Design, synthesis, and AMPA receptor antagonistic activity of a novel 6-nitro-3-oxoquinoxaline-2-carboxylic acid with a substituted phenyl group at the 7 position

Bioorganic & Medicinal Chemistry 13 (2005) 5841–5863

Design, synthesis, and AMPA receptor antagonistic activity of

a novel 6-nitro-3-oxoquinoxaline-2-carboxylic acid with

a substituted phenyl group at the 7 position

Yasuo Takano,^*Futoshi Shiga, Jun Asano, Naoki Ando, Hideharu Uchiki,

Kazunori Fukuchi and Tsuyosi Anraku

Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd, 2399-1, Nogi, Nogi-machi, Simotsuga-gun,

Tochigi 329-0114, Japan

Received 28 April 2005; revised 13 May 2005; accepted 14 May 2005

Available online 1 July 2005

Abstract—We describe the design, synthesis, and biological properties of a novel series of 7-substituted 6-nitro-3-oxoquinoxaline-2-

carboxylic acids. After designing, studying the structure–activity relationships, and evaluating the properties of various compounds,

we found that 7-heterocyclic-6-nitro-3-oxoquinoxaline-2-carboxylic acids that contain a substituted phenyl group linked through

urethane at the 7 position possess good a-amino-3-hydroxy-5-methylisoxazole propionate receptor (AMPA-R) antagonistic activity.

Among the compounds tested, compound 29p (GRA-293), which has a 4-carboxy group on the terminal phenyl moiety, exhibited

high potency and selectivity for the AMPA-R in vitro and good neuroprotective eﬃcacy in vivo. It also showed good aqueous

solubility.

1. Introduction

inedione compounds with competitive AMPA-R antag-

onistic activity have been synthesized and tested against

each of the EAA receptor subtypes. Although the

AMPA-R antagonists do not produce side eﬀects such

as schizophrenia,⁹and eﬀectively protect against neuro-

nal death even in postischemic animal models,²none of

these compounds have yet been marketed as therapeutic

agents for conditions such as acute cerebral ischemia.

The previously described AMPA-R antagonistic quin-

oxalinedione derivatives can be divided into ﬁrst gener-

ation agents (e.g., NBQX and YM-90K^10,11) and second

generation agents (e.g., YM-872 [zonampanel]^12,13).

Both the ﬁrst and second generation agents are potent,

selective AMPA-R antagonists, and have been shown

to exhibit good neuroprotective eﬀects in animal models

of global and focal cerebral ischemia (Fig. 1).^2,11,13

Unfortunately, the ﬁrst-generation compounds caused

renal toxicity as a result of their physicochemical prop-

erties, particularly very low solubility in water,¹⁴and

were thus rejected after reaching clinical trials. In second

generation agents such as YM-872, these undesirable

physicochemical properties have been ameliorated by

introducing a hydrophilic functional group (such as ace-

tic acid) into the quinoxalinedione skeleton. Because of

its good aqueous solubility, YM-872 has not caused

renal toxicity and, together with tissue plasminogen

Glutamic acid, an excitatory amino acid (EAA), is a

major excitatory neurotransmitter in the mammalian

central nervous system. Postsynaptic EAA receptors

can be divided into two main subtypes, namely

ionotropic glutamate receptors (iGlu-Rs), such as N-

methyl-^D-aspartate (NMDA), a-amino-3-hydroxy-5-

methylisoxazole propionate (AMPA) and kinate

(KA), and metabotropic glutamate receptors (mGlu-

Rs). The iGlu-Rs are associated with integral cation-

speciﬁc ion channels, which modulate cell excitability

by gating the ﬂow of calcium and sodium ions into

the cell.¹However, overstimulation of postsynaptic

receptors by EAA is known to worsen neurodegenera-

tion in conditions such as ischemic stroke, epilepsy,

Huntingtonꢀs disease, head trauma, Parkinsonꢀs dis-

ease, and Alzheimerꢀs disease.^2–8

Since the discovery of NBQX,²a potent and selective

AMPA receptor (AMPA-R) antagonist, many quinoxal-

Keywords: Excitatory amino acid; Competitive AMPA-R antagonist;

3-Oxoquinoxaline-2-carboxylic acid; Cerebral ischemia.

*

Corresponding author. Tel.: +81 280 56 2201; fax: +81 280 57

1293; e-mail: yasuo.takano@mb.kyorin-pharm.co.jp

doi:10.1016/j.bmc.2005.05.030

5842

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

Figure 1. Known competitive AMPA-R antagonists.

activator (t-PA), is the only agent for the treatment of

cerebral ischemia to have reached the clinical trial stage.

When we started to search for AMPA-R antagonists,

other researchers had already advanced the development

of several improved quinoxalinediones with good solu-

bility, and therefore there appeared to be little scope

for developing new second generation AMPA-R anta-

gonists based on chemical modiﬁcations of the quinoxal-

inedione structure. Thus, we refocused our research

eﬀorts on designing and synthesizing novel third gener-

ation compounds, which would not have a quinoxaline-

dione skeleton, but would have potent AMPA-R

aﬃnity, strong neuroprotective eﬀects in vivo and be

water soluble, thus allowing them to be administered

by injection.

ies of new compounds based on 7-substituted-6-nitro-3-

oxo-quinoxaline-2-carboxylic acids (Fig. 2). In this pa-

per, we describe the design and synthesis of these novel

7-substituted 6-nitro-3-oxoquinoxaline-2-carboxylic

acid derivatives and their biological eﬀects.

2. Chemistry

The 7-halogeno-6-nitro-3-oxoquinoxaline-2-carboxylic

acid derivatives were prepared using two methods (routes

A and B), as shown in Scheme 1. In the case of the 7-chloro

compound produced via route A, 4-chloro-1,2-phenylen-

ediamine (1) was condensed with diethyl ketomalonate,

then separated by column chromatography to give ethyl

7-chloro-3-oxoquinoxaline-2-carboxylate (2a) and its iso-

mer (3). The esters 2a and 3 were converted to the 6- and 7-

nitro compounds (4a and 5) by selective nitration (2a,

fuming HNO₃, AcOH; 3, KNO₃, concd H₂SO₄) followed

by hydrolysis to give 6- and 7-nitro carboxylic acids (6a

and 7, respectively). Compound 6a could also be synthe-

sized from 4-chloro-2-nitroaniline (8a) using an improve-

ment of the standard procedure,¹⁶as shown in route B.

The nitroaniline 8a was treated with ethyl 3-chloro-3-oxo-

propionate, followed by intramolecular cyclization and

deoxygenation to give ethyl 7-chloro-3-oxoquinoxaline-

2-carboxylate (2a); the chemical shift and coupling con-

stants seen on ¹H NMR spectroscopy indicated that this

product was consistent with compound 2a, as obtained

from 4-chloro-1,2-phenylenediamine (1) and diethyl

ketomalonate via route A. In the case of the 7-ﬂuoro

and 7-bromo derivatives, quinoxalines 2b and c were pre-

pared from 4-ﬂuoro- and 4-bromo-2-nitroaniline (8b and

c) as shown, following the same procedures used to

produce ethyl 7-chloro-3-oxoquinoxaline-2-carboxylate

(2a) from 8a. These intermediates were then converted

to the nitro compounds 4b and c by selective nitration

(fuming HNO₃, AcOH) at the 6 position, followed by

hydrolysis to give the 7-ﬂuoro and 7-bromo carboxylic

acids (6b and c, respectively).

In an AMPA-R pharmacophore model, three important

interaction units have already been identiﬁed for the

binding of quinoxalinediones, namely (1) the 2,3-dione

moiety of quinoxalinedione is needed to form a coulom-

bic interaction between the tautomeric 2-oxo moiety and

the AMPA-R, (2) a hydrogen bond donor at 4 position

of the quinoxalinedione molecule is needed to interact

with the AMPA-R, and (3) the nitro group at 6 position

undergoes a speciﬁc interaction with the AMPA-R.¹⁵

However, it also appeared to us that the acetic acid

group introduced into the quinoxalinedione skeleton

of YM-872 resulted in improved aﬃnity for the

AMPA-R compared with YM-90K. We therefore decid-

ed that an acetic acid group on the quinoxaline skeleton

would aﬀect the ability of the compound to interact with

the AMPA-R as well as improve the water solubility.

With these attributes in mind, we concluded that 3-oxo-

quinoxaline-2-carboxylic acids bearing a hydrophilic

group on a quinoxaline nucleus, instead of a quinoxalin-

edione structure with a hydrophilic group, would pro-

vide a good nucleus for a new generation of AMPA-R

antagonists with both good AMPA-R activity and solu-

bility. Consequently, we designed and synthesized a ser-

A quinoxaline methylated at the 4 position (14) was pre-

pared from the 6-nitro compound 4b by methylation

with iodomethane, followed by hydrolysis of the ester

group. The 6-amino compound 15 and 2-carboxamide

compound 16 were prepared from the imino ether 11a,

which reacted with the 7-F ester 4b and iodomethane

in the presence of silver(I) oxide. Hydrogenation or ami-

dation of the imino ether 11a followed by hydrolysis

Figure 2. Synthetic targets for

antagonists.

a new generation of AMPA-R

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5843

Scheme 1. 7-Halogeno-6-nitro-3-oxoquinoxaline-2-carboxylic acid derivatives. Reagents: (a) Diethyl ketomalonate, EtOH and then separation by

chromatography; (b) fuming HNO₃, AcOH; (c) KNO₃, concd H₂SO₄; (d) 1 N KOH, EtOH; (e) ethyl 3-chloro-3-oxopropionate, toluene or Et₃N-

DMF; (f) KO^tBu, DMF; (g) PBr₃, DMF; (h) NaH, Mel, DMF; (i) Mel or EtBr, Ag₂O, toluene; (j) H₂, 10% Pd–C, EtOH; (k) NH₄OH, MeOH; (l) 1 N

NaOH, EtOH followed by 48% HBr, AcOH; (m) 48% HBr or concd HCl, AcOH.

gave compounds 15 and 16. Treatment of the 7-F ester

4b with bromoethane instead of iodomethane in the

presence of silver(I) oxide yielded the imino ether 11b.

alicyclic, and heterocyclic amino substituted derivatives

19a–l. The imino ether 11b was also treated with 4-meth-

oxybenzylamine in THF, followed by deprotection of

both the 4-methoxybenzyl (PMB) group and the imino

ether, then hydrolyzed to give the 7-amino compound

21. The 7-pyrrolyl compound 22 was prepared

from the 7-amino compound 21 using 2,5-dimeth-

oxytetrahydrofuran in AcOH.

The syntheses of the novel 7-heterocyclic-6-nitro-3-oxo-

quinoxaline-2-carboxylic acid derivatives are outlined in

Scheme 2. The imino ethers 11a or 11b, derived from the

7-ﬂuoro ester 4b, were reacted with commercially avail-

able amines ðR⁰₂-NHÞ such as dialkyl, alicyclic, and het-

erocyclic amines in THF, MeCN, or DMF to produce

nucleophilic substitution of the ﬂuoro group at the 7

position, followed by hydrolysis to form the 7-dialkyl,

Next, 4-urethane linked imidazole derivatives (28a–f,

29a–y, and 30a–c) were prepared from the key inter-

mediate 7-[4-(hydroxymethyl)imidazolyl]quinoxaline (18k),

Scheme 2. 7-Heterocyclic-6-nitro-3-oxoquinoxaline-2-carboxylic acid derivatives. Reagents: (a) ðR⁰₂-NHÞ, Et₃N or none, THF or MeCN or DMF;

(b) concd HCl; (c) KOH or NaOH, EtOH–H₂O followed by 3 N HCl or 48% HBr; (d) concd HCl or 48% HBr, AcOH; (e) concd HCl, AcOH

followed by LiOH, H₂O; (f) 4-methoxybenzyl amine, Et₃N, THF; (g) CF₃CO₂H, anisole; (h) 1 N NaOH, EtOH followed by concd HCl, EtOH; (i)

2,5-dimethoxytetrahydrofuran, AcOH.

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Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

Scheme 3. 4-Urethane linked imidazole derivatives. (a) 4-[HO(CH₂)_n]imidazole, Et₃N, MeCN or DMA; (b) R⁴-NCO, CH₂Cl₂or benzene or MeCN;

(c) R⁴CO₂H, DPPA, Et₃N, benzene; (d) 48% HBr or concd HCl, AcOH; (e) concd HCl, AcOH followed by 1 N LiOH or 2 N NaOH; (f) 1 N KOH,

EtOH followed by 48% HBr.

Scheme 4. 3-Urethane linked pyridone derivatives. (a) n-BuLi, ⁱPr₂NH, THF followed by DMF; (b) NaBH₄, EtOH; (c) NaOH, H₂O; (d) compound

11b, DMF; (e) R⁴-NCO, CH₂Cl₂; (f) consd HCl, AcOH; (g) concd HCl, AcOH followed by LiOH, H₂O.

which was synthesized from imino ether 11b and 4-

(hydroxymethyl)imidazole. Treatment with various

isocyanates followed by hydrolysis provided the corre-

sponding 4-urethane linked derivatives shown in Scheme

3. Compound 31 was also prepared from 7-[4-(2-

hydroxyethyl)imidazolyl]quinoxaline (23), which was

obtained from imino ether 11b using 4-(2-hydroxyeth-

yl)imidazole¹⁷instead of 4-(hydroxymethyl)imidazole

in the above procedure.

3.1. 7-Halogeno derivatives

The structures and AMPA-R aﬃnities of the 7-halo-

geno compounds are shown in Table 1. Previous stud-

ies on quinoxalinedione compounds with known

AMPA-R antagonistic activity^10,13have indicated that

a nitro group at the 6 position on the quinoxalinedione

nucleus confers high aﬃnity for the AMPA-R. To pro-

vide initial conﬁrmation that the 3-oxoquinoxaline-2-

The 3-urethane linked pyridone derivatives 38a and b

were prepared from 7-[3-(hydroxymethyl)-4-pyridon-1-

yl]quinoxaline (36) as the key intermediate following

the same procedure as for the imidazole derivatives. 3-

(Hydroxymethyl)-4-pyridone (35), the starting material

for the heterocycle at the 7 position, was synthesized

by hydroxylation of 3-(hydroxymethyl)-4-chloropyri-

dine (34) with NaOH solution. This was in turn pre-

pared by lithiation of 4-chloropyridine (32) followed

by treatment with N,N-dimethylformamide and reduc-

tion with sodium borohydride (Scheme 4).

Table 1. 7-Halogenated derivatives

Compound

R

R¹

R²

R³

AMPA-R aﬃnity^25,26

K_i(nM)

6a

7

Cl NO₂OH

NO₂Cl OH

H

1300

6700

4a

6b

6c

14

15

16

Cl

F

NO₂OEt

NO₂OH

NH₂OH

NO₂NH₂

>10,000

2000

690

3. Results and discussion

Br

F

Me >10,000

The structures and characteristics of the 7-substituted-6-

nitro-3-oxoquinoxaline-2-carboxylic acid derivatives are

shown in Tables 1–6.

F

H

>10,000

F

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5845

carboxylic acid nucleus would be suitable for the gen-

eration of AMPA-R antagonists, we introduced a nitro

group by carrying out electron-withdrawing at the 6 or

7 position in the 3-oxoquinoxaline-2-carboxylic acid.

of a strong electron-withdrawing group at the 6

position, a carboxyl group at the 2 position, and a

hydrogen atom at the 4 position of the 3-oxoquinoxa-

line-2-carboxylic acid.

Accordingly,

3-oxoquinoxaline-2-carboxylic

acid,

which has a nitro group at the 6 position (6a), exhibit-

ed good AMPA-R aﬃnity. However, 6-chloro-7-nitro-

3-oxoquinoxaline-2-carboxylic acid (7), which contains

anti-substituted compounds at the 6 and 7 positions,

was approximately 5-fold less potent than the parent

compound 6a. These binding-aﬃnity results can be

ascribed to the presence of a chloro group, a weak elec-

tron-withdrawing group, rather than a nitro group at

the 6 position. On the other hand, replacing the halo-

gen atoms at the 7 position (ﬂuorine [6b] or bromine

[6c]) produced nearly the same activity as in 6a. An

electron-donating group such as an amino group (15)

at the 6 position in the 3-oxoquinoxaline-2-carboxylic

acid also resulted in loss of AMPA-R aﬃnity. In par-

ticular, in the 3-oxoquinoxaline-2-carboxylic acids with

a nitro group at the 6 position, conversion of the car-

boxyl group to an ester group (4a) or an amide group

(16) and replacement of the intramolecular amide moi-

ety by a methyl group (14) resulted in markedly de-

creased AMPA-R aﬃnity. These results conﬁrmed

that potent AMPA-R aﬃnity depended on the presence

3.2. 7-Dialkyl, alicyclic, and heterocyclic amino

derivatives

As the next approach, based on the results obtained with

the 7-halogeno-3-oxoquinoxaline-2-carboxylic deriva-

tives, we selected a compound with a nitro group at the

6 position and performed chemical modiﬁcations which

introduced a variety of amines (such as dialkyl, alicyclic,

and heterocyclic amines) at the 7 position. The structures

and AMPA-R aﬃnities of these 7-(substituted)amino

compounds are shown in Table 2. The dialkyl and alicy-

clic amino derivatives (19a–f) showed similar AMPA-R

aﬃnity to the 7-halogeno derivatives. However, introduc-

tion of aheterocyclicamine, asin the 4-pyridon-1-yl (19g),

imidazol-1-yl (19h), and pyrrol-1-yl (22) compounds con-

siderably enhanced AMPA-R binding aﬃnity. Conse-

quently, substitution of a ﬂat heterocyclic structure

bearing p-electrons at the 7 position on the 6-nitro-3-oxo-

quinoxaline-2-carboxylic acid appears to be essential for

increasing AMPA-R aﬃnity.

3.3. 7-(Substituted)imidazole derivatives and their molec-

ular modeling

Table 2. 7-Substituted amino and 7-heterocyclic 2-quinoxalinecarb-

oxylic acid derivatives

Next, we compared the eﬀects of substitution onto the

imidazole moiety on AMPA-R aﬃnity using commer-

cially available imidazoles. Compared to a nonsubstitut-

ed imidazole, AMPA-R aﬃnity was reduced by the

introduction of substituents at the 2 or 4 position on

the imidazole moiety (19h vs 19i–19l). Substitution at

the 2 position produced a particularly marked decrease

in AMPA-R aﬃnity. Among the compounds with sub-

stituents at the 4 position, however, the carboxymethyl

compound 19l maintained AMPA-R aﬃnity equal to

that of the nonsubstituted imidazole compound 19h.

