High-Potency Phenylquinoxalinone Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activators

Article abstract of DOI:10.1021/acs.jmedchem.6b01759

We previously identified phenylquinoxalinone CFTR_act-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC₅₀ of ～200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC₅₀ down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

Full text of DOI:10.1021/acs.jmedchem.6b01759

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Article
High-potency phenylquinoxalinone cystic fibrosis
transmembrane conductance regulator (CFTR) activators
Jung-Ho Son, Jie S. Zhu, Puay-Wah Phuan, Onur Cil, Andrew P.
Teuthorn, Colton K. Ku, Sujin Lee, Alan S Verkman, and Mark J. Kurth
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01759 • Publication Date (Web): 23 Feb 2017
Downloaded from http://pubs.acs.org on February 24, 2017
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High-potency phenylquinoxalinone cystic fibrosis transmembrane con-
ductance regulator (CFTR) activators
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Jung-Ho Son†, Jie S. Zhu†, Puay-Wah Phuan‡, Onur Cil‡, Andrew P. Teuthorn†,
Colton K. Ku†, Sujin Lee‡, Alan S. Verkman‡, Mark J. Kurth†*
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†Department of Chemistry, University of California, Davis, CA, 95616, USA
‡Departments of Medicine & Physiology, University of California San Francisco, CA 94143,
USA
ABSTRACT: We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic
fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ~200 nM, and
demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity
studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with
improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core
was generally accomplished by condensation of 1,2-phenylenediamines with substituted phe-
nyloxoacetates. Structure-activity studies established, among other features, the privileged
nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed
improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater
metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry
eye, cholestatic liver diseases, and inflammatory lung disorders.
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INTRODUCTION
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chlo-
ride channel expressed in mammalian epithelia in the respiratory, gastrointestinal, and repro-
ductive systems, as well as in exocrine glands and other tissues.1 Loss-of-function mutations in
CFTR cause cystic fibrosis, and CFTR over activation causes certain secretory diarrheas in-
cluding cholera and Travelers’ diarrhea.2 CFTR is considered an important drug target, with ac-
tivators of CFTR of potential benefit for constipation,3,4 dry eye,5 inflammatory lung disorders,6
and cholestatic liver disease; inhibitors of wildtype CFTR may be useful for treatment of certain
secretory diarrheas and polycystic kidney disease;7,8 and correctors and potentiators of mutant
CFTRs for treatment of cystic fibrosis.9
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We previously identified by high-throughput screening the phenylquinoxalinone CFTRact-
J027 (4; Figure 1) as a CFTR activator and demonstrated its efficacy in normalizing stool output,
hydration, and intestinal transit in a mouse model of opioid-induced constipation.3 Phenylquinox-
alinone 4 activated CFTR chloride conductance with an EC50 of ~200 nM and showed no ap-
parent off-target actions or toxicity following chronic administration in mice. In a follow-up study,4
4 was shown by patch-clamp and biochemical studies to target CFTR directly, and was demon-
strated to activate CFTR in human enterocytes and normalize stool parameters in mouse mod-
els of acute and chronic constipation. Side-by-side comparisons of intestinal fluid secretion and
H2N
N
NO2
N
O
CFTRact-J027 (4)
EC50 = 200 nM
Figure 1. A phenylquinoxalinone CFTR activator identified by high-throughput screening.
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stool output in constipation models showed greater efficacy of 4 than supramaximal doses of
the FDA-approved drugs lubiprostone and linaclotide.
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Here, motivated by the potential therapeutic utility of phenylquinoxalinone-based CFTR ac-
tivators in constipation and other diseases, we synthesized 36 analogs of phenylquinoxalinone
4 in order to establish structure-activity relationships and to identify compounds with greater
potency. Also, while the rapid hepatic metabolism of 4 results in minimal systemic exposure
following oral administration in mice, which is desirable for treatment of constipation, we also
sought phenylquinoxalinone CFTR activators with greater metabolic stability for treatment of
lung and liver disorders where systemic exposure is necessary.
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RESULTLS AND DISCUSSION
Chemistry. General synthesis of phenylquinoxalinones. Most of the phenylquinoxali-
none in this study were expediently synthesized in four steps starting from acetophenones
(Scheme 1). We generated the phenylquinoxalinone core by condensing o-phenylenediamines
with substituted phenyloxoacetates (6), which were synthesized following literature methods.10
Scheme 1. Synthesis of phenylquinoxalinones 1-3.
R1
R1
R1
a
b
O
O
O
Br
Br
5
MeO
O
6
NH2
R2
c
NH2
N
R1
R1
N
d
R2
R2
N
R3
1-3
O
N
H
O
7
(a) Br2, 1,4-dioxane; (b) DMSO, D; MeOH; (c) toluene, D; (d) R-Br, K2CO3, DMF.
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Briefly, substituted acetophenone was doubly brominated with bromine in 1,4-dioxane to give 5,
then heated in DMSO followed by addition of methanol to give 6. Phenylquinoxalinone 7 was
N1-alkylated using K2CO3 and R-X in DMF11 and pure products (1-3) were obtained via column
chromatography.
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Phenylquinoxalinone 1a was prepared as outlined in Scheme 2. Treatment of methyl 3-
fluoro-4-nitrobenzoate (8) with methyl 2-cyanoacetate under basic conditions delivered interme-
diate 9.12 Subsequent copper(I) iodide-catalyzed aerobic oxidation12 delivered methyl 2-oxo-2-
phenylacetate 10 and, from here, target 1a was prepared in parallel to the chemistry employed
in Scheme 1. With 1a in hand, saponification and nitro reduction were accomplished as outlined
in Scheme 3 to deliver analogs 1b, 1j, and 1k.
Scheme 2. Synthesis of phenylquinoxalinone 1a.
O2N
F
O2N
NC
O2N
O
a
b
NC
CO2Me
CO2Me
CO2Me
CO2Me
9
CO2Me
MeO
O
8
10
NH2
R2
c
NH2
O2N
N
O2N
N
CO2Me
R2
d
CO2Me
R2
N
O
N
H
O
1a (R2 = H)
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(a) Cs2CO3, DMSO; (b) CuI, 1,10-phen, ACN, O2; (c) toluene, D; (d) K2CO3, DMF, BnBr.
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Scheme 3. Diversification from phenylquinoxalinone 1a.
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O2N
H2N
N
N
COOR
COOR
a
N
O
N
O
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1a (R = Me)
1b (R = H)
1j (R = Me)
1k (R= H)
b
c
(a) H2, Pd/C, MeOH; (b) NaOH, EtOH; (c) KOH, MeOH.
Constrained ring phenylquinoxalinone analogs 16 and 20 were prepared as outlined in
Scheme 4. 2-Amino-3-nitrophenol was N- and O-alkylated with 1,2-dibromoethane to deliver
intermediate 14 and subsequent nitro reduction and condensation of the resulting diamine with
1-acetyl-5-nitroindoline-2,3-dione led smoothly to analog 16.13 Employing 2-bromo-1-phenyle-
than-1-one in place of 1,2-dibromoethane and 5-nitroindoline-2,3-dione in place of 1-acetyl-5-
nitroindoline-2,3-dione delivered analog 20.14 Interestingly, the reaction of N-benzyl-1,2-dia-
minobenzene with 5-fluoroisation (in analogy with the protocol employed to prepare compounds
16, 20 and 4) led to 22 and the attempted de-acylation of 21 (X = NO2) led to 23.
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Scheme 4. Synthesis of constrained ring phenylquinoxalinone analogs.
