Benzimidazole compounds

This invention relates to compounds useful in treating 5-HT.sub.4 and/or 5-HT.sub.3 mediated conditions of the formula ##STR1## or a pharmaceutically acceptable salt thereof wherein R.sub.1 and R.sub.2 are independently selected from the group consisting of hydrogen, alkoxy, halogen, amino, monoalkylamino, dialkylamino, acylamino and alkylsulfonylamino; R.sub.3 is selected from the group consisting of H, alkyl and cycloalkyl; X is NH or O; and Z is selected from the group consisting of ##STR2## pharmaceutical compositions including the compounds and a method for treating serotonin mediated conditions with the compositions which act as 5-HT.sub.4 agonists or antagonists and/or 5-HT.sub.3 antagonists.

BACKGROUND OF THE INVENTION 
The present invention relates to pharmaceutical agents (compounds) which 
act as 5-HT.sub.4 agonists or antagonists and/or 5-HT.sub.3 antagonists in 
mammals. As 5-HT.sub.4 agonists these agents are useful in the treatment 
of hypomotility disorders of the gastrointestinal (GI) tract including 
reflux esophagitis, non-ulcer dyspepsia, gastroparesis, ileus, irritable 
bowel syndrome (constipation predominant) and constipation. Additionally, 
5HT.sub.4 agonists are useful for the treatment of learning and memory 
disorders and as cardiovascular inotropic agents. As 5-HT.sub.4 
antagonists these compounds are useful in the treatment of motility 
disorders of the GI tract such as diarrhea and irritable bowel syndrome 
(diarrhea predominant). As 5-HT.sub.3 antagonists these compounds are 
useful for treating emesis (caused by cancer chemotherapy or 
post-operative), anxiety, cognitive disorders, drug abuse (either cravings 
or withdrawal syndrome) and irritable bowel syndrome (diarrhea 
predominant). 
Serotonin (5-hydroxytryptamine; 5-HT) functions as a neurotransmitter in 
the mammalian central nervous system (CNS) and in the periphery. 
Serotonergic neurons regulate a wide variety of sensory, motor and 
cortical functions. Additionally serotonin regulates enteric reflexes, 
mediates contraction of the vascular smooth muscle and platelet shape 
change and aggregation and as such effects such diverse systems as the 
cardiovascular system and gastrointestinal system, in addition to the 
central nervous system. 
Pharmacological and physiological studies show that the activity of 
serotonin is mediated by several distinct cell surface receptor subtypes. 
These receptor subtypes either transduce extracellular signals by 
activating GTP-binding proteins (G-protein-coupled receptor subtypes) or 
activate the opening of nonselective cation channels to promote fast, 
depolarizing responses in neurons (ligand-gated ion channel receptor 
subtypes). 5-HT.sub.4 belongs to the former category while 5-HT.sub.3 
belongs to the latter. Agents which interact with these receptors thereby 
modulate a variety of ion channels and intracellular messenger signaling 
pathways thereby extending the flexibility of serotonin's activity and 
eliciting a multitude of cellular and physiological responses. P. Bonate, 
Clinical Neuropharmacology, Vol. 14, No. 1, pp. 1-16 (1991). 
European Patent application 309,423 discloses azabicyclo substituted 
benzimidazoline-2-oxo-1-carboxylic acid derivatives which are useful as 
5-HT receptor antagonists. 
Dumuis et al., Nauyn-Schmiedeberg's Arch Pharmacol, (1991) 343: 245-251 
disclose azabicycloalkyl benzimidazolone derivatives as potent agonists at 
the 5-HT.sub.4 receptor. 
In Pharmacological Research, Vol. 22, Supplement 2, (1990) Schiantarelli et 
al. disclose two benzimidazolone compounds useful as 5-HT.sub.3 
antagonists and 5-HT.sub.4 agonists. 
