 1H), 8.21–8.13 (m, 2H), 7.33–7.26 (m, 2H), 6.94 (d, J = 4.4 Hz, 1H), 6.87 (d, J = 4.4 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −58.87 (s, 3F); ¹³C NMR (101 MHz, acetone-d6) δ 152.4, 150.0, 144.3–144.2 (m), 140.1, 122.3, 121.6 (q, J_CF = 254.3 Hz), 121.5, 120.9, 116.6; HRMS (ESI⁺) calcd for C₁₁H₉F₃N₃O₂ (M + H)⁺ 272.0641, found 272.0627.

3-((3,5-Bis(trifluoromethyl)phenyl)amino)pyrazin-2-ol (16d).

Synthesized by procedure C to yield 16d to yield in 36% as a yellow solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 11.02 (s, 1H), 9.18 (s, 1H), 8.86–8.79 (m, 2H), 7.68–7.56 (m, 1H), 7.02 (d, J = 4.5 Hz, 1H), 6.97 (d, J = 4.5 Hz, 1H); ¹⁹F NMR (376 MHz, acetone-d₆) δ −63.53 (s, 6F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.2, 150.0, 142.8, 132.3 (q, J_CF = 33.2 Hz), 125.9 (q, J_CF = 272.3 Hz), 121.1, 119.8–119.1 (m), 117.9, 115.5–115.1 (m); HRMS (ESI⁺) calcd for C₁₂H₈F₆N₃O (M + H)⁺ 324.0566, found 324.0556.

3-((4-(Trifluoromethyl)phenyl)amino)pyrazin-2-ol (16e).

Synthesized by procedure C to yield 16e to yield in 37% as an off white solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.98 (s, 1H), 8.87 (s, 1H), 8.30–8.21 (m, 2H), 7.73–7.62 (m, 2H), 6.98 (d, J = 4.5 Hz, 1H), 6.92 (d, J = 4.4 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz) δ −62.14 (s, 3F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.3, 150.0, 144.3 (d, J_CF = 1.8 Hz), 126.7 (q, J_CF = 4.1 Hz), 125.7 (q, J_CF = 270.7 Hz), 123.8 (q, J_CF = 32.3 Hz), 121.3, 119.5, 117.2; HRMS (ESI⁺) calcd for C₁₁H₉F₃N₃O (M + H)⁺ 256.0692, found 256.0698.

3-((3-(Trifluoromethyl)phenyl)amino)pyrazin-2-ol (16f).

Synthesized by procedure C to yield 16f to yield in 35% as a brown solid: ¹H NMR ((CD₃)₂CO, 400 MHz) δ 10.86 (s, 1H), 8.87 (s, 1H), 8.61 (s, 1H), 8.22 (dd, J = 8.3, 2.2 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 7.7, 1H), 6.98 (d, J = 4.4 Hz, 1H), 6.91 (d, J = 4.5 Hz, 1H); ¹⁹F NMR ((CD₃)₂CO, 376 MHz, acetone-d₆) δ −63.13 (s, 3F); ¹³C NMR ((CD₃)₂CO, 101 MHz) δ 152.4, 150.1, 141.7, 131.2 (d, J_CF = 31.9 Hz), 130.3, 125.4 (q, J_CF = 271.4 Hz), 123.2 (d, J_CF = 1.5 Hz), 121.5–121.3 (m), 119.1 (q, J_CF = 4.3 Hz), 116.9, 115.9 (q, J_CF = 4.2 Hz); HRMS (ESI⁺) calcd for C₁₁H₉F₃N₃O (M + H)⁺ 256.0692, found 256.0687.

3-((3′-Methoxy-[1,1′-biphenyl]-4-yl)amino)pyrazin-2-ol (16g).

Synthesized by procedure C to yield 16g to yield in 30% as a brown solid: ¹H NMR ((CD₃)₂SO, 500 MHz) δ 11.97 (s, 1H), 9.20 (s, 1H), 8.17–8.02 (m, 2H), 7.69–7.53 (m, 2H), 7.35 (t, J = 7.9 Hz, 1H), 7.26–7.20 (m, 1H), 7.18 (t, J = 2.1 Hz, 1H), 6.92 (d, J = 4.3 Hz, 1H), 6.89 (dd, J = 8.2, 2.5 Hz, 1H), 6.82 (d, J = 4.3 Hz, 1H), 3.82 (s, 3H); ¹³C NMR ((CD₃)₂SO, 101 MHz) δ 159.7, 151.4, 148.9, 141.4, 139.6, 133.4, 129.9, 126.7, 120.5, 119.4, 119.3, 118.5, 115.7, 112.3, 111.7, 55.1; HRMS (ESI⁺) calcd for C₁₇H₁₆N₃O₂ (M + H)⁺ 294.1237, found 294.1236

3-((4′-Fluoro-[1,1′-biphenyl]-4-yl)amino)pyrazin-2-ol (16h).