3
NO2
4
5
6
7
8
9
NH2
NH
12
NO2
NH
NH2
Br
13
a
b
OH
Br
O
O
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Ac
N
O
c
NO2
NO2
O
12
NO2
NH
NH2
Br
H
N
OH
Ac
N
d
O
NO2
O
N
O
O
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17
16
b
H2N
N
H
N
O
NO2
NH2
NH
NO2
O
N
O
O
c
O
19
20
H
F
N
O
F3C
NH2
NH
F3C
N
N
F
O
N
c
22
H
N
NO2
Ac
F3C
N
F3C
N
N
NO2
e
N
N
O
21
23
(a) KOH, DMF; (b) H2, Pd/C, MeOH; (c) HOAc, toluene; (d) K2CO3, ACN (e) HCl, MeOH.
Modifications of the 3-aryl ring. Compound 4 contains a 2-amino-5-nitro phenyl ring at the
3-position of the quinoxalinone core (Figure 1) and our first effort was to modify this ring. Several
compounds (Table 1) were rationally synthesized and their activities were determined using a
plate reader assay. The most active compound was CFTRact-J125 (1c), which only lacks the 2-
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amino group at C2 of the 3-aryl ring compared to 4, and it showed an ~10-fold increased potency
when compared to 4. We then synthesized a series of analogs retaining this amino group dele-
tion. In contrast to the high activity of 1c, compounds without the 3-nitro group had significantly
lower activity (1h), indicating the privileged nature of the 3-nitro group in 1c.
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Table 1. CFTR activation with variation in the 3-aryl ring (EC50 reported in µM ± S.E.M.).
R1
N
3
N
O
O2N
O2N
NO2
CO2Me
CO2H
1a
EC50 = 9.9
1b
EC50 = >10
1c (J125)
EC50 = 0.009
±
2
±
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CF3
O2N
NO2
1d
1e
1f
EC50 = 1
±
0.2
EC50 = 1
±
±
0.1
EC50 = 1
±
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F
Br
NO2
1g
1h
EC50 = 5
1i
EC50 = 0.33
±
0.07
0.4
EC50 = 0.69 ± 0.22
Nitro bioisosteres
H2N
H2N
CO2Me
0.1
CO2H
1j
1k
EC50 = >10
EC50 = 0.55
±
N
O
N
CN
0.4
1l
1m
EC50 = 1
±
EC50 = 0.018 ± 0.006
Bioisosteric compounds containing –COOR replacements of the –NO2 moiety in the 3-aryl
group were synthesized according to Schemes 2 and 3. It was found that carboxylic acid analog
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1k was inactive, while methyl ester analog 1j was moderately potent. Combining a 2-NO2 with
either a 5-COOMe or a 5-COOH (1a and 1b, respectively) resulted in very low activity. Positional
deviation of the nitro group also resulted in reduced activity, as with 2-nitro or 4-nitro analogs 1d
and 1e, respectively. Introduction of other functional groups, such as 4-CF3 or 3-Br, in place of
the 3-NO2 of 1c also showed low activity (1f and 1g, respectively). Introducing a fluorine atom
into 1c (i.e., 1i) reduced activity. Replacements of the nitro group with a bioisosteric nitrile (1l)
greatly reduced activity, but interestingly, the bioisosteric benzoxadiazole (1m) showed compa-
rable potency to the nitro analog (1c).
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Modifications of the quinoxalinone core. Moving forward with 1c as the lead, quinoxali-
none backbone modifications were undertaken (Table 2). Halogen substitution at the 6-position,
such as 6-Cl (2b) or 6-Br (2f), showed good activity albeit less than that of 1c. Substitution at
the 5-, 7-, or 8-positions generally resulted in lower activity. Disubstitution at the 6- and 7-posi-
tions (dichloro; 2d / difluoro; 2g / dimethyl; 2i) reduced activity.
Table 2. CFTR activation with variation in the quinoxalinone core (EC50 reported in µM ± S.E.M.).
N
NO2
R2
N
O
Cl
Cl
Cl
2a
2b
2c
EC50 = 0.76 ± 0.38
EC50 = 0.07 ± 0.03
EC50 = 0.11 ± 0.04
Cl
Cl
Br
Br
2d
2e
2f
EC50 = >10
EC50 = Inactive
EC50 = 0.063 ± 0.03
F
F
2g
2h
2i
EC50 = >10
EC50 = >10
EC50 = >10
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Modifications of the N1-substituent. We next modified the N1-benzyl group (Table 3). We
reported previously that placing substituents on the benzyl ring was not tolerated.4 However,
heterocycle analogs such as thiophenyl (3j), furanyl (3i), and pyridyl (3h) showed comparable
activity. Among short chains, allyl and ethyl groups showed moderate activity, but methyl and
propyl groups had poor activity (3a-e). Replacing the phenyl with a larger aromatic group, such
as naphthyl, significantly reduced activity (3f and 3g).
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Table 3. CFTR activation with variation in the N1-substituent (EC50 reported in µM ± S.E.M.).
N
NO2
N
O
R3
H
CH3
3a
3b
3c
3d
EC50 = >10
EC50 = >10
EC50 = 0.065 ± 0.02 EC50 = 0.177 ± 0.048
3e
3f
3g
EC50 = Inactive
EC50 = 0.055 ± 0.01
EC50 = 4.3 ± 0.6
N
O
S
3h
3i
3j (J170)
EC50 = 0.052 ± 0.02
EC50 = 0.02 ± 0.01
EC50 = 0.01 ± 0.005
Compounds with constrained rings. Motivated by our prior findings that constrained rings
can enhance the activity of a CFTR corrector,15 we next examined two different types of con-
strained analogs (Scheme 4) with hindered rotation of the N1-alkyl group (16 and 20) or phenyl
ring (22 and 23). As previously shown, the N-acetylated version of 4 has very low activity;4 we
therefore attempted to synthesize 16 without an N-acyl group, but both deacylation of 16 and
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condensation with 5-nitroisatin failed. The activities of neither 20 (EC50 = >10 µM) nor 16 (EC50
= 0.82 µM) were greater than that of 4 (see Table 4). Phenyl-ring constrained compounds 22
and 23 were unexpected by-products of the deacylation reaction (see Scheme 4 and Table 4).
These compounds are purple and red, respectively – a consequence of their extended aromatic
systems – and those colors are different from the bright yellow color of most derivatives of 4.
Intramolecular heterocycle formation in this system might have been facilitated by the presence
of the electron withdrawing CF3 group in the quinoxalinone backbone under these acidic deac-
ylation conditions. With other substituents in the quinoxalinone backbone, only small amounts
of uncharacterized reddish byproduct formed during the deacylation step, suggesting a minimal
amount of by-product formation.
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Table 4. CFTR activation with constrained ring analogs (EC50 reported in µM ± S.E.M.).
H2N
H
N
N
N
NO2
Ac
N
O
NO2
O
N
O
O
20 EC50 = >10
16 EC50 = 0.82 ± 0.25
X
F3C
N
N
N
X = F; 22 EC50 = >10
X = NO2; 23 EC50 = >10
Biology. In vitro characterization of phenylquinoxalinones. Phenylquinoxalinone CFTR
activators with the highest potency as determined by plate-reader assay, 1c and CFTRact-J170
(3j; Table 3), were further characterized. Short-circuit current measurements were done using
CFTR-expressing FRT cells in the presence of a transepithelial chloride gradient and with per-
meabilization of the cell basolateral membrane; consequently, current is a direct, linear measure
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of CFTR chloride conductance. Compounds 1c and 3j were added to the apical side of the
monolayer. Representative data in Figure 2 for 1c and 3j shows a small increase in current
following addition of a low concentration of forskolin, followed by concentration-dependent in-
creases in current following activator additions. EC50 values were determined to be 21 ± 4 and
70 ± 11 nM for 1c and 3j, respectively. The potency for CFTR inhibition by 1c was similar in non-
permeabilized and permeabilized CFTR-expressing FRT cells (data not shown).
