New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Part 2: Synthesis and structure-activity relationships in the S-3578 series

Article abstract of DOI:10.1016/j.bmc.2004.05.022

Among the prepared novel cephalosporin derivatives related to S-3578, a series of 7β-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetamido] -3-[1-(aminoalkyl)-1H-pyrazolo[4,3-b]pyridinium-4-yl]methyl-3-cephem-4- carboxylate showed potent activity against both MRSA and Pseudomonas aeruginosa, and displayed good water solubility.

Full text of DOI:10.1016/j.bmc.2004.05.022

Bioorganic & Medicinal Chemistry 12 (2004) 4211–4219
New broad-spectrum parenteral cephalosporins exhibiting potent
activity against both methicillin-resistant Staphylococcus aureus
(MRSA) and Pseudomonas aeruginosa. Part 2: Synthesis and
structure–activity relationships in the S-3578 series
Hidenori Yoshizawa,* Tadatoshi Kubota, Hikaru Itani, Hiroyuki Ishitobi, 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
Abstract—Among the prepared novel cephalosporin derivatives related to S-3578, a series of 7b-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-
(Z)-ethoxyiminoacetamido]-3-[1-(aminoalkyl)-1H-pyrazolo[4,3-b]pyridinium-4-yl]methyl-3-cephem-4-carboxylate showed potent
activity against both MRSA and Pseudomonas aeruginosa, and displayed good water solubility.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
bacteria including P. aeruginosa, and displayed good
water solubility.1
Methicillin-resistant Staphylococcus aureus (MRSA)
and Pseudomonas aeruginosa are nosocomial pathogens
associated with serious infections and considerable
mortality. From the need for antibacterial agents
exhibiting highly potent activity against these patho-
gens, we began a search for novel parenteral C-30 qua-
ternary ammonium cephalosporins exhibiting potent
activity against both MRSA and P. aeruginosa.
In the course of studies of S-3578, we found that
introduction of an aminoalkyl group to the 1-position of
imidazo[4,5-b]pyridinium enhanced anti-MRSA activity
(1 and 2 in Fig. 1, and their antibacterial activity
in Table 1) and led to good water solubility.1 As an
aminoalkyl group, the 3-aminopropyl group was the
best match for antibacterial activity against MRSA and
P. aeruginosa.
In our previous paper, we reported that 7b-[2-(5-amino-
1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido]-3-[1-
(3-methylaminopropyl)-1H-imidazo[4,5-b]pyridinium-4-
yl]methyl-3-cephem-4-carboxylate sulfate (S-3578, Fig.
1) showed extremely potent activity against Gram-
positive bacteria including MRSA and Gram-negative
These findings prompted us to extend our synthetic
program to various other cephalosporins in which the 1-
(3-aminopropyl)-1H-imidazo[4,5-b]pyridinium group of
2 was replaced by the 2-(3-aminopropyl)-1H-imi-
dazo[4,5-b]pyiridinium group (3a–b, Table 1) or another
S
N
O
H
N
H2N
1: R=
2: R=
H
S
N
.
NH2
HCl
Me
N
N
O
N
R
N
O
O
S-3578: R=
.
N
H
N
H2SO4
Et
O
Figure 1. Structures of cephalosporin derivatives 1, 2, and S-3578.
* Corresponding author. Tel.: +81-6-6458-5861; fax: +81-6-6458-0987; e-mail: hidenori.yoshizawa@shionogi.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.022

4212
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
Table 1. Antibacterial activities (MIC, lg/mL) of 1-5f, S-3578, CZOP, CFSL, and VCM
S
N
O
H
N
H2N
Q =
N
N
N
S
N
Me
NH
N
N
N
N
N
O
Q
3a
3b
O
R
O
R
Et
O
R
N
H
N
N
N
R
.
N
N
3a-b,4a-c,5a-b:
5c :
NH2 HCl
R=
R=
N
N
.
NH2
R
HCl
4a
4b
4c
H
.
N
HCl
5d :
5e :
R=
R=
Me
Me
N
N
.
N
H
HCl
OH
R
N
N
R
N
N
5f :
.
R=
N
HCl
H
5a
5b-f
Compound
S. a.
MRSA 1
MRSA 2
E. c.
0.2
P. a. 1
1.56
0.78
P. a. 2
1
2
1.56
0.78
12.5
3.13
12.5
3.13
12.5
0.39
3.13
3a
3b
1.56
1.56
6.25
6.25
6.25
6.25
0.2
0.78
0.78
3.13
3.13
12.5
4a
4b
4c
3.13
3.13
1.56
25
25
25
25
6.25
0.2
0.78
1.56
0.78
3.13
6.25
1.56
0.39
0.05
6.25
5a
5b
5c
5d
5e
5f
1.56
0.78
0.78
0.78
0.78
0.78
12.5
6.25
6.25
12.5
6.25
6.25
12.5
6.25
6.25
12.5
0.39
0.2
3.13
0.78
0.78
1.56
1.56
1.56
12.5
3.13
3.13
6.25
6.25
6.25
0.2
0.39
0.2
6.25
6.25
0.2
S-3578
CZOP
CFSL
VCM
0.78
0.78
0.78
1.56
3.13
50
25
3.13
0.39
0.05
0.05
1.56
0.39
3.13
6.25
1.56
6.25
50
25
1.56
0.78
>100
>100
>100
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.
condensed-heterocyclic pyridinium derivative bearing
the aminopropyl group such as the aminopropyl-imi-
dazo[4,5-c]pyridinium (4a–c) or the aminopropyl-pyr-
azolo[4,3-b]pyridinium (5a–b) group. We also prepared
a few other 1-(aminoalkyl)-pyrazolo[4,3-b]pyridinium
cephalosporin derivatives (5c–f). Here we report on the
synthesis and structure–activity relationships of novel
cephalosporins related to S-3578.
cephems 3a–b, 4a–c, 5a, and 5c–f were prepared by a
method similar to that for preparing of 5b using 6 and
the corresponding condensed-heterocyclic pyridine
derivatives 3a0–b0, 4a0–c0, 5a0, and 5c0–f0. (Structures of
3a0–b0, 4a0–c0, and 5a0 are shown in Schemes 2–6, and
those of 5c0–f0 are shown in Fig. 2.)
The methods of synthesizing condensed-heterocyclic
pyridine derivatives are shown in Schemes 2–6. 2-(Am-
inoalkyl)-imidazo[4,5-b]pyridine derivatives 3a0 and 3b0
were synthesized as shown in Scheme 2. Compound 9
was prepared by regio-selective acylation of 2,3-diamino-
pyridine (8) with the corresponding acid using water-
soluble carbodiimide hydrochloride (WSCDÆHCl). The
cyclization of compound 9 was performed by heating in
nitrobenzene to afford compound 10 followed by treat-
ment with di-tert-butyl dicarbonate (Boc2O) in the
presence of 4-(dimethylamino)pyridine (DMAP) to give
compound 3a0. On the other hand, 9 was treated
with methyl iodide in the presence of sodium hydride to
afford a mixture of 11 and 3b0, which was purified by
chromatography on silica gel.
