Orally active benzoxazole derivative as 5-HT3 receptor partial agonist for treatment of diarrhea-predominant irritable bowel syndrome

Article abstract of DOI:10.1021/jm050209t

During our search for therapeutic agents to treat diarrhea-predominant IBS, we found that 2-substituted benzoxazole derivatives have a characteristic 5-HT₃ receptor partial agonist activity with high affinity. Some of these compounds showed high in vitro metabolical stability, and 6g showed marked antidiarrhetic activity with little side effect of constipation in in vivo tests. Our results indicate that 5-HT₃ receptor partial agonists might be superior as therapeutic agents to the drugs currently used for IBS treatment.

Full text of DOI:10.1021/jm050209t

J. Med. Chem. 2005, 48, 7075-7079
7075
Brief Articles
Orally Active Benzoxazole Derivative as 5-HT3 Receptor Partial Agonist for
Treatment of Diarrhea-Predominant Irritable Bowel Syndrome
Satoshi Yoshida,* Sojiro Shiokawa, Ken-ichi Kawano, Tomoko Ito, Hiroshi Murakami, Hisashi Suzuki, and
Yasuo Sato
Pharmaceutical Research Department Meiji Seika Kaisha, Ltd., 760 Morooka-Cho, Kohoku-ku, Yokohama 222-8567 Japan
Received March 8, 2005
During our search for therapeutic agents to treat diarrhea-predominant IBS, we found that
2-substituted benzoxazole derivatives have a characteristic 5-HT3 receptor partial agonist
activity with high affinity. Some of these compounds showed high in vitro metabolical stability,
and 6g showed marked antidiarrhetic activity with little side effect of constipation in in vivo
tests. Our results indicate that 5-HT3 receptor partial agonists might be superior as therapeutic
agents to the drugs currently used for IBS treatment.
Chart 1
Introduction
5-Hydroxytryptamine (1, serotonin, 5-HT) is involved
in various biological pathways.1 Seven classes of 5-HT
receptors are present in both the central and peripheral
nervous systems.2-5 Intensive studies have been focused
on subtype-selective 5-HT receptor ligands, since they
are not only attractive biological tools, but also novel
candidates as therapeutic agents for a variety of dis-
eases. Particular interest has been focused on the
serotonin type 3 (5-HT3) receptor,6,7 because selective
5-HT3 receptor antagonists effectively prevent the nau-
sea and vomiting that commonly occur during cytotoxic
cancer chemotherapy and/or radiation therapy. 8,9 More-
over, alosetron (2), a potent novel 5-HT3 antagonist, was
approved as the first therapeutic agent for diarrhea-
predominant irritable bowel syndrome (IBS).10 Although
it possesses potent antidiarrhetic efficacy, it has several
side effects, including constipation, which is seen in
approximately 30% of patients treated.11-13 Ondansetron9
(3) improved stool consistency in patients with diarrhea-
predominant IBS, but some of the patients reported
discomfort and constipation.14 In the treatment of
diarrhea-predominant IBS, 5-HT3 antagonists may
potentially cause these side effects by inhibiting normal
lower bowel function.15 We proposed that 5-HT3 receptor
partial agonists might control gastroenteric motility
without completely blocking 5-HT3-sensitized nerve
function.16,17 This characteristic could make 5-HT3
receptor partial agonists useful for the treatment of
gastroenteric disorders, exemplified by IBS. In a previ-
ous study, our benzoxazole derivatives with a nitrogen-
containing heterocyclic substituent at the 2-position
were found to act as selective 5-HT3 receptor partial
agonists.16,17 Among them, compound 4 showed high
antidiarrhetic activity in an in vivo test (ID50 ) 0.3 mg/
kg, sc) without preventing normal colonic function even
at high doses of up to 30 mg/kg, sc.16 Here, we describe
our further efforts to find a drug candidate with high
efficacy, particularly in in vivo tests. We found some
analogues of 2-(1-piperazinyl)benzoxazole (6b, 6d, 6f)
and 2-(1-homopiperazinyl)benzoxazole (6g) possessing
similar or superior 5-HT3 receptor affinity to the cor-
responding 2-(4-methyl-1-piperazinyl)benzoxazole de-
rivatives (6a, 6c, 6e). Compounds which have an NCH3-
containing heterocyclic substituent, such as 4-methyl-
1-piperazine, are known to be metabolized by P450 2D6
(CYP2D6) and other enzymes.18 Therefore, we expected
that benzoxazole derivatives with an NH-containing
heterocyclic substituent would be candidate therapeutic
agents with improved metabolic stability, if they retain
favorable 5-HT3 partial agonist characteristics.
