Synthesis, antibacterial and anticonvulsant evaluations of some cyclic enaminones

Article abstract of DOI:10.1016/j.ejmech.2008.07.005

Several cyclic enaminone esters were synthesized, characterized, and evaluated for anticonvulsant and antibacterial activities using standardized tests. A series of enaminones were mainly phenyl analogs of anticonvulsant enaminones, while a second series

Full text of DOI:10.1016/j.ejmech.2008.07.005

European Journal of Medicinal Chemistry 44 (2009) 967–975
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antibacterial and anticonvulsant evaluations
of some cyclic enaminones
,
a
b
a
a
Ivan O. Edafiogho a , Oludotun A. Phillips , Edet E. Udo , Santosh Samuel , Beigy Rethish
*
a Faculty of Pharmacy, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
b Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
a r t i c l e i n f o
a b s t r a c t
Article history:
Several cyclic enaminone esters were synthesized, characterized, and evaluated for anticonvulsant and
antibacterial activities using standardized tests. A series of enaminones were mainly phenyl analogs of
anticonvulsant enaminones, while a second series comprised of compounds bearing the oxazolidinone
pharmacophoric moiety found in the synthetic antibacterial linezolid. The enaminone ester bearing an
unsubstituted anilino analog showed class 2 anticonvulsant activity. This represents a first report of an
unsubstituted anilino enaminone with anticonvulsant activity. The enaminone esters gave interesting UV
data, and four analogs displayed potent anticonvulsant activities, while another four compounds showed
moderate anticonvulsant activities. Surprisingly, none of the enaminone esters had any significant
antibacterial activity.
Received 30 March 2008
Received in revised form 2 June 2008
Accepted 8 July 2008
Available online 15 July 2008
Keywords:
Anticonvulsant
Enaminone esters
Oxazolidinones
UV data
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
class of agents [3,4]. It is effective against Gram-positive pathogenic
bacteria including methicillin-resistant Staphylococcus aureus
Epilepsy is a brain disorder in which clusters of nerve cells, or
neurons, in the brain sometimes signal abnormally. In epilepsy,
the normal pattern of neuronal activity becomes disturbed causing
strange sensations, emotions, and behaviors or sometimes
convulsions, muscle spasms, and loss of consciousness. Anticon-
vulsant drugs are an important part of the treatment program for
epilepsy [1]. Once epilepsy is diagnosed, it is important to begin
treatment as soon as possible. This is because seizures can be
controlled with modern medicines and surgical techniques in
about 80% of those diagnosed with epilepsy.
The search for antiepileptic compounds with more selective
activity and lower toxicity is an area under intensive investigation;
hence one of the objectives of this research was to synthesize
enaminones with anticonvulsant activity. Enaminones (Fig. 1) are
chemical compounds consisting of an amino group linked through
a C]C bond to a keto group [2]. The second objective of this
research was to synthesize enaminones containing the oxazolidi-
none moiety, and evaluate them for anticonvulsant and antibac-
terial activities.
(MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE),
Streptococcus pneumoniae (S. pn), Streptococcus pyogenes (S. py), and
vancomycin-resistant Enterococcus faecium (VREF) [5,6]. Early
structure–activity relationship (SAR) on oxazolidinones indicated
that the C5-acylamino group was essential for good antibacterial
activity [7]. However, compounds bearing thio-amides, thioureas,
halogen-substituted methyl-amides, ureas, and N-carbamates have
comparable or superior activity to linezolid [8,9]. Furthermore,
radical modifications involving the introduction of O-linked 2a and
N-linked 2b and PH-027 (Fig. 1) heterocyles at the C5-position have
been shown to give compounds with strong antibacterial activity
[9,10]. In this regard, it was hypothesized that the oxazolidinone
pharmacophoric moiety bearing enaminones at the C5-position
would also exhibit antimicrobial activity.
In addition, it was expected that some of the secondary enam-
inones bearing the oxazolidinone moiety would display anticon-
vulsant activity, similar to the anilino and benzylamino enaminone
derivatives.
Oxazolidinone antibacterial agents exemplified by linezolid 1
are totally synthetic, structurally distinct and mechanistically novel
2. Results and discussions
2.1. Chemistry
* Corresponding author. Tel.: þ965 498 6916; fax: þ965 534 2807.
E-mail addresses: ivanoe@hsc.edu.kw (I.O. Edafiogho), dphillips@hsc.edu.kw
(O.A. Phillips), edet@hsc.edu.kw (E.E. Udo), santhosh_samuel@hotmail.com (S.
Samuel), beigy_01@yahoo.com (B. Rethish).
2.1.1. Synthesis
The cyclohexanone intermediates 5a,b were prepared from the
cyclization reactions between the ketones 3a,b and the esters 4a,b
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.005

968
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
R4
R3
N
O
O
H
N
O
N
N
CH
R2
3
O
F
Y
CO R1
1
O
2
O
Enaminone
O
HO
O
O
N
N
N
X
N
N
O
O
N
N
S
N
F
2a: X = O; Y = H
2b: X = NH; Y = F
N
N
F
PH-027
Fig. 1. Chemical structures of enaminones and oxazolidinones.
(i) Firstly, some enaminones showed slight hypsochromic shift in
acid, and bathochromic shift in alkaline solutions when
compared to their absorptions in neutral solutions. Enami-
nones 6a, 6h, 6m, 6n, 6w, 7c, 7d, and 7m had UV absorption
ranges from 282 to 317 nm in neutral solutions, 259 to 311 nm
in acidic solutions, and 283 to 319 nm in alkaline solutions.
(ii) Secondly, some enaminones showed hypsochromic shift in
acid, and no change in alkaline solutions when compared to
absorptions in neutral solutions. Enaminones 6b, 6c, 6e, 6f, 6g,
6i, 6j, 6p, 6s, 6t, 6y, 6z, 7b, 7h, 7i, and 7j had UV absorption
ranges from 283 to 308 nm in neutral solutions, 259 to 296 nm
in acidic solutions, and 283 to 308 nm in alkaline solutions.
(iii) Lastly, some of the enaminones showed hypsochromic shifts
both in acidic and alkaline solutions when compared to their
absorptions in neutral solutions. Enaminones 6d, 6k, 6l, 6q,
6u, 6v, and 6x had UV absorption ranges from 306 to 315 nm in
neutral solutions, 271 to 309 nm in acidic solutions, and 281 to
313 nm in alkaline solutions.
