Synthesis and antimicrobial activity of 2,3-bis(bromomethyl)quinoxaline derivatives

Article abstract of DOI:10.1016/j.bioorg.2011.12.002

We synthesized 12 derivatives of 2,3-bis(bromomethyl)quinoxaline with substituents at the 6- and/or 7-positions, and evaluated their activities against bacteria and fungi. Of the 12 compounds, nine (1a-h, 1j, and 1k) showed antibacterial activity. The derivative 1g, which bears a trifluoromethyl group at the 6-position, showed the highest activity against Gram-positive bacteria, while 1c, which has a fluoro-group at the 6-position, showed the widest antifungal activity spectrum. However, only the derivative with an ethyl ester substitution, 1k showed activity against Gram-negative bacteria.

Full text of DOI:10.1016/j.bioorg.2011.12.002

Bioorganic Chemistry 41-42 (2012) 1–5
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and antimicrobial activity of
2,3-bis(bromomethyl)quinoxaline derivatives
Hisato Ishikawa a, Takayuki Sugiyama b, Keisuke Kurita a, Akihiro Yokoyama a,
⇑
a Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1, Kichijoji-kitamachi, Musashino, Tokyo 180-8633, Japan
b Japan EnviroChemicals, Ltd., 5-11-61, Torishima, Konohana-ku, Osaka 554-0051, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 November 2011
Available online 29 December 2011
We synthesized 12 derivatives of 2,3-bis(bromomethyl)quinoxaline with substituents at the 6- and/or
7-positions, and evaluated their activities against bacteria and fungi. Of the 12 compounds, nine (1a-h,
1j, and 1k) showed antibacterial activity. The derivative 1g, which bears a trifluoromethyl group at the
6-position, showed the highest activity against Gram-positive bacteria, while 1c, which has a fluoro-group
at the 6-position, showed the widest antifungal activity spectrum. However, only the derivative with an
ethyl ester substitution, 1k showed activity against Gram-negative bacteria.
Keywords:
Quinoxaline
Bromomethyl
Antibacterial activity
Antifungal activity
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
compounds with antimicrobial activity. Because the bromo-
methyl-imino group would function in a manner chemically
Antimicrobial agents are essential for the treatment of a num-
ber of diseases of bacterial or fungal origin, and many such agents
are used in clinically and industrially. Recently, however, bacteria
have acquired drug resistance and so the development of new anti-
microbial agents is required [1–3].
A number of compounds based on nitrogen-containing hetero-
cycles show antimicrobial activity and have been developed for
clinical use [4]. Among the various classes of heterocyclic units,
the quinoxaline ring has frequently been used as a component of
various antibiotic molecules, such as hinomycin, levomycin, and
actindeutin, which inhibit the growth of Gram-positive bacteria
and are active against various transplantable tumors [5–7]. In addi-
tion, many reports describe a variety of biological activities of
quinoxaline derivatives including anticancer, antibacterial, anti-
fungal, antiviral and antiprotozoal activities [8–18]. Thus, the quin-
oxaline ring is an important structural unit in these bioactive
compounds.
equivalent to the bromoacetyl group, we designed a derivative of
2,3-bis(bromomethyl)quinoxaline.
Previously, Nielsen and co-workers reported that the activity of
40-carboxychalcones against Staphylococcus aureus increased when
lipophilic substitutions were made [23]. Furthermore, Sanna and
co-workers reported that 2-quinoxalinone derivatives bearing
electron-withdrawing groups at the 6- or 7-positions showed
antibacterial, antifungal, and anticancer activities [8–11]. Hence,
lipophilicity and the electronic properties of the substituents affect
the biological activities of these compounds.
In this paper, we report the synthesis of 2,3-bis(bromo-
methyl)quinoxaline derivatives 1a–l bearing various substituents
at the 6- and/or 7-positions (Fig. 1), and investigate the effect of
lipophilicity and electronic properties of substituents on their
antibacterial and antifungal activities. Among the synthesized
quinoxaline derivatives, trifluoromethyl-substituted compound
1g showed the highest activity against Gram-positive bacteria,
while fluoro-substituted 1c showed the broadest-acting antifungal
activity.
