Anti-Helicobacter pylori agents. 1. 2-(Alkylguanidino)-4-furylthiazoles and related compounds

Article abstract of DOI:10.1021/jm970407n

A series of 2-(alkylguanidino)-4-[5-(acetamidomethyl)furan-2- yl]thiazoles and related compounds were synthesized and evaluated for antimicrobial activity against Helicobacter pylori, inhibitory effect on gastric acid secretion, and histamine H₂

Full text of DOI:10.1021/jm970407n

2462
J . Med. Chem. 1997, 40, 2462-2465
An ti-Helicoba cter pylor i Agen ts. 1. 2-(Alk ylgu a n id in o)-4-fu r ylth ia zoles a n d
Rela ted Com p ou n d s
Yousuke Katsura,* Tetsuo Tomishi, Yoshikazu Inoue, Kazuo Sakane, Yoshimi Matsumoto,
Hirohumi Ishikawa, and Hisashi Takasugi
New Drug Research Laboratories, Fujisawa Pharmaceutical Company, Ltd., 2-1-6, Kashima, Yodogawa-ku Osaka 532, J apan
Received J une 20, 1997X
A series of 2-(alkylguanidino)-4-[5-(acetamidomethyl)furan-2-yl]thiazoles and related compounds
were synthesized and evaluated for antimicrobial activity against Helicobacter pylori, inhibitory
effect on gastric acid secretion, and histamine H2-receptor antagonist activity. Introduction of
alkyl substituents on the guanidino moiety resulted in a significant increase in antimicrobial
activity, which was associated with the alkyl chain length. Of the compounds obtained, the
n-hexylguanidino derivative 13 demonstrated a 250-fold improvement in activity (MIC ) 0.11
µg/mL) over the unsubstituted guanidino derivative 7. Alkyl-substituted guanidino derivatives
also displayed gastric antisecretion and H2-antagonist activities. However, a simple correlation
between the alkyl chain length and the activities was not found in these assays. Replacement
of the guanidine with other bioisosteric groups (thiourea, urea, or (dimethylamino)methyl)
resulted in loss of all activities tested. Thus the guanidino moiety was found to be essential
for activity in this series of compounds.
Since the initial isolation from gastric disease pa-
tients,1 the clinical importance of Helicobacter pylori (H.
pylori) has been pursued for over 10 years. As a result,
it is now widely accepted that H. pylori is a major
causative factor in peptic ulcer disease, and eradication
of the organism results in a dramatic decrease in the
recurrence rate in peptic ulcer patients.2-14 Recently,
the National Institutes of Health consensus conference
on H. pylori stated that all ulcer patients with H. pylori
infection should be treated with antimicrobial agents
in addition to gastric antisecretory drugs.15 Although
the optimal protocol for eradication has not been
established, a variety of drugs with susceptibility for
H. pylori, such as antibiotics (amoxicillin and clarithro-
mycin), bactericidal agents (bismuth salt), and antipro-
tozoal agents (metronidazole), have been effective in the
clinic. On the other hand, adverse effects (e.g., nausea,
vomiting, and diarrhea) and acquired resistance have
been problematic in these drugs.9-14 Therefore the
development of novel types of anti-H. pylori agents is
of importance.
In the course of research on novel histamine H2-
receptor antagonists (H2-antagonists), we found com-
pound 7 possessed significant anti-H. pylori activity16
which had not been observed in known H2-antagonists
before the report was published.17 As a novel chemical
lead consisting of a different structure from known
antimicrobial agents, we focused on 7 to carry out
chemical modifications to improve the antimicrobial
potency. In this paper we describe the synthesis and
the pharmacological evaluation, antimicrobial activity
against H. pylori and the effects on gastric acid secretion
and H2-antagonism, of some 2-(alkylguanidino)-4-fu-
rylthiazoles and related compounds.
cyclization of halo ketones (1a 18 or 1b) with thioamide,
amidinothiourea, or (dimethylamino)methylthioamide,19
respectively. Treatment of 2 with benzoyl isothiocyan-
ate gave the benzoylthiourea derivative 5, which was
hydrolyzed with sodium hydroxide to yield the thiourea
derivative 6. After methylation of 6 with methyl iodide,
reaction with amine or diamine afforded the guanidine
(8-13) and imidazolidine (14) derivatives, respectively.
The urea derivative 15 was prepared by reacting 2 with
n-butyl isocyanate.