2. Chemistry
Novel C-30 condensed-heterocyclic pyridinium cephems
3a–b, 4a–c, and 5a–f were synthesized by a method
similar to one reported.1 Cephalosporin derivative 5b
was synthesized as shown in Scheme 1. The C-3
iodomethyl cephalosporin intermediate 61 was displaced
by pyrazolopyridine 5b0 to afford iodide salt 7, which
was treated with AlCl3-anisole. Purification by reversed
phase (HP-20) column chromatography yielded cephalo-
sporin derivative 5b as a hydrochloride salt. The other

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
4213
S
N
N
O
H
BocHN
N
S
N
+
a
N
NHBoc
N
N
O
I
O
O
N
Et
OPMB
5b'
6
S
N
O
H
H2N
S
N
S
N
I
b
N
O
N
N
N
O
N
N
N
NBoc
N
N
NH2
HCl
O
O
.
Et
OPMB
O
5b
7
Scheme 1. Reagents and conditions: (a) DMF, RT, 3 h; (b) (i) AlCl3-anisole, CH2Cl2, )30 to 0 °C, 1 h, (ii) purification by HP-20 chromatography.
Boc
N
H
N
NH2
NH2
NHCO
R
a
c
b
R
R
N
N
10
N
R
N
N
N
NH2
9
8
3a'
d
Me
Me
N
R
NCO
R=
NHBoc
+
N
N
N
NH2
11
3b'
Scheme 2. Reagents and conditions: (a) 4-tert-butoxycarbonylamino-butyric acid, WSCDÆHCl, DMF; (b) nitrobenzene, mole sieve, 195 °C, 5 h; (c)
(Boc)2O, DMF, cat. DMAP; (d) NaH, MeI, DMF, 0 °CꢀRT, 18 h.
O
H
H
N
NH2
N
N
R
NH2
NH2
a
b
c
R
N
N
N
N
N
N
12
13
4c'
14
R=
NHBoc
OMe
OMe
Scheme 3. Reagents and conditions: (a) p-anisaldehyde (1.1 equiv), 4 N NaOH (1.1 equiv), EtOH, 18 h; (b) 4-tert-butoxycarbonylamino-butyric acid,
DMF, WSCDÆHCl; (c) (i) 2 N HCl–MeOH (1:2), RT, 1 h, (ii) TFA, 100 °C, 30 min, (iii) (Boc)2O, Et3N.
R
R
HN
N
R
N
HN
N
NH2
OEt
HN
N
NH2
NO2
NO2
NO2
b
c
d
a
N
N
.
HCl
N
15
16
17
18
4a'
R=
NHBoc
Scheme 4. Reagents and conditions: (a) 1,3-diaminopropane (1.5 equiv), EtOH, 130 °C in sealed tube, 2 h; (b) (Boc)2O, THF; (c) H2, Pd (C), MeOH;
(d) (EtO)2CH(OAc), reflux, EtOH.
2-(Aminoalkyl)-imidazo[4,5-c]pyridine derivative 4c0
was prepared, as shown in Scheme 3. The amino group
at the 3-position of diaminopyridine 12 was selectively
protected as imine using p-anisaldehyde. The imine 13
was acylated with carboxylic acid using WSCDÆHCl to
give compound 14. After hydrolysis of the imino group
of 14, the cyclization was achieved by heating in TFA
and the resulting amine was protected using Boc2O to
give compound 4c0.
As shown in Scheme 4, 1-(aminoalkyl)-imidazo[4,5-
c]pyridine derivative 4a0 was prepared. The ethoxy
group of nitropyridine 15 was displaced by 1,3-diamino-
propane to give the amine 16, which was then treated

4214
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
NO2
NO2
NH2
H
N
H
N
Cl
N
N
a
c
R
b
R
R=
NHBoc
N
N+
O-
N+
O-
N
R
19
20
21
4b'
Scheme 5. Reagents and conditions: (a) (i) 1,3-diaminopropane (4 equiv), MeOH, reflux, (ii) dioxane–H2O, Et3N, Boc2O; (b) H2 (4 kgf/cm2), Raney-
Ni, AcOH, MeOH; (c) HC(OEt)3, Ac2O, 90 °C.
H
N
R
N
F
N
N
b
a
N
N
R +
R=
NHBoc
N
CHO
22
N
N
N
23
5b'
5a'
Scheme 6. Reagents and conditions: (a) hydrazine hydrate, 0 °C ꢀ RT (16 h), 130 °C (8 h); (b) R-OSO2Me, NaH, DMF.
R
Boc
Me
N
OH
NHBoc
N
Me
Boc
R=
N
N
N
Boc
5c'
5d'
5e'
5f'
N
Figure 2. Structures of 1-(aminoalkyl)-pyrazolo[4,3-b]pyridine derivatives (5c0–f0).
with Boc2O to give 17. This nitro compound 17 was
reduced using palladium-catalyzed hydrogenation in
methanol to give compound 18, which was treated with
diethoxymethyl acetate in ethanol to give the imidazo-
pyridine derivative 4a0.
Most of the cephalosporin derivatives prepared in this
study showed good antibacterial activity against Gram-
positive bacteria including MRSA and Gram-negative
bacteria including P. aeruginosa, although their anti-
MRSA activity was inferior to that of parent compound
2 and S-3578.
3-(Aminoalkyl)-imidazo[4,5-c]pyridine derivative 4b0
was prepared by the route shown in Scheme 5. The
chloride 192 was substituted by amine and the resulting
amine was protected using Boc2O to give 20. This nitro
compound 20 was reduced by Raney-Ni catalyzed
hydrogenation to give compound 21. Next, the amine 21
was treated with triethyl orthoformate in the presence of
acetic anhydride to afford imidazopyridine 4b0.
3.1. 2-(Aminopropyl)-imidazo[4,5-b]-pyridinium 3a–b
Comparison of 2 and 3a indicates that the 1-substituted
derivative 2 is preferred for activity against Gram-po-
sitive bacteria including MRSA over the 2-substituted
derivative 3a, and the substitution position of the am-
inopropyl group influences the antibacterial activity of
cephalosporins. Introduction of a methyl group at the 1-
position of the imidazopyridine moiety of 3a was ex-
pected to improve antibacterial activity based on our
earlier SAR studies for S-3578 in which introduction of
a methyl group at the 1-position of C-30 imidazo[4,5-
b]pyridine moiety enhanced antibacterial activity.1
However, 3b showed a decrease of activity against
Gram-negative bacteria including P. aeruginosa without
improvement of anti-MRSA activity.
As shown in Scheme 6, 1- and 2-(aminoalkyl)-pyraz-
olo[4,3-b]pyridines, 5a0, and 5b0, were synthesized. Pyr-
azolo[4,3-b]pyridine 23 was prepared by treatment of
3-fluoro-pyridine-2-carbaldehyde 223 with hydrazine
hydrate. The alkylation of pyrazolopyridine 23 using
alkyl methanesulfonate in the presence of sodium hy-
dride gave a mixture of compound 5a0 and 5b0, and they
were purified by chromatography. Other 1-(amino-
alkyl)-pyrazolopyridine derivatives 5c0–e0 were synthe-
sized by a procedure similar to that described for the
preparation of 5b0. Compound 5f0 was derived by
alkylation of 5b0 (see experimental section).