Compounds 19j and 19k showed decreased activity com-

pared to 19h. Interestingly, the AMPA-R aﬃnity of

compound 19j was increased by introducing a carboxy

group onto the 4-methyl group on the imidazole ring.

From these results, we concluded that a carboxymethyl

group on the imidazole moiety may facilitate interaction

Compound

R

AMPA-R aﬃnity^25,26

K_i(nM)

19a

(CH₃)₂N–

910

3800

2000

2500

3700

2600

330

19b

19c

19d

19e

19f

19g

19h

22

Table 3. 7-Substituted imidazole derivatives

Compound

R²

R³

AMPA-R aﬃnity^25,26

K_i(nM)

19h

19i

19j

19k

19l

H

560

>10,000

1300

560

H

CH₃

H

CH₂OH

CH₂CO₂H

H

2600

530

920

H

5846

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

Figure 3. Modeled structure of AMPA-R bound to ligands. (a) The bound conformation of 19h in the active site of the modeled AMPA-R. (b) The

bound conformation of compound 19l in the active site of modeled AMPA-R.

with the AMPA-R, and that a carboxyl group would be

needed as a proton acceptor and/or donor to interact

with the AMPA-R (Table 3).

acceptors would be the most favorable substituents at

the 4 position on the imidazole group.

3.4. 7-Urethane linked imidazole derivatives

Based on the binding-aﬃnity results, we studied the

impact of structural conformation on molecular model-

ing. We divided the structure of compound 19h into three

parts (Fig. 3a). The ﬁrst part was the 3-oxo-2-carboxylic

acid moiety of the 6-nitro-3-oxoquinoxaline-2-carboxyl-

ic acid. This interacts with the AMPA-R through two

potential hydrogen bonds to Thr504 and Thr506, and

an ion pair to Arg509. The second part consists of the

amide nitrogen of 19h, which forms a hydrogen bond

with the backbone carbonyl group of Pro502. The third

part consists of the 6-nitro group on the quinoxaline

ring, which forms a hydrogen bond with the side chains

of Tyr756 and Asn733. These modeling results convinced

us that the three individual parts of the molecule were

essential for interaction of the AMPA-R with 6-nitro-

3-oxoquinoxaline-2-carboxylic acids. In the model, the

7-imidazole ring of compound 19h lies near the side chain

of Thr707, and the nitrogen atom at the 3 position on the

7-imidazole ring could form a weak hydrogen bond with

the hydroxyl group of Thr707.

Based on the information yielded by molecular model-

ing, we synthesized a series of carbonyl compounds

with urethane-containing groups attached through

the hydroxymethyl group on the imidazole moiety of

19k, which could in turn be readily synthesized. The

structures and aﬃnities of these 7-urethane linked

imidazole derivatives for the AMPA-R and NMDA-

R are shown in Table 4. We found that compounds

with the urethane linkage exhibited more potent

AMPA-R aﬃnity than the nonsubstituted compound

19h and alcohol and the carboxylic compounds 19k

and l. In particular, urethane compounds with a ter-

minal lipophilic ring, such as a cyclohexyl (28c) or

phenyl ring (28d) exhibited good AMPA-R aﬃnity

and selectivity. However, when the methylene chain

between the urethane linkage and the terminal phenyl

group was extended, AMPA-R aﬃnity was reduced

(28e and f). We also attempted the introduction of a

similar substituent group, a phenyl group linked

through a urethane moiety, to a 4-pyridone compound

(19g), which exhibited good AMPA-R aﬃnity. Despite

having the same urethane-linked phenyl group as a

substituent at the 7 position on the quinoxaline ring,

the 4-pyridone compound 38a showed weaker

AMPA-R aﬃnity than the imidazole compound 28d.

Therefore, we concluded that an imidazole group at

the 7 position on the quinoxaline ring is optimal,

and that the imidazole group is needed to confer func-

tional activity through the urethane linkage to en-

hance AMPA-R aﬃnity. We further concluded that

the urethane moiety would function in a similar way

to the carboxyl group of compound 19l, and that a

terminal lipophilic ring (such as phenyl ring) would

be needed to allow good interaction with the

AMPA-R, as suggested by molecular modeling.

We next attempted a docking study, in which compound

19l (which possesses a carboxymethyl group at the 4

position on the imidazole ring) was superimposed on

the modeled AMPA-R structure (Fig. 3b). Because com-

pound 19l had greater AMPA-R aﬃnity compared to

compounds 19k and 19j, we concluded that the carbonyl

oxygen of its carboxymethyl group produced the most

favorable interaction with the side chain of Thr707.

Moreover, we believed that it would be possible to in-

crease the AMPA-R aﬃnity of compound 19l by intro-

ducing substituents at the 4 position on the imidazole

ring directed toward another interaction site, which in-

cludes Val714, Leu728, and Tyr735. Because this region

is predominantly hydrophobic in nature, lipophilic

groups joined through various links with hydrogen

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

Table 4. 7-Urethane linked imidazole/pyridone derivatives

5847

Compound

Ring A

R⁴

AMPA-R aﬃnity^25,26

K_i(nM)

NMDA-R aﬃnity²⁷

K_i(nM)

Selectivity

NMDA-R

AMPA-R

ꢀ

ꢁ

28a

28b

28c

28d

28e^a

28f

n-Bu

82

100

39

8900

NT

110

NT

iso-Pr

cycl.Hex

Ph

>10,000

7200

>260

86

67

>120

110

NT

PhCH₂

PhCH₂CH₂

Ph

170

NT

>10,000

38a

>59

NT, not tested.

^aNa salt.

3.5. Conﬁrmation of AMPA-R binding of an imidazole

with a 7-urethane linked phenyl group (28d) by molecular

modeling

a terminal lipophilic ring, such as a phenyl ring, are

needed for good interaction with AMPA-R. Our struc-

tural modeling experiments on the binding of the

AMPA-R to essential components of compound 19h,

thus, eventually led to the synthesis of compound 28d,

which shows high AMPA-R aﬃnity. This implies that

rational drug design using homology modeling and

docking strategies is useful.

A model of the binding between compound 28d and the

AMPA-R is shown in Figure 4a. The quinoxaline nucle-

us of compound 28d exhibited the same interactions as

in the three essential parts of compound 19h. Moreover,

we conﬁrmed that the ether oxygen on the urethane link-

age of compound 28d formed a hydrogen bond with the

side chain of Thr707, and that the terminal phenyl moi-

ety interacted with the side chains of Val714, Leu728,

and Tyr735. A substituent introduced at the 4 position

on the imidazole group produced a signiﬁcant increase

in AMPA-R aﬃnity when a urethane linkage was pres-

ent. Indeed, AMPA-R aﬃnity increased to a subnanom-

olar level, 7-fold stronger than that of compound 19h.

Therefore, it became clear that a urethane linkage and

Recently, the soluble ligand binding core of GluR2

(GluR2-S1S2J) was created and the X-ray crystallogra-

phy structure of the S1S2J-DNQX complex (PDB entry:

1FTL) was determined as shown in Figure 4b.¹⁸When

we compared our model structure with the X-ray struc-

ture, several diﬀerences were apparent in some of the

loop regions, which were generally diﬃcult to model

correctly. However, the amino acid residues near the

ligand-binding regions in our model structure were con-

Figure 4. Modeled and X-ray structures of AMPA-R bound to ligands. (a) The bound conformation of 28d in the active site of the modeled AMPA-

R. (b) The bound conformation of DNQX in the X-ray structure of AMPA-R active site.

5848

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

sistent with those seen in the X-ray structure. This con-

ﬁrmed that our AMPA-R model structure was useful for

the design of novel, highly potent inhibitors of the

AMPA-R.

the other compounds. Although the reason for this is

not clear, we assume that the interaction of terminal

phenyl group on the compound 29n against the

AMPA-R might be diﬀerent from other carboxylic com-

pounds (29o, p) because of intramolecular hydrogen

bonding formed between 2-carboxy group and urethane

moiety. In particular, compounds with strong electron-

withdrawing groups substituted at the 3 or 4 position

exhibited excellent AMPA-R aﬃnity and selectivity

(29l, m, o, p). On the other hand, except when a carbox-

ylic acid or triﬂuoromethyl group was introduced to the

phenyl ring (29m and p), compounds with substituents

at the 4 position showed low selectivity (29c, f, i, j).

Moreover, di-substituted compounds with substituents

at the 4 position also showed low selectivity, even if

the substituents introduced at the 2 or 3 position were

associated with good selectivity (29s, v, y vs 29r, t, u,

w, x). Although a non-substituted benzyl compound

(28e) exhibited slightly less selectivity for the AMPA-R

3.6. 7-Urethane linked imidazole derivatives with a

substituted phenyl group

Next, we investigated the eﬀects of substituents in the

terminal phenyl group attached through the urethane

linkage in order to identify an active compound with

high AMPA-R aﬃnity and better selectivity for the

AMPA-R than the NMDA-R (Table 5). There was a

signiﬁcant increase in AMPA-R aﬃnity after the intro-

duction of various substituents into the terminal phenyl

ring. Among these modiﬁcations, mono-substitution at

the 2 or 3 position resulted in signiﬁcantly better selec-

tivity, together with good AMPA-R aﬃnity, except for

the 2-carboxylic compound 29n, which was inferior to

Table 5. 7-Imidazole/pyridone derivatives with a urethane linkage

Compound

R⁴

n

AMPA-R aﬃnity^25,26

K_i(nM)

NMDA-R aﬃnity²⁷

K_i(nM)

Selectivity

NMDA-R

AMPA-R

ꢀ

ꢁ

28d

29a

29b

29c

29d

29e^a

29f

29g^a

29h

29i

29j^b

29k

29I

29m

29n

29o

29p

29q

29r

29s

29t

Ph

2-Cl-Ph

1

86

20

>10,000

4100

6600

>120

210

370

43

1

2

1

—

3-Cl-Ph

4-Cl-Ph

18

30

1300

8300

2-Br-Ph

3-Br-Ph

30

16

280

440

38

7000

1200

4-Br-Ph

2-Me-Ph

32

16

5900

6300

370

290

70

3-Me-Ph

4-Me-Ph

22

30

2100

2000

4-MeO-Ph

2-CF₃-Ph

3-CF₃-Ph

4-CF₃-Ph

2-CO₂H-Ph

3-CO₂H-Ph

4-CO₂H-Ph

4-CO₂Et-Ph

2,3-Cl₂-Ph

2,4-Cl₂-Ph

2,5-Cl₂-Ph

2,6-Cl₂-Ph

3,4-Cl₂-Ph

3,5-Cl₂-Ph

1-Naphtyl

2-Naphtyl

4-CO₂H-Ph

2-Br-PhCH2

3-Br-PhCH2

4-Br-PhCH2

4-CO₂H-Ph

30

32

67

9300

290

>500

>370

>10

>370

>450

>170

260

41

20

27

>10,000

4400

>1000

27

22

60

17

32

15

1300

4100

270

190

77

29u

29v^a

29w

29x

29y

31

17

31

3200

2400

14

3200

8600

230

610

63

32

2000

NT

270

29

NT

160

69

30a

30b

30c

38b

4700

6900

100

47

2900

>10,000

62

>83

120

NT, not tested.

^aHCl salt.

^bNa salt

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5849

than the phenyl compound 28d, we investigated the

inﬂuence of the length of the methylene chain between

the terminal phenyl ring and the urethane group on

the side chain at the 7 position in an attempt to enhance

AMPA-R aﬃnity and selectivity. Unfortunately, benzyl

compounds with bromine substituents on the terminal

phenyl group exhibited reduced AMPA-R selectivity

or aﬃnity (29d–f vs 30a–c). Moreover, when a methy-

lene group was inserted between the imidazole group

and the urethane group there was also signiﬁcantly low-

er AMPA-R aﬃnity (29p vs 31). Consequently, we con-

sider that extending the methylene chain between the

urethane linkage and the terminal phenyl group, or

between the imidazole ring and the urethane linkage,

adversely inﬂuences AMPA-R aﬃnity and selectivity.

Therefore, we concluded that the best side chain to at-

tach to the 4 position of the imidazole ring is a ꢁphenyla-

minocarbonyloxymethyl groupꢀ, which forms a very

important core for interaction with the AMPA-R. In

addition, a urethane linkage and a terminal phenyl

group are both important interaction units that may

function as proton acceptors during interaction with

the AMPA-R. On the other hand, we attempted the

introduction of carboxy group at the 4 position on the

terminal phenyl group of pyridine 38a, because the 4-

carboxylic compound 29p exhibited more potent

AMPA-R aﬃnity than the nonsubstituted compound

28d. However, the 4-pyridone 38b with 4-carboxyphenyl

group linked through a urethane also showed weak

AMPA-R aﬃnity, and the result fell short of our

expectation.

administered at an iv infusion rate of 20–40 mg/kg/h

for 4 h. However, the nonsubstituted imidazole 19h did

not show neuroprotective eﬀects at the same iv infusion

rate. Compounds 29o and 29p showed better neuropro-

tective eﬀects in vivo, as well as AMPA-R activity in vi-

tro, than NBQX, YM-90K, and YM-872. It was

noteworthy that the AMPA-R aﬃnity and neuroprotec-

tive eﬀects of 19h were inferior to those of 29o and 29p,

despite the presence of a 7-imidazolyl-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid nucleus. Compound 19h

lacked the urethane-linked phenyl ring substituent at

the 7 position on the imidazole group, indicating that

this novel substituent not only confers potent AMPA-

R aﬃnity but also contributes to therapeutic eﬃcacy

in animal models. In a comparison of the biological

activities of compounds 29o and 29p, both of which have

a carboxyl group on the terminal phenyl group, the

4-carboxyphenyl compound 29p exhibited markedly

more potent activity in vivo than the 3-carboxyphenyl

compound 29o. In particular, 29p showed neuroprotec-

tive eﬀects superior to those of any of the previously

reported quinoxalinedione compounds, and this was

achieved with a relatively low iv infusion rate of at least

2.5 mg/kg/h for 4 h. Furthermore, compound 29p

showed the best aqueous solubility among the 4-ure-

thane linked imidazole derivatives, which was higher

than aqueous solubility of the ﬁrst generation

compounds (compound 29p: 4.9 mg/mL, NBQX and

YM-90K: <1 mg/mL at pH 7.4).²⁰These characteristics

warrant further investigation of this compound for use

3.7. Neuroprotective eﬀects in permanent focal ischemia in

rats

The neuroprotective eﬀects of the selected compounds,

19g, 19h, 29o, and 29p, and those of the reference com-

pounds, NBQX, YM-90K, and YM-872, were examined

using the rat permanent focal ischemia model described

by Tamura et al.¹⁹The neuroprotective eﬀects, as as-

sessed using a four-point damage score, are shown in

Table 6 and Figure 5. Among our selected compounds,

the 4-pyridone compound 19g was highly soluble in an

aqueous solution (8.1 mg/mL at pH 7.4),²⁰and showed

good, dose-dependent neuroprotective eﬀects when

Figure 5. Four-point damage score in focal ischemia model.

Table 6. Pharmacological data for 7-heterocyclic quinoxalinecarboxylic acid derivatives

Compound

AMPA-R

aﬃnity^25,26

K_i(nM)

NMDA-R

aﬃnity²⁷

K_i(nM)

Selectivity

AMPA-R

antagonism²⁸

(D.C. potential)

Protective eﬀects in

focal ischemia model¹⁹

(Dose:mg/kg/h for 4 h, iv)

ꢀ

>1300

ꢁ

NMDA ꢀ R

AMPA ꢀ R

NBQX

YM-90K

YM-872

19g

65

100

62

>84,500

43,000

15,000

27,000

NT

(+)

2.3 (30)

2.8 (15)

0.5 (30)

2.2 (20)

2.8 (40)

0.2 (20)

2.0 (2.5)

2.3 (5.0)

1.6 (1.25)

3.0 (2.5)

3.0 (3.0)

n = 4

n = 7

n = 6

n = 5

n = 6

n = 3

n = 5

n = 3

n = 2

430

240

330

190

19h

29o

560

27

63,000

>10,000

480

>370

(+)

29p

22

>10,000

>450

(+)

NT, not tested.

Control; <1.0.

5850

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

as an injectable formulation in the treatment of acute

cerebral ischemia.

J = 2.4 Hz, 1H), 7.59 (dd, J = 8.6, 2.4 Hz, 1H), 7.41 (d,

J = 8.6 Hz, 1H), 4.55 (q, J = 7.3 Hz, 2H), 1.48 (t,

J = 7.3 Hz, 3H). Compound 3: H NMR (DMSO-d₆)

1

d: 12.23 (br s, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.44 (d,

J = 1.8 Hz, 1H), 7.38 (dd, J = 8.6, 2.4 Hz, 1H), 4.56 (q,

J = 7.3 Hz, 2H), 1.49 (t, J = 7.3 Hz, 3H).

4. Conclusion

Molecular design and synthesis of novel third generation

AMPA-R antagonists based on the 6-nitro-3-oxoqui-

noxaline-2-carboxylic acid nucleus led to the creation

of novel quinoxaline compounds with good neuropro-

tective activities in vivo. In particular, we found that

3-oxoquinoxaline-2-carboxylic acids with both a nitro

group at the 6 position (as an electron-withdrawing

group) and a ﬂat heterocyclic group bearing p-electrons

at the 7 position formed a very important scaﬀold for

AMPA-R antagonistic activity, and that a urethane

linkage played an important role as a proton acceptor

in interactions with the AMPA-R. After investigating

the structure–activity relationships and evaluating the

properties of various compounds, we identiﬁed com-

pound 29p (GRA-293) as a novel AMPA-R antagonist

that possesses high potency and good selectivity in vitro

as well as more potent neuroprotective eﬀects in an

5.1.3. Ethyl 7-chloro-3,4-dihydro-6-nitro-3-oxoquinoxa-

line-2-carboxylate (4a). To a solution of compound 2a

(200 mg, 792 lmol) in AcOH (2 mL), fuming HNO₃

(65.5 lL, 1.58 mmol), was added at 80 ꢁC and the solu-

tion was stirred for 2 h at the same temperature. The

reaction was poured into ice water and the precipitate

was collected by ﬁltration, washed with water, and dried

under vacuum. The precipitate was puriﬁed by ﬂash col-

umn chromatography (SiO₂, n-hexane:AcOEt, 2:1) to

give the title compound as yellow powder (119 mg,

1

50%); H NMR (DMSO-d₆) d: 13.24 (br s, 1H), 8.29

(s, 1H), 7.91 (s, 1H), 4.40 (q, J = 7.3 Hz, 2H), 1.33 (t,

J = 7.3 Hz, 3H).

