Preparation of 6-substituted quinoxaline JSP-1 inhibitors by microwave accelerated nucleophilic substitution

Article abstract of DOI:10.3390/11120988

A small library of 6-aminoquinoxalines has been prepared by nucleophilic substitution of 6-fluoroquinoxaline with amines and nitrogen-containing heterocycles under computer-controlled microwave irradiation. Some compounds were found to be potent inhibitors of JNK Stimulatory Phosphatase-1 (JSP-1) in an in vitro biological assay.

Full text of DOI:10.3390/11120988

Molecules 2006, 11, 988-999
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Preparation of 6-Substituted Quinoxaline JSP-1 Inhibitors by
Microwave Accelerated Nucleophilic Substitution
Li Zhang 1, Beiying Qiu 2, Xin Li 1, Xin Wang 1, Jingya Li 2, Yongliang Zhang 1, Jian Liu 1,
Jia Li 2 and Jingkang Shen 1,*
1 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, P.R.
China; E-mail: zhangli@mail.shcnc.ac.cn
2 The National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, P.R. China;
E-mail: byqiu@mail.shcnc.ac.cn, E-mail: jli@mail.shcnc.ac.cn
* Author to whom correspondence should be addressed; E-mail: jkshen@mail.shcnc.ac.cn
Received: 27 November 2006; in revised form: 17 December 2006 / Accepted: 18 December 2006 /
Published: 21 December 2006
Abstract: A small library of 6-aminoquinoxalines has been prepared by nucleophilic
substitution of 6-fluoroquinoxaline with amines and nitrogen-containing heterocycles
under computer-controlled microwave irradiation. Some compounds were found to be
potent inhibitors of JNK Stimulatory Phosphatase-1 (JSP-1) in an in vitro biological
assay.
Keywords: Microwave irradiation; nucleophilic substitution; 6-substituted quinoxalines;
JSP-1.
Introduction
Quinoxalines are an important class of nitrogen-containing heterocycles with a variety of
biological activities, specifically as AMPA/GlyN receptor antagonists [1-3], angiotensin II receptor
antagonists [4-5], anticancer agents [6-9], antiinfection agents [10-11] and immunomodulating agents
[12-13].
Through a high-throughput screening of our sample collection, an aminoquinoxaline derivative I
(Figure 1) was found to show inhibitory activity towards JNK Stimulatory Phosphatase-1 (JSP-1) in an
in vitro biological assay [14]. JSP-1, also known as VHX/MKPX [15] or JKAP [16], is a special

Molecules 2006, 11
989
member of the so-called Dual-Specificity Protein Phosphatases (DSPs) family [17], a class of protein
tyrosine phosphatases (PTPs), which are a family of intracellular enzymes that dephosphorylate
proteins with phosphate on serine, threonine and/or tyrosine, and play important regulative roles in
cellular signal transduction. Unlike other DSPs, which directly inactivate mitogen-activated protein
kinases (MAPK), JSP-1 is a selective activator of the MAPK Jun NH2-terminal kinase (JNK). The
JNK signal transduction pathway is implicated in many pathological conditions, including cancer,
diabetes and neurodegenerative diseases [18-19], therefore, JSP-1 might be a novel potential
therapeutic target for treating diseases associated with dysfunctional JNK signaling.
Figure 1.
N
F
N
N
HO
OH
I IC50 = 19.86 ± 1.63 µM
According to the literature 6-aminoquinoxalines could be prepared by condensation of
corresponding ortho-phenylenediamines with 1,2-dioxo compounds [20-21] or by nucleophilic
substitution of haloquinoxalines [22-26]. Since appropriately substituted ortho-phenylenediamines are
not always readily available, the former route is not very useful for the construction of 6-amino-
quinoxaline libraries and consequently, the latter one was considered a more attractive option to
synthesize the target compounds. However, the nucleophilic substitution reaction, which affords 26-
88% yields of the target compounds after reactions at high temperature for 3-22 hours, usually requires
an electron-withdrawing substituent such as a fluorine atom or nitro group at the ortho-position of the
6-halo substituted quinoxalines [22-25]. There is one report claiming that 2,3-dimethyl-6-
chloroquinoxaline or methyl-6-fluoroquinoxaline reacted with methylamine in sealed tube for 16-26
hours to produce the expected product in 63-80% yield [26].