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Figure 2. Short-circuit current measurement of CFTR activation by 1c and 3j. A. Measurements
done in FRT cells expressing human wild-type CFTR showing responses to indicated concen-
trations of forskolin, 1c or 3j, and 10 µM CFTR inhibitor CFTRinh-172. B. Concentration-depend-
ent activation of CFTR (mean ± S.E.M., n=3).
The CFTR specificity of the most potent compound, 1c, was further studied. At 10 µM, 1c
did not affect the cellular cAMP level (Figure 3A), nor did it elevate cytoplasmic calcium or inhibit
the ATP-stimulated elevation in cytoplasmic calcium (Figure 3B). In addition, 1c at 10 µM neither
inhibited nor activated calcium-activated chloride channels in HT-29 cells (Figure 3C) or in FRT
cells expressing TMEM16A (Figure 3D).
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Figure 3. In vitro characterization of 1c. A. Cellular cAMP in FRT cells in response to incubation
for 10 min with 10 µM 1c without or with 90 nM forskolin (fsk). Positive controls included fsk (100
nM and 20 µM) and fsk + IBMX (20 µM + 100 µM) (mean ± S.E.M., n = 4). B. Cytoplasmic
calcium measured by Fluo-4 fluorescence. FRT cells were pretreated for 5 min with 10 µM 1c
(or control), with 100 µM ATP added as a calcium agonist as indicated. C. CaCC activity meas-
ured in HT-29 cells expressing YFP showing no activation (iodide addition) or inhibition (iodide
+ ATP addition) by 10 µM 1c. D. TMEM16A activity measured in FRT cells expressing YFP
showing no activation (iodide addition) or inhibition (iodide + ATP addition) by 10 µM 1c.
Efficacy of 1c in a loperamide-induced mouse model of acute constipation. We previ-
,
ously demonstrated the efficacy of 4 in a loperamide-induced mouse model of constipation.3 4
Here, the efficacy of 1c was tested. Phenylquinoxalinone 1c was administered orally to mice 1
h prior to loperamide and 3-h stool samples were collected after loperamide. Figure 4 shows
that orally administered 1c fully normalized stool weight, pellet number and hydration with half-
maximal effective dose (ED50) < 1 mg/kg.
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Figure 4. Efficacy of 1c in a mouse model of acute constipation. A. Experimental protocol. Mice
were treated with 1c (orally) or vehicle 1 hour before loperamide (0.3 mg/kg, intraperitoneal).
Weight, pellet number and water content were determined for stool collected at 3 h. B-D. Stool
weight, pellet number and water content after treatment with 0, 0.1, 0.3, 1, 3, 10 mg/kg of 1c
(mean ± S.E.M., 4 mice per group). The control was without treatment with loperamide and 1c.
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Phenylquinoxalinone 4 was shown previously to have minimal oral bioavailability and rapid
,
metabolism.3 4 Though these properties are favorable for ‘topical’ applications in the treatment
of constipation and dry eye in which systemic exposure is not needed, they are not favorable for
treatment of liver and lung diseases where systemic exposure and organ accumulation are de-
sired. Figure 5 shows substantially slower in vitro hepatic microsomal metabolism of 1c com-
pared with 4, with nearly 80% of 4 metabolized at 60 min compared with <40% metabolism of
1c.
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Figure 5. In vitro metabolic stability of 1c. Remaining 1c following incubation with human hepatic
microsomes in the presence of NADPH after incubation for specified times (0, 15, 60 min), com-
paring with reference compound 4 (mean ± S.E.M., n=3).
Indeed, a number of important structure-activity relationships have been revealed in the pre-
sent study. Previously, the amino group on the 3-aryl ring of phenylquinaxolinones – present in
all of the 175 analogs reported in our preliminary study – was assumed to be a central structural
feature (see 4 with an EC50 = 200 nM).4 Through the series of compounds synthesized and
assayed in this work, we determined that deletion of this amino group generally improves the
potency of this class of CFTR activators (compare 1c – the amino-deleted analog of 4 – with an
EC50 = 21 nM). Additional amino-deleted analogs further established that an unsubstituted
quinoxalinone core affords the best potency (compare 1c with the quinoxalinone core analogs
depicted in Table 2). Indeed, there appears to be a delicate balance between electronic and
steric effects, especially considering that the highest performing analog with a substituted core
(2f) underperforms by nearly an order of magnitude compared to unsubstituted 1c. Quinoxali-
none N-substituent effects were also thoroughly explored in this work (see Table 3) and, in gen-
eral, N-benzyl or N-heteroaromatic groups provide optimal potency. Finally, structure-activity re-
sults reported here establish that in vitro hepatic microsomal metabolism is also dramatically
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variable – see Figure 5 comparing the 60 min metabolic results for 1c (<40% metabolized) with
that for 4 (nearly 80% metabolized).
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Small molecule CFTR activators have potential utility in various diseases involving epithelial
fluid secretion. Compounds with minimal systemic absorption are of interest for treatment of dry
eye disorders5 with topical (eye drop) compound administration, and for constipation3 with oral
administration, as CFTR is expressed in surface-exposed epithelial cells in these tissues. How-
ever, compounds that are absorbed and accumulate in relevant tissues are needed to treat dis-
orders of cholestasis16 (by increasing bile flow) and inflammatory lung diseases (by increasing
airway surface liquid volume).17
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CONCLUSIONS
In conclusion, synthesis of 36 phenylquinoxalinones established structure-activity relation-
ships and identified compounds with ~10-fold improved potency and greater metabolic stability
than reference compound 4. The most potent analog, 1c, showed CFTR selectivity and efficacy
in a mouse model of acute opioid-induced constipation. CFTR activation by phenylquinoxali-
nones may have utility in constipation and dry eye, as supported by prior experimental animal
data,3,4 as well as in inflammatory lung disorders and hepatic cholestasis.
EXPERIMENTAL SECTION
General Experimental: All compounds described in this manuscript have ≥95% purity. The
1
analytical method used to determine purity was H NMR (see the accompanying Supporting
Information file which provides the 1H- and 13C-NMR for the thirty-six compounds assayed) and
HPLC/HRMS. For HRMS analysis, samples were analyzed by flow-injection analysis into a
Thermo Fisher Scientific LTQ Orbitrap (San Jose, CA) operated in the centroided mode. Sam-
ples were injected into a mixture of 50% MeOH/H2O and 0.1% formic acid at a flow of 0.2
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mL/min. Source parameters were 5.5kV spray voltage, capillary temperature of 275 ºC and
sheath gas setting of 20. Spectral data were acquired at a resolution setting of 100,000 FWHM
with the lockmass feature, which typically results in a mass accuracy <2ppm.
