Quinoxalin-2(1 h)-one derivatives as inhibitors against hepatitis C virus

ARTICLE

pubs.acs.org/jmc

Quinoxalin-2(1H)-One Derivatives As Inhibitors Against

Hepatitis C Virus

Rui Liu,^†Zhuhui Huang,^§Michael G. Murray,^§Xiaoyong Guo,^†and Gang Liu*^,†,‡

^†Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 2A Nanwei Road,

Xicheng District, Beijing 100050, P. R. China

^‡Tsinghua-Peking Center for Life Sciences and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine,

Tsinghua University, Haidian District, Beijing 100084, P. R. China

^§Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701, United States

S Supporting Information

b

ABSTRACT: Hepatitis C virus (HCV) infection is a serious problem world-

wide, but no eﬀective drugs are currently available. Through screening of our

privileged structure library, quinoxalin-2(1H)-one derivative N-(7-(cyclohexyl-

(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-6-ylcarbamothioyl)benzamide

(compound 1) was identiﬁed as potent HCV inhibitor in vitro. Subsequently,

a structureꢀactivity relationship analysis was carried out that showed

N-(7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-6-ylcarbamothioyl)-

furan-2-carboxamide (compound 11, EC₅₀= 1.8 μM, SI = 9.6), 6-(cyclohexyl-

(methyl)amino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-2(1H)-one (compound

33, EC₅₀= 1.67 μM, SI = 37.4), 2-(cyclohexyl(methyl)amino)-3-(4-phenylthia-

zol-2-ylamino)-7,8,9,10-tetrahydro-5H-pyrido[1,2-a]quinoxalin-6(6aH)-one

(compound 60, EC₅₀= 1.19 μM, SI = 9.27), 8-(cyclohexyl(methyl)amino)-

7-(4-phenylthiazol-2-ylamino)pyrrolo[1,2-a]quinoxalin-4(5H)-one (compound 65,

EC₅₀= 1.82 μM, SI = 9.9), and 6-(diethylamino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-2(1H)-one (compound 78, EC₅₀

=

1.27 μM, SI = 17.9) acted against HCV. The data from the structureꢀactivity relationship study suggests that quinoxalin-2(1H)-one

derivatives exhibited potent activity against HCV.

’ INTRODUCTION

Several small-molecule inhibitors of NS3/4A protease are cur-

rently in preclinical or clinical studies, such as ITMN-191,

telaprevir, and boceprevir^a(Figure 1).⁷HCV NS5B polymerase

is another attractive target for antiviral therapy. The inhibitors of

NS5B polymerase include nucleoside (such as NM-283 and

R7128) and nonnucleoside polymerase inhibitors (such as PF-

00868554 and VCH-916) (Figure 1).⁸However, the emergence

of drug resistance is the major limitation for anti-HCV therapies

targeted at viral protease or viral polymerase.⁹Therefore, there is

an urgent need to develop new classes of antiviral agents for the

treatment of HCV infection.

Antiviral screening of our privileged structure library was

carried out, and compound 1, N-(7-(cyclohexyl(methyl)amino)-

3-oxo-3,4-dihydroquinoxalin-6-ylcarbamothioyl)benzamide

(Figure 2), was identiﬁed as a potent HCV inhibitor in vitro.

Quinoxalinone is an important subunit in drugs with many

biological activities, such as antitumor, antimicrobial, and

antithrombotic functions.¹⁰However, as one type of small

molecular heterocyclic compound, quinoxalin-2(1H)-one has

not been reported for its anti-HCV activity. As the compound

Hepatitis C virus (HCV) belongs to the ﬂaviviridae family and

is a positive single-stranded RNA virus.¹It was ﬁrst identiﬁed in

1989 as the pathogen responsible for non-A and non-B hepatitis.²

Currently, HCV infection is a signiﬁcant health problem world-

wide. An estimated 3% of the world’s population (i.e., more than

170 million people) is infected by HCV, with 3ꢀ4 million new

cases being reported each year.³Approximately 60ꢀ80% of these

infections lead to the chronic form of hepatitis C virus that may

lead to ﬁbrosis, cirrhosis, and hepatocellular carcinoma. Unfortu-

nately, there is no vaccine or speciﬁc antiviral drugs available

against HCV. Interferon-based therapy was the main strategy for

treatment of this disease for more than two decades.⁴Treatment

with interferon R or PEG-interferon R, either alone or in

combination with a broad spectrum antiviral ribavirin, is the

current standard of care. However, this treatment regimen is only

eﬀective for 40ꢀ60% of people infected with HCV genotype-1,

which accounts for the majority of infections in the U.S., Europe,

and Asia.^5,6Meanwhile, serious adverse eﬀects, such as depres-

sion and ﬂu-like symptoms, also limit its application. Recently,

much eﬀort has been devoted toward developing drugs that

inhibit viral replication. The most promising agents under

development are viral protease and polymerase inhibitors.

Received: April 3, 2011

Published: July 15, 2011

r

2011 American Chemical Society

5747

dx.doi.org/10.1021/jm200394x J. Med. Chem. 2011, 54, 5747–5768

|

Journal of Medicinal Chemistry

ARTICLE

Figure 1. Candidate drugs under development for treatment of hepatitis C virus infection.

of segment A were considered from its three pharmacophores,

including phenyl, thiourea, and acyl groups. To examine the

effect of the phenyl group for antiviral activity, compound 4 was

reacted with various acyl isothiocyanates with different R₁

groups, including electron-withdrawing, electron-donating, and

large aromatic hydrocarbon groups, to provide compounds

5ꢀ13 (Scheme 2). The selection of various acyl isothiocyanates

was further considered, including halogenations, aromatic het-

erocycles, and double bonds, for their substituent influence on

the benzoyl group of segment A. Second, the acyl thiourea was

replaced by thiourea (compounds 14ꢀ17) or urea (compounds

18ꢀ19) through the reaction of 4 with isothiocyanates or

isocyanates to simplify the acyl thiourea group. The guanidino

group, a classic bioisostere of the thiourea group, is an important

pharmacophore in many drugs. Therefore, the acyl thiourea

group was replaced by a guanidino group mimicking thiourea

and urea. As illustrated in Scheme 3i, the key intermediate

cyanamide 20 was predicted to form from the reaction of

cyanogen bromide with compound 4. However, the reactivity of

the 7-amino group of compound 4 was too weak to obtain the

target compound, so the alternative Scheme 3ii was designed and

was successfully performed. Compound 1 was first converted

into the free thiourea compound 21 in the presence of K₂CO₃.

The reaction of 21 with CH₃I in ethanol afforded the methylated

compound 22 that offered a route to compounds 24ꢀ30 by

refluxing 22 with primary amines in pyridine.¹²However, when

intermediate 22 was reacted with aniline, compound 23 did not

form, even in the presence of HgCl₂.¹³A thiohydantoin group

was introduced to block the NH group H-bonding donor of the

thiourea moiety and to fix the steric configuration. As shown in

Scheme 4i, from the reaction of compound 14 with 2-chloroa-

cetyl chloride in the presence of Et₃N, a compound with four

possible structures, 31, 31⁰, 31⁰⁰, or 31⁰⁰⁰, was obtained.¹⁴From

the ¹H NMR spectra obtained, the two H atoms of the methylene

group of thiohydantoin showed geminal coupling 4.11 (d, 1H, J =

17.4 Hz) and 4.19 (d, 1H, J = 17.4 Hz), indicating that the

Figure 2. Structure of compound 1.

contains a unique quinoxalin-2(1H)-one scaﬀold amenable to

chemical modiﬁcation, we carried out a structureꢀactivity

relationship (SAR) investigation of compound 1 with the goal

to develop compounds with improved eﬃcacy against HCV

and lower cytotoxicity properties. The results are reported in

this article.

’ RESULTS AND DISCUSSION

Chemistry. To facilitate the synthesis and subsequent SAR

analysis, compound 1 was characterized from the four major

segments indicated in Figure 2. To modify segment A, the core

structure 4 was synthesized according to our previous report

(Scheme 1).¹¹In the presence of diisopropylethylamine (DIPEA),

two fluoro atoms of 1,5-difluoro-2,4-dinitrobenzene (DFDNB)

were substituted by an equivalent methyl 2-aminoacetate

hydrochlorate and subsequently N-methylcyclohexanamine to

give compounds 2 and 3. After reduction of the two nitro

groups using 10% palladium on activated carbon, followed by a

self-cyclization, the key intermediate 4 was obtained in total

yield of 68%.

Segment A Modifications (Schemes 2ꢀ5). To investigate

the medicinal potential, a series of compounds with modifications

5748

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Scheme 1^a

^aReagents and conditions used: (a) NH₂CH₂COOCH₃HCl, DIPEA, THF, room temperature; (b) N-methylcyclohexanamine, DIPEA, THF, reﬂux;

3

(c) 10% Pd/C, NH₄COOH, THF/CH₃CH₂OH, room temperature.

Scheme 2^a

^aReagents and conditions used: (a) R₁CONCS, acetone, reﬂux; (b) R₂NCX, THF, reﬂux.

Scheme 3^a

^aReagents and conditions used: (a) K₂CO₃, CH₃OH/H₂O, 75 °C; (b) CH₃I, CH₃CH₂OH, 50 °C; (c) R₃-NH₂, pyridine, reﬂux.

methylene group of the thiohydantoin was adjacent to a stereo

7-N atom. This observation excluded the possibility of com-

pounds 31⁰⁰and 31⁰⁰⁰. Further structural identification using

nuclear Overhauser effect spectroscopy (NOESY) revealed that

the methylene group correlated with 6-N-methylcyclohexana-

mine, but not with a phenyl group (Figure 3, 31, the NOESY is

5749

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Scheme 4^a

^aReagents and conditions used: (a) Et₃N, 1,4-dioxane, reﬂux.

Scheme 5^a

The thiocarbonyl group is a reactive group that can have

potential adverse eﬀects on patients who take the drug over a

long period of time. Therefore, we further cyclized it into a

thiazole moiety. Compounds 33ꢀ45 were prepared from the

reaction of 21 with various commercially available R-bromo

substituted ketones that oﬀer thiazole analogues of compound 1

(Scheme 5).

Segment B Modifications (Schemes 6ꢀ8). Segment B was

studied because it is located directly in the quinoxalin-2(1H)-one

core structure. Alkyl, aryl, or thioether groups were introduced to

examine the effect of steric hindrance, πꢀπ interactivity, and the

change in hydrophobicity (compounds 51ꢀ55, Scheme 6).

Intermediates 48aꢀ48d were synthesized according to a proce-

dure similar to Scheme 1, where the R₅group was introduced

using different amino acid alkyl esters. The difference was that

compound 48e was obtained from a reduction reaction via

SnCl₂/HCl. Accordingly, 49aꢀ49e were obtained after reaction

of benzoyl isothiocyanate, which further afforded free thiourea

compounds 50aꢀ50e after treatment with K₂CO₃. On cycliza-

tion of 50aꢀ50e with 2-bromo-1-phenylethanone, compounds

51ꢀ55 were obtained.

^aReagents and conditions used: (a) CH₃CH₂OH, reﬂux.

Notably, two tricyclic compounds 60 and 65 were constructed

(Schemes 7 and 8), which provided a large hydrophobic region

and a mimic of the 3,4-double bond of segment B. As illustrated

in Scheme 7, starting from 46, methyl piperidine-2-carboxylate

was used to prepare the disubstituted compound 56, which was

subsequently reduced via catalytic hydrogenation to obtain

compound 57. Condensation of 57 with benzoyl isothiocyanate

was followed by hydrolysis of the resulting compound 58 and

subsequent cyclization of 59 with 2-bromo-1-phenylethanone to

give the desired compound 60.

Figure 3. Three dimensional structures of compounds 31, 32, and 32⁰

plotted using the Chem3D software package.

shown in the Supporting Information). Taking all this informa-

tion together, the final compound was determined to be com-

pound 31. However, in the reaction of compound 17 with

2-chloroacetyl chloride, two final products were obtained in

yields of 59% (32) and 34% (32⁰) (Scheme 4ii). Interestingly,

NOE data was not indicated in NOESY experiments on 32 and

While attempting to prepare the anticipated compound 65⁰,

intermediate 62⁰could not be obtained. Further investigation

showed that 62⁰was converted into 62 under the experimental

conditions used,¹⁵which allowed the synthesis of compound 65

(Scheme 8).

Segment C Modifications (Scheme 9). The significance of

the N-methylcyclohexanamine moiety for anti-HCV activity was

examined using segment C. The role of any electronic effect was

explained by introducing an N-methylbenzenamine group (70).

Steric hindrance and the necessity of the N-methyl moiety on the

1

32⁰. The difference in the H NMR spectra arises from the

chemical shift of the methylene group of thiohydantoin 32 4.31

(d, 1H, J = 17.1 Hz) and 4.51 (d, 1H, J = 17.1 Hz), which is

shifted to a lower field compared to 32⁰4.14 (d, 1H, J = 17.1 Hz)

and 4.23 (d, 1H, J = 17.1 Hz). It is thought that the naphthalene

group deshields the methylene group of thiohydantoin 32, and

so it is concluded that the methylene group of compound 32,

but not compound 32⁰, is closed to the naphthalene group

(Figure 3).

5750

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Scheme 6^a

^aReagents and conditions used: (a) N-methylcyclohexanamine, DIPEA, THF, room temperature; (b) R₅CH(NH₂)COOCH₃HCl, DIPEA, THF,

3

reﬂux; (c) 10% Pd/C, NH₄COOH, THF/CH₃CH₂OH, room temperature; (d) PhCONCS, acetone, reﬂux; (e) K₂CO₃, CH₃OH/H₂O, 75 °C;

(f) PhCOCH₂Br, CH₃CH₂OH, reﬂux. ^bReagents and conditions used: (c) SnCl₂2H₂O, 36% HCl, CH₃CH₂OH, reﬂux.

3

Scheme 7^a

^aReagents and conditions used: (a) DIPEA, THF, reﬂux; (b) 10% Pd/C, NH₄COOH, THF/CH₃CH₂OH, room temperature; (c) PhCONCS,

acetone, reﬂux; (d) K₂CO₃, CH₃OH/H₂O, 75 °C; (e) PhCOCH₂Br, CH₃CH₂OH, reﬂux.

N-methylcyclohexanamine group were investigated by introdu-

cing an N-ethylcyclohexanamine group (71). Considering the drug-

like properties of the entire molecule, the N-methylcyclohexanamine

group was replaced by various six-member rings, such as piper-

idine, morpholine, and 1-methylpiperazine (72ꢀ76). Mimicking

N-cyclohexanamine using a chain alkyl amine was also investi-

gated (77ꢀ79). When the N atom was replaced by an O atom, a

substituent of cyclohexanol could be introduced (80).

As shown in Scheme 9, the reaction of methyl-2-(5-ﬂuoro-2,4-

dinitrophenylamino)acetate (2) with various commercially

5751

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Scheme 8^a

^aReagents and conditions used: (a) DIPEA, THF, reﬂux; (b) 10% Pd/C, cyclohexene, reﬂux; (c) PhCONCS, acetone, reﬂux; (d) K₂CO₃, CH₃OH/H₂O,

75 °C; (e) PhCOCH₂Br, CH₃CH₂OH, reﬂux.

Scheme 9^a

^aReagents and conditions used: (a) secondary amine or sodium cyclohexanolate, DIPEA, THF, reﬂux; (b) 10% Pd/C, NH₄COOH, THF/CH₃CH₂OH,

room temperature; (c) PhCONCS, acetone, reﬂux; (d) K₂CO₃, CH₃OH/H₂O, 75 °C; (e) PhCOCH₂Br, CH₃CH₂OH, reﬂux.

available secondary amines or sodium alcoholate in the pres-

ence of a base gave the corresponding intermediates 66aꢀ66k,

followed by a reduction and then self-cyclization to aﬀord the key

structures 67aꢀ67k. Similar to the previous schemes shown,

5752

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Scheme 10^a

^aReagents and conditions used: (a) HOCH₂COOCH₃or HSCH₂COOCH₃, DIPEA, THF, reﬂux; (b) 10% Pd/C, NH₄COOH, THF/CH₃CH₂OH,

room temperature; (c) PhCONCS, acetone, reﬂux; (d) K₂CO₃, CH₃OH/H₂O, 75 °C; (e) PhCOCH₂Br, CH₃CH₂OH, reﬂux. ^bReagents and

conditions used: (b) SnCl₂2H₂O, 36% HCl, CH₃CH₂OH, reﬂux.