5.1.4. Ethyl 6-chloro-3,4-dihydro-7-nitro-3-oxoquinoxa-

line-2-carboxylate (5). To a solution of compound 3

(200 mg, 792 lmol) in concd H₂SO₄(4 mL), a solution

of KNO₃(160 mg, 1.58 mmol) in concd H₂SO₄(2 mL)

was added, and the solution was stirred for 2 h at room

temperature. The reaction was poured into ice water and

the precipitate was collected by ﬁltration, washed with

water, and dried under vacuum. The precipitate was

puriﬁed by ﬂash column chromatography (SiO₂, n-hex-

ane:AcOEt, 2:1) to give the title compound as pale

animal model

compounds.

than

known

quinoxalinedione

5. Experimental section

5.1. Chemistry

1

5.1.1. General. All reagents, starting materials, and sol-

vents were purchased from commercial suppliers and

used as received. Evaporation was carried out on a rota-

ry evaporator at bath temperatures <45 ꢁC and reduced

pressure. Column chromatography was performed with

silica gel (Merck: silica gel 60 with particle size 0.040–

0.063 mm). Reactions were monitored by TLC on silica

gel 60 F₂₅₄(Merck). Melting points were determined

with a YANAKO MP-500D and are uncorrected. Pro-

ton NMR spectra were recorded on a JEOL JNM-

EX400 and JEOL JNM-ECA400 with tetramethylsilane

as an internal standard. Chemical shifts are given in

parts per million (d) and splitting patterns are designat-

ed as follows: s, singlet; d, doublet; dd, double doublet;

dt, double triplet; t, triplet; td, triple doublet; q, quartet;

m, multiplet; br, broad; and br s, broad like singlet.

HRMS and FABHRMS data were recorded on a JEOL

JMS-SX102A. Elemental analyses were carried out on a

YANAKO CHN CORDER MT-5.

yellow powder (102 mg, 43%); H NMR (DMSO-d₆) d:

13.26 (br s, 1H), 8.62 (s, 1H), 7.49 (s, 1H), 4.39 (q,

J = 7.3 Hz, 2H), 1.32 (t, J = 7.3 Hz, 3H).

5.1.5. 7-Chloro-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (6a). To a suspension of compound 4a

(108 mg, 363 lmol) in EtOH (2 mL), 1 N KOH

(1.09 mL, 1.09 mmol) was added and the solution was

reﬂuxed for 3 h. After cooling, the reaction was diluted

with water and adjusted to pH 4 with AcOH, and

concentrated. A small amount of water was added to

the residue and the precipitate was collected by ﬁltra-

tion, washed with water, and dried under vacuum to

give the title compound as yellow powder (55.8 mg,

57%); mp 227–229 ꢁC (decomp.); ¹H NMR (DMSO-

d₆) d: 8.28 (s, 1H), 7.92 (s, 1H); HRMS 268.9824

(ꢀ1.5 mmu).

5.1.6. 6-Chloro-3,4-dihydro-7-nitro-3-oxoquinoxaline-2-

carboxylic acid (7). Following the procedure described

for compound 6a, the title compound was prepared

from compound 5, pale yellow powder (92%); mp

5.1.2. Ethyl 7-chloro-3,4-dihydro-3-oxoquinoxaline-2-car-

boxylate (2a) and ethyl 6-chloro-3,4-dihydro-3-oxoqui-

noxaline-2-carboxylate (3) prepared from compound 1

(route A). To a solution of compound 1 (1.00 g,

7.01 mmol) in EtOH (20 mL), diethyl ketomalonate

(1.12 mL, 7.36 mmol), was added and the solution was

reﬂuxed for 5 h. After cooling, the reaction was concen-

trated and the residue was puriﬁed by ﬂash column

chromatography (SiO₂, n-hexane:AcOEt, 2:1) to give

compound 2a (610 mg, 34%) as yellow powder and com-

pound 3 (500 mg, 28%) as yellow powder. Compound

1

>300 ꢁC; H NMR (DMSO-d₆) d: 8.61 (s, 1H), 7.73 (s,

1H); FAB(ꢀ)HRMS 267.9721 (ꢀ4.0 mmu).

5.1.7. Ethyl 3-[(4-chloro-2-nitrophenyl)amino]-3-oxopro-

pionate (9a). A solution of compound 8b (20.0 g,

116 mmol)

and

ethyl

3-chloro-3-oxopropionate

(15.7 mL, 122 mmol) in toluene (200 mL) was reﬂuxed

for 24 h. After cooling, the reaction was concentrated

and Pr₂O was added to the residue. The precipitate

i

1

2a: H NMR (DMSO-d₆) d: 12.69 (br s, 1H), 7.97 (d,

was collected by ﬁltration, washed with ⁱPr₂O, and dried

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5851

under vacuum to give the title compound as brown pow-

der (63%); ¹H NMR (CDCl₃) d: 8.71 (d, J = 9.0 Hz, 1H),

8.19 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 9.0, 2.4 Hz, 1H),

4.30 (q, J = 7.3 Hz, 2H), 3.56 (s, 2H), 1.21 (t,

J = 7.3Hz, 3H).

described for compound 2a (route B, step 1), the title

compound was prepared from compound 9b, brown

powder (31%); ¹H NMR (CDCl₃) d: 8.09–7.96 (m,

1H), 7.51–7.42 (m, 2H), 4.56 (q, J = 7.0 Hz, 2H), 1.46

(t, J = 7.0 Hz, 3H).

5.1.8. Ethyl 3-[(4-ﬂuoro-2-nitrophenyl)amino]-3-oxopro-

pionate (9b). Following the procedure described for

compound 9a, the title compound was prepared from

compound 8b, yellow powder (88%); ¹H NMR (CDCl₃)

d: 8.71 (dd, J = 9.7, 4.8 Hz, 1H), 7.91 (dd, J = 8.4,

3.1 Hz, 1H), 7.49–7.27 (m, 1H), 4.30 (q, J = 7.0 Hz,

2H), 3.56 (s, 2H), 1.33 (t, J = 7.0 Hz, 3H).

5.1.11.2. Step 2: Ethyl 3,4-dihydro-7-ﬂuoro-3-oxoqui-

noxaline-2-carboxylate (2b). Following the procedure

described for compound 2a (route B, step 2), the title

compound was prepared from ethyl 3,4-dihydro-7-ﬂuo-

ro-3-oxoquinoxaline-2-carboxylate 1-oxide, pale yellow

powder (75%); ¹H NMR (CDCl₃) d: 7.70–7.59 (m,

1H), 7.48–7.38 (m, 2H), 4.55 (q, J = 7.0 Hz, 2H), 1.48

(t, J = 7.0 Hz, 3H).

5.1.9. Ethyl 3-[(4-bromo-2-nitrophenyl)amino]-3-oxopro-

pionate (9c). To a solution of compound 8c (82.7 g,

381 mmol) in DMF (500 mL), ethyl 3-chloro-3-oxopro-

pionate (51.2 mL, 400 mmol) and Et₃N (55.8 mL,

400 mmol) were added at ice cooling, and the solution

was stirred for 30 min at the same temperature followed

by for 2 h at room temperature. The reaction was

poured into ice water and the precipitate was collected

by ﬁltration, washed with water, and dried under vacu-

um to give the title compound as brown powder (121 g,

96%); ¹H NMR (DMSO-d₆) d: 10.63 (br s, 1H), 8.18 (d,

J = 2.5 Hz, 1H), 7.93 (dd, J = 8.8, 2.4 Hz, 1H), 7.67 (d,

J = 8.8 Hz, 1H), 4.13 (q, J = 6.9 Hz, 2H), 3.53 (s, 2H),

1.21 (t, J = 6.8 Hz, 3H).

5.1.12. Ethyl 7-bromo-3,4-dihydro-3-oxoquinoxaline-2-

carboxylate (2c)

5.1.12.1. Step 1: Ethyl 7-bromo-3,4-dihydro-3-oxoqui-

noxaline-2-carboxylate 1-oxide. Following the procedure

described for compound 2a (route B, step 1), the title

compound was prepared from compound 9c, brown

powder (51%); ¹H NMR (DMSO-d₆) d: 8.22 (d,

J = 2.5 Hz, 1H), 7.91 (dd, J = 8.8, 2.0 Hz, 1H), 7.36 (d,

J = 8.8 Hz, 1H), 4.39 (q, J = 6.9 Hz, 2H), 1.31 (t,

J = 6.9 Hz, 3H).

5.1.12.2. Step 2: Ethyl 7-bromo-3,4-dihydro-3-oxoqui-

noxaline-2-carboxylate (2c). Following the procedure de-

scribed for compound 2a (route B, step 2), the title

compound was prepared from ethyl 7-bromo-3,4-dihy-

dro-3-oxoquinoxaline-2-carboxylate 1-oxide, yellow

powder (65%); ¹H NMR (DMSO-d₆) d: 8.06 (d,

J = 2.0 Hz, 1H), 7.80 (dd, J = 8.8, 2.0 Hz, 1H), 7.30 (d,

J = 8.8 Hz, 1H), 4.37 (q, J = 7.3 Hz, 2H), 1.32 (t,

J = 7.3 Hz, 3H).

5.1.10. Ethyl 7-chloro-3,4-dihydro-3-oxoquinoxaline-2-car-

boxylate (2a) prepared from compound 9a (route B)

5.1.10.1. Step 1: Ethyl 7-chloro-3,4-dihydro-3-oxoqui-

noxaline-2-carboxylate 1-oxide. To a solution of com-

pound 9a (6.00 g, 21.0 mmol) in DMF (30 mL), a

suspension of KO^tBu (4.70 g, 41.8 mmol) in DMF

(20 mL) was added at 0 ꢁC and the solution was stirred

for 1.5 h at the same temperature. The reaction was adjust-

edtopH 4with1 NHCl,extractedwithCH₂Cl₂, dried over

Na₂SO₄, and evaporated. The residue was puriﬁed by ﬂash

column chromatography (SiO₂, CH₂Cl₂:MeOH, 4:1) to

give the title compound (1.59 g, 28%) as pale yellow pow-

5.1.13. Ethyl 3,4-dihydro-7-ﬂuoro-6-nitro-3-oxoquinoxa-

line-2-carboxylate (4b). Following the procedure de-

scribed for compound 4a, the title compound was

prepared from compound 2b, yellow powder (45%);

¹H NMR (CDCl₃) d: 8.16 (d, J = 6.2 Hz, 1H), 7.89 (d,

J = 10.6 Hz, 1H), 4.58 (q, J = 7.0 Hz, 2H), 1.49 (t,

J = 7.0 Hz, 3H).

1

der; H NMR (CDCl₃) d: 8.33 (d, J = 2.2 Hz, 1H), 7.65

(dd, J = 8.8, 2.2 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 4.56

(q, J = 6.9 Hz, 2H), 1.46 (t, J = 6.9 Hz, 3H).

5.1.14. Ethyl 7-bromo-3,4-dihydro-6-nitro-3-oxoquinoxa-

line-2-carboxylate (4c). Following the procedure de-

scribed for compound 4a, the title compound was

5.1.10.2. Step 2: Ethyl 7-chloro-3,4-dihydro-3-oxoqui-

noxaline-2-carboxylate (2a). To a solution of ethyl 7-chlo-

ro-3,4-dihydro-3-oxoquinoxaline-2-carboxylate 1-oxide

(1.80 g, 6.70 mmol) in DMF (20 mL), PBr₃(1.20 mL,

13.4 mmol) was added, and the solution was stirred for

1.5 h at room temperature. The reaction was poured into

ice water, extracted with CHCl₃, dried over Na₂SO₄, and

evaporated. The residue was washed with ⁱPr₂O, and dried

under vacuum to give the title compound (1.55 g, 92%) as

pale yellow powder; ¹H NMR (CDCl₃) d: 7.94 (d,

J = 2.0 Hz, 1H), 7.58 (dd, J = 8.8, 2.0 Hz, 1H), 7.41 (d,

J = 8.8 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.33 (t,

J = 7.1 Hz, 3H).

1

prepared from compound 2c, yellow powder (93%); H

NMR (DMSO-d₆) d: 13.24 (br s, 1H), 8.40 (s, 1H),

7.86 (s 1H), 4.40 (q, J = 7.3 Hz, 2H), 1.33 (t,

J = 7.3 Hz, 3H).

5.1.15. Ethyl 3,4-dihydro-7-ﬂuoro-4-methyl-6-nitro-3-

oxoquinoxaline-2-carboxylate (10). To a solution of

compound 4c (345 mg, 1.23 mmol) in DMF (10 mL),

sodium hydride (50% in oil, 61.3 mg, 1.54 mmol) was

added, and the solution was stirred for 30 min at room

temperature. Iodomethane (95.5 lL, 1.54 mmol) was

added to the reaction and the solution was stirred for

2 h at room temperature. The reaction was poured into

ice water, extracted with AcOEt, dried over Na₂SO₄,

and evaporated. The residue was puriﬁed by ﬂash col-

umn chromatography (SiO₂, n-hexane:AcOEt, 5:2) to

5.1.11. Ethyl 3,4-dihydro-7-ﬂuoro-3-oxoquinoxaline-

2-carboxylate (2b)

5.1.11.1. Step 1: Ethyl 3,4-dihydro-7-ﬂuoro-3-oxoqui-

noxaline-2-carboxylate 1-oxide. Following the procedure

5852

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

give the title compound as pale yellow powder (272 mg,

75%); ¹H NMR (DMSO-d₆) d: 8.04 (d, J = 6.3 Hz, 1H),

7.87 (d, J = 10.3 Hz, 1H), 4.53 (q, J = 7.3 Hz, 2H), 3.77

(s, 3H), 1.45 (t, J = 7.3 Hz, 3H).

from compound 4c, yellow powder (64%); mp 218–

1

220 ꢁC (decomp.); H NMR (CDCl₃) d: 8.38 (s, 1H),

7.88 (s, 1H); HRMS 312.9358 (+2.4 mmu). Anal. Calcd

2

for C₉H₄BrN₃O₅ꢁ H₂O: C, 33.15%; H, 1.65%; N,

12.89%. Found: C, ³33.04%; H, 1.89%; N, 12.83%.

5.1.16. Ethyl 7-ﬂuoro-3-methoxy-6-nitroquinoxaline-2-

carboxylate (11a). A suspension of compound 4b

(1.00 g, 3.56 mmol), iodomethane (440 lL, 7.07 mmol),

and silver(I) oxide (990 mg, 4.31 mmol) in toluene

(100 mL) was stirred for 2 h at 100 ꢁC. After cooling,

the reaction was ﬁltered through Celite^ꢂ, and the ﬁltrate

was concentrated. The residue was puriﬁed by ﬂash col-

umn chromatography (SiO₂, CH₂Cl₂:AcOEt, 4:1) to

give the title compound as pale yellow powder

(580 mg, 55%); ¹H NMR (CDCl₃) d: 8.57 (d,

J = 7.3 Hz, 1H), 7.95 (d, J = 10.8 Hz, 1H), 4.55 (q,

J = 7.3 Hz, 2H), 4.18 (s, 3H), 1.47 (t, J = 7.3 Hz, 3H).

5.1.22. 3,4-Dihydro-7-ﬂuoro-4-methyl-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid (14). To a solution of com-

pound 10 (207 mg, 701 lmol) in AcOH (15 mL), concd

HCl (1 mL) and water (1 mL) were added, and the solu-

tion was stirred for 1 h at room temperature followed by

3 h at 80 ꢁC. After cooling, the reaction was concentrat-

ed. The residue was washed with MeCN and dried under

vacuum to give the title compound as brown powder

1

(78.4 mg, 41%); mp 173–175 ꢁC; H NMR (DMSO-d₆)

d: 8.32 (d, J = 6.8 Hz, 1H), 8.16 (d, J = 11.2 Hz, 1H),

3.67 (s, 3H); FAB(+)HRMS 268.0366 (ꢀ0.4 mmu).

1

5

Anal. Calcd for C₁₀H₆FN₃O₅ꢁ H₂O: C, 44.36%; H,

5.1.17. Ethyl 7-ﬂuoro-3-methoxy-6-nitroquinoxaline-2-

carboxylate (11b). Following the procedure described

for compound 11a, the title compound was prepared

from compound 4b and bromoethane, pale yellow pow-

der (68%); ¹H NMR (CDCl₃) d: 8.63 (d, J = 7.8 Hz, 1H),

8.33 (d, J = 11.7 Hz, 1H), 4.57 (q, J = 7.3 Hz, 2H), 4.47

(q, J = 6.8 Hz, 2H), 1.41 (t, J = 7.3 Hz, 2H), 1.36 (t,

J = 6.8 Hz, 3H).

2.38%; N, 15.52%. Found: C, 44.33%; H, 2.25%; N,

15.79%.

5.1.23. 6-Amino-3,4-dihydro-7-ﬂuoro-3-oxoquinoxaline-

2-carboxylic acid (15). To a solution of compound 12

(50.0 mg, 189 lmol) in MeOH (1 mL), 1 N NaOH

(500 lL) was added, and the solution was stirred for

1 h at room temperature. The reaction was concentrated

and the residue was dissolved in AcOH (3 mL). Forty-

seven percent of HBr (1 mL) was added to the solution

and stirred overnight. The reaction was concentrated,

and the residue was dissolved in 1 N NaOH and eluted

through synthetic adsorbent Sepabeads^ꢂSP850 (water).

The elution was concentrated and made acidic with 1 N

HCl. The precipitate was collected by ﬁltration, washed

with water, and dried under vacuum to give the title

compound as brown powder (10.2 mg, 23%); mp

5.1.18. Ethyl 6-amino-7-ﬂuoro-3-methoxyquinoxaline-2-

carboxylate (12). To a solution of compound 11a

(300 mg, 1.02 mmol) in EtOH (50 mL), 10% Pd–C

(60 mg) was added, and the solution was stirred for

2 h at room temperature under hydrogen atmosphere.