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CFTR functional assays. Fischer Rat Thyroid (FRT) cells stably co-expressing human
wildtype CFTR and the halide-sensitive yellow fluorescent protein (YFP)-H148Q were cultured
as described.18 Fluorescence platereader assays of CFTR function were done as described,18
in which 96-well plates containing near-confluent cell cultures were washed with phosphate-
buffered saline (PBS) and incubated for 10 min with PBS containing test compound and 125 nM
forskolin. Assays of iodide influx into cells were done in single wells by continuous measurement
of YFP fluorescence just for 2 s before (for baseline) and 12 s after addition of an iodide con-
taining solution (final 140 mM iodide). TMEM16A activity assay was done similarly, as de-
scribed,19 using FRT cells co-expressing YFP and TMEM16A. Activity of non-TMEM16A CaCC
activity was assayed as described20 in HT-29 cells expressing YFP. In each assay, iodide influx
rate and concentration-dependent curves were computed as described.18-20 For short-circuit cur-
rent measurement cells were cultured on porous filters and current was measured in the pres-
ence of a transepithelial chloride gradient and following permeabilization of the basolateral mem-
brane, as described.21 Cyclic AMP and cytoplasmic calcium measurement were done as de-
scribed.19,22
Loperamide model of acute constipation in mice. All mouse experiments were approved
by the UCSF Institutional Animal Care and Use Committee (approval number: AN108711-02A)
and were conducted in accordance with the NIH guidelines for the care and use of animals. As
described,3 CD1 mice (age 8-10 weeks) were administered 0.3 mg/kg loperamide intraperitone-
ally (ip) and placed in metabolic cages with free access to food and water. Stool samples were
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collected for 3 h for determination of total stool weight, number of fecal pellets, and stool water
content (by wet and dry weight measurements). Compound 1c (or vehicle control) was admin-
istered orally 1 h prior to loperamide. The vehicle consisted of saline containing 5% DMSO and
10% Kolliphor HS 15.
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In vitro metabolic stability. Test compound (at 5 µM) was incubated for specified times at
37 °C with mouse liver microsomes (1 mg protein/ml; Sigma-Aldrich, St. Louis, MO) in potassium
phosphate buffer containing 1 mM NADPH, as described.3 Following ethyl acetate extraction,
non-metabolized parent compound was assayed by LC/MS.
Statistical analysis. Data are presented as meanꢀ±ꢀS.E.M. Comparisons between two
groups were performed using the unpaired Student's t-test. Pꢀ<ꢀ0.05 was considered as statisti-
cally significant.
General procedure for synthesis of dibromophenylpropanedione derivatives (5). A
flask was charged with 1,4-dioxane (15 mL) was bubbled with N2 for 10 min with stirring. Br2 (2
mL, 39 mmol) was added and the solution was stirred for 30 min with slow N2 bubbling. Substi-
tuted acetophenone (12 mmol) was dissolved in 1,4-dioxane (20 mL) and added. The mixed
solution was stirred for 3 h, poured in water, and extracted with ethyl acetate. The organic layer
was washed with water (3x) and brine, then dried over magnesium sulfate. Solvent was removed
in vacuo to yield reddish oil of 5, which was used for next step without further purification.
General procedure for synthesis of oxophenylacetate derivatives (6). Anhydrous DMSO
(15 mL) was added to the oily product of 5, and heated at 75 °C overnight. The solution was
cooled to RT, and methanol (10 mL) was added and stirred overnight. The solution was poured
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in water, extracted with ethyl acetate. The organic layer was washed with water (3x) and brine,
and dried over magnesium sulfate. The brown oily product was used in the next step without
purification.
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General procedure for synthesis of N-H phenylquinoxalinone derivatives (7). Substi-
tuted phenyloxoacetate (6, 1 mmol) was mixed with o-phenylenediamine (1 mmol) in toluene
(20 mL), and heated at 70 °C overnight. The precipitate that formed was collected by filtration,
triturated with toluene and hexane, and used in the next step without purification.
General procedure for N-alkylation of phenyquinaxolinone (1-3). Compound 7 (0.5
mmol) was dissolved in DMF (20 mL), benzyl bromide (0.6 mmol) and K2CO3 (1 mmol) were
added, and the mixture was stirred overnight. The solution was diluted with water, and extracted
with ethyl acetate. The organic layer was washed with water three times. The organic layer was
washed with brine and dried over magnesium sulfate. The final product obtained after solvent
evaporation was purified by flash column chromatography.
Methyl 3-(4-benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)-4-nitrobenzoate (1a). Methyl 3-
fluoro-4-nitrobenzoate (8, 1.0 g, 5 mmol) was mixed with Cs2CO3 (3.3 g, 10 mmol) in DMSO (20
mL). Methyl cyanoacetate (1.0 g, 10 mmol) was added and the solution was heated at 130 °C
for 4 h and then maintained at 90 °C overnight. Upon cooling, the reaction mixture was extracted
with ethyl acetate and the organic layer was washed with 1N HCl, water (3x), and brine. After
drying over magnesium sulfate and filtration, the solvent was removed in vacuo to yield 9 as a
purple oil, which was used without purification in the next step.
Crude intermediate 9 (1.8 g, 6.5 mmol) was dissolved in acetonitrile (20 mL), and CuI (1 g,
5.3 mmol) and 1,10-phenanthroline (0.23 g, 1.3 mmol) were added. The mixture was reacted at
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50 °C overnight with an O2 balloon overnight. After cooling, the solution was filtered through
Celite and concentrated in vacuo. The product was purified by flash column chromatography to
yield colorless 10 after solvent evaporation. Yield = 0.66 g (38 %).
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9
Intermediate 10 (163 mg, 0.61 mmol) was mixed with o-phenylenediamine (78 mg, 0.72
mmol) in toluene (30 mL) and heated at 70 °C overnight. The resulting tan precipitate of 11 was
collected by filtration, triturated with toluene and hexane, and air dried; it was used in the next
step without purification. Yield = 185 mg (93 %).
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Intermediate 11 (185 mg, 0.57 mmol) was mixed with benzyl bromide (150 mg, 0.88 mmol)
and K2CO3 (170 mg, 1.2 mmol) in DMF (10 mL) and stirred overnight at RT. After dilution with
water, the solution was extracted with ethyl acetate, washed with water (3x) and brine, and dried
over MgSO4. Solvent removal and purification by column chromatography gave 1a. Yield = 149
mg (63 %). 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 1.8 Hz, 1H), 8.22 (dd, J = 8.5, 1.9 Hz, 1H),
8.13 (d, J = 8.4 Hz, 1H), 7.89 (dd, J = 8.0, 1.6 Hz, 1H), 7.50 – 7.37 (m, 1H), 7.36 – 7.06 (m, 7H),
5.44 (s, 2H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.83, 154.65, 154.19, 151.57, 134.82,
134.73, 133.30, 133.17, 132.98, 131.80, 131.61, 131.28, 130.79, 129.05, 127.83, 126.89,
124.22, 124.20, 114.82, 52.91, 46.25. HRMS [C23H17N3O5+H]+ : calcd 416.1247 / found
416.1253.
3-(4-Benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)-4-nitrobenzoic acid (1b). Compound 1a
(10 mg, 0.024 mmol) was dissolved in hot ethanol (30 mL). Sodium hydroxide (0.1 g, 2.5 mmol)
dissolved in water (5 mL) was added and the mixture stirred for 1 h. The cooled solution was
acidified with 1 N HCl and extracted with ethyl acetate. The organic layer was washed with water
and brine, then dried over MgSO4. Solvent was removed in vacuo, and product was purified by
1
flash column chromatography. Yield = 9 mg (93 %). H NMR (400 MHz, DMSO-d6) δ 8.39 (s,
1H), 8.30 (d, J = 1.4 Hz, 2H), 7.98 (dd, J = 8.0, 1.4 Hz, 1H), 7.62 (ddd, J = 8.6, 7.2, 1.5 Hz, 1H),
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7.56 – 7.39 (m, 2H), 7.39 – 7.09 (m, 5H), 5.53 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.89,
154.75, 154.16, 151.48, 135.90, 133.43, 133.14, 132.95, 132.23, 131.82, 131.49, 130.53,
129.19, 127.93, 127.19, 124.88, 124.65, 115.88, 45.52. HRMS [C22H15N3O5+H]+ : calcd
402.1090 / found 402.1085.