There is a need in the area of serotonin regulation for agents with broad 
clinical usefulness. Serotonin is one of the newer neurotransmitters to be 
recognized for physiological importance and agents which interact with 
5-HT receptors are currently the focus of much research. P. Bonate, 
Clinical Neuropharmacology, Vol. 14, No. 1, pp. 1-6 (1991). 
Accordingly, it is the object of this invention to produce compounds for 
use as pharmaceutical agents which will exhibit 5-HT.sub.4 agonist or 
antagonist and/or 5-HT.sub.3 antagonist activity in mammals. The compounds 
of the present invention meet the need for an agent which has broad 
clinical usefulness for treating serotonin mediated conditions in mammals 
by administering a therapeutically effective amount of the compounds to 
act as 5-HT.sub.4 agonists or antagonists and/or 5-HT.sub.3 antagonists. 
SUMMARY OF THE INVENTION 
This invention relates to compounds of the Formula I 
##STR3## 
or a pharmaceutically acceptable salt thereof wherein R.sub.1 and R.sub.2 
are independently selected from the group consisting of hydrogen, alkoxy, 
halogen, amino, monoalkylamino, dialkylamino, acylamino and 
alkylsulfonylamino; 
R.sub.3 is selected from the group consisting of H, alkyl and cycloalkyl; 
X is NH or O; and 
Z is selected from the group consisting of 
##STR4## 
The present invention also provides pharmaceutical compositions comprised 
of a therapeutically effective amount of the compounds of Formula I in 
combination with a pharmaceutically acceptable carrier and a method for 
treating serotonin mediated conditions with said compositions which act as 
5-HT.sub.4 agonists or antagonists and/or 5-HT.sub.3 antagonists.

The following examples illustrate the methods used to prepare the compounds 
of this invention. These examples are given by way of illustration only 
and are not meant to be construed as limiting the invention in spirit or 
scope, as many modifications in materials and methods will be apparent 
from this disclosure to one having ordinary skill in the art. 
EXAMPLE 1 
##STR9## 
5-chloro-2-nitroaniline (8.6 g; 0.05 mole), 2,2-dimethoxypropane (10.0 ml; 
0.09 mole) and trifluoroacetic acid [TFA] (4.0 ml; 0.005 moles) were 
dissolved in toluene (100 ml) and stirred for 1 hr. A boron/pyridine 
complex (hereinafter BH.sub.3 * pyridine) (5.0 ml; 0.05 moles) was added 
in 1.0 ml increments. The reaction was exothermic, and the reaction 
progress was monitored by tlc (40% EtOAc/hexane). Additional TFA, BH.sub.3 
* pyridine, and 2,2-dimethoxypropane were added until the tlc indicated 
that the 5-chloro-2-nitroaniline was consumed. The reaction mixture was 
placed on a bed of silica and eluted with toluene. The product was the 
first major component to elute, which produced 6.6 g (61%) of a Yellow 
crystalline solid. 
______________________________________ 
C.sub.9 H.sub.10 ClN.sub.2 O.sub.2 M.W. 213.63 
Elements Calc Found 
______________________________________ 
Carbon 50.36 50.27 
Hydrogen 5.17 5.17 
Nitrogen 13.05 12.97 
Chlorine 16.52 15.92 
______________________________________ 
EXAMPLE 2 
##STR10## 
N-isopropyl-5-chloro-2-nitroaniline (6.5 g; 0.03 mole) was dissolved in 
MeOH (330 ml) and hydrogenated at room temperature, at 5.0 psi over Ra-Ni 
for 1. 5 hr. at which time the theoretical hydrogen uptake had been 
achieved. The reaction mixture was filtered and the filtrate was 
concentrated. The residue was dissolved in CH.sub.2 Cl.sub.2 (250 ml) and 
triphosgene (3.06 g; 0.01 mole) dissolved in 25 ml of CH.sub.2 Cl.sub.2 
was added. The reaction was exothermic. The reaction mixture was stirred 
for 1 hr. and then concentrated. The residue was placed on a bed of silica 
and eluted with EtOH/CH.sub.2 Cl.sub.2 using a gradient from 5% 
.fwdarw.40% EtOH. The fractions containing the product were combined and 
concentrated to yield 2.7 g (43%) of a purple-white solid. 