(iv) Other enaminones showed bathochromic shifts in acidic
solutions, and hypsochromic shifts in alkaline solutions when
compared to their absorptions in neutral solutions. Enami-
nones 6o, 7a, 7f, 7g, 7k, and 7l had UV absorption ranges from
317 to 332 nm in neutral solutions, 319 to 336 nm in acidic
solutions, and 293 to 315 nm in alkaline solutions. Similar to
a few enaminones previously reported [12,21], these enami-
nones uniquely display acidic properties.
in alcohol and sodium alkoxide in the respective alcohol (see also
Scheme 1). The condensation of the cyclohexanone intermediates
5a,bwith avarietyof amino compoundsyielded the finalenaminone
esters (Scheme 1 Tables 1 and 2). The 5-aminomethyl oxazolidinone
and the morpholine and piperazine related amino derivatives were
prepared according to literature methods [10,11]. The enaminone
esters 6a–z and 7a–m were completely characterized by UV, IR,
NMR, MS, and elemental analyses. The recorded data were as
expected for secondary and tertiary enaminones [2], and confirmed
the chemical structures of the compounds in Tables 1 and 2.
2.1.2. Ultraviolet absorption of enaminones
In this study the wavelength of maximum absorption (lmax) and
absorptivity (3) values for selected enaminones were determined.
The enaminone derivatives produced intense absorption bands
under UV spectroscopy and where a compound exhibited more
than one band the most intense bands having molar absorptivity (3)
values of >14 000 were selected and reported. The UV data of the
enaminones were quite unique for the compounds. While the UV
data of enaminones in 95% EtOH and water indicated absorptions in
neutral media; those in, 1 M HCl and 1 M NaOH indicated their
absorptions in acidic and alkaline media, respectively. Most of the
enaminones are basic in nature, and therefore existed in the
partially protonated form in the neutral medium. Deprotonation of
these enaminones in alkaline solution (1 M NaOH) had small hyp-
sochromic or no significant effect on their lmax values when
compared to neutral solutions. The enaminones that normally
behave as weak bases exhibited hypsochromic shift on moving
from neutral to acidic medium. In acidic medium (1 M HCl), the
enaminones were usually protonated. Tertiary enaminones dis-
played UV absorption at longer wavelengths than secondary
enaminones in acidic, alkaline, and neutral solutions. The tertiary
enaminones absorbed UV light at the higher end and secondary
enaminones towards the lower end of the UV wavelength range
259–336 nm in aqueous media.
(v) The enaminone 6r showed bathochromic shift in both acidic
solution (283 nm) and alkaline solution (282 nm), when
compared to its absorption in a neutral medium (261 nm).
(vi) The enaminone 7e showed no change in acidic solution
(318 nm) when compared to its neutral solution (318 nm).
However, enaminone 7e showed hypsochromic shift in alka-
line solution (311 nm) when compared to its absorption in
a neutral solution (318 nm).
It was observed that the enaminones that showed anticon-
vulsant activities were within sub-groups (i)–(iii), whereas enam-
inones in sub-groups (iv)–(vi) including all the heterocyclic analogs
From the UV data of the enaminones (Table 3), interesting UV
absorption patterns were noted, and generally classified into six
sub-groups as follows.
R4
R3
OH
N
O
CO R1
2
(i)
(ii)
+
R2
O
CO R1
2
R2
O
R2
CO R1
2
CO R1
2
5
R1 = Me, Et
R2 = Me, Ph
3
2
R = Me, Ph
4
R1 = Me, Et
6a-z; 7a-m
(R1, R2, R3 R4)
as shown in Tables 1 and 2
Scheme 1. Synthesis of heterocyclic enaminones. (i) Na/MeOH or Na/EtOH; (ii) R3R4NH (1ꢀ or 2ꢀ amine)/EtOH/heat 4–6 h.

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
969
Table 1
Physical constants and anticonvulsant activity of cyclic enaminones
R3
R4
N
R2
O
CO2 R1
Compound
R1
R2
R3
R4
4-NO2Ph
C log P
Mþ ion peaks
381.1
Mp (ꢀC)
132–136
ADDa
3
6a
–C2H5
–Ph
–H
4.570
O
N
6b
6c
–C2H5
–C2H5
–Ph
–Ph
–
2.197
4.498
330.1
140–142
3
N
Cl
–H
371.1
144–145
3
6d
6e
–C2H5
–C2H5
–Ph
–H
–
4-OHPh
3.512
1.308
352.1
268.1
197–199
133–135
3
2
N
O
–CH3
6f
6g
6h
6i
–C2H5
–CH3
–CH3
–Ph
–Ph
–Ph
–Ph
–H
–
3.606
2.611
3.140
4.495
3.966
302.2
300.1
314.2
364.1
350.1
131–132
172–174
156–158
132–135
162–164
1
3
2
3
3
N
N
–C2H5
–C2H5
–CH3
–
–H
–H
6j
6kb
–C2H5
–CH3
–H
4.306
321.1
130–131
1
Cl
6lc
–C2H5
–C2H5
–CH3
–CH3
–CH3
–Ph
–H
–H
–H
4-BrPh
4-FPh
4-FPh
4.456
3.736
4.625
354
292.1
340.1
147–149
142–144
214–215
1
1
3
6mc
6n
N
Cl
6o
6p
–CH3
–CH3
–Ph
–Ph
–H
–H
3.969
4.345
357.1
364.2
226–229
153–154
3
3
6q
6r
–CH3
–C2H5
–Ph
–Ph
–H
–C2H5
Ph
–C2H5
3.650
3.573
322.1
316.3
183–187
114–116
2
3
6s
–CH3
–Ph
–H
4.874
379.2
158–159
3
6t
–CH3
–Ph
–Ph
–Ph
–Ph
–Ph
–C2H5
–H
–H
–H
–H
–C2H5
4-ClPh
4-BrPh
4-FPh
Ph
3.044
5.195
5.345
4.625
4.179
312.3
370.1
415
354.2
336
140–141
220–222
211–214
208–209
171–172
3
2
3
3
3
6u
6v
6w
6x
–C2H5
–C2H5
–C2H5
–C2H5
(continued on next page)

970
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
Table 1 (continued )
Compound
R1
R2
R3
R4
C log P
Mþ ion peaks
379.1
Mp (ꢀC)
65–73
ADDa
3
6y
–C2H5
–Ph
–H
4.874
6z
–C2H5
–Ph
–H
5.403
392.2
136–140
3
a
ADD indicates Anticonvulsant Drug Development program classification for potency of evaluated compounds: class 1 > class 2 > class 3. Class 1, active at 100 mg/kg or less;
class 2, active at doses >100 mg/kg and up to 300 mg/kg; class 3, inactive at 300 mg/kg dose. Four enaminones exhibited potent activity: 6f (class 1 in MES), 6k (class 1 in MES
and SCMET), 6l (class 1 in MES and SCMET), 6m (class 1 in MES and SCMET); while another four enaminones showed moderate activity: 6e (class 2 in SCMET), 6h (class 2 in
MES), 6q (class 2 in SCMET), and 6u (class 2 in MES).
b
Ref. [13].