* To whom correspondence should be addressed. Tel: +81-45-545-
3133. Fax: +81-45-541-0667. E-mail: satoshi_yoshida@meiji.co.jp.
10.1021/jm050209t CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/12/2005

7076 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Brief Articles
Table 1
Table 2. In Vitro Activity and Metabolic Stability of 5-HT3
Receptor Partial Agonists
contraction activity
5-HT3 receptor metabolic act.a
binding
(Ki, nM)
(nmol/min/mg
protein)
compd pD2 ( SEM ia ( SEM
6a
6b
6c
6d
6e
6f
4
6g
5.01 ( 0.13 0.62 ( 0.12
5.74 ( 0.10 0.59 ( 0.08
6.32 ( 0.13 0.62 ( 0.12
7.07 ( 0.07 0.13 ( 0.03
6.70 ( 0.13 0.24 ( 0.07
7.56 ( 0.11 0.14 ( 0.01
7.76 ( 0.14 0.12 ( 0.02
40
20
n.t.b
n.t.
n.t.
0.80 ( 0.02
1.30 ( 0.05
0.55 ( 0.02
1.18 ( 0.05
0.57 ( 0.03
-
6.0
3.4
5.5
4.7
1.1
2.52
1.13
7.48 ( 0.11 0.12 ( 0.03
c
alosetron
-
-
a Metabolic activity was measured by using rat S9; mean ( SD,
n ) 3. b n.t.; not tested. c Alosetron did not induce a contractile
response.
indicates that the compounds of the NH series generally
show greater 5-HT3 receptor partial agonist potency
(higher pD2 values) than those of the NCH3 series. The
influence of methyl substituents at the 5 and 7 positions
of the benzoxazole nucleus seems to be similar in both
series, i.e., this modification markedly improved the
affinity for the 5-HT3 receptor in both series (6c, 6d vs.
6a, 6b). Further increase of the affinity was observed
when a chlorine atom was introduced at the 5 position
of the benzoxazole ring. Our previous study on the SAR
of 2-(4-methylpiperazinyl)benzoxazole derivatives indi-
cated that introduction of small lipophilic substituents
such as CH3 and Cl at the 5- and 7-position of the
benzoxazole ring greatly increased the affinity for the
5-HT3 receptor, 16 and this was also the case for the NH
series. However, the influence of these substituents on
the intrinsic activity was not the same in the two series.
In the NCH3 series, the 5,7-substituted compound 6c
(ia ) 0.62) retains the intrinsic activity of the unsub-
stituted compound 6a (ia ) 0.62), while in the NH
series, the 5,7-substituted compound 6d (ia ) 0.13) has
only 25% of the intrinsic activity of the corresponding
unsubstituted compound 6b (ia ) 0.59). Modification of
the 5-methyl substituent to a 5-chloro substituent
(compound 6e) dramatically lowered the intrinsic activ-
ity in the NCH3 series. However, in case of the NH
series, the same modification led to retention of intrinsic
activity (6d, ia ) 0.13; 6f, ia ) 0.14). We consider that
the intrinsic activity in both series is basically deter-
mined by the balance of electronic and steric charac-
teristics of the substituents on the benzoxazole ring.
