Synthesis and structure-activity relationships of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids as HB-EGF shedding inhibitors

Article abstract of DOI:10.1016/S0968-0896(02)00426-1

HB-EGF Shedding inhibitors have been expected to become effective medicines for skin diseases caused by the proliferation of keratinocytes. In order to discover novel HB-EGF shedding inhibitors and clarify their structure-activity relationships, 5,6,7,8-tetrahydronaphthylidine-based hydroxamic acid and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids have been synthesized. Among the synthesized compounds, the ethoxyethoxy derivative 3o and the methoxypropoxy derivative 3p exhibited much more potent HB-EGF shedding inhibitory activity than CGS 27023A. The structural modification of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids enabled us to establish the following structure-activity relationships; the existences of the hydroxamic acid, the sulfonamide, and the phenyl moieties are crucial for a potent HB-EGF shedding inhibitory activity, and the stereochemistry of the alpha carbon of hydroxamic acid is also important. In addition, from the comparison of their HB-EGF shedding inhibitory activities with their MMPs inhibitory activities, we found that the S1′ pocket of the responsible enzyme for HB-EGF shedding is deep unlike that of MMP-1.

Full text of DOI:10.1016/S0968-0896(02)00426-1

Bioorganic & Medicinal Chemistry 11 (2003) 433–450
Synthesis and Structure–Activity Relationships of
5,6,7,8-Tetrahydropyrido[3,4-b]pyrazine-based Hydroxamic
Acids as HB-EGF Shedding Inhibitors
Kazuya Yoshiizumi,* Minoru Yamamoto, Tomohiro Miyasaka, Yasuko Ito,
Hiroshi Kumihara, Masaaki Sawa, Takao Kiyoi, Takeshi Yamamoto, Fumio Nakajima,
Ryoichi Hirayama, Hirosato Kondo, Etsuko Ishibushi, Hiroshi Ohmoto,
Yoshimasa Inoue and Kohichiro Yoshino
R&D Laboratories, Nippon Organon K.K., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, Japan
Received 5 November 2001; accepted 26 August 2002
Abstract—HB-EGF Shedding inhibitors have been expected to become effective medicines for skin diseases caused by the pro-
liferation of keratinocytes. In order to discover novel HB-EGF shedding inhibitors and clarify their structure–activity relationships,
5,6,7,8-tetrahydronaphthylidine-based hydroxamic acid and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids have
been synthesized. Among the synthesized compounds, the ethoxyethoxy derivative 3o and the methoxypropoxy derivative 3p
exhibited much more potent HB-EGF shedding inhibitory activity than CGS 27023A. The structural modification of 5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-based hydroxamic acids enabled us to establish the following structure–activity relationships; the exis-
tences of the hydroxamic acid, the sulfonamide, and the phenyl moieties are crucial for a potent HB-EGF shedding inhibitory
activity, and the stereochemistry of the alpha carbon of hydroxamic acid is also important. In addition, from the comparison of
their HB-EGF shedding inhibitory activities with their MMPs inhibitory activities, we found that the S10 pocket of the responsible
enzyme for HB-EGF shedding is deep unlike that of MMP-1.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
is synthesized as a membrane-binding form, and it is
shed to give the soluble active form, HB-EGF.2 It has
been reported that a disintegrin and metalloproteinases
(ADAMs) are associated with the shedding of HB-
EGF.4 ADAMs consist of more than 30 enzymes, but it
is still unclear which enzyme is responsible for HB-EGF
shedding. It was already found that N-{dl-{2-(hydroxy-
aminocarbonyl)methyl} - 4 - methylpentanoyl} - l - 3 - (20-
naphtyl)-alanyl-l-alanine 2-aminoethylamide (TAPI), a
peptide-type matrix metalloproteinase (MMP) inhi-
bitor, inhibited the proteolytic cleavage of proHB-
EGF.5 Moreover, Tokumaru et al. have reported that
other peptide-type MMP inhibitors suppressed the pro-
liferation of keratinocytes in both in vitro and in vivo
models.3 Therefore, we have expected that MMP inhi-
bitors would become effective medicines for skin dis-
eases caused by the proliferation of keratinocytes.
Keratinocytes are main components in the epidermis,
and they play a central role in the barrier function of
skin. In various skin diseases, such as psoriasis, atopic
disease, and epidermal cancers, the aberrant prolifera-
tion of keratinocytes is observed. The proliferation of
keratinocytes is driven by a number of growth factors,1
including epidermal growth factor (EGF), transforming
growth factor-a (TGF-a), amphiregulin (AR), heparin-
binding epidermal growth factor-like growth factor
(HB-EGF), fibroblast growth factor-1 (FGF-1), and
hepatocyte growth factor (HGF). Recently, it has been
reported that, among these growth factors, HB-EGF2
might be the most important for the proliferation of
keratinocytes.3 The precursor of HB-EGF (proHB-EGF)
*Corresponding author at current address: Medicinal Chemistry
Department, Organon Laboratories Ltd., Newhouse, Motherwell,
Lanarkshire ML1 5SH, UK. Tel.: +44-1698-736000; fax: +44-1698-
736001; e-mail: k.yoshiizumi@organon.nhe.akzonobel.nl
In general, peptide-type MMP inhibitors have draw-
backs concerning bioavailability, for example poor oral
absorption. Such poor pharmacokinetic profiles of
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00426-1

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
Figure 1. Design of bicyclic compounds from CGS 27023A.
peptide-type MMP inhibitors made us select another
type of MMP inhibitors as our lead compound. CGS
27023A, a sulfonamide-type MMP inhibitor, is known
to have a better pharmacokinetic profile,6 so we chose
CGS 27023A as our lead compound. The study on the
conformation of CGS 27023A by NMR has showed
that the pyridymethyl moiety and the isopropyl moiety
of CGS 27023A are located close to each other.7 Based
on this information, we designed bicyclic compounds
1a–d and 2 shown in Figure 1. Then, we synthesized
them and evaluated their HB-EGF shedding inhibitory
activity in order to discover novel HB-EGF shedding
inhibitors with excellent potency. In addition, we tried to
clarify their structure–activity relationships (SARs),
because no research groups have not reported on the SARs
of HB-EGF shedding inhibitors. Moreover, many of the
synthesized compounds were tested for MMPs inhibitory
activities in order to elucidate whether they are selective
HB-EGF shedding inhibitors or not. In this paper, at first,
we describe the synthesis of compounds 1a–d and 2.
Then, the structural requirements for a potent HB-EGF
shedding inhibitory activity are presented. From the
comparison of the HB-EGF shedding activities with the
MMPs inhibitory activities, we describe the S10 pocket of
the responsible enzyme for HB-EGF shedding.
known that compound 10 can be easily synthesized
from phenylalanine 9 and formaldehyde in the presence
of hydrochloric acid (Pictet–Spengler reaction8). We
applied the typical reaction condition of Pictet–Spengler
reaction to the synthesis of 12b, 12c, and 14. However,
the treatment of pyridylalanine or pyrazylalanine with
formaldehyde in the presence of hydrochloric acid did
not provide the target compounds 12b, 12c, and 14 at
all.
As a result of exploring alternative synthetic routes for
12a–d and 14, we found the synthetic route shown in
Scheme 2. At first, 2,3-pyridinedicarboxylic acid (15) was
converted into 2,3-bis(chloromethyl)pyridine (18) b y
esterification, reduction, and chlorination. The alkyl-
ation of diethyl acetamidomalonate with compound 18
in the presence of sodium hydride gave bicyclic diester
as a mixture of regioisomers 19a and 19b. Then, this
mixture was heated with 6 N hydrochloric acid to afford
a mixture of bicyclic amino acids 12a and 12b. Ester-
ification of this mixture, and subsequent sulfonylation
Chemistry
We selected compounds 12a–d and 14 (Fig. 1) as start-
ing materials for the synthesis of bicyclic compounds
1a–d and 2. However, these bicyclic amino acids 12a–
d and 14 had not been synthesized yet. Therefore, first
of all, we tried to synthesize them. Compounds 12a–d
and 14 are structurally related to 1,2,3,4-tetra-
hydroisoqiunoline-3-carboxylic acid 10 (Scheme 1). It is
Scheme 1. Reagents: (a) formaldehyde, hydrochloric acid.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
435
Scheme 2. Reagents: (a) conc H2SO4, MeOH; (b) NaBH4; (c) (i) SOCl2; (ii) NaHCO3 aq; (d) diethyl acetamidomalonate, NaH; (e) 6 N HCl;
(f) SOCl2, MeOH; (g) 4-methoxybenzenesulfonyl chloride, 4-dimethylaminopyridine.
with 4-methoxybenzenesulfonyl chloride gave two sul-
fonylamino esters, which were separated by column
chromatography to yield compounds 21a and 21b. The
structures of 21a and 21b were confirmed by the
measurement of their HMBC spectra.
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine skeleton. Hydro-
lysis and decarboxylation of 26 was performed by heat-
ing 26 in hydrochloric acid, followed by esterification
with thionyl chloride to give amino ester 27. Sulfonyl-
ation of 27 with 4-methoxybenzenesulfonyl chloride
afforded sulfonylamino ester 28, which was converted
into the target compound 2 in the same manner as 1a
(Scheme 3).
Hydrolysis of 21a under an alkaline condition, followed
by condensation with O-benzylhydroxylamine in the
presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (EDCI) and 1-hydroxybenzo-
triazole hydrate (HOBt), and hydrogenolysis to give the
target compound 1a (Scheme 3). Compound 1b, a
regioisomer of 1a, was synthesized from sulfonylamino
ester 21b in the same manner as 1a. 3,4-Pyridine-
dicarboxylic acid was converted into compounds 1c and
1d in the same manner as 2,3-pyridinedicarboxylic acid
(the route is not shown here).
Schemes 5 shows the synthetic routes for optically
active derivative of 2 (3a) and their related compounds
(compounds 3c, 3e–h, 3j, 3p, 3r–s, and 5a) listed in
Tables 2 and 3. First of all, diester 26 was heated with
6 N HCl, followed by acylation with di-tert-butyl
Next, this new synthetic method was applied to the
synthesis of 2 with tetrahydropyrido[3,4-b]pyrazine ske-
leton (Scheme 4). To begin with, 2,3-dimethylpyrazine
(24) was chlorinated into 2,3-bis(chloromethyl)pyrazine
(25) by treatment with N-chlorosuccimide (NCS) in
carbon tetrachloride. Then, the cyclization between di-
ethyl acetamidomalonate and compound 25 in the pre-
sence of cesium carbonate gave compound 26 with
Scheme 4. Reagents: (a) N-chlorosuccimide, benzoyl peroxide; (b)
diethyl acetamidomalonate, Cs2CO3; (c) 6 N HCl; (d) SOCl2, MeOH;
(e) 4-methoxybenzenesulfonyl chloride, 4-dimethylaminopyridine.
Scheme 5. Reagents: (a) 6 N HCl; (b) di-tert-dicarbonate, NaOH; (c)
(S)-(ꢀ)-1-phenylethylamine or (R)-(+)-1-phenylethylamine; (d) citric
acid.
.
Scheme 3. (Method A). Reagents: (a) NaOH aq; (b) H2NOBn HCl,
EDCI, HOBt, Et3N; (c) Pd/C, H2.

436
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
.
Scheme 6. (Method B). Reagents: (a) H2NOBn HCl, EDCI, HOBt, Et3N; (b) 4 N HCl/ethyl acetate solution; (c) benzenesulfonyl chloride or
4-substituted benzenesulfonyl chloride Et3N; (d) NaOH aq; (e) Pd/C, H2; (f) 4-methoxybenzoyl chloride, Et3N.
Scheme 7. (Method C). Reagents: (a) 4 N HCl/ethyl acetate solution; (b) 4-substituted benzenesulfonyl chloride; (c) (i) ClCOCOCl, (ii) H2NOH aq.
Scheme 8. (Method D). Reagents: (a) SOCl2, MeOH; (b) bromobenzenesulfonyl chloride, Et3N; (c) Et3N, Ph3P, CuI, trimethylsilylacetylene;
(d) KOH aq; (e) ClCOCOCl; (f) H2NOH aq.
dicarbonate to afford (ꢁ)-tert-butoxycarbonylamino
acid 31, as shown in Scheme 5. Optical resolution of 31
by use of (S)-(ꢀ)-1-phenylethylamine gave (ꢀ)-tert-
butoxycarbonylamino acid 32a, and on the other hand,
that by use of (R)-(+)-1-phenylethylamine gave (+)-
tert-butoxycarbonylamino acid 32b.
The target compounds 3a, 3c, 3e–h, 3j, 3p, and 3r–s were
prepared from (ꢀ)-tert-butoxycarbonylamino acid 32a
according to Method B shown in Scheme 6. At first, 32a
was condensed with O-benzylhydroxylamine in the pre-
sence of EDCI and HOBt to afford N-benzyloxy amide
33a. Removal of the tert-butoxycarbonyl (Boc) moiety