Ref. [14].
c
in Table 2 were inactive. These results indicated that specific elec-
tronic properties of the enaminones contributed to, but were not
the only factors responsible for the anticonvulsant activities of the
active enaminones. We had suggested in previous reports that
electron-withdrawing properties of moieties, partition coefficient
(exemplified by C log P values), hydrogen bonding, hydrophilic–
hydrophobic bonding portions, and steric conformations for
receptor binding are important determinants for anticonvulsant
activity of enaminones [2,12,16].
values and anticonvulsant activity of the enaminones. Moreover,
the eight anticonvulsant enaminones have a variety of electron-
withdrawing effects that contributed to their anticonvulsant
activity.
The C log P values of the heterocyclic enaminones in Table 2
ranged from 2.386 to 5.548, yet none of the 13 enaminones was
anticonvulsant. This observation indicated that C log P data were
not very important for the anticonvulsant activity of the enami-
nones. Instead, the steric hindrance due to the bulky amino
groups in these enaminones probably prevented suitable binding
with putative receptor sites resulting in compounds that were
inactive.
2.2. Pharmacology
2.2.1. Anticonvulsant activities of cyclic enaminones
2.2.3. Structure–activity relationship (SAR) of cyclic enaminones
The presence of the enaminone system is essential for anti-
convulsant activity, and our current data support our observation
that the enaminone pharmacophore existing in a sterically favored
conformation is very important for anticonvulsant activity. In this
series of enaminones, we observed the rarity of tertiary enami-
nones (6e and 6h) displaying anticonvulsant activity, although
these were only class 2 anticonvulsants and thus were moderately
active. However, the NH moiety is still very important for highly
potent anticonvulsant activity of secondary enaminones (6f, 6k–
m). These enaminones were potent class 1 anticonvulsants. The
anticonvulsant enaminones in this study were active against
SCMET seizures when the substitution at C-6 was phenyl; and
protective against MES seizures when the substitution at C-6 was
a methyl group. However, the enaminone ester 6q was an unsub-
stituted anilino analog which was a class 2 anticonvulsant. The
substitution at C-6 in 6q was a phenyl group. This is the first time
we are reporting an unsubstituted anilino enaminone as possessing
anticonvulsant activity. The enaminone ester 6u was a 4-chlor-
ophenylamino analog which was expected to be very highly potent,
but it was only a class 2 anticonvulsant. The active compounds were
mainly anilino derivatives which have been shown to elevate
gamma aminobutyric acid (GABA) levels in the brain, and to reduce
the firing frequency of certain neurons in vitro in brain cells or
slides [2,13–20].
Eight anticonvulsant enaminones in this study offer new
compounds with different mechanisms of action and minimal side
effects. We had shown previously that enaminones inhibit excit-
atory postsynaptic currents (EPSCs) in the brain by enhancing
extracellular GABA levels [17]; inhibit tetrodotoxin (TTX)-sensitive
currents to modulate excessive firing in individual neurons [18];
and elicit anticonvulsant activity on neuronal network responses
by suppressing epileptiform activity corresponding to both ictal
and interictal events representative of clinical seizures [20]. From
our previous findings, part of the anticonvulsant activity is at the
synaptic level involving GABA [17], while part is direct on the
The anticonvulsant evaluation of these enaminones in the
Anticonvulsant Drug Development (ADD) program [1] indicated
that a total of eight of enaminones displayed’ anticonvulsant
activity, among which four enaminones 6f, 6k, 6l, and 6m, showed
class 1 activity and the other four active enaminones 6e, 6h, 6q and
6u showed class 2 activity. Class 1 anticonvulsant enaminones are
more potent than class 2 anticonvulsant enaminones, whereas class
3 analogs are inactive at 300 mg/kg doses. The enaminones did not
display selective anticonvulsant activity. Compound 6e exhibited
class 2 activity against SCMET seizures, compound 6f exhibited
class 1 activity against MES seizures, compound 6h exhibited class
2 activity against MES seizures, compounds 6k, 6l, and 6m showed
class 1 activity against MES and SCMET seizures, compound 6q
exhibited class 2 activity against SCMET seizures, and compound 6u
exhibited class 2 activity against MES seizures. Thus, the anticon-
vulsant profile of the enaminones was qualitatively different
among its analogs. Generally, anticonvulsant enaminones that are
active against the MES test, but not the SCMET test, show a phar-
macological profile similar to phenytoin, whereas enaminones that
are active against SCMET, but not against the MES test, show
a pharmacological profile similar to ethosuximide. Enaminones
that are active against both MES and SCMET tests show a pharma-
cological profile similar to valproate. However, we recently repor-
ted that anticonvulsant enaminones have several unique
mechanisms of action, as explained below in the structure–activity
relationship (SAR) of cyclic enaminones. Enaminones 7a–m
including those containing the oxazolidinone moiety were inactive
in the ADD evaluation.