Resu lts a n d Discu ssion
The compounds obtained were evaluated for antimi-
crobial activity against H. pylori, antisecretory activity
on histamine-stimulated gastric acid secretion in lumen-
perfused anesthetized rats, and H2-antagonist activity
using the histamine-stimulated chronotoropic response
of the isolated guinea pig right atrium.16 The results
are summarized in Table 1. In anti-H. pylori activity,
compound 3 which introduced a methyl group on the
thiazole nucleus showed 3-fold less activity than the lead
compound 7. In contrast, introduction of a methyl group
to the guanidino moiety (8) resulted in a 2-fold increase
in activity over 7. This encouraging result led us to
prepare a series of compounds which contained an alkyl
group on the guanidino moiety. The anti-H. pylori
activity was enhanced as the alkyl chain length in-
creased, and the improvement in activity was as fol-
lows: 9-fold for ethyl (9), 9-fold for isopropyl (11), 16-
fold for n-propyl (10), 129-fold for n-butyl (12), and 250-
fold for n-hexyl (13) in comparison with 7. The potencies
of these compounds were superior to those of the
bactericidal drugs bismuth salt and metronidazole,
which have been widely used for eradication therapy
in H. pylori infection. On the other hand, imidazolidine
derivative 14, a structural rigid analog of 9, dramati-
cally decreased the activity. Flexibility at this position
was required to show the activity.
Ch em istr y
The synthetic pathways to the target compounds are
shown in Scheme 1. Compounds 2-4 were obtained by
* Address for correspondence: Research Planning, Research Divi-
sion, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-
ku, Osaka 532, J apan.
Concerning gastric acid antisecretory activity, ethyl
(9) and n-propyl (10) derivatives showed strong activity
over 7 and the referenced H2-antagonist cimetidine.
X Abstract published in Advance ACS Abstracts, J uly 15, 1997.
S0022-2623(97)00407-X CCC: $14.00 © 1997 American Chemical Society

Anti-Helicobacter pylori Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2463
Sch em e 1a
a
Reagents: (a) R1C(dS)NH2/EtOH/reflux; (b) PhCONCS/acetone/reflux; (c) NaOH/MeOH (90%)-H2O (10%)/60 °C; (d) MeI/MeOH/
reflux; (e) R3-NH2/EtOH/reflux; (f) H2N(CH2)2NH2/EtOH/reflux; (g) n-C4H9NCO/DMF/60 °C.
Ta ble 1. Antimicrobial Activity against H. pylori
inhibition (%)
MIC (µg/mL)b
range
gastric secretionc
(rat, 1 mg/kg iv)
H2-antagonismd
compd
R1
R2
mean
76
>200
>200
(1 × 10-6 g/mL)
3
4
(H2N)2CdNH-
CH3
H
H
H
H
H
H
H
H
25-200
3
4
10
6
(CH3)2NCH2-
6
H2NCSNH-
11
68
24
79
97
32
45
44
18
0
7a
8
(H2N)2CdN-
27
12.5
3.13
1.67
2.9
0.21
0.11
25-50
85
25
51
81
27
78
79
8
CH3HN(H2N)CdN-
C2H5HN(H2N)CdN-
n-C3H7HN(H2N)CdN-
i-C3H7HN(H2N)CdN-
n-C4H9HN(H2N)CdN-
n-C6H13HN(H2N)CdN-
12.5
9
1.56-6.25
0.78-3.13
1.56-6.25
0.1-0.39
0.05-0.2
10
11
12
13
14
H
H
>100
15
n-C4H9NHCONH-
H
>100
9
0
bismuth subcitrate
metronidazole
cimetidine
18
12.5-25
1.56-25
800-1600
5.4
1130
53
43
a
b
Reference 16. Minimum inhibitory concentration (MIC) was determined as the lowest drug concentration that inhibited macroscopic
colonial growth. Mean MIC and range of MICs were obtained from the results of 10 different strains. c Inhibition of histamine-stimulated
gastric acid secretion in lumen-perfused stomach of anesthetized rats (n ) 2). Inhibition of the histamine-stimulated chronotoropic
response in the isolated guinea pig right atrium.
d
n-Butyl (12) and n-hexyl (13) derivatives exhibited mar-
ginal activity despite having potent H2-antagonist activ-
ity. Methyl (8) and isopropyl (11) derivatives showed
weak activities on both gastric secretion and H2-anta-
gonism assays. Thus in contrast with the result in the
anti-H. pylori activity, lengthening the alkyl chain on
the guanidino moiety was not crucial for gastric acid
antisecretory and H2-antagonist activities.
