Studies on anti-Helicobacter pylori agents. Part 3: A novel, efficacious cephem derivative, FR193879

Article abstract of DOI:10.1016/j.bmcl.2004.02.068

The synthesis, therapeutic efficacy against H. pylori, and preliminary safety of the novel cephem derivative, FR193879 (8a) are described. Compound 8a having a (4-carbamoylmethylthiazol-2-yl)thio moiety at the 3-position and a phenylacetamido at the 7-position was found to have good safety showing a nontoxic dose of 100mg/kg in dogs in a 4-week repeat dose toxicity study and extremely potent therapeutic efficacy against H. pylori, showing 30 times superior activity compared to AMPC, and did not display cross-resistance with CAM or MNZ.

Full text of DOI:10.1016/j.bmcl.2004.02.068

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2627–2631
Studies on anti-Helicobacter pylori agents. Part 3: A novel,
efficacious cephem derivative, FR193879
Yoshiki Yoshida,a,* Keiji Matsuda,a Hiroshi Sasaki,a Yoshimi Matsumoto,b
Satoru Matsumoto,b Shuichi Tawarab and Hisashi Takasugia
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima,
Yodogawa-ku, Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
Received 18 December 2003; accepted 17 February 2004
Abstract—The synthesis, therapeutic efficacy against H. pylori, and preliminary safety of the novel cephem derivative, FR193879
(8a) are described. Compound 8a having a (4-carbamoylmethylthiazol-2-yl)thio moiety at the 3-position and a phenylacetamido at
the 7-position was found to have good safety showing a nontoxic dose of >100 mg/kg in dogs in a 4-week repeat dose toxicity study
and extremely potent therapeutic efficacy against H. pylori, showing 30 times superior activity compared to AMPC, and did not
display cross-resistance with CAM or MNZ.
Ó 2004 Elsevier Ltd. All rights reserved.
Since its discovery in the gastric mucosa of humans, the
clinical importance of eradication of Helicobacter pylori
(H. pylori) has increased significantly due to its rela-
tionship to many diseases.1–3 While to date only a small
number of multidrug therapy regimens are used for
eradication of H. pylori, 4 these therapies are not entirely
successful, and furthermore, problems remain such as
drug resistance,5;6 side effects,7;8 and noncompliance.9
Since there are currently no new anti-H. pylori com-
pounds that show superior therapeutic eradication effi-
cacy compared to AMPC and CAM, which are
generally used for eradication, the need for alternative
and novel structural types is evident and has stimulated
the search for novel agents that have potent therapeutic
efficacy and resolve the problems associated with current
treatment regimens. In a previous paper, we reported the
structure–activity relationships of certain cephem
derivatives,10 and from these studies, compound 1 was
found to have extremely potent in vitro anti-H. pylori
activity, superior therapeutic efficacy compared to
AMPC and CAM, no cross resistance between CAM or
MNZ and low potential for causing diarrhea due to
instability to b-lactamase.11 However, we found that the
nontoxic dose for 1 in dogs in a 4-week repeat dose
toxicity study was <32 mg/kg, which was considered to
be too low in comparison to other oral cephem deriva-
tives on the market, which are generally >100 mg/kg.12;13
Since we postulated that the greater toxicity originated
from decomposition of the 3-position side chain, we
sought to obtain novel derivatives with comparable
features to 1, but with improved safety by 3-position
modification. In this communication, we wish to report
improvement of the toxicity profile as well as studies on
therapeutic efficacy for a novel potent anti-H. pylori
compound 8a (Fig. 1).
In this research program, we designed novel compounds
aiming for the following features: (1) good stability lead-
ing to a good recovery rate, (2) moderate lipophilicity
H
N
S
N
N
O
N
CH3
S
S
O
O
OH
1
H
N
S
CONH2
N
O
N
S
S
O
O
OH
8a
* Corresponding author. Tel.: +81-6-6390-1226; fax: +81-6-6304-1264;
e-mail: yoshiki_yoshida@po.fujisawa.co.jp
Figure 1.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.068