2.2.2. Attempted correlation of anticonvulsant activity
with C log P data
The C log P data for the eight anticonvulsant enaminones (6e, 6f,
6h, 6k, 6l, 6m, 6q, and 6u) in Table 1 ranged from 1.308 to 5.195,
which also covered the range for the C log P values for most of the
inactive enaminones in Table 1. In support of our previous report
[12], no direct correlation could be established between C log P

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
971
Table 2
Physical constants and anticonvulsant activity of heterocyclic enaminones
R3
R4
N
R2
O
R1
CO2
Compound
R1
R2
R3
R4
C log P
Mp (ꢀC)
189–193
ADDa
O
O
O
N
7a
–CH3
–CH3
–H
3.078
3
F
O
O
O
N
N
7b
7c
–CH3
–CH3
–H
–H
2.386
2.915
206–209
202–204
3
3
F
O
N
N
–C2H5
–CH3
F
O
O
O
O
N
N
7d
7e
–CH3
–Ph
–H
–H
3.275
3.607
210–212
3
3
F
N
–C2H5
–CH3
164
F
O
O
N
N
7f
–CH3
–Ph
–Ph
–H
–H
3.967
4.496
220
216
3
3
F
F
7g
–C2H5
N
N
O2N
O2N
7h
7i
–C2H5
–CH3
–
–
3.268
3.628
162–163
201–202
3
3
F
N
N
–CH3
–Ph
F
N
N
O2N
7j
–C2H5
–CH3
–Ph
–Ph
–
4.157
5.019
166–167
222–223
3
3
F
O
7k
–H
O
N
N
F
(continued on next page)

972
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
Table 2 (continued )
Compound
R1
R2
R3
R4
C log P
Mp (ꢀC)
196–197
ADDa
O
O
N
N
O
O
N
7l
–C2H5
–Ph
–H
5.548
3
F
F
N
7m
–CH3
–CH3
–H
4.130
184–185
3
a
ADD indicates Anticonvulsant Drug Development program classification for potency of evaluated compounds: class 1 > class 2 > class 3. Class 1, active at 100 mg/kg or less;
class 2, active at doses >100 mg/kg and up to 300 mg/kg; class 3, inactive at 300 mg/kg dose.
postsynaptic cell [18]. Enaminones depress population spike (PS)
amplitude, and reversibly reduce the number of single PS being
transformed into multiple PS in the brain. The multiple spikes
following afferent stimulation represent ictal events, while the
spontaneous bursts (SB) represent interictal events in seizures. In
addition, enaminones suppress the frequency of spontaneous
bursts (SB) and suppress afterdischarges (AD) in the brain. The
anticonvulsant enaminones in this present study may be acting
by one or more of these mechanisms. These properties of
enaminones make them good candidates to abort seizures by
synaptic and non-synaptic mechanisms, and to prevent additional
seizure attacks [20].
2.2.4. Antibacterial activity of selected compounds
The minimum inhibitory concentrations (MIC, mg/ml values) for
the selected cyclic enaminones (7b–e and 7g) in comparison to
linezolid, PH-027 and vancomycin are shown in Table 4. Linezolid
and PH-027 are 5-methylacetamido and 5-methyltriazolyl oxazo-
lidines that showed potent antibacterial activity. Unfortunately,
none of the new compounds evaluated showed significant
Table 3
UV data of cyclic enaminones in neutral, acidic and alkaline media
Compound
Mol. formula
Mol. wt.
95% EtOH
H2O
1 M HCl
1 M NaOH
lmax
3
lmax
3
lmax
3
lmax
3
6a
6b
6c
6d
6e
6f
6g
6h
6i
C21H20O5N2
C19H23O4N
C20H19O3N2
380.38
329.37
370.84
351.96
267.20
301.18
299.37
313.40
363.46
349.43
319.67
352.03
291.11
339.34
356.79
363.44
321.35
315.4
284
303
284
285
300
290
303
302
284
290
313
314
302
303
334
292
311
283
291
284
315
316
303
310
292
291
316
287
287
288
318
319
319
301
303
302
320
319
318
34 819
282
308
283
306
306
295
306
305
283
295
311
314
305
307
332
297
310
261
296
283
314
315
306
311
297
296
317
292
291
293
318
318
319
306
308
308
319
319
317
28 229
33740
41208
32 096
38 288
32 600
35 204
34 914
30 932
21679
31242
28 421
24 303
27 010
29 275
29 096
28 323
24787
37331
27 916
26 563
30 067
24714
25 450
32 385
32 000
29 696
44 495
32 638
32 216
25 318
23 921
24794
22738
25 336
21300
28 766
22 914
27 968
259
296
260
271
292
284
286
285
259
286
302
306
299
303
336
285
305
283
284
259
308
309
302
304
285
284
319
285
285
289
318
321
320
291
294
293
321
321
311
24 243
24 260
28 662
29 217
28 515
22 661
24 270
24 300
21574
15 579
26 590
21696
20 018
22 672
20 071
22 432
22748
27276
28 388
18 794
20 922
23 658
20 657
20 260
23 996
24 546
24 375
38 432
28 295
28 968
20 993
21355
23 403
11759
12 382
10 773
26 052
20 393
27 068
283
308
283
281
306
295
306
306
283
295
309
311
307
309
315
297
308
282
296
283
312
313
309
309
297
296
310
292
293
294
311
293
314
306
308
308
313
313
319
38 029
31244
42731
26104
36 058
33 026
34700
34 377
37679
24162
29 968
25 679
23 265
26159
19 854
30 621
26 519
26 946
36 204
27653
24 956
29 029
23 811
24 333
31935
31281
29 007
43 232
31533
31153
25 078
27633
24 926
20 446
20 568
18 582
28 966
23 043
22 359
29752
28 889
43 895
34 469
31209
33 413
33590
32 042
20197
30103
28 575
22 247
24 403
30 068
26 279
26 358
27448
35 350
28 813
22 533
28 367
22 668
23 873
31473
30 604
27 739
47 541
34 805
35 274
23 602
22 296
22 670
23 549
25150
21050
27350
21881
26 282
C
21H21O4N
C14H21O4N
C18H23O3N
C18H21O3N
C19H23O3N
C23H25O3N
C22H23O3N
C16H18O3ClN
6j
6k
6l
C
16H18O3BrN
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
7a
7b
7c
7d
7e
7f
C16H18O3FN
C20H18O3NF
C19H17O3N2Cl
C23H25O3N
C20H19O3N
C19H25ON
C
24H28ON
378.47
311.4
C18H23O3N
C21H20O3NCl
C21H20O3NBr
C21H20O3NF
C21H21O3N
C24H28O3N
C25H29O3N
369.84
414.29
353.37
335.38
378.47
391.49
362.40
461.49
475.52
523.57
376.43
424.48
438.50
405.43
453.46
467.48
523.58
536.60
460.51
C
19H23FN2O4
C23H28FN3O6
C24H30FN3O6
C28H30FN3O6
C20H25FN2O4
C24H25FN2O4
C25H27FN2O4
C20H24FN3O5
7g
7h
7i
7j
7k
7l
C
24H24FN3O5
C25H26FN3O5
C29H34FN3O5
C30H35FN3O5
C24H31FN3O5
7m

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
973
Table 4
Antibacterial activity of heterocyclic enaminones against susceptible and resistant clinical isolates
Compound C log Pa (measured)b MIC (
m
g/ml) ranges for
MSSA (n ¼ 11) MRSA (n ¼ 10) MS-CNS (n ¼ 8) MR-CNS (n ¼ 3) H. influenzae (n ¼ 7) S. pn (n ¼ 6) VSE (n ¼ 7) VRE (n ¼ 4)
7b
7c
7d
7e
2.3865
2.9155
3.2755
3.6074
4.4964
0.6308
0.76 (0.5321)
n.d.