Synthesized by procedure C to yield 16h to yield in 16% as an off brown solid: ¹H NMR ((CD₃)₂SO, 400 MHz) δ 11.97 (s, 1H), 9.21 (s, 1H), 8.12–8.03 (m, 2H), 7.69–7.61 (m, 2H), 7.62–7.52 (m, 2H), 7.33–7.17 (m, 2H), 6.90 (m, 1H), 6.80 (m, 1H); ¹⁹F NMR ((CD₃)₂SO, 376 MHz) δ −116.47 (tt, J = 8.9, 5.4 Hz, 1F); ¹³C NMR (101 MHz, DMSO-d₆) δ 161.5 (d, J_CF = 243.5 Hz), 151.5, 148.9, 139.4, 136.4 (d, J_CF = 3.0 Hz), 132.5, 128.0 (d, J_CF = 8.0 Hz), 126.6, 120.5, 119.5, 115.7, 115.6 (d, J_CF = 21.4 Hz); HRMS (ESI⁺) calcd for C₁₆H₁₃FN₃O (M + H)⁺ 282.1037, found 282.1037.

Oxygen Consumption Rate Seahorse Assay.

Oxygen consumption rate (OCR) was measured using an Agilent Seahorse XF24 or XFe96 Analyzer (Agilent Technologies, Santa Clara, CA). L6 myoblasts were seeded in a Seahorse 24- or 96-well tissue culture plate at a density of 3.5 × 10⁴ cells/well. The cells were then allowed to adhere overnight. Prior to the assay, the medium was changed to unbuffered DMEM containing pyruvate and glutamine (Gibco no. 12800–017, pH = 7.4 at 37 °C) and the cells were equilibrated for 1 h at 37 °C without CO₂. Compounds were injected during the assay, and OCR was measured using 2 min measurement periods. Cells were treated with a single drug concentration per well and measured over a 90 min period. Two wells were used per condition, and where applicable, results from multiple plates were averaged together. The first three measurements after injection for each concentration were averaged to produce a dose curve. EC₅₀ values were calculated using GraphPad Prism’s nonlinear regression built-in equation, Y = Bottom + (XHillslope)(Top − Bottom)/(XHillSlope + EC₅₀^HillSlope), with the Bottom constrained to the 100% baseline. Area under curve (AUC) values were also calculated using the same software.

Acute Dose Experiments.

Acute dose experiments were performed at UNSW and approved by the UNSW Animal Care and Ethics Committee (Project Approval 17–66B). Mice were purchased from Australian BioResources (Moss Vale, NSW, Australia). Mice were housed at 22 °C in a light/dark cycle of 12 h. Unless otherwise stated, mice were provided with ad libitum access to water and standard chow diet (Gordons Specialty Feeds, NSW, Australia).

To measure pharmacokinetics for SHS4121705 (12i), 12ah, 12az, 12bh, 12bj, and 12ce, compounds were administered as a bolus to 9–12 week-old male C57BL/6 mice by oral gavage at a dose of 10 mg/kg body weight. Compounds were delivered in a mixture containing 90–91% (v/v) methylcellulose (Sigma, M0512), 2–3% (v/v) Tween 80 (Sigma, P6474), and 6–8% (v/v) DMSO (Sigma, D5879). Blood samples were collected from the tail tip at the time points shown in heparinized capillary tubes. Plasma was collected by centrifugation at 2000g for 10 min. Samples were processed for LC–MS/MS by extraction in 100 μL acetonitrile:methanol (9:1) for 7 μL plasma, followed by centrifugation at 18 000g for 10 min and collection of the supernatant for analysis. Standards were prepared by spiking in known amounts (1–100 ng) of compound into plasma from untreated mice and processed the same way. LC–MS/MS was performed on a Shimadzu Prominence LCMS-8030 (Shimazdu, Japan). Chromatographic separation was achieved using an ACUITY UPLC BEH, C18 column (Waters, WT186002350, USA). Mobile phase A consisted of 0.1% v/v formic acid in HLPC grade water. Mobile phase B consisted of 0.1% v/v formic acid in acetonitrile. The analyte was eluted with a gradient of 5–80% mobile phase B at a flow rate of 0.4 mL/min with 10 μL injection volume electrosprayed into the mass spectrometer. ESI was performed in negative mode. Identification was achieved by the following m/z transitions and retention times: 313 → 201, 284 at 5.3 min for SHS4121705 (12i); 301 → 243, 215 at 4.7 min for 12ah; 279 → 167, 125 at 4.8 min for 12az; 285 → 174 at 5 min for 12bh; 284 → 173 at 4.6 min for 12bj. Quantification was determined by measuring peak areas using LabSolutions software on the instrument. Concentration of test samples was interpolated from a standard curve derived from the integrated intensity values of standards.