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Y. Yoshida et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2627–2631
S
a
N
CONH2
NH4
H2N
S
HS
HS
b
S
O
N
CO2Et
4
Cl
CO2Et
HS
S
c
3
N
CO2H
2
S
5
R
No.
RSH
d
H
N
H
N
H
N
S
N
S
S
e
N
CONH2
CO2H
7a, 8a
7b, 8b
O
O
N
R
O
N
R
S
OMs
S
S
O
O
O
CO2CHPh2
CO2CHPh2
CO2H
N
6
7
8
S
Scheme 1. Reagents: (a) water–EtOH; (b) NH4OH, NH4Cl, water; (c) 1 N–NaOH, dioxane; (d) RSH, t-BuOK, THF–DME; (e) TFA, anisole,
CH2Cl2.
Table 1. Recovery rate after iv administration
so that compound can enter into the phospholipid layer
of the stomach, and (3) good water solubility under
acidic conditions.
H
N
S
O
N
N
R
S
CO2H
O
The derivatives described in this paper, 7-phenylacet-
amido derivatives having various heterocyclic thio
moieties at the 3-position, were synthesized by the routes
shown in Scheme 1. Coupling reactions of 3-mesylated
compound 6 with heterocyclic mercaptans using potas-
sium t-butoxide, followed by deprotection of the benz-
hydryl ester, afforded 8a,b. Preparation of new
heterocyclic mercaptans was performed according to the
route shown in Scheme 1. Treatment of 2 with ammo-
nium dithiocarbamate gave mercaptothiazole 3,14 which
was treated with ammonium hydroxide and ammonium
chloride to yield amide compound 4. Saponification of 3
afforded carboxylic acid 5. Compounds 1 and 9–12 were
prepared by the same methods as described in our pre-
vious paper.10 Selected data for compound 8a: IR (KBr)
Compound
R
Recovery ratea (%)
N
1
33
9
CH3
S
N
N
9
S
N
10
11
21
63
N
S
N
S
a Urineþbile, 20 mg/kg, iv, rat.
3440, 3284, 1776, 1664, 1539, 1357 cmꢀ1
(200 MHz, DMSO-d6)
;
1H NMR
d
3.52 (dd, 2H, J ¼ 13:9,
thiazole ring and prepared various thiazole derivatives
in order to maintain stability, avoid toxicity, and max-
imize potency.
17.8 Hz), 3.55 (s, 2H), 3.50 and 3.74 (ABq, 2H,
J ¼ 17:6 Hz), 5.20 (d, 1H, J ¼ 4:9 Hz), 5.74 (dd, 1H,
J ¼ 4:9, 8.4 Hz), 6.99 (br s, 1H), 7.1–7.4 (m, 5H), 7.43
(br s, 1H), 7.53 (s, 1H), 9.21 (d, 1H, J ¼ 8:4 Hz); FAB-
MS m=z 491.0 (MHþ).
In addition to stability, we also screened for physico-
chemical properties with the objective of determining
the main factors that influence treatment efficacy. Ini-
tially, we had taken notice of lipophilicity/hydrophilicity
and evaluated high performance liquid chromatography
(HPLC) retention times as an index of polarity, for
compound 1 and especially 12, which hardly showed
therapeutic efficacy, in spite of superior in vitro activity
to 1. As a result, 12 was shown to be much more
hydrophilic compared to 1 (Table 2). Therefore, we
concluded that too high hydrophilicity was the reason
12 hardly displayed therapeutic efficacy.
For biological evaluation, we attempted to determine
the cause of the low nontoxic dose and estimated total
recovery rate (urine and bile) of compound 1 after
intravenous administration as a surrogate marker of
stability, since we postulated that the toxicity displayed
by 1 originated from decomposition of the 3-position
side chain. As a result, we found that the recovery rate
of 1 was only 33%. Therefore, the recovery rate after
intravenous administration was investigated for various
compounds bearing thiazole or thiadiazole rings at the
3-position and which had good anti-H. pylori activity
(Table 1). Consequently, 11 having a thiazole ring at the
3-position had a 63% recovery rate, whilst the three
thiadiazole compounds 1, 9, and 10 had low recovery
rates (9–33%). This data indicated that a compound
having a thiazole ring at the 3-position was more stable
than compounds having a thiadiazole ring. Accordingly,
we decided to search for novel compounds having a
Next, we speculated that the most important action
route for these cephem compounds for therapeutic effi-
cacy against H. pylori was direct action from the gastral
cavity, since the oral bioavailability of compounds
showing excellent therapeutic effect was not high. As a
result, it was expected that the state of a compound in
the stomach was an important factor for therapeutic
effect. Accordingly, we concluded that a compound

