New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Part 3: 7β-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2- ethoxyiminoacetamido] cephalosporins beari

Article abstract of DOI:10.1016/j.bmc.2004.05.021

Among the prepared C-3′ substituted-pyridinium cephalosporins, a series of 7β-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido] cephalosporins bearing 4-[3-(aminoalkyl)-ureido]-1-pyridinium at C-3′ showed highly potent antibacterial activity agai

Full text of DOI:10.1016/j.bmc.2004.05.021

Bioorganic & Medicinal Chemistry 12 (2004) 4221–4231
New broad-spectrum parenteral cephalosporins exhibiting potent
activity against both methicillin-resistant Staphylococcus aureus
(
MRSA) and Pseudomonas aeruginosa. Part 3: 7b-[2-(5-Amino-
1
,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido] cephalosporins
0
bearing 4-[3-(aminoalkyl)-ureido]-1-pyridinium at C-3
*
Hidenori Yoshizawa, Tadatoshi Kubota, Hikaru Itani, Kyoji Minami, Hideaki Miwa
and Yasuhiro Nishitani
Shionogi Research Laboratories, Shionogi & Co., Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
Received 7 April 2004; revised 13 May 2004; accepted 13 May 2004
Available online 24 June 2004
0
Abstract—Among the prepared C-3 substituted-pyridinium cephalosporins, a series of 7b-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-
ethoxyiminoacetamido] cephalosporins bearing 4-[3-(aminoalkyl)-ureido]-1-pyridinium at C-3 showed highly potent antibacterial
0
activity against MRSA and Pseudomonas aeruginosa.
Ó 2004 Elsevier Ltd. All rights reserved.
1
. Introduction
replaced by other condensed-heterocyclic pyridinium
derivatives bearing an aminoalkyl group, showed also
potent antibacterial activity against MRSA and P.
0
In our previous papers, we reported that C-3 1-(amino-
alkyl)-imdazo[4,5-b]pyridinium cephalosporins, such as
S-3578 and 1a (Fig. 1), exhibit potent antibacterial
activity against both MRSA and Pseudomonas aerugin-
osa, and display good water solubility. In the course of
studies of S-3578, we found that introduction of a basic
functionality such as an aminoalkyl group to the 1-po-
2
aeruginosa.
0
These findings led interested in not only C-3 condensed-
heterocyclic pyridinium cephalosporins such as S-3578
0
but also structurally more simple C-3 pyridinium
cephalosporins bearing a basic functionality.
0
sition of C-3 imidazo[4,5-b]pyridinium moiety enhances
1
anti-MRSA activity.
0
C-3 unsubstituted-pyridinium cephalosporin 1b (Fig. 1) has
been reported to show potent antibacterial activity against
Subsequently, we found that 1a analogs, in which the
aminoalkyl-imidazo[4,5-b]pyridinium moiety of 1a is
Gram-positive and Gram-negative bacteria including P.
3
aeruginosa (anti-MRSA activity of 1b was not reported).
S
N
O
Q
=
H2N
H
N
N
S
N
N
O
Q
N
Me
N
O
N
N
H
O
N
NH2
.HCl
N
N
Et
.
N
O
H SO
2
4
S-3578
1a
1b
Figure 1. Structures of S-3578, 1a, and 1b.
*
0
Corresponding author. Tel.: +81-6-6458-5861; fax: +81-6-6458-0987; e-mail: hidenori.yoshizawa@shionogi.co.jp
968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.021

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H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
Table 1. Antibacterial activities (MIC, lg/mL) of 2a–k, S-3578, CZOP, CFSL and VCM
S
N
O
H2N
H
N
R1
N
S
Q =
N
N
R2
N
N
O
Q
O
O
2a-i
2j-k
Et
O
(2a-k; hydrochloride salt)
O
H
N
H
NH2
Me
O
N
NH2
NH2
R1=
NH2
NH2
N
H
2a
2b
2c
2d
2e
H
H
H
H
H
N
N
N
NH2
N
O
N
NH2
NH2
NH2
O
O
O
O
2f
2g
2h
2i
O
Me
NH2
H
N
R2=
NH2
N
H
O
2j
2k
ꢀ
Compound
S. a.
MRSA 1
MRSA 2
E. c.
P. a. 1
P. a. 2
2
2
2
2
2
2
2
2
2
2
2
a
b
c
d
e
f
1.56
0.78
0.2
12.5
25
12.5
25
0.2
0.78
1.56
0.78
3.13
1.56
1.56
0.78
0.78
0.78
1.56
1.56
1.56
0.39
0.39
3.13
>100
3.13
6.25
3.13
6.25
3.13
3.13
3.13
3.13
3.13
6.25
6.25
6.25
1.56
1.56
6.25
>100
0.05
0.05
0.05
0.2
12.5
6.25
12.5
12.5
6.25
6.25
3.13
12.5
12.5
3.13
12.5
50
12.5
6.25
12.5
12.5
6.25
6.25
6.25
12.5
12.5
3.13
12.5
50
0.2
1.56
0.78
1.56
1.56
0.78
3.13
1.56
0.78
1.56
0.78
0.78
1.56
0.05
0.1
g
h
i
0.1
0.1
j
0.78
0.39
0.39
0.05
0.05
0.05
>100
k
S-3578
1
b
CZOP
CFSL
VCM
25
25
0.78
1.56
ꢀ
Abbreviations: S. a., Staphylococcus aureus SMITH; MRSA 1, S. aureus SR3626; MRSA 2, S. aureus SR3637; E. c., Escherichia coli NIHJ JC-2; P. a. 1,
Pseudomonas aeruginosa SR24; P. a. 2, P. aeruginosa SR5393; CZOP, cefozopran; CFSL, cefoselis; VCM, vancomycin.
We prepared a series of 7b-[2-(5-amino-1,2,4-thiadiazol-
2a–h and 2j–s were synthesized from iodomethy 3 and
0
0
0
0
3
-yl)-2-ethoxyiminoacetamido]-3-pyridinomethyl-cephal-
the corresponding pyridine derivative 2a –h and 2j –s
by a method similar to that for preparing of 2i (the
0
osporins bearing a basic functionality on the C-3
pyridinium ring (2a–s in Tables 1 and 2) in order to
discover novel cephalosporins exhibiting potent anti-
bacterial activity against MRSA and P. aeruginosa.
0
0
0
0
0
structures of 2a –c , 2h , and 2r are shown in Schemes
0
0
0
0
2–5, and those of 2d –g , 2j –q , and 2s are shown in
Fig. 2).
Here we report the synthesis and antibacterial activity of
0
The syntheses of selected pyridine derivatives are shown
in Schemes 2–5.
novel C-3 pyridinium cephalosporins.
0
Pyridine derivative 2a was prepared as shown in
Scheme 2. Alkene 4 was reduced by palladium-catalyzed
hydrogenation in ethanol, affording ester 5, which was
hydrolyzed by sodium hydroxide to give acid 6. This
2
. Chemistry
0
0
Novel C-3 substituted-pyridinium cephems (2a–s) were
1
acid 6 was converted to 2a using a 2-methyl-2-propanol
and diphenylphosphoryl azide (DPPA)–triethylamine
synthesized by a method similar to one reported. The
0
4
method for the preparation of C-3 pyridinium cephem
3
(Et N) system.
2
sporin intermediate 3 was displaced by pyridine deriv-
i is shown in Scheme 1. The C-3 iodomethyl cephalo-
1
0
0
Pyridine derivative 2b and 2c were synthesized as
shown in Scheme 3. Chloropyridine 7 was treated with
2-aminoethanol in the presence of sodium hydride, and
0
0
ative 2i to afford iodide salt 3 , which was treated with
an AlCl -anisole system. Purification by reversed phase
3
(HP-20) column chromatography yielded cephalosporin
derivatives 2i as the hydrochloride salt. Other cephems
the resulting amine was allowed to react with di-tert-
0
butyl dicarbonate (Boc
O) to give 2b . On the other
2

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4223
Table 2. Antibacterial activities of 21–s
S
N
O
H2N
H
N
H
N
H
N
N
S
R
N
N
O
N
O
O
O
(2l-s; hydrochloride salt)
Et
O
NH2
Me
R=
NH2
NH2
N
H
NH
2
l Me
2m
2o OH
2n
2p
NH
NH
NH2
H
N
NH
N
N
H
2q
2r NH2
2s
ꢀ
Compound
S. a.
MRSA 1
MRSA 2
E. c.
P. a. 1
P. a. 2
2
2
2
2
2
2
2
2
l
0.78
0.78
1.56
1.56
0.78
0.78
0.78
0.78
3.13
3.13
6.25
6.25
3.13
6.25
1.56
3.13
3.13
3.13
6.25
6.25
3.13
6.25
3.13
3.13
0.1
1.56
1.56
1.56
1.56
0.78
1.56
1.56
1.56
3.13
3.13
6.25
3.13
3.13
6.25
3.13
3.13
m
n
o
p
q
r
0.05
0.1
0.1
0.05
0.1
0.1
0.05
s
ꢀ
Abbreviations: see footnote in Table 1.