Bromomethyl-substituted compounds, such as 4-bromomethyl-
[2,2]-p-cyclophane and simple peptides with bromomethyl groups,
showed antibacterial and antifungal activities [19–22]. Similarly,
bromoacetyl groups can be found in industrial antimicrobial agents
such as benzyl bromoacetate, 1,4-bis(bromoacetoxy)-2-butene and
2-bromo-1-(4-hydroxyphenyl)ethanone.
2. Results and discussion
2.1. Synthesis
Based on these data, we hypothesized that the combination of a
quinoxaline ring with a bromoacetyl group would produce novel
2,3-Bis(bromomethyl)quinoxaline derivatives 1a–i were synthe-
sized by the condensation reaction of the corresponding 1,2-phenyl-
enediamine derivatives 2a–i and 1,4-dibromo-2,3-butanedione 3
according to the method reported by Carta [11] (Scheme 1). The
⇑
Corresponding author. Fax: +81 422 37 3871.
E-mail address: ayokoyama@st.seikei.ac.jp (A. Yokoyama).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.12.002

2
H. Ishikawa et al. / Bioorganic Chemistry 41-42 (2012) 1–5
Fig. 1. Structure of the 2,3-bis(bromomethyl)quinoxaline derivatives 1a–l.
Scheme 2. Synthesis of the methyl ester 1j and ethyl ester 1k.
target compounds 1a–i were obtained in 69–93% yields, and the
electronic properties of the substituent at the 4-position of the
1,2-phenylenediamine derivatives did not significantly affect the
efficiency of this reaction. When the reaction was completed, the
compounds1a–f, 1h and 1i were precipitated from the reaction mix-
ture; 1g was not precipitated due to the difficulty of solidification.
After usual work-up, all products were purified by silica gel column
chromatography.
The carboxylic acid 1i was converted into the esters 1j and 1k.
The esterification of 1i was carried out in methanol and ethanol in
the presence of a catalytic amount of sulfuric acid, and the methyl
ester 1j and ethyl ester 1k were obtained in good yields (Scheme
2).
Scheme 3. Synthesis of compound 1l.
Table 1
Antibacterial activities of 1a–l.
R1
R2
MIC (
lg/mL)
Gram-positive
Gram-negative
b
c
d
e
B. s.a
S. a.
E. c.
P. a.
S. m.
The 6-hydroxyquinoxaline 1l was synthesized by treating the
methoxy compound 1f with boron tribromide. The demethylation
reaction proceeded without any side-reaction to produce an excep-
tional yield of 1l (Scheme 3). Boron tribromide-assisted demethyl-
ation of the methoxy group on the benzene ring of compounds such
as dimethoxytoluene and dimethoxyisopropylbenzene was com-
plete within several hours [24,25]; however, demethylation of 1f
required 5 days for completion, indicating that cleavage of the
CAO bond of the methoxy group at the 6-position of the quinoxa-
line ring is more difficult than in the equivalent benzene analog.
1a
1b
1c
1d
1e
1f
1g
1h
1i
CH3
CN
F
Cl
Br
OCH3
CF3
NO2
CO2H
CO2CH3
CO2CH2CH3
OH
CH3
H
H
H
H
H
H
H
H
50
25
25
50
25
25
50
25
50
50
50
50
12.5
50
>100
50
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
12.5
25
>100
50
25
>100
1j
1k
1l
H
H
H
>100
>100
>100
>100
a
b
c
Bacillus subtilis.
Staphylococcus aureus.
Escherichia coli.
Pseudomonas aerugianosa.
Serratia marcescens.
