Enaminones 12. An explanation of anticonvulsant activity and toxicity per Linus Pauling's clathrate hypothesis

Article abstract of DOI:10.1016/j.ejmech.2012.02.003

The x-ray crystal structure of 3-((5-methylisoxazol-3-yl)amino)-5- methylcyclohex-2-enone (12b) and 3-((5-methylisoxazolyl-3-yl)amino)-5,5- dimethylcyclohex-2-enone (12c) were determined and correlated to their anticonvulsant activity in mice and rats. A hypothesis for the toxicity of the analogs are advanced. In addition, a series of 5-methyl-N-(3-oxocyclohex-1-enyl) -isoxazole-3-carboxamides were synthesized and evaluated for anticonvulsant activity. These compounds were compared to the activity of the corresponding amino and aminomethyl enaminones. Additional investigation involved the synthesis and evaluation of a trifluoromethyl analog of the active isoxazole tert-butyl 4-(5-methisoxazol-3-yl-amino)-6-methyl-2-oxo-cyclohex-3-ene carboxylate (4f).

Full text of DOI:10.1016/j.ejmech.2012.02.003

European Journal of Medicinal Chemistry 51 (2012) 42e51
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enaminones 12. An explanation of anticonvulsant activity and toxicity per Linus
Pauling’s clathrate hypothesis
,
,
Patrice L. Jackson a, Clive D. Hanson b 1, Alanna K. Farrell b 2, Raymond J. Butcher c, James P. Stables d,
,
Natalie D. Eddington e, K.R. Scott b
*
a Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Eastern Shore, 1 Backbone Rd., Princess Anne, MD 21853, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, Howard University, 2300 4th Street, N.W., Washington, DC 20059, USA
c Department of Chemistry, Graduate School, Howard University, 525 College Street, N.W., Washington, DC 20059, USA
d Neuroscience Center, National Institute of Neurological Disorders and Stroke, NIH, 6001 Executive Blvd., MSC 9523, Bethesda, MD 20892, USA
e Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The x-ray crystal structure of 3-((5-methylisoxazol-3-yl)amino)-5-methylcyclohex-2-enone (12b) and 3-
((5-methylisoxazolyl-3-yl)amino)-5,5-dimethylcyclohex-2-enone (12c) were determined and correlated
to their anticonvulsant activity in mice and rats. A hypothesis for the toxicity of the analogs are advanced.
In addition, a series of 5-methyl-N-(3-oxocyclohex-1-enyl)-isoxazole-3-carboxamides were synthesized
and evaluated for anticonvulsant activity. These compounds were compared to the activity of the cor-
responding amino and aminomethyl enaminones. Additional investigation involved the synthesis and
evaluation of a trifluoromethyl analog of the active isoxazole tert-butyl 4-(5-methisoxazol-3-yl-amino)-
6-methyl-2-oxo-cyclohex-3-ene carboxylate (4f).
Received 13 September 2011
Received in revised form
29 January 2012
Accepted 1 February 2012
Available online 9 February 2012
Keywords:
Isoxazole enaminones
Clathrate
MES
Published by Elsevier Masson SAS.
scPTZ
GABA
1. Introduction
toxic side effects. Estimates suggest that available medications
control the seizures in only 50% of patients or decrease the inci-
Epilepsy is currently one of the most prevalent neurological
disorders worldwide [1e3]. Despite the optimal use of available
antiepileptic drugs, many patients with epilepsy fail to experience
seizure control and others do so only at the expense of significant
dence in only 75% of patients [4]. The isoxazoles was our project of
interest. Recently, gaboxadol (1) has shown to be a potent, selective
extrasynaptic GABAA receptor agonist and has been investigated for
its effect on a variety of clinical conditions (e.g. epilepsy, feeding
disorders and pain [5e7]). In addition, Jansen et al. synthesized 5-
(4-piperidyl)-3-isoxazolol (4-PIOL) (2), which proved to be a low-
efficacy GABA mimetic, which was a weak partial agonist or
antagonist, depending on the brain area and the GABAA receptor
composition [8e10]. The isoxazole, N-[4,4-bis(3-methyl-2-
thienyl)-3-butenyl]-3-hydroxy-4-methylamino-4,5,6,7-tetrahydro-
benzo[d]isoxazol-3-ol (EF1 502, 3) has been shown to selectively
inhibit GAT1, GABA transporter 1, and GAT2/BGT-1, and also dis-
played anticonvulsant properties [11]. Recently our group [12]
investigated the isoxazoles to see whether this group (4, 5)
would have anticonvulsant properties in the enaminone series. We
found that this series of analogs indeed provided anticonvulsant
activity. Martinez and his group have generated a number of papers
on the molecular modeling aspects of the data [13e15].
Abbreviations: MES, maximal electroshock seizure; TD50, toxic dose for 50% of
test animals; ED50, effective dose for 50% of test animals; ip, intraperitoneal; PI,
protective index, TD50/ED50; scPTZ, subcutaneous pentylenetetrazol; Tox, neuro-
logic toxicity; ADD, Antiepileptic Drug Development; NINDS, National Institutes of
Neurological Disorders and Stroke; ASP, Anticonvulsant Screening Program.
* Corresponding author. Tel.: þ1 202 806 7288; fax: þ1 202 806 4636.
E-mail addresses: pljackson@umes.edu (P.L. Jackson), hansonclive@hotmail.com
(C.D. Hanson), alanna_farrell@hotmail.com (A.K. Farrell), rbutcher99@yahoo.com
(R.J. Butcher), StablesJ@ninds.nih.gov (J.P. Stables), neddingt@rx.umaryland.edu
(N.D. Eddington), kscott@howard.edu (K.R. Scott).
1
Holy Cross Hospital,
1500 Forest Glen Road, Silver Spring, MD 20910-1484, USA.
2
St. Luke’s Roosevelt Hospital Center, 1000 10th Ave., New York, NY 10027, USA.
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2012.02.003

P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
43
Carabesat (SB-204269, 6), a novel fluorobenzoylamino benzo-
pyran has been introduced which displays excellent
electroshock seizure (MES) animal test model, which is comparable
to tonic-clonic seizures in humans. Compound 7 had an ED50(rat) of
5.5 mg kgꢀ1, a TD50 > 500 mg kgꢀ1, and a protective index
(PI ¼ ED50/TD50) > 90.9. Compound 8 provided an ED50(rat) of
8.9 mg kgꢀ1, a TD50 > 500 mg kgꢀ1 and a PI > 56.2. In the isomeric
benzamide series 9 and 10, however, the results were less clear.