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
437
Table 1. HB-EGF Shedding inhibitory activity of tetrahydro-
naphthylidine-based hydroxamic acids 1a–d, tetrahydropyrido[3,4-b]
pyrazine-based hydroxamic acid 2, and CGS 27023A
of 33a by treatment with hydrochloric acid gave the
unprotected compound 34a, which was sulfonylated
with various sulfonyl chlorides in the presence of a ter-
tially amine to yield sulfonamides 35. Finally, hydro-
genolysis of 35 in the presence of palladium–carbon
(Pd/C) gave the target hydroxamic acids. The hydroxy
derivative 3f and the carboxyl derivative 3s were syn-
thesized by treating the pivaloyloxy derivative 35u and
the ethyl ester derivative 35r, respectively, with alkali,
followed by hydrogenolysis of the obtained compounds
35f and 35s. The hexanesulfonamide derivative 6 was
prepared in the same manner as benzenesulfonamide
type compounds (the scheme is not shown here). The
enantiomer of 3a (3b) was synthesized from (+)-Boc-
amino acid 32b in the same manner as 3a (the scheme is
not shown here). The carboxamide derivative 5a was
prepared from 34a and 4-methoxybenzoyl chloride via
N-benzyloxyamide 36a.
Compd
X
Synthetic Sign of HB-EGF Shedding
method optical inhibitory activitya
rotation
IC50 (mM)b
0.094
1a
A
ꢁ
1b
A
A
A
A
ꢁ
ꢁ
ꢁ
ꢁ
2.5
0.37
0.83
0.80
1.2
1c
Scheme 7 shows the synthetic route (Method C) for
compounds 3a, 3d, 3i, 3k, 3n, 3o, 3q, and 4a. The Boc
moiety of (ꢀ)-Boc-carboxylic acid 32a was removed by
hydrochloric acid treatment to give amino acid 37a. The
sulfonylation of 37a by various kinds of sulfonyl chlo-
ride gave sulfonylamino acids 4. Finally, the carboxylic
acid moiety of 4 was converted into hydroxamic acid
moiety via carboxylic acid chloride.
1d
2
CGS 27023A
aThe details of the assay are described in the Experimental.
bConcentration required for 50% inhibition of HB-EGF shedding. All values
represent the mean of two determinations.
The synthetic route of the ethynyl derivative 3m is
shown in Scheme 8. First, amino acid 37a was treated
Table 2. HB-EGF Shedding inhibitory activity and MMPs inhibitory activity of tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acid and
related compounds
Compd
Y
L
Synthetic
method
Sign of
optical
rotation
HB-EGF Shedding
inhibitory activitya
MMPs Inhibitory
activity, Ki (nM)c
IC50 (mM)b
MMP-1
61
MMP-3
15
MMP-9
45
3a
-CONHOH
B, C
+
0.35
3b
-CONHOH
-CO2H
B
C
B
B
ꢀ
+
+
+
>10d
>10e
>10f
4.4
>850
>850
>850
560
>650
>650
>650
>650
9.8
>790
>790
>790
740
4a
5a
-CONHOH
-CONHOH
6
-SO2-(CH2)5CH3
CGS 27023A
1.2
28
36
aThe details of the assay are described in the Experimental.
bConcentration required for 50% inhibition of HB-EGF shedding. All values represent the mean of at least two determinations.
cKi Values were calculated from percent inhibition and Km value of each MMPs to the substrate. All values represent the mean of two determinations. The details of
the assays are described in the Experimental.
d15% Inhibition at 10 mM.
e7.9% Inhibition at 10 mM.
f<5% Inhibition at 10 mM.

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K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
Table 3. HB-EGF Shedding inhibitory activity and MMPs inhibitory activities of tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acid and
related compounds
Compd
R
Synthetic
method
Sign of
optical
rotation
HB-EGF Shedding
inhibitory activitya
MMPs Inhibitory
activity, Ki (nM)c
IC50 (mM)b
MMP-1
MMP-3
MMP-9
3a
3c
3d
3e
3f
3g
3h
3i
–OCH3
B, C
B
C
B
B
B
B
C
B
C
D
C
C
B
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.35
0.51
1.5
2.7
0.30
1.8
0.81
2.2
40
1.1
0.13
0.12
0.036
0.053
0.10
1.1
61
190
170
150
42
460
15
22
20
15
270
31
300
120
220
>650
4.8
>650
46
14
0.20
77
45
180
250
130
52
390
370
15
210
44
5.1
0.70
43
–H
–SCH3
–CH3
–OH
–NH2
–F
–COCH3
–CF3
–CH=CH2
3j
3k
3m
3n
3o
3p
3q
3r
3s
35
14
–CꢂCH
–OCH2CH2CH2CH2CH3
–OCH2CH2OCH2CH3
–OCH2CH2CH2OCH3
–OCH2CH2CH2SCH3
–OCH2CH2CH2CO2CH2CH3
–OCH2CH2CH2CO2H
290
>850
380
110
260
>850
9.2
0.90
4.8
5.4
0.60
29
C
B
B
95
500
200
aThe details of the assay are described in the Experimental.
bConcentration required for 50% inhibition of HB-EGF shedding. All values represent the mean of at least two determinations.
cKi Values were calculated from percent inhibition and Km value of each MMPs to the substrate. All values represent the mean of two determina-
tions. The details of the assays are described in the Experimental.
with thionyl chloride in methanol to give methyl ester
38a. The sulfonylation of 38a with 4-bromobenzene-
sulfonyl chloride to give sulfonylamino ester 39. The
cross coupling between 39 and trimethylsilylacetylene in
the presence of palladium catalysis gave the trimethyl-
silylethynyl derivative 40. The removal of trimethylsilyl
and methyl group of 40 was simultaneously accom-
plished by treatment with alkali to give ethynyl car-
boxylic acid 4m. Finally, 4m was treated with oxalyl
chloride, followed by addition of hydroxylamine to give
hydroxamic acid 3m.
Then, we investigated the HB-EGF shedding inhibitory
activities of optically active derivatives of 2 (3a and 3b).
Among them, the inhibitory activity of (+)-enantiomer
3a was more potent than that of CGS 27023A, while the
inhibitory activity of (ꢀ)-enantiomer 3b was con-
siderably weaker. This result means that the stereo-
chemistry of the alpha carbon of the hydroxamic acid
group is crucial to inhibit HB-EGF shedding. The cor-
responding carboxylic acid derivative of 3a (4a) did not
exhibit a strong inhibitory activity compared to 3a,
indicating that the hydroxamic acid group of 3a plays
an important role in the inhibition of HB-EGF shed-
ding. The conversion of the sulfonamide group of 3a
into carboxamide group (5a) caused a large decrease in
the inhibitory activity. The conversion of the 4-methoxy-
phenyl moiety of 3a into n-hexyl moiety (6) diminished
the inhibitory activity.
HB-EGF Shedding inhibitory activity and MMPs
inhibitory activities
All the hydroxamic acids synthesized here were evaluated
in HB-EGF shedding inhibition assay by use of HT1080
cell. In addition, many of them were tested in MMPs
inhibition assays. The results are shown in Tables 1–3.
The inhibitory activities of compounds 3a, 3b, 4a, 5a,
and 6 against MMP-1, -3, and 9 enabled us to establish
the same SARs as those observed in the HB-EGF
shedding inhibition. Thus, the existences of the hydro-
xamic acid, the sulfonamide, and the 4-methoxyphenyl
moieties were important for potent MMPs inhibitory
activities, and the stereochemistry of the alpha carbon
of the hydroxamic acid group was also crucial. The
attempt to get a singular crystal of 3a for X-ray struc-
tural analysis resulted in a failure, so we have not
determined the absolute configuration of compound 3a
yet. However, Novartis’ research group has reported that
CGS 27023A (R isomer) was much more active as an
MMP-3 inhibitor than the other enantiomer (S isomer).
Among four tetrahydronaphthylidine-base hydroxamic
acids 1a–d, compound 1a exhibited the most potent
HB-EGF shedding inhibitory activity (Table 1). Inter-
estingly, compound 1b, one of regioisomers of 1a,
showed 25 times weaker inhibitory activity than 1a.
Tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acid
2 exhibited a moderate HB-EGF shedding inhibitory
activity, which was not so potent as that of 1a. How-
ever, we selected compound 2 as our lead compound,
because the synthesis of 1a was accompanied by the
formation of regioisomer. In addition, compound 1a
was not stable in water compared to 2.

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
439
Therefore, it was considered that the stereochemistry of
the asymmetric carbon of 3a is R configuration.
Next, our attention was focused on the optimization of
the substituent on the benzene ring. We synthesized
derivatives of 3a bearing various kinds of substituents
on the benzene ring, and evaluated their HB-EGF
shedding inhibitory activity. The results are shown in
Table 3. Both of the deletion of the methoxy group of
3a (3c) and the deletion of the methyl group from the
methoxy group (3f) had no influence on the HB-EGF
shedding inhibitory activity. On the other hand, the
replacement of the oxygen atom of the methoxy group
with sulfur atom (3d) and the deletion of the oxygen
atom from the methoxy group (3e) weakened the
potency. Amino group is an electron-donating group as
well as methoxy group (the sp value of methoxy group:
ꢀ0.27, the sp value of amino group: ꢀ0.66), but the
inhibitory activity of the amino derivative 3g was not as
strong as that of 3a. Among three compounds having an
electron-withdrawing group, the fluoro derivative 3h
exhibited a moderate HB-EGF shedding inhibitory
activity, while the acetyl derivative 3i and the tri-
fluoromethyl derivative 3j did not show a strong inhibi-
tory activity. Interestingly, the replacement of the
methoxy group with ethynyl group (3m) improved the
inhibitory activity, whereas the replacement of that with
ethenyl group (3k) weakened the inhibitory activity. It
is obscure what property of ethynyl group contributed
to the excellent inhibitory activity of 3m. But, as one
possibility, the improved inhibitory activity can be attri-
buted to the straight orientation of the ethynyl group. The
elongation of the methoxy group led to the discovery of
the most potent HB-EGF shedding inhibitors, including
the ethoxyethoxy derivative 3o (IC50=36 nM,
SD=21nM, n=3) and the methoxypropoxy derivative 3p
(IC50=53 nM, SD=17nM, n=4), which were much
more stronger than CGS 27023A (IC50=1200 nM,
SD=37 nM, n=3). The ethyl ester derivative 3r was a
moderately potent inhibitor, but hydrolysis of 3r resulted
in a large reduction in the inhibitory activity (3s).
Figure 2. Structure of CGS 27023A and its related compounds.
activity because of the steric hindrance between the long
substituent and the S10 pocket. As a result of MMP-1
inhibition assay, all of the lengthy derivatives 3n–r
exhibited Ki values above 100 nM against MMP-1. This
result means that our compounds bind to MMP-1 in the
same binding mode as CGS 25966.
Novartis’ researchers have reported that the elongation
of the methoxy group of compound 40 (a compound
related to CGS 27023A, see Fig. 2) to butoxy group (41)
strengthened the MMP-3 inhibitory activity, but that
the replacement of the methoxy group of 40 with
ethoxyethoxy group (42) weakened the inhibitory activ-
ity.6 Among the lengthy derivatives 3n–r, the pentyloxy
derivative 3n exhibited a potent MMP-3 inhibitory
activity whose Ki values were below 1 nM, while the
ethoxyethoxy derivative 3o was less active than the
methoxy derivative 3a. These results were consistent
with Novartis’ ones. Interestingly, the methoxypropoxy
derivative 3p exhibited a strong MMP-3 inhibitory
activity comparable to that of 3a, and moreover, the
methylthiopropoxy derivative 3q and the ethoxy-
carbonylpropoxy derivative 3r showed more potent
MMP-3 inhibitory activity than 3a. The MMP-9 inhi-
bitory activities of 3n–r allowed us to establish the same
SAR as that from the MMP-3 inhibitory activities.
Thus, the ethoxyethoxy derivative 3o was a poor MMP-9
inhibitor compared to 3n and 3p–r.
Then, compounds 3c–s were subjected to the assays for
MMPs inhibitory activities. The results are shown in
Table 3. Novartis’ research group explored the binding
mode of CGS 27023A to MMP-3 by use of NMR
spectroscopy, and concluded that the 4-methoxyphenyl
group of CGS 27023A occupies the S10 pocket of
MMP-3.7 On the other hand, Agouron’s research group
performed an X-ray crystallographic analysis of the
complex of CGS 25966 (a benzyl analogue of CGS
27023A. See Fig. 2) and MMP-1, and found that CGS
25966 binds to MMP-1 in the same binding mode that
CGS 27023A does to MMP-3.9 Thus, when CGS 25966
binds to MMP-1, its 4-methoxyphenyl group lies in the
S10 pocket of MMP-1. Many research group reported
that the S10 pocket of MMP-1 is comparatively shallow,
while the S10 pockets of MMP-3 and MMP-9 are deep
and hydrophobic. If our 5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-based hydroxamic acids bind to MMP-1 in the
same binding mode as CGS 25966, the compounds
bearing a long substituent on the benzene ring, for
example 3n, should not show a strong MMP-1 inhibitory
Both of compounds 3o and 3p have an alkoxyalkoxy
group, but the MMP-3 and MMP-9 inhibitory activities
of 3o were less potent than those of 3p. Novartis’
researchers have reported that the low inhibitory activ-
ity of 42 against MMP-3 must be due to its low hydro-
phobicity compared to that of the butoxy derivative 41.6
However, the calculated logP (ClogP) value of 3o was a
little greater than that of 3p (3o: ꢀ0.288 versus 3p:
ꢀ0.434), meaning that the hydrophobicity of 3o is
higher than that of 3p. Therefore, the poor MMP-3 and
MMP-9 inhibitory activities of 3o are not due to the low
hydrophobicity of 3o. The only structural difference
between 3o and 3p is the position of oxygen atom in the
alkoxyalkoxy group, so it can be considered that the
difference in oxygen position is associated with the dif-
ference in the MMP-3 and MMP-9 inhibitory activities
between 3o and 3p.