2.2. Antibacterial activity
d
e
The antibacterial activity of the compounds 1a–l was evaluated
by means of the minimum inhibitory concentration (MIC), as
determined by the dilution method. The results are summarized
in Table 1. Nine compounds (1a–h, 1j and 1k) showed antibacterial
activities (MIC = 25–50 lg/mL). The order of electron-withdrawing
activities (MIC 6 100 lg/mL), indicating that the 2,3-bis(bromo-
abilities of the halogen atoms is Br < Cl < F, and the lipophilicity fol-
lows the order F < Cl < Br; it is possible, therefore, that the elec-
tronic and the lipophilic effects of these halogens on antibacterial
activity may cancel each other out.
methyl)quinoxaline is the structural unit that exerts antibacterial
activities. Among these nine compounds, the most active com-
pound against Gram-positive bacteria was 1g, which has a strong
electron-withdrawing and highly lipophilic trifluoromethyl group
The methyl ester 1j and ethyl ester 1k both showed antibacte-
rial activities, but the carboxylic acid 1i did not. Similarly, the
methoxy-substituted 1f showed some activity, while the phenol
1l did not. These results indicate that increasing the lipophilicity
improves the antibacterial activity, but an increase in hydrophilic-
ity decreases activity. In general, the lipophilic property of a com-
pound plays an important role in allowing the compound to
permeate the bacterial cell membrane [26]. Therefore, it is likely
that the penetration of 1i and 1l through bacterial cell membranes
is inhibited by the hydrophilic substituents of these compounds.
at the 6-position (MIC = 12.5 lg/mL). However, the activity of 1b
and 1h, which bear hydrophilic and electron-withdrawing cyano
and nitro groups, respectively, was lower than that of 1g, and
almost identical to that of 1a and 1f which have lipophilic and elec-
tron-releasing methyl and methoxy groups, respectively
(MIC = 25–50 lg/mL). These results suggest that both electron-
withdrawing ability and lipophilicity of the substituents at the
6-position are need for high antibacterial activity.
The halogen-substituted compounds 1c, 1d, and 1e, which have
F, Cl, and Br at the 6-position, respectively, showed similar
Scheme 1. Synthesis of the 2,3-bis(bromomethyl)quinoxaline derivatives 1a–i.

H. Ishikawa et al. / Bioorganic Chemistry 41-42 (2012) 1–5
3
Table 2
Antifungal activities of 1a–l.
R1
R2
MIC (
Mold
A. n.a
lg/mL)
Yeast
b
c
d
e
f
g
h
i
P. c.
C. c.
A. p.
A. s.
M. s.
G. v.
R. r.
S. c.
1a
1b
1c
1d
1e
1f
1g
1h
1i
CH3
CN
F
Cl
Br
OCH3
CF3
NO2
COOH
CO2CH3
CO2CH2CH3
OH
CH3
H
H
H
H
H
H
H
H
>100
50
50
50
100
50
>100
25
50
>100
25
50
25
50
50
100
50
>100
100
>100
>100
>100
50
>100
25
50
>100
>100
100
100
25
>100
>100
>100
>100
>100
25
25
50
50
50
25
50
>100
25
>100
>100
100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
100
100
>100
50
50
>100
>100
100
50
50
>100
50
100
>100
100
>100
>100
100
50
100
>100
50
100
100
>100
50
100
>100
100
>100
>100
>100
>100
>100
100
>100
>100
>100
100
50
100
>100
50
>100
>100
1j
1k
1l
H
H
H
100
>100
100
>100
>100
a
b
c
d
e
f
Aspergillus niger.
Penicillium citrinum.
Cladosporium cladosporiodes.
Aureobasidium pullelans.
Alternaria sp.
Mucor spinescens.
Gliocladium virens.
g
h
i
Rhodotorula rubra.
Saccharomyces cerevisiae.