S
N
O
BocHN
N
H
N
H
N
H
N
H
N
H
N
S
I
R
R
a
+
N
N
O
I
N
O
N
O
.
O
O
NHBoc
Et
2i'
OPMB
R=
3'
3
S
N
O
H2N
H
N
H
N
H
N
N
S
b
NH2
HCl
N
N
O
N
O
.
O
O
Et
2i
O
0
Scheme 1. Reagents and conditions: (a) 2i , MeCN, rt, 90 min; (b) (i) AlCl
3
-anisole, CH
2
2
Cl , )40–0 °C, (ii) purification by HP-20 chromatography.
CO2Et
CO2Et
CO2H
NHBoc
a
c
b
N
N
N
N
4
5
6
2a'
2 3
Scheme 2. Reagents and conditions: (a) H , Pd(C), EtOH; (b) 2 N NaOH, EtOH; (c) 2-methyl-2-propanol, DPPA, Et N, reflux.
Boc
N
b
a
O
N
NHBoc
N
Cl
N
NHBoc
2c'
2b'
7
Scheme 3. Reagents and conditions: (a) (i) 2-aminoethanol, NaH, (ii) Boc
ii) Boc O, H O, THF.
2
O, EtOAc, H
2
2 3
O; (b) (i) ethylenediamine, K CO , 130 °C, 3 days,
(
2
2
CO2H
NCO
H
a
NHBoc
b
N
O
N
NHBoc
+
HO
N
N
O
8
9
10
2h'
Scheme 4. Reagents and conditions: (a) DPPA, Et
3
N, MeCN, 75 °C, 2 h; (b) Et N, DMF, rt.
3

4224
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
O
O
H
N
H
N
H
N
H
N
CO2H
b
NHBoc
N
N
a
N
O
N
O
2i'
O
O
11
12
c
NBoc
H
N
H
N
N
H
NHBoc
N
O
2r'
Scheme 5. Reagents and conditions: (a) (i) DPPA, Et
H
3
N, DMF, 60 °C, (ii) 4-aminopyridine, rt; (b) (i) hydrazine hydrate, EtOH, 80 °C, (ii) Boc
O, THF; (c) (i) hydrazine hydrate, EtOH, 80 °C, (ii) 1H-pyrazole-1-[N,N -bis(tert-butoxycarbonyl)]carboxamidine, DMF, DMSO.
2
O,
0
2
O
Me
NHBoc
Boc
N
H
N
H
N
NHBoc
NHBoc
NHBoc
R1 R1=
N
H
N
O
O
2
d' (4-substituted)
2g' (4-substituted)
2e' (4-substituted)
j' (3-substituted)
2f' (4-substituted)
2k' (3-substituted)
2
NHBoc
Me
Boc
R2=
NHBoc
N
NHBoc
OSiEt3
H
N
H
N
Me
R2
2
l'
2m'
2q'
2n'
2o'
N
O
NBoc
NBoc
R2=
NBoc
N
N
H
NHBoc
2
p'
2s'
0
0
0
0
0
Figure 2. Structures of pyridine derivatives 2d –g , j –q , s .
0
hand, 7 was treated with ethylenediame in the presence
of potassium carbonate in DMF at 130 °C for 3 days,
the 3- or 4-position of the C-3 pyridinium ring with a
variety of linkers between the aminoalkyl group and the
pyridinium ring.
and the resulting diamine was treated with Boc
0
2
O to
yield 2c .
Most of these compounds (2a–k) showed good anti-
bacterial activity against Gram-positive bacteria
including MRSA and Gram-negative bacteria including
P. aeruginosa, although these anti-MRSA activities were
inferior to S-3578.
0
5
Pyridine derivative 2h was prepared as shown in
Scheme 4. Isocyanate 9 was synthesized from carbox-
ylic acid 8 using the DPPA–Et
was allowed to react with alcohol 10 in the presence of
0
3
N system. The isocyanate
9
Et N to afford 2h .
3
Among the 4-substituted compounds (2a–i), amino-
linked pyridinium 2d, amide-linked pyridinium 2g,
carbamate-linked pyridinium 2h, and ureido-linked py-
ridinium 2i showed more potent activity against MRSA
than the reference compound 1b. Comparison of amino-
linked pyridinium 2c and 2d indicates that elongation of
0
0
Pyridine derivative 2i and 2r were prepared as shown
in Scheme 5. 4-Aminopyridine was allowed to react
with the isocyanate generated in situ from carboxylic
3
acid 11 using the DPPA–Et N system to yield the
pyridine 12. After deprotection by treatment of 12 with
hydrazine hydrate, the resulting amine was treated with
Boc 1H-pyrazole-1-[N,N -bis(tert-butoxycar-
0
bonyl)]carboxamidine to give ureido-pyridine 2i or 2r ,
0
the spacer carbon chain between the C-3 pyridinium
0
2
O
or
moiety and the terminal amino group enhances anti-
MRSA activity, but significantly decreases antibacterial
activity against P. aeruginosa SR24.
0
respectively.
On the other hand, 3-substituted pyridinium 2j and 2k
were less active against Staphylococcus aureus SMITH
and Gram-negative bacteria including P. aeruginosa
than the corresponding 4-subsituted pyridinium 2e and
3
. Antibacterial activities
2f, respectively. Both anti-MRSA activities of 2j and 2k
were as weak as those of 2e and 2f.
0
Table 1 shows the antibacterial activities (MICs) of C-3
substituted-pyridinium cephalosporins (2a–k), and ref-
6
erence compounds 1b, cefozopran (CZOP), cefoselis
7
Among compounds 2a–k, ureido-linked pyridinium 2i
showed the most potent activity against MRSA and P.
aeruginosa. Therefore, we continued modification of the
aminoalkyl moiety of 2i to try to improve anti-MRSA
activity.
(
CFSL), S-3578, and vancomycin (VCM). MICs were
determined by the standard serial twofold agar dilution
method using Mueller–Hinton agar.
0
All C-3 substituted-pyridinium cephalosporins 2a–k
contain an aminoalkyl group as a basic functionality at
Table 2 shows the antibacterial activity of 2i analogs.

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4225
Anti-MRSA activity could be enhanced by elongating
the carbon chain of the aminoalkyl moiety (2i vs 2l, 2m,
J ¼ 7:8 Hz), 4.23 (2H, q, J ¼ 8 Hz), 7.14 (2H, d,
J ¼ 6:4 Hz), 8.51 (2H, d, J ¼ 6:4 Hz).
2
p), or by replacing the amino group with another basic
group, guanidine (2i vs 2r, 2s). Anti-MRSA activity was
reduced by introducing another polar group such as a
hydroxy group on the spacer carbon chain (2m vs 2o),
introducing a methyl group on the terminal amine (2m
vs 2n) or replacing the amino group with imidazole as a
basic function (2i vs 2q). The antibacterial activities
against P. aeruginosa, except for that of 2p, were re-
duced compared to that of 2i, but still strong compa-
rable to that of S-3578.
4.3. 3-Pyridin-4-yl-propionic acid (6)
To a solution of 5 (134 mg, 0.75 mmol) in EtOH
(0.6 mL) was added 2 N NaOH (0.41 mL) and the
reaction mixture was stirred at room temperature for
30 min. After addition of 1 N HCl (0.83 mL), the mix-
ture was concentrated and the precipitate was filtered to
give 6 as a crystalline solid (71 mg, 63% yield); mp 227–
1
2
30 °C; H NMR (DMSO-d ) d 2.58 (2H, t, J ¼ 6:6 Hz),
6
Among the prepared novel cephalosporins, 2i, 2l, 2m, 2p,
2
2.83 (2H, t, J ¼ 6:6 Hz), 7.25 (2H, d, J ¼ 5:8 Hz), 8.45
8 9 2 2
r, and 2s showed highly potent antibacterial activity
(2H, d, J ¼ 5:8 Hz); Anal Calcd for C
H NO Æ0.1H O:
C, 62.82; H, 6.06; N, 9.16. Found: C, 62.9; H, 6.04; N
against Gram-positive bacteria including MRSA and
Gram-negative bacteria including P. aeruginosa. In par-
ticular, guanidine derivative 2r showed more potent anti-
MRSA activity than S-3578. However, subsequent
evaluation of 2i, 2l, 2m, 2p, 2r, and 2s revealed that
their water solubility was inferior to that of S-3578 and
their mouse acute toxicity (i.v.) was stronger than that of
S-3578.
9.32.
