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Table 3
The analgesic profiles of compound 44r
In vivo pain models
44r
Morphine
Ketorolac
Acetic acid-induced writhing test in mice
Hot plate test in mice
Formalin test in mice (late phase)
0.94ip 2.65po
2.86po
4.77po
0.96ip 5.57po
13.5po
9.15po
0.59ip 39.1po
N.E# up to 100ip
12.9ip 17.9po
a
Carrageenan-induced mechanical hyperalgesia in rats
0.68po
0.60po
36.4%⁄
NE#: Not effective /All data were represented as ED50 (mg/kg).
a
% inhibition at 100 mg/kg po (⁄p <0.05).
Table 4
binding was observed to most receptors tested including opioids,
ion channels, COXs, 5-HT/NE transporters, and other 5-HT recep-
tors. However, to explain mechanism of action of 44r more in de-
tail, in vitro functional assays were necessary and it was
determined that compound 44r only exhibit weak 5-HT2A func-
Pharmacokinetic data of compound 44r after single administration in rats
PK parameters
iva (15 mg/kg, n = 3)
po (15 mg/kg, n = 3)
Tmax, h
1.1
2200
13159
Cmax, ng/mL
AUCall, ng h/mL
CL, mL/kg/h
Vss, mL/kg
T1/2, h
Bioavailability,%
9953
17825
846
1769
1.8
tional antagonist activity (IC50 = 5.8 lM). To determine the 5-
HT2A antagonistic effects of the compound 44r in vivo, its ability
to inhibit the (+)-l-(4-iodo-2,5-dimethoxyphenyl)-2-propanamine
(DOI)-induced head twitches and 5-hydroxytryptophan (5-HTP)-
induced head twitches in mice were employed.18 Compound 44r
2.6
73.8
a
Intravenous.
dose-dependently blocked DOI-induced head twitches (40.0%⁄⁄
,
61.4%⁄⁄ and 89.3%⁄⁄ of the antagonism at 0.3, 1 and 3 mg/kg, ip,
respectively, ⁄⁄p <0.01) and blocked the 5-HTP-induced head
twitches (respectively, 46.2%⁄⁄ and 100%⁄⁄ of the antagonism at 3
and 10 mg/kg ip, ⁄⁄p <0.01) demonstrating that compound 44r
might exhibit its analgesic activities to some degree through 5-
HT2A antagonistic mechanism.
In summary, we have disclosed the development of a novel
arylpiperazine series for analgesic agents. Starting from hit com-
pound 1 which was moderate in vitro binding assay (5-HT1A and
5-HT2A) we were able to develop 44r by combining the phenotypic
in vivo screening. It is a potent analgesic agent that has favorable
pharmacokinetic properties in rats and most importantly, has a
wide safety margin. We demonstrated with in vitro and in vivo
functional assays that its analgesic activity might be through 5-
HT2A antagonism to some extent. This work further prompted by
discovery of other more promising lead series in this scaffold,
which will be reported in due course.
might be notably from other different mechanism as well as 5-
HT2A mechanism. The compound 44r showed better metabolic sta-
bility in both species compared to compound 43r (44r, HLMT1/
2 = 319 min, RLMT 1/2 = 91 min; 43r, HLMT 1/2 = 62 min; RLMT 1/
2 = 8 min, respectively).
Considering all these data, although compound 44r was weakly
active in receptor binding assays as depicted in Table 2, the analge-
sic potential of the compound 44r was encouraged us to further
evaluate it for analgesic profiles in both anti-nociceptive and
anti-inflammatory pain models using reference drugs (Morphine
and Ketorolac, respectively).
As depicted in Table 3, compound 44r emerged as the most ac-
tive analgesic agent. The anti-nociceptive activities of compound
44r in acetic acid-induced writhing test, hot plate test and formalin
test17 in mice were orally more potent when compared to the ref-
erence compound morphine. Moreover, its activity was not
blocked in the hot plate test after the subcutaneous administration
of the opioid antagonist naloxone (0.1–10 mg/kg) demonstrating it
might act through a non-opioid mechanism of action (data not
shown). And also compound 44r reversed carrageenan-induced
mechanical hyperalgesia in a dose-dependent manner in a rat
model of persistent inflammatory pain.
Acknowledgment
This research was supported by Biostar Project Grant from the
Korean Ministry of Knowledge Economy.
References and notes
In addition to its potent analgesic activities, the median neuro-
toxic doses of compound 44r in the rotarod test were determined
at 52.7 mg/kg (ip) and 139.0 mg/kg (po), suggesting no significant
drug-related side effects such as myorelexation or sedation at the
effective doses. These observation could be of relevance in terms
of the side effect profile of the compound 44r indicating a wide
margin of safety. With the promising in vivo efficacy results in
hand, the pharmacokinetic properties of 44r were evaluated, which
in summarized in Table 4.
After oral administration of a 15 mg/kg dose of 44r to rats, Cmax
of 2200 ng/mL was observed at 1.1 h. The elimination half-life for
44r following oral administration was 2.6 h in rats. And compound
44r showed good oral bioavailability (73.8%).
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Moderate binding was observed to Sigma 1 receptor (Ki = 392 nM),
a1-receptor (Ki = 863 nM). At 10 lM concentration, no significant