>16
>16
>16
>16
>16
0.5–1
2
>16
>16
>16
>16
>16
>16
>16
>16
>16
1
>16
>16
>16
>16
>16
0.5–1
0.5–1
2
>16
>16
>16
>16
>16
32
>16
>16
>16
>16
>16
>16
>16
>16
>16
0.5–2
1–2
>16
>16
>16
>16
>16
0.5–2
1–2
7g
>16
>16
PH-027
Lzd
Van
0.5–2
0.5–2
0.5–2
0.5–1
0.5–1
0.25–1
0.5–1
1–2
8
>32
1–2
1–4
8 to >32
a
Ref. [24].
b
Value obtained from the Pharmacia & Upjohn Material Safety Data Sheet January 2000.
antibacterial activity against standard and clinical isolates of Gram-
positive and Gram-negative bacteria used in the present study.
Surprisingly, compounds 7b–d containing the oxazolidinone phar-
macophoric group and the morpholino moiety found in linezolid
were also devoid of antibacterial activity. This could be due to
the bulkiness and lack of planarity of the enaminone ring struc-
ture. In addition, compounds 7e–g having the arylmorpholino
moiety found in linezolid were also inactive. The enaminones 7b–e
a UV-3101PC spectrophotometer equipped with computerized
programs to plot the spectrum as the sample was being run and to
printout the (lmax) value for each enaminone. Molar absorptivity (3)
was then calculated for each compound and
nearest hundred [21–23].
3 value rounded to
Infrared (IR) spectra were recorded on Perkin Elmer System
2000 FT-IR spectrometer using KBr pellets. Column chromatog-
raphy was carried out with silica gel [Kieselgel 60, 70–230 mesh
(Aldrich)]. TLC was conducted on 0.25 mm precoated silica gel
plates (60F254, Merck). All extracted solvents were dried over
Na2SO4, followed by evaporation in vacuum. The calculated parti-
tion coefficient (C log P) values were determined by using the CS
ChemDraw Ultra version 6.01, computer software by
CambridgeSoft.Com [24].
and 7g showed MIC values >16 mg/ml compared to MIC ranges
between 0.5 and 8 for linezolid, PH-027 and vancomycin, respec-
tively (Table 4).
3. Conclusion
In conclusion, four enaminones showed potent anticonvulsant
activity, and another four analogs showed moderate anticonvulsant
activity against experimentally induced seizures in mice. These
anticonvulsant enaminones included tertiary enaminones 6e and
6h, and unsubstituted anilino enaminone 6q that we are reporting
to be active for the first time. Enaminone 6f was particularly potent
and selective against MES test. Most of the enaminones exhibited
hypsochromic shifts in acidic solutions and bathochromic shifts in
alkaline solutions compared to their neutral solutions. Thus, they
generally behaved like basic compounds in solution, although a few
enaminones exhibited neutral or acidic properties in solution
[12,21]. Our current results support our previous report [12] that
the anticonvulsant activity of enaminones is not due to electronic
effect alone, but it results from a combination of factors which
include electronic and steric effects, lipophilicity, and hydrogen
bonding. The anticonvulsant enaminones in this study may be
acting by synaptic and non-synaptic mechanisms in similar
manners as reported recently [2,20]. However, none of the cyclic
enaminones showed significant antibacterial activity, probably due
to the presence of the bulky 5-methylenaminone and its lack of fit
at the receptor site.
4.2. Syntheses
4.2.1. General procedure for the preparation of the enaminone
derivatives
The cyclohexanedione esters (3.312 mmol) were prepared
[12,16] and refluxed with the appropriate amino compounds
(2.548 mmol) in absolute ethanol or isopropanol (25 ml) as solvent
(Scheme 1). 5-Aminomethyl oxazolidinone was prepared accord-
ing to literature methods [10,11]. Then, the reaction mixture was
evaporated to give a residue, which was recrystallized using suit-
able organic solvents (isopropanol or ethanol) to give crystalline
solids. The following are representative reaction conditions, yields
and spectroscopic data of representative compounds.
4.2.2. Methyl 4-(3-fluoro-4-morpholinophenylamino)-6-methyl-2-
oxocyclohex-3-ene-1-carboxylate (7a)
Recrystallization from isopropanol gave a crystalline solid
734 mg (80%), mp 189–193 ꢀC. 1H NMR (DMSO-d6, 400 MHz):
d 9.03
(s, 1H, –NH), 6.95–7.08 (m, 3H, phenyl H), 5.20 (s, 1H, vinyl]CH),
3.74 (t, 4H, J ¼ 5.0 Hz, morpholine H), 3.63 (s, 3H, OCH3), 2.99 (t, 4H,
J ¼ 5.0 Hz, morpholine H), 2.33–2.51 (m, 4H, cyclohexyl), 1.02 (d,
3H, J ¼ 6.0 Hz, CH3). Anal. CHN (%): 62.9, 6.43, 7.73, found (%): 63.31,
6.38, 7.80.