Against Gram-negative bacteria, only the ethyl ester 1k showed
When the antifungal activities of halogenated compounds 1c
(R1 = F), 1d (R1 = Cl), and 1e (R1 = Br) were compared, activity in-
creased in the order of 1e < 1d < 1c, which is the same order as
the substituent’s electron-withdrawing ability. Of the synthesized
compounds, fluoro-substituted 1c showed the widest antifungal
spectrum. These results indicate that the antifungal activities of
the 2,3-bis(bromomethyl)quinoxaline derivatives are greater when
strong electron-withdrawing and moderately lipophilic substitu-
ents are introduced at the 6-position.
any activity. Sanna and co-workers examined the antibacterial
activities of 3-ethoxycarbonyl- and 3-carboxy-2-quinoxalinone
derivatives, and reported that only 3-ethoxycarbonyl-2-quinoxali-
none derivatives showed activities against Gram-negative bacteria
(Escherichia coli and Pseudomonas aerugianosa) [8]. Therefore, it is
possible that it is the ethoxycarbonyl group that exerts a specific
action against Gram-negative bacteria. However, the methyl ester
1j does not show any activity against Gram-negative bacteria,
and so the activity against Gram-negative bacteria of 1k may be
due to the increase in the alkyl chain length.
3. Conclusions
We synthesized 12 kinds of 2,3-bis(bromomethyl)quinoxaline
derivatives, evaluated their activities against bacteria and fungi,
and found that the compounds bearing strongly electron-releas-
ing/withdrawing and moderately lipophilic substituents showed
high antimicrobial activities. Among them, trifluoromethyl-
substituted 1g showed the highest activity against Gram-positive
bacteria. However, cyano-substituted 1b and fluoro-substituted
1c showed the greatest and widest-ranging activities against fungi
respectively. These results demonstrate that 2,3-bis(bromo-
methyl)quinoxaline framework is a good candidate for novel
industrial antimicrobial agent. The synthesis and evaluation of
the antibacterial and antifungal activities of quinoxaline deriva-
tives bearing halomethyl groups other than the bromomethyl
group at the 2- and the 3-positions are in progress.
2.3. Antifungal activity
As with the antibacterial activity, we evaluated antifungal activ-
ity by means of MIC observations, and the results are summarized
in Table 2. Antifungal activity (MIC 6 100 lg/mL) was seen in eight
of the 2,3-bis(bromomethyl)quinoxaline derivatives: 1b–h, 1j and
1k. When the effects of electron-withdrawing substituents were
compared (1b (R1 = CN), 1g (R1 = CF3), 1h (R1 = NO2), 1j
(R1 = CO2Me), and 1k (R1 = CO2Et)), the compounds with stronger
electron-withdrawing groups such as 1b, 1g and 1h showed wider
antifungal spectra than 1j or 1k. Of these, 1b exhibited the highest
activity. Among the electron-releasing group-substituted com-
pounds, 1a, with a methyl group at the 6-position, did not show
any antifungal activity, but 1f, having a stronger electron-releasing
methoxy group, showed some activity. These results suggest that,
as in the case of antibacterial activity, the electronic effect of the
substituent at the 6-position influences both breadth of antifungal
spectrum and degree of activity, and the compounds bearing stron-
ger electron-releasing or withdrawing substituents show improved
antifungal spectra and activity.
As for the carboxylic acid derivatives, the methyl ester 1j
showed antifungal activities and spectra similar to 1b, 1g and 1h.
However, the more lipophilic ethyl ester 1k exhibited lower anti-
fungal activity and a narrower spectrum, while the carboxylic acid
1i did not show any activity. These results indicate that moderate
lipophilicity is needed to exert antifungal activities. A similar trend
was also observed when compounds 1f and 1l were compared: the
methoxy-substituted 1f showed some activity, but the phenol 1l
did not.