Smaller and electron-donating substituents tend to
increase the intrinsic activity, but bulkier and/or electron-
withdrawing substituents lower it. Further SAR study
of the substituent effect is under way to confirm this
hypothesis. The distinction between the NH and NCH3
series as regards their intrinsic activity appears to be
a Elemental analysis for C, H, N.
Chemistry. All synthesized compounds and the
synthetic procedures are summarized in Table 1 and
Scheme 1. Compounds substituted at the 5 and 7
positions of the benzoxazole ring were synthesized from
commercially available or known o-nitrophenols. Reduc-
tion of the nitro group was performed by hydrogenation,
and the obtained aminophenols were treated with
carbon disulfide and potassium hydroxide in ethanol
without isolation. 19 The cyclized thiol compounds were
then coupled with amines by heating in toluene (com-
pounds 6c-g; procedure D). Compounds 6a and 6b were
obtained by the coupling of 2-chlorobenzoxazole (10)
with corresponding amines.
Results and Discussion
To determine the characteristics of each compound,
agonist activity for the 5-HT3 receptor in the gut was
examined in contraction tests using isolated guinea pig
ileum.20 Contraction induced by the test compounds was
completely blocked by a 5-HT3 receptor antagonist,
granisetron (5).8 The values of potency (pD2) and
intrinsic activity (ia; % efficacy ratio to the contraction
obtained using 5-HT at 10 µM) presented are each the
mean ( SEM of five independent determinations. Alos-
etron which did not induce a contractile response at all
(no intrinsic activity) in this test could be identified as
a 5-HT3 receptor antagonist and is different from our
compounds possessing a wide range of intrinsic activ-
ity.21
A radioligand binding assay using 5-HT3 receptor22
isolated from the rat cortex was also performed for all
compounds. These results are summarized in Table 2.
Comparison of pD2 and Ki values between the NH series
(6b, 6d, 6f) and NCH3 series (6a, 6c, 6e) clearly
Scheme 1a
a (A) Pd/C, EtOH; (B) Pt on sulfide carbon, EtOH; (C) CS2, KOH, EtOH, reflux; (D) amine, toluene, reflux; (E) amine, chloroform

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7077
Table 3. Binding Affinities for 6g to Other 5-HT Receptors
from the control even at 30 mg/kg, though alosetron
markedly delayed colonic propulsion at 20 mg/kg and
higher (p < 0.01 compared to vehicle control). The
results indicate that the therapeutic index (ratio of the
efficacious dose to the maximum tolerable dose) of 6g
is greater than in the case of alosetron and strongly
support our concept that 5-HT3 partial agonists may be
useful to treat diarrhea-predominant IBS without the
side effect of constipation.
receptor
radioligand
% inhibition (10-5 M)a
5-HT1A
5-HT1B
5-HT2A
5-HT4
5-HT5A
5-HT6
5-HT7
[3H]-8-OH-DPAT
17.5
[
125I]-iodocyanopindolol
93 (Ki ) 786 nM)
[3H]-ketanserin
[3H]-GR113808
7.6
73 (Ki ) 3120 nM)
[
[
[
125I]-LSD
125I]-LSD
125I]-LSD
7.8
8.3
18.9
a % Inhibition are the means of two experiments.
Conclusion
Table 4. Inhibition of 5-HT-Induced Diarhea and Effect on
Colonic Propulsion
As part of our search for novel therapeutic agents to
treat IBS, we synthesized 2-(1-piperazinyl)benzoxazole
and 2-(1-homopiperazinyl)benzoxazole derivatives and
investigated their activities as 5-HT3 receptor partial
agonists. Compound 6g exhibited favorable profiles both
in vitro and in vivo. This compound possessed not only
high affinity and appropriate intrinsic activity for the
5-HT3 receptor, together with high metabolic stability
in an in vitro study, but also exhibited antidiarrhetic
activity without preventing normal bowel function in
vivo. These results suggest that compounds of this type
are promising candidates for the treatment of irritable
bowel syndrome, having satisfactory antidiarrhetic
activity without causing constipation.
effects on colonic propulsion (s)b
inhibition of
5-HT-induced
diarrhea,a ID50
(mg/kg po)
compd
6g
(95% CL)
control 10 mg/kg 20 mg/kg 30 mg/kg
370 ( 38 394 ( 67
0.86 (0.00-14.6) 258 ( 20 332 ( 47
alosetron 1.16 (0.48-3.68) 258 ( 20 424 ( 69 479 ( 73** 549 ( 69
a Twelve animals were used at each dose. CL: confidence limits.