4. Experimental
4.1. Characterization
4.2.3. Methyl 4-(((S)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)methylamino)-6-methyl-2-oxocyclohex-3-
enecarboxylate (7b)
Melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. The Science Analytical Facilities
(SAF), Faculty of Science, Kuwait University, performed all the
analyses. Elemental analyses were determined on LECO elemental
analyzer CHNS 932 apparatus and were within ꢁ0.4% of the
calculated values. 1H NMR spectra were recorded on Bruker DPX
400 NMR (400 MHz) spectrometer using tetramethylsilane (TMS)
as an internal standard. High-resolution mass spectra were
measured on a Finnigan MAT INCOS XL mass spectrometer. Ultra-
violet (UV) spectroscopy of the enaminones was determined on
Recrystallization from ethanol gave a crystalline yellow solid
538 mg (77%), mp 207–209 ꢀC. 1H NMR (DMSO-d6, 400 MHz):
d
7.54 (br s, 1H, –HN, exchangeable with D2O), 7.52 (dd, 1H, J ¼ 3.0,
15.0 Hz, phenyl H), 7.17 (dd, 1H, J ¼ 1.0, 10.0 Hz, phenyl H), 7.08 (t,
1H, J ¼ 10.0 Hz, phenyl H), 5.03 (s, 1H, vinyl H), 4.82 (br s, 1H,
oxazolidinone C5H), 4.12 (t, 1H, J ¼ 9.0 Hz, C4H oxazolidinone), 3.74
(t, 4H, J ¼ 4.5 Hz, morpholinyl), 3.72 (dd, 1H, C4H oxazolidinone,
overlaps partly with morpholinyl H), 3.62 (s, 3H, OCH3), 3.40–3.44

974
I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
(m, 2H, –CH2), 2.97 (t, 4H, J ¼ 4.5 Hz, morpholinyl), 2.33–2.51 (m,
4H, cyclohexyl), 0.93 (d, 3H, J ¼ 5.0 Hz, CH3). Anal. CHN (%): 59.86,
6.12, 9.11, found (%): 59.47, 6.13, 9.12.
16.5 Hz, enaminone H), 2.60 (dd, 1H, J ¼ 4.3, 16.6 Hz, enaminone H),
1.03 (t, 3H, CO2CH2CH3). Anal. CHN (%): 68.48, 6.21, 6.39, found (%):
68.31, 6.13, 6.79.
4.2.4. Ethyl 4-(((S)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)methylamino)-6-methyl-2-oxocyclohex-
3-ene-1-carboxylate (7c)
4.2.9. Ethyl 4-(4-(2-fluoro-4-nitrophenyl)piperazin-1-yl)-6-
methyl-2-oxocyclohex-3-ene-1-carboxylate (7h)
Recrystallization from isopropanol gave a yellow crystalline
Recrystallization from ethanol gave a crystalline pale yellow
solid 1.42 g (79% yield), mp 162–163 ꢀC. 1H NMR (CDCl3, 400 MHz):
solid 406 mg (63%), mp 202–204 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
8.03 (dd, 1H, J ¼ 2.5, 8.6 Hz, phenyl H), 7.96 (dd, 1H, J ¼ 2.6,
d
7.46 (dd, 1H, J ¼ 2.0, 14.0 Hz, phenyl H), 7.10 (dd, 1H, J ¼ 2.0, 9.0 Hz,
12.9 Hz, phenyl H), 6.94 (t, 1H, J ¼ 8.7 Hz, phenyl H), 5.36 (s, 1H,
vinyl H), 4.27 (q, 2H, J ¼ 7.1, 14.2 Hz, CO2CH2CH3), 3.52–3.63 (m, 4H,
piperazinyl H), 3.36–3.43 (m, 4H, piperazinyl H), 3.03 (d, 1H,
J ¼ 11.0 Hz, enaminone CH), 2.69 (dd, 1H, J ¼ 4.7, 16.0 Hz, enami-
none H), 2.57–2.64 (m, 1H, enaminone H), 2.17 (dd, 1H, J ¼ 10.2,
16.0 Hz, enaminone H), 1.37 (t, 3H, J ¼ 7.12 Hz, CO2CH2CH3), 1.14 (d,
3H, J ¼ 6.4 Hz, CH3). Anal. CHN (%): 63.14, 7.21, 7.22, found (%):
63.02, 6.97, 7.50.
phenyl H), 6.94 (t, 1H, J ¼ 9.0 Hz, phenyl H), 5.41 (br s, 1H, NH), 5.21
(s, 1H, vinyl H), 4.89 (m, 1H, oxazolidinone C5H), 4.26 (q, 2H,
J ¼ 7.1 Hz, CO2CH2CH3), 4.10 (tt, 1H, J ¼ 2.6, 9.1 Hz, C4H oxazolidi-
none), 3.89 (t, 4H, J ¼ 4.5 Hz, morpholinyl H), 3.69–3.74 (m, 1H,
oxazolidinone H), 3.55–3.58 (m, 1H), 3.43–3.47 (m, 1H), 3.07 (t, 4H,
J ¼ 4.5 Hz, morpholinyl H), 3.02 (t, 1H, J ¼ 11.0 Hz), 2.57–2.60 (m,
1H), 2.40–2.44 (m, 1H), 2.22–2.32 (m, 1H), 1.31 (t, 3H, J ¼ 7.1 Hz,
CO2CH2CH3), 1.09 (dd, 3H, J ¼ 2.8, 6.4 Hz, CH3). Anal. CHN (%): 60.62,
6.36, 8.84, found (%): 60.79, 6.32, 8.90.
4.2.10. Methyl 4-(4-(2-fluoro-4-nitrophenyl)piperazin-1-yl)-
2-oxo-6-phenylcyclohex-3-ene-1-carboxylate (7i)
4.2.5. Methyl 4-(((S)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)methylamino)-2-oxo-6-phenylcyclohex-
3-ene-1-carboxylate (7d)
Recrystallization from isopropanol gave a yellow crystalline
solid 1.08 g (67% yield), mp 201–203 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
8.03 (dd, 1H, J ¼ 2.5, 8.6 Hz, phenyl H), 7.95 (dd, 1H, J ¼ 2.6,
Recrystallized from ethanol gave a yellow crystalline solid
12.9 Hz, phenyl H), 7.28–7.39 (m, 5H, phenyl H), 6.93 (t, 1H,
J ¼ 8.7 Hz, phenyl H), 5.45 (s, 1H, vinyl H), 3.53–3.75 (m, 6H,
piperazinyl H and enaminone H, overlaps with CH3 signal at
3.59 ppm), 3.59 (s, 3H, CH3, overlaps with piperazinyl and enami-
none H at 3.53–3.75 ppm), 3.38–3.41 (m, 4H, piperazinyl H), 2.85
(dd, 1H, J ¼ 4.3, 16.0 Hz, enaminone H), 2.64 (dd, 1H, J ¼ 10.4,
16.0 Hz, enaminone H). Anal. CHN (%): 63.57, 5.33, 9.27, found (%):
63.27, 5.79, 9.47.