Recently, microwave-enhanced organic reactions have become quite attractive in organic synthesis
[27-33]. The main advantages of this protocol are the potential to improve the yields and dramatically
shorten the reaction times. Herein, we would like to report a microwave-assisted synthetic method to
synthesize 6-aminoquinoxalines using 6-fluoroquinoxalines and amines or nitrogen-containing
heterocycles in a single-mode microwave synthesizer.
Scheme 1. Nucleophilic substitution of 6-fluoroquinoxalines with amines or nitrogen-
containing heterocycles.
R2
R1
R1
R1
N
F
N
N
N
N
NH2
NH2
NH2
R3
a
b
c
R1
F
NO2
F
2a-d
3-22
1
R1 = H, methyl, 2-furyl,
4-methylphenyl
NR2R3 = pyrrolidine, piperidine, homopiperidine,
4-methoxybenzylamine,
pyrrole, imidazole, pyrazole
Reagents: (a) H2, 10% Pd/C, EtOH. (b) R1COCOR1, EtOH, reflux. (c) HNR2R3, base.

Molecules 2006, 11
Results and Discussion
Chemistry
990
The synthetic route used is outlined in Scheme 1. 4-Fluoro-2-nitroaniline was reduced by catalytic
hydrogenation in the presence of 10% Pd/C to afford 4-fluoro-o-phenylenediamine (1) [34].
Condensation of compound 1 with 1,2-dioxo compounds gave the 6-fluoroquinoxalines 2a-d. As a
model reaction to optimize the reaction conditions for preparing compounds 3-22, the synthesis of 6-
(1-pyrrolidinyl)-quinoxaline (3) was examined in some detail. First, 1 equivalent of 6-fluoro-
quinoxaline (2a) was refluxed with 2 equivalents of pyrrolidine in the presence of 2 equivalents of
potassium carbonate in dimethyl sulfoxide (DMSO). After 30 min, the resulting complex reaction
mixture was checked by HPLC, which showed 21% of unreacted starting material 2a and 16% of the
expected product 3 (Table 1, entry 1). When the reflux time was prolonged to 3 hours (Table 1, entry 2),
the disappearance of the starting material 2a was confirmed by HPLC, but the yield of substituted
product 3 was only slightly increased to 22% and more side products were formed.
Table 1. Optimization of reaction conditions.
2a
Pyrrolidine
T
t
(min)
30
Yield of 3a
(%)
16b
Entry
Base
Solvent
(°C)
189
189
120
200
200
200
200
200
180
220
200
(equiv.)
(equiv.)
1
2
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1
2
2
2
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
DBU
DMSO
DMSO
DMF
180
30
22b
22c
55c
93c
30c
80c
30c
71c
3
4
NMP
30
5
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
30
6
30
7
NaOH
K2CO3
K2CO3
K2CO3
K2CO3
30
8
30
9
30
10
30
83c
70c
11
a
20
The yields were determined by HPLC and LC–MS spectroscopy of the crude
reaction mixture.
b
c
The reactions were carried out with stirring under refluxing in an oil bath.
The reactions were carried out with stirring under microwave irradiation.
To improve the nucleophilic substitution reaction, microwave irradiation was then employed, and
the optimum reaction conditions for this reaction were investigated. As the results presented in Table 1
show, when a mixture of 1 equivalent of 6-fluoroquinoxaline (2a), 2 equivalents of pyrrolidine and 2
equivalents of potassium carbonate in N,N-dimethylformamide (DMF) was exposed to microwave
irradiation at 120 °C for 30 min (Table 1, entry 3), the yield of substituted product 3 was not improved
and considerable starting material 2a was detected. When the reaction temperature was raised to 200
°C and the solvent was changed from DMF to N-methyl pyrrolidone (NMP) or DMSO, a moderate

Molecules 2006, 11
991
yield improvement was observed for the reaction in NMP (Table 1, entry 4), but the reaction was
complete within 30 min in DMSO to give the best yield (93%) (Table 1, entry 5). Based on these
reactions DMSO was selected as the solvent for subsequent work. The effect of the base, sodium
hydroxide or 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), was also investigated, but no yield
improvements were observed (Table 1, entries 6-7) and more side products were detected in both cases.