______________________________________ 
C.sub.10 H.sub.11 ClN.sub.2 O M.W. 210.65 
Elements Calc Found 
______________________________________ 
Carbon 57.01 56.61 
Hydrogen 5.26 5.29 
Nitrogen 13.30 13.19 
Chlorine 16.83 17.20 
______________________________________ 
EXAMPLE 3 
##STR11## 
4-chloro-2-nitroaniline (8.6 g; 0.05 mole), 2,2-dimethoxypropane (10.0 ml; 
0.9 mole) and trifluoroacetic acid [TFA] (4.0 ml; 0.005 moles) were 
dissolved in toluene (100 ml) and stirred for 1 hr. BH.sub.3 * pyridine 
(5.0 ml; 0.05 moles) was added in 1.0 ml increments. The reaction was 
exothermic, and the reaction progress was monitored by tlc 40% 
EtOAc/Hexane. Additional TFA, BH.sub.3 * pyridine and 2,2-dimethoxypropane 
were added until the tlc indicated that the 4-chloro-2-nitroaniline was 
consumed. The reaction mixture was placed on a bed of silica and eluted 
with toluene. The product was the first major component to elute, which 
produced 8.4 g (78%) of a yellow crystalline solid. 
______________________________________ 
C.sub.9 H.sub.10 ClN.sub.2 O.sub.2 M.W. 213.63 
Elements Calc Found 
______________________________________ 
Carbon 50.36 50.44 
Hydrogen 5.17 5.26 
Nitrogen 13.05 12.96 
Chlorine 16.52 16.22 
______________________________________ 
EXAMPLE 4 
##STR12## 
N-isopropyl-4-chloro-2-nitroaniline (8.3 g; 0.0389 mole) was dissolved in 
MeOH (330 ml) and hydrogenated at room temperature, at 5.0 psi over Ra--Ni 
for 1.5 hr. at which time the theoretical hydrogen uptake had been 
achieved. The reaction mixture was filtered and the filtrate was 
concentrated. The residue was dissolved in CH.sub.2 Cl.sub.2 (250 ml) and 
triphosgene (3.9 g; 0.014 mole) dissolved in 25 ml of CH.sub.2 Cl.sub.2 
was added. The reaction was exothermic. The reaction mixture was stirred 
for 1 hr. then concentrated. The solid was triturated with water and 
filtered, washed with Et.sub.2 O and suction dried which produced 5.7 g 
(69.5%) of a purple-white solid. 
______________________________________ 
C.sub.10 H.sub.11 ClN.sub.2 O M.W. 210.65 
Elements Calc Found 
______________________________________ 
Carbon 57.01 56.89 
Hydrogen 5.26 5.28 
Nitrogen 13.30 13.29 
Chlorine 16.83 16.54 
______________________________________ 
EXAMPLE 5 
##STR13## 
5-chloro-2-nitroaniline (17.2 g; 0.1 mole), acetal aldehyde diethyl acetal 
(Acetal) (21.1 ml; 0.15 mole) and TFA (7.7 ml; 0.1 moles) were dissolved 
in toluene (500 ml) and stirred for 1 hr. BH.sub.3 * pyridine (10.0 ml; 
0.1 moles) was added in 1.0 ml increments. The reaction was exothermic, 
and the reaction progress was monitored by tlc 40% EtOAc/Hexane. 
Additional TFA, BH.sub.3 * pyridine and acetal were added until the tlc 
indicated that the 5-chloro-2-nitroaniline was consumed. The reaction 
mixture was washed 2X with water and placed on a bed of silica and eluted 
with toluene. The product was the first major component to elute. The 
product was crystallized from MeOH which produced 17.6 g (88%) of a yellow 
crystalline solid. 