The catalyst was removed by ﬁltration through Celite^ꢂ

and the ﬁltrate was concentrated to give the title com-

pound as yellow needles (260 mg, 96%); ¹H NMR

(CDCl₃) d: 7.65 (d, J = 11.2 Hz, 1H), 7.03 (d,

J = 8.8 Hz, 1H), 4.50 (q, J = 7.3 Hz, 2H), 4.45 (br s,

2H), 4.10 (s, 3H), 1.45 (t, J = 7.3 Hz, 3H).

1

>300 ꢁC; H NMR (DMSO-d₆) d: 7.59 (d, J = 11.7 Hz,

1H), 6.91 (br s, 2H), 6.63 (d, J = 8.3 Hz, 1H);

FAB(+)HRMS 224.0516 (+4.4 mmu). Anal. Calcd for

3

C₉H₆FN₃O₃ꢁ H₂O: C, 46.20%; H, 3.10%; N, 17.96%.

5.1.19. 7-Fluoro-3-methoxy-6-nitroquinoxaline-2-carbox-

amide (13). To a suspension of compound 11a (542 mg,

1.84 mmol) in MeOH (20 mL), 28% NH₃aq (1.5 mL)

was added, and the solution was reﬂuxed for 3 h. After

cooling, the reaction was concentrated and water was

added to the residue. The precipitate was collected by ﬁl-

tration, dissolved in AcOEt, dried over Na₂SO₄, and

Found: C, 46.⁵32%; H, 3.02%; N, 17.77%.

5.1.24. 3,4-Dihydro-6-nitro-7-ﬂuoro-3-oxoquinoxaline-2-

carboxamide (16). To a solution of compound 13

(108 mg, 406 lmol) in AcOH (3 mL), 48% HBr

(0.6 mL) was added at 0 ꢁC, and the reaction was stirred

for 1 h at room temperature followed by for 1.5 h at

60 ꢁC. The reaction was poured into ice water, and the

solution was stirred for 20 min. The precipitate was col-

lected by ﬁltration and dried under vacuum to give the

title compound as brown powder (69.7 mg, 68%); mp

i

evaporated. The residue was washed with Pr₂O, and

dried under vacuum to give the title compound as red-

1

dish brown powder (369 mg, 76%); H NMR (DMSO-

d₆) d: 8.56 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 10.7 Hz,

1H), 4.18 (s, 3H).

1

>300 ꢁC; H NMR (DMSO-d₆) d: 13.01 (s, 1H), 8.22

(br s, 1H), 8.09 (d, J = 11.7 Hz, 1H), 8.03 (d,

J = 6.8 Hz, 1H), 7.99 (br s, 1H); FAB(+)HRMS

251.0221 (+0.5 mmu). Anal. Calcd for C₉H₅FN₄O₄: C,

42.87%; H, 2.00%; N, 22.22%. Found: C, 42.89%; H,

2.03%, N, 21.96%.

5.1.20. 3,4-Dihydro-7-ﬂuoro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (6b). Following the procedure described

for compound 6a, the title compound was prepared

from compound 4b, yellow powder (77%); mp 213–

215 ꢁC (decomp.); ¹H NMR (CDCl₃) d: 8.10 (d,

J = 11.7 Hz, 1H), 8.07 (d, J = 7.3 Hz, 1H); HRMS

253.0162 (+2.7 mmu).

5.1.25. 3,4-Dihydro-6-nitro-3-oxo-7-(4-pyridon-1-yl)qui-

noxaline-2-carboxylic acid (19g)

5.1.25.1. Step 1: Ethyl 3-methoxy-6-nitro-7-(4-pyri-

don-1-yl)quinoxaline-2-carboxylate (17g). To a solution

of compound 11a (180 mg, 610 lmol) in THF (20 mL),

4-pyridone (290 mg, 3.05 mmol) was added, and the

5.1.21. 7-Bromo-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (6c). Following the procedure described

for compound 6a, the title compound was prepared

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5853

solution stirred for 4 h at 100 ꢁC followed by 18 h at

90 ꢁC in a sealed tube. After cooling, the reaction was

concentrated and the residue was puriﬁed by ﬂash col-

umn chromatography (SiO₂, CHCl₃:EtOH, 40:1 to

20:1) to give the title compound as pale yellow oil

d: 7.72 (s, 1H), 7.70 (s, 1H), 2.93 (br t, J = 4.9 Hz,

2H), 1.66–1.57 (m, 4H), 1.56–1.48 (m, 2H); HRMS

318.0977 (+1.3 mmu).

5.1.28. 3,4-Dihydro-7-morpholino-6-nitro-3-oxoquinoxa-

line-2-carboxylic acid (19c). A solution of compound

11a (506 mg, 1.71 mmol) and morpholine (749 lL,

8.56 mmol) in MeCN (2 mL) was stirred for 6 h at

80 ꢁC. After cooling, the reaction was concentrated and

the residue was puriﬁed by ﬂash column chromatography

(SiO₂, n-hexane:AcOEt, 2:1) to give crude compound 17c

as red oil. The obtained compound 17c was dissolved into

MeOH (2 mL) and 5% NaOH aq (5 mL) was added. After

stirring for 24 h at room temperature, the reaction

was adjusted to pH 3 with 3 N HCl, extracted with

CH₂Cl₂, dried over Na₂SO₄, and evaporated. HCl (3 N,

5 mL) was added to the residue and stirred for 65 h.

The precipitate was collected by ﬁltration, washed with

water, and dried under vacuum to give the title compound

1

(70.0 mg, 31%); H NMR (CDCl₃) d: 8.60 (s, 1H), 8.22

(s, 1H), 7.38 (d, J = 7.8 Hz, 2H), 6.52 (d, J = 7.8 Hz,

2H), 4.56 (q, J = 7.2 Hz, 2H), 4.24 (s, 3H), 1.47 (t,

J = 7.2 Hz, 3H).

5.1.25.2. Step 2: 3,4-Dihydro-6-nitro-3-oxo-7-(4-pyri-

don-1-yl)quinoxaline-2-carboxylic acid (19g). To a solu-

tion of compound 17g (1.34 g, 3.62 mmol) in EtOH

(40 mL), water (10 mL) and 1 N KOH (10.9 mL) were

added, and the reaction was reﬂuxed for 4 h. After cool-

ing, cation-exchange resin Dowex^ꢂXFS43279.00 was

added to neutralize. The resin was removed by ﬁltration,

and the ﬁltrate was concentrated. The residue was dis-

solved into 3 N HCl (70 mL) and the solution was stir-

red for 4 h at room temperature. The solution was

concentrated, and the residue was washed with water

and dried under vacuum to give the title compound

as yellow powder (1.00 g, 81%); mp 283–285 ꢁC;

¹H NMR (DMSO-d₆) d: 8.35 (s, 1H), 8.11 (s, 1H),

7.83 (d, J = 7.3 Hz, 2H), 6.24 (d, J = 7.3 Hz, 2H);

FAB(+)HRMS 329.0542 (+ 2.0 mmu). Anal. Calcd for

1

as red powder (275 mg, 48%); mp 213.5–214.5 ꢁC; H

NMR (DMSO-d₆) d: 7.82 (s, 1H), 7.73 (s, 1H), 3.69 (t,

J = 4.9 Hz, 2H), 2.98 (t, J = 4.9 Hz, 2H); FAB(ꢀ)HRMS

319.0688 (+0.9 mmu). Anal. Calcd for C₁₃H₁₂N₄O₆ꢁ

9

₁₀H₂O: C, 46.41%; H, 4.13%; N, 16.65%. Found: C,

46.66%; H, 4.00%; N, 16.32%.

4

C₁₄H₉N₄O₆ꢁ H₂O: C, 49.07%, H, 2.82%; N, 16.35%.

5.1.29. 3,4-Dihydro-7-(4-hydoxypiperidino)-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (19d)

5

Found: C, 48.84%; H, 2.62%; N, 16.05%.

5.1.29.1. Step 1: Ethyl 7-(4-hydoxypiperidino)-3-meth-

oxy-6-nitroquinoxaline-2-carboxylate (17d). Following

the procedure described for compound 19g (step 1),

the title compound was prepared from compound 11a

and 4-hydroxypiperidine, red gum (83%); ¹H NMR

(CDCl₃) d: 8.15 (s, 1H), 7.73 (s, 1H), 4.54 (q,

J = 7.3 Hz, 2H), 4.14 (s, 3H), 3.97–3.88 (m, 1H), 3.38–

3.28 (m, 2H), 2.99–2.91 (m, 2H), 2.09–2.00 (m, 2H),

1.82–1.72 (m, 2H), 1.46 (t, J = 7.3 Hz, 3H).

5.1.26. 3,4-Dihydro-7-dimethylamino-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid (19a)

5.1.26.1. Step 1: Ethyl 7-dimethylamino-3-methoxy-6-

nitroquinoxaline-2-carboxylate (17a). Following the pro-

cedure described for compound 19g (step 1), the title

compound was prepared from compound 11a and dim-

1

ethylamine, brown oil (36%); H NMR (CDCl₃) d: 8.29

(s, 1H), 7.66 (s, 1H), 4.43 (q, J = 7.3 Hz, 2H), 4.05 (s,

3H), 2.86 (s, 6H), 1.35 (t, J = 7.3 Hz, 3H).

5.1.29.2. Step 2: 3,4-Dihydro-6-nitro-3-oxo-7-piperidi-

noquinoxaline-2-carboxylic acid (19d). Following the

procedure described for compound 19g (step 2), the title

compound was prepared from compound 17d, brown

5.1.26.2. Step 2: 3,4-Dihydro-7-dimethylamino-6-nitro-

3-oxoquinoxaline-2-carboxylic acid (19a). Following the

procedure described for compound 19g (step 2), the title

compound was prepared from compound 17a, dark

brown powder (15%); mp 194.5–196.5 ꢁC; ¹H NMR

(DMSO-d₆) d: 7.72 (s, 1H), 7.62 (s, 1H), 2.79 (s, 6H);

HRMS 278.0641 (ꢀ1.0 mmu).

1

powder (38%); mp 253–255 ꢁC; H NMR (DMSO-d₆)

d: 7.71 (s, 2H), 4.72 (br s, 1H), 3.20–3.00 (m, 2H),

2.85–2.75 (m, 2H), 1.80–1.90 (m, 2H), 1.55–1.45 (m,

2H); FAB(+)HRMS 334.0894 (ꢀ1.9 mmu). Anal. Calcd

3

10

for C₁₄H₁₄N₄O₆ꢁ H₂O: C, 49.50%; H, 4.33%; N,

5.1.27. 3,4-Dihydro-6-nitro-3-oxo-7-piperidinoquinoxa-

line-2-carboxylic acid (19b)

16.49%. Found: C, 49.76%; H, 4.19%; N, 16.32%.

5.1.27.1. Step 1: Ethyl 3-methoxy-6-nitro-7-piperidi-

noquinoxaline-2-carboxylate (17b). Following the proce-

dure described for compound 19g (step 1), the title

compound was prepared from compound 11a and piper-

5.1.30. 7-(4-Acetylpiperazin-1-yl)-3,4-dihydro-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (19e)

5.1.30.1. Step 1: Ethyl 7-(4-acetylpiperazin-1-yl)-3-

methoxy-6-nitroquinoxaline-2-carboxylate (17e). Follow-

ing the procedure described for compound 19g (step 1),

the title compound was prepared from compound 11a

and 1-acetylpiperazine, red gum (69%); ¹H NMR

(CDCl₃) d: 8.20 (s, 1H), 7.76 (s, 1H), 4.54 (q, J = 7.3 Hz,

2H), 4.15 (s, 3H), 3.83–3.77 (m, 2H), 3.67–3.62 (m, 2H),

3.13–3.06 (m, 4H), 2.15 (s, 3H), 1.47 (t, J = 7.3 Hz, 3H).

1

idine, red oil (88%); H NMR (CDCl₃) d: 8.13 (s, 1H),

7.69 (s, 1H), 4.53 (q, J = 7.3 Hz, 2H), 4.13 (s, 3H),

3.05 (br t, J = 4.8 Hz, 2H), 1.77–1.71 (m, 4H), 1.64–

1.58 (m, 2H), 1.46 (t, J = 7.3 Hz, 3H).

5.1.27.2. Step 2: 3,4-Dihydro-6-nitro-3-oxo-7-piperidi-

noquinoxaline-2-carboxylic acid (19b). Following the

procedure described for compound 19g (step 2), the title

compound was prepared from compound 17b, purple

powder (50%); mp >300 ꢁC; ¹H NMR (DMSO-d₆)

5.1.30.2. Step 2: 7-(4-Acetylpiperazin-1-yl)-3,4-dihy-

dro-6-nitro-3-oxoquinoxaline-2-carboxylic acid (19e).

Following the procedure described for compound 19g

5854

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

(step 2), the title compound was prepared from

compound 17e, with 47% HBr instead of 3 N HCl,

brown powder (57%); mp 212–214 ꢁC; ¹H NMR

(DMSO-d₆) d: 7.81 (s, 1H), 7.76 (s, 1H), 3.60–3.50 (m,

4H), 3.10–2.90 (m, 4H), 2.03 (s, 3H); FAB(+)HRMS

362.1153 (+5.2 mmu).

5.1.33. 3,4-Dihydro-7-(2-methylimidazol-1-yl)-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (19i)

5.1.33.1. Step 1: Ethyl 3-ethoxy-7-(2-methylimidazol-

1-yl)-6-nitroquinoxaline-2-carboxylate (18i). 2-Methyl-

imidazole (438 mg, 5.33 mmol) and Et₃N (223 lL,

1.60 mmol) were added, to a solution of compound

11b (330 mg, 1.07 mmol) in DMF (2 mL) and stirred

for 12 h at 110 ꢁC. After cooling, the reaction was

poured into ice water, extracted with AcOEt, dried over

Na₂SO₄, and evaporated. The residue was puriﬁed by

ﬂash column chromatography (SiO₂, n-hexane:AcOEt,

1:2) to give the title compound as light brown amor-

5.1.31. 3,4-Dihydro-7-[4-(4-ﬂuorophenyl)piperazin-1-yl]-

6-nitro-3-oxoquinoxaline-2-carboxylic acid (19f)

5.1.31.1. Step 1: Ethyl 7-[4-(4-ﬂuorophenyl)piperazin-

1-yl]-3-methoxy-6-nitroquinoxaline-2-carboxylate (17f).

Following the procedure described for compound 19g

(step 1), the title compound was prepared from com-

pound 11a and 1-(4-ﬂuorophenyl)piperazine, red gum

1

phous solid (268 mg, 68%); H NMR (CDCl₃) d: 8.42

(s, 1H), 8.12 (s, 1H), 7.01 (d, J = 1.5 Hz, 1H), 6.97 (d,

J = 1.5 Hz, 1H), 4.67 (q, J = 7.3 Hz, 2H), 4.55 (q,

J = 7.3 Hz, 2H), 2.28 (s, 3H), 1.53 (t, J = 7.3 Hz, 3H),

1.47 (t, J = 7.3 Hz, 3H).

1

(87%); H NMR (CDCl₃) d: 8.18 (s, 1H), 7.80 (s, 1H),

7.00 (t, J = 8.8 Hz, 2H), 6.94 (dd, J = 8.8, 4.4 Hz, 2H),

4.55 (q, J = 7.3 Hz, 2H), 4.15 (s, 3H), 3.27 (s, 8H),

1.47 (t, J = 7.3 Hz, 3H).

5.1.33.2. Step 2: 3,4-Dihydro-7-(2-methylimidazol-1-

yl)-6-nitro-3-oxoquinoxaline-2-carboxylic acid (19i). To a

solution of compound 18i (218 mg, 587 lmol) in AcOH

(10 mL), concd HCl (700 lL) was added, and the reac-

tion was stirred for 24 h at room temperature. The reac-

tion was concentrated, and the residue was washed with

water and dried under vacuum to give the title com-

pound as brown powder (97.4 mg, 53%); mp 268–

271 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d: 8.31 (s,

1H), 8.17 (s, 1H), 7.50 (s, 1H), 7.27 (s, 1H), 2.30 (s,

3H); FAB(ꢀ)HRMS 314.0514 (ꢀ1.2 mmu).

5.1.31.2. Step 2: 3,4-Dihydro-7-[4-(4-ﬂuorophenyl)pip-

erazin-1-yl]-6-nitro-3-oxoquinoxaline-2-carboxylic acid

(19f). Following the procedure described for com-

pound 19e (step 2), the title compound was prepared

from compound 17f, pale yellow powder (55%); mp

235.5–237.5 ꢁC; ¹H NMR (DMSO-d₆) d: 7.86 (s,

1H), 8.75 (s, 1H), 7.07 (t, J = 9.3 Hz, 2H), 7.01 (dd,

J = 9.3, 4.9 Hz, 2H), 3.19 (d, J = 5.4 Hz, 4H), 3.15

(d, J = 5.4 Hz, 4H); FAB(+)HRMS 414.1188

7

(ꢀ2.6 mmu). Anal. Calcd for C₁₉H₁₆FN₅O₅ꢁ H₂O:

10

C, 53.57%; H, 4.12%; N, 16.44%. Found: C, 53.74%;

H, 3.77%; N, 16.15%.

5.1.34. 3,4-Dihydro-7-(4-methylimidazol-1-yl)-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (19j)

5.1.34.1. Step 1: Ethyl 3-ethoxy-7-(4-methylimidazol-

1-yl)-6-nitroquinoxaline-2-carboxylate (18j). A solution

of compound 11b (2.00 g, 6.47 mmol) and 4-methylimi-

dazole (2.66 g, 32.4 mmol) in MeCN (15 mL) was stirred

for 8 h at 100 ꢁC in a sealed tube. After cooling, the

reaction was diluted with CH₂Cl₂, washed with water,

dried over Na₂SO₄, and evaporated. The residue was

puriﬁed by ﬂash column chromatography (SiO₂, n-hex-

ane:AcOEt, 1:1) to give the title compound as yellow

powder (1.20 g, 50%); ¹H NMR (CDCl₃) d: 8.38 (s,

1H), 8.12 (s, 1H), 7.60 (d, J = 1.5 Hz, 1H), 6.83 (s,

1H), 4.66 (q, J = 7.0 Hz, 2H), 4.55 (q, J = 7.2 Hz, 2H),

2.31 (s, 3H), 1.52 (t, J = 7.1 Hz, 3H), 1.47 (t,

J = 7.3 Hz, 3H).