3.2. Aminopropyl-imidazo[4,5-c]pyridinium 4a–c
Both 1-substituted derivative 4a and 3-substituted
derivative 4b showed much less activity against MRSA
than 2, but good activity against Gram-negative bacte-
ria. On the other hand, the 2-substituted derivative 4c
showed 4 times more potent activity against MRSA
than 4a and 4b, and potent activity against Gram-neg-
ative bacteria including P. aeruginosa.
3. Antibacterial activities
The antibacterial activities (MICs) of 3a–b, 4a–c, 5a–f,
and reference compounds, 1, 2, S-3578, cefozopran
(CZOP),4 cefoselis (CFSL),5 and vancomycin (VCM),
against various selected Gram-positive and Gram-neg-
ative bacteria are shown in Table 1. MICs were deter-
mined by the standard serial two-fold agar dilution
method using Mueller–Hinton agar.
The great differences of anti-MRSA activity among 2,
4a, 4b, and 4c suggest that the manner in which the

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
4215
substituted-imidazole ring fuses to the C-30 pyridinium
ring and the substitution position of the aminopropyl
group are important for anti-MRSA.
ceutical, Tokyo, Japan). The overnight cultures of bac-
terial strains in Mueller Hinton broth (Becton
Dickinson) were diluted to about 106 CFU/mL. Bacte-
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.
3.3. Aminopropyl-pyrazolo[4,3-b]pyridinium 5a–b
The 2-substituted derivative 5a showed relatively weak
activity against all bacteria, especially against P. aeru-
ginosa. In contrast, the 1-substituted derivative 5b
showed potent activity equal to 2 against Staphylococcus
aureus SMITH and Gram-negative bacteria including
P. aeruginosa, although anti-MRSA activity of 5b was
slightly inferior to 2. The potent antibacterial activity of
5b might be due to the structural resemblance to that of 2.
4.2. [3-(2-Amino-pyridin-3-ylcarbamoyl)-propyl]-car-
bamic acid tert-butyl ester (9)
To a solution of 2,3-diaminopyridine (3.6 g, 33 mmol) in
DMF (45 mL) was added 4-tert-butoxycarbonylamino-
butyric acid (8.0 g, 40 mmol) and water-soluble carbo-
diimide hydrochloride (WSCDÆHCl) (7.64 g, 40 mmol)
under cooling in an ice-water bath. The reaction mixture
was stirred at room temperature for 3 days. After
evaporation of the solvent, the residue was extracted
with methyl ethyl ketone and washed with brine.
The organic layer was dried over Na2SO4 and fil-
tered. The filtrate was concentrated in vacuo and crys-
tallization was performed from the residue with
MeCN and Et2O to obtain compound 9 (6.0 g, 65%
yield); MP 150–152 °C; 1H NMR (CDCl3) d 1.46 (9H, s),
1.87 (2H, m), 2.40 (2H, m), 3.28 (2H, m), 4.92 (1H, m),
5.06 (2H, brs), 6.70 (1H, dd, J ¼ 4:8 Hz, 7.8 Hz), 7.84
(1H, d, J ¼ 7:8 Hz), 7.91 (1H, d, J ¼ 4:8 Hz), 8.54
(1H, s).
Among of 3a–b, 4a–c, and 5a–b, 1-(aminopropyl)-py-
razolopyridinium 5b showed the best balanced activity
against Gram-positive bacteria including MRSA and
Gram-negative bacteria including P. aeruginosa, and
displayed also good water-solubility (>50 mg/mL at
neutral pH). Therefore, we prepared a few analogs of
compound 5b. We modified the length of the carbon
chain of the aminopropyl group (5c), and introduced a
substituent, such as a methyl group (5d, 5e) or a hy-
droxyethyl group (5f), on the primary amine in a man-
ner similar to our earlier SAR studies for S-3578.
Antibacterial activity did not improve, but 5c, 5e, and 5f
displayed the same activity against Gram-positive bac-
teria including MRSA as compound 5b while retaining
potent activity against Gram-negative bacteria including
P. aeruginosa and acceptable water solubility (>50 mg/
mL at neutral pH) for intravenous administration.
4.3. [3-(1H-Imidazo[4,5-b]pyridin-2-yl)-propyl]-carbamic
acid tert-butyl ester (10)
A solution of 9 (3.0 g, 10 mmol) in nitrobenzene (15 mL)
was heated at 195–200 °C in the presence of molecular
sieves for 5 h. After evaporation of the solvent, the res-
idue was purified by silica-gel column chromatography
In conclusion, the described chemical modification made
by changing the substitution position of the aminoalkyl
function and a change of the C-30 condensed-heterocy-
clic pyridinium moiety of compound 2 demonstrated
that 5b, 5c, 5e, and 5f displayed potent antibacterial
activity and good water solubility.
1
to give 10 as a colorless oil (290 mg); H NMR (CDCl3)
d 1.44 (9H, s), 2.05 (2H, m), 3.06 (2H, m), 3.26 (2H, m),
5.08 (1H, brs), 7.20 (1H, m), 7.95 (1H, d, J ¼ 7:8 Hz),
8.36 (1H, d, J ¼ 4:8 Hz).
4. Experimental
4.4. 2-(3-tert-Butoxycarbonylamino-propyl)-imidazo[4,5-
b]pyridine-1-carboxylic acid tert-butyl ester (3a0)
MPs were determined on a Yanagimoto micro melting
point apparatus and uncorrected. IR spectra were taken
on JASCO IR-700. 1H NMR spectra were recorded with
a Varian Gemini-300 (300 MHz) or Varian Gemini-200
(200 MHz) spectrometer. Chemical shifts are reported in
ppm from 2,2-dimethyl-2-silapentane-5-sulfonate (DSS
in D2O) or TMS (in CDCl3 and DMSO-d6) as internal
standard. The following abbreviations are used: s sin-
glet, d doublet, dd double doublet, t triplet, q quartet, m
multiplet, ABq AB quartet, brs broad singlet. Column
chromatography was carried out on Merck Kieselgel
and Mitsubushi Chemical HP-20.
To a solution of 10 (290 mg, 1.0 mmol) in DMF (3 mL)
was added DMAP (10 mg) and Boc2O (0.54 mL,
2.3 mmol), and the reaction mixture was stirred at room
temperature for 3 days. After evaporation of the solvent,
the residue was purified by silica-gel column chroma-
tography to give 3a0 as a pale yellow oil (320 mg, 8.8%
1
from 9); H NMR (CDCl3) d 1.42 (9H, s), 1.72 (9H, s),
2.14 (2H, m), 3.30 (4H, m), 4.87 (1H, brs), 7.25 (1H, m),
8.16 (1H, d, J ¼ 8:4 Hz), 8.82 (1H, m).
