P. Hewawasam et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1117–1120
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Table 1. Structure and physical properties of 1,3-diaryl 1,2,4-(4H)-triazol-5-one derivatives 10a–g, effect on maxi-K-mediated outward current in
Xenopus laevis oocytes expressing the cloned maxi-Kchannels mSlo and relaxation of isolated rat bladder strips pre-contracted with carbachol
Compd
R1
R2
R3
R4
R5
Mp
(ꢀC)a
% Increase in
mSlo current
@ 20 mM
% Inhibition
of force
@ 3 mMc
10a
10b
H
H
H
H
H
H
H
H
H
276–278
167–170
Insol. @ 20 mM
91.4Æ5.9 @ 1 mM
117.1Æ4.3
35.0Æ6.3
89.4 @ 20 mM
34.0Æ3.0
CF3
90Æ2 @ 20 mM
10c
10d
H
H
CF3
H
H
CF3
H
H
H
H
240–245
252–255
161.5Æ4.6 @ 10 mMb
42 (n=2)e
196.8Æ17.7
87Æ3 @ 20 mM
22Æ7 @ 1 mM
15 (n=2)
5 (n=2)
46 (n=2)
10e
10f
10g
H
H
H
CF3
Cl
H
H
CF3
CF3
CF3
H
H
H
H
CF3
250–253
220–224
270–275
154.2Æ4.8
198.5Æ10.5
175.5Æ9.4
131.8Æ12.8d
94Æ1 @ 20 mM
1 (NS-004)
8Æ6 @ 20 mM
aAll new compounds exhibited spectroscopic and combustion data in accord with the designated structure.
bData obtained from Xenopus laevis oocytes expressing hSlo.
cPercentage inhibition of isometric force in response to a test compound in isolated rat bladder strips pre-contracted with 10 mM carbachol.
dReference compound 1 (NS-004) shown to have identical effects on mSlo and hSlo-mediated maxi-Kcurrents.. 20
eStandard deviation not determined for n=2.
The structure–activity relationships described in Table 1
provide a basic understanding of the triazolone maxi-K
channel opening pharmacophore. For this preliminary
SAR investigation, the substitution pattern of the 1-aryl
moiety was restricted to the p-chlorophenol element
present in the prototypical maxi-Kchannel opener
NS-004 and the optimal substitution pattern of 3-aryl
moiety probed. From the results presented in Table 1, it
appears that an electron withdrawing substituent is
essential for the expression of maxi-Kchannel opening
activity, a structure–activity relationship similar to that
observed with a series of benzimidazolone derivatives.21
The introduction of a CF3 substituent markedly enhan-
ces channel opening efficacy as demonstrated by the
three regioisomers 10b–d. Furthermore, both the para
regioisomer 10d and the meta regioisomer 10c were
found to be superior to the ortho CF3 substituted ana-
logue 10b. Similarly, both the 3,5-bis-CF3 and the
2,4-bis-CF3 derivatives 10e and 10g, respectively, exhib-
ited good efficacy as did the 3-Cl, 4-CF3 analogue 10f.
10g, inhibited the contractile response by almost 90% at
20 mM. Compound 10d was found to be particularly
potent with a significant relaxant effect recorded at
concentrations as low as 100 nM. For comparison,
reference compound 1 (NS-004) was not effective in this
assay. However, as can be seen from the data presented
in Table 1, the correlation between maxi-Kchannel
opening activity and the ability to inhibit the force of
carbachol-induced contractions in rat bladder strip assay
is poor. In an attempt to reverse the rat bladder relaxation
with iberiotoxin, a selective maxi-Kchannel blocker, only
partial reversal was observed implicating a role for addi-
tional biochemical mechanisms in the tissue assay.
In summary, we have identified a novel class of maxi-K
channel opener and demonstrated that channel opening
activity is sensitive to both the nature and pattern of
substitution of the 3-phenyl ring. The preference for
electron-withdrawing substituents is reminiscent of SAR
observed for a series of benzimidzolone-based maxi-K
openers.21 Whilst effective relaxants of pre-contracted
rat bladder strips in vitro, the lack of correlation
between this functional effect and maxi-Kchannel
opening activity suggest that alternate mechanisms are
operative. Nevertheless, these triazolone derivatives
offer potential as agents for the treatment of urinary
incontinence. A more detailed analysis of SAR for this
series along with effects of these maxi-Kchannel open-
ers on bladder contractile function will be described in
future publications.
The triazolones 10a–g were evaluated in vitro for their
ability to inhibit the contractile response induced
by carbachol in rat bladder strips isolated from male
Sprague–Dawley rats (250 to 350 g). The contractile
response to 10 mM carbachol was measured twice and,
following the second washout, the tissues were exposed
to the test compound (1, 3, or 20 mM) for 1 h. A third
contractile response to carbachol was then determined
followed by washing away the test compound and
assessing the contractile response to a fourth applica-
tion of carbachol. For all experimental trials, one blad-
der strip per animal served as a paired vehicle control.
The results are expressed as the percentage reduction of
the second carbachol-induced response and were cor-
rected for any spontaneous changes observed in the
paired, vehicle control.
References and Notes
1. Abrams, P.; Blaivas, J. G.; Stanton, S. L.; Andersen, J. T.
Scand. J. Urol. Nephrol. Suppl. 1988, 114, 5.
2. Urinary Incontinence in Adults: Acute and Chronic Man-
Several of the compounds in Table 1 produced greater
than a 30% reduction of contractile force at concentra-
tion of 3 mM and four compounds, 10a, 10b, 10d, and
agement. Clinical Practice Guideline Number
Update); AHCPR publication No. 96–0682; March, 1996.
3. Knapp, P. M. Postgrad. Med. 1998, 103, 279.
2 (1996