Pharmacokinetics analysis for compound 12ce was performed by GVK Biosciences (Hyderabad, India). Swiss Albino mice were administered 10 mg/kg 12ce by oral gavage in a triturated formulation with Tween 80 and 0.5% (w/v) methylcellulose. Blood samples were collected at time points indicated in the figure, with three replicates per time point. Plasma was separated by centrifugation. 50 μL of plasma was precipitated with 200 μL of acetonitrile containing internal standard of telmisartan at 200 ng/mL. Samples were vortexed for 5 min at 850 rpm and centrifuged at 4000 rpm for 5 min at 4 °C. From this, 110 μL of supernatant was diluted with 150 μL of methanol:water (1:1, v/v). Calibration standards were prepared using 2.0 μL of calibration curve standard added to 48 μL of blank matrix, which was processed in the same way as 50 μL of sample plasma. Liquid chromatography–tandem mass spectrometry was performed on a 5500 QTRAP (SCIEX, USA). Chromatographic separation was achieved using a Kinetex Evo, C18, 50 mm × 4.6 mm, 5 μm. Mobile phase A consisted of 10 mM ammonium acetate with 0.1% v/v formic acid in Milli-Q water. Mobile phase B consisted of acetonitrile:methanol (50:50). The analyte was eluted with a gradient of 5–95% mobile phase B at a flow rate of 1 mL/min with 15 μL injection volume. Electrospray ionization (ESI) was performed in negative mode. Transition of m/z 286 → 125, and 1.64 min retention time were used to identify 12ce, and transition of m/z 513.2 → 287.1, and 1.59 min retention time were used to identify telmisartan as an internal standard. Concentration of test samples was interpolated from a standard curve derived from the intensity values of standards (1–1000 ng/mL). Animal studies performed by GVK Biosciences were approved by its Institutional Animal Ethics Committee.

To measure body temperature, mice were similarly administered an oral bolus of SHS4121705 (12i), except that doses ranged from 0 (vehicle) to 50 mg/kg body weight. Core body temperature was measured with a rectal probe thermometer (Braintree, TW2–107) at the time points shown.

To measure tissue distribution, mice were similarly orally administered a bolus of SHS4121705 (12i) at 10 mg/kg body weight. Mice were euthanized by cervical dislocation after 1 h, and tissues were dissected, rinsed in PBS, and snap-frozen in liquid nitrogen. To process the tissues for LC–MS/MS analysis, frozen tissues were powdered in liquid nitrogen using a tissue pulverizer (Cellcrusher, USA), then homogenized with a motorized pellet pestle homogenizer. Homogenate was centrifuged (10 000g for 10 min) and supernatant collected. Samples were processed and analyzed as described for plasma samples above.

Mouse Study.

The STAM mouse model was performed by SMC Laboratories, Inc. (Tokyo, Japan). Compound SHS4121705 (12i) was administered in diet by premixing high-fat diet (HFD, 57 kcal % fat, catalog no. HFD32, CLEA Japan Inc., Japan) at SMC Laboratories. Telmisartan (Micardis) was purchased from Boehringer Ingelheim GmbH (Germany) and dissolved in pure water. NASH was induced in 24 male mice by a single subcutaneous injection of 200 μg of streptozotocin (Sigma-Aldrich, USA) solution 2 days after birth and feeding with HFD after 4 weeks of age. From 6 weeks of age, SHS4121705 (12i) was administered orally by feeding with HFD to achieve a dose of 25 mg kg⁻¹ d⁻¹, and telmisartan was administered orally in a volume of 10 mL/kg to achieve a dose of 10 mg kg⁻¹ d⁻¹. The viability, clinical signs, and behavior were monitored daily. Body weight was recorded before the treatment. Mice were observed for significant clinical signs of toxicity, moribundity, and mortality approximately 60 min after each administration. The animals were sacrificed at 9 weeks of age (day 21) by exsanguination through direct cardiac puncture under isoflurane anesthesia.