Compound 9, 2,6-dimethyl-N-(5-methylisoxazol-3-yl)benzamide,
had an ED50(rat) of 9.2 mg kgꢀ1, a TD50 > 500 mg kgꢀ1, and
a PI > 54.3. Compound 10 was found inactive. Compound 9 (D2916)
anticonvulsant activity and efficacy comparable to existing anti-
convulsants [3,16,17]. The benzamido pharmacophore as noted in
structure 6 has been reported to be active against seizures [17].
Earlier, Lepage and coworkers [18,19] reported on a series of N-aryl
isoxazolecarboxamides and their isomeric N-isoxazolylbenzamides
(7e10) that were evaluated as potential anticonvulsants. In the
carboxamide series 7 and 8, were the most active in the maximal

44
P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
and spatially, they are relatively the same size, but electroni-
cally, these groups are quite different, with the trifluoromethyl
group being electronegative and the methyl group being
electropositive.
Linus Pauling, in order to explain the diversity of structures
that provided general anesthesia, put forth the clathrate (L. cla-
thare, to furnish with a lattice) hypothesis. Pauling hypothesized
that all of the compounds that possessed general anesthetic
properties were capable of producing clathrates in the brain,
which interfered with normal brain transmission [22e24]. We
noted that
methylisoxazolyl-3-yl)
a
clathrate had formed (Fig. 1) in 3-((5-
amino)-5,5-dimethylcyclohex-2-enone
12c. This structure may be the cause of the toxicity noted in the
animal testing of the compound.
2. Results and discussion
2.1. Chemistry
Fig. 1. X-ray crystal structure of 3-((5-methylisoxazol-3-yl)amino)-5,5-dimethyl-
cyclohex-2-enone (12c).
The 5-methyl isoxazole enaminone analogs were synthesized as
shown in Scheme 1 and have been previously documented in our
laboratories [12] and elsewhere [26]. Ketones 11a (R ¼ R1 ¼ H), 11b
(R ¼ CH3, R1 ¼ H), 11c (R ¼ R1 ¼ CH3), were either commercially
available (11a and 11c) or synthesized (11b) via an ester hydrolysis
and decarboxylation reaction of the tert-butyl ester diketone
[25,26].
was advanced for further evaluation and displayed different phar-
macokinetic profiles in male and female rats while compound 7
(D2624) was advanced to preclinical testing [19,20].
In our continuing investigations on the enaminones, we
evaluated a series of comparable 3- and 5-methyl isoxazoles
[12]. Our results paralleled those of Lepage and coworkers in
that the 5-methyl isoxazole compounds, 4, were highly active.
The properties of products 12aec and 13a,b are provided in
Table 1. Scheme 2 shows the process by which we produced the 3-
carboxamide isoxazole analogs. We recently reported on the base-
catalyzed solution phase synthesis of vinylic benzamides [27] that
was employed in the current synthesis. Employing sodium hydride
(NaH), N-acylation provided a smoother condensation reaction
without the need for extensive workup and column chromatog-
raphy as was previously reported using triethylamine [21]. The
properties of products 15aec are provided in Table 2. The ketones
in Scheme 1 were used in this synthesis. They were aminated by
means of ammonium acetate (NH4OAc) in refluxing benzene or
toluene using a DeaneStark trap, providing 3-amino vinylogous
amide analogs 14aec. The use of ammonium acetate was more
efficient and less hazardous from our previous method that
utilized gaseous ammonia [21]. After purification, the amines were
subsequently condensed with 5-methyl-isoxazole-3-carbonyl
chloride, prepared as reported by Lepage and coworkers [18],
forming the desired 5-methly-N-(3-oxocyclohex-1-enyl)-iso-
xazole-3-carboxamides 15aec.
Compound 4e, R ¼ C2H5 had an ED50(rat) of 68.9 mg kgꢀ1
,
a TD50 > 500 mg, and a PI > 7.3. For compound 4f, R ¼ C(CH3)3,
the ED50(rat) was 28.1 mg kgꢀ1, the TD50 > 500 mg kgꢀ1, and the
PI > 17.8. For the isomeric 3-methyl series, 5a, R ¼ CH3 and 5c,
R ¼ C(CH3)3 were orally active at 30 mg kgꢀ1. However as
a group, the 3-methyl isoxazoles were the more toxic entities. It
was of interest to determine whether the amino enaminone
pharmacophore could be extended to the amide series as well.
Earlier, Foster et al. investigated the anticonvulsant activity of
vinylic benzamides and their comparable benzylamines [21]. The
evaluation revealed significant differences in anticonvulsant
activity between the two entities. In addition to noting the
pharmacological changes in converting the enaminones into
amide derivatives, Foster was interested in evaluating substitu-
ents at position 6 of the cyclic enaminone system. We found the
6-methyl substituent to have augmented activity. In continuing
with our extensive SAR study, we replaced the 6-methyl group
with a 6-trifluoromethyl group. While both groups are lipophilic
The synthesis of the trifluoromethyl analog 19 was a variation of
the Friary procedure [26] utilizing ethyl 4,4,4-trifluorocrotonate 16
which was condensed with tert-butyl acetoacetate 17 in the presence
Scheme 1. Synthesis of the 5-methyl-isoxazole enaminones. Conditions: a/b EtOH:EtOAc (1:1),
D 6 h.

P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
45
Table 1
determined at the time of peak effect for 12b by the method of
Litchfield and Wilcoxon [29].
5-methyl isoxazole enaminones 12aec, 13a,b.
CH3
2.3. X-ray crystallography
x
O
N
As a verification of our previously document role of x-ray
analysis in the evaluation of anticonvulsant activity [30e35], an x-
ray diffraction study of the representative 5-methyisoxazoles was
undertaken. This data is shown in Figs. 1 and 2 for 12c and 12b, and
Tables 4 and 5, respectively.