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1-Benzyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (1c). Yield = 150 mg (70 %). H NMR
(400 MHz, CDCl3) δ 9.37 (t, J = 2.0 Hz, 1H), 8.88 (ddd, J = 7.9, 1.7, 1.1 Hz, 1H), 8.37 (ddd, J =
8.3, 2.3, 1.1 Hz, 1H), 8.03 (dd, J = 8.0, 1.5 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.55 (ddd, J = 8.6,
7.3, 1.6 Hz, 1H), 7.47 – 7.30 (m, 7H), 5.63 (s, 2H). 13C NMR (151 MHz, CDCl3) δ 154.51, 151.07,
148.25, 137.44, 135.48, 135.02, 133.08, 132.97, 131.27, 130.92, 129.00, 128.93, 127.82,
126.92, 124.70, 124.13, 114.50, 109.99, 46.23. HRMS [C21H15N3O3+H]+ : calcd 358.1192 / found
358.1188.
1
1-Benzyl-3-(2-nitrophenyl)quinoxalin-2(1H)-one (1d). Yield = 108 mg (69 %). H NMR
(400 MHz, CDCl3) δ 8.18 (dd, J = 8.1, 1.1 Hz, 1H), 7.97 (dd, J = 8.0, 1.5 Hz, 1H), 7.87 – 7.73
(m, 2H), 7.65 (ddd, J = 8.7, 7.0, 2.0 Hz, 1H), 7.48 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.42 – 7.24 (m,
7H), 5.52 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 155.76, 154.35, 149.16, 135.01, 133.82, 133.30,
132.94, 131.72, 131.66, 131.00, 130.58, 129.04, 127.78, 126.96, 126.92, 124.09, 124.08,
114.83, 46.19. HRMS [C21H15N3O3+H]+ : calcd 358.1192 / found 358.1187.
1-Benzyl-3-(4-nitrophenyl)quinoxalin-2(1H)-one (1e). Yield = 94 mg (49 %). 1H NMR (400
MHz, CDCl3) δ 8.76 – 8.56 (m, 2H), 8.42 – 8.23 (m, 2H), 8.01 (dd, J = 8.0, 1.5 Hz, 1H), 7.56
(ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.47 – 7.29 (m, 7H), 5.62 (s, 2H). 13C NMR (101 MHz, CDCl3) δ
154.55, 151.50, 148.67, 141.76, 134.97, 133.20, 133.02, 131.58, 131.08, 130.67, 129.05,
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127.90, 126.93, 124.25, 123.14, 114.58, 46.29. HRMS [C21H15N3O3+H]+ : calcd 358.1192 / found
358.1187.
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1-Benzyl-3-(4-(trifluoromethyl)phenyl)quinoxalin-2(1H)-one (1f). Yield = 120 mg (83 %).
1H NMR (600 MHz, CDCl3) δ 8.53 (d, J = 8.2 Hz, 2H), 7.98 (dd, J = 8.0, 1.6 Hz, 1H), 7.75 (d, J
= 8.2 Hz, 2H), 7.50 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), 7.42 – 7.26 (m, 7H), 5.59 (s, 2H). 13C NMR
(151 MHz, CDCl3) δ 154.61, 152.55, 139.23, 135.16, 133.25, 132.96, 131.91 (q, JC-F= 33 Hz),
130.90, 130.85, 129.97, 128.93, 127.74, 126.92, 124.88 (br), 124.23 (q, JC-F=325 Hz), 123.92,
114.40, 46.18. HRMS [C22H15F3N2O+H]+ : calcd 381.1215 / found 381.1206.
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1-Benzyl-3-(3-bromophenyl)quinoxalin-2(1H)-one (1g). Yield = 295 mg (75 %). 1H NMR
(400 MHz, CDCl3) δ 8.62 (t, J = 1.8 Hz, 1H), 8.42 (dt, J = 7.9, 1.3 Hz, 1H), 7.99 (dd, J = 8.0, 1.5
Hz, 1H), 7.65 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 7.50 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.44 – 7.29
13
(m, 8H), 5.60 (s, 2H). C NMR (101 MHz, CDCl3) δ 154.58, 152.33, 137.85, 135.18, 133.32,
133.20, 132.83, 132.49, 130.82, 130.76, 129.60, 129.00, 128.35, 127.78, 126.93, 123.99,
122.29, 114.42, 46.20. HRMS [C21H15BrN2O+H]+ : calcd 391.0446 / found 391.0442.
1
1-Benzyl-3-phenylquinoxalin-2(1H)-one (1h). Yield = 62 mg (44 %). H NMR (400 MHz,
CDCl3) δ 8.40 (ddd, J = 6.3, 2.9, 1.5 Hz, 2H), 7.99 (dd, J = 8.0, 1.5 Hz, 1H), 7.53 (tt, J = 3.9, 2.4
Hz, 3H), 7.48 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.40 – 7.18 (m, 7H), 5.61 (s, 2H). 13C NMR (101
MHz, CDCl3) δ 154.80, 154.23, 136.01, 135.37, 133.38, 132.76, 130.60, 130.45, 130.32, 129.66,
128.95, 128.13, 127.71, 126.99, 123.81, 114.36, 46.15. HRMS [C21H16N2O+H]+ : calcd 313.1341
/ found 313.1341.
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1-Benzyl-3-(4-fluoro-3-nitrophenyl)quinoxalin-2(1H)-one (1i). Yield = 127 mg (71 %). 1H
NMR (600 MHz, CDCl3) δ 9.31 (dd, J = 7.5, 2.3 Hz, 1H), 8.87 (ddd, J = 8.8, 4.3, 2.3 Hz, 1H),
7.98 (dd, J = 8.0, 1.5 Hz, 1H), 7.52 (ddd, J = 8.6, 7.2, 1.5 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.36 –
7.31 (m, 3H), 7.28 (dt, J = 9.7, 3.1 Hz, 3H), 5.59 (s, 2H). 13C NMR (151 MHz, CDCl3) δ 158.19,
154.54 (d, JC-F=12 Hz), 149.65, 136.75, 136.63, 134.89, 132.92 (br), 132.79 (br), 131.41,
130.86, 129.03, 127.88, 127.67 (br), 126.87, 124.28, 118.06, 117.79, 114.56. 46.25. HRMS
[C21H14FN3O3+H]+ : calcd 376.1098 / found 376.1092.
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Methyl 4-amino-3-(4-benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)benzoate (1j). Compound
1a (120 mg, 0.29 mmol) was dissolved in hot ethanol (50 mL). After cooling, a saturated NH4Cl
solution (30 mL) and Zn dust (1 g) were added and the mixture stirred for 3 h. The solution was
filtered through Celite, concentrated, and purified by flash column chromatography. Yield = 100
1
mg (90 %). H NMR (600 MHz, CDCl3) δ 9.26 – 8.97 (m, 1H), 7.91 (ddd, J = 8.6, 2.1, 0.9 Hz,
1H), 7.83 (dt, J = 8.0, 1.2 Hz, 1H), 7.45 (ddt, J = 8.5, 7.2, 1.2 Hz, 1H), 7.38 – 7.11 (m, 7H), 6.77
(dd, J = 8.6, 0.8 Hz, 1H), 6.18 (s, 2H), 5.59 (s, 2H), 3.87 (d, J = 0.9 Hz, 3H). 13C NMR (151 MHz,
CDCl3) δ 154.68, 154.23, 151.69, 135.24, 134.67, 132.68, 132.44, 132.17, 130.20, 129.51,
128.90, 127.67, 126.94, 123.79, 118.49, 117.26, 116.47, 114.44, 51.61, 46.44. HRMS
[C23H19N3O3+H]+ : calcd 386.1505 / found 386.1509.