4.4. (2-Pyridin-4-yl-ethyl)-carbamic acid tert-butyl ester
(2a )
0
To a mixture of 6 (453 mg, 3 mmol) and 2-methyl-2-
propanol (9 mL) was added diphenylphoshorylazide
(
0.72 mL, 3.3 mmol) and Et
this was refluxed for 4 h. After addition of H
EtOAc, the organic layer was washed with brine, dried
over Na SO , filtered, and concentrated in vacuo. The
residue was chromatographed on a silica gel column to
3
N (0.46 mL, 3.3 mmol) and
4. Experimental
2
O and
Mps were determined on a Yanagimoto micro melting
point apparatus and uncorrected. IR spectra were taken
2
4
1
0
1
on JASCO IR-700. H NMR spectra were recorded with
a Varian Gemini-300 (300 MHz) or Varian Gemini-200
give 2a (370 mg, 55% yield) as a colorless oil; H NMR
(CDCl
3
) d 1.43 (9H, s), 2.81 (2H, t, J ¼ 6:9 Hz), 3.41
(
200 MHz) spectrometer. Chemical shifts are reported in
ppm from 2,2-dimethyl-2-silapentane-5-sulfonate (DSS
in D O) or TMS (in CDCl and DMSO-d ) as internal
(2H, m), 4.60 (1H, m), 7.13 (2H, d, J ¼ 5:7 Hz), 8.52
(2H, d, J ¼ 5:7 Hz).
2
3
6
standard. The following abbreviations are used: s sin-
glet, d doublet, dd double doublet, t triplet, q quartet, m
multiplet, ABq AB quartet, br s broad singlet. Column
chromatography was carried out on Merck Kieselgel
and Mitsubushi Chemical HP-20.
4.5. [2-(Pyridin-4-yloxy)-ethyl]-carbamic acid tert-butyl
ester (2b )
0
To 2-aminoethanol (2 mL) was added 60% NaH
(480 mg) under cooling on ice-water bath. The mixture
was stirred at room temperature for 40 min. To this was
added 4-chloropyridine (7) (600 mg, 4 mmol), and the
reaction mixture was stirred for 16 h and stirred at 60 °C
for 1 h. After evaporation, the residue was dissolved in a
4
.1. Measurement of in vitro antibacterial activity
MIC was determined by a serial twofold dilution
method with Sensitivity Disk Agar-N (Nissui Pharma-
ceutical, Tokyo, Japan). The overnight cultures of bac-
terial strains in Mueller–Hinton broth (Becton
mixture of H O (4 mL) and EtOAc (4 mL). To this was
2
added Boc
stirred at room temperature for 2 h. After addition of
EtOAc and H O, the organic phase was washed with
brine, dried over Na SO , filtered, and concentrated.
2
O (2.3 mL), and the reaction mixture was
6
Dickinson) were diluted to about 10 CFU/mL. Bacte-
2
rial suspensions of 1 lL were spotted onto agar plates
containing various concentrations of an antibiotic and
incubated for 20 h at 37 °C before the MICs were scored.
2
4
The residue was purified by silica gel column chroma-
0
tography to give the title compound 2b (712 mg, 75%
1
yield) as a colorless oil; H NMR (CDCl ) d 1.45 (9H, s),
3
3
.55 (2H, m), 4.07 (2H, t, J ¼ 5:1 Hz), 5.01 (1H, br s),
4
.2. 3-Pyridin-4-yl-propionic acid ethyl ester (5)
6.81 (2H, d, J ¼ 6 Hz), 8.44 (2H, d, J ¼ 6 Hz).
To a solution of (E)-3-pyridin-4-yl-acrylic acid ethyl
ester (4) (177 mg, 1 mmol) in EtOH was added 10% Pd–
C (26 mg). The mixture was stirred under hydrogen
atmosphere for 30 min under ambient pressure and at
room temperature. The catalyst was filtered off and the
4.6. (2-tert-Butoxycarbonylamino-ethyl)-pyridin-4-yl-car-
bamic acid tert-butyl ester (2c )
0
To a mixture of 4-chloropyridine (7) hydrochloride
(30 g, 0.2 mol) and ethylenediamine (150 mL) was added
filtrate was concentrated in vacuo to give 5 (177 mg, 99%
1
yield) as a colorless oil; H NMR (CDCl
3
) d 1.23 (3H, t,
K
2
CO
(55 g, 0.4 mol). The reaction mixture was stirred
3
J ¼ 8 Hz), 2.64 (2H, t, J ¼ 7:8 Hz), 2.95 (2H, t,
at 130 °C for 3 days and concentrated in vacuo. To the

4226
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
residue was added, in succession, concd HCl (13 mL),
O (100 mL), THF (60 mL), and Boc O (230 mL). The
mixture was stirred at room temperature for 1 h. After
addition of EtOAc and H O, the organic layer was
washed with brine, dried over Na SO , filtered, and
20 mmol) using N-Boc-b-alanine (4.16 g, 22 mmol),
WSCDÆHCl (4.21 g, 22 mmol), and THF as the solvent
by a similar procedure to that for the synthesis of 2e ;
H
2
2
0
1
2
mp 121–122 °C; H NMR (CDCl
(1H, m), 2.60 (2H, m), 3.44 (2H, m), 5.35 (1H, m), 7.55
3
) d 1.43 (9H, s), 2.15
2
4
concentrated in vacuo. The residue was purified by silica
gel column chromatography to give the title compound
(2H, m), 8.43 (2H, m).
0
2
1
3
c (39.7 g, 59% yield) as a crystalline solid; mp 103–
1
04 °C; H NMR (CDCl ) d 1.41 (9H, s), 1.49 (9H, s),
4.11. 4-Isocyanato-pyridine (9)
3
.45 (2H, m), 3.82 (2H, t, J ¼ 6:6 Hz), 4.78 (1H, m), 7.26
(
2H, d, J ¼ 6:3 Hz), 8.52 (2H, d, J ¼ 6:3 Hz).
To a mixture of isonicotinic acid (24.6 g, 0.2 mol), Et
(29.2 mL, 0.21 mL), and MeCN (170 mL) was added
3
N
dropwise DPPA (45.2 mL, 0.21 mol) under cooling on
an ice-water bath, and the mixture was stirred at the
same temperature for 30 min and stirred at 75 °C for 2 h.
The reaction mixture was cooled to room temperature,
and the resulting precipitate was collected by filtration
and rinsed with EtOAc. The amorphous solid was dried
under reduced pressure to give 9 (13.4 g, 56% yield),
which was used for synthesis without further purifi-
cation. Using this compound 9 and aniline, 1-phenyl-
3-pyridin-4-yl-urea was prepared by the following
procedure to confirm the structure of 9. To a mixture of
4.7. (3-tert-Butoxycarbonylamino-propyl)-pyridin-4-yl-
carbamic acid tert-butyl ester (2d )
0
0
Compound 2d was obtained as a colorless oil in 41%
yield from 4-chloropyridine hydrochloride (3.6 g) using
1
,3-diaminopropane (20 mL) and K CO (6.6 g) by a
2 3
0
1
similar procedure to that for the synthesis of 2c ; H
NMR (CDCl ) d 1.44 (9H, s), 1.48 (9H, s), 1.74 (2H, m),
.14 (2H, m), 3.78 (2H, t, J ¼ 6:9 Hz), 4.85 (1H, m), 7.20
2H, m), 8.53 (2H, m).
3
3
(
9
(
(120 mg, 1 mmol) and THF (1.5 mL) was added aniline
0.18 mL, 2 mmol), and the mixture was stirred at room
4.8. {2-[(Pyridine-4-carbonyl)-amino]-ethyl}-carbamic
0
acid tert-butyl ester (2e )
temperature for 4 h. After evaporation of the solvents,
the residue was chromatographed on silica gel to give
1
-phenyl-3-pyridin-4-yl-urea (136 mg, 63% yield) as a
5b
To a mixture of isonicotinic acid (1.23 g, 10 mmol) and
CH Cl (25 mL) was added water-soluble carbodiimide
crystalline solid; mp 160–161 °C (lit. mp 150 °C, 166–
167 °C; ) H NMR (CDCl
7.26–7.38 (6H, m), 8.05 (1H, br s), 8.35 (2H, d,
5c 1
2
2
3
) d 7.07 (1H, t, J ¼ 7:2 Hz),
hydrochloride (WSCDÆHCl) (2.3 g, 12 mmol) under
cooling in an ice-water bath. The mixture was stirred at
room temperature for 1 h. To this was added a solution
of (2-amino-ethyl)-carbamic acid tert-butyl ester (1.92 g,
J ¼ 5:7 Hz), 8.69 (1H, br s); Anal Calcd for C H N O:
1
2
11
3
C, 67.59; H, 5.20; N, 19.71. Found: C, 67.31; H, 5.23; N,
O [(M+H) ]:
þ
19.50; HR-FABMS calcd for C12
214.0980, found: 214.0975.