R
R1
O
Compound
R
R1
x
% Yield
mp, ꢁC
Clog Pa
2.4. Structureeactivity correlations
12a
12b
12c
13a
13b
H
H
H
CH3
H
CH3
0
0
0
1
1
80
51
51
73
62
225e226
205e206
204e207
119e120
167e168
ꢀ0.03
0.49
1.01
0.30
0.82
CH3
CH3
CH3
CH3
2.4.1. Enaminones (12aec, 13a,b)
Enaminones 12aec provides dramatic variations in activity and
toxicity. In the Phase I evaluation in mice, 12a was inactive in the
MES evaluation while showing exclusive activity in the scPTZ test,
a departure from our previous reported profiles of the enaminone
derivatives. This compound, 12a, when evaluated in the rat
provided broad protection in both test models, i.e. MES and scPTZ. It
was also noted that the protection was greater by the intraperito-
neal (ip) route than orally, indicating a possible absorption
problem. CLog P data [36] (Table 1) indicates that 12a is the most
hydrophilic of the analogs evaluated. The addition of a methyl
group to the cyclohexene ring, as in 12b, produces a compound
with dual protection in mice, however, with increased neurological
toxicity in mice. Compound 12b was evaluated in the rat (ip) and
provided an MES ED50 66 mg kgꢀ1, a TD50 >120 mg kgꢀ1 and a PI
1.8. Solubility was a problem with this compound as well. In the ip
evaluation in rats at the highest dose, the volume of the injected
solution affected the apparent activity of the compound (2/8 pro-
tected at the smaller volume vs. 6/8 protected with a doubled
volume). The dimethyl substituted analog, 12c, produced
a compound that was exclusively MES active and toxic. It should be
noted that the starting amines 14aec (Table 3) were all quite toxic
as well. Analysis of the x-ray structures of 12b and 12c display
significant differences. In Fig. 2, 12b shows the characteristic
intramolecular hydrogen binding of the vinyl proton at C2 with the
a
Calculated from reference [36].
of sodium ethoxide (NaOEt), producing tert-butyl 6-(tri-
fluoromethyl)-4-hydroxy-2-oxocyclohex-3-ene carboxylate 18,
which
methylisoxazole under previously reported conditions [25] to yield
tert-butyl 4-(5-methylisoxazole-3-ylamino)-6-(trifluoromethyl)-2-
oxocyclohex-3-ene carboxylate 19. The synthesis is shown in
Scheme 3.
High field NMR analyses on the reported compounds were
consistent with the assigned structures.
was
subsequently
condensed
with
3-amino-5-
2.2. Pharmacology
Pharmacological testing of the compounds listed in Tables 1 and
2 have been provided by the Antiepileptic Drug Development
(ADD) Program, Epilepsy Branch, Neurological Disorders Program,
National Institute of Neurological Disorders and Stroke (NINDS)
[28]. Phase I evaluation of the reported compounds involved three
tests: maximal electroshock seizure (MES), subcutaneous pentyl-
enetetrazol (scPTZ), and neurologic toxicity (Tox) in mice. Phase I
data for the compounds are shown in Table 3. As previously
reported [28], to differentiate the results between different rodent
species, the most active class 1 analogs in Phase I were subse-
quently evaluated for oral (po) activity (Phase VIA, Phase VIB) in the
rat. All data is shown in Table 3. Phase VIB determined the median
dose (ED50) and median toxic dose (TD50) in male SpragueeDawley
rats. The ED50 and TD50 values and their confidence limits were
isoxazole ring nitrogen, N2, producing
a pseudo three-ring
configuration as previously reported [21].
However, in Fig. 1, a more complicated structural configuration
occurs. The dimethyl analog, 12c, is assembled as a head-to-tail
dimer, exhibiting both intramolecular hydrogen bonding (C2B-
N2B and N2A-C2A), but also intermolecular hydrogen bonding with
the carbonyl oxygen (O1B and the isoxazoles proton, C10A, and the
proton on the secondary amine, N1A) producing a pocket between
these molecules. Further, this clathrate formation effectively blocks
Scheme 2. Synthesis of the 3-carboxamide 5-methyl-isoxazole analogues. Conditions: (a) NH4OAc,
D
1 h. (b) NaH, dry THF, 20 ꢁC.

46
P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
Table 2
favorable for anticonvulsant activity but the effective interacting
bond distance is apparently the limiting factor.
3-carboxamide isoxazole enaminones 15aec.
Clog P determines both transportability through biological
membranes and pharmacological effects by binding to the
receptor [13]. The optimum Clog P should be less inhibited in their
movement through the aqueous and lipophilic phases of living
tissue. We note the Clog P for our compounds is in the optimum
range [41].
O
CH3
HN
O
N
R
R1
O
2.4.2. Amides (15aec)
Cyclohexyl amide 15a showed some protective activity in the
scPTZ model, but it occurred at a dose that produced toxic side
effects. The 5-methyl derivative, 15b, was also inactive and
produced more toxic effects (Table 3). The 5,5-dimethyl analog, 15c
was also inactive, but without any neurotoxicity. Clearly the amides
did not offer any notable anticonvulsant leads.
Compound
R
R1
% Yield
mp, ꢁ
C
CLog Pa
15a
15b
15c
H
CH3
CH3
H
H
CH3
23
21
35
132e134
175e177
144e146
ꢀ0.22
0.30
0.82
a
Calculated from reference [36].
2.4.3. Trifluoromethyl analog (19)
The trifluoromethyl analog, 19, resulted in an inactive and non-
toxic compound. Therefore, replacement of the methyl group with
an electronegative substituent at the position 6 with the same
approximate size abolishes activity. To resolve the actual lip-
ophilicity of the 6-trifluoromethyl intermediate 18, (Clog P 1.41 vs.
2.00 for the 6-methyl analog), we experimentally determined these
analogs. The actual log P was 0.28 for 19 vs. ꢀ0.16 for the 6-methyl
analog, 4f. Earlier we had shown [25a] that replacement of the
methyl group with a bulky substituent, e.g. phenyl, abolishes
activity, while replacement with hydrogen maintains activity. It is
therefore evident that substitution on the 6 position is critical for
anticonvulsant activity.
access to the proposed active site [37e39] by virtue of the dimethyl
substituents at both ends of the pocket, although hydrogen
bonding has been correlated with anticonvulsant activity. Poupert
and coworkers observed that the anticonvulsant activity of
phenytoin [40] was greatly reduced when the H-bonding groups
(C]O or NH) were removed from this compound. In contrast to the
previous active analogs, the increase in chain length abolished
activity. It is worth noting that in the homologous benzene series,
i.e. in going from aniline to benzylamine [21,25a] activity increased.
However, in contrast with the previous series, no toxicity was
observed.
Molecular modeling studies were undertaken to determine the
probability of the pseudo ring system forming in this series as
compared with the previously reported isoxazole analogs.