When the amount of pyrrolidine used was reduceded to one equivalent, the yield also decreased (Table
1, entry 8). Distinctly, the concentration of nucleophile played an important role in the outcome of the
reactions. The yield was not improved regardless of whether the reaction temperature was raised or
decreased. It was observed that the reaction was incomplete at temperatures lower than 200 °C (Table
1, entry 9) and more by-products were formed at higher temperature (Table 1, entry 10). Shortening
the reaction time also resulted in a decrease in the yield (Table 1, entry 11).
Following the results described above, it is suggested that the optimal reaction conditions involve a
mixture of one equivalent of 6-fluoroquinoxaline (2a), two equivalents of pyrrolidine and two
equivalents of potassium carbonate in DMSO exposed to microwave irradiation at 200°C for 30 min
and that microwave irradiation might be a pivotal driving force to accelerate the nucleophilic
substitution.
To extend the utility of the reaction, the above method was employed to synthesize other 6-amino-
quinoxalines, and the reactions of substituted 6-fluoroquinoxalines and various amines were
investigated (Table 2). It could be shown that the reaction yields depended on the character of the
nucleophilic reagents used. When nitrogen-containing aromatic heterocycles were employed as
nucleophiles, excellent yields (88-97%) were obtained. When aliphatic amines were reacted with
6-fluoroquinoxalines, both nucleophilic and steric effects influenced the yields and reaction rates. No
good reaction ocurred with primary amines, except in the case of 4-methoxybenzylamine, but when
secondary amines were used, moderate to good yields were obtained. Changing from pyrrolidine to
six- membered cyclic secondary amines, a decrease in yields was noted, which may be attributed to
steric hindrance. Meanwhile, an effect of the substituents on the quinoxaline ring was also observed.
2,3-Difuryl substituted 6-fluoroquinoxaline reacted with cyclic secondary amines to give excellent
yields of product within 30 min following the general procedure, but the reaction times had to be
prolonged to complete the reaction of 2,3-dimethyl- or 2,3-di-p-tolyl- substituted 6-fluoroquinoxaline
with amines.
Table 2. Nucleophilic substitution of 6-fluoroquinoxalines with amines and nitrogen-
containing aromatic heterocycles.
Entry Compound
R1
NR2R3
Yield (%)a
1
3
H
Pyrrolidine
93
2
3
4
5
6
7
4
5
6
7
8
9
H
H
Piperidine
4-Methoxybenzylamine
Imidazole
85
45
92b
88b
88c
50c
H
H
Pyrazole
Methyl
Methyl
Pyrrolidine
Piperidine

Molecules 2006, 11
992
Table 2. Cont.
Entry Compound
R1
Methyl
NR2R3
Imidazole
Pyrazole
Yield (%)a
8
10
11
12
13
14
15
16
17
18
19
20
21
22
88
88
93
90
73
80
94
97
97
90c
50c
96
96
9
Methyl
10
11
12
13
14
15
16
17
18
19
20
2-Furyl
Pyrrolidine
Piperidine
2-Furyl
2-Furyl
Homopiperidine
4-Methoxybenzylamine
Pyrrole
2-Furyl
2-Furyl
2-Furyl
Imidazole
2-Furyl
Pyrazole
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
Pyrrolidine
Homopiperidine
Pyrrole
Pyrazole
a Isolated yields of pure compounds.
b Microwave irradiation, 200 oC, 5 min
c Microwave irradiation, 200 oC, 60 min
Biological activity
All the synthesized compounds were assayed in vitro for their inhibiting activities towards JSP-1,
and a number of these 2,3-diaryl substituted quinoxalines exhibited potent inhibitory activity, with
compound 16 displaying the most potent activity.