Nonfasting blood glucose was measured in whole blood using Stat Strip glucose meter (Nipro Corporation, Japan). Readings greater than 900 mg/dL (one mouse in telmisartan group) were recorded as 900 mg/dL. For plasma biochemistry, nonfasting blood was collected in polypropylene tubes with anticoagulant (Novo-Heparin, Mochida Pharmaceutical Co. Ltd., Japan) and centrifuged at 1000g for 15 min at 4 °C. The supernatant was collected and stored at −80 °C until use. Plasma ALT level was measured by FUJI Dri-Chem 7000 (Fujifilm, Japan).

For liver samples, the left lateral lobe was collected and cut into 6 pieces. Two pieces of left lateral lobe, left and right medial lobes, and caudate lobe were snap frozen in liquid nitrogen and stored at −80 °C. The other 2 pieces of left lateral lobe were fixed in Bouin’s solution and then embedded in paraffin. Samples were stored at room temperature for histology. Samples were stored at −80 °C. The right lobe was snap frozen in liquid nitrogen and stored at −80 °C for liver biochemistry.

Liver total lipid-extracts were obtained by Folch’s method. Liver samples were homogenized in chloroform–methanol (2:1, v/v) and incubated overnight at room temperature. After washing with chloroform–methanol–water (8:4:3, v/v/v), the extracts were evaporated to dryness and dissolved in isopropanol. Liver triglyceride content was measured by triglyceride E-test (Wako Pure Chemical Industries, Ltd., Japan).

For histopathology staining, sections were cut from paraffin blocks of liver tissue prefixed in Bouin’s solution and stained with Lillie-Mayer’s hematoxylin (Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (Wako Pure Chemical Industries). NAFLD activity score (NAS) was calculated according to the criteria of Kleiner.⁹³ To visualize collagen deposition, Bouin’s fixed liver sections were stained using Picro-Sirius Red solution (Waldeck, Germany). For quantitative analysis of fibrosis areas, bright field images of Sirius Red-stained sections were captured around the central vein using a digital camera (DFC295; Leica, Germany) at 200-fold magnification, and the positive areas in 5 fields/section were measured using ImageJ software (National Institutes of Health, USA).

The animals were maintained in a SPF facility under controlled conditions of temperature (23 ± 2 °C), humidity (45 ± 10%), lighting (12 h artificial light and dark cycles; light from 8:00 to 20:00), and air exchange. A high pressure was maintained in the experimental room to prevent contamination of the facility. All animals used in the study were housed and cared for in accordance with the Japanese Pharmacological Society Guidelines for Animal Use.

This STAM experiment using SHS4121705 (12i) was conducted as part of a study using the same control groups (vehicle and termisartan) as previously published.⁷⁸

Plasma Biochemistry.

Plasma triglyceride was measured by a colorimetric assay through reaction with GPO reagent (Pointe Scientific T7532) according to the manufacturer’s protocol. A standard curve was constructed using serial dilutions of glycerol standard (Sigma G7793). In brief, samples were incubated with GPO at 37 °C for 5–20 min until absorbance values at 500 nm had stabilized. Sample plasma triglyceride concentrations were determined through interpolation from the standard curve. Plasma cholesterol was measured by a colorimetric assay using Infinity cholesterol liquid stable reagent (ThermoFisher TR13421) according to the manufacturer’s protocol. A standard curve was constructed using serial dilutions of cholesterol standard (Pointe Scientific C7509). In brief, samples were incubated with Infinity cholesterol reagent at 37 °C for 5–20 min until absorbance values at 500 nm had stabilized. Absorbance at 660 nm was subtracted from absorbance at 500 nm to correct for background. Sample plasma cholesterol concentrations were determined through interpolation from the standard curve. Plasma AST was measured using Infinity AST (GOT) liquid stable reagent (ThermoFisher TR70121) according to the manufacturer’s protocol. In brief, samples were incubated with the Infinity AST reagent at 37 °C and absorbance was measured at 340 nm every minute for 20 min. Activity in U/L was calculated by multiplying the change in absorbance per minute by a correction factor, which was defined as total volume of reaction divided by the product of the molar absorption coefficient of NADH, the sample volume added, and the path length of absorption.

Statistical Analysis.

All data are presented as the mean ± standard error of the mean (SEM). Statistical testing was carried out using Prism (version 8.1.2; GraphPad Software), where the threshold for significance was designated as p < 0.05, compared to vehicle. For normally distributed data, differences between groups were examined using a one-way analysis of variance (ANOVA) with Dunnett’s post hoc test for multiple comparisons. For nonparametric data, the Kruskal–Wallis test was conducted with Dunn’s post hoc test for multiple comparisons.