440
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
As mentioned above, all of the lengthy derivatives 3n–r
showed a strong HB-EGF shedding activity. On the
other hand, all of 3n–r showed a week MMP-1 inhibi-
tory activity. The responsible enzyme for HB-EGF
shedding has not been identified yet, but these results
mean that the S10 pocket of the HB-EGF shedding
responsible enzyme is deep unlike that of MMP-1.
washed with water, dried over magnesium sulfate, and
concentrated in vacuo to give the title compound as a
brown solid (21.0 g, Y=72%). 1H NMR (CDCl3) d 3.94
(s, 3H), 4.00 (s, 3H), 7.50 (dd, J=4.8, 7.9 Hz, 1H), 8.18
(dd, J=1.6, 7.9 Hz, 1H), 8.77 (dd, J=1.6, 4.8 Hz, 1H).
2,3-Bis(hydroxymethyl)pyridine (17). Sodium borohydride
(15.0 g, 397 mmol) was added in portions to a solution
of dimethyl 2,3-pyridinedicarboxylate (16) (15.5 g,
79.4 mmol) in ethanol (200 mL) under ice-cooling, and
then the resulting mixture was refluxed for 17 h. After
ethanol (200 mL) had been added to the hot reaction
mixture, insoluble matter was removed by filtration
while the diluted mixture was still hot. The filtrate was
concentrated in vacuo. The obtained residue was pur-
ified by column chromatography (eluent: chloroform/
methanol/triethylamine=15:5:1) to give the title com-
pound as an orange oil (9.40 g, Y=85%). 1H NMR
(CDCl3) d 4.52 (s, 2H), 4.60 (s, 2H), 5.63 (br s, 2H), 7.08
(dd, J=4.9, 7.4 Hz, 1H), 7.62 (d, J=7.4 Hz, 1H), 8.10–
8.40 (m, 1H).
Through HB-EGF shedding inhibition and MMPs
inhibition assays, we discovered three kinds of HB-EGF
shedding inhibitors which are different in the selectivity.
First one is selective-type HB-EGF shedding inhibitor,
such as 3o, whose MMPs inhibitory activities were
comparatively weak compared to its potent HB-EGF
shedding inhibitory activity. Second one is broad-type
HB-EGF shedding inhibitor, such as 3m, which also
showed potent inhibitory activities against all of MMP-1,
MMP-3, and MMP-9. Final one is HB-EGF shedding
inhibitor with potent MMP-3 and MMP-9 inhibitory
activity, such as 3n. Recently, Tokumaru et al. have
reported that HB-EGF plays a crucial role in the pro-
liferation of keratinocytes,3 which means that HB-EGF
shedding inhibitors would become effective medicines for
skin diseases caused by the proliferation of keratino-
cytes, such as psoriasis. On the other hand, the effect of
each MMP on the proliferation of keratinocytes has not
been clarified sufficiently yet. Therefore, all of the above
three types of HB-EGF shedding inhibitors will be
characterized in both in vitro keratinocytes proliferation
inhibition test and in vivo psoriasis model.
2,3-Bis(chloromethyl)pyridine (18). Thionyl chloride
(50 mL) was added to a solution of 2,3-bis(hydroxy-
methyl)pyridine (17) (9.40 g, 67.6 mmol) in dichloro-
methane (20 mL) under ice-cooling, and then the
resulting mixture was stirred at 75 ꢃC for 1 h. After
having been cooled to room temperature, the reaction
mixture was concentrated in vacuo to give hydro-
chloride salt of the title compound as a brown pow-
der. The powder was dissolved in a saturated aqueous
solution of sodium hydrogen carbonate, and then the
solution was extracted with ethyl acetate. The organic
layer was washed with a saturated aqueous solution of
sodium chloride, dried over magnesium sulfate, and
concentrated in vacuo. Finally, the obtained residue
was purified by column chromatography (eluent: ethyl
acetate) to give the title compound as a dark brown
oil (3.80 g, Y=26%). 1H NMR (CDCl3) d 4.73 (s,
2H), 4.82 (s, 2H), 7.25 (dd, J=4.8, 7.8 Hz, 1H), 7.72
(dd, J=1.6, 7.8 Hz, 1H), 8.52 (dd, J=1.6, 4.8 Hz,
1H).
In conclusion, we succeeded in the discovery of potent
HB-EGF shedding inhibitors with 5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine skeleton, such as com-
pounds 3o and 3p. In addition, we clarified their SARs,
which would be very useful for design of more effective
HB-EGF shedding inhibitors.
Experimental
Melting points were determined on a Yamato MR-21
capillary melting point apparatus and are uncorrected.
1H NMR spectra of all the compounds synthesized here
were obtained on a Brucker DPX-250 spectrometer at
250 MHz (tetramethylsilane as an internal standard).
MALDI-TOF MS spectra were obtained on a PerSep-
tive Biosystems Voyager-DE RP spectrometer. Column
chromatography was performed using silica gel (YMC-
GEL SIL-60A) under medium pressure. No attempt
was made to maximize the yields.
Ethyl 6-acetyl-7-ethoxycarbonyl-5,6,7,8-tetrahydro[1,6]-
naphthylidine-7-carboxylate (19a) and ethyl 7-acetyl-6-
ethoxycarbonyl - 5,6,7,8 - tetrahydro[1,7]naphthylidine - 6-
carboxylate (19b). Diethyl acetamidomalonate (4.69 g,
21.6 mmol) and sodium hydride (60%, 860 mg,
21.5 mmol) were successively added at room tempera-
ture to a solution of 2,3-bis(chloromethyl)pyridine (18)
(3.80 g, 21.6 mmol) in dimethylformamide (DMF)
(20 mL), and then the resulting mixture was stirred at
room temperature for 30 min. Sodium hydride (60%,
860 mg, 21.5 mmol) was further added to the reaction
mixture, and then the resulting mixture was stirred at
room temperature for 14 h. The reaction mixture was
diluted with water (500 mL), and then the resulting
mixture was extracted with ethyl acetate. The organic
layer was washed with a saturated aqueous solution of
sodium chloride, dried over magnesium sulfate, and
concentrated in vacuo. The obtained oil was purified
with column chromatography (eluent: ethyl acetate/
methanol=20:1) to give a mixture of the title compounds
Dimethyl 2,3-pyridinedicarboxylate (16)
Concentrated sulfuric acid (10 mL) was added dropwise
in 5 min to a mixture of 2,3-pyridinedicarboxylic acid
(25.0 g, 150 mmol) and methanol (100 mL) under ice-
cooling, and then the resulting mixture was refluxed for
20 h. After cooling, the reaction mixture was con-
centrated in vacuo. A saturated solution of sodium
hydrogen carbonate was added to the obtained residue
carefully until pH showed 8, and then the whole was
extracted with ethyl acetate. The organic layer was

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
441
(19a/19b=5:1) as a brown oil (2.50 g, Y=36%). 1H
NMR (CDCl3) d 1.15–1.30 (m, 6H), 2.30 (s, 3H), 3.47
(s), 3.65 (s), 4.05–4.30 (m, 4H), 4.73 (s), 4.81 (s), 7.15–
7.25 (m, 1H), 7.40–7.55 (m, 1H), 8.40–8.55 (m, 1H).
chloride, dried over magnesium sulfate, and concentrated
in vacuo. The obtained oil was purified by column
chromatography (eluent: ethyl acetate) to give the title
compounds, methyl (ꢁ)-6-(4-methoxybenzenesulfonyl)-
5,6,7,8-tetrahydro[1,6]naphthylidine-7-carboxylate (21a)
as a colorless oil (1.00 g), and methyl (ꢁ)-7-(4-methoxy-
benzenesulfonyl)-5,6,7,8-tetrahydro[1,7]naphthylidine-6-
carboxylate (21b) as a colorless oil (200 mg).
(ꢁ) - 5,6,7,8 - Tetrahydro[1,6]naphthylidine -7 -carboxylic
acid dihydrochloride (12a) and (ꢁ)-5,6,7,8-tetrahydro
[1,7]naphthylidine-6-carboxylic acid dihydrochloride (12b).
Hydrochloride (6 N, 15 mL) was added to a mixture of
ethyl 6-acetyl-7-ethoxycarbonyl-5,6,7,8-tetrahydro[1,6]
naphthylidine-7-carboxylate (19a) and ethyl 7-acetyl-6-
ethoxycarbonyl-5,6,7,8-tetrahydro[1,7]naphthylidine-6-
carboxylate (19b) (1.80 g, 5.62 mmol), and then the
resulting mixture was refluxed for 3 h. After having been
cooled to room temperature, the reaction mixture was
concentrated in vacuo. Dioxane (150 mL) was added to
the obtained residue, and the resulting mixture was
concentrated in vacuo to give a mixture of the title
compounds (12a/12b=5:1) as a brown powder (1.40 g,
Physicochemical data of 21a. 1H NMR (CDCl3) d
3.30–3.50 (m, 2H), 3.51 (s, 3H), 3.87 (s, 3H), 4.57 (d,
J=15.8 Hz, 1H), 4.79 (d, J=15.8 Hz, 1H), 5.10–5.20 (m,
1H), 6.98 (d, J=8.8 Hz, 2H), 7.14 (dd, J=4.7, 7.8 Hz,
1H), 7.35–7.45 (m, 1H), 7.80 (d, J=8.8 Hz, 2H), 8.35–
8.50 (m, 1H). Anal. calcd for C17H18N2O5S: C, 56.34;
H, 5.01; N, 7.73; found: C, 56.11; H, 4.75; N, 7.90.
1
Y=quant). H NMR (DMSO-d6–D2O) d 3.10–3.65 (m,
Physicochemical data of 21a. 1H NMR (CDCl3) d
3.10–3.35 (m, 2H), 3.42 (s, 3H), 3.84 (s, 3H), 4.48 (d,
J=16.5 Hz, 1H), 4.83 (d, J=16.5 Hz, 1H), 5.10 (dd,
J=3.0, 5.6 Hz, 1H), 6.96 (d, J=8.9 Hz, 2H), 7.09 (dd,
J=4.7, 7.7 Hz, 1H), 7.41 (d, J=7.7 Hz, 1H), 7.79 (d,
J=8.9 Hz, 2H), 8.39 (d, J=4.7 Hz, 1H). Anal. calcd for
C17H18N2O5S: C, 56.34; H, 5.01; N, 7.73; found: C,
56.31; H, 5.09; N, 7.80.
2H), 4.30–4.60 (m, 3H), 7.42 (dd, J=4.9, 7.7 Hz), 7.65
(dd, J=5.1, 7.8 Hz), 7.82 (d, J=7.7 Hz), 8.08 (d,
J=7.8 Hz), 8.45 (d, J=4.9 Hz), 8.60 (d, J=5.1 Hz).
Methyl (ꢁ)-5,6,7,8-tetrahydro[1,6]naphthylidine-7-carb-
oxylate dihydrochloride (20a) and methyl (ꢁ)-5,6,7,8-
tetrahydro[1,7]naphthylidine-6-carboxylate dihydrochloride
(20b). Methanol (10 mL) and thionyl chloride (2.00 g,
16.8 mmol) were successively added at room tempera-
Method A: (ꢁ)-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,6]naphthylidine- 7-carboxylic acid hydro-
chloride (22a). An aqueous solution of sodium hydrox-
ide (0.5 N, 11.0 mL, 5.52 mmol) was added to a solution
of methyl (ꢁ)-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,6]naphthylidine-7-carboxylate (21a) (1.00g,
2.76 mmol) in dioxane (11 mL), and then the resulting
mixture was stirred at room temperature for 25 h. pH of
the reaction mixture was adjusted to 2 by addition of
concentrated hydrochloric acid. The resulting mixture
was concentrated in vacuo. Dioxane (30 mL) was added
to the obtained residue, and then the resulting mixture
was concentrated in vacuo to dryness. The obtained
residue was stirred with a mixture of chloroform and
methanol (10:1), and then insoluble matter was removed
by filtration. The filtrate was concentrated in vacuo to
give the title compound as an yellow solid (1.10 g,
ture to
a
mixture of (ꢁ)-5,6,7,8-tetrahydro[1,6]
naphthylidine-7-carboxylic acid dihydrochloride (12a)
and (ꢁ)-5,6,7,8-tetrahydro[1,7]naphthylidine-6-carboxylic
acid dihydrochloride (12b) (1.40 g, 5.58 mmol), and then
the resulting mixture was refluxed for 20 h. After having
been cooled to room temperature, the reaction mixture
was concentrated in vacuo. Dioxane (50 mL) was
added to the obtained residue, and then the resulting
mixture was concentrated in vacuo to give a mixture of
the title compounds (20a/20b=4:1) as a brown solid
1
(1.50 g, Y=quant). H NMR (DMSO-d6) d 3.20–3.70
(m, 2H), 3.82 (s), 3.84 (s), 4.35–4.80 (m, 3H), 7.45–7.55
(m), 7.65–7.85 (m), 7.91 (d, J=7.6 Hz), 8.20 (d,
J=7.7 Hz), 8.56 (d, J=4.3 Hz), 8.70 (d, J=4.9 Hz),
10.60 (br s, 3H).
1
Methyl (ꢁ)-6-(4-methoxybenzenesulfonyl)-5,6,7,8-tetra-
hydro[1,6]naphthylidine- 7-carboxylate (21a) and methyl
(ꢁ)-7-(4 - methoxybenzenesulfonyl) - 5,6,7,8 - tetrahydro
[1,7]naphthylidine-6-carboxylate (21b). 4-Dimethylamino-
pyridine (2.06 g, 16.9 mmol) and 4-methoxybenzene-
sulfonly chloride (1.68 g, 8.37 mmol) were successively
added at room temperature to a suspension of methyl
(ꢁ)-5,6,7,8-tetrahydro[1,6]naphthylidine-7-carboxylate
dihydrochloride (20a) and methyl (ꢁ)-5,6,7,8-tetra-
hydro[1,7]naphthylidine-6-carboxylate dihydrochloride
(20b) (1.50 g, 5.66 mmol) in DMF (15 mL), and then the
resulting mixture was stirred at room temperature for 5
days. After the reaction mixture had been poured into
water (200 mL), pH of the resulting mixture was adjus-
ted to 8 by addition of a saturated aqueous solution of
sodium hydrogen carbonate. The neutralized mixture
was extracted with ethyl acetate. The organic layer was
washed with a saturated aqueous solution of sodium
Y=quant). H NMR (DMSO-d6) d 3.05–3.50 (m, 2H),
3.81 (s, 3H), 4.46 (d, J=16.4 Hz, 1H), 4.71 (d,
J=16.4 Hz, 1H), 4.98 (dd, J=3.0, 6.3 Hz, 1H), 7.06 (d,
J=8.8 Hz, 2H), 7.30 (dd, J=4.8, 7.8 Hz, 1H), 7.65–7.75
(m, 1H), 7.77 (d, J=8.8 Hz, 2H), 8.35–8.45 (m, 1H).
.
Anal. calcd for C16H16N2O5S 0.5H2O: C, 53.77; H,
4.79; N, 7.84; found: C, 54.02; H, 5.08; N, 8.15.
(ꢁ)-N-Benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,6]naphthylidine-7-carboxamide (23a). EDCI
(997 mg, 5.20 mmol) and HOBt (796 mg, 5.20 mmol)
were added to
a
solution of (ꢁ)-6-(4-methoxy-
benzenesulfonyl)-5,6,7,8-tetrahydro[1,6]naphthylidine-7-
carboxylic acid hydrochloride (22a) (1.00 g, 2.60 mmol)
in DMF (10 mL), and then the resulting mixture was
stirred at room temperature for 15 min. A mixture of
O-benzylhydroxylamine
5.20 mmol), triethylamine (789 mg, 7.80 mmol), and
hydrochloride
(830 mg,