4-Amino-3-(4-benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)benzoic acid (1k). Compound 1j
(20 mg, 0.052 mmol) was dissolved in methanol (100 mL) at 85 °C. KOH (0.2 g, 3.6 mmol) and
water (30 mL) were added and the solution refluxed overnight. Solvent was removed in vacuo,
and acidified with 1 N HCl. The product was extracted with dichloromethane. Yield = 13 mg (67
%). 1H NMR (600 MHz, acetone-d6) δ 9.20 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.0, 1.5 Hz, 1H),
7.84 (dd, J = 8.6, 2.1 Hz, 1H), 7.51 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.47 (dd, J = 8.5, 1.4 Hz, 1H),
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7.43 – 7.30 (m, 5H), 7.30 – 7.19 (m, 1H), 7.08 (s, 2H), 6.93 (d, J = 8.7 Hz, 1H), 5.67 (s, 2H). 13C
NMR (151 MHz, acetone-d6) δ 166.89, 154.55, 154.34, 152.99, 136.23, 135.27, 132.57, 132.28,
132.11, 130.02, 129.31, 128.70, 127.32, 126.98, 123.52, 116.81, 116.56, 115.85, 114.84, 45.73.
HRMS [C22H17N3O3+H]+ : calcd 372.1348 / found 372.1351.
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3-(4-Benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)benzonitrile (1l). Yield = 27 mg (62 %). 1H
NMR (400 MHz, CDCl3) δ 8.81 (t, J = 1.7 Hz, 1H), 8.72 (dt, J = 8.1, 1.5 Hz, 1H), 7.97 (dd, J =
8.0, 1.6 Hz, 1H), 7.76 (dq, J = 7.7, 1.4 Hz, 1H), 7.60 (td, J = 7.9, 1.8 Hz, 1H), 7.51 (ddd, J = 8.6,
7.3, 1.6 Hz, 1H), 7.41 – 7.26 (m, 7H), 5.58 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 154.54, 151.35,
137.00, 135.02, 133.85, 133.45, 133.13, 132.93, 131.27, 130.90, 129.04, 128.92, 127.88,
126.94, 124.20, 118.73, 114.53, 112.46, 46.26. HRMS [C22H15N3O+H]+ : calcd 338.1293 / found
338.1290.
3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-benzylquinoxalin-2(1H)-one (1m). Yield = 30 mg (75
%). 1H NMR (400 MHz, CDCl3) δ 9.37 – 9.25 (m, 1H), 8.83 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 8.34
(ddd, J = 8.2, 2.4, 1.1 Hz, 1H), 8.00 (dd, J = 8.0, 1.4 Hz, 1H), 7.71 – 7.55 (m, 3H), 7.42 (ddd, J
= 8.3, 7.0, 1.4 Hz, 1H), 7.25 (dd, J = 5.2, 1.3 Hz, 2H), 7.20 (dt, J = 3.5, 1.0 Hz, 1H), 6.97 (dd, J
= 5.1, 3.5 Hz, 1H), 5.70 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 154.04, 151.18, 148.25, 137.33,
136.88, 135.52, 133.15, 132.49, 131.35, 131.18, 129.04, 127.58, 126.85, 126.01, 124.84,
124.70, 124.31, 113.96, 41.24. HRMS [C21H14N4O2+H]+: calcd 355.1195 / found 355.1207.
1-Benzyl-7-chloro-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2a). Yield = 31 mg (15 %). 1H
NMR (400 MHz, CDCl3) δ 9.35 (t, J = 2.0 Hz, 1H), 8.86 (dt, J = 7.9, 1.3 Hz, 1H), 8.36 (ddd, J =
8.2, 2.4, 1.1 Hz, 1H), 7.99 – 7.85 (m, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.44 – 7.30 (m, 7H), 5.56 (s,
13
2H). C NMR (101 MHz, CDCl3) δ 154.27, 151.03, 148.24, 137.44, 137.07, 135.52, 134.44,
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133.77, 131.97, 131.58, 129.19, 129.09, 128.12, 126.95, 125.02, 124.79, 124.71, 114.47, 46.42.
HRMS [C21H14ClN3O3+H]+ : calcd 392.0802 / found 392.0810.
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1-Benzyl-6-chloro-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2b). Yield = 56 mg (31 %). 1H
NMR (400 MHz, CDCl3) δ 9.37 (t, J = 2.0 Hz, 1H), 8.87 (dt, J = 7.9, 1.3 Hz, 1H), 8.39 (ddd, J =
8.2, 2.4, 1.1 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.49 (dd, J = 9.0, 2.4 Hz,
1H), 7.42 – 7.20 (m, 6H), 5.60 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 154.22, 152.30, 148.24,
137.00, 135.59, 134.61, 133.57, 131.59, 131.31, 130.09, 129.60, 129.15, 129.11, 128.06,
126.85, 125.19, 124.86, 115.71, 46.44. HRMS [C21H14ClN3O3+H]+: calcd 392.0802 / found
392.0819.
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1-Benzyl-5-chloro-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2c). Yield = 8 mg (8 %). H
NMR (400 MHz, CDCl3) δ 9.51 (t, J = 2.0 Hz, 1H), 8.96 (dt, J = 7.9, 1.4 Hz, 1H), 8.39 (ddd, J =
8.2, 2.4, 1.1 Hz, 1H), 7.71 (t, J = 8.1 Hz, 1H), 7.57 – 7.40 (m, 2H), 7.40 – 7.18 (m, 6H), 5.63 (s,
13
2H). C NMR (101 MHz, CDCl3) δ 154.25, 150.92, 148.29, 137.14, 135.84, 135.73, 134.66,
134.45, 131.23, 129.73, 129.16, 129.12, 128.01, 126.80, 125.26, 125.10, 125.03, 113.41, 46.67.
HRMS [C21H14ClN3O3+H]+ : calcd 392.0802 / found 392.0813.
1-Benzyl-6,7-dichloro-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2d). Yield = 13 mg (31 %).
1H NMR (400 MHz, CDCl3) δ 9.35 (t, J = 2.0 Hz, 1H), 8.86 (ddt, J = 7.9, 2.8, 1.3 Hz, 1H), 8.46 –
8.28 (m, 1H), 8.11 (s, 1H), 7.70 (td, J = 8.1, 3.8 Hz, 1H), 7.46 (s, 1H), 7.43 – 7.28 (m, 5H), 5.56
13
(d, J = 6.8 Hz, 2H). C NMR (101 MHz, CDCl3) δ 154.00, 152.23, 148.25, 136.73, 135.57,
134.16, 132.21, 132.08, 131.98, 131.53, 129.28, 129.18, 128.27, 126.90, 125.35, 124.85,
124.73, 115.94, 46.56. HRMS [C21H13Cl2N3O3+H]+ : calcd 426.0412 / found 426.0405.