Employing our previous documented analysis [38] the following
vinyl proton to the isoxazole nitrogen bond distances were deter-
2.5. Mechanism of action
We had earlier shown that for the enaminones methyl 4-[(40-
chlorophenyl)-amino]-6-methyl-2-oxocyclohex-3-ene-1-carboxylate
20a, and methyl 4[(40-trifluoromethoxyphenyl)amino]-6-methyl-2-
oxocyclohex-3-ene-1-carboxylate 20b, were active as inhibitors of
voltage-gated sodium channels [25c]. New information has recently
mined: 12b 2.50 Å vs. 13a 3.66 Å (D 1.16 Å) and 12c 2.38 Å vs. 13b
4.49 Å ( 2.11 Å). These significant differences may account for the
D
lack of activity in this series. Clog P determinations (Table 1) show
an increase in lipophilicity in the 13a,b series which would be
Table 3
Anticonvulsant screening project (ASP): Anticonvulsant results.
Compound
Anticonvulsant resultsa
12a
Phase I (mice): Class 1 MES test-inactive up to 300 mg kgꢀ1; scPTZ test-active 4/5 animals protected at 300 mg kgꢀ1 at 30 min; Tox test-
no toxicity up to 300 mg kgꢀ1 at 30 min and 4 h.
Phase VIA (i.p. rats): MES test-active 2/3 animals protected at 30 mg kgꢀ1 at 1 h; scPTZ test-active 1/3 animals protected at 30 mg kgꢀ1 at
4 h; Tox test-0/6 animals displayed toxicity at 30 mg kgꢀ1 up to 4 h.
12b
12c
Phase I (mice): Class 1 MES test-active 1/3 animals protected at 100 mg kgꢀ1 at 15 min, 2/3 at 300 mg kgꢀ1 at 1 h; scPTZ test-active 4/5
animals protected at 300 mg kgꢀ1 at 30 min; Tox test-active 3/8 animals were unable to grasp rotorod at 100 mg kg-1 at 30 min and 3/4
unable o grasp rotorod at 300 mg kg-1 at 15 min.
Phase VIB (i.p. rats): MES ED50 66 mg kgꢀ1, TD50 > 120 mg kgꢀ1; PI 1.8.
Phase I (mice): Class 1 MES test-active 3/7 animals protected at 100 mg kgꢀ1 at 30 min, 4/5 animals protected to 300 mg kgꢀ1 at 30 min
and at 4 h; scPTZ test-inactive up to 300 mg kgꢀ1; Tox test-2/8 animals were toxic at 100 mg kgꢀ1 at 30 min, 3/4 toxic at 300 mg kgꢀ1 at
30 min and 1/2 toxic at 300 mg kgꢀ1 at 4 h.
13a
13b
14a
Phase I (mice): Class 3
Phase I (mice): Class 3
Phase I (mice): Class 3 (in Tox evaluation-8/8 animals displayed muscle spasms, and unable to grasp rotorod at 100 mg kgꢀ1; 4/4 animals
showed the same toxicity at 300 mg kgꢀ1 at 30 min)
14b
14c
Phase I (mice): Class 3 (in Tox evalution-8/8 animals displayed muscle spasms at 100 mg kgꢀ1; 4/4 animals displayed muscle spasms and
unable to grasp rotorod at 300 mg kgꢀ1 at 30 min)
Phase I (mice): Class 3 (in scPTZ evaluation-1/1 animals died following continuous seizure at 30 mg kgꢀ1 at 4 h; 1/1 animals died
following continuous seizure at 30 mg kgꢀ1 at 30 min; 1/5 animals underwent myoclonic jerks at 100 mg kgꢀ1 at 4 h; (in Tox evaluation-
4/8 animals displayed muscle spasms at 100 and 300 mg kgꢀ1 at 30 min)
15a
15b
15c
19
Phase I (mice): Class 3
Phase I (mice): Class 3; 4/4 mice unable to grasp the rotorod at 300 mg kgꢀ1 at 30 min; 2/2 mice toxic at 300 mg kgꢀ1 at 4 h
Phase I (mice): Class 3
Phase I (mice): Class 3
a
Phase I in mice activity-class 1 ¼ activity at 100 mg kgꢀ1 or <; class 2 ¼ activity between 100 and 300 mg kgꢀ1 class 3 ¼ no activity at 300 mg kgꢀ1
.

P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
47
been reported by Kombian et al. [39] that two of our analogs, methyl 4-
(40-bomophenylamino)-6-methyl-2-oxocyclohex-3-ene carboxylate
(ADD 206038) 20c [25b], and 3-(40-chlorophenylamino)cyclohex-3-
enone 21a, support an indirect action on the GABA system, producing
GABA-dependent synaptic depression. Thus, these enaminones may
act to potentiate extracellular GABA concentration that will promote
GABA acting on GABAA receptors, located on presynaptic glutamate
terminals to decrease EPSC amplitude [39]. We have recently evalu-
ated similar analogs that we used in the sodium channel inhibition
(trifluoromethoxy)phenyl)amino)cyclohex-2-enone 21b for GABA
activity using mouse olfactory bulb slices and found that the latter
Table 4
Atomic coordinates (ꢂ104) and equivalent isotropic displacement parameters
(A2
ꢂ
103) for 3-((5-methyisoxazol-3-yl)amino)-5,5-dimethylcyclohex-2-enone
(12c). U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
x
y
z
U(eq)
O(1A)
O(2A)
O(1B)
O(2B)
N(1A)
N(2A)
N(1B)
N(2B)
C(1A)
C(2A)
C(3A)
C(4A)
C(5A)
C(6A)
C(7A)
C(8A)
C(9A)
C(10A)
C(11A)
C(12A)
C(1B)
C(2B)
C(3B)
C(4B)
C(5B)
C(6B)
C(7B)
C(8B)
C(9B)
C(10B)
C(11B)
C(12B)
9359(4)
715(4)
543(5)
ꢀ3545(2)
ꢀ4235(2)
ꢀ8416(2)
8474(2)
6360(2)
6673(2)
9011(2)
7016(2)
6781(2)
7928(2)
8476(2)
7529(2)
7765(2)
829(2)
8608(2)
8639(2)
7795(2)
9320(2)
8808(3)
6684(2)
6214(2)
6034(2)
5585(2)
7437(2)
7321(2)
6812(2)
6453(2)
6248(2)
7024(2)
5526(3)
5998(3)
8408(2)
8867(2)
9230(2)
9793(2)
78(1)
64(1)
90(1)
75(1)
54(1)
65(1)
60(1)
73(1)
45(1)
51(1)
55(1)
58(1)
57(1)
55(1)
92(2)
97(2)
46(1)
51(1)
50(1)
63(1)
51(1)
59(1)
62(1)
66(1)
55(1)
57(1)
82(1)
81(1)
54(1)
62(1)
62(1)
78(1)
studies
plus
an
additional
compound
5-methyl-3-((4-
6300(4)
3351(5)
2523(5)
1228(5)
4459(5)
5231(5)
6291(5)
8308(6)
9177(5)
7461(6)
6109(6)
5984(7)
8595(7)
2015(5)
ꢀ12(5)
ꢀ754(5)
ꢀ10,220(2)
ꢀ6267(2)
ꢀ4516(2)
ꢀ11,531(2)
ꢀ10,190(2)
ꢀ5908(2)
ꢀ4821(3)
ꢀ4532(3)
ꢀ5451(3)
ꢀ6497(3)
ꢀ6863(3)
ꢀ6207(4)
ꢀ7448(3)
ꢀ5618(3)
ꢀ6090(3)
ꢀ5201(3)
ꢀ5065(3)
ꢀ10,962(3)
ꢀ9907(3)
ꢀ9366(3)
ꢀ9960(3)
ꢀ11,243(3)
ꢀ11620(3)
ꢀ11,622(3)
ꢀ11,776(3)
ꢀ11,191(3)
ꢀ11,880(3)
ꢀ11,243(3)
ꢀ11,417(3)
ꢀ2771(6)
213(5)
1017(6)
ꢀ183(6)
ꢀ2466(6)
ꢀ2745(6)
ꢀ1968(5)
ꢀ1445(7)
ꢀ5160(6)
3225(6)
4144(6)
6029(6)
7822(6)
Scheme 3. Synthesis of the 3-amino-5-methyl-isoxazole trifluoromethyl analog.