4. Experimental
4.1. General
All common reagents and solvents were obtained from Wako
Pure Chemical Industries, Tokyo Chemical Industry, and Sigma–
Aldrich, and used without further purification. Column chromatog-
raphy was carried out using silica gel (Silica Gel 60 N, 63–210 lm,
Kanto Chemical). Thin layer chromatography (TLC) was conducted
on Merck Silica Gel 60 F254. Melting points were determined on an
SMP3 melting point apparatus (Bibby Scientific Limited) and were
uncorrected. 1H and 13C NMR spectra were recorded on JEOL JNM-
LA400D and JNM-ECA-500, respectively, using DMSO-d6 and CDCl3
as solvents. Chemical shifts (d) were reported as parts per million

4
H. Ishikawa et al. / Bioorganic Chemistry 41-42 (2012) 1–5
(ppm) relativeto tetramethylsilane (TMS) as an internal standard for
1H NMR and the midpoints of CDCl3 (77.16 ppm) and DMSO-d6
(39.52 ppm) for 13C NMR. IR spectra were recorded with a JASCO
FT/IR-470. Elemental analyses for C, H and N were performed using
a Perkin Elmer 2400 analyzer series II. All compounds were charac-
terized by the above techniques.
4.2.6. 2,3-Bis(bromomethyl)-6-methoxyquinoxaline 1f
Yield: 76%, mp: 133–134 °C. 1H NMR (400 MHz, CDCl3) d: 3.97
(3H, s), 4.88 (2H, s), 4.89 (2H, s), 7.35 (1H, d, J = 2.7 Hz), 7.44 (1H,
dd, J = 2.7 and 9.3 Hz), 7.94 (1H, d, J = 9.3 Hz). 13C NMR
(126 MHz, CDCl3) d: 30.6 (CH2), 30.9 (CH2), 56.1 (OCH3), 106.3
(CH), 124.6 (CH), 130.1 (CH), 137.9 (C), 143.5 (C), 148.1 (C), 151.0
(C), 161.7 (C). IR (KBr, cmꢀ1): 3017, 2962, 1358, 1238, 1019, 906,
835, 514. Anal. Calcd for C11H10N2OBr2: C, 38.18; H, 2.91; N, 8.10.
Found: C, 38.28; H, 2.64; N, 8.03.
4.2. Preparation of 2,3-bis(bromomethyl)quinoxalines 1a–i
A solution of equimolar amounts (7.0 mmol) of the correspond-
ing 1,2-phenylenediamine (2a–i) and 1,4-dibromo-2,3-butanedi-
one (3) in MeOH (10 mL) was refluxed for 2 h. After cooling to
room temperature, the precipitates of 1a–f, 1h and 1i were col-
lected by suction filtration, or in the case of 1g the solvent was
evaporated. The crude product was purified by column chromatog-
raphy on silica gel with CHCl3 for compounds 1a–h or 6:1 CHCl3/
MeOH for 1i.
4.2.7. 2,3-Bis(bromomethyl)-6-(trifluoromethyl)quinoxaline 1g
Yield: 69%, mp: 59–61 °C. 1H NMR (400 MHz, CDCl3) d: 4.93 (2H,
s), 4.94 (2H, s), 7.97 (1H, dd, J = 1.8 and 8.8 Hz), 8.20 (1H, d,
J = 8.8 Hz), 8.40 (1H, d, J = 1.8 Hz). 13C NMR (126 MHz, CDCl3) d:
30.7 (CH2), 123.5 (q, JCF = 274 Hz, CF3), 126.7 (d, JCF = 3.6 Hz, CH),
127.2 (d, JCF = 4.8 Hz, CH), 130.5 (CH), 132.7 (q, JCF = 34 Hz, C),
140.7 (C), 142.7 (C), 152.6 (C), 153.2 (C). IR (KBr, cmꢀ1): 3040,
2979, 1339, 1281, 906, 819, 668. Anal. Calcd for C11H7N2F3Br2ꢁ0.1
H2O: C, 34.25; H, 1.88; N, 7.26. Found: C, 33.95; H, 1.77; N, 7.10.