4.5. [3-(1-Methyl-1H-imidazo[4,5-b]pyridin-2-yl)-propyl]-
carbamic acid tert-butyl ester (3b0)
4.1. Measurement of in vitro antibacterial activity
MIC was determined by a serial twofold dilution
method with Sensitivity Disk Agar-N (Nissui Pharma-
To a solution of 9 (5.88 g, 20 mmol) in DMF (59 mL)
was added 60% NaH (880 mg, 22 mmol) under cooling

4216
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
in an ice-water bath, and the mixture was stirred at
room temperature for 1 h. To the mixture was added
dropwise MeI (3.1 g, 21 mmol) under cooling in an ice-
water bath. The reaction mixture was stirred at the same
temperature for 2 h and then stirred at room tempera-
ture for 16 h. After addition of a mixture of brine and
EtOAc, the organic layer was washed with brine, and
dried over Na2SO4, and filtered. The filtrate was con-
centrated in vacuo. The residue was purified by silica-gel
column chromatography. The fraction that was eluted
first with CHCl3/MeOH ¼ 9/1 was concentrated to give
11 (1.3 g, 21% yield) as a crystalline solid; MP 148–
4.8. [3-(1H-Imidazo[4,5-b]pyridin-2-yl)-propyl]-carbamic
acid tert-butyl ester (4c0)
To a mixture of 14 (6.1 g, 14.7 mmol) and MeOH
(30 mL) was added 2 N HCl (15 mL), and the mixture
was stirred at room temperature for 1 h. After addition
of 2 N NaOH (15 mL) and CHCl3, the organic layer was
washed with H2O, dried over MgSO4, and filtered. The
filtrate was concentrated and the residue was chroma-
tographed on silica gel column to give [3-(3-amino-
pyridin-4-ylcarbamoyl)-propyl]-carbamic acid tert-butyl
1
ester (2.94 g) as a crystalline solid; MP 149–151 °C; H
1
149 °C; H NMR (CDCl3) d 1.41 (9H, s), 1.76 (2H, m),
NMR (CDCl3) d 1.45 (9H, s), 1.87 (2H, m), 2.43 (2H,
m), 3.27 (2H, m), 4.18 (2H, brs), 4.93 (1H, m), 7.72 (1H,
d, J ¼ 4:8 Hz), 8.00 (1H, d, J ¼ 4:8 Hz), 8.11 (1H, s),
9.01 (1H, brs). This compound (2.0 g, 6.79 mmol) was
dissolved in TFA (2.6 mL) and the solution was stirred
at 100 °C for 30 min. After concentration in vacuo, the
residue was chromatographed on HP-20 resin. The
solution containing the target material was concentrated
and lyophilized to give 3-(1H-imidazo[4,5-c]pyridin-2-
yl)-propylamine TFA salt (2.67 g) as an amorphous
powder. To a suspension of 3-(1H-imidazo[4,5-c]pyri-
din-2-yl)-propylamine TFA salt (1.7 g, 4.1 mmol) in
DMF (15 mL) and CHCl3 (15 mL) was added Et3N
(1.16 mL, 8.2 mmol) under cooling in an ice-water bath,
followed by dropwise addition of Boc2O (0.96 mL,
4.1 mmol). The reaction mixture was stirred at room
temperature for 16 h. After addition of ice-cold water,
sat. NaHCO3 and EtOAc, the organic layer was washed
with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Crystallization was done using MeCN
and Et2O to give compound 4c0 (0.89 g, 50% yield from
2.12 (2H, m), 3.09 (2H, m), 3.17 (3H, m), 4.72 (1H, m),
4.74 (2H, brs), 6.71 (1H, dd, J ¼ 4:8 Hz, 7.8 Hz), 7.28
(1H, d, J ¼ 7:8 Hz), 8.09 (1H, d, J ¼ 4:8 Hz).
The fraction that was eluted second with
CHCl3:MeOH ¼ 9:1 was concentrated to give 3b0 (1.1 g,
1
18%) as a crystalline solid; MP 99–100 °C; H NMR
(CDCl3) d 1.40 (9H, s), 1.24 (2H, m), 2.12 (2H, m), 3.27
(2H, m), 3.74 (3H, s), 5.05 (1H, m), 7.15 (1H, dd,
J ¼ 4:8 Hz, 7.8 Hz), 7.58 (1H, d, J ¼ 7:8 Hz), 8.48 (1H,
d, J ¼ 4:8 Hz).
4.6. 3-(4-Methoxy-benzylidene)-pyridine-3,4-diamine (13)
To
a
suspension of 2,3-diaminopyridine (21.8 g,
200 mmol) in EtOH (200 mL) was added p-anisaldehyde
(29.9 g, 220 mmol) and 4 N NaOH (55 mL), and the
mixture was stirred at room temperature for 16 h. The
mixture was neutralized to pH 7 using 5 N HCl (44 mL).
After addition of EtOAc, the organic layer was washed
with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Crystallization was performed from
the residue with Et2O and hexane to give compound
13 (23.6 g, 52% yield); MP 124–125 °C; 1H NMR
(DMSO-d6) d 3.83 (3H, s), 5.91 (2H, s), 6.60 (1H, d,
J ¼ 5:7 Hz), 7.06 (2H, d, J ¼ 8:7 Hz), 7.89 (1H, d,
J ¼ 5:4 Hz), 7.94 (2H, d, J ¼ 8:7 Hz), 7.98 (1H, s), 8.59
(1H, s).
1
14); MP 114–115 °C; H NMR (CDCl3) d 1.49 (9H, s),
1.94 (2H, m), 3.01 (2H, m), 3.25 (2H, m), 5.01 (1H, m),
7.51 (1H, d, J ¼ 5:4 Hz), 8.38 (1H, d, J ¼ 5:4 Hz), 8.96
(1H, s).
4.9. 1-(3-Nitro-pyridin-4-yl)-propane-1,3-diamine (16)
To a solution of 15 (4.2 g, 22 mmol) in EtOH (15 mL)
was added 1,3-diaminopropane (2.45 g, 33 mmol) and
the mixture was stirred at 130 °C in a sealed tube for 2 h.
After evaporation of the solvent, the residue was diluted
with THF (40 mL). The precipitate was filtered off, the
filtrate was concentrated, and the residue was chroma-
tographed on silica gel column to give 16 (4.14 g, 94%
yield) as a reddish oil; 1H NMR (CDCl3) d 1.87 (2H, m),
2.91 (2H, m), 3.44 (2H, m), 6.75 (1H, d, J ¼ 6:2 Hz),
8.28 (1H, d, J ¼ 6:2 Hz), 8.65 (1H, brs), 9.20 (1H, s).