Conditions: (a) Na, dry EtOH, 2.5h; (b) EtOAc: EtOH (1:1), 6 h.
D
D

48
P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
Methylisoxazole-3-yl)methaneamine was obtained from ASDI
Biosciences, Inc. (http://www.asdi.net), or prepared synthetically
[43]. 5-Methylcyclohexane-1,3-dione, [12,26] 3-aminocyclohex-2-
enone (14a, R ¼ R ¼ H), [17,27] 3-amino-5-methylcyclohex-2-
enone (14b, R1
¼
CH3,
R
¼
H) [18,27] and 3-amino-5,5-
dimethylcyclohex-2-enone (14c, R1
¼
R
¼
CH3) [44] were
prepared by literature methods.
3.2. General procedure for isoxazole enaminones
3.2.1. 3-((5-Methylisoxazol-3-yl)amino)cyclohex-2-enone (12a)
Cyclohexane-1,3-dione (11a, R1 ¼ R ¼ H, 3.03 g, 27 mmol) and 3-
amino-5-methylisoxazole (3.24 g, 33 mmol) were added to
a mixture of absolute EtOH (100 mL) and EtOAc (100 mL), and the
solution was refluxed and stirred for 6 h. During that time, one-half
of the solvents were slowly removed, via a DeaneStark trap, and
after cooling, replaced with an equal volume of anhydrous Et2O.
The mixture became cloudy while stirring and was allowed to
continue overnight whereupon crystals spontaneously deposited.
Recrystallization from EtOAc yielded a tan powder, 4.15 g (80%); mp
Fig. 2. X-ray crystal structure of 3-((5-methylisoxazol-3-yl)amino)-5-methylcyclohex-
2-enone (12b).
compound acts as a positive allosteric modulator of GABAA receptors
and enhances GABA affinity [42].
225e226 ꢁC; 1H NMR (DMSO-d6
ring), 3.4 (3H, s, CH3), 6.0 (1H, s, ]CH), 6.3 (1H, s, isoxazole H), 9.5
) d 1.1e2.2 (6H, m, cyclohexene
(1H, br s, NH). 13C (DMSO-d
) d 12.30, 17.75, 21.96, 38.43, 96.22,
3. Experimental section
6
106.22, 158.06, 160.18, 168.95. 197.81. IR (KBr) 3339.41 cmꢀ1 (NH
stretch), 3140.90 cmꢀ1 (5-methylisoxazole stretch), and
1685.35 cmꢀ1 (C]O stretch). Anal. (C, H, N).
3.1. Chemistry
Melting points were determined on a Thomas-Hoover capillary
melting point apparatus and were uncorrected. Observed boiling
points were also uncorrected. Analytical TLC was performed using
silica gel with a fluorescent indicator coated on 1 ꢂ 3 inch glass
3.2.2. 3-((5-Methylisoxazole-3-yl)amino)-5-methylcyclohex-2-
enone (12b)
Following the general procedure for 12a, ketone (11b, R1 ¼ CH3,
R ¼ H) and 3-amino-5-methylisoxazole produced 12b as pale
yellow crystals from EtOAc, 2.81 g (50.5%); mp 205e206 ꢁC; 1H
plates with 0.2 mm thickness (Whatman MKGF silica gel 200 m). IR
spectra were obtained on a Nicolet Magna-IR 560 spectrometer.
The samples were recorded with KBr pellets. The 1H and 13C NMR
spectra were determined on a Bruker 1 Ultra Shield-400 MHz NMR
spectrometer. The samples were dissolved either in deuterated
NMR (DMSO-d6
cyclohexene ring), 3.3 (3H, s, CH3 on isoxazole ring), 6.0 (1H, s,
]CH), 6.3 (1H, s, isoxazole H), 9.4 (1H, br s, NH). 13C (DMSO-d
)
d
1.0 (3H, d ¼ 6.4 Hz, CH3), 1.9e2.4 (5H, m,
)
6
chloroform (CDCl3) or dimethylsulfoxide (DMSO- ) containing
d
11.21, 32.08, 42.03, 46.04, 49.98, 53.36, 95.46, 102.90, 105.15,
d6
168.81, 197.40. IR (KBr) 3342.75 cmꢀ1 (NH stretch), 3139.76 cmꢀ1
(5-methylisoxazole stretch), and 1680.15 cmꢀ1 (C]O stretch). Anal.
(C, H, N).
0.03% tetrametylsilane (TMS) as an internal reference. Elemental
analyses (C, H, and N) were determined by Schwarzkopf Microan-
alytical Laboratory, Woodside, NY 11377, USA. The analytical results
for the elements were within ꢃ0.4% of the theoretical values.