442
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
DMF (10 mL) was added to the reaction mixture, and
then the resulting mixture was stirred at room tempera-
ture for 64 h. The reaction mixture was poured into
water (500 mL), and then the resulting mixture was
extracted with ethyl acetate. The organic layer was
washed with water, dried over magnesium sulfate, and
concentrated in vacuo. The obtained residue was pur-
ified by column chromatography (eluent: chloroform/
methanol=10:1) to give an yellow powder. The powder
was stirred with ethyl acetate (20 mL) for 10 min, and
insoluble solid was collected by filtration to give the title
2H), 7.04 (dd, J=4.8, 7.7 Hz, 1H), 7.20–7.40 (m, 6H),
7.67 (d, J=9.0 Hz, 2H), 8.30–8.40 (m, 1H), 9.59 (s, 1H).
Anal. calcd for C23H23N3O5S: C, 60.91; H, 5.11; N,
9.27; found: C, 60.90; H, 4.89; N, 9.20.
(ꢁ)-N-Hydroxy-7-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,7]naphthylidine-6-carboxamide (1b). A col-
1
orless powder. H NMR (DMSO-d6) d 2.90–3.00 (m,
2H), 3.81 (s, 3H), 4.45–4.70 (m, 3H), 7.04 (d, J=8.9 Hz,
2H), 7.15 (dd, J=5.0, 7.6 Hz, 1H), 7.45–7.55 (m, 1H),
7.72 (d, J=8.9 Hz, 2H), 8.25–8.40 (m, 1H), 8.82 (s, 1H),
1
.
compound as an yellow powder (590 mg, Y=50%). H
10.78 (s, 1H). Anal. calcd for C16H17N3O5S 0.5H2O: C,
NMR (DMSO-d6) d 2.85–3.10 (m, 2H), 3.79 (s, 3H),
4.45–4.75 (m, 5H), 7.04 (d, J=8.7 Hz, 2H), 7.10–7.25
(m, 1H), 7.25–7.45 (m, 5H), 7.56 (d, J=7.9 Hz, 1H),
7.74 (d, J=8.7 Hz, 2H), 8.25–8.35 (m, 1H), 11.43 (s,
1H). Anal. calcd for C23H23N3O5S: C, 60.91; H, 5.11;
N, 9.27; found: C, 60.78; H, 5.02; N, 9.39.
51.60; H, 4.87; N, 11.28; found: C, 51.63; H, 4.84; N,
11.44. MALDI-TOF MS (Mr=363.39): 364 [M+H]+,
386 [M+Na]+, 402 [M+K]+.
Compounds 1c and 1d were synthesized from 3,4-pyr-
idinedicarboxylic acid in the same manner as 1a and 1b.
The physicochemical data of compounds 1c and 1d are
shown below.
(ꢁ)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,6]naphthylidine-7-carboxamide (1a). (ꢁ)-N-
Benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-tetra-
hydro[1,6]naphthylidine-7-carboxamide (23a) (560 mg,
1.23 mmol) was dissolved in a mixture of methanol
(45 mL) and dioxane (90 mL) with heating. A mixture of
Pd/C (10%, 1.31 g) and dioxane (30 mL) was added to
the solution, and then the resulting mixture was stirred
at room temperature under hydrogen atmosphere for
17 h. The reaction mixture was diluted with methanol
(500 mL), and then Pd/C was removed by filtration. The
obtained filtrate was concentrated in vacuo, and then
the obtained residue was purified by HPLC (eluent:
water/acetonitrile=2:1) to give the title compound as a
colorless powder (210 mg, Y=47%). 1H NMR (DMSO-
d6) d 2.90–3.05 (m, 2H), 3.81 (s, 3H), 4.50–4.75 (m, 3H),
7.02 (d, J=8.9 Hz, 2H), 7.15 (dd, J=4.8, 7.8 Hz, 1H),
7.50–7.60 (m, 1H), 7.72 (d, J=8.9 Hz, 2H), 8.20–8.35
(m, 1H), 8.84 (s, 1H), 10.79 (s, 1H). Anal. calcd for
(ꢁ)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[2,6]naphthylidine-7-carboxamide (1c). A col-
orless powder. H NMR (DMSO-d6) d 2.75–3.10 (m,
1
2H), 3.80 (s, 3H), 4.40–4.75 (m, 3H), 7.02 (d, J=8.9 Hz,
2H), 7.09 (d, J=5.0 Hz, 1H), 7.73 (d, J=8.9 Hz, 2H),
8.25 (d, J=5.0 Hz, 1H), 8.34 (s, 1H), 8.85 (s, 1H), 10.76
.
(s, 1H). Anal. calcd for C16H17N3O5S 0.5H2O: C, 51.60;
H, 4.87; N, 11.28; found: C, 51.82; H, 4.99; N, 11.28.
(ꢁ)-N-Hydroxy-7-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[2,7]naphthylidine-6-carboxamide (1d). A col-
1
orless powder. H NMR (DMSO-d6) d 2.80–3.30 (m,
2H), 3.82 (s, 3H), 4.50–4.90 (m, 3H), 7.05 (d, J=8.9 Hz,
2H), 7.42 (d, J=5.0 Hz, 1H), 7.74 (d, J=8.9 Hz, 2H),
8.20–8.60 (m, 2H), 8.83 (br s, 1H), 10.83 (s, 1H). Anal.
.
calcd for C16H17N3O5S 0.5H2O: C, 51.60; H, 4.87; N,
11.28; found: C, 51.62; H, 4.86; N, 11.53.
.
C16H17N3O5S 0.5H2O: C, 51.60; H, 4.87; N, 11.28;
found: C, 51.87; H, 4.91; N, 11.24. MALDI-TOF MS
(Mr=363.39): 364 [M+H]+, 386 [M+Na]+, 402
[M+K]+.
2,3-Bis(chloromethyl)pyrazine (25). N-Chlorosuccimide
(205 g, 1.54 mol) and benzoyl peroxide (3.00 g) were
added to a solution of 2,3-dimethylpyrazine (75.0 g,
0.720 mol) in carbon tetrachloride (750 mL), and then
the resulting mixture was refluxed for 21h. After removal
of precipitating solid by filtration, the filtrate was con-
centrated in vacuo. The obtained oil was purified by col-
umn chromatography (eluent: n-hexane/acetone=10:1) to
give the title compound as a pale brown oil (55.7 g,
Y=44%). 1H NMR (CDCl3) d 4.86 (s, 4H), 8.54 (s, 2H).
Compound 1b was synthesized from compound 21b via
compounds 22b and 23b in the same manner as com-
pound 1a. The physicochemical data of compounds 22b,
23b, and 1b are shown below.
(ꢁ)-7-(4-Methoxybenzenesulfonyl)-5,6,7,8-tetrahydro[1,7]
naphthylidine-6-carboxylic acid hydrochloride (22b). A
colorless powder. 1H NMR (DMSO-d6) d 3.00–3.30
(m, 2H), 3.82 (s, 3H), 4.54 (d, J=17.1 Hz, 1H), 4.78 (d,
J=17.1 Hz, 1H), 4.94 (dd, J=2.5, 6.3 Hz, 1H), 7.07
(d, J=8.9 Hz, 2H), 7.44 (dd, J=5.1, 7.6 Hz, 1H), 7.77
(d, J=8.9 Hz, 2H), 7.89 (d, J=7.6 Hz, 1H), 8.45–8.55
Ethyl 6-acetyl-7-ethoxycarbonyl-5,6,7,8-tetrahydropyrido
[3,4-b]pyrazine-7-carboxylate (26). Cesium carbonate
(514.0 g, 1.58 mol) and diethyl acetamidomalonate
(171.4 g, 0.789 mol) were successively added in potions
at 70 ꢃC to a solution of 2,3-bis(chloromethyl)pyrazine
(25) (140.0 g, 0.791 mol) in acetonitrile (2500 mL) with
vigorous stirring. After completion of the addition, the
mixture was stirred at reflux temperature for 4 h. Cesium
carbonate (80.0 g, 0.246 mol) was further added to the
reaction mixture, and then the resulting mixture was
refluxed for 1 h with vigorous stirring. After the reaction
mixture had been cooled to room temperature, insoluble
matter was removed by filtration. The insoluble matter
.
(m, 1H). Anal. calcd for C16H16N2O5S 0.5H2O: C, 53.77;
H, 4.79; N, 7.84; found: C, 53.99; H, 5.04; N, 7.54.
(ꢁ)-N-Benzyloxy-7-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydro[1,7]naphthylidine-6-carboxamide (23b).
A
1
colorless powder. H NMR (CDCl3) d 2.64 (dd, J=6.5,
15.9 Hz, 1H), 3.10–3.35 (m, 1H), 3.79 (s, 3H), 4.42 (d,
J=16.7 Hz, 1H), 4.55–4.85 (m, 4H), 6.85 (d, J=9.0 Hz,