5.1.32. 3,4-Dihydro-7-(imidazol-1-yl)-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid (19h)

5.1.32.1. Step 1: Ethyl 7-(imidazol-1-yl)-3-methoxy-

6-nitroquinoxaline-2-carboxylate (17h). A solution of

compound 11a (1.41 g, 4.78 mmol) and imidazole

(1.63 g, 23.9 mmol) in MeCN (10 mL) was stirred for

9 h at 50 ꢁC. The reaction was diluted with CH₂Cl₂,

washed with brine, dried over Na₂SO₄, and evaporat-

ed. The residue was puriﬁed by ﬂash column chromat-

ography [SiO₂, n-hexane:AcOEt (1:1) to AcOEt] to

give the title compound as orange oil (423 mg, 26%);

¹H NMR (CDCl₃) d: 8.46 (s, 1H), 8.18 (s, 1H), 7.72

(s, 1H), 7.27 (s, 1H), 7.15 (t, J = 1.5 Hz, 1H), 4.56

(q, J = 7.3 Hz, 2H), 4.23 (s, 3H), 1.47 (t, J = 7.3 Hz,

3H).

5.1.34.2. Step 2: 3,4-Dihydro-7-(4-methylimidazol-1-

yl)-6-nitro-3-oxoquinoxaline-2-carboxylic acid (19j).

Following the procedure described for compound 19e

(step 2), the title compound was prepared from com-

pound 18j, brown powder (69%); mp 210–212 ꢁC (de-

5.1.32.2. Step 2: 3,4-Dihydro-7-(imidazol-1-yl)-6-nitro-

3-oxoquinoxaline-2-carboxylic acid (19h). A solution of

compound 17h (423 mg, 1.23 mmol) in 3 N HCl

(20 mL) was stirred for 6 h at 80 ꢁC. To the reaction,

concd HCl (2 mL) was added, and the reaction was stir-

red for 10 h at 80 ꢁC. After cooling, the precipitate was

collected by ﬁltration, washed with water and MeOH,

and dried under vacuum to give the title compound as

1

comp.); H NMR (DMSO-d₆) d: 8.19 (s, 1H), 8.08 (s,

2H), 7.23 (s, 1H), 2.19 (s, 3H); FAB(+)HRMS

316.0688 (ꢀ1.6 mmu).

5.1.35. 3,4-Dihydro-7-[4-(hydroxymethyl)imidazol-1-yl]-

6-nitro-3-oxoquinoxaline-2-carboxylic acid (19k)

1

brown powder (166 mg, 44%); mp >300 ꢁC; H NMR

(DMSO-d₆) d: 8.24 (s, 1H), 8.22 (s, 1H), 8.06 (s, 1H),

7.56 (s, 1H), 7.25 (s, 1H); FAB(ꢀ)HRMS 300.0347

5.1.35.1. Step 1: Ethyl 3-ethoxy-7-[4-(hydroxymeth-

yl)imidazol-1-yl]-6-nitroquinoxaline-2-carboxylate (18k).

To a solution of compound 11b (6.90 g, 22.3 mmol) in

MeCN (70 mL), 4-(hydroxymethyl)imidazole hydro-

chloride (15.1 g, 112 mmol) and Et₃N (23.4 mL,

1

2

(ꢀ2.2 mmu). Anal. Calcd for C₁₂H₇N₅O₅ꢁ H₂O: C,

46.46%; H, 2.60%; N, 22.58%. Found: C, 46.17%; H,

2.44%; N, 22.61%.

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5855

168 mmol) were added, and the solution was reﬂuxed for

16 h. After cooling, the reaction was diluted with

CH₂Cl₂, washed with water, dried over Na₂SO₄, and

evaporated. The residue was puriﬁed by ﬂash column

chromatography (SiO₂, n-hexane:AcOEt, 1:1) to give

5.1.38. 7-Amino-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (21)

5.1.38.1. Step 1: Ethyl 7-amino-3-ethoxy-6-nitroqui-

noxaline-2-carboxylate. To a solution of compound 20

(2.09 g, 4.90 mmol) in anisole (5 mL) was added

CF₃CO₂H (5 mL). After stirring for 6 h at room temper-

ature, the reaction was concentrated. The residue was

puriﬁed by ﬂash column chromatography (SiO₂,

CH₂Cl₂) to give the title compound as purple powder

(1.20 g, 80%); ¹H NMR (DMSO-d₆) d: 8.41 (s, 1H),

8.49 (s, 1H), 7.12 (s, 2H), 4.47 (d, J = 6.8 Hz, 2H),

4.43 (q, J = 7.3 Hz, 2H), 1.37 (t, J = 7.3 Hz, 3H), 1.35

(t, J = 6.8 Hz, 3H).

1

the title compound as brown power (3.69 g, 43%); H

NMR (CDCl₃) d: 8.43 (s, 1H), 8.15 (s, 1H), 7.68 (d,

J = 1.5 Hz, 1H), 7.09 (s, 1H), 4.71 (s, 2H), 4.66 (q,

J = 7.2 Hz, 2H), 4.55 (q, J = 7.2 Hz, 2H), 1.53 (t,

J = 7.1 Hz, 3H), 1.47 (t, J = 7.1 Hz, 3H).

5.1.35.2. Step 2: 3,4-Dihydro-7-[4-(hydroxymethyl)imi-

dazol-1-yl]-6-nitro-3-oxoquinoxaline-2-carboxylic

acid

(19k). A solution of compound 18k (100 mg, 258 lmol)

in 3 N HCl (2 mL) was stirred for 2 h at room tempera-

ture. The reaction was concentrated, and the residue

was puriﬁed by synthetic adsorbent Sepabeads^ꢂSP850

(water) to give the title compound as orange powder

(36.7 mg, 43%); mp 240–242 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 8.16 (s, 1H), 8.05 (s, 1H), 7.97 (s, 1H),

7.28 (s, 1H), 5.07 (br s, 1H), 4.42 (s, 2H); FAB(ꢀ)HRMS

330.0511 (+3.6 mmu).

5.1.38.2. Step 2: 7-Amino-3,4-dihydro-6-nitro-3-oxo-

quinoxaline-2-carboxylic acid (21). To a solution of ethyl

7-amino-3-ethoxy-6-nitroquinoxaline-2-carboxylate

(200 mg, 653 lmol) in EtOH (10 mL), 1 N NaOH

(1.96 mL, 1.96 mmol) and water (2 mL) were added,

and the solution was reﬂuxed for 30 min. After cooling,

the reaction was adjusted to pH 4 with 10% HCl, diluted

with brine, extracted with CHCl₃, dried over MgSO₄, and

evaporated. The residue was dissolved into EtOH

(10 mL), and concd HCl (2 mL) was added. After stirring

for 24 h at room temperature, the reaction was concen-

5.1.36. 7-[4-(Carboxymethyl)imidazol-1-yl]-3,4-dihydro-

6-nitro-3-oxoquinoxaline-2-carboxylic acid (19l). A solu-

tion of compound 11b (450 mg, 1.46 mmol) and methyl

imidazole-4-acetate (617 mg, 4.40 mmol) in MeCN

(5 mL) was stirred for 15 h at 110 ꢁC in a sealed tube.

After cooling, the reaction was concentrated and the res-

idue was puriﬁed by ﬂash column chromatography

(SiO₂, CH₂Cl₂) to give crude compound 18l. The ob-

tained compound 18l was dissolved into AcOH–concd

HCl (5:1, 3 ml) and stirred overnight. The reaction

was concentrated and the residue was dissolved into

MeOH. A solution of LiOH monohydrate (119 mg,

2.84 mmol) in water (1 mL) was added to the reaction,

and stirred overnight. The reaction was concentrated,

made acidic with 0.5 N HCl, and concentrated again.

Water was added to the residue, the precipitate was col-

lected by ﬁltration, washed with water and AcOEt, and

dried under vacuum to give the title compound as brown

powder (149 mg, 27%); mp >300 ꢁC; ¹H NMR (DMSO-

d₆) d: 8.19 (s, 1H), 8.04 (s, 1H), 8.01 (s, 1H), 7.33 (s, 1H),

3.56 (s, 2H); FAB(ꢀ)HRMS 358.0425 (+0.2 mmu).

Anal. Calcd for C₁₄H₉N₅O₇ÆH₂O: C, 44.57%; H,

2.94%; N, 18.56%. Found: C, 44.64%; H, 3.11%; N,

18.64%.

i

trated, washed with Pr₂O, and dried under vacuum to

give the title compound as brown powder (156 mg,

95%); mp >300 ꢁC; ¹H NMR (DMSO-d₆) d: 7.94 (s,

1H), 7.43 (s, 1H), 7.17 (s, 2H); HRMS 250.0311

(ꢀ2.7 mmu).

5.1.39. 3,4-Dihydro-6-nitro-3-oxo-7-(pyrrole-1-yl)quinox-

aline-2-carboxylic acid (22). To a solution of compound

21 (50.0 mg, 200 lmol) in AcOH (5 mL), 2,5-dim-

ethoxytetrahydrofuran (31.7 mg, 240 lmol) was added,

and stirred for 4 h at 80 ꢁC. The reaction was concen-

i

trated, washed with water and Pr₂O, and dried under

vacuum to give the title compound as brown powder

1

(28.0 mg, 47%); mp > 300 ꢁC; H NMR (DMSO-d₆) d:

8.07 (s, 1H), 7.91 (s, 1H), 6.99 (t, J = 2.0 Hz, 2H), 6.27

(t, J = 2.0 Hz, 2H); HRMS 300.0502 (+0.7 mmu).

5.1.40. Ethyl 3-ethoxy-7-[4-(2-hydroxyethyl)imidazolyl]-

6-nitroquinoxaline-2-carboxylate (23). To a solution of

compound 11b (309 mg, 999 lmol) in DMA (10 mL),

4-(2-hydroxyethyl)imidazole (270 mg, 2.41 mmol) and

Et₃N (1 mL) were added. After stirring for 15 h at

120 ꢁC, the reaction was concentrated. The residue was

puriﬁed by ﬂash column chromatography [SiO₂, CH₂Cl₂

to CH₂Cl₂:MeOH (10:1)] to give the title compound as

brown powder (114 mg, 28%); ¹H NMR (CDCl₃) d:

8.42 (s, 1H), 8.15 (s, 1H), 7.66 (d, J = 1.0 Hz, 1H),

6.94 (d, J = 1.0 Hz, 1H), 4.66 (q, J = 7.3 Hz, 2H), 4.55

(q, J = 6.8 Hz, 2H), 3.97 (t, J = 5.9 Hz, 2H), 2.92–2.87

(m, 2H), 2.81 (d, J = 4.4 Hz, 1H), 1.53 (t, J = 7.3 Hz,

3H), 1.47 (t, J = 6.8 Hz, 3H).

5.1.37. Ethyl 3-ethoxy-7-(4-methoxybenzyl)amino-6-

nitroquinoxaline-2-carboxylate (20). To a solution of com-

pound 11b (2.00 g, 6.47 mmol) in THF (15 mL), 4-meth-

oxybenzylamine (1.06 g, 7.76 mmol) and Et₃N (785 mg,

7.76 mmol) were added, and the solution was reﬂuxed

for 24 h. After cooling, the reaction was diluted with

AcOEt, washed with brine, dried over MgSO₄, and evap-

orated. The residue was puriﬁed by ﬂash column chro-

matography [SiO₂, CH₂Cl₂to CH₂Cl₂:AcOEt (1:1)] to

give the title compound as purple powder (2.09 g, 76%);

¹H NMR (DMSO-d₆) d: 8.49 (s, 1H), 8.08 (t, J = 6.3 Hz,

1H), 7.37 (d, J = 8.8 Hz, 2H), 7.22 (s, 1H), 6.91 (d,

J = 8.8 Hz, 2H), 4.77 (d, J = 6.3 Hz, 2H), 4.58 (q,

J = 7.3 Hz, 2H), 4.41 (q, J = 7.3 Hz, 2H), 3.72 (s, 3H),

1.37 (t, J = 7.3 Hz, 3H), 1.32 (t, J = 7.3 Hz, 3H).

5.1.41. 7-[4-((n-Butylamino)carbonyloxymethyl)imidazol-

1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-carboxylic

acid (28a). To a solution of compound 18k (100 mg,

258 lmol) in CH₂Cl₂(1 mL), n-butyl isocyanate

(43.6 lL, 387 lmol) was added . After stirring for 8 h

at room temperature, the reaction was concentrated to

5856

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

give crude compound 24a. The obtained compound 24a

was dissolved into AcOH (3 mL) and concd HCl

(0.6 mL). After stirring for 24 h at room temperature,

the reaction was concentrated. The residue was dis-

solved into 2 N NaOH, and the precipitate was removed

by ﬁltration. The solution was neutralized with concd

HCl. The precipitate was collected by ﬁltration, washed

with water and AcOEt, and dried under vacuum to give

the title compound as yellow powder (31.2 mg, 28%);

mp 194–196 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d:

8.19 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H), 7.48 (s, 1H),

7.20 (t, J = 5.6 Hz, 1H), 4.91 (s, 2H), 2.98 (q,

J = 6.5 Hz, 2H), 1.41–1.34 (m, 2H), 1.31–1.23 (m, 2H),

0.86 (t, J = 7.3 Hz, 3H); FAB(ꢀ)HRMS 429.1161

5.1.44. 3,4-Dihydro-6-nitro-3-oxo-7-[4-((phenylamino)car-

bonyloxymethyl)imidazol-1-yl]quinoxaline-2-carboxylic

acid (28d). Following the procedure described for com-

pound 28a, the title compound was prepared from com-

pound 18k and phenyl isocyanate, dark brown powder

1

(52%); mp 241–243 ꢁC (decomp.); H NMR (DMSO-

d₆) d: 9.81 (s, 1H), 8.37 (s, 1H), 8.27 (s, 1H), 8.08 (s,

1H), 7.72 (s, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.28 (t,

J = 8.1 Hz, 2H), 6.99 (t, J = 7.3 Hz, 1H), 5.12 (s, 2H);

FAB(+)HRMS 451.1008 (+0.5 mmu).

5.1.45. Sodium 7-[4-((Benylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylate (28e). To a solution of compound 18k

(100 mg, 258 lmol) in CH₂Cl₂(3 mL), benzyl isocyanate

(47.8 ll, 387 lmol) was added. After stirring for 6 h at

room temperature, the reaction was concentrated to give

crude compound 24e. The obtained compound 24e was

dissolved into AcOH (3 mL) and concd HCl (0.6 mL).

After stirring for 36 h at room temperature, the reaction

was concentrated. The residue was dissolved into 2 N

NaOH, and washed with AcOEt. The aqueous layer was

concentrated, and the precipitate was collected by ﬁltra-

tion, washed with water and CHCl₃, and dried under vac-

uum to give the title compound as yellow powder

(49.0 mg, 36%); mp 222–224 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 8.21 (s, 1H), 8.13 (s, 1H), 7.95 (s, 1H),

7.81 (t, J = 6.6 Hz, 1H), 7.48 (s, 1H), 7.34–7.21 (m, 5H),

4.95 (s, 2H), 4.10 (d, J = 6.4 Hz, 1H); FAB(+)HRMS

487.0998 (+2.0 mmu).

1

2

(+0.2 mmu). Anal. Calcd for C₁₈H₁₈BrN₆O₇ꢁ H₂O: C,

49.20%; H, 4.36%; N, 19.13%. Found: C, 49.06%; H,

4.23%; N, 18.92%.

5.1.42. 3,4-Dihydro-7-[4-((isopropylamino)carbonyloxy-

methyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline-2-carbox-

ylic acid (28b). Following the procedure described for

compound 28a, the title compound was prepared from

compound 18k and isopropyl isocyanate, yellow powder

(64%); mp 199–201 ꢁC (decomp.); ¹H NMR (DMSO-d₆)

d: 8.17 (s, 1H), 8.03 (s, 1H), 7.94 (s, 1H), 7.45 (s, 1H),

7.13 (d, J = 7.3 Hz, 1H), 4.90 (s, 2H), 3.63–3.57 (m,

1H), 1.05 (d, J = 6.8 Hz, 6H); FAB(ꢀ)HRMS 415.1001

(ꢀ0.2 mmu). Anal. Calcd for C₁₇H₁₆N₆O₇ÆH₂O: C,

47.01%; H, 4.18%; N, 19.35%. Found: C, 47.19%; H,

3.91%; N, 19.40%.

5.1.43. 7-[4-((Cyclohexylamino)carbonyloxymethyl)imi-

dazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-car-

boxylic acid (28c)

5.1.46.

3,4-Dihydro-6-nitro-3-oxo-7-[4-(((2-phenylethyl)

amino)carbonyloxymethyl)imidazol-1-yl]quinoxaline-2-car-

boxylic acid (28f)

5.1.43.1. Step 1: Ethyl 7-[4-((cyclohexylamino)carbon-

yloxymethyl)imidazol-1-yl]-3-ethoxy-6-nitroquinoxaline-2-

carboxylate (24c). To a solution of compound 18k

(100 mg, 258 lmol) in benzene (10 mL), cyclohexyl isocy-

anate (65.9 lL, 516 lmol) was added, and the solution

was reﬂuxed for 2 h. After cooling, the reaction was con-

centrated and the residue was puriﬁed by ﬂash column

chromatography (SiO₂, n-hexane:AcOEt, 1:1) to give the

title compound as brown amorphous solid (61.0 mg,

5.1.46.1. Step 1: Ethyl 3-ethoxy-6-nitro-7-[4-(((2-phen-

ylethyl)amino)carbonyloxymethyl)imidazol-1-yl]quinoxa-

line-2-carboxylate (24f). To a solution of compound 18k

(150 mg, 387 lmol) and 3-phenylpropionic acid (116 mg,

774 lmol) in benzene (12 mL), diphenylphosphoryl azide

(167 lL, 774 lmol) and Et₃N (108 lL, 774 rmumol) were

added, and the solution was reﬂuxed for 3 h. After cooling,

the reaction was concentrated and the residue was puriﬁed

by ﬂash column chromatography (SiO₂, n-hexane:AcOEt,

1:1) to give the title compound as light brown amorphous

solid (123 mg, 59%); ¹H NMR (CDCl₃) d: 8.43(s, 1H), 8.14

(s, 1H), 7.67 (d, J = 1.5 Hz, 1H), 7.31–7.27 (m, 2H), 7.22–

7.17 (m, 3H), 5.12 (s, 2H), 4.85 (br s, 1H), 4.66 (q,

J = 6.9 Hz, 2H), 4.55 (q, J = 7.3 Hz, 2H), 3.47 (q,

J = 6.9 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H), 1.53 (t,

J = 6.9 Hz, 3H), 1.47 (t, J = 7.3 Hz, 3H).