Cyclohexane-1,3-dione, 5,5-dimethylcyclohexane-1,3-dione and 3-
amino-5-methyisoxazole were obtained from Aldrich Chemical
Company and used without further purification. (5-
3.2.3. 3-((5-methylisoxazol-3-yl)amino)-5,5-dimethylcyclohex-2-
enone (12c)
Following the general procedure for 12a, ketone (11c,
R1 ¼ R ¼ CH3) and 3-amino-5-methylisoxazole produced 12c as
light yellow crystals from EtOAc, 3.06 g (51.4%); mp 204e207 ꢁC;
Table 5
1H NMR (DMSO-d6
)
d
1.0 (6H, s, gem CH3), 2.0 (2H, s, C4 CH2), 2.5
(2H, s, C6 CH2), 3.3 (3H, s, CH3 on isoxazole ring), 6.0 (1H, s, ]CH),
6.2 (1H, s, isoxazole H), 9.4 (1H, br s, NH). 13C (DMSO-d
5.02,
Atomic coordinates (ꢂ104) and equivalent isotropic displacement parameters
(A2 ꢂ 103) for 3-((5-methyisoxazol-3-yl)amino)-5-methylcyclohex-2-enone (12b).
U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
)
d
6
27.34, 41.94, 43.43, 45.47.47.23, 97.09, 102.34, 105.25, 155.33, and
197.05. IR (KBr) 3340.45 cmꢀ1 (NH stretch), 3143.68 cmꢀ1 (5-
methylisoxazole stretch), and 1678.55 cmꢀ1 (C]O stretch). Anal.
(C, H, N).
x
y
z
U(eq)
O(1)
O(2)
N(1)
N(2)
C(1)
ꢀ1274(1)
ꢀ1456(2)
1307(2)
ꢀ887(2)
1119(2)
ꢀ73(2)
ꢀ143(2)
5913(2)
3667(2)
4572(2)
2289(2)
1709(2)
311(2)
3070(2)
952(2)
64(1)
72(1)
48(1)
68(1)
43(1)
47(1)
48(1)
52(1)
51(1)
48(1)
52(1)
51(1)
48(1)
71(1)
45(1)
52(1)
53(1)
71(1)
2452(2)
1549(2)
2824(2)
2619(2)
3133(2)
3783(2)
3403(2)
3489(2)
3470(60)
4210(40)
4980(30)
4143(3)
1847(2)
1469(2)
918(2)
3.2.4. 3-[(5-Methylisoxazol-4-yl)methylamino]-5-methylcyclohex-
2-enone (13a)
C(2)
C(3)
ꢀ195(2)
1015(3)
2124(2)
2358(2)
1010(60)
2300(30)
2180(30)
3382(2)
373(2)
Into a 50 mL single neck flask, 5-methyl-1,3-cyclohexanedione
(0.88 g, 7 mmol) and 5-methyl-3-isoxazolemethylamine (0.95 g
8.5 mmol), were added to a mixture of absolute EtOH (25 mL) and
EtOAc (25 mL), and the solution was refluxed and stirred for 6 h.
During refluxing, one-half of the solvent mixture was slowly
removed, via a DeaneStark trap. Once cooled to r.t. an equal volume
of anhydrous Et2O was added. After the addition of ether, the
mixture became cloudy and was allowed to stir overnight at room
temperature whereupon crystals spontaneously deposited. The
mixture was filtered and dried to give the title compound, 13a, as
C(4A)
C(5A)
C(6A)
C(4B)
C(5B)
C(6B)
C(7)
C(8)
C(9)
C(10)
C(11)
ꢀ6164(4)
ꢀ237(2)
1403(2)
ꢀ600(80)
160(40)
1420(40)
ꢀ1093(3)
4736(2)
6152(2)
6798(2)
8253(3)
668(2)
ꢀ499(2)
ꢀ931(3)
310(2)

P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
49
yellow crystals, mp 119e120 ꢁC, unchanged after recrystallization
from EtOAc (1.12 g, 73%); 1H NMR (DMSO-d
1.1 (3H, d, CH3), 1.8
3.2.10. 5-Methylisoxazole-3-carbonyl chloride
mixture of 5-methylisoxazole-3-carboxylic acid (3.81 g,
)
d
A
6
(2H, s, C4 CH2), 2.1e2.4 (5H, m, cyclohexene ring), 2.5 (3H, s, iso-
xazole CH3), 4.4 (1H, s, cyclohexene CH]), 5.2 (1H, s, isoxazole
CH]), 6.0 (1H, s, NH). 13C (DMSO-d6) 12.45, 20.35, 29.29, 36.44,
44.27, 96.07, 100.01, 159.89, 165.38, 170.44 and 197.29. IR (KBr)
3347.25 cmꢀ1 (NH stretch), 3183.44 cmꢀ1 (5-methylisoxazole
stretch) and 1688.45 cmꢀ1 (C]O stretch). Anal. (C, H, N).
0.03 mol) in 150 mL of toluene was dried via azeotropic distillation
of some of the toluene into a two-neck 500 mL round bottom flask
fitted with a DeaneStark trap and a magnetic stirrer. The mixture
was allowed to cool to about 90 ꢁC, and dimethylformamide, 1 mL,
was added. Thionyl chloride (4.8 g, 0.04 mol) was added dropwise
over 15 min and the mixture was refluxed for 5 h with continuous
stirring. The reaction was allowed to cool to room temperature, and
then the solvent was evaporated in vacuo to yield the product,
a dark-brown oil, 80%, which crystallized on standing. It was kept
under vacuum until further use.