Y. Yoshida et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2627–2631
Table 2. Polarity of cephalosporin derivatives
2629
H
N
S
R
N
N
O
N
CH3
S
S
O
CO2H
Compound
R
MIC (lg/mL)a
Therapeutic efficacy (ratio)b
LC-RT (min)c
FP1757
Doses (mg/kg)
0.32
0.1
1
0.00156
0.00078
5/5
2/5
10.0
1.0
N
12
1/8
0/7
H2N
S
a MIC (lg/mL), brucella agar þ7% horse blood, 37 °C, 72 h, 10%-CO2, stamp method.
b Mouse; PO, infection; H. pylori FP1757, therapy; 2/day ꢁ 4 days, termination; 2 weeks after final therapy.
c Compound 1 ¼ 10.0, 0.1%TFA–H2O/CH3CN ¼ 75/25, column: ODS-80TM, flow rate: 1 mL/min, detection: 254 nm.
Table 3. Therapeutic efficacy and toxicity of cephalosporin derivatives
H
N
S
O
N
R
S
CO2H
O
Compound
R
MIC
(lg/mL)a
Therapeutic efficacy (ratio)
Gastric mucosa LC-RT
(min)e
Solubility Recovery Nontoxic
rate (%)f
concentration
(lg/g)d
(mg/mL)
dose
(mg/kg)g
FP1757
FP1836
Mouseb
Guinea pigc
Hour (h)
pH
Dose (mg/kg)
0.5
1.0
2.0
4.0
2.0
4.0
0.32
0.1
2.0
0.5
0.032
0.125
N
N
0.00156
0.00313
5/5
2/5
1/5
5/5
2/5
0/5
89
83
48
23
10.0
6.2
0.053
1.05
33
68
<32
>100
NTh
NT
1
CH3
S
0.00078
0.00313
5/5
4/5
1/5
5/5
3/5
0/5
385
163
68
0.431
2.21
CONH2
N
N
8a
8b
S
S
38
0.00039
0.00313
5/5
1/5
2/5
2/5
0/5
0/5
377
150
111
63
9.9
0.125
1.82
50
CO2H
AMPC
0.0125
0.05
1/8
0/8
NT
NT
33
12
NT
NT
NT
<6
NT
a MIC (lg/mL), brucella agar þ7% horse blood, 37 °C, 72 h, 10%-CO2, stamp method.
b PO, infection; H. pylori FP1757, therapy; 2/day ꢁ 4 days, termination; 2 weeks after final therapy.
c PO, infection; H. pylori FP1836, therapy; 3/day ꢁ 4 days, termination; 1 day after final therapy.
d Rat, 20 mg/kg (PO), gastric mucosa was scraped from the stomach and homogenated in phosphate buffer.
e Compound 1¼10.0, 0.1%TFA–H2O/CH3CN¼75/25, column: ODS-80TM, flow rate: 1 mL/min, detection: 254 nm.
f Urineþbile, 20 mg/kg, iv, rat.
g Dog, orally administered for 4 weeks.
h NT ¼ not tested.