4.7. [3-(3-{[1-(4-Methoxy-phenyl)-methylidene]-amino}-
pyridin-4-ylcarbamoyl)-propyl]-carbamic acid tert-butyl
ester (14)
To a solution of 13 (3.0 g, 13.2 mmol) in DMF (35 mL)
was added 4-tert-butoxycarbonylamino-butyric acid
(3.68 g, 18 mmol) and WSCDÆHCl (3.47 g, 18 mmol)
under cooling in an ice-water bath. The reaction mixture
was stirred at room temperature for 2 days. The reaction
mixture was poured into H2O. After addition of a
mixture of EtOAc and CHCl3, the organic layer was
washed with H2O, dried over Na2SO4, and filtered. The
filtrate was concentrated in vacuo and crystallization
was performed from the residue with EtOH, Et2O and
hexane to give compound 14 (5.2 g, 95% yield); MP 129–
4.10. [3-(3-Nitro-pyridin-4-ylamino)-propyl]-carbamic
acid tert-butyl ester (17)
To a solution of 16 (4.1 g, 21 mmol) in THF (50 mL) was
added dropwise Boc2O (5.3 mL, 23 mmol) and the
reaction mixture was stirred at room temperature for
16 h. After evaporation, the residue was chromato-
graphed on silica gel column to give 17 (4.68 g, 72%
yield) as a pale yellow oil; 1H NMR (CDCl3) d 1.44 (9H,
s), 1.90 (2H, m), 3.28 (2H, m), 3.41 (2H, m), 4.72 (1H,
1
130 °C; H NMR (CDCl3) d 1.40 (9H, s), 1.93 (2H, m),
2.50 (2H, m), 3.24 (2H, m), 3.90 (3H, s), 4.80 (1H, m),
7.03 (2H, d, J ¼ 8:7 Hz), 7.89 (2H, d, J ¼ 8:7 Hz), 8.32
(1H, 2), 8.38 (1H, s), 8.52 (1H, s), 8.62 (1H, s).

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
4217
m), 6.70 (1H, d, J ¼ 6:3 Hz), 8.29 (1H, d, J ¼ 6:3 Hz),
organic layer was concentrated and the residue was
chromatographed on silica gel column to give 21 (750 mg,
87% yield) as a pale yellow oil; H NMR (DMSO-d6) d
9.21 (1H, s).
1
1.38 (9H, s), 1.70 (2H, m), 3.02 (2H, m), 3.35 (2H, m),
4.39 (1H, m), 5.39 (2H, s), 6.41 (1H, d, J ¼ 5:1 Hz), 6.87
(1H, m), 7.52 (1H, d, J ¼ 5:1 Hz), 7.53 (1H, s).
4.11. [3-(3-Amino-pyridin-4-ylamino)-propyl]-carbamic
acid tert-butyl ester (18)
To a solution of 17 (4.67 g, 15.8 mmol) in MeOH
(150 mL) was added 10% Pd–C (1.0 g). The mixture was
stirred under hydrogen atmosphere for 90 min under the
ambient pressure at room temperature. After filtration
of the catalyst, the filtrate was concentrated in vacuo to
give 18 (4.67 g) as a pale yellow oil; 1H NMR (CDCl3) d
(9H, s), 1.78 (2H, m), 3.22 (2H, m), 3.27 (2H, m), 3.38
(2H, brs), 4.59 (1H, m), 4.72 (1H, m), 6.44 (1H, d,
J ¼ 5:7 Hz), 7.88 (1H, s), 7.94 (1H, d, 5.7 Hz).
4.15. (3-Imidazo[4,5-c]pyridin-3-yl-propyl)-carbamic acid
tert-butyl ester (4b0)
To a mixture of 21 (714 mg, 2.68 mmol) and triethyl
orthoformate (5 mL) was added acetic anhydride
(1 mL), and the mixture was stirred at 90 °C for 15 min
and concentrated in vacuo. The residue was chromato-
graphed on silica gel column to give 4b0 (580 mg, 88%
1
yield) as a pale yellow oil; H NMR (DMSO-d6) d 1.38
(9H, s), 1.95 (2H, m), 2.94 (2H, m), 4.35 (2H, m), 6.98
(1H, m), 7.66 (1H, d, J ¼ 5:6 Hz), 8.34 (1H, d,
J ¼ 5:6 Hz), 8.44 (1H, s), 9.01 (1H, s).
4.12. (3-Imidazo[4,5-c]pyridin-1-yl-propyl)-carbamic acid
tert-butyl ester (4a0)
A mixture of 18 (4.67 g) and acetic acid diethoxymethyl
ester (13 mL) was stirred at room temperature for 16 h.
After concentration of the mixture, the residue was
dissolved in EtOH (15 mL) and the mixture was refluxed
for 90 min, and concentrated in vacuo. The residue was
chromatographed on silica gel column to give 4a0 (3.5 g,
62% yield from 17) as a colorless oil; 1H NMR (CDCl3)
d 1.46 (9H, s), 2.11 (2H, m), 3.20 (2H, m), 4.28 (2H, m),
4.82 (1H, m), 7.38 (1H, d, J ¼ 6:6 Hz), 8.09 (1H, s), 8.47
(1H, d, J ¼ 6:6 Hz), 9.13 (1H, s).
4.16. 1H-Pyrazolo[4,3-b]pyridine (23)
3-Fluoro-pyridine-2-carbaldehyde (22) (6.67 g, 53 mmol)
was dissolved in hydrazine monohydrate (40 mL) under
cooling in an ice-water bath, and the mixture was stirred
at the same temperature for 5 min. After removal of the
ice-water bath, the mixture was stirred at room tem-
perature overnight and then was heated at 130 °C for
5 h. The reaction mixture was concentrated in vacuo and
to the residue was added H2O and NaHCO3 (4.45 g,
52.9 mmol). The aqueous layer was extracted with
EtOAc. The organic layer was dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was
purified by silica-gel column chromatography to give
compound 23 (3.6 g, 57% yield) as a crystalline solid;
4.13. [3-(4-Nitro-1-oxy-pyridin-3-ylamino)-propyl]-
carbamic acid tert-butyl ester (20)
To a solution of 3-chloro-4-nitro-pyridine 1-oxide (19)
(3.0 g, 17.2 mmol) in MeOH (30 mL) was added a mix-
ture of 1,3-diaminopropane (5.7 mL, 68.7 mmol) and
MeOH (110 mL) and the reaction mixture was stirred at
room temperature for 30 min and refluxed for 1 h. After
evaporation of the solvents, the residue was dissolved in
a mixture of dioxane (5 mL) and H2O (5 mL), followed
by addition of Et3N (2.6 mL) and Boc2O (7.4 mL,
34 mmol). The reaction was stirred for 16 h and con-
centrated in vacuo. The residue was chromatographed
on silica gel column to give 20 (1.0 g, 18% yield) as a pale
1
MP 95–96 °C (lit.6 MP 105–106 °C); H NMR (DMSO-
d6) 7.36 (1H, dd, J ¼ 4:2 Hz, 8.7 Hz), 8.01 (1H, d,
J ¼ 8:7 Hz), 8.29 (1H, s), 8.51 (1H, d, J ¼ 4:2 Hz); Anal.
Calcd for C6H5N3: C, 60.5; H, 4.23; N, 35.27. Found: C,
60.75; H, 4.19; N, 35.25.