**p < 0.01 vs vehicle control. b Each result represents the mean
( SE of eight animals. The test compound was treated po.
based on a fundamental difference of their natures, i.e.,
the intrinsic activity of the NCH3 series is dominated
by the electronic effect rather than the steric effect of
the substituents, whereas that of the NH series is more
susceptible to the steric character of the substituents.
Therefore, the weakly electron-donating CH3 groups in
6c allow retention of intrinsic activity equal to that of
the unsubstituted analogue 6a, while the electron-
withdrawing chlorine atom in 6e lowers the intrinsic
activity. In the cases of 6d and 6f, either substituent
(5,7-dimethyl of 6d and 5-chloro-7-methyl of 6f) equally
increases the steric bulk of the molecule, and conse-
quently both lower the intrinsic activity.23 Compound
6g, a homopiperazine-containing NH compound derived
from compound 4, was also synthesized and evaluated.
This compound showed similar or superior activity at
the 5-HT3 receptor to the other compounds and had the
same intrinsic activity as 6f and 4.
The metabolic stability of 6d-g in the presence of rat
liver microsomes was also evaluated,24 and the results
are summarized in Table 2. Compounds possessing a
NH-containing heterocycle showed superior stability to
those in the NCH3-containing heterocycle series. Com-
pound 4 had the strongest affinity for the 5-HT3 receptor
in both the contraction test and binding assay, but its
metabolic stability was inferior to that of 6g. We chose
compound 6g for further investigation since it possessed
not only a suitable profile at the 5-HT3 receptor, but
also had high metabolic stability. Binding study of 6g
to other 5-HT receptors revealed that this compound is
selective for the 5-HT3 receptor (Table 3).25 We exam-
Experimental Section
Chemistry. All melting points are uncorrected. IR spectra
were recorded on Shimadzu FT-IR 8100 spectrometers. NMR
spectra were obtained on JEOL GX-400 FT- NMR spectrom-
eters. The following abbreviations are used: s ) singlet, d )
doublet, t ) triplet, q ) quartet, quin ) quintet, m ) multiplet,
and br ) broad. MS were measured with Hitachi M-80B and
JEOL JMS-700 instruments. Serotonin (1) was a commercial
product, and alosetron (2) was synthesized according to the
procedure reported previously. The synthesis and character-
ization of compound 6a, 6b, 6c, 6e were represented in our
previous report.16
5,7-Dimethyl-2-(1-piperazinyl)benzoxazole (6d). Pro-
cedures A, C, and D. 2,4-Dimethyl-6-nitrophenol28 (6 g, 35.9
mmol) was dissolved in ethanol (50 mL), and 10% palladium-
carbon (600 mg) was added to the solution. The reaction
mixture was stirred under a hydrogen atmosphere for 24 h,
and the palladium-carbon was removed by filtration. The
solution was concentrated in vacuo, then the crude 2-amino-
4,6-dimethylphenol was refluxed for 8 h with potassium
hydroxide (4.7 g, 84 mmol) and carbon disulfide (33 mL) in
ethanol (100 mL). The reaction mixture was concentrated in
vacuo, and 5 N aqueous hydrochloric acid (18 mL) and ethyl
acetate (100 mL) were added to the residue. The organic layer
was washed with water (100 mL), dried over MgSO4, and
concentrated in vacuo. 2-Mercapto-5,7-dimethylbenzoxazole
(9d) was obtained as a pale-brownish solid (6.0 g) and used in
the next step without further purification. 9d (2.43 g, 13.6
mmol) and piperazine (2.3 g, 27.1 mmol) were dissolved in dry
toluene (15 mL), and the mixture was refluxed for 16 h. The
solvent was removed under reduced pressure, and the residue
was chromatographed on silica gel with dichloromethane-
methanol (10:1) to give 6d (9.66 mmol) as a yellow solid. mp.