______________________________________ 
C.sub.8 H.sub.9 ClN.sub.2 O.sub.2 M.W. 200.63 
Elements Calc Found 
______________________________________ 
Carbon 47.89 47.69 
Hydrogen 4.52 4.36 
Nitrogen 13.96 13.92 
Chlorine 17.67 17.71 
______________________________________ 
EXAMPLE 6 
##STR14## 
N-ethyl-5-chloro-2-nitroaniline (17.6 g; 0.0877 mole) was dissolved in MeOH 
(1.0 l) and hydrogenated at room temperature, at 5.0 psi over Ra--Ni for 
1.9 hr. at which time the theoretical hydrogen uptake had been achieved. 
The reaction mixture was filtered and the filtrate was concentrated. The 
residue was dissolved in CH.sub.2 Cl.sub.2 (500 ml) and triphosgene (8.6 
g; 0.029 mole) dissolved in 50 ml of CH.sub.2 Cl.sub.2 was added. The 
reaction was exothermic. The reaction mixture was stirred for 1 hr. and 
then concentrated. The solid was triturated with water and filtered, 
washed with Et.sub.2 O and suction dried which produced 7.9 g (46%) of a 
blue-white solid. 
______________________________________ 
C.sub.9 H.sub.9 ClN.sub.2 O M.W. 196.64 
Elements Calc Found 
______________________________________ 
Carbon 52.57 52.18 
Hydrogen 4.90 4.68 
Nitrogen 13.62 13.68 
Chlorine 17.24 17.17 
______________________________________ 
EXAMPLE 7 
##STR15## 
4-chloro-2-nitroaniline (8.6 g; 0.05 mole), acetaldehyde (5.5 ml; 0.05 
mole) and HOAc (4.0 ml; 0.1 moles) were dissolved in toluene (100 ml) and 
stirred for 1 hr. BH.sub.3 * pyridine (5.0 ml; 0.01 moles) was added in 
1.0 ml increments. The reaction was exothermic, and the reaction progress 
was monitored by tlc (40%EtOAc/Hexane). Additional TFA, BH.sub.3 * 
pyridine and acetaldehyde were added until the tlc indicated that the 
4-chloro-2-nitroaniline was consumed. The reaction mixture was washed 2X 
with water and placed on a bed of silica and eluted with 20% EtOAc/Hexane. 
The product was the first major component to elute. The product was 
crystallized from MeOH which produced 5.0 g (50%) of a yellow crystalline 
solid. 
______________________________________ 
C.sub.8 H.sub.9 ClN.sub.2 O.sub.2 M.W. 200.63 
Elements Calc Found 
______________________________________ 
Carbon 47.89 47.57 
Hydrogen 4.52 4.41 
Nitrogen 13.96 14.07 
Chlorine 17.67 17.77 
______________________________________ 
EXAMPLE 8 
##STR16## 
15 N-ethyl-4-chloro-2-nitroaniline (8.7 g; 0.0436 mole) was dissolved in 
MeOH (330 ml) and hydrogenated at room temperature, at 5.0 psi over Ra--Ni 
for 3.1 hr. at which time the theoretical hydrogen uptake had been 
achieved. The reaction mixture was filtered and the filtrate was 
concentrated. The residue was dissolved in CH.sub.2 Cl.sub.2 (200 ml) and 
triphosgene (4.3 g; 0.0145 mole) dissolved in 25 ml of CH.sub.2 Cl.sub.2 
was added. The reaction was exothermic. The reaction mixture was stirred 
for 1 hr. then concentrated. The solid was triturated with water and 
filtered, washed with Et2O and suction dried which produced 5.5 g (64%) of 
a blue-white solid. 
