α-fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide, a novel potent anticonvulsant derivative of a cyclic analogue of valproic acid

Article abstract of DOI:10.1021/jm900017f

2,2,3,3-Tetramethylcyclopropanecarboxylic acid (TMCA, 4) is a cyclic analogue of the antiepileptic drug (AED) valproic acid (VPA) (1). α-F, α-Cl, R-Br, and R-methyl derivatives of 4 and their amides were synthesized and tested in rodent models for anticonvulsant potency and AED-induced teratogenicity. In the anticonvulsant rat-maximal electroshock (MES) and subcutaneous metrazol (scMet) tests, α-Cl-TMCD (17) had ED ₅₀ values of 97 and 27 mg/kg, respectively. α-F-TMCD (11) was 120 times more potent than VPA in the rat-scMet test (ED₅₀) 6 mg/kg) and had a protective index (PI) TD₅₀/ED₅₀) of 20. In the 6 Hz psychomotor mouse model 11 had ED₅₀ values of 57 mg/kg (32 mA) and 59 mg/kg (44 mA). The ED50 values of 11 in the hippocampal-kindled rat model and in the pilocarpine-induced-status rat model were 30 and 23 mg/kg, respectively. Unlike 1, 11 was nonteratogenic in mice. This novel compound has the potential to become a candidate for development as a new potent and safe antiepileptic and CNS drug.

Full text of DOI:10.1021/jm900017f

J. Med. Chem. 2009, 52, 2233–2242
2233
r-Fluoro-2,2,3,3-Tetramethylcyclopropanecarboxamide, a Novel Potent Anticonvulsant
Derivative of a Cyclic Analogue of Valproic Acid
Neta Pessah,† Meir Bialer,†,‡ Bogdan Wlodarczyk,§ Richard H. Finnell,§ and Boris Yagen*,‡,|
Department of Pharmaceutics and Department of Natural Products and Medicinal Chemistry, School of Pharmacy, Faculty of Medicine, and
The DaVid R. Bloom Centre for Pharmacy, The Hebrew UniVersity of Jerusalem, Jerusalem, Israel, and Center for EnVironmental and Genetic
Medicine, Institute of Biosciences and Technology, Texas A & M Health Science Center, Texas A & M UniVersity, Houston, Texas
ReceiVed January 8, 2009
2,2,3,3-Tetramethylcyclopropanecarboxylic acid (TMCA, 4) is a cyclic analogue of the antiepileptic drug
(AED) valproic acid (VPA) (1). R-F, R-Cl, R-Br, and R-methyl derivatives of 4 and their amides were
synthesized and tested in rodent models for anticonvulsant potency and AED-induced teratogenicity. In the
anticonvulsant rat-maximal electroshock (MES) and subcutaneous metrazol (scMet) tests, R-Cl-TMCD (17)
had ED50 values of 97 and 27 mg/kg, respectively. R-F-TMCD (11) was 120 times more potent than VPA
in the rat-scMet test (ED50 ) 6 mg/kg) and had a protective index (PI ) TD50/ED50) of 20. In the 6 Hz
psychomotor mouse model 11 had ED50 values of 57 mg/kg (32 mA) and 59 mg/kg (44 mA). The ED50
values of 11 in the hippocampal-kindled rat model and in the pilocarpine-induced-status rat model were 30
and 23 mg/kg, respectively. Unlike 1, 11 was nonteratogenic in mice. This novel compound has the potential
to become a candidate for development as a new potent and safe antiepileptic and CNS drug.
Introduction
Epilepsy is a common neurological disorder characterized by
recurrent seizures that affects about 1% of the world’s popula-
tion.1 Despite the availability of >20 antiepileptic drugs (AEDsa),
there is still a substantial need for the development of more
effective and safer AEDs, since about 30% of epileptic patients
are not seizure-free with the existing AEDs.2 Furthermore, most
of the currently utilized AEDs have side effects, which are
sometimes severe and in some cases even fatal.3
Valproic acid (VPA, 1, Figure 1), an eight-carbon branched
short chain fatty acid, has a broad spectrum of anticonvulsant
activities and is highly efficient in the treatment of various types
of epilepsies. 1, one of the four most prescribed AEDs, is also
Figure 1. Structures of compounds 1-3.
used for the treatment of bipolar disorders and migraine
prophylaxis and is being currently tested as a possible anticancer
(3) (Figure 1), possessing a terminal double bond in their
drug.4-7 The clinical utilization of 1 is limited by different side
structure.15,16 These metabolites are further biotransformed in vivo
effects with the most serious of those being teratogenicity8-10
to chemically reactive intermediates that bind to cellular macro-
and life-threatening hepatotoxicity.11,12
molecules and enzymes involved in fatty acids metabolism.16-18
Since the teratogenicity of these compounds is strictly
structure dependent, numerous analogues and derivatives of 1
were investigated in an attempt to find new nonteratogenic and
The induced teratogenicity of 1 is caused primarily by the
parent compound,13,14 but its induced hepatotoxicity is caused
primarily by 1’s metabolites 4-ene-VPA (2) and 2,4-diene-VPA
nonhepatotoxic CNS-active follow-up compounds that can
become second generation to VPA.13,14,19-28 Recent studies
* To whom correspondence should be addressed. Address: Department
of Natural Products and Medicinal Chemistry, School of Pharmacy, Faculty
have revealed that further branching of 1 by the addition of a
of Medicine, The Hebrew University of Jerusalem, P.O.B 12065 Ein Karem,
methyl group in the R position to the carboxyl group or to one
Jerusalem 91120, Israel. Phone: 972-2-6758606. Fax: 972-2-6757246.
of its side chains reduced the teratogenic and embryotoxic
potency of these branched analogues but did not decrease their
anticonvulsant activity.14 The R-methylation of 1 led to a
compound with improved anticonvulsant activity in the subcu-
taneous metrazol seizure threshold test (scMet).14 The R-fluo-
rination of 1 resulted in a reduced anticonvulsant activity profile
and loss of hepatotoxicity.15,17 R-Fluoro-VPA hydroxamic acid
was found to be a nonteratogenic derivative of 1 with improved
anticonvulsant activity compared to R-fluoro-VPA (20, Figure
4).29
E-mail: yagen@cc.huji.ac.il.
†
Department of Pharmaceutics, The Hebrew University of Jerusalem.
The David R. Bloom Centre for Pharmacy, The Hebrew University of
‡
Jerusalem.
§
Texas A & M University.
Department of Natural Products and Medicinal Chemistry, The Hebrew
|
University of Jerusalem.
a Abbreviations: AED, antiepileptic drug; VPA, valproic acid; CNS,
central nervous system; SAR, structure-activity relationship; scMet,
subcutaneous metrazol; MES, maximal electroshock seizure; SE, status
epilepticus; PI, protective index; LEV, levetiracetam; TMCA, 2,2,3,3-
tetramethylcyclopropanecarboxylic acid; TMCD, 2,2,3,3-tetramethylcyclo-
propanecarboxamide; TMCU, 2,2,3,3-tetramethylcyclopropanecarbonyl urea;
VPD, valpromide; THF, tetrahydrofuran; LDA, lithiumdiisopropylamine;
NFSI, n-fluorobenzenesulfonimide; BuLi, butyllithium; NTD, neural tube
defects.
2,2,3,3-Tetramethylcyclopropanecarboxylic acid (TMCA, 4,
Figure 2) is a cyclic analogue of 1. The two quaternary carbons
in the ꢀ-position to the carboxyl group prevent its biotransfor-
10.1021/jm900017f CCC: $40.75
2009 American Chemical Society
Published on Web 03/18/2009