In order to identify the importance of the guanidino
moiety, we prepared and examined the compounds in

2464 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Katsura et al.
the residue was dissolved in AcOEt-water, and the mixture
was acidified to pH 1 with 6 N HCl. The aqueous layer that
separated was basified to pH 10 with 4 N NaOH and extracted
with AcOEt. The extract was dried over MgSO4 and concen-
trated to give a residue, which was recrystallized from MeOH-
dioxane to afford 3 (3.5 g, 21%): mp 247-248 °C; IR 3420,
which the guanidine was replaced by nonbasic bioiso-
steric functional groups: thiourea (6) or urea (15). We
also tried to investigate the derivative with a (dimeth-
ylamino)methyl group (4) which has been known as a
surrogate base for guanidine that could be found in the
relationship between nizatidine20 and famotidine.21
However, these derivatives displayed a dramatic de-
crease in the activities for all assays tested. This result
seems to be in contrast with that of our previous study
on the conversion of the acetamido part at the 5-position
of the furan ring. The acetamido group could be
replaced by other functions such as cyanoguanidine or
urea without a marked decrease in the activities.16
Therefore it is conceivable that the guanidino moiety is
essential for the anti-H. pylori, gastric acid antisecre-
tory, and H2-antagonist activities in these types of
compounds.
3350, 1660 cm-1 1H NMR δ 1.85 (3H, s), 2.40 (3H, s), 4.24
;
(2H, d, J ) 5 Hz), 6.22 (1H, d, J ) 3 Hz), 6.72 (4H, s), 8.20
(1H, t, J ) 5 Hz). Anal. (C12H15N5O2S) C, H, N.
4-[5-(Acetam idom eth yl)fu r an -2-yl]-2-[(dim eth ylam in o)-
m eth yl]th ia zole (4). A solution of 2-(acetamidomethyl)-5-
(chloroacetyl)furan (1a )18 (12.0 g, 56 mmol) and (dimethylami-
no)methylthioamide19 (7.9 g, 67 mmol) in EtOH (120 mL) was
refluxed for 2.5 h with stirring. After removal of the solvent,
the residue was added to AcOEt-water, and the resulting
mixture was acidified to pH 1 with 6 N HCl. The aqueous
layer that separated was basified to pH 9 with 20% aqueous
K2CO3 and extracted with AcOEt. The extract was dried over
MgSO4 and concentrated to give a residue, which was chro-
matographed on silica gel eluting with CHCl3-MeOH (40/1)
and recrystallized from AcOEt-hexane-IPE to afford 4 (2.6
Con clu sion s
1
g, 17%): mp 111-113 °C; IR 1630 cm-1; H NMR δ 1.89 (3H,
s), 2.30 (6H, s), 3.76 (2H, s), 4.29 (2H, d, J ) 5 Hz), 6.31 (1H,
d, J ) 3 Hz), 6.66 (1H, d, J ) 3 Hz), 7.61 (1H, s), 8.28 (1H, br
s); MS m/z 279 (M+). Anal. (C13H17N3O2S) C, H, N.
In conclusion, some structure-activity relationships
for anti-H. pylori, gastric acid antisecretory, and H2-
antagonist activities in a novel series of 4-furyl-2-
modified thiazoles have been obtained. Introduction of
several alkyl substituents on the guanidino moiety at
the 2-position of the thiazole ring enhanced the anti-H.
pylori activity in order of increasing alkyl chain length
in the range of the investigation, C1 to C6. Of the
compounds obtained, the n-hexyl (13) derivative dem-
onstrated the highest activity (MIC ) 0.11 µg/mL).
Alkylguanidino derivatives also showed gastric acid
antisecretory and H2-antagonist activities. However, no
simple correlation between the alkyl chain length and
the activities was observed. Replacement of the guani-
dino moiety with other bioisosteric functional groups
resulted in loss of all activities tested. These data
indicate that the guanidino moiety is essential for the
activity and provide information on a promising chemi-
cal modification to potentiate the anti-H. pylori activity
of prototype compound 7. Further extensive structure-
activity relationships in this type of compound will be
reported in due course.