Supplementary Material

supporting info

NIHMS1984181-supplement-supporting_info.pdf^{(239.7KB, pdf)}

ACKNOWLEDGMENTS

We thank Continuum Biosciences, Inc. and VBHRC Virginia Catalyst for financial support. C.J.G. is supported by a training grant from NIH (Grant R25GM072767). We thank Carter J. Gottschalk for assistance in creating the cover art.

ABBREVIATIONS USED

ATP: adenosine triphosphate
pmf: proton motive force
UCP: uncoupling protein
T2D: type 2 diabetes
NASH: non-alcoholic steatohepatitis
FDA: Food and Drug Administration
DNP: 2,4-dinitrophenol
NAFLD: nonalcoholic fatty liver disease
FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
SAR: structure–activity relationship
STZ: streptozotocin
STAM: Stelic animal model

Footnotes

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.0c00542

The authors declare the following competing financial interest(s): W.L.S. and K.L.H. are inventors of small molecule mitochondrial uncouplers that are licensed to Continuum Biosciences, Inc. S.P.T. has equity position at Continuum Biosciences, Inc.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00542.

Experimental procedures and characterization data (¹H, ¹³C, ¹⁹F NMR and HRMS) for aniline intermediates, hERG assay data, HPLC chromatograms of lead compounds (PDF)

Molecular formula strings and some data (CSV)

Contributor Information

Joseph M. Salamoun, Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States.

Christopher J. Garcia, Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States.

Stefan R. Hargett, Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia 22908, United States

Jacob H. Murray, Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

Sing-Young Chen, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia.

Martina Beretta, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia.

Stephanie J. Alexopoulos, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

Divya P. Shah, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

Ellen M. Olzomer, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

Simon P. Tucker, Continuum Biosciences, Pty Ltd., Sydney 2035, Australia; Continuum Biosciences Inc., Boston, Massachusetts 02116, United States

Kyle L. Hoehn, Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia 22908, United States; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia

Webster L. Santos, Departments of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States

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PERMALINK

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

Joseph M Salamoun

Christopher J Garcia

Stefan R Hargett

Jacob H Murray

Sing-Young Chen

Martina Beretta

Stephanie J Alexopoulos

Divya P Shah

Ellen M Olzomer

Simon P Tucker

Kyle L Hoehn

Webster L Santos

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

Figure 2.

RESULTS AND DISCUSSION

Scheme 1a.

Scheme 2a.

Table 1.

Table 5.

Table 2.

Table 3.

Table 6.

Table 4.

Figure 3.

Figure 4.

Figure 5.

CONCLUSION

EXPERIMENTAL SECTION

General Material and Synthetic Procedures.

General Procedure A.

General Procedure B.

General Procedure C.

Characterizations. 6-((2-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12a).

6-((3-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12b).

6-((4-Fluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12c).

6-((2-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12d).

6-((3-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12e).

6-((4-(Trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12f).

6-((2-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12g).

6-((3-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12h).

6-((4-(Trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (SHS4121705, 12i).

6-((2-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12j).

6-((4-Chlorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12k).

6-((4-(Difluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12l).

6-((4-(1,1,2,2-Tetrafluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12m).

6-((4-(Chlorodifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12n).

6-((2,2-Difluorobenzo[d][1,3]dioxol-5-yl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12o).

6-((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).

6-((2,3-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12q).

6-((2,6-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12r).

6-((3,5-Difluorophenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12s).

6-((2-Fluoro-5-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12t).

6-((2-Fluoro-4-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12u).

6-((3-Fluoro-4-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12v).

6-((2-Fluoro-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12w).

6-((3,5-Bis(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12x).

6-((2-Fluoro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12y).

6-((3-Fluoro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12z).

6-((3-Chloro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aa).

6-((2-Chloro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ab).

6-((2-Methoxy-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ac).

6-((3-Nitro-4-(trifluoromethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ad).

6-((2-Fluorophenyl)(methyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ae).

6-((3-Methoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12af).

6-((3-Butoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ag).

6-((4-Butoxyphenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ah).

6-((4-(2,2,2-Trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ai).

6-((3-Methyl-4-(2,2,2-trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aj).

6-((3-Fluoro-4-(2,2,2-trifluoroethoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ak).

6-((4-(2,2,2-Trifluoroethoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12al).

6-((3-Fluoro-4-(4,4,4-trifluorobutoxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12am).

6-((4-(4,4,4-Trifluorobutoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12an).

6-((4-(Cyclopropylmethoxy)-3-(trifluoromethyl)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol) (12ao).

6-(Benzo[d][1,3]dioxol-5-ylamino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12ap).

6-((4-(Benzyloxy)phenyl)amino)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol (12aq).

Scheme 1^a.

Scheme 2^a.