H
12
N
3
1
2 mmol) in CH Cl (5 mL) under cooling on an ice-
2 2
water bath. The mixture was stirred for 3 days. After
addition of EtOAc and H O, the organic phase was
washed with brine, dried over Na SO , filtered, and
2
2
4
4.12. Pyridin-4-yl-carbamic acid 2-tert-butoxycarbonyl-
amino-ethyl ester (2h )
0
concentrated in vacuo. The residue was purified by silica
gel column chromatography to give the title compound
0
1
2
e (1.1 g, 41%) as a crystalline solid; mp 129–130 °C; H
To a solution of (2-hydroxyethyl)-carbamic acid tert-butyl
ester (10) (1.61g, 10mmol) in DMF (10 mL) was added
NMR (CDCl ) d 1.43 (9H, s), 3.43 (2H, m), 3.55 (2H, m),
3
5
.08 (1H, m), 7.68 (2H, m), 7.75 (1H, m), 8.73 (2H, m).
Et N (1.4 mL, 10mmol) and 4-isocyanato-pyridine (9)
3
(1.2g, 10mmol). The reaction mixture was stirred at room
temperature for 16h. After evaporation of the solvent, the
residue was purified by silica gel column chromatography
4
.9. [(R)-1-(Pyridin-4-ylcarbamoyl)-ethyl]-carbamic acid
tert-butyl ester (2f )
0
0
to give the title compound 2h (2.0 g, 71% yield) as a pale
1
yellow oil; H NMR (DMSO-d
6
) d 1.1.37 (9H, s), 3.22
(2H, m), 4.11 (2H, m), 6.96 (1H, m), 7.43 (2H, d,
0
Compound 2f (1.61 g) was obtained as a crystalline
solid in 60% yield from 4-aminopyridine (0.94 g,
1
J ¼ 5:7 Hz), 8.36 (2H, d, J ¼ 5:7 Hz), 10.1 (1H, s).
0 mmol) using N-Boc-
D-alanine (2.08 g, 11 mmol) and
WSCDÆHCl (2.11 g, 11 mmol) by a similar procedure to
0
1
that for the synthesis of 2e ; mp 164–165 °C; H NMR
4.13. 1-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethyl]-3-
pyridin-4-yl-urea (12)
(
(
(
DMSO-d
1H, m), 7.17 (1H, d, J ¼ 6:6 Hz), 7.57 (2H, m), 8.42
6
) d 1.26 (3H, d, J ¼ 6:9 Hz), 1.38 (9H, s), 4.11
2H, m), 10.3 (1H, s).
To a mixture of 3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-
propionic acid (11) (245 mg, 1.12 mmol) in MeCN
(5 mL) was added Et N (0.1 mL, 1.23 mmol) and diph-
3
4
tert-butyl ester (2g )
.10. [2-(Pyridin-4-ylcarbamoyl)-ethyl]-carbamic acid
0
enylphosphorylazide (0.265 mL, 1.23 mmol), and the
reaction mixture was stirred at 60 °C for 80 min. To the
reaction mixture was added 4-aminopyridine (100 mg,
1.06 mmol) under cooling in an ice-water bath, and the
mixture was stirred at room temperature for 16 h. After
0
Compound 2g (4.66 g) was obtained as a crystalline
solid in 60% yield from 4-aminopyridine (1.9 g,

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4227
addition of EtOAc and H
washed with brine, dried over Na
concentrated in vacuo. The residue was purified by silica
gel column chromatography to give the title compound
2
O, the organic layer was
SO , filtered, and
1,3-dihydro-isoindol-2-yl)-butyric acid (4.66 g, 20 mmol)
0
2
4
by a similar procedure to that for the synthesis of 2i as a
1
crystalline solid; mp 173–174 °C; H NMR (CDCl ) d
3
1.19 (3H, d, J ¼ 6:3 Hz), 1.42 (9H, s), 3.1–3.5 (2H, m),
3.72 (1H, m), 4.85 (1H, m), 6.0 (1H, m), 7.41 (2H, m),
8.20 (1H, m), 8.37 (2H, d, J ¼ 6 Hz).
1
1
2 (225 mg, 68% yield) as a crystalline solid; mp 181–
1
83 °C; H NMR (DMSO-d ) d 3.37 (2H, m), 3.70 (2H,
6
m), 6.52 (1H, m), 7.27 (2H, d, J ¼ 6:4 Hz), 7.82–8.25
(
4H, m), 8.24 (2H, d, J ¼ 6:4 Hz), 8.96 (1H, s).
4.18. [3-(3-Pyridin-4-yl-ureido)-propyl]-carbamic acid
tert-butyl ester (2m )
0
4
.14. [2-(3-Pyridin-4-yl-ureido)-ethyl]-carbamic acid tert-
butyl ester (2i )
0
0
Compound 2m (3.76 g) was obtained in 82% yield from
4-aminopyridine (1.47 g, 15.6 mmol) using 4-tert-but-
oxycarbonylamino-butyric acid (3.95 g, 19.5 mmol) by a
To a mixture of 12 (2.32 g, 7.48 mmol) and EtOH
80 mL) was added hydrazine hydrate (3.64 mL,
(
similar procedure to that of the synthesis of 12 as a
1
7
5 mmol), and the reaction mixture was stirred at 80 °C
colorless oil; H NMR (CDCl ) d 1.44 (9H, s), 1.63 (2H,
3
for 150 min and cooled in an ice-water bath. After fil-
tration of the precipitate, the filtrate was concentrated in
vacuo to give 1-(2-amino-ethyl)-3-pyridin-4-yl-urea,
m), 3.20 (2H, m), 3.28 (2H, m), 5.88 (1H, m), 6.03 (1H,
m), 7.36 (2H, m), 8.34 (2H, d, J ¼ 5:7 Hz).
which was dissolved in H O (4 mL) and THF (2 mL)
2
2
then Boc O (1.84 mL, 8 mmol) was added to the solu-
4.19. Methyl-[3-(3-pyridin-4-yl-ureido)-propyl]-carbamic
acid tert-butyl ester (2n )
tion. The reaction mixture was stirred at room temper-
ature for 120 min. After addition of EtOAc and H O,
the organic layer was washed with brine, dried over
0
2
To a solution of (3-amino-propyl)-methyl-carbamic acid
tert-butyl ester (0.94 g, 5 mmol) in DMF (5 mL) was
added 4-isocyanato-pyridine (9) (0.60 g, 5 mmol). The
reaction mixture was stirred at room temperature for
2 4
Na SO , filtered, and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography to
0
give the title compound 2i (850 mg, 39% from 12) as a
1
crystalline solid; mp 146–148 °C; H NMR (CDCl
3
) d
.42 (9H, s), 3.28 (2H, m), 3.36 (2H, m), 5.25 (1H, m),
3
was washed with brine, dried over Na SO , filtered, and
h. After addition of EtOAc and H
2
O, the organic layer
1
6
2
4
.13 (1H, m), 7.35 (2H, d, J ¼ 6:4 Hz), 8.30 (2H, d,
concentrated in vacuo. The residue was purified by silica
gel column chromatography to give the title compound
J ¼ 6:4 Hz), 8.34 (1H, m).
0
1
2
n (0.90 g, 59%) as a crystalline solid; mp 93–94 °C; H
4.15. {2-[(Pyridine-3-carbonyl)-amino]-ethyl}-carbamic
0
acid tert-butyl ester (2j )
NMR (CDCl ) d 1.45 (9H, s), 1.69 (2H, m), 2.83 (3H, s),
3
3.22 (2H, m), 3.31 (2H, m), 6.25 (1H, m), 7.36 (2H, d,
J ¼ 4:6 Hz), 7.72 (1H, br s), 8.36 (2H, m); Anal Calcd
0
Compound 2j (1.70 g) was obtained as a crystalline solid
in 64% yield from nicotinic acid (1.23 g, 10 mmol) using
for C H N O : C, 58.42; H, 7.84; N, 18.17. Found: C,
24
15
4
3
58.47; H, 7.78; N, 17.94.
(
2-amino-ethyl)-carbamic acid tert-butyl ester (2.40 g,
1
procedure to that for the synthesis of 2e ; mp 163–
2 mmol) and WSCDÆHCl (2.3 g, 12 mmol) in a similar
0
4.20. [3-(3-Pyridin-4-yl-ureido)-2-triethylsilanyloxy-prop-
0
1
1
3
8
65 °C; H NMR (CDCl
3
) d 1.42 (9H, s), 3.45 (2H, m),
.55 (2H, m), 5.15 (1H, m), 7.38 (1H, m), 7.65 (1H, m),
yl]-carbamic acid tert-butyl ester (2o )
.17 (1H, m), 8.72 (1H, m), 9.06 (1H, m).