Table 3. JSP-1 inhibiting activities of 6-aminoquinoxalines 12-22.
Entry
Compound
R1
NR2R3
Pyrrolidine
Piperidine
JSP-1 IC50 (µM)a
7.51±0.81
4.18±0.03
5.37±0.53
6.44±1.02
2.61±0.34
4.77±0.32
8.03±0.99
31.62±1.84
9.20±0.65
9.23±0.90
42.44±4.80
1
2
12
13
14
15
16
17
18
19
20
21
22
2-Furyl
2-Furyl
3
2-Furyl
Homopiperidine
4-Methoxybenzylamine
Pyrrole
4
2-Furyl
5
2-Furyl
6
2-Furyl
Imidazole
7
2-Furyl
Pyrazole
8
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
Pyrrolidine
Homopiperidine
Pyrrole
9
10
11
Pyrazole
3-((5-((4-fluorophenyl)methylene)-4-oxo-2-
thioxo)-3-thiazolidinyl)-benzoic acid [35]
12
Reference
15.37±0.13
a Data are means of three independent experiments

Molecules 2006, 11
993
Conclusions
In summary, an efficient method was developed to prepare 6-aminoquinoxalines in moderate to
excellent yields within 5-60 min under microwave irradiation, whereas with standard heating this
reaction could not be completed even after prolonged reaction times and a complex mixture of by-
products was formed. To extend the broad applicability of this procedure, this microwave assisted
nucleophilic substitution was employed to construct a small parallel library of 6-substituted
quinoxalines, which exhibited JSP-1 inhibiting activities in an in vitro enzymatic assay.
Experimental Section
General
The reagents were purchased from Lancaster (Morecambe, England), Aldrich (St. Louis, MO,
USA), Acros (Geel, Belgium) and Shanghai Chemical Reagent Company (Shanghai, China), and were
used without further purification. The single-mode microwave synthesizer employed for this work was
an Initiator from Biotage (Uppsala, Sweden), which is equipped with an internal probe that monitors
reaction temperature and pressure, and maintains the desired temperature by computer control.
Reactions were conducted in the 5 mL sealed vials. Analytical thin-layer chromatography was
performed on HSGF 254 plates (150–200 µm thickness; Yantai Huiyou Company, Yantai, Shandong,
China). Preparative thin-layer chromatography was carried out on HSGF 254 plates (400–500 µm
thickness; Yantai Huiyou Company, Yantai, Shandong, China). Column chromatography was
performed using 200-300 mesh silica gels (Qingdao Haiyang Chemical Company, Qingdao, Shandong,
China). Yields were not optimized. Melting points were recorded in a capillary tube on a SGW X-4
1
melting point apparatus without correction. H-NMR was recorded in CDCl3 on a Varian AMX-300
(300 MHz) NMR spectrometer using tetramethylsilane as an internal standard. Chemical shifts were
reported in parts per million (ppm, δ). Proton coupling patterns were described as singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m), and broad (br). Mass spectra (MS) were measured by the
electric ionization (EI) method with a Finnigan MAT-95 instrument (Finnigan, Santa Clara, CA,
USA).
General procedure for the preparation of 6-fluoroquinoxalines 2a-d: Preparation of 6-fluoro-
quinoxaline (2a)
4-Fluoro-2-nitroaniline (2.0 g, 0.013 mol), dissolved in ethanol (80 mL), was hydrogenated in the
presence of 10% Pd/C (800 mg) under 1 atm H2 at room temperature. When the hydrogen uptake was
complete the catalyst was removed by filtration and a mixture of the filtrate and 40% glyoxal in water
(1.5 mL, 0.013 mol) was heated at reflux for 24 hours. The solvent was evaporated under vacuum to
give the crude product, which was further purified by flash column chromatography to give the desired
compound 2a (1.0 g, yield 53 %) as a yellow solid. mp: 33-35 °C; 1H-NMR: δ 8.87 (1H, d, J=1.8 Hz),
8.85 (1H, d, J=1.8 Hz), 8.12～8.17 (1H, m), 7.74~7.78 (1H, m), 7.56~7.63 (1H, m); EIMS m/z: 148
(M+). The following compounds were similarly prepared:

Molecules 2006, 11
994
1
2,3-dimethyl-6-fluoroquinoxaline (2b): A yellow solid, yield 57%; mp: 108-110°C; H-NMR: δ
7.95~8.00 (1H, m), 7.59~7.63 (1H, m), 7.41~7.48 (1H, m), 2.74 (3H, s), 2.73 (3H, s); EIMS m/z: 176
(M+).