______________________________________ 
C.sub.9 H.sub.9 ClN.sub.2 O M.W. 196.64 
Elements Calc Found 
______________________________________ 
Carbon 52.57 52.83 
Hydrogen 4.90 4.90 
Nitrogen 13.62 13.74 
Chlorine 17.24 17.21 
______________________________________ 
EXAMPLE 9 
##STR17## 
4-Cyanoquinuclidine (610 mg, 4.4 mmol; H. P. Fischer and G. A. Grob, 
Helvitica Chemica Acta (1968), 51, 153) was dissolved in tetrahydrofuran 
(THF) (5 mL), and lithium aluminum hydride (10 mL, 10 mmol; 1.0M solution 
in THF) was added. The solution was heated to reflux for 1.5 h. The 
solution was cooled to 0.degree. C. and quenched according to Fieser and 
Fieser work-up (Fieser and Fieser, Reagents for Organic Synthesis, John 
Wiley and Sons, Inc. (New York) 1967, p. 584). Concentration in vacuo 
afforded the title compound as a yellow oil, 549 mg (88%). 
.sup.1 HNMR (CDC13): 1.36 ppm (t, 6H); 2.42 (s, 2H): 2.89 (t, 6H). 
.sup.13 CMR: 28.75 ppm, 28.56; 47.41, 51.67. 
EXAMPLE 10 
##STR18## 
60% NaH/Mineral oil (80 mg; 0.002) was washed with hexane and suspended in 
THF. 1-isopropyl-2-benzimidazolone (176 mg; 0.001 mole) was added to the 
suspension. This mixture was stirred 15 minutes before adding to a mixture 
of 2.5 ml (0.004 mole) 20% phosgene in toluene/2.5 ml THF. The resulting 
mixture was filtered through celite and concentrated. The residue was 
dissolved in THF (50 ml) and the compound of Example 9 (70 mg; 0.0005 
mole) was added with 0.5 ml of Et.sub.3 N. This mixture was stirred 1 hr., 
filtered and the filtrate concentrated. The residue was chromatographed on 
a prep tlc plate, eluting with 20% MeOH/CHCl.sub.3. The product was washed 
from the silica with 5% NH.sub.4 OH/MeOH. The filtrate was concentrated 
and the residue was dissolved in CHCl.sub.3 and filtered through celite 
and concentrated. 144 mg (87%) of product was isolated. The product was 
converted to the HCl salt by dissolving 37.2 .mu.l of acetyl chloride in 
5.0 ml of MeOH and adding this solution to the product, then concentrating 
to dryness. 
______________________________________ 
C.sub.10 H.sub.26 N.sub.4 O.sub.2 * 1.2 HCl * 0.5 H.sub.2 O M.W. 395.20. 
Elements Calc Found 
______________________________________ 
Carbon 57.75 57.4 
Hydrogen 7.19 7.12 
Nitrogen 14.18 14.04 
Chlorine 10.76 10.57 
______________________________________ 
EXAMPLE 11 
##STR19## 
4-fluoro-2-nitroaniline (15.6 g; 0.1 mole), 2,2-dimethoxypropane (24.6 ml; 
0.2 mole) and trifluoroacetic acid [TFA] (23.1 ml; 0.005 moles) were 
dissolved in toluene (500 ml) and stirred for 1 hr. BH.sub.3 * pyridine 
(10.0 ml; 0.1 moles) was added in 1.0 ml increments. The reaction was 
exothermic, and the reaction progress was monitored by tlc 40% 
EtOAc/Hexane. Additional TFA, BH.sub.3 * pyridine and 2,2-dimethoxypropane 
were added until the tlc indicated that the 4-fluoro-2-nitroaniline was 
consumed. The reaction mixture was placed on a bed of silica and eluted 
with 10% methyl-t-butylether/hexane. The product was the first major 
component to elute, which produced 11.2 g (58%) of a yellow oil. 
______________________________________ 
Elements Calc Found 
______________________________________ 
Carbon 54.54 55.09 
Hydrogen 5.59 5.63 
Nitrogen 14.13 14.00 
______________________________________ 
EXAMPLE 12 
##STR20## 
4-fluoro-N-isopropyl-2-nitroaniline (11.1 g; 0.0564 mole) was dissolved in 
MeOH (330 ml) and hydrogenated at room temperature, at 5.0 psi over Ra--Ni 
for 1.5 hr. at which time the theoretical hydrogen uptake had been 
achieved. The reaction mixture was filtered and the filtrate was 
concentrated. The residue was dissolved in CH.sub.2 Cl.sub.2 (200 ml) and 
triphosgene (5.6 g; 0.0566 mole) dissolved in 25 ml CH.sub.2 Cl.sub.2 was 
added. The reaction was exothermic. The reaction mixture was stirred for 1 
hr. and then concentrated. The solid was triturated with water and 
filtered, washed with Et.sub.2 O and suction dried. 5.2 g (47.5%) of a 
purple-white solid. 