4.17. (3-Pyrazolo[4,3-b]pyridin-1-yl-propyl)-carbamic
acid tert-butyl ester (5b0) and (3-pyrazolo[4,3-b]pyridin-2-
yl-propyl)-carbamic acid tert-butyl ester (5a0)
1
yellow oil; H NMR (DMSO-d6) d 1.37 (9H, s), 1.67
(2H, m), 3.03 (2H, m), 3.37 (2H, m), 6.92 (1H, m), 7.48
(1H, d, J ¼ 7:2 Hz), 8.02 (1H, d, J ¼ 7:2 Hz), 8.17 (1H,
s), 8.24 (1H, m).
To a solution of 1H-pyrazolo[4,3-b]pyridine (23)
(357 mg, 3 mmol) in DMF (4 mL) was added 60% NaH
(144 mg, 3.6 mmol) and the mixture was stirred at room
temperature for 20 min. To the mixture was added
a solution of 3-tert-butoxycarbonylaminopropyl me-
thanesulfonate (911 mg, 3.6 mmol) in DMF (2 mL). The
reaction mixture was stirred at the same temperature
for 1 h. After addition of EtOAc and ice water, the or-
ganic phase was washed with water and brine, dried
over Na2SO4, and filtered. After concentration of the
filtrate, the residue was chromatographed on silica gel
column. The fraction eluted with EtOAc/toluene (2/1)
was concentrated to give 5b0 (330 mg, 39% yield) as a
4.14. [3-(4-Amino-pyridin-3-ylamino)-propyl]-carbamic
acid tert-butyl ester (21)
To a solution of 20 (1.0 g, 3.2 mmol) in MeOH (60 mL)
and AcOH (1.5 mL) was added Raney-Ni (1 mL, slurry
in water). The mixture was stirred under hydrogen
atmosphere at the pressure of 4 kgf/cm2. After filtration
of the catalyst, the filtrate was concentrated in vacuo.
The residue was dissolved in H2O, alkalized by addition
of saturated NaOH and extracted with Et2O. The
1
pale yellow oil; H NMR (CDCl3) d 1.42 (9H, s), 2.12
(2H, m), 3.11 (2H, m), 4.46 (2H, t, J ¼ 9:9 Hz), 4.8 (1H,

4218
H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
brs), 7.29 (1H, m), 7.77 (1H, d, J ¼ 12:9 Hz), 8.24 (1H,
s), 8.58 (1H, d, J ¼ 6:6 Hz).
water (2 mL), and AcOH (1.7 mL), then this was stirred
at room temperature for 18 h. The reaction mixture was
poured into a mixture of ice water and EtOAc. The
aqueous layer was adjusted to pH 8 with Na2CO3 and
extracted with EtOAc. The organic layer was dried over
Na2SO4, filtered, and concentrated. The residue was
chromatographed on silica gel column to obtain 5f0 as a
pale yellow oil in 82% yield from 5b0; 1H NMR (CDCl3)
d 1.38 (9H, s), 2.22 (2H, m), 3.32 (2H, m), 3.37 (2H, m),
3.70 (2H, m), 4.41 (2H, t, J ¼ 6:8 Hz), 7.30 (1H, m), 7.77
(1H, d, J ¼ 8:6 Hz), 8.24 (1H, s), 8.58 (1H, m).
Concentration of the fraction eluted with EtOAc/tolu-
ene (4/1) gave a regioisomer 5a0 (260 mg, 31% yield) as a
1
pale yellow oil; H NMR (CDCl3) d 1.43 (9H, s), 2.21
(2H, m), 3.16 (2H, m), 4.53 (2H, t, J ¼ 9:9 Hz), 4.8 (1H,
brs), 7.22 (1H, m), 8.03 (1H, d, J ¼ 14:2 Hz), 8.24 (1H,
s), 8.57 (1H, d, J ¼ 7:8 Hz).
The pyrazolo[4,3-b]pyridine derivatives, 5c0, 5d0 and 5e0
were prepared by the procedure similar to that described
for the preparation of 5b0. In the preparation of 5c0, 5d0,
and 5e0, an undesired regioisomer, 2-(substituted)-pyr-
azolo[4,3-b]pyridine was also produced.
4.22. 7b-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxy-
iminoacetamido]-3-[1-(3-aminopropyl)-1H-pyrazolo[4,3-
b]pyridinium-4-yl]methyl-3-cephem-4-carboxylate hydro-
chloride (5b)
4.18. (2-Pyrazolo[4,3-b]pyridin-1-yl-ethyl)-carbamic acid
tert-butyl ester (5c0)
To a solution of pyrazolopyridine (5b0) (330 mg,
1.19 mmol) in DMF (2 mL) was added a solution of the
iodomethyl derivative (6) (1.18 g, 1.55 mmol) in a mix-
ture of DMF (2 mL) and DMSO (1 mL). The reaction
mixture was stirred at room temperature for 3 h, and the
mixture was poured into iPr2O. The precipitate was fil-
tered and dried in vacuo to give pyrazolopyridinium 7
(1.3 g). To a solution of 7 (1.3 g) in CH2Cl2 (20 mL) was
added a solution of AlCl3 (1.15 g, 8.33 mmol) in anisole
(5 mL) at )30 °C and the reaction mixture was stirred
under cooling in an ice-water bath for 1 h. After addi-
tion of a mixture of 1 N HCl (40 mL) and H2O (40 mL),
the aqueous layer was washed with Et2O and concen-
tration in vacuo. The aqueous solution was chromato-
graphed on HP-20 resin. The target product was eluted
with 0.001 N HCl and the solution was concentrated in
vacuo and lyophilized to give 5b hydrochloride (120 mg,
18% yield from 5b0) as an amorphous powder; 1H NMR
(DMSO-d6+D2O) d 1.19 (3H, t, J ¼ 7:2 Hz), 2.22 (2H,
m), 2.88 (2H, m), 3.2 and 3.6 (2H, ABq, J ¼ 19 Hz), 4.13
(2H, q, J ¼ 7:2 Hz), 4.78 (2H, m), 5.18 (1H, d,
J ¼ 7:8 Hz), 5.84 (2H, brs), 5.88 (1H, d, J ¼ 7:8 Hz),
8.15 (1H, m), 8.83 (1H, s), 9.03 (1H, d, J ¼ 9:3 Hz), 9.22
(1H, d, J ¼ 12:6 Hz); IR (KBr) cmꢁ1 1781, 1673, 1631,
1525, 1475, 1448, 1387; Anal. Calcd for
C23H26N10O5S2Æ2.9HClÆ5H2O: C, 35.3; H, 5.01; Cl,
13.14; N, 17.9; S, 8.20. Found: C, 35.2; H, 4.66; Cl,
13.05; N, 17.61; S, 7.92.
Compound 5c0 was obtained as a pale yellow oil in 48%
yield from 23 using methanesulfonic acid 2-tert-but-
oxycarbonylamino-ethyl ester; 1H NMR (CDCl3) d 1.40
(9H, s), 3.64 (2H, q, J ¼ 5:8 Hz), 4.52 (2H, t,
J ¼ 5:2 Hz), 4.80 (1H, bs), 7.29 (1H, m), 7.80 (1H, d,
J ¼ 8:8 Hz), 8.24 (1H, s), 8.58 (1H, m).