1
2,3-di(2-furanyl)-6-fluoroquinoxaline (2c): A yellow solid, yield 72%; mp: 112-114°C; H-NMR: δ
8.12~8.17 (1H, m), 7.76~7.80 (1H, m), 7.64~7.66 (2H, m), 7.51~7.57 (1H, m), 6.66~6.71 (2H, m),
6.57~6.60 (2H, m); EIMS m/z: 280 (M+).
1
2,3-di(4-methylphenyl)-6-fluoroquinoxaline (2d): A yellow solid, yield 76%, mp: 114-116°C; H-
NMR: δ 8.12~8.17 (1H, m), 7.75~7.79 (1H, m), 7.49~7.55 (1H, m), 7.40~7.44 (4H, m), 7.14~7.17
(4H, m), 2.38 (3H, s), 2.37 (3H, s); EIMS m/z: 328 (M+).
General procedure for the preparation of 6-aminoquinoxalines 3-22 under microwave irradiation:
Synthesis of 6-(1-pyrrolidinyl)-quinoxaline (3).
Pyrrolidine (112 µL, 97 mg, 1.36 mmol) was added in a 5 mL microwave vial containing 6-fluoro-
quinoxaline (100 mg, 0.68 mmol) and K2CO3 (188 mg, 1.36 mmol) in DMSO (1.5 mL). The vial was
sealed by a crimped cap and was placed in a Biotage microwave apparatus. The vessel was heated for
o
30 min at 200 C. After completion of the reaction, the reaction mixture was cooled and the mixture
was poured into ice-water (50 mL). The mixture was extracted three times with dichloromethane and
the combined organic layers were washed with brine, dried over anhydrous sodium sulfate and filtered.
The solvent was evaporated under vacuum, and the resulting crude product was purified by preparative
thin-layer chromatography to give 125 mg of the title compound 3 as a yellow solid, yield 93%; mp:
1
77-79°C; H-NMR: δ 8.62 (1H, d, J=1.8 Hz), 8.46 (1H, d, J=1.8 Hz), 7.87~7.91 (1H, m), 7.22~7.26
(1H, m), 6.90 (1H, m), 3.44~3.49 (4H, m), 2.07~2.12 (4H, m); EIMS m/z: 199 (M+); HRMS (EI):
C12H13N3 Calculated: 199.1110; Found 199.1108. The following compounds were similarly prepared:
1
6-(1-piperidinyl)-quinoxaline (4): A yellow oil, yield 85%; H-NMR: δ 8.67 (1H, d, J=1.8Hz), 8.56
(1H, d, J=1.8 Hz), 7.89~7.92 (1H, m), 7.52~7.56 (1H, m), 7.26 (1H, m), 3.39~3.42 (4H, m), 1.66~1.79
(6H, m); EIMS m/z: 213 (M+); HRMS (EI): C13H15N3 Calculated: 213.1266; Found 213.1266.
4-(methoxy)-N-6-quinoxalinyl-benzenemethanamine (5): A yellow solid, yield 45%; mp: 118-120°C;
1H- NMR: δ 8.63 (1H, d, J=1.5 Hz), 8.51 (1H, d, J=1.5 Hz), 7.83~7.85 (1H, m), 7.32~7.34 (2H, m),
7.13~7.16 (1H, m), 7.00 (1H, m), 6.89~6.91 (2H, m), 4.39 (2H, s), 3.80 (3H, s); EIMS m/z: 265 (M+);
HRMS (EI): C16H15N3O Calculated: 265.1215; Found 265.1207.