3.2.5. 3-[(5-Methylisoxazol-4-yl)methylamino]-5,5-
dimethylcyclohex-2-enone (13b)
In a similar manner, the amination reaction proceeds as previ-
ously stated in 13a. After the addition of ether, the mixture became
cloudy and was allowed to stir overnight at room temperature
whereupon crystals spontaneously deposited. The mixture was
filtered and dried to give the title compound,13b, as tan crystals mp
167e168 ꢁC, unchanged after recrystallization from EtOAc (0.49 g,
3.3. General procedure for isoxazole-3-carboxamides
3.3.1. 5-Methyl-N-5-methyl-3-oxocyclohex-1enyl)isoxazole-3-
carboxamide (15b)
62%); 1H NMR (DMSO-d6
) d 1.0 (6H, s, gem CH3), 1.1 (2H, 2, C4 CH2),
To a 250 mL single neck flask equipped with a ST “Y” tube,
condenser and a pressure equalizing dropping funnel containing
85 mL of anhydrous THF, was cautiously added NaH (0.84 g,
35.0 mmol) with constant stirring, maintaining the temperature
below 20 ꢁC with external cooling. After the reaction, 14b (1.9 g,
15.0 mmol) was added over 30 min through the “Y” tube, and after
the addition, a further 10 mL of THF washed the “Y” tube. The
reaction mixture was heated to reflux for 20 min, cooled to room
temperature and 5-methylisoxazolyl carbonylchloride [7] (2.30 g,
16.0 mmol) in anhydrous THF (25 mL) was added dropwise over
5 min. After stirring at room temperature for a further 10 min, the
mixture was quenched with water and transferred to a 250 mL
Erlenmeyer flask, neutralized with concentrated HCl (w10 mL),
diluted with dichloromethane (55 mL) and transferred to a sepa-
ratory funnel. The aqueous layer was discarded and the organic
layer was washed successively with water (55 mL), 10% NaHCO3
(2 ꢂ 55 mL), and water (55 mL). The organic layer was dried over
sodium sulfate, evaporated in vacuo and the residue triturated with
anhydrous Et2O (110 mL). The crude solid was recrystallized from
EtOAc, to give 15b (0.75 g 21%) as cream colored crystals, mp
2.2e2.3 (5H, m, cyclohexene ring), 2.4 (3H, s, isoxazole CH3), 4.3
(1H, s, cyclohexene CH]), 5.2 (1H, s, isoxazole CH]), 6.0 (1H, s,
NH). 13C (DMSO-d ) 11.18, 28.29, 32.33, 38.47, 42.20, 50.44, 95.32,
6
100.44, 159.89, 163.58, 170.22 and 197.32. IR (KBr) 3447.25 cmꢀ1
(NH stretch), 3203.44 cmꢀ1 (5-methylisoxazole stretch), and
1708.45 cmꢀ1 (C]O stretch). Anal. (C, H, N).
3.2.6. 3-Aminocyclohex-2-enone (14a)
Into a 500 mL round bottom flask fitted with a DeaneStark trap
and a magnetic stirrer was added 150 mL of anhydrous benzene,
1,3-cyclohexanedione (5.6 g, 0.05 mol), and ammonium acetate
(7.7 g, 0.1 mol). The mixture was stirred continuously, and refluxed
for 5 h. The reaction was allowed to reach room temperature. The
precipitate was recrystallized with ethyl acetate to afford yellow
crystals (1.73 g, 31% yield). Mp-128e131 ꢁC [lit.128e131 ꢁC (35)]. 1H
NMR Acetonitrile-d3)
s, CH]). 5.3e5.5 (2H, s, br, NH2).
d
ꢀ1.8e2.4 (6H, m, cyclohexene ring), 5.1 (1H,
132e134 ꢁC 1H NMR (CDCl3)
m, cyclohexene ring 5H, and the isoxazole ring CH3), 6.4 (1H, s,
d
1.0 (3H, J ¼ 6.4 Hz, CH3), 2.0e2.7 (8H,
3.2.7. 3-Amino-5-methylcyclohex-2-enone (14b)
In a like manner as previously stated in 14a, the title compound,
14b, was produced in 80% yield, yellow powder, mp, 175e177 ꢁC, 1H
CH]), 6.8 (1H, s (split), CH] on isoxazole ring), 8.3 (1H, br s, N-H).
13C NMR (CDCl3)
d 12.63, 20.94, 29.11, 36.68, 44.95, 101.46, 112.97,
(DMSO-d6)
d
0.8e1.2 (3H, d, J ¼ 2.4 Hz, CH3), 1.7e2.6 (5H, m,
152.95, 154.03, 157.31, 172.28 and199.72. IR (KBr) 3397.41 cmꢀ1 (NH
stretch), 3143.68 cmꢀ1 (5-methylisoxazole stretch), 1685.35 cmꢀ1
and 1618.99 cmꢀ1 (two C]O stretches). Anal. (C, H, N).
cyclohexene ring), 4.9 (1H, s, CH]) 6.5e7.0 (2H, s, br, NH2).
3.2.8. 3-Amino-5,5-dimethylcyclohex-2-enone (14c)
In a like manner as previously stated in 14a, the title compound,
14c, was produced in 36% yield as light yellow crystals, mp
156e158 ꢁC, 1H NMR (acetone-d6) 1.0 (6H, s, gem CH3), 2.2 (2H, s, C6
CH2), 2.9 (2H, s, C4 CH2) 6.0e6.2 (2H, s, br, NH2).
3.3.2. 5-Methyl-N-(3-oxocyclohex-1enyl)isoxazole-3-carboxamide
(15a)
The above procedure was repeated using amino ketone 14a
(1.33 g, 12 mmol), to give 15a (0.60 g, yield, 23%), as yellow crystals,
mp 132e134 ꢁC (from EtOAc) 1H NMR (CDCl3)
d 1.9e2.7 (6H, m,
3.2.9. 5-Methylisoxazole-3-carboxylic acid
Sodium bicarbonate (13.2 g, 0.157 mol), hydroxylamine hydro-
chloride (10.91 g, 0.157 mol) and ethyl 2,4-dioxopentanoate (25 g,
01.57 mol) were added to 107 mL of ethanol in a 500 mL round
bottom flask. The mixture was refluxed for 4 h. The precipitate was
filtered and the filtrate concentrated in vacuo to yield the ester. The
ester was dissolved in 53.5 mL of EtOH to which was added sodium
hydroxide (59 mL, 10% solution). The solution stirred overnight at
r.t.. The solvent was evaporated under reduced pressure. The
sodium salt was dissolved in water and acidified with concentrated
hydrochloric acid to precipitate the title compound. Recrystalliza-
tion of the crude product from EtOAc yielded the product, white
crystals, mp 172e174 ꢁC (lit. 182 ꢁC (7), 79%, 1H NMR (DMSO-d6) 2.3
(3H, s, CH3), 6.6 (1H, s, CH]), 7.0 (1H, s, COOH). IR spectrum (KBr)
5-methylisoxazole stretch at 3149 cmꢀ1 and C]O stretch at
1655.35 cmꢀ1. Anal: (C, H, N, O).
cyclohexene ring), 2.4 (3H, s, CH3), 6.4 (1H, s, CH]), 6.8 (1H, s,
(split), H on the isoxazole ring), 8.2 (1H, s, br, N-H). 13C NMR (CDCl3)
d
11.48, 24.32, 32.89, 42.57, 50.52, 101.45, 112.26, 151.32, 157.36,
158.23, 172.30 and 199.50. IR (KBr) 3285.07 cmꢀ1 (NH stretch),
3153.70 cmꢀ1 (5-methylisoxozole stretch), 1675.11 cmꢀ1 and
1623.90 cmꢀ1 (two C]O stretches). Anal. (C, H, N).