______________________________________ 
C.sub.10 H.sub.11 FN.sub.2 O * 0.1 H.sub.2 O M.W. 197.8. 
Elements Calc Found 
______________________________________ 
Carbon 61.28 61.06 
Hydrogen 5.76 5.52 
Nitrogen 14.29 14.22 
______________________________________ 
EXAMPLE 13 
##STR21## 
4-methoxy-2-nitroaniline (16.8 g; 0.1 mole), 2,2-dimethoxypropane (24.6 ml; 
0.2 mole) and trifluoroacetic acid [TFA] (23.1 ml; 0.005 moles) were 
dissolved in toluene (500 ml) and stirred for 1 hr. BH.sub.3 * pyridine 
(10.0 ml; 0.1 moles) was added in 1.0 ml increments. The reaction was 
exothermic, and the reaction progress was monitored by tlc 40% 
EtOAc/Hexane. Additional TFA, BH.sub.3 * pyridine and 2,2-dimethoxypropane 
were added until the tlc indicated that the 4-fluoro-2-nitroaniline was 
consumed. The reaction mixture was placed on a bed of silica and eluted 
with 10% methyl-t-butylether/hexane. The product was the first major 
component to elute, which produced 13.2 g (62%) of a yellow solid. 
______________________________________ 
C.sub.10 H.sub.14 N.sub.2 O.sub.3 M.W. 210.22. 
Elements Calc Found 
______________________________________ 
Carbon 57.13 57.15 
Hydrogen 6.71 6.78 
Nitrogen 13.32 13.33 
______________________________________ 
EXAMPLE 14 
##STR22## 
N-isopropyl-4-methoxy-2-nitroaniline (13.1 g; 0.0627 mole) was dissolved in 
MeOH (330 ml) and hydrogenated at room temperature, at 5.0 psi over Ra--Ni 
for 1.5 hr. at which time the theoretical hydrogen uptake had been 
achieved. The reaction mixture was filtered and the filtrate was 
concentrated. The residue was dissolved in CH.sub.2 Cl.sub.2 (250 ml) and 
triphosgene (6.3 g; 0.06447 mole) dissolved in 25 ml CH.sub.2 Cl.sub.2 was 
added. The reaction was exothermic. The reaction mixture was stirred for 1 
hr. then concentrated. The solid was triturated with water and filtered, 
washed with Et.sub.2 O and suction dried. 9.9 g (76%) of a purple-white 
solid. 
______________________________________ 
C.sub.11 H.sub.14 N.sub.2 O.sub.2 * 0.1 H.sub.2 O M.W. 208.05. 
Elements Calc Found 
______________________________________ 
Carbon 63.51 63.31 
Hydrogen 6.88 6.93 
Nitrogen 13.46 13.42 
______________________________________ 
The benzimidazolone compounds of Examples 12 and 14 can be used to prepare 
the compounds of the claimed invention in a manner similar to that of 
Example 10. 
SEROTONIN (5-HT.sub.3) 
Procedure 
GR65630 binds to the 5-HT.sub.3 receptor. Brain cortices were obtained from 
male rats and a membrane fraction prepared by standard techniques. 0.04 mg 
of membrane prep was incubated with 0.2 nM [.sup.3 H]-GR656630 for 60 
minutes at 22.degree. C. Non-specific binding g was estimated in the 
presence of 1 uM ICS 205-930. Membranes were filtered and washed 3 times 
and the filters were counted to determine [3H]-GR65630 specifically bound. 