[2-(tert-Butyl-dimethyl-silanyloxy)-3-(3-pyridin-4-yl-ure-
ido)-propyl]- carbamic acid tert-butyl ester (3.83 g,
90% yield) was obtained as a pale yellow oil from 4-
4
.16. [(R)-1-(Pyridin-3-ylcarbamoyl)-ethyl]-carbamic acid
tert-butyl ester (2k )
aminopyridine (0.94 g, 10 mmol) using 4-tert-butoxy-
carbonylamino-3-(tert-butyl-dimethyl-silanyloxy)-butyric
acid (3.34 g, 10 mmol) by a similar procedure to that for
0
0
0
1
Compound 2k (2.25 g) was obtained as a crystalline solid
in 84% yield from 3-aminopyridine (0.94 g, 10mmol)
the synthesis of 2i ; H NMR (CDCl ) d 0.10 (6H, s),
3
0.89 (9H, s), 1.44 (9H, s), 2.8–3.1 (2H, m), 3.2–3.7 (2H,
m), 3.82 (1H, m), 5.04 (1H, m), 5.91 (1H, m), 7.34 (2H,
d, J ¼ 6:2 Hz), 7.80 (1H, m), 8.33 (2H, d, J ¼ 6:2 Hz).
This compound (13.7 g, 32.4 mmol) was dissolved in
MeCN (65 mL). To the solution was added 12 N HCl
(5.41 mL) under cooling in an ice-water bath. The mix-
ture was stirred at the same temperature for 20 min.
using N-Boc-
(
D
-alanine (2.08 g, 11mmol) and WSCDÆHCl
2.11 g, 11mmol) by a similar procedure to that for the
0
1
synthesis of 2e ; mp 195–197 °C; H NMR (DMSO-d ) d
6
1
(
.27 (3H, d, J ¼ 6:9 Hz), 1.32 (9H, s), 4.12 (1H, m), 7.12
1H, d, J ¼ 6:9 Hz), 7.34 (1H, m), 8.05 (1H, m), 8.26 (1H,
m), 8.75 (1H, d, 1H, J ¼ 2:7 Hz), 10.1 (1H, s).
2 2
After addition of Et O and H O, the aqueous layer was
alkalized with NaHCO3 (6.81 g). After addition of
EtOAc, the organic layer was washed with brine, dried
over Na SO , filtered, and concentrated in vacuo to
4.17. [1-Methyl-2-(3-pyridin-4-yl-ureido)-ethyl]-carbamic
0
acid tert-butyl ester (2l )
2
4
give [2-hydroxy-3-(3-pyridin-4-yl-ureido)-propyl]-carbamic
1
0
Compound 2l (0.96 g) was obtained in 61% yield from
4
acid tert-butyl ester (3.10 g, 31%) as a pale yellow oil; H
NMR (CDCl +DMSO-d ) d 1.44 (9H, s), 3.18 (2H, m),
-aminopyridine (1.51 g, 16 mmol) using 3-(1,3-dioxo-
3
6

4228
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
0
3
7
.30 (2H, m), 3.73 (1H, m), 5.65 (1H, m), 6.31 (1H, m),
.35 (2H, d, J ¼ 6:3 Hz), 8.32 (2H, d, J ¼ 6:3 Hz), 8.76
4.24. 1-[3-N,N -Bis(tert-butoxycarbonyl)guanidino-prop-
yl]-3-pyridin-4-yl-urea (2s )
0
(
5
1H, br s). To a solution of this compound (1.73 g,
.57 mmol) in THF (17 mL) was added Et
3
N (0.85 mL,
.1 mmol) and chlorotriethylsilane (1.03 mL, 6.1 mmol)
[3-(3-Pyridin-4-yl-ureido)-propyl]-carbamic acid tert-
0
6
butyl ester (2m ) (1.47 g, 5 mmol) was dissolved in TFA
and the mixture was stirred at room temperature for
6 h. After addition of Et N (0.19 mL) and chlorotri-
ethylsilane (0.15 mL), the mixture was stirred for 3 h.
(15 mL), and the solution was stirred at room tempera-
ture for 30 min. After evaporation, the residue was dis-
solved in THF (10 mL). After successive addition of
1
3
0
After addition of EtOAc and H O, the organic layer was
2
Et N (3.5 mL) and 1H-pyrazole-1-[N,N -bis(tert-butox-
3
washed with brine, dried over Na SO , filtered, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography to give the title compound
2
4
ycarbonyl)]carboxamidine (1.48 g, 4.99 mmol), the
solution was stirred for 5 h. After addition of EtOAc
and H O, the organic layer was washed with brine, dried
2
0
1
2
o (2.43 g, 99%) as a pale yellow oil; H NMR (CDCl )
3
over Na SO , filtered, and concentrated in vacuo. The
2
4
d 0.63 (6H, q, J ¼ 7:2 Hz), 0.96 (9H, t, J ¼ 7:2 Hz), 1.45
9H, s), 2.7–3.0 (2H, m), 3.4–3.7 (2H, m), 3.83 (1H, m),
.06 (1H, m), 5.89 (1H, m), 7.34 (2H, d, J ¼ 6 Hz), 7. 60
1H, br s), 8.37 (2H, d, J ¼ 6 Hz).
residue was purified by silica gel column chromatogra-
0
(
5
(
phy to give the title compound 2s (1.83 g, 84%) as a pale
1
yellow oil; H NMR (CDCl ) d 1.51 (9H, s), 1.54 (9H, s),
3
1.72 (2H, m), 3.27 (2H, q, J ¼ 5:4 Hz), 3.49 (2H, q,
J ¼ 6:6 Hz), 6.88 (1H, br s), 7.35 (2H, d, J ¼ 6:6 Hz),
7
.51 (1H, m), 8.38 (2H, d, J ¼ 6:6 Hz), 8.54 (1H, m).
.25. 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyi-
4.21. 3-(3-Pyridin-4-yl-ureido)-pyrrolidine-1-carboxylic
0
acid tert-butyl ester (2p )
4
0
Compound 2p (0.85 g) was obtained in 57% yield from
minoacetamido]-3-{4-[3-(2-aminoethyl)-ureido]-pyridi-
nium-1-yl}methyl-3-cephem-4-carboxylate hydrochloride
(2i)
4
-isocyanato-pyridine (9) (0.64 g, 5.4 mmol) using 3-
amino-pyrrolidine-1-carboxylic acid tert-butyl ester
0.91 g, 4.9 mmol) in a similar procedure to that for the
(
0
1
synthesis of 2n as a crystalline solid; mp 182–184 °C; H
NMR (CDCl ) d 1.50 (9H, s), 2.08 (2H, m), 3.24–3.56
4H, m), 4.43 (1H, m), 6.22 (1H, m), 7.35 (2H, d,
J ¼ 4:8 Hz), 8.28 (1H, m), 8.36 (2H, m).
To a solution of iodomethyl 3 (1.48 g, 1.95 mmol) in
0
3
MeCN (2 mL) was added pyridine derivative 2i (420 mg,
(
1.5 mmol) under cooling in ice-water bath and the reac-
tion mixture was stirred at room temperature for 90 min.
After addition of MeCN (2 mL), the mixture was poured
into i-Pr O (200 mL). The precipitate was filtered and
2
0
4
boxylic acid tert-butyl ester (2q )
.22. 4-[2-(3-Pyridin-4-yl-ureido)-ethyl]-imidazole-1-car-
0
dried in vacuo to quantitatively give pyridinium salt 3 .
0
To a solution of 3 in CH (20 mL) was added a
solution of AlCl (1.2 g, 9 mmol) in anisole (10 mL) at
)30 °C and the reaction mixture was stirred under cool-
2
ing in an ice-water bath for 60 min. After addition of H O
(30 mL) and MeOH (10 mL), aqueous layer was washed
with Et O. It was then concentrated and chromato-
graphed on HP-20 resin. The target product was eluted
with 4% MeCN/0.001 N HCl. The fraction containing
the target product was concentrated and lyophilized to
2
Cl
2
3
0
Compound 2q (0.84 g) was obtained in 36% yield from
4
-aminopyridine (0.65 g, 6.9 mmol) using 4-(2-carboxy-
ethyl)-imidazole-1-carboxylic acid tert-butyl ester
1.68 g, 7 mmol) in a similar procedure to that of the
(
2
1
synthesis of 12 as a pale yellow oil; H NMR (CDCl
3
) d
.57 (9H, s), 2.66 (2H, m), 3.46 (2H, m), 6.25 (1H, m),
.0 (1H, m), 7.10 (1H, s), 7.27 (2H, m), 7.98 (1H, d,
1
7
0
J ¼ 1:2 Hz), 8.20 (2H, d, J ¼ 6:3 Hz).