467 mg (66%), mp 210–212 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 6.90–
7.45 (m, 8H, phenyl H), 5.74 (br s, 1H, NH), 5.30 (s, 1H, vinyl H), 4.89
(m, 1H, oxazolidinone C5H), 4.10 (t, 1H, J ¼ 9.0 Hz, C4H oxazoli-
dinone), 3.89 (t, 4H, J ¼ 4.5 Hz, morpholinyl H), 3.42–3.74
(m, 8H), 3.06 (t, 4H, J ¼ 4.5 Hz, morpholinyl H), 2.72–2.81 (m, 1H),
2.47–2.59 (m, 1H). Anal. CHN (%): 64.23, 5.78, 8.03, found (%): 64.21,
5.84, 8.04.
4.2.6. Ethyl 4-(3-fluoro-4-morpholinophenylamino)-6-methyl-2-
oxocyclohex-3-ene-1-carboxylate (7e)
4.2.11. Ethyl 4-(4-(2-fluoro-4-nitrophenyl)piperazin-1-yl)-2-oxo-6-
phenylcyclohex-3-ene-1-carboxylate (7j)
Recrystallized from ethanol to give an off-white crystalline
Recrystallized from propanol to give a yellow crystalline solid
solid 500 mg (43.4% yield), mp 164–166 ꢀC. 1H NMR (CDCl3,
1.26 g (91% yield), mp 166–167 ꢀC. 1H NMR (CDCl3, 400 MHz):
400 MHz):
d
6.90–6.92 (m, 3H, phenyl H), 6.19 (s, 1H, NH), 5.48
d
8.02 (dd, 1H, J ¼ 2.5, 8.6 Hz, phenyl H), 7.95 (dd, 1H, J ¼ 2.6,
(s, 1H, vinyl H), 4.26 (q, 2H, J ¼ 7.12 Hz, CO2CH2CH3), 3.89 (t, 4H,
J ¼ 4.5 Hz, morpholinyl H), 3.05–3.10 (m, 5H, enaminone CH and
morpholinyl H), 2.62–2.69 (m, 1H, enaminone H), 2.50 (dd, 1H,
J ¼ 4.6, 16.5 Hz, enaminone H), 2.37 (dd, 1H, J ¼ 10.9, 16.3 Hz,
enaminone H), 1.31 (t, 3H, J ¼ 7.12 Hz, CO2CH2CH3), 1.12 (d, 3H,
J ¼ 6.5 Hz, CH3). Anal. CHN (%): 63.82, 6.69, 7.44, found (%): 63.62,
6.47, 7.80.
12.9 Hz, phenyl H), 7.26–7.38 (m, 5H, phenyl H), 6.94 (t, 1H,
J ¼ 8.7 Hz, phenyl H), 5.44 (s, 1H, vinyl H), 4.27 (q, 2H, J ¼ 7.1,
14.2 Hz, CO2CH2CH3), 3.68–3.76 (m, 2H, enaminone H), 3.52–3.65
(m, 4H, piperazinyl H), 3.22–3.43 (m, 4H, piperazinyl H), 2.86 (dd,
1H, J ¼ 4.3, 16.0 Hz enaminone H), 2.64 (dd, 1H, J ¼ 10.4, 16.0 Hz,
enaminone H), 1.04 (t, 3H, J ¼ 7.12 Hz, CO2CH2CH3). Anal. CHN (%):
64.23, 5.61, 8.99, found (%): 64.10, 5.79, 9.17.
4.2.7. Methyl 4-(3-fluoro-4-morpholinophenylamino)-2-oxo-6-
phenylcyclohex-3-ene-1-carboxylate (7f)
4.2.12. tert-Butyl 4-(2-fluoro-4-(3-hydroxy-4-(methoxycarbonyl)-
5-phenylcyclohex-1-enylamino)phenyl)piperazine-1-carboxylate
(7k)
Recrystallization from ethanol gave an off-white crystalline
solid 740 mg (57% yield), mp 220–222 ꢀC. 1H NMR (CDCl3,
Recrystallized from isopropanol to give a greenish crystalline
400 MHz): d 7.28–7.37 (m, 5H, phenyl H), 6.91–6.96 (m, 3H, phenyl
solid,1.29 g (79% yield), mp 222–223 ꢀC. 1H NMR (CDCl3, 400 MHz):
H), 6.40 (s, 1H, NH), 5.55 (s, 1H, vinyl H), 3.89 (t, 4H, J ¼ 4.5 Hz,
morpholinyl H), 3.75 (dd, 1H, J ¼ 4.3, 12 Hz, enaminone CH), 3.68 (d,
1H, J ¼ 12.0 Hz, enaminone H), 3.55 (s, 3H, CO2CH3), 3.09 (t, 4H,
J ¼ 4.5 Hz, morpholinyl H), 2.87 (dd, 1H, J ¼ 12, 16.5 Hz, enaminone
H), 2.61 (dd,1H, J ¼ 4.3,16.6 Hz, enaminone H). Anal. CHN (%): 67.91,
5.60, 6.60, found (%): 67.23, 5.92, 7.01.
d 6.92–7.37 (s, 9H, phenyl H and NH), 5.63 (s, 1H, vinyl H), 3.69–3.79
(q, 2H, enaminone H), 3.63 (br s, 4H, piperazinyl H), 3.57 (s, 3H,
CO2CH3), 3.05 (br s, 4H, piperazinyl H), 2.86 (dd, 1H, J ¼ 4.3, 16.0 Hz,
enaminone H), 2.64 (dd, 1H, J ¼ 10.4, 16.0 Hz, enaminone H), 1.50 (s,
9H, C(CH3)3). Anal. CHN (%): 66.52, 6.55, 8.03, found (%): 66.35, 6.44,
8.17.
