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C. Yang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
tively showed improved Racine’s score. This comparison indicates
that 4-nitrophenyl on R2 position provides the best activity in the
pilocarpine model. In a similar fashion, compounds with cyclo-
propyl group in the R1 position, in combination with 4-nitrophenyl
(5m), 4-chlorophenyl (5n), and 2-ethylphenyl (5o) were compared.
Within this series, the 4-nitrophenyl group provided the best anti-
convulsant activity.
In both cases, activities of compounds containing a 4-nitro-
phenyl group in R2 position, and having a small hydrophobic group
(5g and 5m) are the same in contributing to the lower Racine’s
score (Racine’s score = 0.4) than the compound with phenyl group
at R2 position (5r, Racine’s score = 3.5). A similar SAR is also con-
firmed by comparing 5h, 5n with 5s, where 4-chlorophenyl group
in R2 position, in combination with aliphatic groups proved to be
more effective.
Figure 1. Structures of phenytoin and the spirohydantoins.
Once we had prepared all the derivatives (5a–5s), we proceeded
to evaluate their anticonvulsant activities. They were tested at a
dose of 100 mg/kg in a pilocarpine model of temporal lobe epi-
lepsy, a model of status epilepticus.17–19 Besides synthesized com-
pounds, phenytoin and dimethyl sulfoxide (DMSO) were included
as a clinical drug control, and the vehicle control, respectively. In
order to compare the activity of newly synthesized compounds
to our previously published findings, 5d was resynthesized and
tested in this pilocarpine model. The anticonvulsant activities were
evaluated using the average Racine’s score13 at the endpoint of 2 h
after injection of pilocarpine. The latency of onset time of motor
seizure (Racine’s score P 3), duration of severity (suffering from
motor seizure), and survival rates were also determined and
recorded.
Ten out of eighteen compounds significantly reduced the aver-
age Racine’s score from 4.5 to less than 2.0, compared to DMSO
(Table 1). Their activities were much better than phenytoin
(Racine’s score = 3.3). Among them 5b, 5c, 5d, 5g, 5h, 5i, 5m and
5n exhibited best activities, with significantly reduced average
Racine’s score compared to DMSO (Table 1). Their activities were
noticeably better than that of phenytoin. To better understand
the structure activity relationship of these new compounds, we
studied the groups on N-10 and N-30 positions. With an n-propyl
group in the R1 position, 5g, 5h, and 5i, where R2 is substituted
with 4-nitrophenyl, 4-chlorophenyl and 2-ethylphenyl, respec-
It is interesting to note that, when a phenyl group is in R2 posi-
tion, compounds 5e, 5k and 5p showed potent sedative effect. Rat
pups lost righting reflex within 10 min after injection of these com-
pounds, and they did not recover in the following four hours. This
is a unique finding from this study, and may provide a new direc-
tion for further research in the future.
DMSO and phenytoin had a motor seizure onset time of 6 min
and 12 min, respectively, where five of the synthetic spirohydan-
toins (5f, 5g, 5l, 5r and 5s) considerably delayed the seizure onset
time. Interestingly, compound 5r delayed the onset time from
6 min to 104 min. Moreover, compound 5c completedly protected
the rats from motor seizures.
As Pilocarpine induces status epilepticus in our experimental
model, we monitor progression of seizure over a 120 min period.
After injection of pilocarpine, DMSO group rapidly reached stage
5, the average Racine’s score were not decreased in the following
two hours which means the rats would suffer from constant sei-
zure. Thus, shortening duration of severity or total prevention of
motor seizure is very important. Most tested compounds reduced
duration of motor seizures compared to DMSO with 114 min dura-
tion. Eight compounds shorten the duration of severity to less than
half duration time of severity of phenytoin. For the most promising
candidates, we monitored the progression of seizure every 30 min
up to 120 min (Table 2). The results indicate that the test animals
recovered from seizure within 90–120 min when treated with
compounds 5c, 5d, 5g and 5m. Additionally, compound 5c was
effective in preventing motor seizure induced by pilocarpine, and
the rats recovered from the pre-stage of motor seizure within
30 min. As constant seizure occurs in pilocarpine model, the sur-
vival rates were also evaluated. Most of the tested compounds
increased the survival rate of the rats. They could largely increase
survival rate to more than 80% when compared to DMSO (53% sur-
vival rate) and phenytoin (67% survival rate).
Table 1
SAR and activity profile of the synthetic spirohydantoins
Compounds
N-30 position (R1)
N-10-position (R2)
Racine’s scorea,b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
Ethyl
Ethyl
Ethyl
Ethyl
Propyl
Propyl
Propyl
Propyl
Methyl-4-benzoyl
2,6-Diethylphenyl
2,4-Dimethoxyphenyl
4-Nitrophenyl
Phenyl
4-Ethylphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Ethylphenyl
2,6-Diethylphenyl
Phenyl
4-Ethylphenyl
4-Nitrophenyl
4-Chlorophenyl
2-Ethylphenyl
Phenyl
4-Ethylphenyl
4-Nitrophenyl
4-Chlorophenyl
1.8⁄
1.6⁄⁄
1.0
1.0⁄⁄
Sedative
3.5⁄⁄⁄
0.4
0.8⁄⁄
0.8⁄⁄
1.3⁄
Propyl
Propyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Phenyl
Phenyl
Phenyl
Phenyl
Sedative
3.0⁄⁄⁄
0.4
1.0⁄⁄⁄
Sedative
Sedative
3.0
5r
5s
3.5
5.0
DMSO
Phenytoin
4.5
3.3
a
Racine’s score: stage 0 —normal nonepileptic activity; stage 1— facial move-
ments, wet dog shakes, and scratching; stage 2— head nodding, tremor; stage
3—forelimb clonus and forelimb extension; stage 4—rearing and salivation; stage
5— falling and status epilepticus. Animals dead from status epilepticus during
experiments were recorded as stage 5 in the following observing time points.
b
One way ANOVA followed by the first post hoc, LSD test was performed and
data are expressed, n = 5. SEM was not listed. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 are
Scheme 1. Synthesis of the N-10, N-30-disubstituted spirohydantoins.
considered to be significantly different from DMSO alone.