give 2i (185 mg, 18% yield from 2i ) as an amorphous
1
powder; H NMR (DMSO-d
6
+D
2
O) d 1.23(3H, t,
J ¼ 6:9 Hz), 2.96(2H, t, J ¼ 6:0 Hz), 3.22 and 3.59 (2H,
ABq, J ¼ 17:7 Hz), 3.41 (2H, t, J ¼ 6:0 Hz), 4.16 (2H, q,
J ¼ 6:9 Hz), 4.94 and 5.27 (2H, ABq, J ¼ 15:3 Hz), 5.10
0
4
3
.23. 1-[2-N,N -Bis(tert-butoxycarbonyl)guanidino-ethyl]-
-pyridin-4-yl-urea (2r )
0
(
1H, d, J ¼ 4:8 Hz), 5.76 (1H, d, J ¼ 4:8 Hz), 7.83 (2H, d,
ꢁ
1
1
-(2-Amino-ethyl)-3-pyridin-4-yl-urea, which was ob-
tained by the treatment of 12 (0.20 g, 0.64 mmol) with
J ¼ 7:8 Hz), 8.80 (2H, d, J ¼ 7:8 Hz); IR (KBr) cm
1772, 1714, 1608, 1518, 1465, 1390; Anal Calcd for
N
hydrazine hydrate, was dissolved in DMF (2 mL) and
0
C
22
H
26
10
O
6
S
2
2
Æ1HClÆ3.4H O: C, 38.39; H, 4.95; Cl, 5.15;
DMSO (2 mL), then 1H-pyrazole-1-[N,N -bis(tert-
N, 20.35; S, 9.32. Found: C, 38.32; H, 4.89; Cl, 5.24; N,
20.45; S, 9.29.
butoxycarbonyl)]carboxamidine (0.414 g, 1.4 mmol) was
added to the solution. The reaction mixture was stirred
at room temperature for 16 h. After addition of EtOAc
O, the organic layer was washed with brine, dried
SO , filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatogra-
phy to give the title compound 2r (0.212 g, 78% from
2) as a pale yellow oil; H NMR (CDCl ) d 1.50 (9H, s),
.52 (9H, s), 3.47 (2H, m), 3.54 (2H, m), 7.03 (1H, m),
The other cephalosporins 2a–h, j–s, and reference com-
pound 1b were prepared by a procedure similar to that
described for the preparation of 2i.
and H
over Na
2
2
4
0
1
1
1
7
3
4.26. Compound 1b
.31 (2H, d, J ¼ 6:2 Hz), 8.39 (2H, d, J ¼ 6:2 Hz), 8.71
1b was obtained as an amorphous powder in 31% yield
1
(
1H, m).
from pyridine; H NMR (DMSO-d
6
+D
2
O) d 1.20 (3H,

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4229
ꢁ
1
t, J ¼ 7:2 Hz), 3.05 and 3.49 (2H, ABq, J ¼ 18:0 Hz),
8.61 (1H,m); IR (KBr) cm 1773, 1649, 1556, 1523,
1460, 1385; Anal Calcd for C22 Æ1.1HClÆ4H O:
4
.11 (2H, q, J ¼ 7:2 Hz), 5.04 (1H, d, J ¼ 4:2 Hz), 5.13
H
27
N
9
O
5
S
2
2
and 5.61 (2H, ABq, J ¼ 13:8 Hz), 5.69 (1H, d,
C, 39.22; H, 5.4; Cl, 5.79; N, 18.71; S, 9.52. Found: C,
39.16; H, 5.34; Cl, 5.90; N, 18.85; S, 9.43.
J ¼ 4:2 Hz), 8.05–8.2 (2H, m), 8.45–8.6 (1H, m), 9.25–
ꢁ
1
9
1
C
.4 (2H, m); IR (KBr) cm 1775, 1666, 1617, 1526,
485,
1455,
Æ2.6H
1.96. Found: C, 42.3; H, 4.58; N, 18.55; S, 11.94.
1390;
Anal
Calcd
for
19
H
19
N
7
O
5
S
2
2
O: C, 42.55; H, 4.55; N, 18.28; S,
4.31. Compound 2e
1
2e was obtained as an amorphous powder in 48% yield
from 2e ; H NMR (D
0
1
2
O) d 1.34 (3H, t, J ¼ 7:2 Hz), 3.3
4
2
.27. Compound 2a
and 3.75 (2H, ABq, J ¼ 18 Hz), 3.34 (2H, t, J ¼ 6:3 Hz),
3
.82 (2H, t, J ¼ 6:3 Hz), 4.39 (2H, q, J ¼ 7:2 Hz), 5.34
a was obtained as an amorphous powder in 19% yield
0
(1H, d, J ¼ 5:1 Hz), 5.48 and 5.73 (2H, ABq,
1
6 2
from 2a ; H NMR (DMSO-d +D O) d 1.21 (3H, t,
J ¼ 15 Hz), 5.93 (1H, d, J ¼ 5:1 Hz), 8.43 (2H, d,
ꢁ
1
J ¼ 7:5 Hz), 3.2–3.25 (4H, m), 3.05 and 3.55 (2H, ABq,
J ¼ 18 Hz), 4.12 (2H, q, J ¼ 7:5 Hz), 5.1 (1H, d,
J ¼ 4:8 Hz), 5.12 and 5.62 (2H, ABq, J ¼ 13:5 Hz), 5.73
J ¼ 6:6 Hz), 9.19 (2H, d, J ¼ 6:6 Hz); IR (KBr) cm
1775, 1668, 1616, 1527, 1456, 1393; Anal Calcd for
Æ1.3HClÆ4.5H O: C, 37.53; H, 5.05; Cl,
6.55; N, 17.90; S, 9.11. Found: C, 37.48; H, 4.98; Cl,
C
22
H
25
N
9
O
6
S
2
2
(
1H, d, J ¼ 4:8 Hz), 8.10 (2H, d, J ¼ 6:6 Hz), 9.31 (2H,
ꢁ
1
d, J ¼ 6:6 Hz); IR (KBr) cm 1773, 1616, 1521, 1466,
6.63; N, 18.17; S, 8.95.
1
3
3
395; Anal Calcd for C21
9.13; H, 5.16; Cl, 6.05; N, 17.38; S, 9.95. Found: C,
H
24
N
8
O
5
S
2
2
Æ1.1HClÆ4H O: C,
9.18; H, 5.17; Cl, 6.13; N, 17.26; S, 9.95.
4.32. Compound 2f
2f was obtained as an amorphous powder in 16% yield
from 2f ; H NMR (D
0
1
4
2
.28. Compound 2b
2
O) d 1.34 (3H, t, J ¼ 6:9 Hz), 1.70
(
3H, d, J ¼ 7:5 Hz), 3.30 and 3.70 (1H, ABq,
b was obtained as an amorphous powder in 12% yield
0
J ¼ 17:7 Hz), 4.36 (2H, q, J ¼ 6:9 Hz), 4.4 (1H, m), 5.26
and 5.50 (2H, ABq, J ¼ 14:4 Hz), 5.33 (1H, d,
J ¼ 4:8 Hz), 5.93 (1H, d, J ¼ 4:8 Hz), 8.18 (2H, d,
1
from 2b ; H NMR (DMSO-d
6 2
+D O) d 1.21 (3H, t,
J ¼ 6:9 Hz), 3.05 and 3.52 (2H, ABq, J ¼ 18 Hz), 3.32
ꢁ
1
(
2H, m), 4.16 (2H, q, J ¼ 6:9 Hz), 4.57 (2H, m), 5.0 and
J ¼ 7:2 Hz), 8.79 (2H, d, J ¼ 7:2 Hz); IR (KBr) cm
5
5
9
1
.45 (2H, ABq, J ¼ 14:1 Hz), 5.08 (1H, d, J ¼ 5:1 Hz),
1777, 1727, 1669, 1608, 1517, 1465, 1397; Anal Calcd for
C H N O S Æ1.6HClÆ4.2H O: C, 37.24; H, 4.95; Cl,
.73 (1H, d, J ¼ 5:1 Hz), 7.66 (2H, d, J ¼ 7:5 Hz),
22
25
9
6
2
2
ꢁ
1
.15 (2H, d, J ¼ 7:5 Hz); IR (KBr) cm 1773, 1642,
7.99; N, 17.76; S, 9.04. Found: C, 37.03; H, 5.02; Cl,
7.91; N, 17.85; S, 8.92.
519,
1484,
1456,
1398;
Anal
Calcd
for
C H N O S Æ1.1HClÆ5.2H O: C, 36.96; H, 5.24; Cl,
21
24
8
6
2
2
5.71; N, 16.42; S, 9.4. Found: C, 36.69; H, 5.18; Cl, 5.63;
N, 16.40; S, 9.36.