4.2.1. 2,3-Bis(bromomethyl)-6,7-dimethylquinoxaline 1a
Yield: 70%, mp: 147 °C (decomp.). 1H NMR (400 MHz, CDCl3) d:
2.50 (6H, s), 4.90 (4H, s), 7.81 (2H, s). 13C NMR (126 MHz, CDCl3) d:
20.6 (CH3), 30.9 (CH2), 128.1 (CH), 140.7 (C), 141.9 (C), 149.9 (C). IR
(KBr, cmꢀ1): 3022, 2970, 2922, 1361, 861, 534. Anal. Calcd for
4.2.8. 2,3-Bis(bromomethyl)-6-nitroquinoxaline 1h
Yield: 90%, mp: 113–115 °C. 1H NMR (400 MHz, CDCl3) d: 4.93
(2H, s), 4.95 (2H, s), 8.23 (1H, d, J = 9.3 Hz), 8.57 (1H, dd, J = 2.4
and 9.3 Hz), 8.98 (1H, d, J = 2.4 Hz). 13C NMR (126 MHz, CDCl3) d:
29.8 (CH2), 29.9 (CH2), 124.4 (CH), 125.5 (CH), 130.9 (CH), 140.5
(C), 143.9 (C), 148.5 (C), 153.5 (C), 154.3 (C). IR (KBr, cmꢀ1):
3084, 3036, 2980, 1536, 1364, 1330, 891, 851, 586. Anal. Calcd
for C10H7N3O2Br2: C, 33.27; H, 1.95; N, 11.64. Found: C, 33.41; H,
2.10; N, 11.69.
C12H12N2Br2: C, 41.89; H, 3.52; N, 8.14. Found: C, 41.66; H, 3.59;
N, 7.94.
4.2.2. 2,3-Bis(bromomethyl)-6-cyanoquinoxaline 1b
Yield: 93%, mp: 157–159 °C. 1H NMR (400 MHz, CDCl3) d: 4.91
(2H, s), 4.93 (2H, s), 7.95 (1H, dd, J = 1.7 and 8.8 Hz), 8.18 (1H, d,
J = 8.8 Hz), 8.45 (1H, d, J = 1.7 Hz). 13C NMR (126 MHz, CDCl3) d:
29.8 (CH2), 29.9 (CH2), 114.4 (C), 117.7 (CN), 130.8 (CH), 131.7
(CH), 135.0 (CH), 140.7 (C), 142.9 (C), 153.2 (C), 153.9 (C). IR
(KBr, cmꢀ1): 3022, 2976, 2228, 1359, 912, 805, 509. Anal. Calcd
for C11H7N3Br2: C, 38.74; H, 2.07; N, 12.32. Found: C, 38.79; H,
2.10; N, 12.21.
4.2.9. 2,3-Bis(bromomethyl)quinoxaline-6-carboxylic acid 1i
Yield: 83%, mp: 170 °C (decomp.). 1H NMR (400 MHz, DMSO-d6)
d: 5.06 (4H, s), 8.20 (1H, d, J = 8.8 Hz), 8.34 (1H, dd, J = 1.5 and
8.8 Hz), 8.58 (1H, d, J = 1.5 Hz). 13C NMR (126 MHz, DMSO-d6) d:
30.0 (CH2), 129.1 (CH), 130.3 (CH), 130.4 (CH), 133.0 (C), 140.1
(C), 142.6 (C), 152.2 (C), 153.0 (C), 166.3 (C@O). IR (KBr, cmꢀ1):
3312, 3035, 2981, 1692, 1314, 1276, 910, 826, 635. Anal. Calcd
for C11H8N2O2Br2: C, 36.72; H, 2.24; N, 7.78. Found: C, 36.92; H,
2.06; N, 7.65.