1
6-(1H-imidazol-1-yl)-quinoxaline (6): A yellow solid, yield 92%; mp: 197-199°C; H-NMR: δ 8.93
(1H, d, J=2.1 Hz), 8.91 (1H, d, J=2.1 Hz), 8.27 (1H, d, J=9.0 Hz), 8.13 (1H, d, J=2.7 Hz), 8.08 (1H,
dd, J=2.7 Hz, J=9.0 Hz), 7.87~7.91 (1H, m), 7.49 (1H, m), 7.31 (1H, m); EIMS m/z: 196 (M+); HRMS
(EI): C11H8N4 Calculated: 196.0749; Found 196.0734.

Molecules 2006, 11
995
1
6-(1H-pyrazol-1-yl)-quinoxaline (7): A yellow solid, yield 88%; mp: 75-78°C; H-NMR: δ 8.89 (1H,
d, J=2.0 Hz), 8.85 (1H, d, J=2.0 Hz), 8.35~8.38 (1H, m), 8.30 (1H, m), 8.20~8.23 (1H, m), 8.15 (1H,
m), 7.84 (1H, m), 6.58 (1H, m); EIMS m/z: 196 (M+); HRMS (EI): C11H8N4 Calculated: 196.0749;
Found 196.0746.
2,3-dimethyl-6-(1-pyrrolidinyl)-quinoxaline (8): A yellow solid, yield 88%; mp: 168-171°C; 1H-NMR:
δ 7.80 (1H, d, J=9.0 Hz), 7.13 (1H, dd, J=9.0 Hz, J=2.7 Hz), 6.85 (1H, d, J=2.7 Hz), 3.41~3.46 (4H,
m), 2.66 (3H, s), 2.65 (3H, s), 2.05~2.10 (4H, m); EIMS m/z: 227 (M+); HRMS (EI): C14H17N3
Calculated: 227.1423; Found 227.1420.
1
2,3-dimethyl-6-(1-piperidinyl)-quinoxaline (9): A yellow solid, yield 50%; mp: 104-106°C; H-NMR:
δ 7.80 (1H, d, J=9.1 Hz), 7.44 (1H, dd, J=9.1 Hz, J=2.6 Hz), 7.22 (1H, d, J=2.6 Hz), 3.84~3.87 (2H,
m), 2.81~2.90 (2H, m), 2.67 (3H, s), 2.66 (3H, s), 1.75~1.80 (2H, m), 1.34~1.40 (4H, m); EIMS m/z:
241 (M+); HRMS (EI): C15H19N3 Calculated: 241.1579; Found 241.1584.
2,3-dimethyl-6-(1H-imidazol-1-yl)-quinoxaline (10): A yellow solid, yield 88%; mp: 138-141°C; 1H-
NMR: δ 8.12 (1H, d, J=9.0 Hz), 8.05 (1H, m), 8.02 (1H, d, J=2.4 Hz), 7.75 (1H, dd, J=9.0 Hz, J=2.4
Hz), 7.45 (1H, m), 7.28 (1H, m), 2.77 (3H, s), 2.76 (3H, s); EIMS m/z: 224 (M+); HRMS (EI):
C13H12N4 Calculated: 224.1062; Found 224.1061.
1
2,3-dimethyl-6-(1H-pyrazol-1-yl)-quinoxaline (11): A yellow solid, yield 88%; mp: 112-115°C; H-
NMR: δ 8.24 (1H, dd, J=2.7 Hz, J=9.0 Hz), 8.17 (1H, d, J=2.7 Hz), 8.10 (1H, m), 8.08 (1H, d, J=9.0
Hz), 7.81 (1H, m), 6.55 (1H, m), 2.76 (3H, s), 2.75 (3H, s); EIMS m/z: 224 (M+); HRMS (EI):
C13H12N4 Calculated: 224.1062; Found 224.1066.