(Kilpatrick G. J., Jones B. J. and Tyers M. B. Identification and 
distribution of 5-HT.sub.3 receptors in rat brain using radioligand 
binding. Nature, 330: 746-748, 1987). 
Results 
Kd=2.46 nM 
Bmax=154 fmol/mg protein 
% Specific Binding: 70 
______________________________________ 
Effect of Reference Compounds on 
[H]-GR65630 Bound (0.2 nM) 
Compound IC.sub.50 Ki Hill Coefficient 
______________________________________ 
Quipazine 
0.5 nM 0.18 nM 0.86 
ICS 205-930 
2.2 nM 0.51 nM 1.0 
5-HT 122 nM 0.39 uM 1.0 
RU24969 320 nM 1.85 uM 1.0 
Zacopride 
0.55 nM 0.18 nM 0.86 
Example 10 
8.6 nM 4.3 nM 1.0 
______________________________________ 
BEZOLD-JARISCH REFLEX 
The compound to be evaluated was administered i.p. (mg/kg) to a group of 3 
mice. Thirty minutes later, a 5-HT (0.25 mg/kg i.v.)-induced bradycardia 
was recorded in pentobarbital anesthetized animals. A greater than 50 
percent (&gt;50) reduction in the bradycardic response relative to 
vehicle-treated control mice was considered significant. This method has 
been described by Saxena, P. R. and Lawang, A., Arch. Int. Pharmacodyn., 
277: 235-252, 1985. 
The assay results for the compounds of the present invention and their 
Minimum Effective Dose (MED) and the reference compounds and their MEDs 
are recorded in Table II. 
TABLE II 
______________________________________ 
Minimum Effective Dose 
COMPOUND (MED) mg/kg 
______________________________________ 
Example 10 0.03 
BRL-43694 0.05 
cisapride 5 
cyproheptadine 
5 
domperidone &gt;10 
GR-38032 0.5 
ketanserin &gt;10 
mecamylamine 2.5 
methylsergide 
&gt;10 
metoclopramide 
5 
scopolamine 2.5 
______________________________________ 
IN VITRO FUNCTIONAL ASSAY FOR SEROTONIN 5-HT.sub.4 AGONISM: RAT TMM 
Serotonin 5-HT.sub.4 agonism was measured in the rat esophagus in vitro 
preparation as reported by Baxter et al (Naunyn. Schmied. Arch. Pharmacol. 
1991, 343, 439). Agonist activity was determined utilizing relaxation of 
carbachol-contracted rat tunica muscularis mucosae. One 2 cm segment of 
intrathoracic esophagus proximal to the diaphragm was removed from male 
rats, weighing approximately 300 gm, and the outer muscle layers removed. 
The inner tunica muscularis mucosa was mounted under 0.2-0.3 g of tension 
in a tissue bath containing oxygenated Tyrode's solution at 37.degree. C. 
Cortisterone acetate (30 .mu.M) and fluoxetine (1 .mu.M) were included in 
the buffer to prevent uptake of serotonin, as well as pargyline (10 .mu.M) 
to inhibit monoamine oxidase. Following a 30 min equilibrium period, 
tissues were isometrically contracted with carbachol (3 .mu.M) to obtain a 
tonic contraction. A stable plateau was obtained within 20 min when test 
compound was added cumulatively to relax the muscle strip. EC50 values 
were obtained for each agonist in tissues from 5 rats. EC50 values for 
agonists at this 5-HT.sub.4 receptor are indicated in Table III. 
TABLE III 
______________________________________ 
5-HT.sub.4 Agonism (Rat TMM) In 
Compound Vitro Assay: EC50 Values 
______________________________________ 
Serotonin 9 nM 
Example 10 125 nM 
______________________________________ 
Although this invention has been described with respect to specific 
embodiments, the details of these embodiments are not to be construed as 
limitations. Various equivalents, changes and modifications may be made 
without departing from the spirit and scope of this invention, and it is 
understood that such equivalent embodiments are part of this invention.