C.-B. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4606–4609
4607
O2N
H2N
H2N
a
c
b
N
H
O
N
H
N
N
O
O
S
H
H
2
3
1
H
H
N
H2N
N
Cl
N
d
e
f
O
N
O
N
N
N
R2
N
N
N
N
N
N
R1
R1
R1
6a-s
5
4
Scheme 1. Synthetic scheme for the synthesis of compounds 6a–s. Reagents and conditions: (a) H2SO4, HNO3 (95%); (b) H2, Pd/C, MeOH (85%); (c) P2S5, Et3N, CH3CN (70%); (d)
R1CONHNH2, cyclohexanol, reflux, N2, 4 h (55%); (e) ClCH2COCl, CH2Cl2, rt (98%); (f) 4-substitued piperazine, Na2CO3, MeOH, reflux, 10 h (83–93%).
The method of the measuring left atrial stroke volume was
adopted to evaluate positive inotropic activity of the compounds
synthesized above. An isolated, perfused atrial preparation was
prepared by using the method described previously.7,8 Thus, the at-
rium was perfused with N-2-hydroxyethylpiperazine-N0-2-ethane-
sulfonic acid (HEPES) buffer solution by means of a peristaltic
pump (1.25 mL/min).9 The composition of the buffer was as fol-
lows (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3,
10.0 glucose, 10.0 HEPES (adjusted to pH 7.4 with 1 M NaOH), and
0.1% bovine serum albumin (BSA). Soon after the perfused atrium
was setup, transmural electrical field stimulation with a luminal
electrode was started at 1.5 Hz (duration, 0.3–0.5 ms, voltage
30 V). The changes in the atrial stroke volume were monitored
by reading the lowest level of the water column in the calibrated
atrial cannula during the end diastole. The atria were perfused
for 60 min to stabilize the stroke volume. The atrial beat rate was
fixed at 1.5 Hz, the left atrium stroke volume was recorded at 2-
min interval, and the stimulus effect of the sample was recorded
after a circulation of the control group. Every circulation was
12 min.
The compounds were investigated using the single dose tech-
nique at concentration of 3 ꢀ 10ꢁ5 M. Samples were dissolved in
DMSO and diluted with the HEPES buffer to an appropriate volume.
The biological evaluation data for these compounds was expressed
in means of increased stroke volume percentage as shown in Table
1. Heart rate measurements for those selected compounds were
carried out in isolated rabbit hearts by recording the electrocardio-
gram in the volume conduction model. In order to assess differ-
ences, repeated measurements were compared by means of an
ANOVA test. The statistical significance was defined as P < 0.05
and the data are presented as means SE.
As shown in Table 1, 11 compounds out of the 19 tested
compounds showed inotropic effects on isolated rabbit heart
preparations. Compounds 6a, 6b, 6g, 6j, 6m, 6q, and 6r exhib-
ited more potent effects compared to milrinone (4.7 0.3%,
3 ꢀ 10ꢁ5 M), among which the compound 6j showed the most
potent activity with 13.2 1.3% increased stroke volume. As for
the relationship between inotropic activity and different sub-
stituents on the triazole ring and benzene ring attached to
piperazine (R1, R2), those compounds only having electron-
donating groups on the phenyl ring displayed enhanced effects
for the 1-methyl substituted derivatives, but such a phenome-
non was not found so obvious for those derivatives possessing
1-phenylmethyl substituents on the triazole ring. These results
indicate that the contribution of the substituent R1 on triazole
ring to the biological effect might be more important than that
of the substituent R2 on phenyl ring. Interestingly, in contrast
to the previously evaluated PHR9612 (no data), 6i showed no
efficiency in such a case of structure modification to introduce
triazole ring to the 1,2-position of 3,4-dihydro-2(1H)-quinoli-
none, but for the compound 6j, a significant inotropic effect
was found, in which the only difference is that the methyl
group is replaced by the phenylmethyl group. A similar case
was also observed between 6e and 6f (2.4 0.0%), and the re-
sults seem to support the indication mentioned above. The ad-
verse results, however, were also found between 6a (5.1 0.2%)
and 6b (5.2 0.3%), 6c (1.3 0.3%) and 6d (0.7 0.1%), and 6g
(8.8 0.5%) and 6h (0.5 0.2%), respectively. In latter cases,
the electron-donating effect of the substituent R2 seems still
to exert more dominating influence on the inotropic activity.
As for the influence of the substituents at 4-position of piper-
azine on the inotropic effects, we found that there were no
significant differences between acyl groups and benzyl groups
for the biological efficiency. Nevertheless, among those com-
pounds having more potent effects compared to milrinone, five
compounds (6a, 6b, 6j, 6q, and 6r) possess substituted benzyl
groups and only two compounds (6g and 6m) have substituted
aromatic acyl groups at this position.
On the other hand, we investigated the dynamics of the
tested compounds in perfused beating rabbit atria and found
that compound 6g did not show a desirable biological dynamic
profile, in which the stroke volume of 6g was changed mark-
edly decreasing as the time progressed, in spite of its signifi-
cant increased stroke volume (no figure afforded). Compounds
6a and 6r exhibited similar atrial dynamic profiles to milrinone
with good increased stroke volume (Fig. 1A). Much more desir-
able atrial dynamic profiles were measured for the compounds
6b, 6j, 6m, and 6q (Fig. 1B and C), in which the compound 6j
displayed excellent inotropic effects with the highest increased
stroke volume (Fig. 1B). As shown in Table 2, compounds 6a,
6b, 6j, 6m, 6q, and 6r were also investigated for their chrono-
tropic effects in a beating atrium, and no significant increased
heart rates (P > 0.05) were observed for compounds 6a, 6b,
6m, and 6q at the same concentration. Compounds 6j and
6r, however, showed the changed heart rates unfortunately,
for which further in vivo study was required in order to inves-
tigate their chronotropic effects.
In conclusion, based on the structure of PHR9612, we synthe-
sized 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline derivatives and
tried to find more potent compounds for cardiac contractility with-
out increasing heart rate. As a result, we obtained several com-
pounds having enhanced inotropic effects and desirable
biological profile in our present study, in which the compounds
6a, 6b, 6m, and 6q exhibited more promising cardiovascular pro-
files. These compounds are now undergoing other biological tests
including in vivo evaluation, coronary vasodilating tests, and pos-
sible action mechanism study in order to be selected to the new
candidates for further clinical trials.
Zhang, Chun-Bo