2234 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Pessah et al.
Figure 4. Structures of compounds 20, 21, 22, and 23.
mice strain sensitive to VPA-associated teratogenicity at doses
several times higher than their anticonvulsant effective dose
(ED50) values, none of the above-mentioned derivatives of 4
(Figure 2) appeared to share the degree of teratogenicity caused
by 1.23,33
The amides of 20 and of its analogues have improved the
anticonvulsant profile compared to the corresponding nonflu-
orinated amides29,34 The present study dealt with the syntheses
of the R-F, R-Cl, R-Br, and R-methyl derivatives of 4 and 5
and with the evaluation of their potency in two anticonvulsant
model tests.
Figure 2. Structures of compound 4 and its amide derivatives 5-9.
R-F-TMCD (11), the most potent of the synthesized novel
compounds emerging from this study, was further investigated
for quantification of its anticonvulsant activity values in the
scMet test, in the 6 Hz psychomotor seizure model, in the
hippocampal kindled rat model, and in the pilocarpine induced
status model in rats. Its teratogenic and embryotoxic risks were
determined using NMRI mice. The results are reported here.
Chemistry
Compound 4 was the starting material for the syntheses of
compounds 10-15 (Figure 3). 4 was converted to its corre-
sponding ethyl 2,2,3,3-tetramethylcyclopropanecarboxylate
(TMCE) with ethyl alcohol and sulfuric acid by a standard
esterification method.35 The hydrogen atom at the R-position
to the carbonyl in TMCE was substituted by a fluorine atom
using lithiumdiisopropylamine (LDA) as a base and N-fluo-
robenzensulfonimide (NFSI) as the substituting reagent. R-Methyl-
TMCE was synthesized using LDA and methyl iodide. Both
reactions were performed in dry tetrahydrofuran (THF) under
nitrogen at -8 °C (Scheme 1).
The fluorinated or methylated esters were hydrolyzed by
potassium hydroxide (0.045 mol in water/ethanol (1:1)). The
ethanol was evaporated, the aqueous mixture was acidified to
pH 1 using 1 N HCl, and the corresponding acids were extracted
with ethyl acetate. Purification of the acids was performed by
extraction of the ethyl acetate solution three times with a
saturated solution of sodium bicarbonate, combining the extracts
and acidification of the basic aqueous solution to pH 1 using 1
N HCl, followed by the extraction of the acidic solution with
dichloromethane, yielding the corresponding acids 10 or 14.
2,3-Dimethyl-2-butene was the starting material for com-
pounds 16-19 (Figure 3). This olefin was reacted at 4 °C with
chloroform or bromoform in the presence of potassium tert-
butoxide in tert-butanol (via a dihalo carbene), yielding 1,1-
dichloro or 1,1-dibromo-2,2,3,3-tetramethylcyclopropane cor-
respondingly. The dihalo cyclopropanes were then reacted with
butyllithium (BuLi) and carbon dioxide at -78 °C, yielding the
desired R-chloro or R-bromo-2,2,3,3-tertramethylcyclopropane
carboxylic acid 16 or 18 (Scheme 2).
Figure 3. Structures of R substituted (by F, methyl group, Cl, or Br)
derivatives of 4 (10, 14, 16, 18) and their amides (11-13, 15, 17, and
19).
mation to hepatotoxic metabolite(s) with a terminal double bond.
4 is inactive at the rat anticonvulsant maximal electroshock MES
(ED50 > 150 mg/kg) model.30 In contrast the following amide
derivatives of 4, 2,2,3,3-tetramethylcyclopropanecarboxamide
(TMCD, 5),24,31 N-methyl-TMCD (MTMCD, 6),23,24,31 N-
methoxy-TMCD (N-OCH3-TMCD, 7),28 TMC urea28 (TMCU,
8), and N-(2,2,3,3,-tetramethylcyclopropanecarboxamide)-p-
phenylsulfonamide (9)32 (Figure 2), had potent anticonvulsant
activity (ED50 values ranging between 10 and 90 mg/kg) in the
rat-MES or scMet tests models. Even when administered to a