68-70 °C (ether-hexane). NMR (CDCl3) δ: 1.81 (1H, brs), 2.35
(3H, s), 2.37 (3H, s), 2.99 (4H, m), 3.68 (4H, m), 6.65 (1H, s),
6.97 (1H, s). MS (TSP) m/z 232 (M + 1). Anal. (C13H17N3O) C,
H, N.
ined the effects of 6g on 5-HT-induced diarrhea in mice
16,26
to evaluate the in vivo antidiarrhetic efficacy,
and
the effects on colonic propulsion in normal mice to
estimate the propensity to cause constipation, using
alosetron as a reference compound.16,27 Compounds were
administered orally (Table 4). Compound 6g showed
comparable antidiarrhetic activity to alosetron in the
5-HT-induced diarrhea model in mice. The ID50 values
were 0.86 mg/kg for 6g, and 1.16 mg/kg for alosetron.
In the normal mouse model, 6g did not affect colonic
propulsion, with no statistically significant difference
5-Chloro-7-methyl-2-(1-piperazinyl)benzoxazole (6f).
Procedures B, C, and D. This procedure illustrates the
general method for preparation of compound 6f-g. 4-Chloro-
2-methyl-6-nitrophenol29 (2.0 g, 10.7 mmol) was dissolved in
ethyl acetate (60 mL), and 5% platinum on sulfide carbon (60
mg: Aldrich Chemical Co.) was added to the solution. The
reaction mixture was stirred under a hydrogen atmosphere

7078 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Brief Articles
for 24 h, and the platinum on sulfide carbon was removed by
filtration. The solution was concentrated in vacuo, then the
crude 2-amino-4-chloro-6-methylphenol (8f, 1.68 g, 10.7 mmol)
was treated as described for the preparation of 9d. 5-Chloro-
2-mercapto-7-methylbenzoxazole (9f) was obtained as pale-
brownish needles (893 mg) and used in the next step without
further purification. 9f (200 mg, 1.00 mmol) and piperazine
(172 mg, 2.00 mmol) were dissolved in dry toluene (5 mL), and
the mixture was refluxed for 16 h. The solvent was removed
under reduced pressure, and the residue was chromatographed
on silica gel with dichloromethane-methanol (10:1) to give 6f
(224 mg, 0.85 mmol) as yellow plates. NMR (CDCl3) δ: 1.67
(1H, brs), 2.99 (4H, t, J ) 7 Hz), 3.67 (4H, t, J ) 5 Hz), 6.81
(1H, s), 7.14 (1H, s). MS (TSP) m/z 252 (M + 1). Anal. (C12H14-
ClN3O) C, H, N.
5-Chloro-7-methyl-2-(1-homopiperazinyl)benzox-
azole (6g). Obtained as a brownish solid, 67% yield from
4-chloro-2-methyl-6-nitrophenol. mp. 68-70 °C (CH3CN). NMR
(CDCl3) δ: 1.96 (2H, quin, J ) 6 Hz), 2.37 (3H, s) 2.93 (2H, t,
J ) 6 Hz), 3.07 (2H, m), 3.07 ∼ 3.83 (4H. m), 7.02 (1H, d, J )
1.7 Hz), 6.78 (1H, d, J ) 2 Hz), 7.13 (1H, d, J ) 2 Hz). MS
(TSP) m/z 266 (M + 1). Anal. (C13H16ClN3O) C, H, N.