4.19. Methyl-(2-pyrazolo[4,3-b]pyridin-1-yl-ethyl)-car-
bamic acid tert-butyl ester (5d0)
Compound 5d0 was obtained as a pale yellow oil in 26%
yield from 23 using methanesulfonic acid 2-(tert-butox-
ycarbonyl-methyl-amino)-ethyl ester and THF (as the
1
solvent); H NMR (CDCl3) d 1.44 (9H, s), 2.38 (3H, s),
3.66 (2H, t, J ¼ 6:0 Hz), 4.56 (2H, m), 7.35 (1H, m), 7.70
(1H, m), 8.26 (1H, s), 8.58 (1H, m).
4.20. Methyl-(3-pyrazolo[4,3-b]pyridin-1-yl-propyl)-car-
bamic acid tert-butyl ester (5e0)
Compound 5e0 was obtained as a pale yellow oil in 53%
yield from 23 using methanesulfonic acid 3-(tert-butox-
ycarbonyl-methyl-amino)-propyl ester; 1H NMR
(CDCl3) d 1.40 (9H, s), 2.18 (2H, m), 2.88 (3H, s), 3.28
(2H, t, J ¼ 6:0 Hz), 4.39 (2H, t, J ¼ 6:8 Hz), 7.32 (1H,
m), 7.76 (1H, d, J ¼ 8:8 Hz), 8.24 (1H, s), 8.57 (1H, d,
J ¼ 4:4 Hz).
Compound 3a: 3a was obtained as an amorphous
powder in 33% yield from 3a0; 1H NMR (DMSO-
d6+D2O) d 1.18 (3H, t, J ¼ 7:2 Hz), 2.13 (2H, m), 2.96
(2H, m), 3.11 (2H, m), 3.2–3.6 (2H, m), 4.11 (2H, 2H, q,
J ¼ 7:2 Hz), 5.10 (1H, d, J ¼ 5:1 Hz), 5.19 and 5.66 (2H,
ABq, J ¼ 13:8 Hz), 5.75 (1H, d, J ¼ 5:1 Hz), 8.09 1H, d,
J ¼ 6:6 Hz), 8.86 (1H, d, J ¼ 6:6 Hz), 9.76 (1H, s); IR
(KBr) cmꢁ1 1774, 1614, 1527, 1482, 1461, 1402; Anal.
Calcd for C23H26N10O5S2Æ1.3HClÆ4.4H2O: C, 38.73; H,
5.10; Cl, 6.46; N, 19.64; S, 8.99. Found: C, 38.96; H,
5.05; Cl, 6.40; N, 19.65; S, 8.69.
4.21. (2-Hydroxy-ethyl)-(3-pyrazolo[4,3-b]pyridin-1-yl-
propyl)-carbamic acid tert-butyl ester (5f0)
To a solution of compound 5b0 (460 mg, 1.81 mmol) and
(2-bromo-ethoxy)-triethylsilane (866 mg, 3.62 mmol) in
DMF (5 mL) was added 60% NaH (145 mg, 3.62 mmol)
and the mixture was stirred at room temperature for 1 h
and then stirred at 40 °C for 30 min. Next, it was poured
into a mixture of cold water and EtOAc. The organic
layer was washed successively with water and brine,
dried over Na2SO4, and filtered off. After evaporation,
the residue was dissolved in a mixture of THF (8 mL),
Compound 3b: 3b was obtained as an amorphous
powder in 26% yield from 3b0; 1H NMR (DMSO-
d6+D2O) d 1.20 (3H, t, J ¼ 7:2 Hz), 2.21 (2H, m), 2.99
(2H, m), 3.13 (2H, m), 3.18 and 3.37 (2H, ABq,

H. Yoshizawa et al. / Bioorg. Med. Chem. 12 (2004) 4211–4219
4219
J ¼ 16:8 Hz), 3.87 (3H, s), 4.13 (2H, q, J ¼ 7:2 Hz), 4.84
(1H, d, J ¼ 5:1 Hz), 5.21 and 6.55 (2H, ABq,
J ¼ 13:8 Hz), 5.79 (1H, d, J ¼ 5:1 Hz), 7.78 (1H, m),
8.73 (1H, d, J ¼ 8:1 Hz), 8.90 (1H, d, J ¼ 6:3 Hz); IR
(KBr) cmꢁ1 1769, 1671, 1617, 1524, 1475, 1456; Anal.
Calcd or C24H28N10O5S2Æ1.3HClÆ5.5H2O: C, 38.58; H,
5.44; Cl, 6.17; N, 18.75; S, 8.58. Found: C, 38.84; H,
5.51; Cl, 6.28; N, 18.97; S, 8.35.
5.91 (1H, m), 8.19 1H, m), 8.88 (1H, m), 9.08 (1H, d,
J ¼ 6:0 Hz), 9.20 (1H, d, J ¼ 8:7 Hz); IR (KBr) cmꢁ1
1781, 1673, 1631, 1525, 1475, 1448, 1387; HR-FABMS
calcd for C22H25N10O5S2 [(M+H)þ]: 573.1451, found:
573.1453.
Compound 5d: 5d was obtained as an amorphous
1
powder in 38% yield from 5d0; H NMR (D2O) d 1.00
(3H, t, J ¼ 7:2 Hz), 2.50 (3H, s), 2.96 and 3.26 (2H,
ABq, J ¼ 18 Hz), 3.46 (2H, t, J ¼ 5:7 Hz), 4.04 (1H, q,
J ¼ 7:2 Hz), 4.72 (2H, t, J ¼ 5:7 Hz), 4.90 (1H, d,
J ¼ 5:1 Hz), 5.47 and 5.55 (2H, ABq, J ¼ 14:7 Hz), 5.58
(1H, d, J ¼ 5:1 Hz), 7.77 (1H, m), 8.49 (1H, d,
J ¼ 0:9 Hz), 8.64 (1H, d, J ¼ 8:8 Hz), 8.73 (1H, d,
J ¼ 5:7 Hz); IR (KBr) cmꢁ1 1779, 1663, 1621, 1524,
1470, 1390; Anal. Calcd for C23H26N10O5S2Æ
1.5HClÆ4.8H2O: C, 37.96; H, 5.14; Cl, 7.31; N, 19.24;
S, 8.81. Found: C, 37.7; H, 4.85; Cl, 7.39; N, 19.14; S,
8.80.
Compound 4a: 4a was obtained as an amorphous
1
powder in 36% yield from 4a0; H NMR (DMSO-d6) d
1.20 (3H, t, J ¼ 7:2 Hz), 2.18 (2H, m), 2.85 (2H, m), 3.08
and 3.60 (2H, ABq, J ¼ 18:3 Hz), 4.11 (2H, q,
J ¼ 7:2 Hz), 4.56 (2H, t, J ¼ 6:6 Hz), 5.10 (1H, d,
J ¼ 4:8 Hz), 5.20 and 5.69 (2H, ABq, J ¼ 14:1 Hz), 5.75
(1H, d, J ¼ 4:8 Hz), 8.50 (1H, d, J ¼ 6:6 Hz), 8.97
(1H, s), 9.17 (1H, d, J ¼ 6:6 Hz), 9.94 (1H, s); IR
(KBr) cmꢁ1 1770, 1641, 1517, 1457; HR-FABMS calcd
for C23H27N10O5S2 [(M+H)þ]: 587.1607, found:
587.1606.