1

46%); H NMR (CDCl₃) d: 8.43 (s, 1H), 8.14 (s, 1H),

7.68 (d, J = 1.5 Hz, 1H), 7.18 (d, J = 1.0 Hz, 1H), 5.10

(s, 2H), 4.66 (q, J = 7.3 Hz, 2H), 4.55 (q, J = 7.3 Hz,

2H), 3.52–3.48 (m, 1H), 1.95–1.91 (m, 2H), 1.72–1.67

(m, 2H), 1.60–1.55 (m, 2H), 1.53 (t, J = 7.3 Hz, 3H),

1.47 (t, J = 7.3 Hz, 3H), 1.44–1.30 (m, 2H), 1.25–1.08

(m, 2H).

5.1.43.2. Step 2: 7-[4-((Cyclohexylamino)carbonyl-

oxymethyl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxo-

quinoxaline-2-carboxylic acid (28c). Following the

procedure described for compound 19i, the title com-

pound was prepared from compound 24c, brown powder

(81%); mp 237–239 ꢁC (decomp.); ¹H NMR (DMSO-d₆)

d: 8.19 (s, 1H), 8.03 (s, 1H), 8.02 (s, 1H), 7.49 (s, 1H),

7.17 (d, J = 7.8 Hz, 1H), 4.91 (s, 2H), 1.76–1.65 (m, 4H),

5.1.46.2. Step 2: 3,4-Dihydro-6-nitro-3-oxo-7-[4-(((2-

phenylethyl)amino)carbonyloxymethyl)imidazol-1-yl]qui-

noxaline-2-carboxylic acid (28f). Following the proce-

dure described for compound 19i, the title compound

was prepared from compound 24f, light brown powder

(30%); mp 212–214 ꢁC (decomp.); H NMR (DMSO-

d₆) d: 8.20 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H), 7.45 (s,

1H), 7.32–7.16 (m, 6H), 4.92 (s, 2H), 3.17 (t,

1

1.25–1.05

(m,

6H);

FAB(ꢀ)HRMS

455.1324

J = 7.3 Hz,

2H),

2.72

(t,

J = 7.3 Hz,

2H);

3

4

(ꢀ2.0 mmu). Anal. Calcd for C₂₀H₂₀N₆O₇ꢁ H₂O: C,

FAB(ꢀ)HRMS 477.1151 (ꢀ0.8 mmu). Anal. Calcd for

1

51.12%; H, 4.61%; N, 17.88%. Found: C, 51.35%; H,

4.62%; N, 17.44%.

C₂₂H₁₈N₆O₇ꢁ H₂O: C, 54.21%; H, 3.93%; N, 17.24%.

Found: C, 54.²22%; H, 3.78%; N, 17.21%.

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5857

5.1.47.

7-[4-((2-Chlorophenylamino)carbonyloxymeth-

C, 41.15%; H, 2.76%; N, 14.40%. Found: C, 41.07%; H,

2.67%; N, 14.35%.

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29a). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 2-chlorophenyl isocyanate,

yellow powder (64%); mp 205–207 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.20 (s, 1H), 8.18 (s, 1H), 8.15 (s,

1H), 7.98 (d, J = 1.0 Hz, 1H), 7.59 (d, J = 6.8 Hz, 1H),

7.56 (s, 1H), 7.47 (dd, J = 8.1 Hz, 1H), 7.33 (t,

J = 7.8 Hz, 1H), 7.18 (td, J = 7.3, 1.4 Hz, 1H), 5.07 (s,

2H); FAB(ꢀ)HRMS 483.0466 (+1.0 mmu).

5.1.52.

7-[4-((4-Bromophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29f). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 4-bromophenyl isocyanate,

yellow powder (60%); mp 270–272 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.95 (s, 1H), 8.20 (s, 1H), 8.03 (s,

1H), 7.99 (s, 1H), 7.57 (d, J = 1.0 Hz, 1H), 7.48–7.43

(m, 4H), 5.07 (s, 2H); FAB(+)HRMS 529.0123

1

2

5.1.48.

7-[4-((3-Chlorophenylamino)carbonyloxymeth-

(+1.5 mmu). Anal. Calcd for C₂₀H₁₃BrN₆O₇ꢁ H₂O: C,

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29b). Following the procedure de-

scribed for compound 28a, the title compound was

prepared from compound 18k and 3-chlorophenyl

isocyanate, yellow powder (36%); mp 215–217 ꢁC (de-

comp.); ¹H NMR (DMSO-d₆) d: 10.02 (s, 1H), 8.19

(s, 1H), 8.05 (s, 1H), 7.98 (d, J = 1.5 Hz, 1H), 7.62

(s, 1H), 7.57 (s, 1H), 7.39 (d, J = 9.3 Hz, 1H), 7.31

(t, J = 8.1 Hz, 1H), 7.06–7.03 (m, 1H), 5.08 (s, 2H);

FAB(ꢀ)HRMS 483.0476 (+2.0 mmu). Anal. Calcd

44.62%; H, 2.62%; N, 15.61%. Found: C, 44.97%; H,

2.51%; N, 15.26%.

5.1.53. 3,4-Dihydro-7-[4-((2-methylphenylamino)carbon-

yloxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline-2-

carboxylic acid hydrochloride (29g). Following the pro-

cedure described for compound 28a, the title compound

was prepared from compound 18k and 2-methylphenyl

isocyanate, brown powder (12%); mp 252–254 ꢁC

1

(decomp.); H NMR (DMSO-d₆) d: 8.96 (s, 1H), 8.20

1

2

for C₂₀H₁₃ClN₆O₇ꢁ HCl ꢁ H₂O: C, 45.30%; H,

(s, 1H), 8.04 (s, 1H), 8.00 (s, 1H), 7.54 (s, 1H), 7.36

(d, J = 7.8 Hz, 1H), 7.19–7.13 (m, 2H), 7.05 (d, J =

7.3 Hz, 1H), 5.04 (s, 2H), 2.20 (s, 3H); FAB(ꢀ)HRMS

463.1009 (+0.7 mmu). Anal. Calcd for C₂₁H₁₆BrN₆O₇ꢁ

HCl: C, 50.36%; H, 3.42%; N, 16.78%. Found: C,

50.38%; H, 3.64%; N, 16.80%.

2.85%; N, 15.85%. Found: C, 45.23%; H, 2.95%; N,

15.84%.

5.1.49.

7-[4-((4-Chlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29c). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 4-chlorophenyl isocyanate, yel-

low powder (63%); mp 250–252 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 9.94 (s, 1H), 8.20 (s, 1H), 8.04 (s, 1H),

8.00 (s, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H),

7.34 (d, J = 9.3 Hz, 2H), 5.07 (s, 2H); FAB(ꢀ)HRMS

483.0451 (ꢀ0.5 mmu).

5.1.54. 3,4-Dihydro-7-[4-((3-methylphenylamino)carbon-

yloxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29h). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 3-methylphenyl isocyanate,

yellow powder (25%); mp 223–225 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.69 (s, 1H), 8.20 (s, 1H), 8.04 (s,

1H), 7.98 (s, 1H), 7.56 (s, 1H), 7.29 (s, 1H), 7.27 (d,

J = 7.8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 6.80 (d,

J = 7.3 Hz, 1H), 5.06 (s, 2H), 2.25 (s, 3H);

5.1.50. 7-[4-((2-Bromophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29d). Following the procedure described

FAB(ꢀ)HRMS 463.0996 (ꢀ0.6 mmu). Anal. Calcd for

1

for compound 28a, the title compound was prepared from

compound 18k and 2-bromophenyl isocyanate, brown

powder (66%); mp 260–262 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 9.12 (s, 1H), 8.23 (s, 1H), 8.08 (s, 1H),

8.04 (s, 1H), 7.64 (dd, J = 8.3, 1.5 Hz, 1H), 7.58 (s, 1H),

7.53 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.13

(td, J = 7.8, 1.5 Hz, 1H), 5.07 (s, 2H); FAB(+)HRMS

529.0084 (ꢀ2.3 mmu). Anal. Calcd for C₂₀H₁₃BrN₆O₇Æ-

H₂O: C, 43.89%; H, 2.76%; N, 15.36%. Found: C,

44.24%; H, 2.66%; N, 15.03%.

C₂₁H₁₆N₆O₇ꢁ H₂O: C, 53.28%; H, 3.62%; N, 17.75%.

Found: C, 53.²27%; H, 3.51%; N, 17.61%.

5.1.55. 3,4-Dihydro-7-[4-((4-methylphenylamino)carbon-

yloxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29i). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 4-methylphenyl isocyanate,

yellow powder (49%); mp 265–267 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.65 (s, 1H), 8.19 (s, 1H), 8.05 (s,

1H), 7.98 (d, J = 1.5 Hz, 1H), 7.55 (d, J = 1.0 Hz, 1H),

7.35 (d, J = 8.3 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H),

5.05 (s, 2H), 2.23 (s, 3H); FAB(+)HRMS 465.1156

(ꢀ0.3 mmu).

5.1.51.

7-[4-((3-Bromophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid hydrochloride (29e). Following the proce-

dure described for compound 28a, the title compound was

prepared from compound 18k and 3-bromophenyl isocy-

anate, yellow powder (50%); mp 266–268 ꢁC (decomp.);

¹H NMR (DMSO-d₆) d: 10.03 (s, 1H), 8.22 (s, 1H), 8.16

(s, 1H), 7.99 (s, 1H), 7.77 (s, 1H), 7.59 (s, 1H), 7.44 (d,

J = 7.8 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H), 7.17 (d,

J = 8.3 Hz, 1H), 5.08 (s, 2H); FAB(+)HRMS 529.0140

(+3.3 mmu). Anal. Calcd for C₂₀H₁₃BrN₆O₇Æ HClÆH₂O:

5.1.56. Sodium 3,4-dihydro-7-[4-((4-methoxyphenylami-

no)carbonyloxymethyl)imidazol-1-yl]-6-nitro-3-oxoqui-

noxaline-2-carboxylate (29j). Following the procedure

described for compound 28e, the title compound was

prepared from compound 18k and 4-methoxylphenyl

isocyanate, yellow powder (53%); mp 265–267 ꢁC (de-

1

comp.); H NMR (DMSO-d₆) d: 9.58 (s, 1H), 8.22 (s,

5858

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

1H), 8.15 (s, 1H), 7.98 (d, J = 1.0 Hz, 1H), 7.56 (s, 1H),

7.37 (d, J = 8.8 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 5.04

(s, 2H), 3.70 (s, 3H); FAB(+)HRMS 503.0913

J = 8.8 Hz, 1H), 7.625 (s, 1H), 7.16 (t, J = 8.8 Hz, 1H),

5.12 (s, 2H), 4.62 (q, J = 7.3 Hz, 2H), 4.48 (q,

J = 7.3 Hz, 2H), 4.32 (q, J = 7.3 Hz, 2H), 1.43 (t,

J = 7.3 Hz, 3H), 1.37 (t, J = 7.3 Hz, 3H), 1.32 (t,

J = 7.3 Hz, 3H).

1

(ꢀ1.4 mmu). Anal. Calcd for C₂₁H₁₅N₆O₈Na ꢁ H₂O:

2

C, 49.32%; H, 3.15%; N, 16.43%. Found: C, 49.51%;

H, 3.08%; N, 16.58%.

5.1.60.2. Step 2: 7-[4-((2-Carboxyphenylamino)car-

bonyloxymethyl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (29n). To a suspension

of compound 25n (97.0 mg, 197 lmol) in EtOH (5 mL),

1 N KOH (592 lL, 592 lmol) and water (1 mL) were

added, and the solution was reﬂuxed for 1 h. After

cooling, the reaction was concentrated, and the residue

was dissolved into 47% HBr (1.6 mL). After stirring for

24 h at room temperature, the reaction was concentrat-

ed. The residue was washed with water and dried under

vacuum to give the title compound as brown powder

(81.0 mg, quant.); mp 216–218 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 10.75 (s, 1H), 8.31 (d,

J = 8.3 Hz, 1H), 8.24 (s, 1H), 8.05 (s, 1H), 7.99–7.97

(m, 2H), 7.62 (t, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.12

(t, J = 7.8 Hz, 1H), 5.11 (s, 2H); FAB(ꢀ)HRMS

493.0752 (+0.8 mmu).

5.1.57. 3,4-Dihydro-6-nitro-3-oxo-7-[4-((2-triﬂuorometh-

ylphenylamino)carbonyloxymethyl)imidazol-1-yl]quinox-

aline-2-carboxylic acid (29k). Following the procedure

described for compound 28a, the title compound was

prepared from compound 18k and 2-triﬂuoromethyl-

phenyl isocyanate, yellow powder (72%); mp 230–

232 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d: 9.21 (s,

1H), 8.20 (s, 1H), 8.04 (s, 1H), 8.00 (s, 1H), 7.72 (d,

J = 7.8 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.53 (s, 1H),

7.52 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 5.04

(s, 2H); FAB(ꢀ)HRMS 517.0704 (ꢀ1.5 mmu). Anal.

1

4

Calcd for C₂₁H₁₃F₃N₆O₇ꢁ H₂O: C, 48.23%; H, 2.60%;

N, 16.07%. Found: C, 47.93%; H, 2.52%; N, 16.09%.

5.1.58. 3,4-Dihydro-6-nitro-3-oxo-7-[4-((3-triﬂuorometh-

ylphenylamino)carbonyloxymethyl)imidazol-1-yl]quinox-

aline-2-carboxylic acid (29l). Following the procedure

described for compound 28a, the title compound was

prepared from compound 18k and 3-triﬂuoromethyl-

phenyl isocyanate, yellow powder (38%); mp 256–

258 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d: 10.18 (s,

1H), 8.21 (s, 1H), 8.04 (s, 1H), 8.01 (s, 1H), 7.93 (s,

1H), 7.69 (d, J = 8.8 Hz, 1H), 7.59 (s, 1H), 7.52 (t,

J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 5.10 (s,

2H); FAB(ꢀ)HRMS 517.0723 (+0.3 mmu). Anal.

Calcd for C₂₁H₁₃F₃N₆O₇ꢁ H₂O: C, 45.46%; H,

2.54%; N, 15.15%. Found: C, 46.13%; H, 2.54%; N,

15.44%.

5.1.61. 7-[4-((3-Carboxyphenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29o). A solution of compound 18k

(500 mg, 1.29 mmol) and ethyl 3-isocyanatobenzoate

(321 lL, 1.94 mmol) in benzene (15 mL) was reﬂuxed

for 2 h. After cooling, the reaction was concentrated

and puriﬁed by ﬂash column chromatography (SiO₂,

n-hexane:AcOEt, 1:1) to give compound 25o as yellow

oil. The obtained compound 25o was dissolved into

AcOH (5 mL) and concd HCl (1 mL), and stirred for

24 h at room temperature. The reaction was concentrat-

ed, and water was added. The precipitate was collected

by ﬁltration, washed with water and AcOEt, and dried

under vacuum. LiOH (1 N, 15 mL) was added and the

solution was stirred for 3 h at room temperature. The

insoluble part was removed by ﬁltration, and the ﬁltrate

was made acidic with concd HCl. The precipitate was

collected by ﬁltration, washed with water, and dried un-

der vacuum to give the title compound as brown powder

(297 mg, 44%); mp 272–274 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 10.00 (s, 1H), 8.22 (s, 1H), 8.13 (s, 1H),

8.08 (s, 1H), 8.04 (s, 1H), 7.69 (t, J = 9.3 Hz, 1H), 7.61

(s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz,

1H), 5.10 (s, 2H); FAB(ꢀ)HRMS 493.0739

5.1.59. 3,4-Dihydro-6-nitro-3-oxo-7-[4-((4-triﬂuorometh-

ylphenylamino)carbonyloxymethyl)imidazol-1-yl]quinox-

aline-2-carboxylic acid (29m). Following the procedure

described for compound 28f, the title compound was

prepared from compound 18k and 4-triﬂuoromethyl-

benzoic acid, yellow powder (11%); mp 276–278 ꢁC (de-

1

comp.); H NMR (DMSO-d₆) d: 10.22 (s, 1H), 8.21 (s,

1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.68 (d, J = 9.3 Hz,

2H), 7.65 (d, J = 9.3 Hz, 2H), 7.59 (s, 1H), 5.11(s, 2H);

FAB(ꢀ)HRMS 517.0703 (ꢀ1.6 mmu).

5.1.60. 7-[4-((2-Carboxyphenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29n)

3

2

(ꢀ0.5 mmu). Anal. Calcd for C₂₁H₁4N₆O₉ꢁ H₂O: C,

48.47%; H, 3.29%; N, 16.15%. Found: C, 48.62%; H,

3.13%; N, 16.27%.

5.1.60.1. Step 1: Ethyl 3-ethoxy-7-[4-((2-ethoxycar-

bonylphenylamino)carbonyloxymethyl)imidazol-1- yl]-6-

nitroquinoxaline-2-carboxylate (25n). To a solution of

compound 18k (200 mg, 516 lmol) in CH₂Cl₂(10 mL),

ethyl 2-isocyanatobenzoate (455 mg, 2.38 mmol) was

added. After stirring for 18 h at room temperature, the

reaction was concentrated. The residue was puriﬁed by

ﬂash column chromatography (SiO₂, n-hexane:AcOEt,

2:1 to 1:3) to give the title compound as pale yellow

5.1.62. 7-[4-((4-Carboxyphenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29p). Following the procedure described

for compound 29o, the title compound was prepared

from compound 18k and ethyl 4-isocyanatobenzoate,

brown powder (37%); mp 268–270 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 10.18 (s, 1H), 8.23 (s, 1H), 8.13

(s, 1H), 8.05 (s, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.63 (s,

1H), 7.58 (d, J = 8.8 Hz, 2H), 5.12 (s, 2H);

FAB(ꢀ)HRMS 493.0769 (+2.5 mmu).