3.4. Trifluoromethyl analog
3.4.1. Tert-Butyl 4-(5-methylisoxazole-3-ylamino)-6-
trifluoromethyl-2-oxocyclohhex-3-ene carboxylate (19)
To a pre-dried 2-neck 250 mL round bottom flask fitted with
a condenser and magnetic stirrer was added 25 mL of anhydrous
EtOH, followed by 1.09 (47.6 g, 47.6 g-atom) of sodium. Complete
solution was achieved by gentle reflux. After cooling to room

50
P.L. Jackson et al. / European Journal of Medicinal Chemistry 51 (2012) 42e51
temperature, tert-butyl acetoacetate, 17 (7.53 g, 47.6 mmol) was
slowly added and the mixture stirred for an additional 30 min. Ethyl
4,4,4-trifluorocrotonate, 16 (8.0 g, 47.6 mmol), dissolved in 25 mL of
anhydrous EtOH was added and the mixture was refluxed for 2.5 h.
Evaporation under vacuum produced an orange gummy crude
residue. The residue was crystallized from EtOAc to provide tert-
butyl 6-(trifluoromethyl)-4-hydroxy-2-oxocyclohex-3-ene carbox-
were shaken for approximately 1 h at ambient temperature, then
gravity filtered to remove any undissolved compound. Aliquots
(w10 mL) of the blank and stock solutions were transferred to test
tubes and centrifuged for approximately 45 min before deter-
mining the absorbance. A 100 ml portion of each stock and blank
solution was volumetrically transferred to individual separatory
funnels, and 5 mL of octanol was added. After the separatory fun-
nels were gently inverted w100 times, the phases were allowed to
separate for about 1 1/2 h. Aliquots (w10 mL) of the partitioned
blank and sample solutions were transferred to test tubes and
centrifuged for approximately 45 min before determining the
absorbance. The absorbance of the aqueous phase was determined
for each solution, before and after partitioning, using a HP 8453 UV/
Vis spectrophotometer and scanning from 400 to 200 nm. The
appropriate blank was used to determine the baseline for each
solution.
ylate, 18 (4.55 g, 34.1%), mp 133e137 ꢁC 1H NMR (DMSO-d
) d 1.9
(9H, m, 3ꢂ CH3), 2.6e3.5 (4H, m, cyclohexene ring), 5.4 6(1H, s,
CH]), 9.0 (1H, s, OH). Anal. (C, H, N, F).
To a 200 mL round bottom flask was added 50 mL of EtOAc and
50 mL of anhydrous EtOH followed by 18 (1.41 g, 5.0 mmol). After
solution, 3-amino-5-methylisoxazole (0.61 g, 6.23 mmol) was
added and after solution, the mixture was refluxed for 6 h. The
mixture was evaporated under reduced pressure to yield a yellow
oil. The crude product was recrystallized from a Et2O/hexane
solvent system to obtain the title compound, 19 (403 mg, 22.4%),
mp 225e226 ꢁC 1H NMR (DMSO-d
CH3 on isoxazole ring), 2.7e3.4 (4H, m, cyclohexene ring), 6.3 (1H, s,
CH]), 9.9 (1H, s, NH). Anal. (C, H, N, F).
)
d
1.4 (9H, m, 3ꢂ CH3), 2.4 (3H, s,
6
4. Conclusion
The mechanism of action of these compounds yielded no posi-
tive results for sodium channel activity. At the present time, the
mode of action at which these compounds elicit their activity is
unknown. Additional tests are underway to evaluate the active
isoxazole analogs’ ability to positively potentiate GABA and this will
be reported shortly.
3.5. Pharmacology
Initial evaluation for anticonvulsant activity was done by the
Epilepsy Branch, National Institute of Neurological Disorders and
Stroke [28]. These tests were performed in male Carworth Farms
no. 1 (CF1) mice. Phase I of the evaluation included three tests:
maximal electroshock (MES), subcutaneous pentylenetetrazole
(scPTZ), and rotorod test for neurological toxicity (Tox). Compounds
were suspended in 30% poly(ethyleneglycol) 400 and were
administered by intraperitoneal injection at three dosage levels
(30, 100 and 300 mg kgꢀ1) with anticonvulsant activity and
neurotoxicity noted 30 min and 4 h after administration. Data is
found in Table 3. Phase VIB determined the median effective dose
(ED50) and median toxic dose (TD50) in male SpragueeDawley rats.
The ED50 and TD50 values and their confidence limits were deter-
mined at the time of peak effect for 12b by the method of Litchfield
and Wilcoxon [29]
Acknowledgments
This research was supported by a grant from the National
Institutes of Health (1 R21 GM634940-02) for which K.R.S. is the
principal investigator and also grant 2 G12 RR003048 from the
RCMI Program, Division of Research Infrastructure, National Center
for Research Resources, NIH. Log P experiments were carried out by
Midwest Research Institute, Kansas City, MO 64110-2299 by Dr.
Linda Seimann and Mr. Matthew Armstrong.
Appendix. Supplementary data
3.6. X-ray crystal analysis
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2012.02.003. These data
include MOL files and InChIKeys of the most important compounds
described in this article.
3-((5-Methylisoxazol-3-yl)amino)-5-methylcyclohex-2-anone
(12b) and 3-((5-methylisoxazol-3-yl)amino)-5,5-dimethylcyclohex-
2-enone (12c) were recrystallized from EtOAc. All experimental
details related to the structural analysis are provided in Figs.1 and 2,
and Tables 4 and 5. The structurewas solved by direct methods of the
SHELXTLPC program and refined by the SHELXTL program [45]. The
full crystallographic data were deposited at the Cambridge Crystal-
lographic Data Center. Copies of the data can be obtained free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (Fax: þ44 1223 336033 or email: deposit@ccdc.camac.uk). The
database numbers are CCDC ID: 280424 (12b) and CCDC ID: 289425
(12c).
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3.7. Log P
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