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
443
was washed with ethyl acetate (2600 mL ꢄ 2). The
obtained filtrate and wash were combined, and con-
centrated in vacuo. The obtained oil was dissolved in
ethyl acetate (200 mL), and the resulting solution was
allowed to stand at room temperature overnight. After
removal of precipitating solid by filtration, the filtrate
was concentrated in vacuo. The obtained oil was pur-
ified by column chromatography (eluent: first, n-hex-
ane/ethyl acetate=1:1, then 1:5) to give the title
(ꢁ)-6-(4-Methoxybenzenesulfonyl)-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxylic acid (29). A colorless
powder. H NMR (CDCl3) d 3.41 (d, J=4.3 Hz, 2H),
3.87 (s, 3H), 4.57 (d, J=17.0 Hz, 1H), 4.88 (d,
J=17.0 Hz, 1H), 5.22 (t, J=4.3 Hz, 1H), 6.96 (d,
J=8.9 Hz, 2H), 7.81 (d, J=8.9 Hz, 2H), 8.38 (s, 2H).
Anal. calcd for C15H15N3O5S: C, 51.57; H, 4.33; N,
12.03; found: C, 51.90; H, 4.33; N, 11.91.
1
1
compound as a red oil (152.3 g, Y=60%). H NMR
(ꢁ)-N-Benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (30). A
(CDCl3) d 1.21 (t, J=7.1 Hz, 6H), 2.31 (s, 3H), 3.70 (s,
2H), 4.10–4.40 (m, 4H), 4.85 (s, 2H), 8.44 (d,
J=2.6 Hz, 1H), 8.46 (d, J=2.6 Hz, 1H). Anal. calcd for
C16H19N3O5: C, 56.07; H, 5.96; N, 13.08; found: C,
56.10; H, 6.12; N, 13.01.
1
colorless powder. H NMR (CDCl3) d 2.86 (dd, J=6.1,
17.2 Hz, 1H), 3.44 (d, J=17.2 Hz, 1H), 3.83 (s, 3H), 4.40
(d, J=18.0 Hz, 1H), 4.70–5.00 (m, 4H), 6.87 (d, J=8.8 Hz,
2H), 7.37 (s, 5H), 7.66 (d, J=8.8 Hz, 2H), 8.33 (s, 2H), 9.17
(s, 1H). Anal. calcd for C22H22N4O5S: C, 58.14; H, 4.88;
N, 12.33; found: C, 58.01; H, 4.89; N, 12.10.
Methyl (ꢁ)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-
carboxylate hydrochloride (27). A mixture of 6 N
hydrochloric acid (40 mL) and ethyl 6-acetyl-7-ethoxy-
carbonyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-car-
boxylate (26) (3.60 g, 11.2 mmol) was refluxed for 1 h.
After the reaction mixture had been concentrated in
vacuo to dryness, methanol (30 mL) was added to the
obtained residue. To the resulting mixture was added
dropwise thionyl chloride (3.0 mL) under ice-cooling,
and then the resulting mixture was refluxed for 5 h. The
reaction mixture was concentrated in vacuo to give the
title compound as a colorless solid (2.40 g, Y=93%).
1H NMR (DMSO-d6) d 3.30–3.50 (m, 2H), 3.85 (s,
3H), 4.30–4.50 (m, 2H), 4.60–4.70 (m, 1H), 8.50–8.70
(ꢁ)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (2).
A
colorless powder. 1H NMR (DMSO-d6) d 2.95 (dd,
J=2.0, 17.4 Hz, 1H), 3.16 (d, J=7.0, 17.4Hz, 1H), 3.82
(s, 3H), 4.59 (d, J=16.8 Hz, 1H), 4.67 (d, J=16.8 Hz,
1H), 4.78 (dd, J=2.0, 7.0 Hz, 1H), 7.05 (d, J=8.9Hz,
2H), 7.75 (d, J=8.9 Hz, 2H), 8.30–8.50 (m, 2H), 8.88
(s, 1H), 10.91 (s, 1H). Anal. calcd for C15H16
.
N4O5S 0.5H2O: C, 48.25; H, 4.59; N, 15.00; found: C,
48.11; H, 4.57; N, 14.71.
(ꢁ)-6-tert-Butoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxylic acid (31). A mixture of ethyl
6-acetyl-7-ethoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxylate (26) (65.0 g, 202 mmol) and 6 N
hydrochloric acid (260 mL) was refluxed for 3 h. After
having been cooled to room temperature, the reaction
mixture was concentrated in vacuo. An aqueous solu-
tion of sodium hydroxide (2 N) was added under ice-
cooling to the obtained residue until pH of the residue
showed above 10. A solution of di-tert-butyl dicarbo-
nate (53.0 g, 243 mmol) in dioxane (130 mL) was added
dropwise in 20 min to the alkaline mixture, and then
the resulting mixture was stirred at room temperature
for 3 days. The reaction mixture was diluted with water
(260 mL), and then the diluted mixture was washed
with diethyl ether twice. The resulting acidic aqueous
layer was acidified with 10% aqueous solution of citric
acid to pH 3, and then the resulting mixture was
extracted with ethyl acetate three times. The organic
layers were combined, washed with a saturated aqu-
eous solution of sodium chloride, and dried over mag-
nesium sulfate. Activated charcoal (3.0 g) was added to
the solution, and then the resulting mixture was stirred
for 5 min. After removal of activated charcoal by fil-
tration, the filtrate was concentrated in vacuo. Diiso-
propyl ether (100 mL) was added to the obtained
residue, and the resulting crystals were collected by fil-
tration to give the title compound as a brown powder
(24.6 g, Y=44%). 1H NMR (CDCl3) d 1.52 (s, 9H),
3.34 (dd, J=6.7, 17.2 Hz, 1H), 3.53 (d, J=17.2 Hz,
1H), 4.50–4.70 (m, 1H), 4.90–5.10 (m, 1H), 5.10–5.50
(m, 1H), 8.40–8.50 (m, 2H). Anal. calcd for
C13H17N3O4: C, 55.91; H, 6.14; N, 15.05; found: C,
56.21; H, 6.11; N, 14.78.
.
(m, 2H). Anal. calcd for C9H12ClN3O2 1.0H2O: C,
43.64; H, 5.70; N, 16.97; found: C, 43.59; H, 5.55; N,
16.69.
Methyl (ꢁ)-6-(4-methoxybenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxylate ( 28). 4-Di-
methylaminopyridine
(0.70 g,
5.73 mmol)
and
4-methoxybenzenesulfonyl chloride (0.60 g, 2.90 mmol)
were successively added to a solution of methyl (ꢁ)-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxylate
(27) (0.64 g, 2.79 mmol) in DMF (10 mL), and then the
resulting mixture was stirred at room temperature
overnight. The reaction mixture was acidified with an
aqueous solution citric acid to pH 3, and then extracted
with ethyl acetate. The organic layer was washed with
water and a saturated aqueous solution of sodium
chloride, and dried over magnesium sulfate. After
removal of magnesium sulfate by filtration, the filtrate
was concentrated in vacuo, and the obtained residue
was purified by TLC (developing solvent: n-hexane/
ethyl acetate=1:1) to give the title compound as an
yellow oil (220 mg, Y=22%). 1H NMR (CDCl3) d
3.30–3.50 (m, 2H), 3.47 (s, 3H), 3.87 (s, 3H), 4.54 (d,
J=16.9 Hz, 1H), 4.90 (d, J=16.9 Hz, 1H), 5.19 (dd,
J=2.8, 5.9 Hz, 1H), 6.97 (d, J=8.9 Hz, 2H), 7.79 (d,
J=8.9 Hz, 2H), 8.39 (s, 2H). Anal. calcd for
C16H17N3O5S: C, 52.88; H, 4.72; N, 11.56; found: C,
52.64; H, 4.77; N, 11.39.
Compound 2 was synthesized from compound 28 via
compounds 29 and 30 in the same manner as compound
1a. The physicochemical data of compounds 29, 30, and
2 are shown below.

444
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
1
(ꢀ)-6-tert-Butoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxylic acid (32a). (ꢁ)-6-tert-Butoxy-
carbonyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-car-
boxylic acid (31) (35.0 g, 125 mmol) was dissolved in
ethyl acetate (900 mL) with heating, and then S-(ꢀ)-1-
phenylethylamine (16.0 mL, 125 mmol) was added to the
solution. The mixture was allowed to stand at room
temperature overnight. The precipitating solid was col-
lected by filtration, and it was dissolved in ethyl acetate
(1000 mL) with heating. The resulting solution was
allowed to stand at room temperature overnight. The
precipitating solid was collected by filtration, and then
the obtained solid was stirred with a 10% aqueous
solution of citric acid (300 mL). The whole was extrac-
ted with ethyl acetate. The organic layers were com-
bined, washed with water and a saturated saline
solution, and dried over magnesium sulfate. Concen-
tration of obtained solution in vacuo gave the title com-
Y=95%). H NMR (DMSO-d6) d 3.19 (dd, J=11.4,
17.7 Hz, 1H), 3.36 (dd, J=5.2, 17.7 Hz, 1H), 4.20–4.40
(m, 2H), 4.50 (d, J=16.6 Hz, 1H), 4.86 (d, J=11.1 Hz,
1H), 4.91 (d, J=11.1 Hz, 1H), 7.30–7.50 (m, 5H), 8.50–
8.60 (m, 2H), 9.90–10.70 (br, 1H), 12.20 (br s, 1H).
.
Anal. calcd for C15H17ClN4O2 1.0H2O: C, 53.18; H,
5.65; N, 16.54; found: C, 53.10; H, 5.57; N, 16.46.
[a]D=+88 (c 1.0, methanol).
(+)-N-Benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(35a).
(+)-N-Benzyloxy-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxamide hydrochloride (34a) (651 mg, 2.03 mmol)
was dissolved in a mixture of water (15 mL) and dioxane
(15 mL). Triethylamine (515 mg, 5.09 mmol) and
4-methoxybenzenesulfonyl chloride (463 mg, 2.24 mmol)
were successively added to the solution under ice-cool-
ing, and then the resulting mixture was stirred at room
temperature for 3 h. After concentration of the reaction
mixture in vacuo, the obtained residue was acidified
with 1 N hydrochloric acid to pH 3, and then the whole
was extracted with ethyl acetate. The organic layers
were combined, washed with 1 N hydrochloric acid,
water, and a saturated aqueous solution of sodium
chloride, and dried over magnesium sulfate. After con-
centration of the solution in vacuo, the obtained residue
was purified by column chromatography (eluent:
chloroform/methanol=30:1) to give the title com-
pound as a pale yellow powder (592 mg, Y=64%).
1H NMR (CDCl3) d 2.86 (dd, J=6.1, 17.2 Hz, 1H),
3.44 (d, J=17.2 Hz, 1H), 3.83 (s, 3H), 4.40 (d,
J=18.0 Hz, 1H), 4.70–5.00 (m, 4H), 6.87 (d, J=8.8 Hz,
2H), 7.37 (s, 5H), 7.66 (d, J=8.8 Hz, 2H), 8.33 (s, 2H),
9.17 (s, 1H). Anal. calcd for C22H22N4O5S: C, 58.14; H,
4.88; N, 12.33; found: C, 58.34; H, 4.85; N, 12.28.
[a]D=+41 (c 1.0, methanol).
1
pound as a colorless solid (9.00 g, Y=26%). H NMR
(DMSO-d6) d 1.52 (s, 9H), 3.34 (dd, J=6.7, 17.2Hz, 1H),
3.53 (d, J=17.2 Hz, 1H), 4.50–4.70 (m, 1H), 4.90–5.10 (m,
1H), 5.10–5.50 (m, 1H), 8.40–8.50 (m, 2H). Anal. calcd
for C13H17N3O4: C, 55.91; H, 6.14; N, 15.05; found: C,
55.96; H, 6.12; N, 14.98. [a]D=ꢀ38 (c 1.0, methanol).
Method B: (+) - N - benzyloxy - 6 - tert - butoxycarbonyl-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(33a). EDCI (1.16 g, 6.05 mmol) and HOBt (927 mg,
6.05 mmol) were successively added to a solution of (ꢀ)-
6-tert - butoxycarbonyl - 5,6,7,8 - tetrahydropyrido[3,4-b]
pyrazine-7-carboxylic acid (32a) (1.30 g, 4.65 mmol) in
DMF (10 mL) under ice-cooling, and then the resulting
mixture was stirred for 1 h under ice-cooling. A mixture
of O-benzylhydroxylamine hydrochloride (966 mg,
6.05 mmol), triethylamine (612 mg, 6.05 mmol), and
DMF (15 mL) were added to the reaction mixture, and
then the resulting mixture was stirred at room tempera-
ture for 48 h. After the reaction mixture had been
poured into water (500 mL), the whole was extracted
with ethyl acetate. The organic layers were combined,
washed with a saturated aqueous solution of sodium
hydrogen carbonate, a 10% aqueous solution of citric
acid, and water, and dried over magnesium sulfate.
After concentration of the solution in vacuo, the
obtained residue was purified by column chromato-
graphy (eluent: first, n-hexane/ethyl acetate=1:1, then
only ethyl acetate) to give the title compound as a col-
orless oil (1.80 g, Y=quant). 1H NMR (CDCl3) d 1.47 (s,
9H), 3.20 (dd, J=6.3, 17.5 Hz, 1H), 3.42 (d, J=17.5 Hz,
1H), 4.36 (d, J=17.8 Hz, 1H), 4.70–5.10 (m, 4H), 7.34 (s,
5H), 8.30–8.50 (m, 2H), 8.92 (br s, 1H). Anal. calcd for
C20H24N4O4: C, 62.49; H, 6.29; N, 14.57; found: C, 62.51;
H, 6.21; N, 14.36. [a]D=+27 (c=1.0, methanol).
(+)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4 - b]pyrazine - 7 - carboxamide (3a).
(+)-N-Benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4 - b]pyrazine - 7 - carboxamide (35a)
(580 mg, 1.28 mmol) was dissolved in a mixture of
methanol (8 mL) and dioxane (8 mL), and then the
solution was stirred with Pd/C (10%, 290 mg) under
hydrogen atmosphere at room temperature overnight.
After removal of Pd/C by filtration, the obtained filtrate
was concentrated in vacuo. The obtained residue was
purified by column chromatography (eluent: chloro-
form/methanol=10:1) to give the title compound as an
1
yellow powder (342 mg, Y=74%). H NMR (DMSO-
d6) d 2.95 (dd, J=2.0, 17.4 Hz, 1H), 3.16 (dd, J=7.0,
17.4 Hz, 1H), 3.82 (s, 3H), 4.59 (d, J=16.8 Hz, 1H), 4.67
(d, J=16.8 Hz, 1H), 4.78 (dd, J=2.0, 7.0 Hz, 1H), 7.05
(d, J=8.9 Hz, 2H), 7.75 (d, J=8.9 Hz, 2H), 8.30–8.50
(m, 2H), 8.88 (s, 1H), 10.91 (s, 1H). Anal. calcd for
(+)-N-Benzyloxy-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxamide hydrochloride (34a).
.
A
solution of
C15H16N4O5S 0.5H2O: C, 48.25; H, 4.59; N, 15.00;
hydrochloric acid in ethyl acetate (4 N, 15 mL) was
added to (+)-N-Benzyloxy-6-tert-butoxycarbonyl-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(33a) (1.80 g, 4.68 mmol) under ice-cooling, and then the
resulting mixture was stirred for 1.5 h under ice-cooling.
The precipitating solid was collected by filtration to give
the title compound as a pale brown powder (1.41 g,
found: C, 48.21; H, 4.62; N, 14.70. MALDI-TOF MS
(Mr=364.38): 365 [M+H]+, 387 [M+Na]+, 403
[M+K]+. [a]D=+30 (c 1.0, methanol).
Compounds 3c, 3e, 3h, 3j, 3p, 3r, and 6 were synthesized
from compound 34a and the corresponding sulfonyl
chloride via the corresponding sulfonylamino benzyloxy-