3

67aꢀ67k were condensed with benzoyl isothiocyanate, followed

by hydrolysis and subsequent cyclization with 2-bromo-1-phe-

nylethanone, to prepare the target compounds 70ꢀ80.

Other mimicking analogues 12 and 13 did not show an improved

antiviral inhibitory potency compared to compound 1. None of

the other compounds had a signiﬁcant selection index.

Further modiﬁcations of segment A are summarized in

Tables 2ꢀ5. Removal of the carbonyl group from 1 gave the

thiourea analogue 14, which showed a greatly reduced activity

against HCV. Introduction of the electron-withdrawing group

4-ﬂuoro-phenyl 15, 3-CF₃-phenyl 16, or large aryl group naphthyl

17 also resulted in inactive compounds (Table 2). These results

imply that simplifying the acyl thiourea moiety into a thiourea

group is not appropriate for anti-HCV activity. Changing the

thiourea moiety to a bioisostere urea group gave compounds 18

and 19, which had a higher toxicity to the virus host cells. The

eﬀect on the anti-HCV activity of the guanidino group is shown

in Table 3. Compound 24, which changed the benzoylthiourea

group to a benzylguanidino group, exhibited an EC₅₀value of

11.44 μM. Furthermore, neither the introduction of an electron-

withdrawing group (F^ꢀ25, Cl^ꢀ26, CF₃^ꢀ27, and CF₃O^ꢀ28)

nor an electron-donating group (CH₃O^ꢀ29) provided an

obvious potency or improvement in the selectivity index. A

pyridine mimic of the phenyl moiety was also disadvantageous

(30). All of these suboptimal results suggest that the introduction

of a urea moiety or an H-bond donor (i.e., the NH group of the

guanidino group) contributed a reduction in potency. Introduc-

tion of a thiohydantoin group (31 and 32) was also unfavorable

for the antiviral potency (Table 4).

Segment D Modifications (Scheme 10). When we fixed the

substituent on segments AꢀC, the role of the quinoxalin-2(1H)-

one core structure was then elucidated by replacing it with other

structures. In the presence of DIPEA in THF, the intermediates

81a and 81b were prepared by coupling 46 with methyl

2-hydroxyacetate or methyl 2-mercaptoacetate, respectively.

The reduction of the nitro groups of 81a and 81b with Pd/C

or SnCl₂, and then self-cyclization, gave compounds 82a and

82b, respectively. The target compounds 85 and 86 were

obtained following reaction steps cꢀe in Scheme 9.

Anti-HCV Activity in Vitro. All the synthesized compounds

were evaluated for anti-HCV activity and cytotoxicity in the HCV

RNA replicon assay in Huh 7 ET cells, as described in a previous

publication.¹⁶The results are summarized in Tables 1ꢀ8. Briefly,

the concentration of compound inhibiting HCV RNA replication

activity by 50% (EC₅₀), the concentration of compound decreas-

ing cell viability by 50% (IC₅₀), and the selective index (SI) is

presented.

Our initial eﬀort to outline the SAR values of segment A

focused on the replacement of substituents of the phenyl group

(Table 1). On replacement of the phenyl group by a 4-ﬂuoro-

phenyl group (5, EC₅₀= 2.97 μM), the potency against the HCV

replicon was retained, compared to compound 1. Similar results

were observed for a 4-CF₃O-phenyl group (6) with an EC₅₀

value of 2.33 μM. In the case of a 3-CF₃-phenyl group (7), a

signiﬁcant reduction in cytotoxicity was observed, however, anti-

HCV activity was lost. Introduction of an electron-donating

substituent (CH₃Oꢀ, 8) or πꢀπ interactions (9 and 10) also

decreased the antiviral activity. Mimicking the phenyl group

using diﬀerent heterocycles gave variable results. The furan

analogue 11 exhibited the most potent antiviral activity in the

series, with an EC₅₀value of 1.8 μM and low cytotoxicity (17.28 μM),

resulting in a selectivity index approaching 10-fold (SI = 9.6).

Cyclization of the thiourea moiety to a thiazole ring provided

compound 33, which had a signiﬁcantly reduced cytotoxicity

compared to compound 1 (Table 5, 33, EC₅₀= 1.67 μM, IC₅₀

=

62.47 μM, SI = 37.4). Various groups were tested to identify the

optimal substituent for the R₄group. The 4-ﬂuoro-phenyl sub-

stitutive compound 34 had an EC₅₀value of 5.26 μM, and the

4-chloro-phenyl substitutive compound 35 (EC₅₀= 5.39 μM)

had a reduced antiviral activity and selection index. These results

show that halogen substituents for R₄may be harmful to the anti-

HCV potency. The same trend was observed for a cyano group,

5753

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 1. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Modiﬁcation of the Phenyl

Group)

^aSI calculated as IC₅₀/EC₅₀.

which is the bioisostere of the chloro atom (36, EC₅₀= 6.07 μM).

Adding electron density to the phenyl group by the addition of an

electron-donating moiety yielded compounds 37 and 38. Both of

these compounds were devoid of antiviral activity. Furthermore,

the replacement of the phenyl group of 33 with a naphthyl moiety

resulted in 39, which exhibited comparable cytotoxicity but had a

lower selectivity index than compound 33. The biphenyl analogue

40 had no measurable potency against HCV but with lower cell

toxicity. We hypothesized that the larger aryl substituent may

reduce the inhibitory activity by increasing the πꢀπ interactions

between the compound and the target. Therefore, the compounds

were tested with further modiﬁcation of the phenyl group into its

bioisosteres, such as furan 41, thiophene 42, and pyridine 43.

None of these analogues showed an improved potency. The

necessity of a phenyl group in R₄was further supported by chang-

ing it into a CF₃group, which also led to a loss in anti-HCV activity

(44). Fixation of the steric conﬁguration between the thiazole

group and the phenyl moiety on 33 by a methylene group yielded

compound 45, which exhibited a marked cytotoxicity. All of the

data in Table 5 supports the conclusion that the thiazoleꢀphenyl

moiety was favorable for anti-HCV potency as well as having lower

cytotoxicity.

Next, modiﬁcations of segment B (R₅) were investigated

in detail (Table 6) when segment A was maintained as the

thiazoleꢀphenyl moiety. In principle, lower alkyl groups

can improve the liposolubility of small molecules and block

5754

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 2. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Replacement of the

Aromatic Acyl Thiourea Group by a Thiourea and Urea Group)

^aSI calculated as IC₅₀/EC₅₀.

metabolism sites. However, methyl (51), isopropyl (52), and

isobutyl (53) group R₅substituents demonstrated lower in vitro

potency than an H atom did (33). The replacement of the R₅

group with an aromatic group (benzyl, 54) also made the

compound inactive. Similarly, introducing a thioether group on

R₅also completely removed the activity (compound 55). Cycliza-

tion at the 3- and 4- positions of 33 with a six-member ring yielded

compound 60, which improved the anti-HCV activity and had a

good selectivity index (EC₅₀= 1.19 μM, IC₅₀= 11.03 μM).

Another tricyclic compound 65 also had a good antiviral potency

and an acceptable cytotoxicity (EC₅₀= 1.82 μM, IC₅₀= 18.01 μM).

From multiple modiﬁcations and the corresponding anti-HCV

results, we concluded that substitution of the R₅group may be

prevented by steric limitations, but the 3,4-double bond could be

mimicked by a cycloalkyl group.

In an eﬀort to obtain an increased potency, we studied the

eﬀect of R₆substitutions (segment C, Table 7). Compounds

70ꢀ80 were initially assessed at a single concentration of 20 μM.

All potentially active or cytotoxic compounds in the primary

assay were then evaluated further in a doseꢀresponse assay.

Replacement of R₆with N-methylbenzenamine (70) reduced the

compound’s activity. We proposed that introducing πꢀπ inter-

actions from an aromatic group may be harmful for the anti-HCV

potency. Changing the N-methylcyclohexanamine group of

compound 33 to N-ethylcyclohexanamine provided compound

71, which showed no activity in the initial single dose screening.

This may be because of steric hindrance at this position. Moving

the N atom of the R₆substituent to various heterocyclic aliphatics

also did not improve the activity against HCV. For example,

compound 72, with a piperidine substitution on R₆, showed only

a 9% inhibition under the test conditions. Introducing an ethyl

ester (75) to compound 72 did not signiﬁcantly change the

activity of the compound. Morpholine substitution (73) resulted

in a cytotoxicity value (IC₅₀) of 58.32 μM but resulted in a near

3-fold loss of inhibitory activity against HCV compared with

compound 33. Moreover, introduction of piperazine analogues

yielded a lower activity of 74 and an inactive 76 under the test

conditions. An alternative strategy to modify R₆with a chain alkyl

amine gave diﬀerent results. Replacement of R₆with dimethyla-

mine (77) led to a complete loss of anti-HCV activity. It is

interesting to note that similar antiviral potency but higher cyto-

toxicity of compound 78 (EC₅₀= 1.27 μM, IC₅₀= 22.78 μM, SI =

17.9) with a diethylamine group at R₆was achieved compared

to the R₆with N-methylcyclohexanamine of compound 33.

However, further extension of the alkyl chain of a dipropylamine

group (79) was disadvantageous with EC₅₀= 3.81 μM. Taking all

5755

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 3. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Replacement of the

Aromatic Acyl Thiourea Group by a Guanidino Group)

^aSI calculated as IC₅₀/EC₅₀.

these results into account, mimicking the N-methylcyclohexana-

mine group at the R₆position with a proper length alkyl chain

and the steric conformation of a lipophilic group may be

promising for improved anti-HCV activity. Further investigation

of substitution of the N atom at the R₆position was also carried

out. Replacement of R₆with cyclohexanol (80) resulted in a loss

of activity.

Finally, two mimics of the quinoxalin-2(1H)-one core struc-

ture were synthesized and evaluated for their anti-HCV activity

(segment D, Table 8). Changing the N atom at the 4- position

of compound 33 by its bioisostere O or S atom gave compounds

85 and 86, respectively. However, both of these compounds

had no potency against HCV. As a result, quinoxalin-2(1H)-

one was considered as the optimal core structure for anti-HCV

activity.

potency and decrease cytotoxic eﬀects. Five compounds, 11,

33, 60, 65, and 78, showed high antiviral potency and an

acceptable selectivity index, with EC₅₀= 1.8 μM and SI = 9.6,

EC₅₀= 1.67 μM and SI = 37.4, EC₅₀= 1.19 μM and SI = 9.27,

EC₅₀= 1.82 μM and SI = 9.9 μM, and EC₅₀= 1.27 μM and

SI =17.9, respectively.

To elucidate the action mechanism of the compound, biochem-

ical assays were carried out essentially as previously described.¹⁷The

results indicated that the compound 33 did not exhibit any

inhibitory eﬀects on HCV protease, helicase, and NS5B poly-

merase (data not show herein). It was concluded that that 33 are

acting on other virus-encoded and/or cellular targets that need to

be further investigated.

In conclusion, quinoxalin-2(1H)-one with a thiazoleꢀphenyl

moiety is optimal for anti-HCV activity; there may be a steric

limitation at 3- position of quinoxalin-2(1H)-one, however, the

3,4-double bond was mimicked by a cycloalkyl group and a

hydrophobic group with proper alkyl chain and steric conforma-

tion at 6- position is necessary for the anti-HCV activity.

Although the mechanism underlying how these compounds

inhibit HCV is unknown, further SAR analysis and mechanistic

studies on this class of compounds against HCV are currently

being investigated.

’ CONCLUSIONS

This study has identiﬁed new quinoxalin-2(1H)-one deriva-

tives as inhibitors of HCV. Compound 1 was ﬁrst identiﬁed for its

anti-HCV activity by screening a library of compounds using the

HCV replicon and cytotoxicity assays. A series of modiﬁcations of

compound 1 were carried out in an eﬀort to improve anti-HCV

5756

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 4. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Replacement of the

Aromatic Acyl Thiourea Group by a Thiohydantoin Group)

^aSI calculated as IC₅₀/EC₅₀.

’ EXPERIMENTAL SECTION

traced by a fast LC-MS system until all of the starting material was

changed to the anticipated compound. Compound 3 (1 g) was dissolved

in a mixed solvent of THF (30 mL) and EtOH (30 mL), followed by the

addition of 10% Pd/C (1 g) and HCOONH₄(2 g). The mixture was

stirred at room temperature for 3 h. The residue solid was filtered off,

and the filtrate was concentrated in vacuo. Water (50 mL) was added to

the resulting product and was further extracted by CH₂Cl₂(3 ꢁ 50 mL).

The organic layers were combined, dried over anhydrous MgSO₄, and

evaporated in vacuo. Intermediate 4 was obtained as a yellow solid after

chromatography through silica gel eluting with CHCl₃ꢀCH₃OH (30:1,

Chemistry. Unless otherwise noted, all materials were obtained

from commercial suppliers and used without purification. All NMR

experiments were carried out on a Varian Mercury 300 or 400 or 500

MHz NMR spectrometer using DMSO-d₆as the solvent. Chemical

shifts were reported in ppm (δ) relative to the solvent, and coupling

constants (J) were reported in Hz. Melting points were determined

without correction with a Yanaco micromelting point apparatus. Auto-

matic HPLC-MS analysis was performed on a Thermo Finnigan LCQ-

Advantage mass spectrometer equipped with an Agilent pump, an

Agilent detector, an Agilent liquid handler, and a fluent splitter. The

column used was a Kromasil C18 column (4.6 μm, 4.6 mm ꢁ 50 mm)

from DIKMA for analysis. The eluent was a mixture of acetonitrile and

water containing 0.05% HCOOH with a linear gradient from 5:95 (v/v)

to 95:5 (v/v) of acetonitrileꢀH₂O within 5 min at a 1 mL/min flow rate

for analysis. The UV detection was carried out at a UV wavelength of

254 nm. The 5% of the eluent was split into the MS system. Mass spectra

were recorded in either positive or negative ion mode using electrospray

ionization (ESI). High resolution LC-MS was carried out by Agilent LC/

MSD TOF using a column of Agilent ZORBAX SB-C18 (rapid

resolution, 3.5 μm, 2.1 mm ꢁ 30 mm) at a flow of 0.40 mL/min. The

solvent is methanol/water = 75:25 (v/v) containing 5 mmol/L ammo-

nium formate. The ion source is electrospray ionization (ESI). Flash

column chromatography was performed with silica gel 60 (200ꢀ300

mesh) from Qingdao Haiyang Chemical Factory. All tested compounds

were purified until the purity was g95%, detected by HPLC under UV

254 nm wavelength, NMR, melting point, and HRLC-MS.

1

v/v). Yield 68%. H NMR (300 MHz, DMSO-d₆): δ 1.03ꢀ1.26 (m,

3H), 1.29ꢀ1.37 (m, 2H), 1.54 (m, 1H), 1.68ꢀ1.77 (m, 4H), 2.55 (s,

3H), 2.65ꢀ2.72 (m, 1H), 5.71 (s, 2H), 6.45 (s, 1H), 7.22 (s, 1H), 7.67

(s, 1H), 11.97 (s, 1H). HRMS calcd for C₁₅H₂₁N₄O (M + H⁺)

273.1710; found 273.1704.