4.2.8. Ethyl 4-(3-fluoro-4-morpholinophenylamino)-2-oxo-6-
phenylcyclohex-3-ene-1-carboxylate (7g)
4.2.13. tert-Butyl 4-(4-(4-(ethoxycarbonyl)-3-oxo-5-
phenylcyclohex-1-enylamino)-2-fluorophenyl)piperazine-1-
carboxylate (7l)
Recrystallization from ethanol gave an off-white crystalline
solid 700 mg (52% yield), mp 216–218 ꢀC. 1H NMR (CDCl3,
Recrystallization from isopropanol gave a greenish crystalline
400 MHz): d 7.28–7.37 (m, 5H, phenyl H), 6.92–6.95 (m, 3H, phenyl
solid, 1.15 g (73% yield), mp 196–197 ꢀC. 1H NMR (CDCl3, 400 MHz):
H), 6.12 (s, 1H, NH), 5.56 (s, 1H, vinyl H), 4.01–4.05 (m, 2H,
CO2CH2CH3), 3.90 (t, 4H, J ¼ 4.5 Hz, morpholinyl H), 3.77 (dd, 1H,
J ¼ 4.3, 12 Hz, enaminone CH), 3.67 (d, 1H, J ¼ 12.0 Hz, enaminone
H), 3.10 (t, 4H, J ¼ 4.5 Hz, morpholinyl H), 2.89 (dd, 1H, J ¼ 12,
d 6.90–7.35 (m, 9H, phenyl H), 6.71 (s, 1H, NH), 5.56 (s, 1H, vinyl H),
4.27 (q, 2H, J ¼ 7.1, 14.2 Hz, CO2CH2CH3), 3.69–3.75 (q, 2H, enami-
none H), 3.62 (s, 4H, piperazinyl H), 3.01 (s, 4H, piperazinyl H), 2.86
(dd, 1H, J ¼ 4.3, 16.0 Hz, enaminone H), 2.62 (dd, 1H, J ¼ 10.4,

I.O. Edafiogho et al. / European Journal of Medicinal Chemistry 44 (2009) 967–975
975
16.0 Hz, enaminone H), 1.50 (s, 9H, C(CH3)3), 1.04 (t, 3H, J ¼ 7.12 Hz,
CO2CH2CH3). Anal. CHN (%): 67.02, 6.75, 7.82, found (%): 67.09, 6.53,
8.10.
The Gram-negative organisms included H. influenzae (n ¼ 7) clinical
isolates and Escherichia coli ATCC 25922. The reference strains, S.
aureus ATCC 25923, S. epidermidis ATCC 12228 and Enterococcus
faecalis ATCC 29212, E. coli (ATCC 25922) and H. influenzae ATCC
49247 were used to control the tests. The final bacterial concen-
tration for inocula was 107 CFU/ml, and was incubated at 35 ꢀC for
18 h. The MIC was defined as the lowest drug concentration that
completely inhibited growth of the bacteria. PH-027, prepared
according to literature methods [10,11], and linezolid and vanco-
mycin obtained from commercial sources were used as reference
antibacterial agents.
4.2.14. tert-Butyl 4-(2-fluoro-4-(4-(methoxycarbonyl)-5-methyl-3-
oxocyclohex-1-enylamino)phenyl)piperazine-1-carboxylate (7m)
Recrystallized from isopropanol gave a crystalline solid, 1.28 g
(67% yield), mp 184–185 ꢀC. 1H NMR (CDCl3, 400 MHz):
d 6.91–6.99
(m, 4H, phenyl H and NH), 5.56 (s, 1H, vinyl H), 3.78 (s, 3H, CO2CH3),
3.62 (s, 4H, piperazinyl H), 3.05–3.12 (m,1H, enaminone H, overlaps
partly with the piperazinyl singlet at 3.05), 3.05 (s, 4H, piperazinyl
H), 2.60–2.68 (m, 1H, enaminone H), 2.57 (dd, 1H, J ¼ 4.3, 16.0 Hz,
enaminone H), 2.38 (dd, 1H, J ¼ 10.4, 16.0 Hz, enaminone H), 1.50 (s,
9H, C(CH3)3), 1.13 (d, 3H, J ¼ 6.4 Hz, CH3). Anal. CHN (%): 62.46, 6.99,
9.12, found (%): 62.33, 6.71, 9.40.
Acknowledgments
The authors thank the Faculty of Pharmacy, Kuwait University
for its support. This was also supported by the Research Adminis-
tration, Kuwait University Instrument Grants GS01/01 and GS03/01
awarded to the Science Analytical Facilities (SAF). We thank James
P. Stables for the in vivo anticonvulsant screening.
4.3. In vivo anticonvulsant evaluation of enaminones
The enaminones synthesized were submitted for evaluation by
the Antiepileptic Drug Development Program, Epilepsy Branch,
National Institute of Neurological Disorders and Stroke [1]. The
antiepileptic drug development (ADD) program initially evaluated
anticonvulsant activity for the newly submitted compounds
following intraperitoneal (i.p.) administration in mice and oral
administration in rats. Two convulsant tests and a toxicity screen
are employed for preliminary evaluation. The Maximal Electro-
shock Seizure (MES) or Maximal Pattern Test is a model for
generalized tonic–clonic seizures, while the Subcutaneous Pentyl-
enetetrazol (metrazol) (SCMET) test is a model that identifies
anticonvulsant compounds that would raise seizure threshold.
Neurotoxicity was evalauated using the standardized rotorod test.
Untreated control mice, when placed on a 6 rpm rotorod, can
maintain their equilibrium for a prolonged period of time. Neuro-
logical impairment can be demonstrated by the inability to main-
tain equilibrium for a minute in each of three successive trials. Data
for the anticonvulsant evaluations are shown in Tables 1 and 2.
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4.4. Microbiology
4.4.1. In vitro antibacterial evaluations
The clinical isolates used in the study were obtained from
culture collection maintained at the MRSA Reference Laboratory,
Faculty of Medicine, Kuwait University. Antibacterial susceptibility
testing was performed by disk diffusion and the agar dilution
methods as described by the National Committee for Clinical
Laboratory Standards (now Clinical Laboratory Standard Institute)
[25]. Minimum inhibitory concentrations (MICs,
determined on Mueller–Hinton (MH) agar with medium containing
dilutions of antibacterial agents ranging from 0.12 to 16 g/ml. The
test compounds were dissolved in 80% DMSO in water while line-
zolid and vancomycin were dissolved in 60% ethanol in water and
water, respectively. The tests were performed using MH agar plates
for all staphylococci and enterococci, and on MH agar plates sup-
plemented with 5% sheep blood to facilitate the growth of S.
pneumoniae, and Haemophilus influenzae. The Gram-positive
organisms utilized in this study consisted of methicillin-resistant S.
aureus (MRSA, n ¼ 10), methicillin-susceptible S. aureus (MSSA,
n ¼ 11), methicillin-resistant coagulase-negative staphylococci
(MR-CNS, n ¼ 3), methicillin-sensitive coagulase-negative staphy-
lococci (MS-CNS, n ¼ 8), S. pneumoniae (n ¼ 6), vancomycin-sensi-
tive (VSE, n ¼ 7) and vancomycin-resistant (VRE, n ¼ 4) enterococci.
mg/ml) were
m
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