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
445
amides 35 by use of reaction conditions similar to those
used for the preparation of 3a. The physicochemical
data of 3c, 3e, 3h, 3j, 3p, 3r, and 6 were shown below.
(+)-N-Hydroxy-6-[4-(3-ethoxycarbonylpropoxy)benzene-
sulfonyl]-5,6,7,8-tetrahydropyrido[3,4- b]pyrazine-7-car-
boxamide (3r). A colorless powder. H NMR (DMSO-
1
d6) d 1.17 (t, J=7.1 Hz, 3H), 1.90–2.10 (m, 2H), 2.45 (t,
J=7.2 Hz, 2H), 2.90–3.00 (m, 1H), 3.16 (dd, J=7.0,
17.3 Hz, 1H), 4.05 (t, J=6.3 Hz, 2H), 4.06 (q,
J=7.1 Hz, 2H), 4.58 (d, J=16.6 Hz, 1H), 4.67 (d,
J=16.6 Hz, 1H), 4.70–4.80 (m, 1H), 7.04 (d, J=8.9 Hz,
2H), 7.74 (d, J=8.9 Hz, 2H), 8.30–8.50 (m, 2H), 8.92
(br s, 1H), 10.95 (br s, 1H). Anal. calcd for
(+)-N-Hydroxy-6-benzenesulfonyl-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxamide (3c). A colorless
powder. 1H NMR (CDCl3) d 2.80–3.00 (m, 1H), 3.41 (d,
J=16.4 Hz, 1H), 4.62 (d, J=17.7 Hz, 1H), 4.82 (d,
J=17.7 Hz, 1H), 4.90–5.10 (m, 1H), 7.30–7.60 (m, 3H),
7.78 (d, J=7.6 Hz, 2H), 8.20–8.40 (m, 2H), 9.91 (br,
.
.
1H). Anal. calcd for C14H14N4O4S 0.25H2O: C, 49.62;
C20H24N4O7S 0.25H2O: C, 51.22; H, 5.27; N, 11.95;
H, 4.31; N, 16.53; found: C, 49.52; H, 4.40; N, 16.32.
MALDI-TOF MS (Mr=334.35): 335 [M+H]+, 357
[M+Na]+, 373 [M+K]+.
found: C, 51.26; H, 5.52; N, 11.75. MALDI-TOF MS
(Mr=464.49): 465 [M+H]+, 487 [M+Na]+, 503
[M+K]+.
(+)-N-Hydroxy-6-(4-methylbenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3e). A color-
less powder. H NMR (DMSO-d6) d 2.34 (s, 3H), 2.93
(+)-N-Hydroxy-6-hexylsulfonyl-5,6,7,8-tetrahydropyrido
[3,4-b]pyrazine-7-carboxamide (6). A colorless powder.
1H NMR (DMSO-d6) d 0.83 (t, J=6.6 Hz, 3H), 1.15–
1.45 (m, 6H), 1.55–1.70 (m, 2H), 3.00–3.50 (m, 4H),
4.55–4.80 (m, 3H), 8.46 (s, 2H), 8.98 (s, 1H), 10.91 (s,
1
(dd, J=1.8, 17.3 Hz, 1H), 3.14 (dd, J=6.8, 17.3 Hz,
1H), 4.58 (d, J=16.8 Hz, 1H), 4.67 (d, J=16.8 Hz, 1H),
4.78 (dd, J=1.8, 6.8 Hz, 1H), 7.34 (d, J=8.2 Hz, 2H),
7.69 (d, J=8.2 Hz, 2H), 8.30–8.50 (m, 2H), 8.91 (br s,
1H), 10.94 (br s, 1H). Anal. calcd for C15H16N4
.
1H). Anal. calcd for C14H22N4O4S 0.25H2O: C, 48.47;
H, 6.54; N, 16.15; found: C, 48.52; H, 6.64; N, 16.06.
MALDI-TOF MS (Mr=342.42): 343 [M+H]+, 365
[M+Na]+, 381 [M+K]+.
.
O4S 0.25H2O: C, 51.06; H, 4.71; N, 15.88; found: C, 51.11;
H, 4.96; N, 15.62. MALDI-TOF MS (Mr=348.38): 349
[M+H]+, 371 [M+Na]+, 387 [M+K]+.
(+)-N-Benzyloxy-6-(4-nitrobenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (35g). The
title compound (a yellow powder) was synthesized from
compound 34a and 4-nitrobenzenesulfonyl chloride by
use of reaction condition similar to that used for the
(+)-N-Hydroxy-6-(4-fluorobenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3h). A color-
1
less powder. H NMR (CDCl3) d 2.80–3.10 (m, 1H),
1
3.37 (d, J=17.8 Hz, 1H), 4.62 (d, J=17.3 Hz, 1H), 4.77
(d, J=17.3 Hz, 1H), 4.90–5.10 (m, 1H), 7.00–7.20 (m,
2H), 7.70–7.90 (m, 2H), 8.10–8.40 (m, 2H), 10.11 (br s,
preparation of 35a. H NMR (CDCl3) d 2.90–3.10 (m,
1H), 3.30–3.50 (m, 1H), 4.40–4.70 (m, 1H), 4.80–5.10
(m, 4H), 7.20–7.50 (m, 5H), 7.94 (d, J=8.9 Hz, 2H),
8.29 (d, J=8.9 Hz, 2H), 8.37 (s, 2H), 8.91 (br s, 1H).
Anal. calcd for C21H19N5O6S: C, 53.73; H, 4.08; N,
14.92; found: C, 53.58; H, 4.06; N, 15.11.
.
1H). Anal. calcd for C14H13FN4O4S 0.25H2O: C, 47.12;
H, 3.81; N, 15.70; found: C, 47.03; H, 3.95; N, 15.48.
MALDI-TOF MS (Mr=352.34): 353 [M+H]+, 375
[M+Na]+, 391 [M+K]+
(+)-N-Hydroxy-6-(4-aminobenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3g). The title
compound (a colorless powder) was synthesized from
compound 35g by use of reaction condition similar to
(+)-N-Hydroxy-6-(4-trifluoromethylbenzenesulfonyl)-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(3j). A colorless powder. H NMR (DMSO-d6) d 2.99
1
1
(dd, J=1.8, 17.4 Hz, 1H), 3.15–3.35 (m, 1H), 4.62 (d,
J=16.5 Hz, 1H), 4.74 (d, J=16.5 Hz, 1H), 4.83 (dd,
J=1.8, 6.9 Hz, 1H), 7.93 (d, J=8.4 Hz, 2H), 8.05 (d,
J=8.4 Hz, 2H), 8.35–8.50 (m, 2H), 8.90 (s, 1H), 10.94
that used for the preparation of 3a. H NMR (DMSO-
d6) d 2.80–3.20 (m, 2H), 4.40–4.70 (m, 2H), 4.70 (dd,
J=1.7, 6.7 Hz, 1H), 6.07 (br s, 2H), 6.40–6.70 (m, 2H),
7.30–7.50 (m, 2H), 8.30–8.50 (m, 2H), 8.91 (br s, 1H),
.
.
(s, 1H). Anal. calcd for C15H13F3N4O4S 0.5H2O: C,
10.93 (br s, 1H). Anal. calcd for C14H15N5O4S 0.75H2O:
43.80; H, 3.43; N, 13.62; found: C, 43.91; H, 3.49; N,
13.62. MALDI-TOF MS (Mr=402.35): 403 [M+H]+,
425 [M+Na]+, 441 [M+K]+
C, 46.34; H, 4.58; N, 19.30; found: C, 46.38; H, 4.82; N,
19.12. MALDI-TOF MS (Mr=349.37): 350 [M+H]+,
372 [M+Na]+, 388 [M+K]+.
(+)-N-Hydroxy-6-[4-(3-methoxypropoxy)benzenesulfonyl]-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(3p). A pale brown colorless powder. 1H NMR
(DMSO-d6) d 1.94 (tt, J=6.3, 6.4 Hz, 2H), 2.80–3.00
(m, 1H), 3.00–3.20 (m, 1H), 3.23 (s, 3H), 3.45 (t,
J=6.3 Hz, 2H), 4.07 (t, J=6.4 Hz, 2H), 4.50–4.70 (m,
2H), 4.70–4.80 (m, 1H), 7.04 (d, J=8.9 Hz, 2H), 7.73 (d,
J=8.9 Hz, 2H), 8.39 (d, J=2.6 Hz, 1H), 8.41 (d,
J=2.6 Hz, 1H), 8.87 (s, 1H), 10.91 (s, 1H). Anal. calcd
(+) - N - Benzyloxy - 6 - (4 - pivaloyloxybenzenesulfonyl)-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
(35u). The title compound (a colorless powder) was
synthesized from compound 34a and 4-pivaloyloxy-
benzenesulfonyl chloride by use of reaction condition
similar to that used for the preparation of 35a. 1H
NMR (CDCl3) d 1.35 (s, 9H), 2.91 (dd, J=6.6,
17.8 Hz, 1H), 3.44 (d, J=17.8 Hz, 1H), 4.43 (d,
J=17.2 Hz, 1H), 4.70–5.00 (m, 4H), 7.15 (d, J=8.5 Hz,
2H), 7.30–7.50 (m, 5H), 7.76 (d, J=8.5 Hz, 2H), 8.35
(s, 2H), 9.15 (s, 1H). Anal. calcd for C26H28N4O6S: C,
59.53; H, 5.38; N, 10.68; found: C, 59.50; H, 5.39; N,
10.70.
.
for C18H22N4O6S 0.6H2O: C, 49.90; H, 5.40; N, 12.93;
found: C, 49.92; H, 5.37; N, 12.63. MALDI-TOF MS
(Mr=422.46): 423 [M+H]+, 445 [M+Na]+, 461
[M+K]+.

446
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
(+)-N-Benzyloxy-6-(4-hydroxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (35f). An
aqueous solution of sodium hydroxide (1 N, 2.0 mL)
was added to a solution of (+)-N-benzyloxy-6-(4-piva-
loyloxybenzenesulfonyl)-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxamide (35u) (350 mg, 0.667 mmol) in
dioxane (5 mL), and then the resulting mixture was
stirred at room temperature for 2 h. The reaction mix-
ture was diluted with a 10% aqueous solution of citric
acid (100 mL), and extracted with ethyl acetate. The
organic layer was washed with water, and a saturated
aqueous solution of sodium chloride, dried over mag-
nesium sulfate, and concentrated in vacuo. The
obtained residue was purified by column chromato-
graphy (eluent: n-hexane/ethyl acetate=1:2) to give the
title compound as a colorless solid (210 mg, Y=71%).
1H NMR (CDCl3) d 2.80–3.00 (m, 1H), 3.40–3.60 (m,
1H), 4.40 (d, J=17.1 Hz, 1H), 4.70–5.00 (m, 4H), 6.81
(d, J=8.5 Hz, 2H), 7.30–7.50 (m, 5H), 7.62 (d,
J=8.5 Hz, 2H), 8.37 (s, 2H), 9.11 (s, 1H). Anal. calcd
for C21H20N4O5S: C, 57.26; H, 4.58; N, 12.72; found: C,
57.01; H, 4.55; N, 13.00.
(s, 1H), 9.71 (s, 1H). Anal. calcd for C23H22N4O4: C,
60.02; H, 5.30; N, 13.39; found: C, 59.73; H, 5.28; N,
13.60.
(+)-N-Hydroxy-6-(4-methoxybenzoyl)-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxamide (5a). The title com-
pound (a colorless powder) was synthesized from
compound 36a by use of reaction condition similar to
1
that used for the preparation of 3a. H NMR (DMSO-
d6) d 3.10–3.50 (m, 2H), 3.81 (s, 3H), 4.50–5.60 (m, 3H),
7.00–7.10 (m, 2H), 7.40–7.60 (m, 2H), 8.45 (s, 2H), 8.92
(s, 1H), 10.90 (s, 1H). Anal. calcd for
.
C16H16N4O4 0.5H2O: C, 56.97; H, 5.08; N, 16.61;
found: C, 56.88; H, 5.26; N, 16.52. MALDI-TOF MS
(Mr=328.32): 329 [M+H]+, 351 [M+Na]+, 367
[M+K]+
(+)-6-tert-Butoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxylic acid (32b). (ꢁ)-6-tert-Butoxy-
carbonyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carb-
oxylic acid (31) was treated with R-(+)-1-
phenylethylamine in the same manner that 32a was
prepared from 31 to give the title compound as a color-
less solid. 1H NMR (DMSO-d6) d 1.52 (s, 9H), 3.34 (dd,
J=6.7, 17.2 Hz, 1H), 3.53 (d, J=17.2 Hz, 1H), 4.50–
4.70 (m, 1H), 4.90–5.10 (m, 1H), 5.10–5.50 (m, 1H),
8.40–8.50 (m, 2H). Anal. calcd for C13H17N3O4: C,
55.91; H, 6.14; N, 15.05; found: C, 55.78; H, 5.99; N,
15.23. [a]D=+38 (c 1.0, methanol).
(+)-N-Hydroxy-6-(4-hydroxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3f). The
title compound (a colorless powder) was synthesized
from compound 35f by use of reaction condition similar
to that used for the preparation of 3a. 1H NMR
(DMSO-d6) d 2.94 (d, J=16.9 Hz, 1H), 3.00–3.30 (m,
1H), 4.50–4.70 (m, 2H), 4.70–4.80 (m, 1H), 6.84 (d,
J=8.4 Hz, 2H), 7.64 (d, J=8.4 Hz, 2H), 8.30–8.50 (m,
2H), 8.90 (br s, 1H), 10.78 (br s, 1H). Anal. calcd for
(ꢀ)-N-Benzyloxy-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxamide hydrochloride (34b). The title compound
(a colorless solid) was synthesized from (+)-6-tert-
butoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxylic acid (32b) via (ꢀ)-N-benzyloxy-6-tert-
butoxycarbonyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxamide (33b) in the same manner as 34a. 1H
NMR (DMSO-d6) d 3.19 (dd, J=11.4, 17.7 Hz, 1H),
3.36 (dd, J=5.2, 17.7 Hz, 1H), 4.20–4.40 (m, 2H), 4.50
(d, J=16.6 Hz, 1H), 4.86 (d, J=11.1 Hz, 1H), 4.91 (d,
J=11.1 Hz, 1H), 7.30–7.50 (m, 5H), 8.50–8.60 (m, 2H),
9.90–10.70 (br, 1H), 12.20 (bs, 1H). Anal. calcd for
.
C14H14N4O5S 0.4H2O: C, 47.03; H, 4.17; N, 15.67;
found: C, 47.25; H, 4.41; N, 15.33. MALDI-TOF MS
(Mr=350.35): 351 [M+H]+, 373 [M+Na]+, 389
[M+K]+.
(+)-N-Hydroxy-6-[4-(3-carboxyproproxy)benzenesulfonyl]-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3s).
The title compound (a colorless powder) was synthe-
sized from compound 34a and 4-(3-ethoxy-
carbonylpropoxy)benzenesulfonyl chloride via compound
35r and the corresponding N-benzyloxyamide 35s by
use of reaction conditions similar to those used for the
preparation of 3f. 1H NMR (DMSO-d6) d 1.85–2.00 (m,
2H), 2.38 (t, J=7.3 Hz, 2H), 2.95 (d, J=15.6 Hz, 1H),
3.00–3.25 (m, 1H), 4.05 (t, J=6.4 Hz, 2H), 4.50–4.80
(m, 3H), 7.00–7.10 (m, 2H), 7.70–7.80 (m, 2H), 8.35–
8.45 (m, 2H), 8.90 (br s, 1H), 10.94 (br s, 1H). Anal.
.
C15H17ClN4O2 1.0H2O: C, 53.18; H, 5.65; N, 16.54;
found: C, 53.04; H, 5.44; N, 16.66. [a]D=ꢀ88 (c 1.0,
methanol).
(ꢀ)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3b). The
title compound (a colorless powder) was synthesized
from (ꢀ)-N-benzyloxy-5,6,7,8-tetrahydro pyrido[3,4-b]
pyrazine-7-carboxamide hydrochloride (34b) via (ꢀ)-N-
benzyloxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (35b) in the
.
calcd for C18H20N4O7S 0.5H2O: C, 48.53; H, 4.75; N,
12.58; found: C, 48.31; H, 4.79; N, 12.60. MALDI-TOF
MS (Mr=436.44): 437 [M+H]+, 459 [M+Na]+, 475
[M+K]+.
1
same manner as 3a. H NMR (DMSO-d6) d 2.95 (dd,
(+)-N-Benzyloxy-6-(4-methoxybenzoyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (36a). The
title compound (a colorless powder) was synthesized
from compound 34a and 4-methoxybenzoyl chloride by
use of reaction condition similar to that used for the
preparation of 35a. 1H NMR (CDCl3) d 3.15–3.35
(m,1H), 3.56 (d, J=17.9 Hz, 1H), 3.85 (s, 3H), 4.39 (d,
J=17.0 Hz, 1H), 4.80–5.00 (m, 3H), 5.43 (br s, 1H), 6.91
(d, J=7.7 Hz, 2H), 7.20–7.40 (m, 7H), 8.35 (s, 1H), 8.45
J=2.0, 17.4 Hz, 1H), 3.16 (dd, J=7.0, 17.4 Hz, 1H),
3.82 (s, 3H), 4.59 (d, J=16.8 Hz, 1H), 4.67 (d,
J=16.8 Hz, 1H), 4.78 (dd, J=2.0, 7.0 Hz, 1H), 7.05 (d,
J=8.9 Hz, 2H), 7.75 (d, J=8.9 Hz, 2H), 8.30–8.50 (m,
2H), 8.88 (s, 1H), 10.91 (s, 1H). Anal. calcd for
.
C15H16N4O5S 0.5H2O: C, 48.25; H, 4.59; N, 15.00;
found: C, 48.11; H, 4.68; N, 14.82. MALDI-TOF MS
(Mr=364.38): 365 [M+H]+, 387 [M+Na]+, 403
[M+K]+. [a]D=ꢀ30 (c 1.0, methanol).