1

powder (199 mg, 67%); H NMR (CDCl₃) d: 10.33 (s,

1H), 8.66 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 8.8 Hz,

1H), 8.05 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.634 (t,

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5859

5.1.63. 3,4-Dihydro-7-[4-((4-ethoxycarbonylphenylami-

no)carbonyloxymethyl)imidazol-1-yl]-6-nitro-3- oxoqui-

noxaline-2-carboxylic acid (29q)

1H), 8.04 (s, 1H), 7.75 (d, J = 2.4 Hz, 1H), 7.61 (s, 1H),

7.51 (d, J = 8.8 Hz, 1H), 7.25 (dd, J = 8.3, 2.4 Hz, 1H),

5.11 (s, 2H); FAB(ꢀ)HRMS 517.0046 (ꢀ2.0 mmu). Anal.

Calcd for C₂₀H₁₂Cl₂N₆O₇ÆH₂O: C, 44.71%; H, 2.63%; N,

15.64%. Found: C, 44.39%; H, 2.40%; N, 15.34%.

5.1.63.1. Step 1: Ethyl 3-ethoxy-7-[4-((4-ethoxycar-

bonylphenylamino)carbonyloxymethyl)imidazol-1-yl]-6-

nitroquinoxaline-2-carboxylate (25p). Following the

procedure described for compound 28c (step 1), the

title compound was prepared from compound 18k

and ethyl 4-isocyanatobenzoate, yellow amorphous

solid (quant.); ¹H NMR (CDCl₃) d: 8.45 (s, 1H),

8.15 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.71 (d,

J = 1.5 Hz, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.25 (d,

J = 1.0 Hz, 1H), 7.17 (s, 1H), 5.25 (s, 2H), 4.66 (q,

J = 6.9 Hz, 2H), 4.55 (q, J = 7.3 Hz, 2H), 4.35 (q,

J = 6.9 Hz, 2H), 1.53 (t, J = 6.9 Hz, 3H), 1.46 (t,

J = 7.3 Hz, 3H), 1.38 (t, J = 6.9 Hz, 3H).

5.1.67. 7-[4-((2,6-Dichlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29u). Following the procedure described

for compound 28c, the title compound was prepared from

compound 18k and 2,6-dichlorophenyl isocyanate,

brown powder (7%); mp 253–255 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.46 (s, 1H), 8.18 (s, 1H), 8.05 (s,

1H), 7.98 (s, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.53 (s, 1H),

7.34 (t, J = 8.3 Hz, 1H), 5.03 (s, 2H); FAB(ꢀ)HRMS

517.0087 (+2.1 mmu).

5.1.63.2. Step 2: 3,4-Dihydro-7-[4-((4-ethoxycarbonyl-

phenylamino)carbonyloxymethyl)imidazol-1-yl]-6-nitro-3-

oxoquinoxaline-2-carboxylic acid (29q). Following the

procedure described for compound 19i (step 2), the title

compound was prepared from compound 25p, brown

powder (80%); mp 207–209 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 10.23 (s, 1H), 8.24 (s, 1H), 8.06 (s, 1H),

7.89 (d, J = 8.8 Hz, 2H), 7.66 (s, 1H), 7.61 (d,

J = 8.8 Hz, 2H), 5.13 (s, 2H), 4.28 (q, J = 7.3 Hz, 2H),

1.30 (t, J = 7.3 Hz, 3H); FAB(ꢀ)HRMS 521.1057

5.1.68. 7-[4-((3,4-Dichlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid hydrochloride (29v). Following the

procedure described for compound 28a, the title com-

pound was prepared from compound 18k and 3,4-

dichlorophenyl isocyanate, yellow powder (36%); mp

1

218–220 ꢁC (decomp.); H NMR (DMSO-d₆) d: 10.15

(s, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.98 (s, 1H), 7.79 (d,

J = 2.4 Hz, 1H), 7.58 (s, 1H), 7.55 (d, J = 8.8 Hz, 1H),

7.42 (dd, J = 9.0, 2.7 Hz, 1H), 5.09 (s, 2H);

FAB(ꢀ)HRMS 517.0062 (ꢀ0.5 mmu). Anal. Calcd for

3

2

(ꢀ0.8 mmu). Anal. Calcd for C₂₃H₁₈N₆O₉ꢁ H₂O: C,

1

2

50.28%; H, 3.85%; N, 15.30%. Found: C, 50.02%; H,

3.62%; N, 14.94%.

C₂₀H₁₂Cl₂N₆O₇ꢁ HCl ꢁ H₂O: C, 42.54%; H, 2.50%; N,

14.88%. Found: C, 42.79%; H, 2.54%; N, 14.95%.

5.1.64. 7-[4-((2,3-Dichlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-

2-carboxylic acid (29r). Following the procedure de-

scribed for compound 28c, the title compound was

prepared from compound 18k and 2,3-dichlorophenyl

isocyanate, brown powder (68%); mp 234–236 ꢁC (de-

5.1.69. 7-[4-((3,5-Dichlorophenylamino)carbonyloxym-

ethyl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxa-

line-2-carboxylic acid (29w). Following the procedure

described for compound 28a, the title compound was

prepared from compound 18k and 3,5-dichlorophenyl

isocyanate, yellow powder (81%); mp 246–248 ꢁC

1

comp.); H NMR (DMSO-d₆) d: 9.43 (s, 1H), 8.22 (s,

(decomp.); H NMR (DMSO-d₆) d: 10.23 (s, 1H), 8.21

1H), 8.05 (s, 1H), 7.59 (dd, J = 8.5, 1.5 Hz, 1H), 7.58 (s,

1H), 7.45 (dd, J = 7.8, 1.5 Hz, 1H), 7.35 (t, J = 8.3 Hz,

1H), 5.09 (s, 2H); FAB(ꢀ)HRMS 517.0043

(ꢀ0.5 mmu). Anal. Calcd for C₂₀H₁₂Cl₂N₆O₇: C,

46.26%; H, 2.33%; N, 16.18%. Found: C, 46.12%; H,

2.38%; N, 15.90%.

(s, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.59 (s, 1H), 7.53 (d,

J = 2.0 Hz, 2H), 7.21 (t, J = 2.0 Hz, 1H), 5.10 (s, 2H);

FAB(ꢀ)HRMS 517.0065 (ꢀ0.2 mmu). Anal. Calcd for

1

2

C₂₀H₁₂Cl₂N₆O₇ꢁ H₂O: C, 45.47%; H, 2.48%; N,

15.91%. Found: C, 45.43%; H, 2.28%; N, 15.95%.

5.1.70.

3,4-Dihydro-7-[4-((1-naphthylamino)carbonyl-

5.1.65. 7-[4-((2,4-Dichlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29s). Following the procedure described

for compound 28c, the title compound was prepared from

compound 18k and 2,4-dichlorophenyl isocyanate,

brown powder (68%); mp 272–274 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.33 (s, 1H), 8.23 (s, 1H), 8.10 (s,

1H), 8.04 (s, 1H), 7.64–7.60 (m, 3H), 7.41 (dd, J = 8.8,

2.4 Hz, 1H), 5.09 (s, 2H); FAB(ꢀ)HRMS 517.0090

(+2.4 mmu).

oxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline- 2-car-

boxylic acid (29x)

5.1.70.1. Step 1: Ethyl 3-ethoxy-7-[4-((1-naphthylami-

no)carbonyloxymethyl)imidazol-1-yl]-6-nitroquinoxaline-

2-carboxylate (25x). Following the procedure described

for compound 28c (step 1), the title compound was pre-

pared from compound 18k and 1-naphthyl isocyanate,

1

yellow amorphous (quant.); H NMR (CDCl₃) d: 8.45

(s, 1H), 8.16 (s, 1H), 7.90–7.72 (m, 3H), 7.66 (d,

J = 8.3 Hz, 1H), 7.53 (dd, J = 6.9, 1.5 Hz, 1H), 7.52–

7.45 (m, 3H), 7.13 (s, 1H), 5.30 (d, J = 1.5 Hz, 2H), 4.66

(q, J = 7.3 Hz, 2H), 4.55 (q, J = 7.3 Hz, 2H), 1.53 (t,

J = 7.3 Hz, 3H), 1.47 (t, J = 7.3 Hz, 3H).

5.1.66. 7-[4-((2,5-Dichlorophenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (29t). Following the procedure described

for compound 28a, the title compound was prepared

from compound 18k and 2,5-dichlorophenyl isocyanate,

yellow powder (53%); mp 203–205 ꢁC (decomp.); ¹H

NMR (DMSO-d₆) d: 9.41 (s, 1H), 8.22 (s, 1H), 8.09 (s,

5.1.70.2. Step 2: 3,4-Dihydro-7-[4-((1-naphthylamino)

carbonyloxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinox-

aline-2-carboxylic acid (29x). Following the procedure

described for compound 19i, the title compound was

5860

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

prepared from compound 25x, brown powder (70%); mp

224–226 ꢁC (decomp.); H NMR (DMSO-d₆) d: 9.70 (s,

FAB(ꢀ)HRMS 541.0113 (+0.6 mmu). Anal. Calcd for

C₂₁H₁₅BrN₆O₇: C, 46.43%; H, 2.78%; N, 15.47. Found:

C, 46.13%; H, 2.80%; N, 15.34.

1

1H), 8.33 (s, 1H), 8.29 (s, 1H), 8.28 (s, 1H), 8.083 (s,

1H), 8.077–8.05 (m, 1H), 7.93 (dd, J = 6.4, 3.4 Hz, 1H),

7.75 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 5.4 Hz, 1H), 7.63

(d, J = 7.8 Hz, 1H), 7.55–7.47 (m, 3H), 5.16 (s, 2H);

FAB(ꢀ)HRMS 499.1031 (+2.9 mmu). Anal. Calcd for

C₂₄H₁₆N₆O₇ÆH₂O: C, 55.60%; H, 3.50%; N, 16.21%.

Found: C, 55.67%; H, 3.39%; N, 15.88%.

5.1.73.

7-[4-((3-Bromobenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (30b)

5.1.73.1. Step 1: Ethyl 7-[4-((3-bromobenylamino)car-

bonyloxymethyl)imidazol-1-yl]-3-ethoxy-6-nitroquinoxa-

line-2-carboxylate (26b). Following the procedure

described for compound 28f (step 1), the title compound

was prepared from compound 18k and 3-bromophenyl-

acetic acid, brown gum (18%); ¹H NMR (CDCl₃) d: 8.44

(s, 1H), 8.15 (s, 1H), 7.68 (d, J = 1.0 Hz, 1H), 7.44–7.38

(m, 2H), 7.24–7.18 (m, 3H), 5.17 (s, 2H), 4.67 (q,

J = 7.3 Hz, 2H), 4.55 (q, J = 7.3 Hz, 2H), 4.37 (d,

J = 5.9 Hz, 2H), 1.53 (t, J = 7.3 Hz, 3H), 1.47 (t,

J = 7.3 Hz, 3H).

5.1.71.

3,4-Dihydro-7-[4-((2-naphthylamino)carbonyl-

oxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxaline- 2-car-

boxylic acid (29y)

5.1.71.1. Step 1: Ethyl 3-ethoxy-7-[4-((2-naphthylami-

no)carbonyloxymethyl)imidazol-1-yl]-6-nitroquinoxaline-

2-carboxylate (25y). Following the procedure described

for compound 28f (step 1), the title compound was pre-

pared from compound 18k and naphthalene-2-carboxyl-

ic acid, brown amorphous (quant.); ¹H NMR (CDCl₃) d:

8.44 (s, 1H), 8.15 (s, 1H), 7.99 (s, 1H), 7.76 (d,

J = 8.8 Hz, 1H), 7.75 (d, J = 7.3 Hz, 1H), 7.71 (d,

J = 1.0 Hz, 1H), 7.46–7.35 (m, 3H), 7.13 (s, 1H), 7.27

(d, J = 1.0 Hz, 1H), 7.01 (s, 1H), 5.28 (s, 2H), 4.66 (q,

J = 6.9 Hz, 2H), 4.54 (q, J = 7.3 Hz, 2H), 1.52 (t,

J = 6.9 Hz, 3H), 1.46 (t, J = 7.3 Hz, 3H).

5.1.73.2. Step 2: 7-[4-((3-Bromobenylamino)carbonyl-

oxymethyl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid (30b). Following the

procedure described for compound 19i, the title com-

pound was prepared from compound 26b, yellow pow-

der (41%); mp 159–161 ꢁC (decomp.); ¹H NMR

(DMSO-d₆) d: 8.16 (s, 1H), 8.08 (s, 1H), 7.95 (s, 1H),

7.84 (t, J = 5.9 Hz, 1H), 7.47–7.42 (m, 3H), 7.29–7.26

(m, 2H), 4.96 (s, 2H), 4.20 (d, J = 5.9 Hz, 2H);

FAB(ꢀ)HRMS 541.0085 (ꢀ2.2 mmu). Anal. Calcd for

C₂₁H₁₅BrN₆O⁷: C, 46.43%; H, 2.78%; N, 15.47%.

Found: C, 46.55%; H, 2.87%; N, 14.92%.

5.1.71.2. Step 2: 3,4-Dihydro-7-[4-((2-naphthylamino)-

carbonyloxymethyl)imidazol-1-yl]-6-nitro-3-oxoquinoxa-

line-2-carboxylic acid (29y). Following the procedure

described for compound 19i, the title compound was pre-

pared from compound 25y, brown powder (58%); mp

1

275–277 ꢁC (decomp.); H NMR (DMSO-d₆) d: 10.01

(s, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 8.05–8.03 (m, 2H),

7.84–7.76 (m, 3H), 7.61 (s, 1H), 7.54 (dd, J = 8.8,

2.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.39–7.35 (m, 1H), 5.13

(s, 2H); FAB(ꢀ)HRMS 499.1021 (+1.9 mmu). Anal.

5.1.74.

7-[4-((4-Bromobenylamino)carbonyloxymeth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (30c)

5.1.74.1. Step 1: Ethyl 7-[4-((4-bromobenylamino)car-

bonyloxymethyl)imidazol-1-yl]-3-ethoxy-6-nitroquinoxa-

line-2-carboxylate (26c). Following the procedure

described for compound 28f (step 1), the title compound

was prepared from compound 18k and 4-bromophenyl-

3

4

Calcd for C₂₄H₁₆N₆O₇ꢁ H₂O: C, 56.09%; H, 3.43%;

N, 16.18%. Found: C, 56.25%; H, 3.36%; N, 15.94%.

5.1.72.

7-[4-((2-Bromobenylamino)carbonyloxymeth-

1

yl)imidazol-1-yl]-3-ethoxy-6-nitroquinoxaline-2-carboxylic

acid (30a)

acetic acid, yellow amorphous solid (52%); H NMR

(CDCl₃) d: 8.44 (s, 1H), 8.14 (s, 1H), 7.68 (s, 1H),

7.45 (d, J = 8.3 Hz, 2H), 7.19 (s, 1H), 7.17 (d,

J = 8.3 Hz, 2H), 5.16 (s, 2H), 4.67 (q, J = 7.3 Hz, 2H),

4.55 (q, J = 7.3 Hz, 2H), 4.34 (d, J = 6.4 Hz, 2H), 1.53

(t, J = 7.3 Hz, 3H), 1.47 (t, J = 7.3 Hz, 3H).

5.1.72.1. Step 1: Ethyl 7-[4-((2-Bromobenylamino)car-

bonyloxymethyl)imidazol-1-yl]-3-ethoxy-6-nitroquinoxa-

line-2-carboxylate (26a). Following the procedure

described for compound 28f (step 1), the title compound

was prepared from compound 18k and 2-bromophenyl-

1

acetic acid, brown powder (34%); H NMR (CDCl₃) d:

5.1.74.2. Step 2: 7-[4-((4-Bromobenylamino)carbonyl-

oxymethyl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoqui-

noxaline-2-carboxylic acid (30c). Following the procedure

described for compound 19i, the title compound was pre-

pared from compound 26c, yellow powder (20%); mp

8.44 (s, 1H), 8.14 (s, 1H), 7.68 (s, 1H), 7.54 (d,

J = 7.8 Hz, 1H), 7.41 (d, J = 6.9 Hz, 1H), 7.33–7.12 (m,

2H), 5.40–5.30 (br, 1H), 5.15 (s, 2H), 4.67 (q, J = 6.9 Hz,

2H), 4.55 (q, J = 7.3 Hz, 2H), 4.46 (d, J = 6.4 Hz, 2H),

1.53 (t, J = 6.9 Hz, 3H), 1.47 (t, J = 7.3 Hz, 3H).

1

217–219 ꢁC (decomp.); H NMR (DMSO-d₆) d: 8.18 (s,

1H), 8.05 (s, 1H), 7.95 (s, 1H), 7.83 (t, J = 5.9 Hz, 1H),

7.51 (d, J = 8.3 Hz, 2H), 7.47 (s, 1H), 7.22 (d,

J = 8.3 Hz, 2H), 4.95 (s, 2H), 4.17 (d, J = 5.9 Hz, 2H);

FAB(ꢀ)HRMS 541.0099 (ꢀ0.8 mmu). Anal. Calcd for

5.1.72.2. Step 2: 7-[4-((2-Bromobenylamino)carbonyl-

oxymethyl)imidazol-1-yl]-3-ethoxy-6- nitroquinoxaline-2-

carboxylic acid (30a). Following the procedure described

for compound 19i, the title compound was prepared

from compound 26a, brown powder (45%); mp 187–

189 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d: 8.18 (s,

1H), 8.07 (s, 1H), 7.96 (s, 1H), 7.86 (t, J = 5.9 Hz,

1H), 7.60 (d, J = 7.8 Hz, 1H), 7.49 (s, 1H), 7.21 (t,

J = 7.8 Hz, 1H), 4.97 (s, 2H), 4.24 (d, J = 5.9 Hz, 2H);

1

2

C₂₁H₁₅BrN₆O₇ꢁ H₂O: C, 45.67%; H, 2.92%; N,

15.22%. Found: C, 45.71%; H, 2.79%; N, 15.20%.