1
2,3-di(2-furanyl)-6-(1-pyrrolidinyl)-quinoxaline (12): A yellow solid, yield 93%; mp: 180-182°C; H-
NMR: δ 7.95 (1H, d, J=9.3 Hz), 7.58~7.62 (2H, m), 7.25 (1H, dd, J=2.7 Hz, J=9.3 Hz), 7.00 (1H, d,
J=2.7 Hz), 6.44~6.56 (4H, m), 3.46~3.51 (4H, m), 2.06~2.13 (4H, m); EIMS m/z: 331 (M+); HRMS
(EI): C20H17N3O2 Calculated: 331.1321; Found 331.1306.
1
2,3-di(2-furanyl)-6-(1-piperidinyl)-quinoxaline (13): A yellow solid, yield 90%; mp: 111-113°C; H-
NMR: δ 7.93 (1H, d, J=9.3 Hz), 7.57~7.60 (2H, m), 7.51 (1H, dd, J=2.7 Hz, J=9.3 Hz), 7.31 (1H, d,
J=2.7 Hz), 6.46~6.56 (4H, m), 3.40~3.44 (4H, m), 1.66~1.73 (6H, m); EIMS m/z: 345 (M+); HRMS
(EI): C21H19N3O2 Calculated: 345.1477; Found 345.1497.
1
2,3-di(2-furanyl)-6-(1-azepanyl)-quinoxaline (14): A yellow solid, yield 73%; mp: 227-230°C; H
NMR: δ 7.92 (1H, d, J=9.6 Hz), 7.56～7.61 (2H, m), 7.35 (1H, dd, J=2.7 Hz, J=9.6 Hz), 7.12 (1H, d,
J=3.0 Hz), 6.41~6.53 (4H, m), 3.61~3.65 (4H, m), 1.85~1.87 (4H, m), 1.56~1.61 (4H, m); EIMS m/z:
359 (M+); HRMS (EI): C22H21N3O2 Calculated: 359.1634; Found 359.1649.
N-2,3-di(2-furanyl)-6-quinoxalinyl-4-(methoxy)-benzenemethanamine (15): A yellow solid, yield 80%;
1
mp: 238-240°C; H-NMR: δ 7.87~7.90 (1H, m), 7.57~7.60 (2H, m), 7.31~7.34 (2H, m), 7.08~7.13

Molecules 2006, 11
996
(2H, m), 6.89~6.92 (2H, m), 6.46~6.55 (4H, m), 4.40 (2H, s), 3.81 (3H, s); EIMS m/z: 397 (M+);
HRMS (EI): C24H19N3O3 Calculated: 397.1426; Found 397.1422.
2,3-di(2-furanyl)-6-(1H-pyrrol-1-yl)-quinoxaline (16): A yellow solid, yield 94%; mp: 146-148°C; 1H-
NMR: δ 8.19 (1H, d, J=9.2 Hz), 8.10 (1H, d, J=2.4 Hz), 7.88 (1H, dd, J=9.2 Hz, J=2.4 Hz), 7.64~7.66
(2H, m), 7.30 (2H, m), 6.71 (1H, m), 6.66 (1H, m), 6.57~6.60 (2H, m), 6.44 (2H, m); EIMS m/z: 327
(M+); HRMS (EI): C20H13N3O2 Calculated: 327.1008; Found 327.0993.
2,3-di(2-furanyl)-6-(1H-imidazol-1-yl)-quinoxaline (17): A yellow solid, yield 97%; mp: 115-117°C;
1H-NMR: δ 8.27 (1H, d, J=9.0 Hz), 8.16 (1H, d, J=2.7 Hz), 8.10 (1H, s), 7.85 (1H, dd, J=2.7 Hz, J=9.0
Hz), 7.66~7.68 (2H, m), 7.49 (1H, m), 7.31 (1H, m), 6.72~6.78 (2H, m), 6.60~6.63 (2H, m); EIMS
m/z: 328 (M+); HRMS (EI): C19H12N4O2 Calculated: 328.0960; Found 328.0956.