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
447
Method C: (+)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-
7-carboxylic acid hydrochloride (37a). A solution of
hydrochloric acid in ethyl acetate (4 N, 15 mL) was
added to (ꢀ)-6-tert-butoxycarbonyl-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxylic acid (32a) (2.00 g,
7.16 mmol) under ice-cooling, and then the resulting
mixture was stirred at room temperature for 4 h. The
precipitating solid was collected by filtration to give the
title compound as a pale brown powder (1.58 g,
Y=quant). H NMR (DMSO-d6) d 3.30–3.50 (m, 2H),
4.30–4.60 (m, 2H), 4.60–4.70 (m, 1H), 8.50–8.70 (m,
.
2H). Anal. calcd for C8H10ClN3O2 1.0H2O: C, 41.12; H,
5.18; N, 17.98; found: C, 41.41; H, 5.10; N, 17.85.
[a]D=+79 (c 0.51, methanol).
The resulting mixture was stirred for 30 min under ice-
cooling and further stirred at room temperature for 2 h.
1,2-Dimethoxyethane (5 mL) and an aqueous solution
of hydroxylamine (50%, 1.5 mL) were successively
added to the reaction mixture under ice-cooling, and the
resulting mixture was stirred for 2.5 h under ice-cooling.
After the reaction mixture had been acidified with dilu-
ted hydrochloric acid to pH 3, the whole was extracted
with ethyl acetate three times. The organic layers were
combined, washed with water and a saturated aqueous
solution of sodium chloride, and dried over magnesium
sulfate. After concentration of the solution in vacuo, the
obtained residue was purified by column chroma-
tography (eluent: chloroform/methanol=10:1) to give
the title compound as a colorless powder (350 mg,
1
1
(+)-6-[4-(3-Methylthiopropoxy)benzenesulfonyl]-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxylic acid (4q).
(+)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxylic
acid hydrochloride (37a) (1.20 g, 5.56 mmol) was dis-
solved in a mixture of water (10 mL) and dioxane
(10 mL). Triethylamine (1.40 g, 13.8 mmol) and 4-(3-
methylthiopropoxy)benzenesulfonyl chloride (1.80 g,
6.41 mmol) were successively added to the solution
under ice-cooling, and then the resulting mixture was
stirred at room temperature for 4 h. After the reaction
mixture had been acidified with diluted hydrochloric
acid to pH 3, the whole was extracted with ethyl acetate.
The organic layers were combined, washed with water
and a saturated aqueous solution of sodium chloride,
and dried over magnesium sulfate. After concentration
of the solution in vacuo, the obtained residue was pur-
ified by column chromatography (eluent: chloroform/
methanol=10:1) to give the title compound as a color-
less powder (790 mg, Y=34%). 1H NMR (CDCl3) d
2.13 (s, 3H), 2.00–2.20 (m, 2H), 2.69 (t, J=7.0 Hz, 2H),
3.40–3.50 (m, 2H), 4.14 (t, J=6.1 Hz, 2H), 4.61 (d,
J=17.4 Hz, 1H), 4.93 (d, J=17.4 Hz, 1H), 5.20–5.30 (m,
1H), 6.98 (d, J=8.8 Hz, 2H), 7.80 (d, J=8.8 Hz, 2H),
8.30–8.50 (m, 2H). Anal. calcd for C18H21N3O5S2: C,
51.05; H, 5.00; N, 9.92; found: C, 49.94; H, 5.02; N,
9.68. [a]D=+8.6 (c 1.0, methanol).
Y=45%). H NMR (DMSO-d6) d 1.90–2.10 (m, 2H),
2.05 (s, 3H), 2.60 (t, J=7.2 Hz, 2H), 2.94 (d, J=17.5 Hz,
1H), 3.15 (dd, J=7.1, 17.5 Hz, 1H), 4.10 (t, J=6.2 Hz,
2H), 4.57 (d, J=16.8 Hz, 1H), 4.67 (d, J=16.8 Hz, 1H),
4.70–4.80 (m, 1H), 7.04 (d, J=8.8 Hz, 2H), 7.73 (d,
J=8.8 Hz, 2H), 8.40–8.50 (m, 2H), 8.91 (s, 1H), 10.94
(s, 1H). Anal. calcd for C18H22N4O5S2: C, 49.30; H, 5.06;
N, 12.78; found: C, 48.99; H, 5.23; N, 12.52. MALDI-
TOF MS (Mr=438.53): 439 [M+H]+, 461 [M+Na]+,
477 [M+K]+. [a]D=+20 (c 0.50, methanol).
Compounds 3a, 3d, 3i, 3k, 3n, and 3o were synthesized
from compound 37a and the corresponding sulfonyl
chloride via the corresponding sulfonylamino acids 4 by
use of reaction conditions similar to those used for the
preparation of 3q. The physicochemical data of 3a, 3d,
3i, 3k, 3n, and 3o were shown below.
(+)-N-Hydroxy-6-(4-methoxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3a). A
colorless powder. 1H NMR (DMSO-d6) d 2.95 (dd,
J=2.0, 17.4 Hz, 1H), 3.16 (dd, J=7.0, 17.4 Hz, 1H),
3.82 (s, 3H), 4.59 (d, J=16.8 Hz, 1H), 4.67 (d,
J=16.8 Hz, 1H), 4.78 (dd, J=2.0, 7.0 Hz, 1H), 7.05 (d,
J=8.9 Hz, 2H), 7.75 (d, J=8.9 Hz, 2H), 8.30–8.50 (m,
2H), 8.88 (s, 1H), 10.91 (s, 1H). MALDI-TOF MS
(Mr=364.38): 365 [M+H]+, 387 [M+Na]+, 403
[M+K]+. The [a]D value (c 1.0, methanol) of 3a pre-
pared by Method C was equal to that of 3a prepared by
Method B.
(+)-6-(4-Methoxybenzenesulfonyl)-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxylic acid (4a). The title
compound (a colorless solid) was synthesized from
compound 37a and 4-methoxybenzenesulfonyl chloride
by use of reaction condition similar to that used for the
preparation of 4q. 1H NMR (DMSO-d6) d 3.10–3.35 (m,
2H), 3.81 (s, 3H), 4.51 (d, J=17.1 Hz, 1H), 4.68 (d,
J=17.1 Hz, 1H), 4.95–5.05 (m, 1H), 7.07 (d, J=9.0 Hz,
2H), 7.79 (d, J=9.0 Hz, 2H), 8.40–8.50 (m, 2H). Anal.
calcd for C18H21N3O6S: C, 53.06; H, 5.20; N, 10.31;
found: C, 52.88; H, 5.19; N, 10.17. MALDI-TOF MS
(Mr=349.36): 350 [M+H]+, 372 [M+Na]+, 388
[M+K]+.
(+)-N-Hydroxy-6-(4-methylthiobenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3d). A
1
colorless powder. H NMR (DMSO-d6) d 2.50 (s, 3H),
2.95 (dd, J=1.7, 17.4 Hz, 1H), 3.19 (dd, J=6.8, 17.4 Hz,
1H), 4.58 (d, J=16.6 Hz, 1H), 4.67 (d, J=16.6 Hz, 1H),
4.78 (dd, J=1.7, 6.8 Hz, 1H), 7.36 (d, J=8.7 Hz, 2H),
7.70 (d, J=8.7 Hz, 2H), 8.39 (d, J=2.7 Hz, 1H), 8.42 (d,
J=2.7 Hz, 1H), 8.91 (d, J=1.6 Hz, 1H), 10.95 (d,
.
J=1.6 Hz, 1H). Anal. calcd for C15H16N4O4S2 0.5H2O:
C, 46.26; H, 4.40; N, 14.39; found: C, 46.46; H, 4.62; N,
14.18. MALDI-TOF MS (Mr=380.44): 381 [M+H]+,
403 [M+Na]+, 419 [M+K]+.
(+) - N - Hydroxy - 6 - [4 - (3 - methylthiopropoxy)benzene-
sulfonyl]-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carb-
oxamide (3q). Oxalyl chloride (260 mg, 2.05 mmol) and
DMF (two drops) were added to a solution of (+)-6-
[4-(3-methylthiopropoxy)benzenesulfonyl]-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxylic acid (4q) (0.75g,
1.77 mmol) in dichloromethane (10 mL) under ice-cooling.
(+)-N-Hydroxy-6-(4-acetylbenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3i). A color-
1
less powder. H NMR (MeOH-d4) d 2.21 (s, 3H), 3.00–
3.30 (m, 2H), 4.67 (d, J=16.9 Hz, 1H), 4.80–5.10 (m,