Compound 5e: 5e was obtained as an amorphous
1
Compound 4b: 4b was obtained as an amorphous
1
powder in 20% yield from 5e0; H NMR (DMSO-d6) d
powder in 33% yield from 4b0; H NMR (DMSO-d6) d
1.20 (3H, t, J ¼ 7:2 Hz), 2.3 (2H, m), 2.5 (3H, s), 2.9
(2H, m), 4.13 (2H, q, J ¼ 7:2 Hz), 4.86 (2H, m), 5.19
(1H, d, J ¼ 7:8 Hz), 5.87 (2H, s), 5.9 (1H, m), 8.16 (2H,
s), 8.3 (1H, m), 8.87 (1H, s), 9.08 (1H, d, J ¼ 7:8 Hz),
9.25 (2H, brs), 9.35 (1H, d, J ¼ 12:9 Hz), 9.59 (1H, d,
J ¼ 12:6 Hz); IR (KBr) cmꢁ1 1779, 1673, 1621, 1524,
1473, 1391; Anal. Calcd for C24H28N10O5S2Æ
2.1HClÆ3.8H2O: C, 38.66; H, 5.10; Cl, 9.98; N, 18.78; S,
8.60. Found: C, 38.75; H, 5.05; Cl, 9.81; N, 18.62; S,
8.53.
1.19 3H, t, J ¼ 7:2 Hz), 2.29 (2H, m), 2.86 (2H, m), 3.43
and 3.55 (2H, ABq, J ¼ 18:9 Hz), 4.12 (2H, q,
J ¼ 7:2 Hz), 4.66 (2H, m), 5.21 (1H, d, J ¼ 5:1 Hz), 5.55
and 5.7 (2H, ABq, J ¼ 14:4 Hz), 5.9 (1H, m), 8.16 (1H,
s), 3.31 (2H, brs), 8.4 (1H, d, J ¼ 6:9 Hz), 9.25 (1H, s),
9.56 (1H, d, J ¼ 8:4 Hz); IR (KBr) cmꢁ1 1780, 1631,
1519, 1479, 1454, 1381; HR-FABMS calcd for
C23H27N10O5S2 [(M+H)þ]: 587.1607, found: 587.1609.
Compound 4c: 4c was obtained as an amorphous
powder in 29% yield from 4c0; 1H NMR (DMSO-
d6+D2O) d 1.18 (3H, t, J ¼ 7:2 Hz), 2.15 (2H, m), 3.0
(2H, m), 3.09 (2H, m), 3.16 and 3.30 (2H, ABq,
J ¼ 17:1 Hz), 4.13 (2H, q, J ¼ 7:2 Hz), 4.93 (1H, s,
J ¼ 4:8 Hz), 5.30 and 6.22 (2H, ABq, J ¼ 13:2 Hz), 5.78
(1H, d, J ¼ 4:8 Hz), 7.65 (1H, m), 8.52 1H, d,
J ¼ 7:5 Hz), 8.77 (1H, d, J ¼ 6:0 Hz); IR (KBr) cmꢁ1
1773, 1614, 1526, 1474; HR-FABMS calcd for
C23H27N10O5S2 [(M+H)þ]: 587.1607, found: 587.1602.
Compound 5f: 5f was obtained as an amorphous pow-
1
der in 16% yield from 5f0; H NMR (DMSO-d6) d 1.20
(3H, t, J ¼ 7:2 Hz), 2.3 (2H, m), 2.9–3.1 (4H, m), 3.2 and
3.6 (2H, ABq, J ¼ 19 Hz), 3.67 (2H, m), 4.14 (2H, q,
J ¼ 7:2 Hz), 4.85 (2H, m), 5.17 (1H, d, J ¼ 4:8 Hz), 5.3
(1H, m), 5.87 (2H, m), 5.9 (1H, m), 8.18 (2H, brs), 8.21
(1H, m), 8.93 (1H, s), 9.15 (1H, d, J ¼ 7:8 Hz), 9.16 (2H,
brs), 9.32 (1H, d, 8.7 Hz), 9.58 (1H, d, J ¼ 8:4 Hz); IR
(KBr) cmꢁ1 1778, 1673, 1617, 1523, 1473, 1455, 1390;
Anal. Calcd for C25H30N10O6S2Æ1.8HClÆ4H2O: C, 39.08;
H, 5.22; Cl, 8.31; N, 18.23; S, 8.35. Found: C, 38.91; H,
5.08; Cl, 8.03; N, 18.36; S, 8.22.
Compound 5a: 5a was obtained as an amorphous
powder in 39% yield from 5a0; 1H NMR (DMSO-
d6+D2O) d 1.21 (3H, t, J ¼ 7:0 Hz), 2.28 (2H, m), 2.95
(2H, m), 3.3 and 3.6 (2H, ABq, J ¼ 19 Hz), 4.12 (2H, q,
J ¼ 7 Hz), 4.78 (2H, m), 5.03 (1H, d, J ¼ 5:2 Hz), 5.65
and 5.82 (2H, ABq, J ¼ 18 Hz), 5.76 (1H, d,
J ¼ 5:2 Hz), 7.98 (1H, m), 9.07 (1H, d, J ¼ 8:8 Hz), 9.26
(1H, d, J ¼ 6 Hz), 9.43 (1H, s); IR (KBr) cmꢁ1 1774,
1670, 1616, 1525, 1457, 1400; Anal. Calcd for
C23H26N10O5S2Æ1.2HClÆ4H2O: C, 39.33; H, 5.05; Cl,
6.06; N, 19.94; S, 9.13. Found: C, 39.20; H, 5.12; Cl,
6.10; N, 10.19; S, 9.07.
References and notes
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.
2. Caldwell, S. R.; Martin, G. E. J. Heterocycl. Chem. 1980,
17, 989.
3. Marasais, F.; Queguiner, G. Tetrahedron 1983, 39, 2009.
4. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura, T.;
Hashimoto, N.; Imada, A. J. Antibiot. 1992, 45, 709.
5. 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.
6. Forster; Hylton, E.; Hurst, J. J. Chem. Soc., Perkin Trans. 1
1973, 2901.
Compound 5c: 5c was obtained as an amorphous
powder in 20% yield from 5c0; 1H NMR (DMSO-
d6+D2O) d 1.20 (3H, t, J ¼ 7:2 Hz), 3.2 and 3.6 (2H,
ABq, J ¼ 19 Hz), 3.50 (2H, m), 4.15 (2H, q, J ¼ 7:2 Hz),
4.96 (2H, m), 5.19 (1H, d, J ¼ 5:1 Hz), 5.89 (2H, m),