General Procedure for the Synthesis of Compounds 1,

5ꢀ13. To a stirred solution of 4 (0.5 mmol) in 20 mL of dried acetone,

various aroyl isothiocyanates (0.5 mmol) were added. The reaction

mixture was refluxed for 4 h. After the reaction was completed, the

solvent was evaporated in vacuo. The final products were characterized

after purification by silica gel column chromatography.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)benzamide (1). Yellow powder in

88% yield; mp 190ꢀ191 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.00ꢀ

1.36 (m, 5H), 1.49ꢀ1.52 (m, 1H), 1.65ꢀ1.69 (m, 2H), 1.76ꢀ1.80 (m,

2H), 2.65 (s, 3H), 2.78 (m, 1H), 7.51ꢀ7.56 (m, 2H), 7.63ꢀ7.69 (m,

2H), 7.95 (d, 2H, J = 7.2 Hz), 8.09 (s, 1H), 8.93 (s, 1H), 11.60 (s, 1H),

12.42 (s, 1H), 13.44 (s, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ 24.8,

25.4, 29.1, 37.5, 61.7, 108.2, 122.6, 128.4, 128.6, 129.6, 132.1, 133.1,

137.0, 140.3, 150.6, 154.9, 167.7, 177.6. HRMS calcd for C₂₃H₂₆N₅O₂S

(M + H⁺) 436.1801; found 436.1815.

Preparation of the Key Intermediate Compound 4. To a

stirred solution of 1,5-difluoro-2,4-dinitrobenzene (DFDNB, 2.04 g, 10

mmol) in THF (50 mL) was added DIPEA (20 mmol) and

NH₂CH₂COOCH₃HCl (10 mmol). After vigorously stirring at room

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-4-fluorobenzamide (5). Light-

3

temperature until the total disappearance of DFDNB, compound 2 was

obtained without purification. Then, N-methylcyclohexanamine

(10 mmol) and DIPEA (10 mmol) were added and stirred under reflux

for 8 h. The solvent was removed under reduced pressure to give

compound 3, which was used directly. The above two reactions were

1

yellow powder in 85% yield; mp 214ꢀ215 °C. H NMR (300 MHz,

DMSO-d₆): δ 1.02ꢀ1.14 (m, 3H), 1.19ꢀ1.33 (m, 2H), 1.50ꢀ1.53

(m, 1H), 1.67ꢀ1.70 (m, 2H), 1.77ꢀ1.81 (m, 2H), 2.65 (s, 3H), 2.75ꢀ

2.82 (m, 1H), 7.36ꢀ7.41 (m, 2H), 7.65 (s, 1H), 8.05ꢀ8.10 (m, 3H),

5757

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 5. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Cyclization of the Thiourea

Moiety to a Thiazole Ring)

^aSI calculated as IC₅₀/EC₅₀. ^bThe structure of compound 45.

8.96 (s, 1H), 11.67 (s, 1H), 12.43 (s, 1H), 13.43 (s, 1H). HRMS calcd for

C₂₃H₂₅N₅O₂FS (M + H⁺) 454.1707; found 454.1723.

DMSO-d₆): δ 0.99ꢀ1.33 (m, 5H), 1.47ꢀ1.50 (m, 1H), 1.64ꢀ1.67 (m,

2H), 1.75ꢀ1.79 (m, 2H), 2.63 (s, 3H), 2.77 (m, 1H), 7.50 (d, 2H, J = 8.1

Hz), 7.63 (s, 1H), 8.07ꢀ8.10 (m, 3H), 8.97 (s, 1H), 11.75 (s, 1H), 12.43

(s, 1H), 13.40 (s, 1H). HRMS calcd for C₂₄H₂₅N₅O₃F₃S (M + H⁺)

520.1624; found 520.1645.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquino-

xalin-6-ylcarbamothioyl)-4-(trifluoromethoxy)benzamide (6).

1

Yellow powder in 87% yield; mp 176ꢀ178 °C. H NMR (300 MHz,

5758

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 6. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Modiﬁcation of Segment B)

^aSI calculated as IC₅₀/EC₅₀. ^bThe structure of compounds 60 and 65.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-3-(trifluoromethyl)benzamide

(7). Pale-yellow solid in 85% yield; mp 187ꢀ189 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.07ꢀ1.28 (m, 5H), 1.51 (m, 1H), 1.66ꢀ1.77 (m, 4H),

2.64 (s, 3H), 2.78 (m, 1H), 7.65 (s, 1H), 7.78 (m, 1H), 8.00ꢀ8.09 (m,

2H), 8.21ꢀ8.31 (m, 2H), 8.98 (s, 1H), 11.96 (s, 1H), 12.43 (s, 1H), 13.40

(s, 1H). HRMS calcd for C₂₄H₂₅N₅O₂F₃S (M + H⁺) 504.1687; found

504.1675.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-3,4,5-trimethoxybenzamide (8).

Pale-yellow powder in 74% yield; mp 110ꢀ111 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.09ꢀ1.32 (m, 5H), 1.52ꢀ1.79(m, 5H), 2.66 (s, 3H), 2.76

(m, 1H), 3.71ꢀ3.88 (m, 9H), 7.22 (s, 1H), 7.35 (s, 1H), 7.62 (s, 1H),

8.10 (s, 1H), 8.83 (s, 1H), 11.61 (s, 1H), 12.41 (s, 1H), 13.39 (s, 1H). ¹³C

NMR (150 MHz, DMSO-d₆): δ 24.9, 25.4, 28.9, 37.1, 55.8, 56.1, 60.1,

61.8, 106.3, 106.5, 108.8, 122.4, 125.8, 126.7, 128.2, 129.6, 136.7, 140.5,

141.6, 150.7, 152.5, 154.8, 166.8, 177.8. HRMS calcd for C₂₆H₃₂N₅O₅S

(M + H⁺) 526.2118; found 526.2140.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-3-phenylacrylamide (9). Light-

yellow powder in 89% yield; mp 232ꢀ233 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.07ꢀ1.34 (m, 5H), 1.53ꢀ1.81 (m, 5H), 2.64 (s, 3H),

2.72ꢀ2.74 (m, 1H), 7.08 (d, 1H, J = 15.9 Hz), 7.46ꢀ7.48 (m, 3H),

7.63ꢀ7.67 (m, 3H), 7.76 (d, 1H, J = 15.9 Hz), 8.08 (d, 1H, J = 1.8 Hz),

8.92 (s, 1H), 11.57 (s, 1H), 12.42 (s, 1H), 13.38 (s, 1H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 24.9, 25.4, 29.1, 37.5, 61.9, 108.0, 119.9,

122.7, 128.2, 128.4, 129.1, 130.7, 134.0, 137.0, 140.2, 144.4, 150.6,

154.9, 165.3, 177.5. HRMS calcd for C₂₅H₂₈N₅O₂S (M + H⁺)

462.1958; found 462.1949.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-1-naphthamide (10). Yellow

powder in 80% yield; mp 169ꢀ170 °C. ¹H NMR (300 MHz, DMSO-

d₆): δ 1.06ꢀ1.21 (m, 3H), 1.33ꢀ1.43 (m, 2H), 1.53ꢀ1.57 (m, 1H),

1.71ꢀ1.75 (m, 2H), 1.82ꢀ1.86 (m, 2H), 2.69 (s, 3H), 2.84ꢀ2.87 (m,

1H), 7.59ꢀ7.66 (m, 4H), 7.80ꢀ7.82 (m, 1H), 8.04ꢀ8.06 (m, 1H),

8.10ꢀ8.15 (m, 3H), 8.96 (s, 1H), 12.03 (s, 1H), 12.45 (s, 1H), 13.49

5759

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 7. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Derivatives on HCV Replication in Huh 7 Cells (Modiﬁcation of Segment C)

^aSI calculated as IC₅₀/EC₅₀. ^bCompounds assessed at a single-concentration of 20 μM with the percentage donating inhibition level compared to the

control cell. ^cND = not detected.

(s, 1H). HRMS calcd for C₂₇H₂₈N₅O₂S (M + H⁺) 486.1958; found

486.1968.

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)-3-methylthiophene-2-carboxa-

1

mide (12). Pale-yellow powder in 85% yield; mp 182ꢀ184 °C. H

N-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroqui-

noxalin-6-ylcarbamothioyl)furan-2-carboxamide (11). Yellow

powder in 81% yield; mp 202ꢀ204 °C. ¹H NMR (300 MHz, DMSO-

d₆): δ 1.00ꢀ1.32 (m, 5H), 1.49ꢀ1.52 (m, 1H), 1.65ꢀ1.69 (m, 2H),

1.76ꢀ1.80 (m, 2H), 2.63 (s, 3H), 2.72ꢀ2.80 (m, 1H), 6.75ꢀ6.77

(m, 1H), 7.64 (s, 1H), 7.87ꢀ7.88 (m, 1H), 8.07ꢀ8.09 (m, 2H), 8.96

(s, 1H), 11.28 (s, 1H), 12.42 (s, 1H), 13.29 (s, 1H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 24.3, 24.9, 28.7, 37.4, 61.2, 107.3, 112.1,

118.3, 122.3, 128.0, 129.0, 136.6, 139.6, 144.0, 147.9, 150.1, 154.4,

156.4, 176.6. HRMS calcd for C₂₁H₂₄N₅O₃S (M + H⁺) 426.1594;

found 426.1605.

NMR (300 MHz, DMSO-d₆): δ 1.07ꢀ1.32 (m, 5H), 1.52ꢀ1.78 (m,

5H), 2.51 (s, 3H), 2.63 (s, 3H), 2.76 (m, 1H), 7.07 (d, 1H, J = 4.8 Hz),

7.63 (s, 1H), 7.84 (d, 1H, J = 4.8 Hz), 8.09 (s, 1H), 8.89 (s, 1H), 11.06 (s,

1H), 12.42 (s, 1H), 13.17 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

15.5, 24.8, 25.4, 29.0, 37.5, 61.7, 108.1, 122.6, 128.4, 128.9, 129.6, 131.1,

131.9, 136.9, 140.3, 143.9, 150.7, 154.9, 162.5, 177.0. HRMS calcd for

C₂₂H₂₆N₅O₂S₂(M + H⁺) 456.1522; found 456.1523.

6-Chloro-N-(7-(cyclohexyl(methyl)amino)-3-oxo-3,4-di-

hydroquinoxalin-6-ylcarbamothioyl)nicotinamide (13). Yel-

low powder in 84% yield; mp 215ꢀ217 °C. ¹H NMR (300 MHz,

5760

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

Table 8. Inhibitory Eﬀects of Quinoxalin-2(1H)-one Deri-

vatives on HCV Replication in Huh 7 Cells (Modiﬁcation of

Segment D)

(s, 1H), 12.39 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.3, 24.9, 29.2,

38.4, 61.1, 122.5, 122.8, 125.9, 126.0, 126.8, 127.0, 128.1, 129.3, 130.0,

133.1, 134.3, 138.2, 138.3, 149.1, 156.0, 178.8. HRMS calcd for C₂₆H₂₈-

N₅OS (M + H⁺) 458.2009; found 458.1993.

General Procedure for the Synthesis of Compounds 18

and 19. To a stirred solution of 4 (0.2 mmol) in 5 mL of dried THF,

various isocyanates (1.0 mmol) were added and the reaction mixture was

refluxed for 10 h. After the reaction was completed, the solvent was

evaporated in vacuo. The final products were characterized after

purification by silica gel column chromatography.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-phenylurea (18). Pale-yellow powder in 89% yield; mp

150ꢀ152 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.02ꢀ1.23 (m, 5H),

1.51ꢀ1.55 (m, 1H), 1.67ꢀ1.70 (m, 2H), 1.87ꢀ1.90 (m, 2H), 2.64

(m, 4H), 6.97ꢀ7.02 (m, 1H), 7.27ꢀ7.33 (m, 2H), 7.49- 7.52 (m, 2H),

7.57 (s, 1H), 7.95 (s, 1H), 8.24 (s, 1H), 8.81 (s, 1H), 9.76 (s, 1H), 12.26

(s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.4, 29.2, 38.8, 62.2,

102.6, 118.1, 118.4, 122.1, 123.5, 127.1, 128.7, 128.8, 130.0, 136.8, 139.3,

139.5, 148.0, 152.0, 155.2. HRMS calcd for C₂₂H₂₆N₅O₂(M + H⁺)

392.2081; found 392.2090.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(naphthalen-1-yl)urea (19). Pale-yellow powder in

79% yield; mp 226ꢀ228 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 0.97ꢀ

1.22 (m, 5H), 1.46ꢀ1.50 (m, 1H), 1.60ꢀ1.77 (m, 4H), 2.55 (s, 3H), 2.63

(m, 1H), 7.47ꢀ7.63 (m, 4H), 7.74 (d, 1H, J = 8.1 Hz), 7.80 (d, 1H, J = 7.2

Hz), 7.94 (d, 1H, J = 1.8 Hz), 7.97(s, 1H), 8.13 (d, 1H, J = 8.1 Hz), 8.21

(s, 1H), 8.98 (s, 1H), 9.61 (s, 1H), 12.27 (s, 1H). HRMS calcd for

C₂₆H₂₈N₅O₂(M + H⁺) 442.2243; found 442.2240.

General Procedure for the Synthesis of Compounds 24ꢀ30.

Compound 1 (1 mmol) and K₂CO₃(1.5 mmol) were dissolved in a mixed

solvent of CH₃OH (20 mL) and H₂O (5 mL). The reaction mixture was

refluxed with stirring for 1 h. After evaporation of the solvent in vacuo,

compound 21 was obtained as a yellow solid after chromatography through

silica gel. The yield was 84%. ¹H NMR (300 MHz, DMSO-d₆): δ

1.01ꢀ1.18 (m, 3H), 1.22ꢀ1.31 (m, 2H), 1.55 (m, 1H), 1.68ꢀ1.79 (m,

4H), 2.62 (s, 3H), 2.65 (s, 1H), 7.47 (s, 1H), 7.99ꢀ8.01 (m, 3H), 8.25 (s,

1H), 9.14 (s, 1H), 12.29 (s, 1H). HRMS calcd for C₁₆H₂₂N₅OS (M + H⁺)

332.1540; found 332.1542.

anti-HCV

activity

cytotoxicity

compd

85

X

EC₅₀(μM)

IC₅₀(μM)

SI^a

O

S

15.36

17.53

1.1

<0.69

>14.3

86

>20

13.85

rIFNR-2b

0.14 IU/mL

^aSI calculated as IC₅₀/EC₅₀.

>2 IU/mL

DMSO-d₆): δ 1.07ꢀ1.24 (m, 5H), 1.49ꢀ1.51 (m, 1H), 1.65ꢀ1.68 (m,

2H), 1.77ꢀ1.80 (m, 2H), 2.63 (s, 3H), 2.77 (m, 1H), 7.65ꢀ7.70 (m,

2H), 8.09 (s, 1H), 8.31ꢀ8.34 (m, 1H), 8.90 (s, 1H), 9.01 (s, 1H), 11.96

(s, 1H), 12.44 (s, 1H), 13.36 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

24.8, 25.4, 29.2, 38.0, 61.8, 107.7, 122.9, 124.0, 127.9, 128.5, 129.6, 137.0,

140.0, 140.1, 150.1, 150.7, 153.8, 154.9, 165.4, 177.1. HRMS calcd for

C₂₂H₂₄N₆O₂SCl (M + H⁺) 471.1364; found 471.1369.

General Procedure for the Synthesis of Compounds 14ꢀ17.

Intermediate 4 (0.2 mmol) was dissolved in dried THF (5 mL). Various

isothiocyanates (1 mmol) were added and the solution was heated at 65 °C

for 24 h. After the reaction was completed, the solvent was evaporated in

vacuo. The final products were characterized after purification by silica gel

column chromatography.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-phenylthiourea (14). Light-yellow powder in 84% yield;

1

mp 131ꢀ132 °C. H NMR (300 MHz, DMSO-d₆): δ 0.82ꢀ1.02 (m,

To a solution of compound 21 (1 mmol) in ethanol (30 mL) was

added methyl iodide (2 mmol). The mixture was heated at 45 °C for 5 h

and then puriﬁed by silica gel column chromatography. Compound 22

5H), 1.22 (m, 1H), 1.48ꢀ1.58 (m, 4H), 2.49 (s, 3H), 2.58 (m, 1H), 7.26ꢀ

7.30 (m, 1H), 7.44ꢀ7.45 (m, 4H), 7.54 (s, 1H), 8.01 (d, 1H, J = 2.4 Hz),

8.74 (s, 1H), 9.74 (s, 1H), 10.44 (s, 1H), 12.35 (s, 1H). HRMS calcd for

C₂₂H₂₆N₅OS (M + H⁺) 408.1853; found 408.1859.