5-HT3 Receptor Binding Assay. The assay was performed
according to the method of Kilpatrick et al.22 Brain cortices
were isolated from male Wistar rats (250-300 g), and a
membrane fraction was prepared by standard techniques. The
membrane fraction (0.04 mg) was incubated with 0.2 nM [3H]-
GR656630 for 60 min at 22 °C. Membranes were collected by
filtration and washed three times. The radioactivity on the
filters was counted to determine [3H]GR656630 specifically
bound. Nonspecific binding was estimated in the presence of
1 mM ICS205-930. For obtaining Ki values, assays were carried
out six times at each dose and displacement curves were fitted
by nonlinear regression. IC50 values were obtained directly.
Ki values were calculated from IC50 values by using the
equation of Cheng and Prusoff.30
Contraction Test.20 Male Hartley guinea pigs weighing
500-600 g were killed by bleeding from the neck, and the
ileum was excised. Pieces (about 20 mm) of ileal longitudinal
muscles were placed in a 5 mL organ bath containing Krebs
solution aerated with 95% O2 and 5% CO2 at 37 °C. The
composition of the solution was as follows (mM): NaCl 118,
KCl 4.7, KH2PO4 1.19, MgSO4 1.2, CaCl2 2.54, NaHCO3 25,
and glucose 11. The solution also contained ritanserin (10-7
M) to inhibit the 5-HT2 receptor. The preparations were
allowed to equilibrate for at least 30 min under 0.5 g of tension.
After equilibration, the preparations were repeatedly exposed
to 3 × 10-7 M 5-HT to desensitize the 5-HT4 receptor.
Compounds were added to the bath, and contractions were
recorded isometrically. The potencies of agonists were ex-
pressed as pD2 values, i.e., the negative logarithum of the
molar concentration which produced 50% of the maximum
contraction obtained from individual concentration-response
curves. The ia of a partial agonist was expressed as the ratio
between the maximum response to a test compound and that
to 5-HT (10-5 M).
on a gently shaking platform maintained at 37 °C. Incubations
were started by the addition of substrate and were stopped
after 0, 15, 30, and 60 min by addition of DMF. Precipitated
proteins were removed by centrifugation and supernatants
were injected into the HPLC system to determine the remain-
ing amount of each compound. HPLC analysis was performed
with an Inertsil C4 (φ4.6 × 250 mm) column (GL Science Inc,
Tokyo, Japan), which was attached to an Inertsil C4 (φ4.0 ×
10 mm) guard column cartridge. The column was developed
with a linear gradient from acetonitrile/50 mM sodium acetate
(pH ) 6.5) 35:65 to 45:55 from 0 to 20 min, and then with a
linear gradient to 80:20 from 20 to 35 min, at a flow rate of 1
mL/min. The column temperature was kept at 40 °C, and the
eluate was monitored at 250 nm.
Inhibition of 5-HT-Induced Diarrhea In Mice.26 Male
mice were starved for 18 h before the experiment. Test
compounds were orally administered 30 min before the injec-
tion of 5-HT (10 mg/kg, sc) or saline as a control. The severity
of diarrhea was scored for 35-min observation period, as
follows; 0: no diarrhea, 1: mild diarrhea (soft or watery feces)
and 2: severe diarrhea (water-like feces). 5-HT (10 mg/kg, sc)
caused diarrhea in 100% of the mice within 35min. The dose
of a test compound required to reduce incidence of the diarrhea
to 50% of the treated animals (ID50) was determined by the
probit analysis, and the confidence limits for p ) 0.95 (95%
CL) were calculated.
Measurement of Transition Time in Distal Colon in
Mice.27 Male mice were starved for 4 h before the experiments.
Thirty minutes following the po administration of test com-
pounds, a glass bead (3 mm in diameter) was inserted into
the distal colon 3 cm above the anus. The time required to
evacuate the bead was measured. The group data were
compared by analysis of variance followed by Steel’s multiple
range test.
Supporting Information Available: Purity data for
6a-g. This material is available free of charge via the Internet
at http://pubs.acs.org.
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