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1-Benzyl-7-bromo-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2e). Yield = 47 mg (15 %). 1H
NMR (400 MHz, CDCl3) δ 9.35 (t, J = 2.0 Hz, 1H), 8.86 (dt, J = 7.9, 1.3 Hz, 1H), 8.37 (dd, J =
8.4, 2.3 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.69 (t, J = 8.1 Hz, 1H), 7.52 (d, J = 8.5 Hz, 2H), 7.46
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– 7.29 (m, 5H), 5.56 (s, 2H). C NMR (101 MHz, CDCl3) δ 154.22, 151.28, 148.25, 137.08,
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135.52, 134.44, 133.89, 132.07, 131.89, 129.20, 129.11, 128.13, 127.65, 126.97, 125.69,
125.05, 124.73, 117.46, 46.40. HRMS [C21H14BrN3O3+H]+ : calcd 436.0297 / found 436.0291.
1-Benzyl-6,7-difluoro-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2g). Yield = 13 mg (10 %).
1H NMR (400 MHz, CDCl3) δ 9.35 (t, J = 2.0 Hz, 1H), 8.85 (dt, J = 7.9, 1.4 Hz, 1H), 8.38 (ddd, J
= 8.2, 2.4, 1.1 Hz, 1H), 7.82 (dd, J = 10.0, 8.2 Hz, 1H), 7.70 (t, J = 8.1 Hz, 1H), 7.47 – 7.22 (m,
5H), 7.15 (dd, J = 11.3, 7.0 Hz, 1H), 5.55 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 154.16, 152.20
(dd, JC-F=257, 15 Hz), 151.47, 148.25, 147.15 (dd, JC-F= 250, 15 Hz), 136.87, 135.49, 134.17,
130.27 (br), 129.41 (br), 129.28, 129.15, 128.26, 126.85, 125.17, 124.73, 118.26 (dd, JC-F = 18,
2 Hz), 103.22 (d, JC-F = 23 Hz), 46.87. HRMS [C21H13F2N3O3+H]+ : calcd 394.1003 / found
394.0996.
1-Benzyl-8-methyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2h). Yield = 36 mg (28 %). 1H
NMR (600 MHz, CDCl3) δ 9.41 (s, 1H), 8.91 (dt, J = 7.9, 1.3 Hz, 1H), 8.43 – 8.10 (m, 1H), 7.66
(t, J = 8.0 Hz, 1H), 7.50 – 7.20 (m, 8H), 5.60 (s, 2H), 2.79 (s, 3H). 13C NMR (151 MHz, CDCl3)
δ 154.44, 148.96, 148.33, 139.84, 137.88, 135.41, 135.20, 133.19, 131.65, 131.10, 128.92,
128.87, 127.70, 126.84, 125.30, 124.69, 124.54, 112.42, 46.31, 17.59. HRMS [C22H17N3O3+H]+:
calcd 372.1348 found 372.1343
1-Benzyl-6,7-dimethyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2i). Yield = 13 mg (31 %).
1H NMR (400 MHz, CDCl3) δ 9.35 (t, J = 2.0 Hz, 1H), 8.86 (dt, J = 7.8, 1.4 Hz, 1H), 8.33 (ddd, J
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= 8.2, 2.4, 1.1 Hz, 1H), 7.77 (s, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.43 – 7.22 (m, 5H), 7.13 (s, 1H),
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5.59 (s, 2H), 2.38 (d, J = 1.2 Hz, 6H). C NMR (101 MHz, CDCl3) δ 154.59, 149.83, 148.21,
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141.67, 137.78, 135.44, 135.23, 133.39, 131.66, 131.12, 130.88, 129.00, 128.93, 127.76,
126.91, 124.59, 124.47, 114.91, 46.11, 20.84, 19.21. HRMS [C23H19N3O3+H]+: calcd 386.1505 /
found 386.1499.
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1-Benzyl-6-bromo-3-(3-nitrophenyl)quinoxalin-2(1H)-one (2j). Yield = 106 mg (34 %). 1H
NMR (400 MHz, CDCl3) δ 9.44 – 9.22 (m, 1H), 8.87 (dt, J = 7.9, 1.4 Hz, 1H), 8.38 (ddd, J = 8.2,
2.4, 1.2 Hz, 1H), 8.18 (d, J = 2.3 Hz, 1H), 7.70 (t, J = 8.1 Hz, 1H), 7.61 (dd, J = 8.9, 2.3 Hz, 1H),
7.44 – 7.26 (m, 5H), 7.23 (d, J = 9.0 Hz, 1H), 5.59 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 154.22,
152.22, 148.24, 136.98, 135.59, 134.58, 134.02, 133.86, 133.18, 132.02, 129.14, 128.07,
126.97, 126.85, 125.20, 124.86, 116.82, 115.98, 46.41. HRMS [C21H14BrN3O3+H]+: calcd
436.0297 / found 436.0290.
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3-(3-Nitrophenyl)quinoxalin-2(1H)-one (3a). Yield = 220 mg (75 %). H NMR (400 MHz,
DMSO-d6) δ 12.76 (s, 1H), 9.31 – 9.12 (m, 1H), 8.78 (ddd, J = 7.9, 1.7, 1.1 Hz, 1H), 8.38 (ddd,
J = 8.2, 2.4, 1.1 Hz, 1H), 7.96 – 7.88 (m, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.71 – 7.48 (m, 1H), 7.47
– 7.25 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 155.02, 152.12, 147.99, 137.43, 135.81,
132.85, 132.34, 131.58, 130.08, 129.52, 125.19, 124.22, 124.15, 115.77. HRMS
[C14H9N3O3+H]+: calcd 268.0722 / found 268.0713. HRMS [C14H9N3O3+H]+: calcd 268.0722 /
found 268.0713.
1-Methyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3b). Yield = 39 mg (74 %). 1H NMR (600
MHz, CDCl3) δ 9.29 (s, 1H), 8.79 (dt, J = 7.9, 1.4 Hz, 1H), 8.33 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H),
7.99 (dd, J = 8.0, 1.5 Hz, 1H), 7.74 – 7.54 (m, 2H), 7.54 – 7.32 (m, 2H), 3.81 (s, 3H). 13C NMR
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(151 MHz, CDCl3) δ 154.43, 151.10, 148.27, 137.51, 135.36, 133.59, 132.86, 131.25, 130.83,
128.89, 124.65, 124.64, 124.05, 113.68, 29.34. HRMS [C15H11N3O3+H]+ : calcd 282.0879 / found
282.0870.
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1-Ethyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3c). Yield = 54 mg (70 %). 1H NMR (400
MHz, CDCl3) δ 9.44 – 9.14 (m, 1H), 8.83 (ddd, J = 7.9, 1.7, 1.1 Hz, 1H), 8.34 (ddd, J = 8.2, 2.4,
1.1 Hz, 1H), 8.09 – 7.88 (m, 1H), 7.77 – 7.58 (m, 2H), 7.53 – 7.36 (m, 2H), 4.44 (q, J = 7.2 Hz,
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2H), 1.47 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl3) δ 153.96, 151.05, 148.19, 137.50,
135.49, 133.16, 132.53, 131.32, 131.10, 128.97, 124.71, 123.96, 113.61, 37.80, 12.44. HRMS
[C16H13N3O3+H]+ : calcd 296.1035 / found 296.1037.
1-Propyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3d). Yield = 13 mg (23 %). 1H NMR (600
MHz, CDCl3) δ 9.30 (t, J = 1.9 Hz, 1H), 8.80 (dt, J = 7.9, 1.3 Hz, 1H), 8.32 (ddd, J = 8.1, 2.3, 1.1
Hz, 1H), 7.99 (dd, J = 8.0, 1.4 Hz, 1H), 7.73 – 7.55 (m, 2H), 7.52 – 7.32 (m, 2H), 4.38 – 4.17 (m,
2H), 1.87 (hept, J = 7.5 Hz, 2H), 1.10 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 154.18,
151.04, 148.25, 137.55, 135.40, 133.11, 132.82, 131.14, 131.04, 128.86, 124.67, 124.61,
123.85, 113.72, 44.19, 20.67, 11.34. HRMS [C17H15N3O3+H]+ : calcd 310.1192 / found 310.1182.