4-[5-(Acet a m id om et h yl)fu r a n -2-yl]-2-(3-b en zoylt h io-
u r eid o)th ia zole (5). Benzoyl chloride (2.7 mL, 23 mmol) was
added dropwise to a solution of NH4SCN (1.9 g, 25 mmol) in
Me2CO (50 mL) under reflux, and the mixture was refluxed
for a further 1 h. 4-[5-(Acetamidomethyl)furan-2-yl]-2-ami-
nothiazole (2)18 (5.2 g, 22 mmol) was added portionwise to the
mixture, and the resulting mixture was refluxed for 2 h. After
removal of the solvent, the residue was added to AcOEt-water.
The resulting precipitate was collected by filtration and
recrystallized from MeOH-IPE to give 5 (5.5 g, 62%): mp
1
213-214 °C; IR 3270, 1675, 1630 cm-1; H NMR δ 1.90 (3H,
s), 4.37 (2H, d, J ) 6 Hz), 6.40 (1H, d, J ) 3 Hz), 6.80 (1H, d,
J ) 3 Hz), 7.42 (1H, s), 7.58-8.17 (5H, m), 8.40 (1H, t, J ) 6
Hz), 12.00 (1H, s), 14.08 (1H, s). Anal. (C18H16N4O3S2) C, H,
N.
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-th iou r eid oth ia z-
ole (6). A solution of NaOH (0.55 g, 14 mmol) in water (5
mL) was added to a suspension of 5 (5.4 g, 14 mmol) in MeOH
(50 mL), and the mixture was stirred at 60 °C for 1 h. After
removal of the solvent, the residue was added to AcOEt-water.
The resulting precipitate was collected by filtration and
recrystallized from MeOH-IPE to give 6 (3.0 g, 74%): mp
231-232 °C; IR 3270, 3190, 3140, 1635 cm-1; 1H NMR δ 1.86
(3H, s), 4.27 (2H, d, J ) 6 Hz), 6.30 (1H, d, J ) 3 Hz), 6.61
(1H, d, J ) 3 Hz), 7.10 (1H, s), 8.28 (1H, t, J )6 Hz), 8.33 (2H,
br s), 11.81 (1H, s). Anal. (C11H12N4O2S2) C, H, N.
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(n -bu tylgu a n id i-
n o)th ia zole (12). Gen er a l P r oced u r e. A suspension of 6
(1.5 g, 5 mmol) and MeI (0.32 mL, 5 mmol) in MeOH (30 mL)
was refluxed for 4 h with stirring. After removal of the solvent,
n-butylamine (5 mL) and EtOH (30 mL) were added to the
residue, and the resulting mixture was refluxed for 42 h. The
solution was concentrated to dryness, and the residue was
dissolved in water. The solution was basified to pH 10 with
20% aqueous K2CO3 and extracted with AcOEt-THF. The
extract was dried over MgSO4 and concentrated to give a
residue, which was recrystallized from AcOEt to afford 12 (0.3
g, 20%): mp 147-148 °C; IR 3460, 3310, 3200, 1640 cm-1; 1H
NMR δ 0.91 (3H, t, J ) 7 Hz), 1.29-1.48 (4H, m), 1.85 (3H, s),
3.16 (2H, q, J ) 7 Hz), 4.26 (2H, d, J ) 5.5 Hz), 6.30 (1H, d,
J ) 3 Hz), 6.54 (1H, d, J ) 3 Hz), 6.77 (1H, s), 7.32 (2H, br s),
8.34 (1H, t, J ) 5.5 Hz); MS m/z 335 (M+). Anal. (C15H21N5O2S)
C, H, N.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. Infrared
(IR) spectra were taken in Nujol using a Hitachi 260-10
spectrometer. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded in dimethyl sulfoxide-d6 (DMSO) with
tetramethylsilane as the internal standard on a Bruker AC-
200P spectrometer. Mass spectral measurements (MS) were
made on a J EOL J MS D-300 mass spectrometer. Analytical
results were within (0.4% of the theoretical values unless
otherwise indicated.
2-(Aceta m id om eth yl)-5-(2-br om op r op ion yl)fu r a n (1b).
AlCl3 (23.0 g, 170 mmol) was added portionwise to a solution
of 2-(acetamidomethyl)furan (10.0 g, 72 mmol) and 2-bro-
mopropionyl bromide (23.0 g, 110 mmol) in CH2Cl2 (150 mL)
at 10 °C, and the mixture was refluxed for 1 h with stirring.