448
K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
.
C9H12ClN3O2 1.0H2O: C, 43.64; H, 5.70; N, 16.97;
found: C, 43.49; H, 5.64; N, 16.73.
2H), 7.79 (d, J=9.1 Hz, 2H), 7.84 (d, J=9.1 Hz, 2H),
8.33 (d, J=2.5 Hz, 1H), 8.38 (d, J=2.5 Hz, 1H). Anal.
.
calcd for C16H16N4O5S 0.5H2O: C, 49.86; H, 4.45; N,
14.54; found: C, 49.50; H, 4.29; N, 14.30. MALDI-TOF
MS (Mr=376.39): 377 [M+H]+, 399 [M+Na]+, 415
[M+K]+.
Methyl (+)-6-(4-bromobenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine- 7-carboxylate (39). Triethy-
lamine (1.62 g, 16.0 mmol) and 4-bromobenzenesulfonyl
chloride (1.77 g, 6.93 mmol) were successively added to a
solution of methyl (+)-5,6,7,8-tetrahydropyrido[3,4-
b]pyrazine-7-carboxylate hydrochloride (38a) (1.15 g,
5.01 mmol) in water (20 mL) and dioxane (20 mL), and
then the resulting mixture was stirred at room tempera-
ture for 15 h. The reaction mixture was diluted with
10% aqueous solution of citric acid (100 mL), and then
the whole was extracted with ethyl acetate three times.
The organic layers were combined, washed with 10%
aqueous solution of citric acid, a saturated aqueous
solution of sodium hydrogen carbonate, and water suc-
cessively, dried over magnesium sulfate, and con-
centrated in vacuo. The obtained oil was purified by
column chromatography (eluent: n-hexane/ethyl ace-
tate=1:2) to give the title compound as a pale yellow oil
(+)-N-Hydroxy-6-(4-vinylbenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3k). A color-
1
less powder. H NMR (DMSO-d6) d 2.97 (d, J=1.7,
17.6 Hz, 1H), 3.19 (dd, J=6.7, 17.6 Hz, 1H), 4.62 (d,
J=16.7 Hz, 1H), 4.72 (d, J=16.7 Hz, 1H), 4.82 (dd,
J=1.7, 6.7 Hz, 1H), 5.46 (d, J=11.0 Hz, 1H), 6.01 (d,
J=17.7 Hz, 1H), 6.80 (dd, J=11.0, 17.7 Hz, 1H), 7.64
(d, J=8.5 Hz, 2H), 7.80 (d, J=8.5 Hz, 2H), 8.35–8.50
(m, 2H), 8.93 (s, 1H), 10.96 (br s, 1H). Anal. calcd for
.
C16H16N4O4S 0.5H2O: C, 52.02; H, 4.64; N, 15.17;
found: C, 52.27; H, 4.76; N, 15.13. MALDI-TOF MS
(Mr=360.39): 361 [M+H]+, 383 [M+Na]+, 399
[M+K]+.
1
(+)-N-Hydroxy-6-(4-pentyloxybenzenesulfonyl)-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazine-7-carboxamide (3n). A
colorless powder. 1H NMR (DMSO-d6) d 0.88 (t,
J=7.1 Hz, 3H), 1.20–1.50 (m, 4H), 1.60–1.80 (m, 2H),
2.80–3.00 (m, 1H), 3.05–3.30 (m, 1H), 4.01 (t,
J=6.4 Hz, 2H), 4.58 (d, J=16.5 Hz, 1H), 4.66 (d,
J=16.5 Hz, 1H), 4.70–4.80 (m, 1H), 7.03 (d, J=8.9 Hz,
2H), 7.72 (d, J=8.9 Hz, 2H), 8.30–8.50 (m, 2H), 8.90 (br
s, 1H), 10.93 (br s, 1H). Anal. calcd for
(950 mg, Y=46%). H NMR (CDCl3) d 3.40–3.50 (m,
5H), 4.53 (d, J=16.9 Hz, 1H), 4.92 (d, J=16.9 Hz, 1H),
5.15–5.25 (m, 1H), 7.60–7.80 (m, 4H), 8.35–8.45 (m,
2H). Anal. calcd for C15H14BrN3O4S: C, 43.70; H, 3.42;
N, 10.19; found: C, 43.49; H, 3.48; N, 9.94.
Methyl (+)-6-(4-trimethylsilylethynylbenzenesulfonyl)-
5,6,7,8 - tetrahydropyrido[3,4 - b]pyrazine - 7 - carboxylate
(40). Triethylamine (1 mL), Pd/C (10%, 36 mg,
33.8 mmol), triphenylphosphine (28 mg, 107 mmol), cop-
per (I) iodide (6.5 mg, 34.1 mmol), and trimethylsilylace-
tylene (80 mg, 814 mmol) were added to a solution of
methyl (+)-6-(4-bromobenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxylate hydrochloride
(39) (280 mg, 679 mmol) in acetonitrile (1 mL), and then
the resulting mixture was refluxed for 2.5 h. After hav-
ing been cooled to room temperature, the reaction mix-
ture was diluted with methanol (20 mL). Insoluble
matter in the resulting mixture was removed by filtra-
tion. The obtained filtrate was concentrated in vacuo,
and the residue was purified by column chroma-
tography (eluent: first, n-hexane/ethyl acetate=3:1, then
2:1) to give the title compound as a colorless crystalline
.
C19H24N4O5S 0.25H2O: C, 53.70; H, 5.81; N, 13.18;
found: C, 53.60; H, 6.01; N, 13.00. MALDI-TOF MS
(Mr=420.49): 421 [M+H]+, 443 [M+Na]+, 459
[M+K]+
(+)-N-Hydroxy-6-[4-(2-ethoxyethoxy)benzenesulfonyl]-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxamide
1
(3o). A colorless powder. H NMR (DMSO-d6) d 1.09
(t, J=7.0 Hz, 3H), 2.93 (dd, J=1.8, 17.4 Hz, 1H), 3.14
(dd, J=7.0, 17.4 Hz, 1H), 3.46 (q, J=7.0 Hz, 2H), 3.60–
3.80 (m, 2H), 4.00–4.20 (m, 2H), 4.57 (d, J=16.8 Hz,
1H), 4.66 (d, J=16.8 Hz, 1H), 4.75 (dd, J=1.8, 7.0 Hz,
1H), 7.04 (d, J=9.0 Hz, 2H), 7.72 (d, J=9.0 Hz, 2H),
8.30–8.50 (m, 2H), 8.86 (br s, 1H), 10.91 (br s, 1H).
1
.
Anal. calcd for C18H22N4O6S 0.25H2O: C, 50.11; H,
solid (190 mg, Y=65%). H NMR (CDCl3) d 0.21 (s,
5.37; N, 12.99; found: C, 50.31; H, 5.44; N, 12.79.
MALDI-TOF MS (Mr=422.46): 423 [M+H]+, 445
[M+Na]+, 461 [M+K]+.
9H), 3.30–3.45 (m, 5H), 4.48 (d, J=16.8 Hz, 1H), 4.89
(d, J=16.8 Hz, 1H), 5.10–5.20 (m, 1H), 7.54 (d,
J=8.5 Hz, 2H), 7.75 (d, J=8.5 Hz, 2H), 8.30–8.40 (m,
2H). Anal. calcd for C20H23N3O4SSi: C, 55.92; H, 5.40;
N, 9.78; found: C, 55.89; H, 5.32; N, 9.84.
Method D: methyl (+)-5,6,7,8-tetrahydropyrido[3,4-b]
pyrazine-7-carboxylate hydrochloride (38a). Thionyl
chloride (3.26 g, 27.4 mmol) was added to a solution of
(+)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carboxylate
(37a) (1.15 g, 5.33 mmol) in methanol (20 mL) under ice-
cooling, and then the resulting mixture was refluxed for
3 h. After having been cooled to room temperature, the
reaction mixture was concentrated in vacuo. Toluene
(40 mL) and methanol (10 mL) were added to the
obtained residue, and the resulting mixture was con-
centrated in vacuo to give the title compound as a
(+) - 6 - (4 - Ethynylbenzenesulfonyl) - 5,6,7,8 - tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxylic acid (4m). An aqu-
eous solution of potassium hydroxide (1 N, 2.30 mL,
2.30 mmol) was added to a solution of methyl (+)-6-(4-
trimethylsilylethynylbenzenesulfonyl)-5,6,7,8-tetrahydro-
pyrido[3,4-b]pyrazine-7-carboxylate
(40)
(190 mg,
0.442 mmol) in dioxane (1.5 mL), and then the resulting
mixture was stirred at room temperature for 2 h. After
the reaction mixture had been diluted with water
(50 mL), pH of the resulting mixture was adjusted to 3
by 10% aqueous solution of citric acid. The whole was
extracted with ethyl acetate three times, and then the
1
brown powder (1.20 g, Y=98%). H NMR (DMSO-d6)
d 3.30–3.50 (m, 2H), 3.85 (s, 3H), 4.30–4.50 (m, 2H),
4.60–4.70 (m, 1H), 8.50–8.70 (m, 2H). Anal. calcd for

K. Yoshiizumi et al. / Bioorg. Med. Chem. 11 (2003) 433–450
449
organic layers were combined, washed with a saturated
aqueous solution of sodium chloride, dried over mag-
nesium sulfate, and concentrated in vacuo. The
obtained residue was purified with column chromato-
graphy (eluent: first, chloroform/methanol=30:1, then
10:1) to give the title compound as an yellow solid
(80 mg, Y=53%). 1H NMR (DMSO-d6) d 3.19 (dd,
J=2.3, 17.4 Hz, 1H), 3.25–3.40 (m, 1H), 4.48 (d,
J=17.0 Hz, 1H), 4.51 (s, 1H), 4.74 (d, J=17.0 Hz, 1H),
5.08 (dd, J=2.3, 6.4 Hz, 1H), 7.65 (d, J=8.3 Hz, 2H),
7.87 (d, J=8.3 Hz, 2H), 8.40–8.50 (m, 2H). Anal. calcd
for C16H13N3O4S: C, 55.97; H, 3.82; N, 12.24; found: C,
55.75; H, 3.79; N, 12.10.
plated into 96-well culture plates and incubated for 24 h.
After aspiration of conditioned media and wash with
PBS (ꢀ), cells were treated with 0.2 mL of compound
solutions in duplicate. After incubation for 30 min at
37 ꢃC, 60 nM TPA containing compounds in MEM was
added. After further incubation for 60 min at 37 ꢃC,
100 mL of conditioned media were transferred from each
well to individual well of new 96-well plates. In order to
inactivate endogenous phosphatase, the plates were
heated at 65ꢃC for 10min. A hundred mL of AP buffer
(1 M diethanolamine, 0.01% MgCl2, 1 mg/mL p-nitro-
phenylphosphate, pH 9.8) were added to each well and
incubated for 60–120min at room temperature. AP activ-
ity was measured as the increase of absorption at 405 nm.
IC50 values in the shedding inhibition were calculated by
using GraphPad Prism Version 3.0 (GraphPad Software,
Inc.) from concentration–inhibition curves.
(+)-N-Hydroxy-6-(4-ethynylbenzenesulfonyl)-5,6,7,8-tetra-
hydropyrido[3,4-b]pyrazine-7-carboxamide (3m). Oxalyl
chloride (148 mg, 1.17 mmol) and DMF (two drops)
were added to a solution of (+)-6-(4-ethylnylbenzene-
sulfonyl)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-7-carb-
oxylic acid (4m) (80 mg, 0.233 mmol) in dichloroethane
(2 mL) under ice-cooling. The resulting mixture was
stirred for 1 h under ice-cooling. 1,2-Dimethoxyethane
(2 mL) and an aqueous solution of hydroxylamine
(50%, 0.924 mL) were successively added to the reaction
mixture under ice-cooling, and then the resulting mix-
ture was stirred for 2 h under ice-cooling. After the
reaction mixture had been diluted with 10% aqueous
solution of citric acid (100 mL), the whole was extracted
with ethyl acetate three times. The organic layers were
combined, washed with a saturated aqueous solution of
sodium chloride, and dried over magnesium sulfate.
After concentration of the solution in vacuo, the
obtained residue was purified by column chromato-
graphy (eluent: first, chloroform/methanol=100:1, then
25:1) to give the title compound as a colorless powder
(42 mg, Y=50%). 1H NMR (DMSO-d6) d 2.90–3.05
(m, 1H), 3.15–3.25 (m, 1H), 4.50–4.60 (m, 1H), 4.61 (d,
J=16.5 Hz, 1H), 4.72 (d, J=16.5 Hz, 1H), 4.75–4.85 (m,
1H), 7.63 (d, J=8.6 Hz, 2H), 7.82 (d, J=8.6 Hz, 2H),
8.40–8.50 (m, 2H), 8.92 (br s, 1H), 10.93 (br s, 1H).
MMP Inhibition assay
DNA fragments coding catalytic domain of human
MMP-1 and human MMP-9 and a DNA fragment
coding from pro-domain to catalytic domain of human
MMP-3 were amplified by polymerase chain reaction
(PCR) from cDNA of HT1080 cells stimulated with
0.01 mM of TPA. The 50-end of each PCR primers was
added a sequence for appropriate restriction enzyme
site. Amplified DNA fragments were cloned into clon-
ing vector, and then introduced into commercially
available expression vector containing His-6 tag
sequence at the end of N-terminus. Recombinant pro-
teins were expressed in Escherichia coli cells and purified
by Ni-NTA resin (Qiagen Inc.) and refolded. Recombi-
nant MMP-3 was activated by incubating with 1 mM
p-aminophenylmercuric acetate for 1 h at 37 ꢃC.
Test compounds were dissolved in DMSO and diluted
with reaction buffer (50 mM Tris, 150 mM NaCl,
10 mM CaCl2, 0.05% Brij-35, pH 7.5). Twenty-five mL
of compound solution was mixed with 25 mL of diluted
enzyme solution in a well of 96-well half-area black
microplate (Costar), and incubated for 10 min at 37 ꢃC.
The reaction was started by adding 50 mL of fluores-
cence-quenching peptide substrate solution to the well,
and incubated for 2 h (MMP-1 and MMP-3) or 3 h
(MMP-9) at 37 ꢃC. Five mM of MOCAc-Pro-Leu-Gly-
Leu-A2pr(Dnp)-Ala-Arg-NH210 (Peptide Institute, Inc.)
was used as a substrate for MMP-1 and MMP-9, and
5 mM of MOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-
.
Anal. calcd for C16H14N4O4S 0.8H2O: C, 51.55; H,
4.22; N, 15.03; found: C, 51.74; H, 4.28; N, 14.65.
MALDI-TOF MS (Mr=358.37): 359 [M+H]+, 381
[M+Na]+, 397 [M+K]+.
Shedding inhibition assay by EGF receptor ligand–AP
fusion protein
Expression vector of HB-EGF fused with human pla-
cental alkaline phosphatase (AP) that were constructed
as described previously3 was a generous gift from Dr.
Higashiyama (School of Medicine, Osaka University,
Osaka, Japan). The vector was transfected into HT1080
cells (American Type Culture Collection, Rockville,
MD, USA) by lipofection using lipofectamine system
(Gibco/BRL, Gaithersburg, MD, USA) according to
the manufacturer’s directions. Stable transfectants were
selected by growth in G418. These transfectants were
maintained in Minimum Essential Medium without
phenol red (Gibco/BRL, Gaithersburg, MD, USA)
supplemented with 10% heat-inactivated fetal calf
serum (MEM). Stable transfectants (2 ꢄ 105 cells/well)
expressing EGF receptor ligand–AP fusion protein were
11
Arg-Lys(Dnp)-NH2 (Peptide Institute, Inc.) was used
as a substrate for MMP-3. After incubation, fluores-
cence intensities (Ex/Em=320/405 nm) of the wells were
measured by fluorescence microplate reader (Polarstar;
BMG LabTechnologies, Germany). Ki values were cal-
culated from percent inhibition and Km value of each
MMPs to the substrate by using GraphPad Prism.
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