4.2.3. 2,3-Bis(bromomethyl)-6-fluoroquinoxaline 1c
Yield: 74%, mp: 142–143 °C. 1H NMR (400 MHz, CDCl3) d: 4.90
(2H, s), 4.91 (2H, s), 7.56–7.61 (1H, m), 7.68–7.71 (1H, m), 8.06–
8.10 ppm (1H, m). 13C NMR (126 MHz, CDCl3) d: 30.3 (CH2), 30.4
(CH2), 112.8 (d, JCF = 22 Hz, CH), 121.5 (d, JCF = 26 Hz, CH), 130.3
(d, JCF = 10 Hz, CH), 138.9 (C), 142.6 (d, JCF = 15 Hz, C), 150.3 (C),
151.9 (C), 163.5 (d, JCF = 254 Hz, C). IR (KBr, cmꢀ1): 3025, 2973,
1330, 1225, 891, 802, 638. Anal. Calcd for C10H7N2FBr2: C, 35.96;
H, 2.11; N, 8.39. Found: C, 36.12; H, 2.09; N, 8.30.
4.3. Preparation of 2,3-bis(bromomethyl)-6-
alkoxycarbonylquinoxaline 1j and 1k
To a 20 mL solution of 1i (1.5 mmol) in MeOH (for 1j) or EtOH
(for 1k), 3 mL conc. H2SO4 was added, and the mixture was re-
fluxed for 2 h in an argon atmosphere. Water (100 mL) was then
added, and the mixture was extracted with CHCl3 (50 mL ꢂ 2).
The CHCl3 layer was washed with H2O (50 mL ꢂ 2) and then dried
over anhydrous Na2SO4. After evaporation of the solvent, the resi-
due was purified by column chromatography on silica gel with
CHCl3.
4.2.4. 2,3-Bis(bromomethyl)-6-chloroquinoxaline 1d
Yield: 73%, mp: 149–151 °C. 1H NMR (400 MHz, CDCl3) d: 4.89
(2H, s), 4.90 (2H, s), 7.74 (1H, dd, J = 2.3 and 9.0 Hz), 8.01 (1H, d,
J = 9.0 Hz), 8.07 (1H, d, J = 2.3 Hz). 13C NMR (126 MHz, CDCl3) d:
30.3 (CH2), 30.4 (CH2), 128.1 (CH), 130.4 (CH), 132.1 (CH), 137.0
(C), 140.8 (C), 141.9 (C), 151.2 (C), 152.0 (C). IR (KBr, cmꢀ1):
3029, 2972, 1358, 879, 836, 626, 547. Anal. Calcd for C10H7N2ClBr2:
C, 34.27; H, 2.07; N, 7.99. Found: C, 34.41; H, 1.93; N, 7.96.
4.3.1. 2,3-Bis(bromomethyl)-6-methoxycarbonylquinoxaline 1j
Yield: 74%, mp: 92–93 °C. 1H NMR (400 MHz, CDCl3) d: 4.02 (3H,
s), 4.93 (4H, s), 8.12 (1H, d, J = 8.8 Hz), 8.39 (1H, dd, J = 1.8 and
8.7 Hz), 8.78 (1H, d, J = 1.8 Hz). 13C NMR (126 MHz, CDCl3) d: 30.1
(CH2), 30.2 (CH2), 52.9 (OCH3), 129.4 (CH), 130.6 (CH), 131.7
(CH), 132.3 (C), 140.9 (C), 143.5 (C), 152.0 (C), 152.8 (C), 166.0
(C@O). IR (KBr, cmꢀ1): 3033, 2951, 1726, 1365, 1263, 911, 802,
637. Anal. Calcd for C12H10N2O2Br2: C, 38.53; H, 2.69; N, 7.49.
Found: C, 38.69; H, 2.61; N, 7.39.