The reaction mixture was poured into ice-water, and the
organic layer that separated was washed with water, dried
over MgSO4, and concentrated to dryness to give 1b (16.2 g,
82%) as an oil: IR (film) 1730, 1660 cm-1; 1H NMR δ 1.71 (3H,
d, J ) 7 Hz), 1.85 (3H, s), 4.30 (2H, d, J ) 5 Hz), 5.43 (1H, q,
J ) 7 Hz), 6.48 (1H, d, J ) 4 Hz), 7.57 (1H, d, J ) 4 Hz), 8.46
(1H, t, J ) 5 Hz).
The following compounds were prepared according to a
similar procedure.
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(m eth ylgu a n id i-
n o)th ia zole (8): yield 34%, mp 188-189 °C (EtOH); IR 3380,
1
4-[5-(Ace t a m id om e t h yl)fu r a n -2-yl]-2-gu a n id in o-5-
m eth ylth ia zole (3). A solution of 1b (16.0 g, 58 mmol) and
amidinothiourea (5.7 g, 49 mmol) in EtOH (150 mL) was
refluxed for 2 h with stirring. After removal of the solvent,
3260, 1635 cm-1; H NMR δ 1.85 (3H, s), 2.74 (3H, d, J ) 5
Hz), 4.26 (2H, d, J ) 5.5 Hz), 6.29 (1H, d, J ) 3 Hz), 6.60 (1H,
d, J ) 3 Hz), 6.77 (1H, s), 7.42 (1H, s), 7.42 (1H, s), 7.42 (2H,
br s), 8.35 (1H, t, J ) 5.5 Hz). Anal. (C12H15N5O2S) C, H, N.

Anti-Helicobacter pylori Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2465
tension of 0.3-0.6 g in an organ bath containing Thyrode
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(eth ylgu a n id in o)-
th ia zole (9): yield 45%, mp 170-171 °C (EtOH-IPE); IR
-5% CO2 gas. The
solution at 30 °C and aerated by 95% O2
3470, 3280, 3100, 1650 cm-1 1H NMR δ 1.10 (3H, t, J ) 7
;
beating rate and the amplitude of contraction of the atrium
were recorded by means of a transducer and a polygraph.
Histamine hydrochloride (1 × 10-6 g/mL) was added to the
beating fluid, and the increase in the beating rate after dosing
was measured. Addition of test compounds (1 × 10-6 g/mL)
was done 30 min after washing out the histamine hydrochlo-
ride. The percent inhibitory effect of the test compound was
calculated by comparing histamine-induced increases in beat-
ing rate before and 30 min after dosing with the test com-
pounds.
Hz), 1.85 (3H, s), 3.12-3.25 (2H, m), 4.26 (2H, d, J ) 5.5 Hz),
6.29 (1H, d, J ) 3 Hz), 6.56 (1H, d, J ) 3 Hz), 6.77 (1H, s),
7.38 (2H, br s), 8.35 (1H, t, J ) 5.5 Hz). Anal. (C13H17N5O2S)
C, H, N.
4-[5-(Acet a m id om et h yl)fu r a n -2-yl]-2-(n -p r op ylgu a n i-
d in o)th ia zole (10): yield 48%, mp 155-156 °C (EtOH-IPE);
IR 3460, 3320, 3210, 1640 cm-1; 1H NMR δ 0.91 (3H, t, J ) 7
Hz), 1.42-1.60 (2H, m), 1.85 (3H, s), 3.08-3.17 (2H, m), 4.26
(2H, d, J ) 5.5 Hz), 6.30 (1H, d, J ) 3 Hz), 6.55 (1H, d, J ) 3
Hz), 6.77 (1H, s), 7.33 (2H, br s), 8.35 (1H, t, J ) 5.5 Hz). Anal.
(C14H19N5O2S) C, H, N.
Ack n ow led gm en t. We are grateful to Dr. H. Tana-
ka for his valuable suggestions and Ms. C. Morinaga
for carrying out the antimicrobial tests. Thanks are also
due to the staff members for our analytical division for
elemental analyses and measurement of spectral data.
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(isop r op ylgu a n i-
d in o)th ia zole (11): yield 43%, mp 104-105 °C (EtOH-IPE);
IR 3420, 3350, 3220, 1630 cm-1; H NMR δ 1.13 (6H, d, J )
1
6.5 Hz), 1.86 (3H, s), 3.81-3.91 (1H, m), 4.27 (2H, d, J ) 5.5
Hz), 6.30 (1H, d, J ) 3 Hz), 6.55 (1H, d, J ) 3 Hz), 6.77 (1H,
s), 7.33 (2H, br s), 8.35 (1H, t, J ) 5.5 Hz). Anal. (C14H19N5O2S)
C, H, N.