1

was obtained as a yellow powder in 88% yield. H NMR (300 MHz,

DMSO-d₆): δ 1.03ꢀ1.22 (m, 4H), 1.31ꢀ1.38 (m, 2H), 1.52ꢀ1.61 (m,

2H), 1.69ꢀ1.72 (m, 2H), 2.36 (s, 3H), 2.60 (s, 3H), 3.11 (m, 1H), 6.39

(s, 2H), 6.53 (s, 1H), 7.13 (s, 1H), 7.92 (s, 1H), 12.11 (s, 1H). HRMS

calcd for C₁₇H₂₄N₅OS (M + H⁺) 346.1696; found 346.1684.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(4-fluorophenyl)thiourea (15). Yellow powder in 85%

yield; mp 137ꢀ138 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.01ꢀ1.09

(m, 5H), 1.48ꢀ1.60 (m, 5H), 2.51 (s, 3H), 2.61 (m, 1H), 7.25ꢀ7.30 (m,

2H), 7.45ꢀ7.50 (m, 2H), 7.54 (m, 1H), 8.02 (s, 1H), 8.69 (s, 1H), 9.67

(s, 1H), 10.40 (s, 1H), 12.35 (s, 1H). HRMS calcd for C₂₂H₂₅FN₅OS

(M + H⁺) 426.1758; found 426.1762.

Compound 22 (0.5 mmol) was dissolved in pyridine (20 mL). Various

amines (0.75 mmol) was added, and the solution was reﬂuxed. After the

reaction was completed, the solution was evaporated in vacuo. The residue

was neutralized with 10% HCl. The mixture was partitioned between

CHCl₃and water. The organic phase was then dried with anhydrous mag-

nesium sulfate and evaporated in vacuo. The ﬁnal products 24ꢀ30 were

characterized after puriﬁcation by silica gel column chromatography.

1-Benzyl-3-(7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihy-

droquinoxalin-6-yl)guanidine (24). Orange powder in 43% yield;

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(3-(trifluoromethyl)phenyl)thiourea (16). Yellow

1

powder in 79% yield; mp 127ꢀ129 °C. H NMR (300 MHz, DMSO-

d₆): δ 1.01ꢀ1.34 (m, 5H), 1.47ꢀ1.66 (m, 5H), 2.58 (s, 3H), 2.64 (m,

1H), 7.54ꢀ7.58 (m, 2H), 7.62ꢀ7.67 (m, 1H), 7.78ꢀ7.81 (m, 1H), 7.98

(s, 1H), 8.04 (s, 1H), 8.44 (s, 1H), 9.70 (s, 1H), 10.66 (s, 1H), 12.35 (s,

1H). HRMS calcd for C₂₃H₂₅F₃N₅OS (M + H⁺) 476.1726; found

476.1731.

1

mp 235ꢀ237 °C. H NMR (300 MHz, DMSO-d₆): δ 1.06ꢀ1.36 (m,

5H), 1.55ꢀ1.69 (m, 5H), 2.63 (s, 3H), 2.70 (m, 1H), 4.49 (d, 2H, J = 5.4

Hz), 7.16 (s, 1H), 7.32ꢀ7.43 (m, 5H), 7.53 (s, 1H), 7.99 (s, 1H), 8.14 (s,

1H), 8.39 (m, 1H), 9.02 (s, 1H), 12.45 (s, 1H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 25.4, 28.5, 34.2, 44.4, 61.8, 111.7, 121.9, 127.2, 127.6,

128.0, 128.5, 130.5, 133.1, 136.7, 142.8, 151.2, 154.5, 154.6. HRMS calcd

for C₂₃H₂₉N₆O (M + H⁺) 405.2397; found 405.2402.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(naphthalen-1-yl)thiourea (17). Yellow powder in

1

82% yield; mp 233ꢀ235 °C. H NMR (300 MHz, DMSO-d₆): δ 0.20

(m, 2H), 0.67 (m, 1H), 0.82ꢀ0.88 (m, 2H), 1.23ꢀ1.35 (m, 5H), 2.00

(s, 3H), 2.32 (m, 1H), 7.45 (s, 1H), 7.59ꢀ7.65 (m, 4H), 7.88ꢀ7.91 (m,

1H), 7.97 (s, 1H), 8.01ꢀ8.04 (m, 2H), 9.12 (s, 1H), 9.72 (s, 1H), 10.57

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(4-fluorobenzyl)guanidine (25). Yellow powder in

5761

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

48% yield; mp 151ꢀ153 °C. ¹H NMR (300 MHz, DMSO-d₆): δ

1.04ꢀ1.22 (m, 3H), 1.30ꢀ1.34 (m, 3H), 1.52ꢀ1.64 (m, 4H), 2.59 (s,

3H), 3.00 (m, 1H), 3.94 (s, 2H), 4.43 (s, 2H), 6.87 (s, 1H), 7.16ꢀ7.29 (m,

4H), 7.37ꢀ7.49 (m, 3H), 7.98 (s, 1H). HRMS calcd for C₂₃H₂₈N₆OF

(M + H⁺) 423.2303; found 423.2310.

1-(4-Chlorobenzyl)-3-(7-(cyclohexyl(methyl)amino)-3-oxo-

3,4-dihydroquinoxalin-6-yl)guanidine (26). Yellow powder in

43% yield; mp 165ꢀ167 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.04ꢀ

1.13 (m, 3H), 1.22ꢀ1.34 (m, 3H), 1.54ꢀ1.75 (m, 4H), 2.62 (s, 3H), 2.68

(m, 1H), 4.47 (d, 2H, J = 6.0 Hz), 7.14 (s, 1H), 7.37 (d, 2H, J = 8.4 Hz),

7.45 (d, 2H, J = 8.4 Hz), 7.52 (s, 1H), 7.98 (s, 1H), 8.14 (s, 1H), 8.36 (m,

1H), 9.07 (s, 1H), 12.45 (s, 1H). HRMS calcd for C₂₃H₂₈N₆OCl

(M + H⁺) 439.2007; found 439.2006.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(3-(trifluoromethyl)benzyl)guanidine (27). Yellow

powder in 47% yield; mp 199ꢀ200 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 1.01ꢀ1.13 (m, 3H), 1.22ꢀ1.28 (m, 3H), 1.50ꢀ1.64 (m, 4H), 2.60 (s,

3H), 2.71 (m, 1H), 4.58 (d, 2H, J = 5.1 Hz), 7.15 (s, 1H), 7.49 (s, 1H),

7.61ꢀ7.73 (m, 4H), 8.00 (s, 1H), 8.14 (s, 1H), 8.39 (m, 1H), 9.08 (s, 1H),

12.45 (s, 1H). HRMS calcd for C₂₄H₂₈N₆OF₃(M + H⁺) 473.2271; found

473.2279.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(4-(trifluoromethoxy)benzyl)guanidine (28). Yel-

low powder in 47% yield; mp 199ꢀ200 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.03ꢀ1.13 (m, 3H), 1.22ꢀ1.33 (m, 3H), 1.51ꢀ1.65 (m,

4H), 2.61 (s, 3H), 2.71 (m, 1H), 4.51 (d, 2H, J = 5.4 Hz), 7.15 (s, 1H),

7.39 (d, 2H, J = 8.4 Hz), 7.48ꢀ7.52 (m, 3H), 7.98 (s, 1H), 8.14 (s, 1H),

8.38(m, 1H),9.08(s,1H),12.46(s,1H).HRMScalcdforC₂₄H₂₈N₆O₂F₃

(M + H⁺) 489.2220; found 489.2223.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(4-methoxybenzyl)guanidine (29). Light-yellow

powder in 53% yield; mp 177ꢀ179 °C. ¹H NMR (300 MHz, DMSO-

d₆): δ 1.03ꢀ1.05 (m, 3H), 1.22ꢀ1.34 (m, 3H), 1.53ꢀ1.67 (m, 4H),

2.61 (s, 3H), 2.69 (m, 1H), 3.74 (s, 3H), 4.41 (d, 2H, J = 3.6 Hz), 6.93

(d, 2H, J = 8.4 Hz), 7.14 (s, 1H), 7.28 (d, 2H, J = 8.4 Hz), 7.51 (s, 1H),

7.99 (s, 1H), 8.13 (s, 1H), 8.39 (s, 1H), 9.05 (s, 1H), 12.46 (s, 1H).

HRMS calcd for C₂₄H₃₁N₆O₂(M + H⁺) 435.2503; found 435.2515.

1-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinox-

alin-6-yl)-3-(pyridin-2-ylmethyl)guanidine (30). Yellow powder

in 44% yield; mp 199ꢀ201 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.06ꢀ

1.11 (m, 3H), 1.22ꢀ1.35 (m, 2H), 1.54ꢀ1.70 (m, 5H), 2.64 (s, 3H), 2.73

(m, 1H), 4.61 (d, 2H, J = 5.1 Hz), 7.20 (s, 1H), 7.36ꢀ7.42 (m, 2H), 7.54

(s, 1H), 7.83ꢀ7.89 (m, 1H), 8.05 (s, 1H), 8.13 (s, 1H), 8.44 (m, 1H), 8.59

(d, 1H, J = 4.8 Hz), 9.27 (s, 1H), 12.43 (s, 1H). HRMS calcd for

C₂₂H₂₈N₇O (M + H⁺) 406.2349; found 406.2354.

Et₃N (2 mmol) and 2-chloroacetyl chloride (1 mmol). The mixture was

refluxed for 8 h. After the reaction was completed, the solution was

evaporated in vacuo. The final product 32 and 32⁰were obtained as yellow

powders after purification by silica gel column chromatography eluting with

CHCl₃ꢀCH₃OH (40:1, v/v).

32: Yield 59%; mp 210ꢀ211 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 0.53ꢀ0.86 (m, 3H), 1.09ꢀ1.22 (m, 4H), 1.37ꢀ1.62 (m, 3H), 2.39

(s, 3H), 2.66 (m, 1H), 4.31 (d, 1H, J = 17.1 Hz), 4.51 (d, 1H, J =

17.1 Hz), 6.68 (s, 1H), 7.19 (s, 1H), 7.55ꢀ7.70 (m, 4H), 7.79ꢀ7.82 (m,

1H), 8.00 (s, 1H), 8.05ꢀ8.11 (m, 2H), 12.22 (s, 1H). ¹³C NMR (150

MHz, DMSO-d₆): δ 25.2, 25.4, 25.5, 28.0, 28.6, 33.0, 33.1, 41.4, 59.5,

106.5, 119.5, 122.3, 125.7, 126.5, 127.0, 127.2, 128.4, 129.3, 129.5, 131.7,

133.9, 141.3, 145.4, 149.4, 154.7, 156.0, 168.5, 171.9. HRMS calcd for

C₂₈H₂₈N₅O₂S (M + H⁺) 498.1963; found 498.1955.

32⁰: Yield 34%; mp 207ꢀ209 °C. ¹H NMR (300 MHz, DMSO-d₆): δ

1.10ꢀ1.34 (m, 5H), 1.41ꢀ1.76 (m, 5H), 2.70 (s, 3H), 2.82 (m, 1H),

4.14 (d, 1H, J = 17.1 Hz), 4.23 (d, 1H, J = 17.1 Hz), 6.94 (d, 1H, J = 7.5

Hz), 7.42ꢀ7.54 (m, 4H), 7.60 (s, 1H), 7.65 (d, 1H, J = 8.4 Hz), 7.87ꢀ

7.92 (m, 2H), 8.20 (s, 1H), 12.58 (s, 1H). HRMS calcd for

C₂₈H₂₈N₅O₂S (M + H⁺) 498.1963; found 498.1953.

General Procedure for the Synthesis of Compounds 33ꢀ45.

Compound 21 (1 mmol) and various R-bromo substituted ketones

(1 mmol) were dissolved in EtOH (30 mL) and refluxed for 2 h. The

mixture was concentrated and the final products 33ꢀ45 were characterized

after purification by silica gel column chromatography.

6-(Cyclohexyl(methyl)amino)-7-(4-phenylthiazol-2-ylami-

no)quinoxalin-2(1H)-one (33). Yellow powder in 80% yield; mp

226ꢀ228 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.08ꢀ1.31 (m, 5H),

1.50ꢀ1.54 (m, 1H), 1.66ꢀ1.70 (m, 2H), 1.84ꢀ1.88 (m, 2H), 2.65

(s, 3H), 2.72 (m, 1H), 7.31ꢀ7.36 (m, 1H), 7.43ꢀ7.48 (m, 3H), 7.58

(s, 1H), 7.96 (s, 1H), 8.08 (d, 2H, J = 7.2 Hz), 8.58 (s, 1H), 9.75 (s, 1H),

12.72 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.4, 29.1, 38.4,

62.0, 101.4, 104.8, 123.3, 126.0, 127.0, 127.6, 128.5, 130.2, 134.2, 136.8,

139.8, 147.4, 150.1, 155.5, 162.0. HRMS calcd for C₂₄H₂₆N₅OS (M +

H⁺) 432.1858; found 432.1841.

6-(Cyclohexyl(methyl)amino)-7-(4-(4-fluorophenyl)thiazol-

2-ylamino)quinoxalin-2(1H)-one (34). Pale-yellow powder in 83%

yield; mp 245ꢀ247 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.08ꢀ1.26

(m, 5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.69 (m, 2H), 1.84ꢀ1.88 (m, 2H),

2.65 (s, 3H), 2.71 (m, 1H), 7.23ꢀ7.29 (m, 2H), 7.46 (s, 1H), 7.57 (s, 1H),

7.96 (s, 1H), 8.12ꢀ8.16 (m, 2H), 8.59 (s, 1H), 9.78 (s, 1H), 12.73 (s, 1H).

HRMS calcd for C₂₄H₂₅FN₅OS (M + H⁺) 450.1758; found 450.1759.

7-(4-(4-Chlorophenyl)thiazol-2-ylamino)-6-(cyclohexyl-

(methyl)amino)quinoxalin-2(1H)-one (35). Pale-yellow pow-

der in 87% yield; mp 278ꢀ280 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 1.08ꢀ1.27 (m, 5H), 1.50ꢀ1.54 (m, 1H), 1.66ꢀ1.70 (m, 2H),

1.85ꢀ1.88 (m, 2H), 2.65 (s, 3H), 2.72 (m, 1H), 7.48 (d, 2H, J = 8.4

Hz), 7.55 (m, 2H), 7.57 (s, 1H), 8.11 (d, 2H, J = 8.4 Hz), 8.58 (s, 1H),

9.81 (s, 1H), 12.74 (s, 1H). HRMS calcd for C₂₄H₂₅ClN₅OS (M +

H⁺) 466.1463; found 466.1455.

6-(Cyclohexyl(methyl)amino)-7-(4-oxo-3-phenyl-2-thiox-

oimidazolidin-1-yl)quinoxalin-2(1H)-one (31). To a stirred

solution of compound 14 (1 mmol) in 1,4-dioxane (50 mL) was added

Et₃N (2 mmol) and 2-chloroacetyl chloride (1 mmol). The mixture was

refluxed for 8 h. After the reaction was completed, the solution was

evaporated in vacuo. The final product 31 was obtained as a yellow

powder after purification by silica gel column chromatography eluting

4-(2-(7-(Cyclohexyl(methyl)amino)-3-oxo-3,4-dihydro-

quinoxalin-6-ylamino)thiazol-4-yl)benzonitrile (36). Light-yel-

low powder in 85% yield; mp 261ꢀ263 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.07ꢀ1.26 (m, 5H), 1.53 (m, 1H), 1.66ꢀ1.69 (m, 2H),

1.85ꢀ1.88 (m, 2H), 2.65 (s, 3H), 2.72 (m, 1H), 7.58 (s, 1H), 7.78(s, 1H),

7.88 (d, 2H, J= 8.4 Hz), 7.97 (s, 1H), 8.28 (d, 2H,J = 8.4 Hz), 8.61 (s, 1H),

9.91 (s, 1H), 12.77 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.5,

29.1, 38.5, 62.1, 101.7, 108.9, 109.6, 119.0, 123.4, 126.6, 127.1, 130.1, 132.6,

136.9, 138.3, 139.7, 147.6, 148.3, 155.5, 162.4. HRMS calcd for C₂₅H₂₅-

N₆OS (M + H⁺) 457.1810; found 457.1792.