4.33. Compound 2g
2g was obtained as an amorphous powder in 53% yield
from 2g ; H NMR (D
0
1
4
2
.29. Compound 2c
2
O) d 1.34 (3H, t, J ¼ 6:9 Hz),
3
.06 (2H, t, J ¼ 6:6 Hz), 3.26 and 3.70 (2H, ABq,
c was obtained as an amorphous powder in 25% yield
0
J ¼ 18 Hz), 3.42 (2H, t, J ¼ 6:6 Hz), 4.37 (2H, q,
J ¼ 6:9 Hz), 5.24 and 5.43 (2H, ABq, J ¼ 14:7 Hz), 5.31
(1H, d, J ¼ 4:5 Hz), 5.91 (1H, d, J ¼ 4:5 Hz), 8.12 (2H,
1
from 2c ; H NMR (DMSO-d
6 2
+D O) d 1.21 (3H, t,
J ¼ 6:9 Hz), 2.99 and 3.50 (2H, ABq, J ¼ 18 Hz), 3.04
ꢁ
1
(
2H, m), 3.61 (2H, m), 4.15 (2H, q, J ¼ 6:9 Hz), 4.70 and
d, J ¼ 7:5 Hz), 8.74 (2H, d, J ¼ 7:5 Hz); IR (KBr) cm
5
5
8
1
.22 (2H, ABq, J ¼ 13:2 Hz), 5.07 (1H, d, J ¼ 5:1 Hz),
1774, 1718, 1604, 1517, 1461, 1396; Anal Calcd for
Æ1.2HClÆ4H O: C, 38.22; H, 4.99; Cl, 6.15;
N, 18.23; S, 9.27. Found: C, 38.55; H, 4.91; Cl, 6.27; N,
.73 (1H, d, J ¼ 5:1 Hz), 6.91 (1H, m), 7.11 (1H, m),
C
22
H
25
N
9
O
6
S
2
2
ꢁ
1
.43 (1H, m), 8.65 (1H, m); IR (KBr) cm 1767, 1649,
1455,
556,
1520,
1389;
Anal
Calcd
for
O: C, 37.41; H, 5.38; Cl, 5.26;
18.47; S, 9.35.
C
N, 18.7; S, 9.51. Found: C, 37.61; H, 5.14; Cl, 5.39; N,
21
H
25
N
9
O
5
S
2
Æ1HClÆ5H
2
1
8.98; S, 9.37.
4.34. Compound 2h
2
h was obtained as an amorphous powder in 14% yield
1
0
4
2
.30. Compound 2d
from 2h ; H NMR (DMS0-d ) d 1.22 (3H, t,
6
J ¼ 7:2 Hz), 3.14 (2H, m), 3.37 and 3.52 (2H, ABq,
J ¼ 17:7 Hz), 4.18 (2H, q, J ¼ 7:2 Hz), 4.43 (2H, m),
5.19 (1H, d, J ¼ 5:1 Hz), 5.30 and 5.41 (2H, ABq,
J ¼ 14 Hz), 5.9 (1H, m), 8.03 (2H, d, J ¼ 6:9 Hz), 8.16
(1H, m), 8.36 (2H, br s), 8.76 (2H, d, J ¼ 6:9 Hz), 9.56
d was obtained as an amorphous powder in 22% yield
0
1
from 2d ; H NMR (DMSO-d +D O) d 1.22 (3H, t,
6
2
J ¼ 7:2 Hz), 1.86 (2H, m), 2.89 (2H, t, J ¼ 7:5 Hz), 2.96
and 3.47 (2H, ABq, J ¼ 18 Hz), 3.36 (2H, t, J ¼ 7:5 Hz),
ꢁ
1
4
.15 (2H, q, J ¼ 7:2 Hz), 4.67 and 5.16 (2H, ABq,
(1H, d, J ¼ 8:1 Hz); IR (KBr) cm 1783, 1752, 1676,
J ¼ 14:1 Hz), 5.04 (1H, d, J ¼ 5:1 Hz), 5.69 (1H, d,
1637, 1593, 1532, 1464, 1383; HR-FABMS calcd for
O
þ
J ¼ 5:1 Hz), 6.88 (1H, m), 6.96 (1H, m), 8.42 (1H, m),
C
22
H
26
N
9
7
S
2
[(M+H) ]: 592.1397, found: 592.1395.

4230
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4
2
.35. Compound 2j
J ¼ 7:2 Hz), 1.80 (2H, m), 2.57 (3H, s), 2.94 (2H,
m), 3.20 and 3.55 (2H, ABq, J ¼ 18 Hz), 3.23 (2H, m),
4.17 (2H, q, J ¼ 7:2 Hz), 4.98 and 5.24 (2H,
ABq, J ¼ 13:2 Hz), 5.11 (1H, d, J ¼ 4:8 Hz), 5.78
(1H, d, J ¼ 4:8 Hz), 7.82 (2H, d, J ¼ 7:2 Hz), 8.73
j was obtained as an amorphous powder in 38% yield
0
1
from 2j ; H NMR (D
and 3.78 (2H, ABq, J ¼ 18 Hz), 3.34 (2H, t, J ¼ 5:7 Hz),
.81 (2H, t, J ¼ 5:7 Hz), 4.38 (2H, q, J ¼ 6:6 Hz), 5.35
1H, d, J ¼ 4:8 Hz), 5.48 and 5.74 (2H, ABq,
J ¼ 14:7 Hz), 5.92 (1H, d, J ¼ 4:8 Hz), 8.28 (1H, m),
.96 (1H, d, J ¼ 8:4 Hz), 9.2 (1H, d, J ¼ 6:3 Hz), 9.47
2
O) d 1.34 (3H, t, J ¼ 6:6 Hz), 3.29
ꢁ
1
3
(
(2H, d, J ¼ 7:2 Hz); IR (KBr) cm
1777, 1713,
1671, 1606, 1518, 1466, 1395; Anal Calcd for
Æ1.35HClÆ4H O: C, 38.96; H, 5.36; Cl,
6.47; N, 18.93; S, 8.67. Found: C, 38.89; H, 5.36; Cl,
C
24
H
30
N
10
O
6
S
2
2
8
ꢁ
1
(
1H, s); IR (KBr) cm 1774, 1670, 1623, 1527, 1457,
6.41; N, 19.1; S, 8.81.
1
3
3
395; Anal Calcd for C22
H
25
N
9
O
6
S
8.22; H, 4.99; Cl, 6.15; N, 18.23; S, 9.27. Found: C,
2
2
Æ1.2HClÆ4H O: C,
7.93; H, 5.00; Cl, 6.40; N, 18.23; S, 9.14.
4.40. Compound 2o
2
o was obtained as an amorphous powder in 44% yield
1
0
4
2
.36. Compound 2k
from 2o ; H NMR (DMSO-d +D O) d 1.23 (3H, t,
6
2
J ¼ 7:2 Hz), 2.69–2.97 (2H, m), 3.15 and 3.56 (2H, ABq,
J ¼ 17:7 Hz), 3.24 (2H, m), 3.6 (1H, m), 4.17 (2H,
q, J ¼ 7:2 Hz), 4.97 and 5.29 (2H, ABq, J ¼ 14:4 Hz),
5.10 (1H, d, J ¼ 4:8 Hz), 5.76 (1H, d, J ¼ 4:8 Hz),
k was obtained as an amorphous powder in 37% yield
0
1
from 2k ; H NMR (D O) d 1.34 (3H, t, J ¼ 6:9 Hz),
2
1
.70 (3H, d, J ¼ 7:2 Hz), 3.29 and 3.72 (2H, ABq,
ꢁ
1
J ¼ 18 Hz), 4.37 (2H, q, J ¼ 6:9 Hz), 4.38 (1H, m), 5.34
1H, d, J ¼ 5:1 Hz), 5.40 and 5.66 (2H, ABq,
J ¼ 14:4 Hz), 5.92 (1H, d, J ¼ 5:1 Hz), 8.11 (1H, m),
.53 (1H, m), 8.78 (1H, d, J ¼ 6:3 Hz), 9.58 (1H,s); IR
7.83 (2H, m), 8.74 (2H, m); IR (KBr) cm
1773,
(
1713, 1606, 1517, 1465, 1396; Anal Calcd for
N
C
23
H
28
10
O
7
S
2
Æ1.2HClÆ4.2H O: C, 37.33; H, 5.12; Cl,
2
8
5.75; N, 18.93; S, 8.66. Found: C, 37.21; H, 5.07; Cl,
5.61; N, 19.03; S, 8.78.
ꢁ
1
(
KBr) cm 1775, 1710, 1669, 1615, 1564, 1505, 1459,
1
3
3
395; Anal Calcd for C H N O S Æ1.3HClÆ4.5H O: C,
7.53; H, 5.05; Cl, 6.55; N, 17.90; S, 9.11. Found: C,
7.52; H, 5.03; Cl, 6.54; N, 18.06; S, 9.10.