2,3-di(2-furanyl)-6-(1H-pyrazol-1-yl)-quinoxaline (18): A yellow solid, yield 97%; mp: 171-173°C;
1H-NMR: δ 8.38 (1H, dd, J=2.7 Hz, J=9.0 Hz), 8.30 (1H, d, J=2.7 Hz), 8.21~8.24 (1H, m), 8.15 (1H,
d, J=9.0 Hz), 7.83 (1H, m), 7.66 (2H, m), 6.68~6.75 (2H, m), 6.58~6.61 (3H, m); EIMS m/z: 328(M+);
HRMS (EI): C19H12N4O2 Calculated: 328.0960; Found 328.0949.
2,3-di(4-methylphenyl)-6-(1-pyrrolidinyl)-quinoxaline (19): A yellow solid, yield 90%; mp: 191-
1
193°C; H-NMR: δ 7.95 (1H, d, J=9.3 Hz), 7.35~7.41 (4H, m), 7.25 (1H, dd, J=9.3 Hz, J=2.7 Hz),
7.09~7.13 (4H, m), 6.99 (1H, d, J=2.7 Hz), 3.47 (4H, m), 3.35 (3H, s), 3.34 (3H, s), 2.09 (4H, m);
EIMS m/z: 379 (M+); HRMS (EI): C26H25N3 Calculated: 379.2048; Found 379.2049.
2,3-di(4-methylphenyl)-6-(1-azepanyl)-quinoxaline (20): A yellow solid, yield 50%; mp: 123-125°C;
1H-NMR: δ 7.93 (1H, d, J=9.3 Hz), 7.33~7.39 (5H, m), 7.08~7.13 (5H, m), 3.62~3.66 (4H, m), 2.35
(3H, s), 2.34 (3H, s), 1.86～1.87 (4H, m), 1.56～1.61 (4H, m); EIMS m/z: 407 (M+); HRMS (EI):
C28H29N3 Calculated: 407.2361; Found 407.2360.
2,3-di(4-methylphenyl)-6-(1H-pyrrol-1-yl)-quinoxaline (21): A yellow solid, yield 96%; mp: 198-
200°C; 1H-NMR: δ 8.20 (1H, d, J=9.0 Hz), 8.11 (1H, d, J=2.4 Hz), 7.87 (1H, dd, J=2.4 Hz, J=9.0 Hz),
7.42~7.46 (4H, m), 7.30~7.32 (2H, m), 7.15~7.18 (4H, m), 6.44~6.46 (2H, m), 2.38 (6H, s); EIMS
m/z: 375 (M+); HRMS (EI): C26H21N3 Calculated: 375.1736; Found 375.1736.
2,3-di(4-methylphenyl)-6-(1H-pyrazol-1-yl)-quinoxaline (22): A yellow solid, yield 96%; mp: 218-
220°C; 1H-NMR: δ 8.31~8.36 (2H, m), 8.22~8.26 (1H, m), 8.15 (1H, m), 7.83 (1H, m), 7.44~7.47 (4H,
m), 7.15~7.19 (4H, m), 6.58 (1H, m), 2.39 (6H, s); EIMS m/z: 376 (M+); HRMS (EI): C25H20N4
Calculated: 376.1688; Found 376.1710.
JSP-1 Inhibition Activities Assay
The JSP-1 activity was determined at room temperature by monitoring the hydrolysis of 3-o-
methylfluorescein phosphate (OMFP). In a typical assay a mixture (100 µL) containing 50 mM

Molecules 2006, 11
997
Bis·Tris, pH 6.5, 1 mM EDTA, 1 mM DTT, 10 µM OMFP and 30 nM purified recombinant GST-
fusion human JSP-1 was used. The enzyme activity was continuously monitored with an excitation
485 nm/emission 535 nm filter set for 3 min and the initial rate of the hydrolysis was determined using
the linear region of the enzymatic reaction kinetic curve. IC50 values were calculated from the non-
linear curve fitting of percent inhibition (% inhibition) vs. inhibitor concentration [I] by using the
equation: % Inhibition = 100/{1+(IC50/[I])k}, where k is the Hill coefficient.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grants
30230400 and 30572255).
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