5.1.75. 7-[4-(2-((4-Carboxyphenylamino)carbonyloxy)eth-

yl)imidazol-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-2-

carboxylic acid (31). To a solution of compound 23

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5861

(110 mg, 274 lmol) in MeCN (5 mL), ethyl 4-iso-

cyanatobenzoate (57.0 mg, 298 lmol) was added, and

the solution was stirred overnight. The reaction was

concentrated and puriﬁed by ﬂash column chromatog-

raphy [SiO₂, CH₂Cl₂to CH₂Cl₂:MeOH (50:1)] to give

crude compound 27. The obtained compound 27 was

dissolved into AcOH–concd HCl (5:1, 6 mL), and stir-

red for 20 min at 80 ꢁC followed by 16 h at room tem-

perature. The reaction was concentrated, and the

residue was dissolved into MeOH (5 mL). LiOH mono-

hydrate (60.0 mg, 1.43 mmol) in water (1 mL) was add-

ed to the solution, and stirred for 2 h at 50 ꢁC. The

reaction was concentrated, adjusted to pH 2 with concd

HCl, and concentrated again. Water was added to the

residue, and the precipitate was collected by ﬁltration,

washed with water and AcOEt, and dried under vacuum

to give the title compound as brown powder (70.5 mg,

J = 7.2 Hz, 2H), 1.54 (t, J = 7.1 Hz, 3H), 1.47 (t,

J = 7.1 Hz, 3H).

5.1.78.

3,4-Dihydro-7-[3-((phenylamino)carbonyloxy-

methyl)-4-pyridon-1-yl]-6-nitro-3-oxoquinoxaline-2-car-

boxylic acid (38a). Following the procedure described for

compound 28a, the title compound was prepared from

compound 36 and phenyl isocyanate, yellow powder

(51%); mp 230–232 ꢁC (decomp.); ¹H NMR (DMSO-d₆)

d: 9.71 (s, 1H), 8.38 (s, 1H), 8.13 (s, 1H), 8.06 (s, 1H),

7.92 (dd, J = 7.3, 2.0 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H),

7.26 (t, J = 7.8 Hz, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.34 (d,

J = 7.8 Hz, 1H), 4.91 (s, 2H); FAB(ꢀ)HRMS 476.0837

3

2

(ꢀ0.6 mmu). Anal. Calcd for C₂₂H₁₅N₅O₈ꢁ H₂O: C,

52.39%; H, 3.60%; N, 13.88%. Found: C 52.70%; H,

3.41%; N, 13.81%.

1

51%); mp >300 ꢁC; H NMR (DMSO-d₆) d: 12.65 (s,

5.1.79. 7-[3-((4-Carboxyphenylamino)carbonyloxymeth-

yl)-4-pyridon-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxaline-

2-carboxylic acid (38b)

1H), 10.05 (s, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 8.02 (s,

1H), 7.86 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 9.2 Hz, 2H),

7.35 (s, 1H), 4.38 (t, J = 7.3 Hz, 2H), 2.95 (t,

J = 7.3 Hz, 2H); FAB(ꢀ)HRMS 507.0902 (+0.1 mmu).

5.1.79.1. Step 1: Ethyl 3-ethoxy-7-[3-((4-ethoxycar-

bonylphenylamino)carbonyloxymethyl)-4-pyridon- 1-yl]-6-

nitroquinoxaline-2-carboxylate (37b). Following the pro-

cedure described for compound 28c (step 1), the title

compound was prepared from compound 36 and ethyl

5.1.76. 4-Chloropyridine-3-methanol (34). To a solution

of ⁱPr₂NH (18.7 mL, 133 mmol) in THF (100 mL)

1.55 M n-BuLi in n-hexane (103 mL, 160 mmol) was

added dropwise at ꢀ78 ꢁC under Ar atmosphere. After

stirring for 15 min at the same temperature, compound

32 (10.0 g, 66.7 mmol) was added to the solution, and

stirred for 30 min. DMF (6.19 mL, 80.0 mmol) was add-

ed dropwise to the solution at ꢀ78 ꢁC. The reaction was

warmed to room temperature slowly, and stirred over-

night. The reaction was quenched with 3 N HCl and stir-

red for 2 h at room temperature. The solution was

neutralized with NaHCO₃, extracted with AcOEt, dried

over Na₂SO₄, and evaporated to give crude compound

33 as brown oil. The obtained compound 33 was dis-

solved into EtOH (100 mL), and NaBH₄(3.03 g,

80.0 mmol) was added to the solution. After stirring

for 3 h at room temperature, the reaction was concen-

trated. Water was added to the residue, extracted with

AcOEt, dried over Na₂SO₄, and evaporated to give the

title compound as pale yellow powder (7.02 g, 73%);

¹H NMR (CDCl₃) d: 8.68 (s, 1H), 8.46 (d, J = 5.4 Hz,

1H), 7.33 (d, J = 5.4 Hz, 1H), 4.84 (s, 2H), 2.23 (s, 1H).

1

4-isocyanatobenzoate, yellow powder (94%); H NMR

(CDCl₃) d: 8.57 (s, 1H), 8.22 (s, 1H), 7.97 (d,

J = 8.8 Hz, 2H), 7.71 (d, J = 2.5 Hz, 1H), 7.43 (d,

J = 8.8 Hz, 2H), 7.39 (dd, J = 7.8, 2.5 Hz, 1H), 6.56 (d,

J = 7.8 Hz, 1H), 5.15 (s, 2H), 4.68 (q, J = 6.9 Hz, 2H),

4.55 (q, J = 7.3 Hz, 2H), 4.34 (q, J = 7.3 Hz, 2H), 1.54

(t, J = 6.9 Hz, 3H), 1.47 (t, J = 7.3 Hz, 3H), 1.37 (t,

J = 7.3 Hz, 3H).

5.1.79.2. Step 2: 7-[3-((4-Ethoxycarbonylphenylami-

no)carbonyloxymethyl)-4-pyridon-1-yl]-3,4-dihydro-6-ni-

tro-3-oxoquinoxaline-2-carboxylic acid. To a solution of

compound 37b (692 mg, 1.14 mmol) in AcOH (12 mL),

concd HCl (3 mL) was added, and stirred for 18 h at

room temperature. Water was added to the reaction,

and the precipitate was collected by ﬁltration, washed

with water, and dried under vacuum to give the title

compound as yellow powder (548 mg, 86%); mp 201–

203 ꢁC (decomp.); ¹H NMR (DMSO-d₆) d: 10.15 (s,

1H), 8.38 (s, 1H), 8.12 (s, 1H), 8.05 (d, J = 2.0 Hz,

1H), 7.91–7.86 (m, 3H), 7.58 (d, J = 8.3 Hz, 2H), 6.30

(d, J = 7.8 Hz, 1H), 4.94 (s, 2H), 4.27 (q, J = 7.3 Hz,

2H), 1.30 (t, J = 7.3 Hz, 3H); FAB(ꢀ)HRMS 548.1053

5.1.77. Ethyl 3-ethoxy-7-[3-(hydroxymethyl)-4-pyridon-1-

yl]-6-nitroquinoxaline-2-carboxylate (36). To a solution

of compound 34 (2.33 g, 16.2 mmol) in water (25 mL),

NaOH (5.20 g, 130 mmol) was added, and the solution

was reﬂuxed for 24 h. After cooling, the reaction was

neutralized with concd HCl, and concentrated to give

crude compound 35. The obtained compound 35 was

dissolved into DMF (20 mL), and compound 11b

(500 mg, 1.62 mmol) was added. After stirring for 4 h

at 110 ꢁC, the reaction was poured into ice water,

extracted with AcOEt, dried over Na₂SO₄, and evapo-

rated. The residue was puriﬁed by ﬂash column chro-

matography (SiO₂, AcOEt) to give the title compound

1

2

(ꢀ0.1 mmu). Anal. Calcd for C₂₅H₁₉N₅O₁₀ꢁ H₂O: C,

53.78%; H, 3.61%; N, 12.54%. Found: C, 53.82%; H,

3.69%; N, 12.60%.

5.1.79.3. Step 3:7-[3-((4-Carboxyphenylamino)carbon-

yloxymethyl)-4-pyridon-1-yl]-3,4-dihydro-6-nitro-3-oxo-

quinoxaline-2-carboxylic acid (38b). To a suspension of

7-[3-((4-ethoxycarbonylphenylamino)carbonyloxymeth-

yl)-4-pyridon-1-yl]-3,4-dihydro-6-nitro-3-oxoquinoxa-

line-2-carboxylic acid (383 mg, 697 lmol) in water

(4 mL), 1 N LiOH (6.97 mL, 6.97 mmol) was added,

and the suspension was stirred for 3 h at room temper-

ature. After puriﬁcation by synthetic adsorbent Sepa-

beads^ꢂSP850 (water–MeCN, 20:1), water was added

and adjusted to pH 2 with concd HCl. The precipitate

1

as yellow powder (410 mg, 61%); H NMR (CDCl₃) d:

8.57 (s, 1H), 8.20 (s, 1H), 7.45 (d, J = 2.4 Hz, 1H),

7.41 (dd, J = 7.3, 2.4 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H),

4.68 (q, J = 7.2 Hz, 2H), 4.59 (s, 2H), 4.56 (q,

5862

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

was collected by ﬁltration, washed with water, and

dried under vacuum to give the title compound as yel-

low powder (253 mg, 65%); mp 231–233 ꢁC (decomp.);

¹H NMR (DMSO-d₆) d: 10.10 (s, 1H), 8.38 (s, 1H),

8.13 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.90 (dd,

J = 7.3, 2.5 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.56 (d,

J = 8.8 Hz, 2H), 6.32 (d, J = 7.3 Hz, 1H), 4.94 (s,

2H); FAB(ꢀ)HRMS 520.0740 (+0.0 mmu). Anal.

Calcd for C₂₃H₁₅N₅O₁₀Æ2H₂O: C, 49.56%; H, 3.44%;

N, 12.56%. Found: C, 49.32%; H, 3.41%; N, 12.47%.

ses were performed with a Hitachi L-6200 pump, a Hit-

achi L-4000 ultraviolet wavelength detector (Hitachi,

Japan) and a Tosoh AS-8010 automatic sample injection

system (Tosoh, Japan).

5.4. Biology

5.4.1. Radioligand receptor-binding assay. Receptor

binding was measured as the percentage displacement

of 5 nM [³H]AMPA and 10 nM [³H]CGS-19755 from

extensively washed rat cortical synaptosomal mem-

branes, as described by Honore et al., Johansen et al.,

and Murphy et al.^25–27Using 50 mM Tris–HCl buﬀer

(pH 7.4), tubes containing the membranes, 5 nM

[³H]AMPA, 2.5 mM CaCl₂, 100 mM KSCN, and the

test compounds were incubated for 30 min at 0 ꢁC, while

other tubes containing 10 nM [³H]CGS-19755 (pH 8.0)

and the test compounds were incubated for 15 min at

4 ꢁC. Nonspeciﬁc binding was determined in the pres-

ence of 0.1 or 1 mM glutamic acid. After stopping the

reaction by suction ﬁltration, the radioactivity on the ﬁl-

ter was measured with a liquid scintillation counter

TR2300 (Packard, Tokyo, Japan). IC₅₀values were cal-

culated and converted to K_ivalues using the Cheng–

Prosoﬀ equation.

5.2. Modeling

5.2.1. Molecular modeling. The crystal structure of bac-

terial lysine/arginine/ornithine-binding protein (LAO-

˚

BP) solved at 1.9 A resolution was chosen as the

template for the AMPA-R homology model (Protein

Data Bank entry: 2LAO²¹) because bacterial LAOBP

is homologous with glutamate receptors of known

three-dimensional structure. The amino acid sequences

of the GluR3 ligand binding core of the AMPA-R and

LAOBP were reported by OꢀHara et al.²²The three-di-

mensional structure of the GluR3 ligand binding core

was modeled using the procedures and strategies out-

lined by Blundell et al.²³The processes employed were

essentially the same as those used by OꢀHara et al.^22,24

The docking experiments were carried out by manually

docking compound 19h, a quinoxaline-2-carboxylic acid

derivative, into our AMPA-R model. Several docking

orientations of the ligand in the binding site of

AMPA-R were obtained and all possible docking model

structures were minimized using the conjugate gradient

method of the CVFF force ﬁeld until a gradient conver-

5.4.2. AMPA-evoked depolarization in rat cortical slices

(DC potential). Coronal sections of rat brain (400 lm

thick) were trimmed to form ꢁwedgesꢀ of tissue contain-

ing cerebral cortex and corpus callosum as described by

Harrison and Simmonds.²⁸After 3 h of incubation in

oxygenated Krebs medium, each slice was placed in a

two-compartment recording chamber. This was

arranged so that the cortical tissue was contained almost

entirely in one compartment and the ventral margin of

the cortex passed through a greased slot so that the cor-

pus callosum was entirely in the other compartment.

The cortical tissue was depolarized by superfusion of

AMPA (5 lM) for 2 min. The DC potential between

the two compartments was monitored via Ag/AgCl elec-

trodes and a high input impedance ampliﬁer. AMPA-in-

duced deviations from this baseline DC potential were

measured at peak amplitude. Each test compound was

applied to the cortical end of the preparation 10 min be-

fore exposure to AMPA, and the ability of the test com-

pound to inhibit AMPA-induced DC potentials was

assessed.

˚

gence of 0.1 kcal/mol A was reached (Ca carbon atoms

were ﬁxed, and the dielectric constant 78 was used).

The docking model structure with the lowest interaction

energy between the ligand and the AMPA-R model was

selected as the AMPA-R complex model among docking

model structures with the low total energy.

5.2.2. Hardware and software. Molecular modeling stud-

ies were carried out using the Insight II/Discover version

98.0 molecular modeling package (Accelrys Inc., San

Diego, CA, USA) on a Silicon Graphics O2 R10K

workstation.

5.3. Solubility measurements

The solubilities of the test compounds were determined

in 0.1 M phosphate buﬀer (pH 7.4) at ambient tempera-

ture as described by Higuchi et al.²⁰About 2 mg of each

compound was mixed with 200 mL phosphate buﬀer

and sonicated for 10 min. After centrifugation, 10 mL

of the supernatant was diluted with 100 mL 50% MeOH

and 10 mL DMSO, in the case of reference samples con-

taining 10 mg/mL of the test compounds. The concen-

tration of the test compound in the solution was

determined by high-performance liquid chromatog-

raphy (HPLC) using a C18 reversed-phase column

(4.6 · 75 mm; Jꢀshpere ODS-H80, YMC, Japan), with

a mobile phase consisting of 30% or 50% MeOH in

50 mM NaH₂PO₄run at a ﬂow rate of 1 mL/min, and

detection at a wavelength of 254 nm. The HPLC analy-

5.4.3. Rat focal ischemia model. Male Wistar rats (300–

350 g) were subjected to permanent occlusion of the

right middle cerebral artery (MCA) under halothane

anesthesia as described by Tamura et al.¹⁹Rectal tem-

perature was maintained at 37 1 ꢁC during the exper-

iment. After 24 h, the brains were removed and sliced

into ﬁve coronal sections (2 mm thick) with the aid of

a rat brain matrix (a manual slicer). The slices were

placed in 2% (w/v) triphenyltetrazolium chloride

(TTC) solution, followed by 10% (v/v) phosphate-buf-

fered formalin. Tissue damage (the area not stained with

TTC) was scored on a four-point damage score as the

infarct volumes (see Fig. 5). On evaluation of four-point

damage score in focal ischemia model, gray areas are in-

farct damage area, then grade 1 damage is ꢁ0ꢀ and grade

Y. Takano et al. / Bioorg. Med. Chem. 13 (2005) 5841–5863

5863

10. Ohmori, J.; Sakamoto, S.; Kubota, H.; Shimizu-Sasamata,

M.; Okada, M.; Kawasaki, S.; Hidaka, K.; Togami, J.;

Furuya, T.; Murase, K. J. Med. Chem. 1994, 37, 467.

11. Shimizu-Sasamata, M.; Kawasaki-Yatsugi, S.; Okada, M.;

Sakamoto, S.; Yatsugi, S.; Togami, J.; Hatanaka, K.;

Ohmori, J.; Koshiya, K.; Usuda, S.; Murase, K. J.

Pharmacol. Exp. Ther. 1996, 276, 84.

4 damage is ꢁ3ꢀ in four-point scores. The ꢁ3ꢀ of grade 4

damage is most eﬀective score against focal ischemia

model. Each test compound was administered by contin-

uous iv infusion for 4 h, starting immediately after

occlusion of the MCA. Control rats received saline only,

and their four-point damage scores were less than 1.0.

12. Shishikura, J.; Tsukamoto, S.; Inami, H.; Fujii, M.;

Okada, M.; Sasamata, M.; Sakamoto, S. WO 9610023;

Chem. Abstr. 1996, 125, 114689.

Acknowledgments

13. (a) Kawasaki-Yatsugi, S.; Yatsugi, S.; Takahashi, M.;

Toya, T.; Ichiki, C.; Shimizu-Sasamata, M.; Yamaguchi, T.;

Minematsu, K. Brain. Res. 1998, 793, 39; (b) Takahashi, M.;

Ni, J. W.; Kawasaki-Yatsugi, S.; Toya, T.; Ichiki, C.;

Yatsugi, S.; Koshiya, K.; Shimizu-Sasamata, M.; Yamag-

uchi, T. J. Pharmacol. Exp. Ther. 1998, 287, 559.

14. Xue, D.; Huang, Z.-G.; Barnes, K.; Lesiuk, H. J.; Smith,

K. E.; Buchan, A. M. J. Cereb. Blood Flow Metab. 1994,

14, 251.

We are grateful to Dr. T. Ishizaki in Kyorin Pharmaceu-

tical Co. Ltd, for many useful suggestions and encour-

agement. We wish to thank Dr. K. Iwase for useful

suggestions for molecular modeling study. We also

thank the staﬀ of the analytical section for spectral mea-

surement and elemental analysis.

15. (a) Bigge, C. F.; Boxer, P. A.; Ortwine, D. F. Curr. Pharm.

Des. 1996, 2, 397; (b) Bigge, C. F.; Nikam, S. S. Expert

Opin. Ther. Patents 1997, 7, 1099.

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Article Doi

Design, synthesis, and AMPA receptor antagonistic activity of a novel 6-nitro-3-oxoquinoxaline-2-carboxylic acid with a substituted phenyl group at the 7 position

DOI: 10.1016/j.bmc.2005.05.030

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Full text of DOI:10.1016/j.bmc.2005.05.030

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