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Y. Yoshida et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2627–2631
Table 4. Activity against clarithromycin- and metronidazole-resistant strains and b-lactamase stability
Compound
MIC (lg/mL)
Stability toward b-lactamase
B. fragilis FP784b
Helicobacter pylori
TEMb
16021a
16043a
15069a
km (lg/mL) Vmax
V
max/kmc
km (lg/mL) Vmax
V
max/kmc
c
c
8a
0.0016
0.025
50
0.0016
0.05
50
0.0063
0.2
19.4
27.3
NTd
NT
0.647
0.0175
NT
1.060
0.0521
NT
34.8
22.0
NT
NT
276
1.18
9.38
11.1
NT
NT
1.0
AMPC
CAM
MNZ
CER
2.94
NT
NT
1.0
25
25
50
NT
50
NT
NT
1.0
NT
1.0
NT
31.7
a Clarithromycin- and metronidazole-resistant strains.
b B. fragilis FP784; cephalosporinase, TEM; penicillinase.
c Relative value (CER ¼ 1.0), Vmax/km; larger values mean more unstable.
d NT ¼ not tested.
having better water solubility under acidic conditions
may show a superior therapeutic effect compared to 1,
since the water solubility of 1 at pH 2.0 and 4.0 was low
(Table 3). Therefore, we decided to search for a com-
pound with moderate polarity and good water solubility
under acidic conditions and prepared various cephem
compounds containing suitable water-solublizing sub-
stituents on the thiazole ring at the 3-position. As a
result, 8a and 8b, which were substituted by carbamoyl
or carboxyl groups, respectively, showed similar polar
character compared to 1 and much improved water
solubility under acidic conditions. In particular, 8a was
8-fold more soluble in a pH 2 medium.
Additionally, we estimated the in vitro effect of 8a
against CAM- and MNZ-resistant strains and instability
to b-lactamase (Table 4). Similar to 1, 8a showed
excellent activity against these strains, which are highly
resistant strains in the clinical environment, and there
was no cross-resistance with CAM and MNZ. More-
over, low potential for causing diarrhea due to insta-
bility to b-lactamase was shown, since 8a showed equal
stability to CEZ, a typical unstable cephem drug,
toward cephalosporinase, and toward penicillinase 8a
was 9-fold more unstable than CEM and almost similar
to AMPC which is a penicillin derivative.
In summary, we have reported the discovery of
FR193879 (8a), a novel, safe, and potent cephalosporin
Next, we estimated gastric mucosa concentration for
these compounds since we speculated this might provide
a rough standard for therapeutic effect (Table 3). As a
result, compounds 8a and 8b showed far higher con-
centrations than 1, especially after a short contact per-
iod. So we evaluated therapeutic efficacies in mice and
guinea pigs. Consequently, 8a showed marginally
improved in therapeutic efficacies in terms of both
eradication ratio in mice and clearance ratio in guinea
pigs. On the other hand, 8b showed decreased thera-
peutic efficacy in both models. We speculated that the
reason for a lack of clearly improved therapeutic efficacy
was as follows: gastric mucosa concentrations showed
that the concentrations of 8a and 8b at 4 h after
administration were not high, while those at a short
contact period were much greater than 1, thus these
compounds may readily dissociate from the mucosal
layer. However, 8a is expected to show better thera-
peutic efficacy in humans, which have thicker gastric
mucosa than model animals. Furthermore, the efficacy
of 8a in mice was at least 30 times superior compared to
AMPC.
anti-H. pylori agent, that contains
a
(4-car-
bamoylmethylthiazol-2-yl)-thio moiety at the 3-position
and a phenylacetamido group at the 7-position. Safety
studies showed a nontoxic dose of >100 mg/kg in dogs in
a 4 week repeated dose study as well as excellent anti-H.
pylori therapeutic efficacy, far superior to AMPC, and
with no cross-resistance to CAM or MNZ. These results
suggest this compound is a suitable candidate for further
development.
Acknowledgements
We are grateful to Dr. David Barrett, Medicinal
Chemistry Research Laboratories, for his help in pre-
paring this manuscript.
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