4.2.5. 6-Bromo-2,3-bis(bromomethyl)quinoxaline 1e
Yield: 77%, mp: 142–144 °C. 1H NMR (400 MHz, CDCl3) d: 4.89
(2H, s), 4.90 (2H, s), 7.87 (1H, dd, J = 1.7 and 9.0 Hz), 7.94 (1H, d,
J = 9.0 Hz), 8.25 (1H, d, J = 1.7 Hz). 13C NMR (126 MHz, CDCl3) d:
30.3 (CH2), 30.4 (CH2), 125.2 (C), 130.4 (CH), 131.5 (CH), 134.6
(CH), 140.4 (C), 142.2 (C), 151.3 (C), 152.0 (C). IR (KBr, cmꢀ1):
3076, 3028, 2972, 1356, 880, 833, 569. Anal. Calcd for
4.3.2. 2,3-Bis(bromomethyl)-6-ethoxycarbonylquinoxaline 1k
Yield: 90%, mp: 72–74 °C. 1H NMR (400 MHz, CDCl3) d: 1.46 (3H,
t, J = 7.2 Hz), 4.47 (2H, q, J = 7.2 Hz), 4.93 (4H, s), 8.12 (1H, d,
J = 8.8 Hz), 8.39 (1H, dd, J = 1.9 and 8.7 Hz), 8.79 (1H, d,
C
10H7N2Br3ꢁ0.4 H2O: C, 29.87; H, 1.96; N, 6.97. Found: C, 29.73;
H, 1.65; N, 6.90.

H. Ishikawa et al. / Bioorganic Chemistry 41-42 (2012) 1–5
5
J = 1.9 Hz). 13C NMR (126 MHz, CDCl3) d: 14.4 (CH3), 30.0 (CH2),
30.1 (CH2), 62.0 (OCH3), 129.2 (CH), 130.7 (CH), 131.5 (CH), 132.8
(C), 140.9 (C), 143.3 (C), 152.1 (C), 152.8 (C), 165.5 (C@O). IR
(KBr, cmꢀ1): 3002, 2940, 1741, 1314, 1195, 1026, 858, 808, 579.
Anal. Calcd for C13H12N2O2Br2ꢁ0.2 H2O: C, 39.87; H, 3.19; N, 7.15.
Found: C, 39.86; H, 3.20; N, 7.16.
glucose bouillon agar medium. Suspensions of each bacterial or
fungal species were prepared that showed 0.05 of turbidity at
660 nm, and suspension of mold species were prepared that con-
tained approximately 106 spore/mL. After incubation at 33 °C for
18 h (antibacterial assay), or at 33 °C for 18 h and 28 °C for 2 days
(antifungal assay), the growth of the bacteria or fungus was ob-
served. The MIC was determined as the lowest concentration of
compound that completely inhibited organism growth.
4.4. Preparation of 2,3-bis(bromomethyl)-6-hydroxyquinoxaline 1l
To a 10 mL solution of compound 1f (39.8 mg/mL, 0.910 mmol)
in dry CH2Cl2, 6 mL of 1 M BBr3 in CH2Cl2 was added at ꢀ30 °C in an
argon atmosphere, and the reaction mixture was stirred for 5 days
at room temperature. The mixture was then cooled to ꢀ30 °C, and
then 36 mL MeOH was added. After evaporation of the solvent, the
residue was dissolved in 50 mL CHCl3, and the CHCl3 solution was
washed with H2O (50 mL ꢂ 3) and then dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure, and
the residue was purified by column chromatography on silica gel
with CHCl3/acetone/EtOH (100/10/4 v/v/v).
Acknowledgment
This work was supported by a grant from Seikei University.
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Yield: 94%, mp: 140 °C (decomp.). 1H NMR (400 MHz, CDCl3) d:
4.87 (2H, s), 4.88 (2H, s), 7.34 (1H, d, J = 2.5 Hz), 7.41 (1H, dd,
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4.5.2. Minimum inhibitory concentration assay
The minimum inhibitory concentration (MIC) was determined
according to the agar plate dilution method using a microplanter
(Sakuma Seisakusho, Tokyo, Japan). Compounds 1a–l were dis-
solved in a water/methyl carbitol (diethylene glycol monomethyl
ether) mixture at a concentration of 1 wt.% (1/water/methyl carb-
itol = 1/79/20 wt/wt/wt). Next, the solution was diluted in a