6-(Cyclohexyl(methyl)amino)-7-(4-(4-methoxyphenyl)thia-

zol-2-ylamino)quinoxalin-2(1H)-one (37). Yellow powder in 83%

yield; mp 226ꢀ228 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.09ꢀ1.27

(m, 5H), 1.50ꢀ1.54 (m, 1H), 1.66ꢀ1.88 (m, 4H), 2.65 (s, 3H), 2.72

1

with CHCl₃ꢀCH₃OH (40:1, v/v). Yield 51%; mp 140ꢀ142 °C. H

NMR (300 MHz, DMSO-d₆): δ 1.07ꢀ1.23 (m, 5H), 1.56ꢀ1.70

(m, 5H), 2.60 (s, 3H), 2.74 (m, 1H), 4.11 (d, 1H, J = 17.4 Hz), 4.19

(d, 1H, J = 17.4 Hz), 6.83ꢀ6.85 (m, 2H), 7.07ꢀ7.12 (m, 1H), 7.25 (s,

1H), 7.32ꢀ7.37 (m, 2H), 7.57 (s, 1H), 8.19 (s, 1H), 12.47 (s, 1H). ¹³C

NMR (125 MHz, DMSO-d₆): δ 25.3, 25.6, 25.7, 28.6, 29.8, 32.9, 33.5,

62.2, 117.2, 120.4, 121.9, 124.2, 127.3, 129.3, 132.9, 145.9, 147.9, 152.6,

154.4, 155.2, 170.9. HRMS calcd for C₂₄H₂₆N₅O₂S (M + H⁺)

448.1802; found 448.1804.

6-(Cyclohexyl(methyl)amino)-7-(3-(naphthalen-1-yl)-5-oxo-

2-thioxoimidazolidin-1-yl)quinoxalin-2(1H)-one (32). To a stir-

red solution of compound 17 (1 mmol) in 1,4-dioxane (50 mL) was added

5762

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

(m, 1H), 3.80 (s, 3H), 6.99 (d, 2H, J = 8.4 Hz), 7.30 (s, 1H), 7.57 (s, 1H),

7.95 (s, 1H), 8.01 (d, 2H, J = 8.4 Hz), 8.57 (s, 1H), 9.71 (s, 1H), 12.71

(s, 1H). HRMS calcd for C₂₅H₂₈N₅O₂S (M + H⁺) 462.1958; found

492.1961.

12.46 (s, 1H). HRMS calcd for C₂₅H₂₆N₅OS (M + H⁺) 444.1853; found

444.1851.

6-(Cyclohexyl(methyl)amino)-3-methyl-7-(4-phenylthiazol-

2-ylamino)quinoxalin-2(1H)-one (51). To a stirred solution of 1,5-

difluoro-2,4-dinitrobenzene (DFDNB, 2.04 g, 10 mmol) in THF (50 mL)

was added DIPEA (10 mmol) and N-methylcyclohexanamine (10 mmol).

After vigorously stirring at room temperature until the total disappearance

of DFDNB, compound 46 was obtained without purification. Then,

7-(4-(Benzo[d][1,3]dioxol-5-yl)thiazol-2-ylamino)-6-(cyclo-

hexyl(methyl)amino)quinoxalin-2(1H)-one (38). Yellow powder

1

in 70% yield; mp 209ꢀ210 °C. H NMR (300 MHz, DMSO-d₆): δ

1.08ꢀ1.27 (m, 5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.88 (m, 4H), 2.65 (s,

3H), 2.71 (m, 1H), 6.06 (s, 2H), 6.96 (d, 1H, J = 7.8 Hz), 7.34 (s, 1H),

7.57 (s, 1H), 7.63ꢀ7.66 (m, 2H), 7.95 (d, 1H, J = 2.1 Hz), 8.53 (s, 1H),

9.71 (s, 1H), 12.74 (s, 1H). HRMS calcd for C₂₅H₂₆N₅O₃S (M + H⁺)

476.1751; found 476.1769.

NH₂CH(CH₃)COOCH₃HCl (10 mmol) and DIPEA (20 mmol) were

3

added and stirred under reflux for 8 h. The solvent was removed under

reduced pressure to give compound 47a that was used directly. The above

two reactions were traced by a fast LC-MS system until all of the starting

material was changed into the anticipated compound. Compound 47a

(1 g) was dissolved in a mixed solvent of THF (30 mL) and EtOH

(30 mL), followed by the addition of 10% Pd/C (1 g) and HCOONH₄

(2 g). The mixture was stirred at room temperature for 3 h. The residue

solid was filtered off, and the filtrate was concentrated in vacuo. Water

(50 mL) was added to the resulting product and then was extracted by

CH₂Cl₂(3 ꢁ 50 mL). The organic layers were combined, dried over

anhydrous MgSO₄, and evaporated in vacuo. Intermediate 48a was

obtained as a yellow solid after chromatography through silica gel eluting

6-(Cyclohexyl(methyl)amino)-7-(4-(naphthalen-1-yl)thiazol-

2-ylamino)quinoxalin-2(1H)-one (39). Yellow powder in 82% yield;

mp 247ꢀ249 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.10ꢀ1.31 (m, 5H),

1.52ꢀ1.55 (m, 1H), 1.69ꢀ1.72 (m, 2H), 1.86ꢀ1.90 (m, 2H), 2.67 (s, 3H),

2.76 (m, 1H), 7.27 (s, 1H), 7.54ꢀ7.63 (m, 4H), 7.87ꢀ7.98 (m, 4H), 8.29

(s, 1H), 8.37ꢀ8.40 (m, 1H), 9.73 (s, 1H), 12.43 (s, 1H). HRMS calcd for

C₂₈H₂₈N₅OS (M + H⁺) 482.2009; found 482.1955.

7-(4-(Biphenyl-4-yl)thiazol-2-ylamino)-6-(cyclohexyl(methyl)-

amino)quinoxalin-2(1H)-one (40). Yellow powder in 85% yield; mp

1

with CHCl₃ꢀCH₃OH (30:1, v/v). Yield 70%. H NMR (300 MHz,

232ꢀ234 °C. H NMR (300 MHz, DMSO-d₆): δ 1.06ꢀ1.28 (m, 5H),

DMSO-d₆): δ 1.03ꢀ1.25 (m, 3H), 1.28ꢀ1.36 (m, 2H), 1.54 (m, 1H),

1.67ꢀ1.74 (m, 4H), 2.26 (s, 3H), 2.54 (s, 3H), 2.65ꢀ2.72 (m, 1H), 5.49

(s, 2H), 6.44 (s, 1H), 7.25 (s, 1H), 11.88 (s, 1H). HRMS calcd for

C₁₆H₂₃N₄O (M + H⁺) 287.1866; found 287.1856.

1.50ꢀ1.54 (m, 1H), 1.67ꢀ1.71 (m, 2H), 1.86ꢀ1.90 (m, 2H), 2.66 (s, 3H),

2.73 (m, 1H), 7.35ꢀ7.40 (m, 1H), 7.47ꢀ7.52 (m, 2H), 7.55 (s, 1H), 7.58

(s,1H),7.72ꢀ7.77 (m, 4H), 7.96 (d, 1H, J= 1.5 Hz), 8.18 (d, 2H, J=8.7Hz),

8.63 (s, 1H), 9.79 (s, 1H), 12.76 (s, 1H). HRMS calcd for C₃₀H₃₀N₅OS(M+

H⁺) 508.2165; found 508.2188.

Intermediate 48a (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated in vacuo to give

compound 49a without puriﬁcation. Intermediate 49a (1 mmol) and

K₂CO₃(1.5 mmol) in CH₃OH (20 mL)/H₂O (5 mL) were heated to

reﬂux for 1 h. After evaporation of the solvent in vacuo, compound 50a

was obtained as a yellow solid after chromatography through silica gel

6-(Cyclohexyl(methyl)amino)-7-(4-(furan-2-yl)thiazol-2-

ylamino)quinoxalin-2(1H)-one (41). Yellow powder in 77%

1

yield; mp 196ꢀ197 °C. H NMR (300 MHz, DMSO-d₆): δ 1.08ꢀ

1.25 (m, 5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.70 (m, 2H), 1.84ꢀ1.88 (m,

2H), 2.64 (s, 3H), 2.72 (m, 1H), 6.63 (dd, 1H, J₁= 1.5 Hz, J₂= 2.7 Hz),

7.11 (s, 1H), 7.14 (d, 1H, J = 2.7 Hz), 7.57 (s, 1H), 7.72 (s, 1H), 7.95

(d, 1H, J = 1.5 Hz), 8.63 (s, 1H), 9.85 (s, 1H), 12.61 (s, 1H). HRMS

calcd for C₂₂H₂₄N₅O₂S (M + H⁺) 422.1645; found 422.1666.

6-(Cyclohexyl(methyl)amino)-7-(4-(thiophen-2-yl)thiazol-

2-ylamino)quinoxalin-2(1H)-one (42). Yellow powder in 75%

1

eluting with CHCl₃ꢀCH₃OH (30:1, v/v). Yield 75%. H NMR (300

MHz, DMSO-d₆): δ 1.07ꢀ1.34 (m, 3H), 1.22ꢀ1.30 (m, 2H), 1.55 (m,

1H), 1.68ꢀ1.79 (m, 4H), 2.35 (s, 3H), 2.61 (s, 3H), 2.65ꢀ2.68 (m, 1H),

7.40 (s, 1H), 7.91 (s, 2H), 8.14 (s, 1H), 9.08 (s, 1H), 12.16 (s, 1H).

HRMS calcd for C₁₇H₂₄N₅OS (M + H⁺) 346.1696; found 346.1680.

50a (0.5 mmol) and 2-bromo-1-phenylethanone (0.5 mmol) were

dissolved in EtOH (30 mL) and reﬂuxed for 2 h. After the reaction was

completed, the solvent was evaporated in vacuo. The ﬁnal product 51

was obtained as a yellow powder after puriﬁcation by silica gel column

chromatography eluting with CHCl₃ꢀCH₃OH (80:1, v/v). Yield 84%;

1

yield; mp 196ꢀ197 °C. H NMR (300 MHz, DMSO-d₆): δ 1.08ꢀ

1.26 (m, 5H), 1.50ꢀ1.54 (m, 1H), 1.66ꢀ1.70 (m, 2H), 1.84ꢀ1.88 (m,

2H), 2.65 (s, 3H), 2.72 (m, 1H), 7.11ꢀ7.14 (m, 1H), 7.22 (s, 1H),

7.49ꢀ7.51 (m, 1H), 7.57 (s, 1H), 7.70ꢀ7.71 (m, 1H), 7.95 (s, 1H), 8.43

(s, 1H), 9.75 (s, 1H), 12.63 (s, 1H). HRMS calcd for C₂₂H₂₄N₅OS₂

(M + H⁺) 438.1417; found 438.1408.

1

mp 229ꢀ231 °C. H NMR (300 MHz, DMSO-d₆): δ 1.08ꢀ1.26 (m,

6-(Cyclohexyl(methyl)amino)-7-(4-(pyridin-3-yl)thiazol-

2-ylamino)quinoxalin-2(1H)-one (43). Yellow powder in 73%

5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.70 (m, 2H), 1.84ꢀ1.88 (m, 2H), 2.36

(s, 3H), 2.64 (s, 3H), 2.71 (m, 1H), 7.33ꢀ7.35 (m, 1H), 7.43ꢀ7.48 (m,

3H), 7.51 (s, 1H), 8.08 (d, 2H, J = 7.2 Hz), 8.55 (s, 1H), 9.68 (s, 1H),

12.62 (s, 1H). HRMS calcd for C₂₅H₂₈N₅OS (M + H⁺) 446.2009; found

446.1987.

1

yield; mp 156ꢀ158 °C. H NMR (300 MHz, DMSO-d₆): δ 1.01ꢀ

1.27 (m, 5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.70 (m, 2H), 1.85ꢀ1.88 (m,

2H), 2.65 (s, 3H), 2.72 (m, 1H), 7.46ꢀ7.50 (m, 1H), 7.58 (s, 1H),

7.66 (s, 1H), 7.96 (s, 1H), 8.43ꢀ8.45 (m, 1H), 8.54ꢀ8.55 (m, 1H),

8.63 (s, 1H), 9.32 (d, 1H, J = 1.5 Hz), 9.87 (s, 1H), 12.74 (s, 1H).

HRMS calcd for C₂₃H₂₅N₆OS (M + H⁺) 433.1805; found 433.1819.

6-(Cyclohexyl(methyl)amino)-7-(4-(trifluoromethyl)thiazol-

2-ylamino)quinoxalin-2(1H)-one (44). Yellow powder in 81% yield;

mp 225ꢀ227 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.06ꢀ1.25 (m, 5H),

1.52ꢀ1.84 (m, 5H), 2.63 (s, 3H), 2.70 (m, 1H), 7.56 (s, 1H), 7.74 (s, 1H),

7.96 (s, 1H), 8.25 (s, 1H), 10.04 (s, 1H), 12.56 (s, 1H). HRMS calcd for

C₁₉H₂₁N₅OF₃S (M + H⁺) 424.1418; found 424.1424.

6-(Cyclohexyl(methyl)amino)-3-isopropyl-7-(4-phenylthia-

zol-2-ylamino)quinoxalin-2(1H)-one (52). Compound 52 was

prepared in a similar manner to the synthesis of compound 51, sub-

stituting NH₂CH(CH(CH₃)₂)COOCH₃HCl for NH₂CH(CH₃)-

3

COOCH₃HCl. The final product 52 was obtained as a yellow solid

3

after purification by silica gel column chromatography. Yield 86%

(the last step); mp 208ꢀ210 °C. ¹H NMR (300 MHz, DMSO-d₆): δ

1.05ꢀ1.27 (m, 11H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.69 (m, 2H), 1.84ꢀ

1.88 (m, 2H), 2.65 (s, 3H), 2.72 (m, 1H), 3.41ꢀ3.46 (m, 1H),

7.33ꢀ7.35 (m, 1H), 7.43ꢀ7.48 (m, 3H), 7.52 (s, 1H), 8.08 (d, 2H, J =

7.2 Hz), 8.55 (s, 1H), 9.68 (s, 1H), 12.62 (s, 1H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 20.1, 24.9, 25.5, 29.2, 29.5, 38.6, 62.1, 101.3, 104.5,

122.8, 126.0, 126.4, 127.5, 128.5, 129.8, 134.2, 136.5, 138.6, 150.1,

154.7, 161.8, 162.1. HRMS calcd for C₂₇H₃₂N₅OS (M + H⁺)

474.2322; found 474.2349.