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1-Allyl-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3e). Yield = 91 mg (79 %). H NMR (400
MHz, CDCl3) δ 9.34 (t, J = 2.0 Hz, 1H), 8.92 – 8.73 (m, 1H), 8.35 (ddd, J = 8.2, 2.4, 1.1 Hz, 1H),
8.02 (dd, J = 8.0, 1.5 Hz, 1H), 7.77 – 7.54 (m, 2H), 7.51 – 7.32 (m, 2H), 6.02 (ddt, J = 17.3, 10.4,
5.2 Hz, 1H), 5.42 – 5.15 (m, 2H), 5.03 (dt, J = 5.2, 1.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
154.02, 151.05, 148.20, 137.41, 135.51, 133.01, 132.81, 131.28, 130.92, 130.36, 128.99,
124.77, 124.71, 124.15, 118.44, 114.30, 44.88. HRMS [C17H13N3O3+H]+ : calcd 308.1035 / found
308.1036.
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1-(Naphthalen-1-ylmethyl)-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3f). Yield = 54 mg (35
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%). H NMR (600 MHz, CDCl3) δ 9.39 (t, J = 2.0 Hz, 1H), 8.89 (dt, J = 7.9, 1.4 Hz, 1H), 8.32
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(ddd, J = 8.3, 2.4, 1.1 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.08 – 8.01 (m, 1H), 7.94 (dd, J = 8.3,
1.2 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.59 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H),
7.46 – 7.33 (m, 2H), 7.32 – 7.22 (m, 1H), 7.08 (dd, J = 8.0, 1.6 Hz, 1H), 6.83 (dd, J = 7.3, 1.2
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Hz, 1H), 6.04 (s, 2H). C NMR (151 MHz, CDCl3) δ 154.50, 150.92, 148.33, 137.43, 135.48,
133.92, 133.15, 133.12, 131.33, 130.85, 130.52, 129.24, 129.17, 128.91, 128.19, 128.17,
126.71, 126.12, 125.38, 124.73, 124.20, 122.30, 122.07, 114.77, 44.04. HRMS [C25H17N3O3+H]+
: calcd 408.1348 / found 408.1342.
1-(Naphthalen-2-ylmethyl)-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3g). Yield = 80 mg
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(75 %). H NMR (400 MHz, CDCl3) δ 9.40 (t, J = 2.0 Hz, 1H), 8.91 (ddd, J = 7.9, 1.7, 1.1 Hz,
1H), 8.38 (ddd, J = 8.2, 2.4, 1.1 Hz, 1H), 8.09 – 7.98 (m, 1H), 7.91 – 7.80 (m, 2H), 7.78 (dd, J =
6.2, 3.4 Hz, 1H), 7.74 – 7.65 (m, 2H), 7.57 – 7.44 (m, 4H), 7.41 (ddd, J = 8.3, 7.6, 1.2 Hz, 2H),
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5.79 (s, 2H). C NMR (101 MHz, CDCl3) δ 154.64, 151.22, 148.26, 137.47, 135.59, 133.33,
133.16, 132.99, 132.85, 132.47, 131.39, 130.98, 129.06, 127.75, 126.53, 126.24, 125.72,
124.86, 124.79, 124.72, 124.27, 114.63, 46.52. HRMS [C25H17N3O3+H]+ : calcd 408.1348 found
408.1339.
3-(3-Nitrophenyl)-1-(pyridin-2-ylmethyl)quinoxalin-2(1H)-one (3h). Yield = 82 mg (90 %).
1H NMR (400 MHz, CDCl3) δ 9.37 (t, J = 2.0 Hz, 1H), 8.86 (dt, J = 7.9, 1.4 Hz, 1H), 8.61 (dt, J =
4.8, 1.4 Hz, 1H), 8.36 (ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 8.01 (dd, J = 8.1, 1.3 Hz, 1H), 7.75 – 7.62
(m, 2H), 7.62 – 7.51 (m, 2H), 7.41 (ddd, J = 8.2, 6.5, 2.0 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.24
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(ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 5.73 (s, 2H). C NMR (101 MHz, CDCl3) δ 155.11, 154.53,
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7
151.10, 149.49, 148.23, 137.43, 137.27, 135.52, 133.10, 131.43, 130.79, 129.03, 124.82,
124.74, 124.31, 122.98, 122.23, 115.06, 48.25. HRMS [C20H14N4O3+H]+ : calcd 359.1144 / found
359.1138.
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1-(Furan-2-ylmethyl)-3-(3-nitrophenyl)quinoxalin-2(1H)-one (3i). Yield = 238 mg (67 %)
1H NMR (400 MHz, CDCl3) δ 9.32 (t, J = 2.0 Hz, 1H), 8.83 (dt, J = 7.8, 1.4 Hz, 1H), 8.34 (ddd, J
= 8.2, 2.3, 1.1 Hz, 1H), 8.00 (dd, J = 8.0, 1.5 Hz, 1H), 7.78 – 7.51 (m, 3H), 7.44 (ddd, J = 8.3,
7.0, 1.5 Hz, 1H), 7.39 (s, 1H), 6.49 (d, J = 3.2 Hz, 1H), 6.36 (dd, J = 3.3, 1.9 Hz, 1H), 5.56 (s,
13
2H). C NMR (101 MHz, CDCl3) δ 154.03, 151.07, 148.57, 148.19, 142.62, 137.34, 135.51,
133.01, 132.82, 131.33, 130.92, 129.00, 124.79, 124.69, 124.26, 114.38, 110.74, 109.85, 39.18.
HRMS [C19H13N3O4+H]+ : calcd 348.0985 / found 348.0984.
3-(3-Nitrophenyl)-1-(thiophen-2-ylmethyl)quinoxalin-2(1H)-one (3j). Yield = 48 mg (35
%). 1H NMR (400 MHz, CDCl3) δ 9.36 – 9.28 (m, 1H), 8.83 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 8.34
(ddd, J = 8.2, 2.4, 1.1 Hz, 1H), 8.06 – 7.95 (m, 1H), 7.72 – 7.55 (m, 3H), 7.42 (ddd, J = 8.3, 7.0,
1.4 Hz, 1H), 7.25 – 7.23 (m, 1H), 7.20 (dq, J = 3.5, 0.9 Hz, 1H), 6.97 (dd, J = 5.1, 3.5 Hz, 1H),
13
5.70 (s, 2H). C NMR (101 MHz, CDCl3) δ 154.05, 151.18, 148.25, 137.33, 136.88, 135.53,
133.15, 132.49, 131.35, 131.18, 129.05, 127.58, 126.86, 126.02, 124.85, 124.70, 124.32,
113.96, 41.24. HRMS [C19H13N3O3S+H]+ : calcd 364.0756 / found 364.0749.
N-(4-nitro-2-(5-oxo-2,3-dihydro-5H-[1,4]oxazino[4,3,2-de]quinoxalin-6-yl)phenyl)acet-
amide (16). 2-Amino-3-nitrophenol (1 g, 6.5 mmol) was dissolved in DMF (15 mL). 1,2-Dibro-
moethane (0.7 mL, 8.1 mmol) and KOH (0.3 g, 5.3 mmol) were added, and the mixture was
refluxed at 160 °C for 3 d. After cooling, the solution was poured into water and extracted with
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