Refer en ces
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(n -h exylgu a n id i-
n o)th ia zole (13): yield 47%, mp 138-139 °C (AcOEt); IR
(1) Warren, J . R.; Marshall, B. J . Unidentified curved bacilli in the
stomach of patients with gastritis and peptic ulceration. Lancet
1984, 1311-1315.
(2) Axon, A. T. R. Eradication of Helicobacter Pylori. Scand. J .
Gastroenterol. 1996, 31, (Suppl. 214), 47-53.
1
3470, 3340, 3225, 1635 cm-1; H NMR δ 0.87 (3H, t, J ) 6.5
Hz), 1.05-1.60 (8H, m), 1.85 (3H, s), 3.16 (2H, q, J ) 6.5 Hz),
4.26 (2H, d, J ) 5.5 Hz), 6.29 (1H, d, J ) 3 Hz), 6.54 (1H, d,
J ) 3 Hz), 7.06 (1H, s), 7.32 (2H, s), 8.34 (1H, t, J ) 5.5 Hz).
Anal. (C17H25N5O2S‚1/2H2O) C, H, N.
(3) Larry, K. L.; Tanaka, K. Therapy of Helicobacter pylori infec-
tions: Current status and future directions. Annu. Rep. Med.
Chem. 1995, 151-158.
4-[5-(Aceta m id om eth yl)fu r a n -2-yl]-2-(im id a zolid in -2-
ylim in o)th ia zole (14): yield 33%, mp 239-240 °C (MeOH-
THF); IR 3290, 3105, 1630 cm-1; 1H NMR δ 1.88 (3H, s), 3.57
(4H, s), 4.33 (2H, d, J ) 6 Hz), 6.33 (1H, d, J ) 3 Hz), 6.82
(1H, s), 6.85 (1H, d, J ) 3 Hz), 7.68 (2H, s), 8.33 (1H, t, J ) 6
Hz); MS m/z 305 (M+). Anal. (C13H15N5O2S) C, H, N.
4-[5-(Aceta m idom eth yl)fu r a n -2-yl]-2-(3-n -bu tylu r eid o)-
th ia zole (15). A solution of 2 (0.50 g, 2.1 mmol) and n-butyl
isocyanate (0.23 g, 2.3 mmol) in DMF (5 mL) was stirred at
70 °C for 15 h. The reaction mixture was poured into water
and extracted with AcOEt. The extract was washed several
times with water, dried over MgSO4, and concentrated to
dryness. The residue was triturated with Et2O and recrystal-
lized from MeOH-AcOEt to give 15 (0.53 g, 75%): mp 186-
188 °C; IR 3340, 1645, 1635 cm-1; 1H NMR δ 0.89 (3H, t, J )
7 Hz), 1.23-1.46 (4H, m), 1.85 (3H, s), 3.08-3.17 (2H, m), 4.27
(2H, d, J ) 5.5 Hz), 6.31 (1H, d, J ) 3 Hz), 6.48 (1H, t, J ) 6
Hz), 6.55 (1H, d, J ) 3 Hz), 7.05 (1H, s), 8.35 (1H, t, J ) 6.5
Hz); MS m/z 337 (M+ + 1). Anal. (C15H20N4O3S) C, H, N.
An tim icr obia l Activity. In vitro antimicrobial activity
against H. pylori was determined by the agar dilution method.
Test strain was precultured in Brucella agar containing 3%
horse serum and 2% starch at 37 °C for 3 days and suspended
in buffered saline to give the turbidity equivalent to McFarland
no. 1; 102-fold dilution of the bacterial suspensions was
inoculated with a multipoint replicator onto a Brucella agar
plus 7% lysed horse blood plate containing serial 2-fold
dilutions of each drug at 37 °C for 3 days. Incubation was
carried out in an atmosphere of 10% CO2. Minimum inhibitory
concentration (MIC) was read after incubation as the lowest
drug concentration that inhibited macroscopic colonial growth.
Mean MIC was determined from the MICs in 10 strains: H.
pylori 8001, 8003, 8004, 8007, 8008, 8009, 8011, 9005, FP1530,
and FP1532.
(4) Marshall, B. J . Helicobacter pylori. Am. J . Gastroenterol. 1994,
89, s116-s128.
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