22
25
9
6
2
2
4.41. Compound 2p
2
p was obtained as an amorphous powder in 31% yield
1
0
4
2
.37. Compound 2l
from 2p ; H NMR (DMSO-d +D O) d 1.23 (3H, t,
6 2
J ¼ 7:0 Hz), 1.8–2.3 (2H, m), 3.09–3.65 (6H, m), 4.16
(2H, q, J ¼ 7:0 Hz), 4.34 (1H, m), 5.06 and 5.27
(2H, ABq, J ¼ 14:4 Hz), 5.15 (1H, d, J ¼ 5:0 Hz), 5.82
(1H, d, J ¼ 5:0 Hz), 7.86 (2H, d, J ¼ 7:0 Hz), 8.72
l was obtained as an amorphous powder in 26% yield
0
1
6 2
from 2l ; H NMR (DMSO-d +D O) d 1.19 (3H, d,
J ¼ 1:8 Hz), 1.22 (3H, t, J ¼ 6:9 Hz), 3.21 and 3.59 (2H,
ABq, J ¼ 17:4 Hz), 3.31–3.35 (3H, m), 4.16 (2H, q,
J ¼ 6:9 Hz), 4.97 and 5.28 (2H, ABq, J ¼ 12:9 Hz), 5.11
ꢁ
1
(2H, d, J ¼ 7:0 Hz); IR (KBr) cm
1778, 1715,
1671, 1608, 1520, 1465, 1396; Anal Calcd for
Æ1.5HClÆ4H O: C, 38.78; H, 5.08; Cl,
(
1H, d, J ¼ 5:4 Hz), 5.77 (1H, d, J ¼ 5:4 Hz), 7.84 (2H, m),
C
24
H
28
N
10
O
6
S
2
2
ꢁ
1
8
1
3
.78 (2H, m); IR (KBr) cm 1773, 1716, 1607, 1519, 1465,
396; Anal Calcd for C23 Æ1.15HClÆ4.7H O: C,
7.78; H, 5.31; Cl, 5.58; N, 19.15; S, 8.77. Found: C, 37.73;
7.15; N, 18.84; S, 8.63. Found: C, 38.68; H, 5.01; Cl,
7.28; N, 18.98; S, 8.40.
H
28
N
10
O
6
S
2
2
H, 5.21; Cl, 5.48; N, 19.37; S, 8.80.
4
.42. Compound 2q
4
2
.38. Compound 2m
2q was obtained as an amorphous powder in 41% yield
0
1
from 2q ; H NMR (D
2
O) d 1.32 (3H, t, J ¼ 7:2 Hz),
m was obtained as an amorphous powder in 22% yield
1
3.02 (2H, t, J ¼ 6:3 Hz), 3.25 and 3.66 (2H, ABq,
J ¼ 18 Hz), 3.58 (2H, t, J ¼ 6:3 Hz), 4.35 (2H, q,
J ¼ 7:2 Hz), 5.14 and 5.35 (2H, ABq, J ¼ 14:7 Hz), 5.30
(1H, d, J ¼ 5:1 Hz), 5.89 (1H, d, J ¼ 5:1 Hz), 7.33 (1H,
d, J ¼ 1:2 Hz), 7.82 (2H, d, J ¼ 7:2 Hz), 8.54 (2H, d,
0
from 2m ; H NMR (DMS-d
6 2
+D O) d 1.23 (3H, t,
J ¼ 7 Hz), 1.78 (2H, m), 2.85 (2H, t, J ¼ 6:6 Hz), 3.22
(
2H, t, J ¼ 6:6 Hz), 3.16 and 3.55 (2H, ABq,
J ¼ 17:6 Hz), 4.16 (2H, q, J ¼ 7 Hz), 5.04 and 5.29 (2H,
ABq, J ¼ 16:2 Hz), 5.13 (1H, d, J ¼ 4:6 Hz), 5.80 (1H,
d, J ¼ 4:6 Hz), 7.88 (2H, m), 8.68 (2H, m); IR (KBr)
ꢁ
1
J ¼ 7:2 Hz), 8.63 (1H, d, J ¼ 1:5 Hz); IR (KBr) cm
1775, 1715, 1671, 1606, 1519, 1466, 1398; Anal Calcd for
Æ1.3HClÆ4H O: C, 39.45; H, 4.81; Cl,
6.06; N, 20.24; S, 8.43. Found: C, 39.20; H, 4.84; Cl,
ꢁ
1
cm 1777, 1710, 1671, 1644, 1606, 1520, 1466, 1398;
C
25
H
27
N
11
O
6
S
2
2
Anal Calcd for C H N O S Æ1.35HClÆ4.7H O: C,
23
28 10
6
2
2
3
3
7.4; H, 5.29; Cl, 6.48; N, 18.96; S, 8.68. Found: C,
7.12; H, 5.01; Cl, 6.60; N, 18.96; S, 8.59.
5.82; N, 20.44; S, 8.43.
4
2
.43. Compound 2r
4
2
.39. Compound 2n
r was obtained as an amorphous powder in 30% yield
1
0
n was obtained as an amorphous powder in 33% yield
0
from 2r ; H NMR (DMSO-d +D O) d 1.22 (3H, t,
J ¼ 7:4 Hz), 3.20–3.40 (4H, m), 3.20 and 3.55 (2H, ABq,
6
2
1
from 2n ; H NMR (DMS-d
6
+D
2
O) d 1.23 (3H, t,

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4221–4231
4231
J ¼ 18 Hz), 4.16 (2H, q, J ¼ 7:4 Hz), 4.92 and 5.30 (2H,
References and notes
ABq, J ¼ 12:9 Hz), 5.12 (1H, d, J ¼ 4:8 Hz), 5.77 (1H,
d, J ¼ 4:8 Hz), 7.93 (2H, m), 8.58 (2H, m); IR (KBr)
1. Yoshizawa, H.; Itani, H.; Ishikura, K.; Irie, T.; Yokoo, K.;
Kubota, T.; Minami, K.; Iwaki, T.; Miwa, H.; Nishitani, Y.
J. Antibiot. 2002, 55, 975.
. Yoshizawa, H.; Kubota, T.; Itani, H.; Ishitobi, H.; Miwa,
H.; Nishitani, Y. Bioorg. Med. Chem. 2004, 12, see
doi:10.1016/j.bmc.2004.05.022.
ꢁ
1
cm 1780, 1716, 1645, 1520, 1464, 1386; Anal Calcd for
C
N, 21.18; S, 8.08. Found: C, 34.59; H, 4.63; Cl, 9.10; N,
23
H
28
N
12
O
7
S
2
Æ2HClÆ4H
2
O: C, 34.81; H, 4.83; Cl, 8.93;
2
2
1.07; S, 8.17.
3
4
. Goto, J.; Sakane, K.; Teraji, T. J. Antibiot. 1984, 37, 557.
. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc.
1
972, 94, 6203.
4
2
.44. Compound 2s
5. (a) Marconi, A.; Pastorin, G.; Ros, T. D.; Spalluto, G.;
Gao, Z.-g.; Jacobson, K. A.; Baraldi, P. G.; Cacciari, B.;
Varani, K.; Moro, S.; Borea, P. A. J. Med. Chem. 2002, 45,
3579; (b) Kong, J. L.; Loach, P. A. J. Heterocycl. Chem.
s was obtained as an amorphous powder in 19% yield
0
1
from 2s ; H NMR (DMSO-d +D O) d 1.23 (3H, t,
6
2
1
980, 17, 733; (c) Pavia, M. R.; Lobbestael, S. J.; Tatlor, C.
P.; Hershenson, F. M.; Miskell, D. L. J. Med. Chem. 1990,
3, 854.
J ¼ 7:2 Hz), 1.70 (2H, m), 3.0–3.3 (4H, m), 3.20 and 3.45
(
2H, ABq, J ¼ 18 Hz), 4.15 (2H, q, J ¼ 7:2 Hz), 4.97
3
and 5.27 (2H, ABq, J ¼ 13:5 Hz), 5.14 (1H, d,
6
. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura, T.;
Hashimoto, N.; Imada, A. J. Antibiot. 1992, 45, 709.
. Mine, Y.; Watanabe, Y.; Sakamoto, H.; Hatano, K.;
Kuno, K.; Higashi, Y.; Kamimura, T.; Matsumoto, Y.;
Tawara, S.; Matsumoto, F.; Kuwahara, S. J. Antibiot.
1993, 46, 71.
J ¼ 5:1 Hz), 5.83 (1H, d, J ¼ 5:1 Hz), 7.82 (2H, m), 8.69
ꢁ
1
(
2H, m); IR (KBr) cm 1774, 1713, 1646, 1521, 1466,
7
1
387; Anal Calcd for C24
H
30
N
8.07; H, 5.1; Cl, 6.09; N, 22.2; S, 8.47. Found: C, 37.90;
12
O
6
S
2
2
Æ1.3HClÆ3.5H O: C,
3
H, 5.07; Cl, 6.18; N, 22.35; S, 8.36.