7-(8H-Indeno[1,2-d]thiazol-2-ylamino)-6-(cyclohexyl-

(methyl)amino)quinoxalin-2(1H)-one (45). Light-yellow

powder in 73% yield; mp 262ꢀ264 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 1.01ꢀ1.31 (m, 5H), 1.50ꢀ1.53 (m, 1H), 1.66ꢀ1.70 (m,

2H), 1.86ꢀ1.89 (m, 2H), 2.66 (s, 3H), 2.73 (m, 1H), 3.86 (s, 2H),

7.20ꢀ7.25 (m, 1H), 7.36ꢀ7.40 (m, 1H), 7.54 (d, 1H, J = 7.5 Hz), 7.57 (s,

1H), 7.67 (d, 1H, J = 7.5 Hz), 7.95 (s, 1H), 8.67 (s, 1H), 9.81 (s, 1H),

5763

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

6-(Cyclohexyl(methyl)amino)-3-isobutyl-7-(4-phenylthia-

zol-2-ylamino)quinoxalin-2(1H)-one (53). Compound 53 was

prepared in a similar manner to the synthesis of compound 51,

was obtained as a yellow powder after puriﬁcation by silica gel column

chromatography eluting with CHCl₃ꢀCH₃OH (50:1, v/v). Yield 75%;

1

mp 130ꢀ131 °C. H NMR (300 MHz, DMSO-d₆): δ 1.15ꢀ1.31 (m,

substituting NH₂CH(CH₂CH(CH₃)₂)COOCH₃HCl for NH₂CH-

5H), 1.59ꢀ1.63 (m, 1H), 1.71ꢀ1.91 (m, 4H), 2.09 (s, 3H), 2.80ꢀ2.85

(m, 2H), 2.94ꢀ2.99 (m, 2H), 3.23 (m, 1H), 3.40 (s, 3H), 5.58 (d, 1H,

J = 8.1 Hz), 6.52 (s, 1H), 7.17 (s, 1H), 7.26ꢀ7.31 (m, 1H), 7.36ꢀ7.43

(m, 2H), 7.57 (s, 1H), 7.88 (d, 2H, J = 7.2 Hz), 11.97 (s, 1H). HRMS

calcd for C₂₇H₃₂N₅OS₂(M + H⁺) 506.2043; found 506.2064.

3

(CH₃)COOCH₃HCl. The final product 53 was obtained as a pale-

3

yellow powder after purification by silica gel column chromatography.

Yield 77% (the last step); mp 223ꢀ224 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 0.92 (d, 6H, J = 6.3 Hz), 1.08ꢀ1.27 (m, 5H), 1.49ꢀ1.53

(m, 1H), 1.66ꢀ1.70 (m, 2H), 1.84ꢀ1.88 (m, 2H), 2.19ꢀ2.24 (m, 1H),

2.61 (d, 2H, J = 7.5 Hz), 2.65 (s, 3H), 2.71 (m, 1H), 7.30ꢀ7.35 (m, 1H),

7.42ꢀ7.48 (m, 3H), 7.53 (s, 1H), 8.07 (d, 2H, J = 7.2 Hz), 8.54 (s, 1H),

9.67 (s, 1H), 12.61 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 22.6,

24.9, 25.5, 26.3, 29.2, 38.4, 41.3, 62.0, 101.3, 104.5, 122.7, 126.0, 126.6,

127.5, 128.5, 129.8, 134.2, 136.5, 138.6, 150.1, 155.4, 157.4, 162.1.

HRMS calcd for C₂₈H₃₄N₅OS (M + H⁺) 488.2479; found 488.2502.

3-Benzyl-6-(cyclohexyl(methyl)amino)-7-(4-phenylthiazol-

2-ylamino)quinoxalin-2(1H)-one (54). Compound 54 was pre-

pared in a similar manner to the synthesis of compound 51, substituting

NH₂CH(CH₂Ph)COOCH₃HCl for NH₂CH(CH₃)COOCH₃HCl.

2-(Cyclohexyl(methyl)amino)-3-(4-phenylthiazol-2-ylamino)-

7,8,9,10-tetrahydro-5H-pyrido[1,2-a]quinoxalin-6(6aH)-one

(60). To a stirred solution of compound 46 (10 mmol) in THF (50 mL),

DIPEA (10 mmol) and ethyl piperidine-2-carboxylate (10 mmol) was

added. After vigorously stirring at 65 °C until the total disappearance of

the starting materials, compound 56 was obtained without purification.

Compound 56 (1 g) was dissolved in a mixed solvent of THF (30 mL)

and EtOH (30 mL), followed by the addition of 10% Pd/C (1 g) and

HCOONH₄(2 g). The mixture was stirred at room temperature for 3 h.

The residue solid was filtered off, and the filtrate was concentrated in

vacuo. Water (50 mL) was added to the resulting product and then was

extracted by CH₂Cl₂(3 ꢁ 50 mL). The organic layers were combined,

dried over anhydrous MgSO₄, and evaporated in vacuo. The intermedi-

ate 57 was obtained as a white solid after purification by silica gel column

chromatography with a yield of 68%. ¹H NMR (300 MHz, DMSO-d₆): δ

1.00ꢀ1.53 (m, 9H), 1.66ꢀ1.79 (m, 6H), 2.00ꢀ2.03 (m, 1H), 2.40ꢀ

2.43 (m, 1H), 2.53 (s, 3H), 2.60ꢀ2.66 (m, 1H), 3.09ꢀ3.13 (m, 1H),

3.50ꢀ3.54 (m, 1H), 4.35 (s, 2H), 6.22 (s, 1H), 6.46 (s, 1H), 10.08 (s,

1H). HRMS calcd for C₁₉H₂₉N₄O (M + H⁺) 329.2336; found 329.2320.

Intermediate 57 (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated to give compound 58

without puriﬁcation. Intermediate 58 (1 mmol) and K₂CO₃(1.5 mmol)

in CH₃OH (20 mL)/H₂O (5 mL) were heated to reﬂux for 1 h.

Compound 59 was obtained as a white solid after evaporation of the

solvent in vacuo and chromatography through silica gel eluting with

CHCl₃ꢀCH₃OH (40:1, v/v). Yield 69%. ¹H NMR (300 MHz, DMSO-

d₆): δ 0.99ꢀ1.17 (m, 4H), 1.29ꢀ1.54 (m, 6H), 1.67ꢀ1.70 (m, 4H),

1.76ꢀ1.80 (m, 2H), 2.00ꢀ2.03 (m, 1H), 2.55 (s, 3H), 2.67ꢀ2.74 (m,

1H), 3.35ꢀ3.39 (m, 1H), 3.68ꢀ3.72 (m, 1H), 6.51 (s, 1H), 7.06 (s, 1H),

7.34 (s, 2H), 8.61 (s, 1H), 10.24 (s, 1H). HRMS calcd for C₂₀H₃₀N₅OS

(M + H⁺) 388.2166; found 388.2157.

3

The final product 54 was obtained as a pale-yellow powder after purifi-

cation by silica gel column chromatography. Yield 71% (the last step); mp

239ꢀ241 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.07ꢀ1.26 (m, 5H),

1.49ꢀ1.52 (m, 1H), 1.65ꢀ1.87 (m, 4H), 2.64 (s, 3H), 2.70 (m, 1H), 4.08

(s, 2H), 7.19ꢀ7.22 (m, 1H), 7.26ꢀ7.35 (m, 5H), 7.42ꢀ7.47 (m, 3H),

7.51 (s, 1H), 8.07 (d, 2H, J = 7.2 Hz), 8.55 (s, 1H), 9.70 (s, 1H), 12.70

(s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.4, 29.2, 38.4, 62.0,

101.3, 104.5, 122.8, 126.0, 126.1, 126.5, 127.5, 128.2, 128.5, 129.0, 130.1,

134.2, 136.7, 138.0, 138.9, 150.1, 155.1, 156.4, 162.1. HRMS calcd for

C₃₁H₃₂N₅OS (M + H⁺) 522.2322; found 522.2345.

6-(Cyclohexyl(methyl)amino)-3-(2-(methylthio)ethyl)-7-(4-

phenylthiazol-2-ylamino)quinoxalin-2(1H)-one (55). To a stir-

red solution of compound 46 (10 mmol) in THF (50 mL) was added

DIPEA (20 mmol) and NH₂CH((CH₂)₂SCH₃)COOCH₃HCl (10

3

mmol). After vigorously stirring at 65 °C until the total disappearance

of the starting materials, compound 47e was obtainedwithout purification.

Compound 47e (5 mmol) and SnCl₂2H₂O (75 mmol) were dissolved in

3

ethanol (75 mL). Then 15 equiv of 12 mol/L hydrochloric acid was added

and the mixture was stirred under reflux for 3 h. The solvent was then

evaporated. The residue was neutralized with 40% NaOH until the pH

reached 8. The mixture was filtered, and the filtrate was extracted with

100 mL of CHCl₃twice. The organic solvent was washed with brine, dried

over anhydrous sodium sulfate, and evaporatedin vacuo. The intermediate

48e was obtained as a yellow solid after purification by silica gel column

chromatography. Yield 51%. ¹H NMR (300 MHz, DMSO-d₆): δ 1.03ꢀ

1.19(m, 3H), 1.26ꢀ1.36(m, 2H),1.54(m, 1H), 1.67ꢀ1.76(m, 4H),2.08

(s, 3H), 2.55 (s, 3H), 2.65ꢀ2.72 (m, 1H), 2.78ꢀ2.84 (m, 2H), 2.89ꢀ2.95

(m, 2H), 5.56 (s, 2H), 6.44 (s, 1H), 7.20 (s, 1H), 11.92 (s, 1H). HRMS

calcd for C₁₈H₂₇N₄OS (M + H⁺) 347.1900; found 347.1889.

Compound 59 (0.5 mmol) and 2-bromo-1-phenylethanone (0.5 mmol)

were dissolved in ethanol (30 mL) and reﬂuxed for 2 h. After the reaction

was completed, the solvent was evaporated in vacuo. The ﬁnal product

60 was obtained as a white solid after puriﬁcation by silica gel column

chromatography eluting with CHCl₃ꢀCH₃OH (50:1, v/v). Yield 73%;

1

mp 200ꢀ202 °C. H NMR (300 MHz, DMSO-d₆): δ 1.07ꢀ1.24 (m,

6H), 1.41ꢀ1.48 (m, 4H), 1.64ꢀ1.68 (m, 2H), 1.74ꢀ1.79 (m, 3H), 2.05

(m, 1H), 2.58 (s, 3H), 2.68 (m, 1H), 3.30 (s, 1H), 3.31ꢀ3.35 (m, 1H),

3.67ꢀ3.71 (m, 1H), 6.67 (s, 1H), 7.23 (s, 1H), 7.26ꢀ7.31 (m, 1H),

7.38ꢀ7.43 (m, 2H), 7.80 (s, 1H), 7.96 (d, 2H, J = 7.8 Hz), 8.94 (s, 1H),

10.59 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 23.0, 23.3, 25.0, 25.5,

26.5, 29.3, 29.6, 37.6, 46.3, 58.7, 61.9, 102.4, 106.1, 107.5, 123.5, 125.9,

127.3, 128.4, 129.3, 130.7, 134.6, 136.3, 150.0, 163.9, 168.0. HRMS calcd

for C₂₈H₃₄N₅OS (M + H⁺) 488.2479; found 488.2495.

Intermediate 48e (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated to give compound

49e without puriﬁcation. Intermediate 49e (1 mmol) and K₂CO₃

(1.5 mmol) in CH₃OH (20 mL)/H₂O (5 mL) were heated to reﬂux

for 1 h. After evaporation of the solvent in vacuo, compound 50e was

obtained as a yellow solid after chromatography through silica gel eluting

8-(Cyclohexyl(methyl)amino)-7-(4-phenylthiazol-2-ylamino)-

pyrrolo[1,2-a]quinoxalin-4(5H)-one (65). To a stirred solution of

compound 46 (10 mmol) in THF (50 mL) was added DIPEA (10 mmol)

and methyl pyrrolidine-2-carboxylate (10 mmol). After vigorously stirring

at 65 °C until the total disappearance of the starting materials, compound

61 was obtained without purification. Compound 61 (5 mmol) and 10%

Pd/C (5 g) were dissolved in ethanol (50 mL). Then cyclohexene

(5 mmol) was added, and the mixture was stirred under reflux for 2 h.

The mixture was filtered, and the filtrate was evaporated in vacuo. The

intermediate 62 was obtained as a white solid after purification by silica gel

1

with CHCl₃ꢀCH₃OH (10:1, v/v). Yield 66%. H NMR (300 MHz,

DMSO-d₆): δ 1.07ꢀ1.13 (m, 3H), 1.27ꢀ1.31 (m, 2H), 1.55 (m, 1H),

1.68ꢀ1.79 (m, 4H), 2.09 (s, 3H), 2.59 (s, 3H), 2.62ꢀ2.65 (m, 1H),

2.83ꢀ2.88 (m, 2H), 3.00ꢀ3.04 (m, 2H), 7.42 (s, 1H), 7.92 (s, 2H), 8.17

(s, 1H), 9.10 (s, 1H), 12.22 (s, 1H). HRMS calcd for C₁₉H₂₈N₅OS₂

(M + H⁺) 406.1730; found 406.1716.

50e (0.5 mmol) and 2-bromo-1-phenylethanone (0.5 mmol) were

dissolved in EtOH (30 mL) and reﬂuxed for 2 h. After the reaction was

completed, the solvent was evaporated in vacuo. The ﬁnal product 55

5764

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

column chromatography. Yield 68%. ¹H NMR (300 MHz, DMSO-d₆): δ

1.12ꢀ1.21 (m, 3H), 1.27ꢀ1.37 (m, 2H), 1.55 (m, 1H), 1.68ꢀ1.78 (m,

4H), 2.60 (s, 3H), 2.72ꢀ2.79 (m, 1H), 4.98 (s, 2H), 6.53ꢀ6.55 (m, 2H),

6.87ꢀ6.88 (m, 1H), 7.57 (s, 1H), 8.04 (m, 1H), 10.89 (s, 1H). HRMS

calcd for C₁₈H₂₃N₄O (M + H⁺) 311.1866; found 311.1864.

After the reaction was completed, the solvent was evaporated in vacuo.

The ﬁnal product 70 was obtained as a light-yellow powder after puri-

ﬁcation by silica gel column chromatography eluting with CHCl₃ꢀ

CH₃OH (50:1, v/v). Yield 70%; mp 262ꢀ264 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 3.15 (s, 3H), 6.62 (d, 2H, J = 7.8 Hz), 6.71ꢀ6.76 (m, 1H),

7.14ꢀ7.20 (m, 2H), 7.31ꢀ7.36 (m, 1H), 7.44ꢀ7.49 (m, 4H), 7.96 (d,

1H, J = 1.8 Hz), 8.11 (d, 2H, J = 7.2 Hz), 8.89 (s, 1H), 10.14 (s, 1H),

12.89 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 39.3, 102.3, 105.3,

114.0, 117.6, 126.1, 127.5, 127.6, 128.6, 128.7, 131.8, 133.4, 134.2, 140.7,

147.9, 149.5, 149.9, 155.6, 162.2. HRMS calcd for C₂₄H₂₀N₅OS (M +

H⁺) 426.1383; found 426.1361.

6-(Cyclohexyl(ethyl)amino)-7-(4-phenylthiazol-2-ylamino)-

quinoxalin-2(1H)-one (71). Compound 71 was prepared in a similar

manner to the synthesis of compound 70, substituting N-ethylcyclohex-

anamine for N-methylaniline and DIPEA for K₂CO₃, respectively. The

final product 71 was obtained as a yellow powder after purification by silica

gel column chromatography. Yield 82% (the last step); mp 247ꢀ249 °C.

¹H NMR (300 MHz, DMSO-d₆): δ 0.85 (t, 3H, J = 6.6 Hz), 1.00ꢀ1.19

(m, 5H), 1.49ꢀ1.52 (m, 1H), 1.66 (m, 2H), 1.91 (m, 2H), 2.80 (m, 1H),

3.07 (q, 2H, J = 6.6 Hz), 7.31ꢀ7.36 (m, 1H), 7.43ꢀ7.49 (m, 3H), 7.58

(s, 1H), 7.96 (s, 1H), 8.09 (d, 2H, J = 7.8 Hz), 8.60 (s, 1H), 9.84 (s, 1H),

12.77 (s, 1H). HRMS calcd for C₂₅H₂₈N₅OS (M + H⁺) 446.2009; found

446.2026.

7-(4-Phenylthiazol-2-ylamino)-6-(piperidin-1-yl)quinoxalin-

2(1H)-one (72). Compound 72 was prepared in a similar manner to the

synthesis of compound 71, substituting piperidine for N-ethylcyclohex-

anamine. The final product 72 was obtained as a yellow powder after

purification by silica gel column chromatography. Yield 79% (the last step);

mp 248ꢀ250 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.55 (m, 2H), 1.78

(m, 4H), 2.81 (m, 4H), 7.33ꢀ7.35 (m, 1H), 7.43ꢀ7.49 (m, 4H), 7.96 (s,

1H), 8.06 (d, 2H, J = 7.2 Hz), 8.54 (s, 1H), 9.52 (s, 1H), 12.71 (s, 1H).

HRMS calcd for C₂₂H₂₂N₅OS (M + H⁺) 404.1540; found 404.1549.

6-Morpholino-7-(4-phenylthiazol-2-ylamino)quinoxalin-

2(1H)-one (73). Compound 73 was prepared in a similar manner to

the synthesis of compound 71, substituting morpholine for N-ethylcy-

clohexanamine. The final product 73 was obtained as a yellow powder

after purification by silica gel column chromatography. Yield 75% (the

last step); mp 234ꢀ236 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 2.86

(br s, 4H), 3.87 (br s, 4H), 7.30ꢀ7.35 (m, 1H), 7.43ꢀ7.48 (m, 2H),

7.50 (s, 1H), 7.54 (s, 1H), 7.97 (s, 1H), 8.07 (d, 2H, J = 7.2 Hz), 8.60 (s,

1H), 9.66 (s, 1H), 12.73 (s, 1H). HRMS calcd for C₂₁H₂₀N₅O₂S (M +

H⁺) 406.1332; found 406.1329.

6-(4-Methylpiperazin-1-yl)-7-(4-phenylthiazol-2-ylamino)-

quinoxalin-2(1H)-one (74). Compound 74 was prepared in a similar

mannerto the synthesisofcompound71, substituting 1-methylpiperazine

for N-ethylcyclohexanamine. The final product 74 was obtained as a

yellow powder after purification by silica gel column chromatography.

Yield 75% (the last step); mp 223ꢀ225 °C. ¹H NMR (300 MHz, DMSO-

d₆): δ 2.89 (s, 3H), 3.15 (br s, 4H), 3.47 (br s, 4H), 7.34ꢀ7.36 (m, 1H),

7.43ꢀ7.48 (m, 2H), 7.53 (s, 1H), 7.58 (s, 1H), 7.99 (s, 1H), 8.08 (d, 2H,

J = 7.2 Hz), 8.64 (s, 1H), 9.59 (s, 1H), 12.79 (s, 1H). HRMS calcd for

C₂₂H₂₃N₆OS (M + H⁺) 419.1649; found 419.1670.

Intermediate 62 (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated to give compound 63

without puriﬁcation. Intermediate 63 (1 mmol) and K₂CO₃(1.5 mmol)

in CH₃OH (20 mL)/H₂O (5 mL) were heated to reﬂux for 1 h.

Compound 64 was obtained as a white solid after evaporation of the

solvent and chromatography through silica gel eluting with CHCl₃ꢀ

1

CH₃OH (40:1, v/v). Yield 69%. H NMR (300 MHz, DMSO-d₆): δ

1.08ꢀ1.16 (m, 3H), 1.31ꢀ1.35 (m, 2H), 1.55 (m, 1H), 1.68ꢀ1.79

(m, 4H), 2.66 (s, 3H), 2.77 (m, 1H), 6.64ꢀ6.65 (m, 1H), 6.97ꢀ6.98

(m, 1H), 7.69 (m, 3H), 7.78 (s, 1H), 8.21 (s, 1H), 8.89 (s, 1H), 11.10

(s, 1H). HRMS calcd for C₁₉H₂₄N₅OS (M + H⁺) 370.1696; found

370.1693.

Compound 64 (0.5 mmol) and 2-bromo-1-phenylethanone (0.5 mmol)

were dissolved in ethanol (30 mL) and reﬂuxed for 2 h. After the reaction

was completed, the solvent was evaporated in vacuo. The ﬁnal product

65 was obtained as a white powder after puriﬁcation by silica gel column

chromatography eluting with CHCl₃ꢀCH₃OH (50:1, v/v). Yield 70%;

mp 248ꢀ250 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.02ꢀ1.32 (m,

5H), 1.50ꢀ1.53 (m, 1H), 1.67ꢀ1.71 (m, 2H), 1.85ꢀ1.88 (m, 2H), 2.69

(s, 3H), 2.74ꢀ2.81 (m, 1H), 6.63ꢀ6.65 (m, 1H), 6.97ꢀ6.99 (m, 1H),

7.29ꢀ7.34 (m, 1H), 7.37 (s, 1H), 7.42ꢀ7.47 (m, 2H), 7.92 (s, 1H),

8.07ꢀ8.09 (m, 2H), 8.20ꢀ8.21 (m, 1H), 8.48 (s, 1H), 9.39 (s, 1H),

11.55 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.5, 29.4, 38.3,

62.0, 103.7, 104.5, 110.6, 110.7, 112.1, 116.8, 117.7, 122.8, 125.4, 126.0,

127.4, 128.5, 134.4, 135.2, 136.0, 150.1, 155.3, 162.7. HRMS calcd for

C₂₇H₂₈N₅OS (M + H⁺) 470.2009; found 470.2028.

6-(Methyl(phenyl)amino)-7-(4-phenylthiazol-2-ylamino)-

quinoxalin-2(1H)-one (70). To a stirred solution of compound 2

(10 mmol) in THF (50 mL) was added K₂CO₃(10 mmol) and

N-methylaniline (10 mmol). The mixture was stirred under reflux for 8 h.

The solvent was removed under reduced pressure to give compound 66a

that was used directly. Compound 66a (1 g) was dissolved in a mixed

solvent of THF (30 mL) and ethanol (30 mL), followed by the addition

of 10% Pd/C (1 g) and HCOONH₄(2 g). The mixture was stirred at

room temperature for 3 h. The residue solid was filtered off, and the

filtrate was concentrated in vacuo. Water (50 mL) was added to the

resulting product and then was extracted by CH₂Cl₂(3 ꢁ 50 mL). The

organic layers were combined, dried over anhydrous MgSO₄, and

evaporated in vacuo. Intermediate 67a was obtained as a yellow solid

after chromatography through silica gel eluting with CHCl₃ꢀCH₃OH

(30:1, v/v). Yield 68%. ¹H NMR (300 MHz, DMSO-d₆): δ 3.11 (s, 3H),

5.85 (s, 2H), 6.56ꢀ6.58 (m, 3H), 6.64ꢀ6.69 (m, 1H), 7.10ꢀ7.15 (m,

2H), 7.25 (s, 1H), 7.69 (s, 1H), 12.08 (s, 1H). HRMS calcd for

C₁₅H₁₅N₄O (M + H⁺) 267.1240; found 267.1230.

Intermediate 67a (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated to give compound

68a without puriﬁcation. Intermediate 68a (1 mmol) and K₂CO₃

(1.5 mmol) in CH₃OH (20 mL)/H₂O (5 mL) were heated to reﬂux

for 1 h. Compound 69a was obtained as a yellow solid after evaporation

of the solvent and chromatography through silica gel eluting with

CHCl₃ꢀCH₃OH (40:1, v/v). Yield 84%. ¹H NMR (300 MHz, DMSO-

d₆): δ 2.07 (s, 3H), 6.65ꢀ6.67 (m, 2H), 6.73ꢀ6.78 (m, 1H), 7.14ꢀ7.19

(m, 2H), 7.47 (s, 1H), 7.87 (s, 2H), 8.02 (s, 1H), 8.52 (s, 1H), 9.23 (s, 1H),

12.42 (s, 1H). HRMS calcd for C₁₆H₁₆N₅OS (M + H⁺) 326.1070; found

326.1055.

Ethyl-1-(2-oxo-7-(4-phenylthiazol-2-ylamino)-1,2-dihydro-

quinoxalin-6-yl)-piperidine-4-carboxylate (75). Compound 75

was prepared in a similar manner to the synthesis of compound 71,

substituting ethyl piperidine-4-carboxylate for N-ethylcyclohexanamine.

The final product 75 was obtained as a yellow powder after purification

by silica gel column chromatography. Yield 71% (the last step); mp

259ꢀ261 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 1.21 (t, 3H, J = 7.5 Hz),

1.89ꢀ1.93 (m, 2H), 2.01ꢀ2.12 (m, 2H), 2.43 (m, 1H), 2.68ꢀ2.75 (m,

2H), 2.98ꢀ3.01 (m, 2H), 4.10 (q, 2H, J = 7.5 Hz), 7.30ꢀ7.35 (m, 1H),

7.42ꢀ7.45 (m, 2H), 7.48ꢀ7.49 (m, 2H), 7.96 (s, 1H), 8.06 (d, 2H, J =

7.5 Hz), 8.59 (s, 1H), 9.61 (s, 1H), 12.72 (s, 1H). HRMS calcd for

C₂₅H₂₆N₅O₃S (M + H⁺) 476.1751; found 476.1772.

Compound 69a (0.5 mmol) and 2-bromo-1-phenylethanone

(0.5 mmol) were dissolved in ethanol (30 mL) and reﬂuxed for 2 h.

5765

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

6-(4-(4-Fluorophenyl)piperazin-1-yl)-7-(4-phenylthiazol-

2-ylamino)quinoxalin-2(1H)-one (76). Compound 76 was pre-

pared in a similar manner to the synthesis of compound 71, substituting

1-(4-fluorophenyl)piperazine for N-ethylcyclohexanamine. The final

product 76 was obtained as a yellow powder after purification by silica

gel column chromatography. Yield 77% (the last step); mp 245ꢀ

247 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 3.02 (br s, 4H), 3.39 (br s,

4H), 7.00ꢀ7.08 (m, 4H), 7.33ꢀ7.36 (m, 1H), 7.43ꢀ7.48 (m, 2H),

7.50 (s, 1H), 7.57 (s, 1H), 7.98 (s, 1H), 8.07 (d, 2H, J = 7.5 Hz), 8.61 (s,

1H), 9.65 (s, 1H), 12.75 (s, 1H). HRMS calcd for C₂₇H₂₄FN₆OS (M +

H⁺) 499.1711; found 499.1735.

6-(Dimethylamino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-

2(1H)-one (77). Compound 77 was prepared in a similar manner to the

synthesis of compound 71, substituting dimethylamine for N-ethylcyclo-

hexanamine. The final product 77 was obtained as a yellow powder after

purification by silica gel column chromatography. Yield 80% (the last

step); mp 221ꢀ223 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 2.66 (s, 6H),

7.30ꢀ7.35 (m, 1H), 7.43ꢀ7.47 (m, 3H), 7.51 (s, 1H), 7.95 (s, 1H), 8.08

(d, 2H, J = 7.5 Hz), 8.68 (s, 1H), 9.99 (s, 1H), 12.74 (s, 1H). HRMS calcd

for C₁₉H₁₈N₅OS (M + H⁺) 364.1227; found 364.1225.

149.9, 163.4, 165.0. HRMS calcd for C₂₄H₂₇N₄O₂S (M + H⁺) 435.1849;

found 435.1868.

7-(Cyclohexyl(methyl)amino)-6-(4-phenylthiazol-2-ylamino)-

2H-benzo[b][1,4]thiazin-3(4H)-one (86). To a stirred solution of

compound 46 (10 mmol) in THF (50 mL) was added DIPEA (10

mmol) and HSCH₂COOCH₃(10 mmol). After vigorously stirring at

65 °C until the total disappearance of the starting materials, compound

81b was obtained without purification. Compound 81b (5 mmol) and

SnCl₂2H₂O (75 mmol) were dissolved in ethanol (75 mL). Then 15

3

equiv of 12 mol/L hydrochloric acid was added and the mixture was

stirred under reflux for 3 h. The solvent was then evaporated. The

residue was neutralized with 40% NaOH until the pH reached 8. The

mixture was filtered, and the filtrate was extracted with 100 mL of

CHCl₃twice. The organic solvent was washed with brine, dried over

anhydrous sodium sulfate, and evaporated in vacuo. The intermediate

82b was obtained as a white solid after purification by silica gel column

chromatography. Yield 49%. ¹H NMR (300 MHz, DMSO-d₆): δ

1.11ꢀ1.18 (m, 3H), 1.21ꢀ1.28 (m, 2H), 1.53 (m, 1H), 1.67ꢀ1.70

(m, 4H), 2.48 (s, 3H), 2.61ꢀ2.64 (m, 1H), 4.86 (s, 2H), 6.32 (s, 1H),

6.81 (s, 1H), 10.20 (s, 1H). HRMS calcd for C₁₅H₂₂N₃OS (M + H⁺)

292.1478; found 292.1472.

6-(Diethylamino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-

2(1H)-one (78). Compound 78 was prepared in a similar manner to the

synthesis of compound 71, substituting diethylamine for N-ethylcyclohex-

anamine. The final product 78 was obtained as a yellow powder after

purification by silica gel column chromatography. Yield 82% (the last step);

Intermediate 82b (1 mmol) and benzoyl isothiocyanate (1 mmol)

were dissolved in dried acetone (30 mL). The reaction mixture was

reﬂuxed with stirring for 4 h and then concentrated to give compound

83b without puriﬁcation. Intermediate 83b (1 mmol) and K₂CO₃

(1.5 mmol) in CH₃OH (20 mL)/H₂O (5 mL) were heated to reﬂux

for 1 h. After evaporation of the solvent in vacuo, compound 84b was

obtained as a white solid after chromatography through silica gel

eluting with CHCl₃ꢀCH₃OH (20:1, v/v). Yield 60%. ¹H NMR (300

MHz, DMSO-d₆): δ 1.06ꢀ1.18 (m, 3H), 1.23ꢀ1.34 (m, 2H),

1.51ꢀ1.54 (m, 1H), 1.68ꢀ1.71 (m, 4H), 2.53 (s, 3H), 2.62ꢀ2.65

(m, 1H), 3.42 (s, 2H), 7.00 (s, 1H), 7.46 (s, 1H), 7.60 (s, 2H), 8.78

(s, 1H), 10.40 (s, 1H). HRMS calcd for C₁₆H₂₃N₄O₂S (M + H⁺)

351.1308; found 292.1294.

1

mp 238ꢀ240 °C. H NMR (300 MHz, DMSO-d₆): δ 0.92 (t, 6H, J =

6.9 Hz), 2.98 (q, 4H, J = 6.9 Hz), 7.31ꢀ7.36 (m, 1H), 7.43ꢀ7.47 (m, 3H),

7.57 (s, 1H), 7.95 (d, 1H, J = 1.8 Hz), 8.09 (d, 2H, J = 7.5 Hz), 8.64 (s, 1H),

9.95 (s, 1H), 12.77 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 11.8, 48.7,

101.2, 104.9, 123.0, 126.0, 127.1, 127.5, 128.5, 130.4, 134.2, 134.3, 141.0,

147.4, 150.0, 155.6, 162.0. HRMS calcd for C₂₁H₂₂N₅OS (M + H⁺)

392.1540; found 392.1557.

6-(Dipropylamino)-7-(4-phenylthiazol-2-ylamino)quinoxalin-

2(1H)-one (79). Compound 79 was prepared in a similar manner to the

synthesis of compound 71, substituting dipropylamine for N-ethylcyclo-

hexanamine. The final product 79 was obtained as a yellow powder after

purification by silica gel column chromatography. Yield 69% (the last

step); mp 215ꢀ217 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 0.81 (t, 6H,

J = 7.5 Hz), 1.30ꢀ1.42 (m, 4H), 2.89 (t, 4H, J = 7.5 Hz), 7.31ꢀ7.36 (m,

1H), 7.43ꢀ7.49 (m, 3H), 7.61 (s, 1H), 7.95 (d, 1H, J = 2.1 Hz), 8.08 (d,

2H, J = 7.2 Hz), 8.59 (s, 1H), 9.79 (s, 1H), 12.75 (s, 1H). HRMS calcd for

C₂₃H₂₆N₅OS (M + H⁺) 420.1853; found 420.1872.

6-(Cyclohexyloxy)-7-(4-phenylthiazol-2-ylamino)quinoxalin-

2(1H)-one (80). Compound 80 was prepared in a similar manner to

the synthesis of compound 71, substituting sodium cyclohexanolate for

N-ethylcyclohexanamine. The final product 80 was obtained as a pale-

yellow solid after purification by silica gel column chromatography. Yield

78% (the last step); mp 215ꢀ216 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 1.22ꢀ1.44 (m, 4H), 1.53ꢀ1.60 (m, 2H), 1.76ꢀ1.80 (m, 2H), 2.02ꢀ

2.05 (m, 2H), 4.47 (m, 1H), 7.30ꢀ7.36 (m, 2H), 7.43ꢀ7.51 (m, 2H),

7.85ꢀ7.87 (m, 1H), 7.96 (s, 1H), 8.08 (d, 2H, J = 7.5 Hz), 8.73 (s, 1H),

9.76 (s, 1H), 12.75 (s, 1H). HRMS calcd for C₂₃H₂₃N₄O₂S (M + H⁺)

419.1536; found 419.1553.

84b (0.5 mmol) and 2-bromo-1-phenylethanone (0.5 mmol) were

dissolved in ethanol (30 mL) and reﬂuxed for 2 h. After the reaction was

completed, the solvent was evaporated in vacuo. The ﬁnal product 86

was obtained as a yellow powder after puriﬁcation by silica gel column

chromatography eluting with CHCl₃ꢀCH₃OH (40:1, v/v). Yield 81%;

1

mp 249ꢀ250 °C. H NMR (300 MHz, DMSO-d₆): δ 1.06ꢀ1.24 (m,

5H), 1.49ꢀ1.52 (m, 1H), 1.65ꢀ1.69 (m, 2H), 1.78ꢀ1.82 (m, 2H), 2.56

(s, 3H), 2.64 (m, 1H), 3.43 (s, 2H), 7.15 (s, 1H), 7.28ꢀ7.34 (m, 2H),

7.40ꢀ7.45 (m, 2H), 7.99 (d, 2H, J = 7.5 Hz), 8.13 (s, 1H), 9.24 (s, 1H),

10.75 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 24.9, 25.4, 29.2, 29.3,

37.9, 61.8, 103.6, 106.5, 110.3, 122.2, 125.9, 127.4, 128.4, 134.3, 134.4,

135.7, 136.1, 150.1, 162.7, 165.4. HRMS calcd for C₂₄H₂₇N₄OS₂(M +

H⁺) 451.1621; found 451.1643.

HCV Antiviral Assay. The Huh7 ET (luc-ubi-neo/ET) cell line

which harbors a dicistronic self-replicating HCV RNA replicon with a

firefly luciferase gene was used for antiviral evaluation.¹⁸The activity of

the luciferase reporter is proportional to HCV RNA levels. Briefly, the

replicon cells were seeded into two identical sets of 96-well plates at a

density of 5000/well in 100 μL of DMEM without G418 overnight.

Compounds were solubilized in DMSO and then dilutions prepared in

DMEM and added to the wells. Following 72 h incubation, one set of the

cells was processed to assess the replicon-derived luciferase activity with

the Steady-Glo luciferase assay system (Promega, Madison, WI) accord-

ing to manufacturer’s instruction. Another set of the plates was used to

determine cytotoxicity of the compounds using a tetrazolium-based

CytoTox-1 cell proliferation assay (Promega, Madison WI). Each data

point represents an average of four replicates to derive EC₅₀(con-

centration inhibiting HCV RNA replication activity by 50%), IC₅₀

(concentration decreasing cell viability by 50%), and SI₅₀(selective

index which is calculated by IC₅₀/EC₅₀) values.

7-(Cyclohexyl(methyl)amino)-6-(4-phenylthiazol-2-ylamino)-

2H-benzo[b][1,4]oxazin-3(4H)-one (85). Compound 85 was pre-

pared in a similar manner to the synthesis of compound 51, substituting

HOCH₂COOCH₃for NH₂CH(CH₃)COOCH₃HCl. The final pro-

3

duct 85 was obtained as a light-yellow powder after purification by silica

gel column chromatography. Yield 86% (the last step); mp 205ꢀ206 °C.

¹H NMR (300 MHz, DMSO-d₆): δ 1.07ꢀ1.24 (m, 5H), 1.53ꢀ1.80 (m,

5H), 2.54 (s, 3H), 2.61 (m, 1H), 4.52 (s, 2H), 6.84 (s, 1H), 7.28ꢀ7.32

(m, 2H), 7.39ꢀ7.44 (m, 2H), 7.97ꢀ8.01 (m, 3H), 9.09 (s, 1H), 10.90 (s,

1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 25.0, 25.5, 29.3, 37.6, 61.8, 66.8,

103.0, 106.0, 111.3, 123.4, 125.9, 127.4, 128.4, 131.9, 134.5, 136.0, 137.7,

5766

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

’ ASSOCIATED CONTENT

C virus polymerase: synthesis and biological characterization of un-

symmetrical dialkyl-hydroxynaphthalenoyl-benzothia-diazines. J. Med.

Chem. 2009, 52, 1659–1669.

S

Supporting Information. The general procedure for the

b

(7) (a) Thompson, A. J. V.; McHutchison, J. G. Review article:

investigational agents for chronic hepatitis C. Aliment. Pharmacol. Ther.

2009, 29, 689–705. (b) Perni, R. B.; Almquist, S. J.; Byrn, R. A.;

Chandorkar, G.; Chaturvedi, P. R.; Courtney, L. F.; Decker, C. J.;

Dinehart, K.; Gates, C. A.; Harbeson, S. L.; Heiser, A.; Kalkeri, G.;

Kolaczkowski, E.; Lin, K.; Luong, Y. P.; Rao, B. G.; Taylor, W. P.;

Thomson, J. A.; Tung, R. D.; Wei, Y. Y.; Kwong, A. D.; Lin, C. Preclinical

proﬁle of VX-950, a potent, selective, and orally bioavailable inhibitor of

hepatitis C virus NS3ꢀ4A serine protese. Antimicrob. Agents Chemother.

2006, 50, 899–909. (c) Njoroge, F. G.; Chen, K. X.; Shih, N. Y.; Piwinski,

J. J. Challenges in modern drug discovery: a case study of Boceprevir, an

HCV protease inhibitor for the treatment of hepatitis C virus infection.

Acc. Chem. Res. 2008, 41, 50–59.

synthesis of intermediates 50bꢀd, 69bꢀk, and 84a. NMR

spectra and HPLC analysis results of all the ﬁnal compounds.

This material is available free of charge via the Internet at http://

pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: +86 10 63167165. Fax: +86 10 63165246. E-mail: gliu@

imm.ac.cn.

’ ACKNOWLEDGMENT

(8) (a) Pierra, C.; Amador, A.; Benzaria, S.; Scott, E. C.; D’Amours,

M.; Mao, J.; Mathieu, S.; Moussa, A.; Bridges, E. G.; Standring, D. N.;

Sommadossi, J. P.; Storer, R.; Gosselin, G. Synthesis and pharma-

cokinetics of Valopicitabine (NM283), an eﬃcient prodrug of the

potent anti-HCV agent 2⁰-C-Methylcytidine. J. Med. Chem. 2006, 49,

6614–6620. (b) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. New NS5B

polymerase inhibitors for hepatitis C. Expert Opin. Invest. Drugs 2010,

19, 963–975. (c) Li, H.; Tatlock, J.; Linton, A.; Gonzalez, J.; Jewell, T.;

Patel, L.; Ludlum, S.; Drowns, M.; Rahavendran, S. V.; Skor, H.; Hunter,

R.; Shi, S. T.; Herlihy, K. J.; Parge, H.; Hickey, M.; Yu, X.; Chau, F.;

Nonomiya, J.; Lewis, C. Discovery of (R)-6-cyclopentyl-6-(2-(2,6-

diethylpyridin-4-yl)ethyl)-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-

2-yl)methyl)-4-hydroxy-5,6-dihydropyran-2-one (PF-00868554) as a

potent and orally available hepatitis C virus polymerase inhibitor.

J. Med. Chem. 2009, 52, 1255–1258. (d) Lawitz, E.; Cooper, C.;

Rodriguez-Torres, M.; Ghalib, R.; Lalonde, R.; Sheikh, A.; Bourgault,

B.; Chauret, N.; Proulx, L.; McHutchison, J. Safety, tolerability and antiviral

activity of VCH-916, a novel non-nucleoside HCV polymerase inhibitor

patients with chronic HCV genotype-1 ingection. J. Hepatol. 2009, 50, S37.

(9) Stauber, R. E.; Kessler, H. H. Drugs in development for hepatitis

C. Drugs 2008, 68, 1347–1359.

This research is supported ﬁnancially by the National Natural

Science Foundation of China (no. 90713045) and National

Institute of Health of the United States, NIAID contract N01-

AI-30047.

’ ABBREVIATIONS USED

HCV, hepatitis C virus; DFDNB, 1,5-diﬂuoro-2,4-dinitrobenzene;

DIPEA, diisopropylethylamine; NOESY, nuclear Overhauser eﬀect

spectroscopy; THF, tetrahydrofuran; EC₅₀, concentration of com-

pound inhibiting HCV replicon by 50%; IC₅₀, concentration of

compound decreasing cell viability by 50%; SI, selectivity index;

SAR, structureꢀactivity relationship

’ ADDITIONAL NOTE

^aTelaprevir and boceprevir were approved by USA FDA recently

for treatment of HCV infection.

(10) (a) Lawrence, D. S.; Copper, J. E.; Smith, C. D. Structure

ꢀactivity studies of substituted quinoxalinones as multiple-drug-resis-

tance antagonists. J. Med. Chem. 2001, 44, 594–601. (b) Ajani, O. O.;

Obafemi, C. A.; Nwinyi, O. C.; Akinpelu, D. A. Microwave assisted

synthesis and antimicrobial activity of 2-quinoxalinone-3-hydrazone

derivatives. Bioorg. Med. Chem. 2010, 18, 214–221. (c) Ries, U. J.;

Priepke, H. W. M.; Hauel, N. H.; Handschuh, S.; Mihm, G.; Stassen,

J. M.; Wienen, W.; Nar, H. Heterocyclic thrombin inhibitors. Part 2:

quinoxalinone derivatives as novel, potent antithrombotic agents. Bioorg.

Med. Chem. Lett. 2003, 13, 2297–2302. (d) Chen, D. B; Bao, W. L. An

eﬃcient domino synthesis of quinoxalin-2(1H)-ones via an S_NAr/

coupling/demesylation reaction catalyzed by copper(I) as key step.

Adv. Synth. Catal. 2010, 352, 955–960.

(11) Zhang, L.; Liu, G.; Zhang, S. D.; Yang, H. Z.; Li, L.; Wu, X. H.;

Yu, J. L.; Kou, B. B.; Xu, S.; Li, J.; Sun, G. C.; Ji, Y. F.; Cheng, G. F. Parallel

approach for solution-phase synthesis of 2-quinoxalinol analogues and

their inhibition of LPS-induced TNF-R release on mouse macrophages

in vitro. J. Comb. Chem. 2004, 6, 431–436.

(12) Hu, L. Y.; Guo, J. Q.; Magar, S. S.; Fischer, J. B.; Burke-Howie,

K. J.; Durant, G. J. Synthesis and pharmacological evaluation of N-(2,5-

disubstituted phenyl)-N⁰-(3-substituted phenyl)-N⁰-methylguanidines

as N-methyl-D-aspartate receptor ion-channel blockers. J. Med. Chem.

1997, 40, 4281–4289.

’ REFERENCES

(1) Tan, S. L.; Pause, A.; Shi, Y.; Sonenberg, N. Hepatitis C

therapeutic: current status and emerging strategies. Nature Rev. Drug

Discovery 2002, 1, 867–881.

(2) (a) Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley,

D. W.; Houghton, M. Isolation of a cDNA clone derived from a blood-

borne non-A, non-B viral hepatitis genome. Science 1989, 244, 359–362.

(b) Huang, Z. H.; Murray, M. G.; Secrist, J. A., III Recent development

of therapeutics for chronic HCV infection. Antiviral Res. 2006, 71,

351–362.

(3) Manns, M. P.; Foster, G. R.; Rockstroh, J. K.; Zeuzem, S.;

Zoulim, F.; Houghton, M. The way of forward in HCV treatment-

ﬁnding the right path. Nature Rev. Drug Discovery 2007, 6, 991–1000.

(4) Hoognagle, J. H. Treatment of chronic non-A, non-B hepatitis

with recombinant human alpha interferon: a preliminary report. N. Engl.

J. Med. 1986, 315, 1575–1578.

(5) Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Maltseva, T.;

Leveque, V. J.-P.; Rajyaguru, S.; Pogam, S. L.; Najera, I.; Benkestock, K.;

Zhou, X. X.; Kaiser, A. C.; Maag, H.; Cammack, N.; Martin, J. A.;

Swallow, S.; Johansson, N. G.; Klumpp, K.; Smith, M. The design,

synthesis, and antiviral activity of monoﬂuoro and diﬂuoro analogues of

4⁰-azidocytidine against hepatitis C virus replication: the discovery of 4⁰-

azido-2⁰-deoxy-2⁰-ﬂuorocytidine and 4⁰-azido-2⁰-dideoxy-2⁰,2,-diﬂuoro-

cytidine. J. Med. Chem. 2009, 52, 2971–2978.

(13) Ferris, A. F.; Schutz, B. A. The preparation of carbodiimides,

isocyanates, and isothiocyanates by metal ion-assisted elimination of

mercaptan. J. Org. Chem. 1963, 28, 71–74.

(14) D€ur€ust, Y.; Nohut, F. A new and convenient synthesis of some

substituted thiohydantoins. Synth. Commun. 1999, 29, 1997–2005.

(15) Chicharro, R.; Castro, S. D.; Reino, J. L.; Ar€an, V. J. Synthesis of

tri- and tetracyclic condensed quinoxalin-2-ones fused across the

C-3ꢀN-4 bond. Eur. J. Org. Chem. 2003, 12, 2314–2326.

(6) Wagner, R.; Larson, D. P.; Beno, D. W. A.; Bosse, T. D.;

Darbyshire, J. F.; Gao, Y.; Gates, B. D.; He, W. P.; Henry, R. F.;

Hernandez, L. E.; Hutchinson, D. K.; Jiang, W. W.; Kati, W. M.; Klein,

L. L.; Koev, G.; Kohlbrenner, W.; Krueger, A. C.; Liu, J. R.; Liu, Y. Y.;

Long, M. A.; Maring, C. J.; Masse, S. V.; Middleton, T.; Montgomery,

D. A.; Pratt, J. K.; Stuart, P.; Molla, A.; Kempf, D. J. Inhibitors of hepatitis

5767

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Journal of Medicinal Chemistry

ARTICLE

(16) Kim, H. J.; Sharon, A.; Bal, C.; Wang, J. N.; Allu, M.; Huang, Z. H.;

Murray, M. G.;Bassit, L.;Schinazi, R. F.;Korba, B.;Chu, C. K. Synthesisand

anti-hepatitis B virus and anti-hepatitis C virus activities of 7-deazanepla-

nocin a analogues in vitro. J. Med. Chem. 2009, 52, 206–213.

(17) (a) Frick, D. N.; Ginzburg, O.; Lam, A. M. A method to

simultaneously monitor hepatitis C virus NS3 helicase and protease

activities. Methods Mol. Biol. 2010, 587, 223–233. (b) Luo, G.; Hamatake,

R. K.; Mathis, D. M.; Racela, J.; Rigat, K. L.; Lemm, J.; Colonno, R. J. De

novo initiation of RNA synthesis by the RNA-dependent RNA polymer-

ase (NS5B) of hepatitis C virus. J. Virol. 2000, 74, 851–863.

(18) Pietschmann, T.; Lohmann, V.; Kaul, A.; Krieger, N.; Rinck, G.;

Rutter, G.; Strand, D.; Bartenschlager, R. Persistent and transient

replication of full-length hepatitis C virus genomes in cell culture.

J. Virol. 2002, 76, 4008–4021.

5768

dx.doi.org/10.1021/jm200394x |J. Med. Chem. 2011, 54, 5747–5768

Article Doi

Quinoxalin-2(1 h)-one derivatives as inhibitors against hepatitis C virus

DOI: 10.1021/jm200394x

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm200394x

Full text of DOI:10.1021/jm200394x

Products guided by the article

R&D Labs maybe for 1325216-75-8

Relevant to this article

Hot Product