Carboxamide AnticonVulsant
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2235
Scheme 1. Synthesis of Compounds 10 and 14 and Their Amide Derivatives 11-13 and 15a
a Reagents and conditions: (i) EtOH, H2SO4, reflux, 48 h. (ii) In the preparation of R-F-TMC ethyl ester (X ) F): LDA, -8 °C, 40 min, NFSI, -8°C,
30 min. In the preparation of R-methyl-TMC ethyl ester (X ) CH3): LDA, -8°C, 40 min, CH3I, -8°C, 30 min. (iii) 0.5 M KOH in H2O/EtOH. (iv) SOCl2,
CH2Cl2, 0°C, 10 h. (v) In preparation of amides 11 and 15 (R ) NH2): 25% NH4OH, 0 °C. In preparation of amide 12 (R ) NHCH3): methylamine, 0 °C.
In preparation of amide 13 (R ) NHCONH2): NH2CONH2 in acetonitrile, 80 °C.
Scheme 2. Synthesis of Compounds 16 and 18 and Their Amide Derivatives 17 and 19a
a Reagents and conditions. In the preparation of 16 (X ) Cl): (i) CHCl3, tBuO-K+, tBuOH, 0 °C, 10 h, room temp; (ii) BuLi, -78 °C, 40 min, CO2, 2 h,
t
t
-78°C. In the preparation of 18 (X ) Br): (i) CHBr3, BuO-K+, BuOH, 0 °C, 10 h, room temp; (ii) BuLi, -78 °C, 40 min, CO2, 2 h, -78°C. In the
preparation of 17 (X ) Cl): (iii) SOCl2, CH2Cl2, 0°C, 10 h; (iv) 25% NH4OH, 0 °C. In the preparation of 19 (X ) Br): (iii) SOCl2, CH2Cl2, 0°C, 10 h; (iv)
25% NH4OH, 0 °C.
Compounds 10, 14, 16, and 18 were converted by SOCl2 to
the corresponding acyl chlorides and then coupled with the
suitable amine in dry acetonitrile or dry dichloromethane to yield
compounds 11, 12, 13, 15, 17, and 19. The synthesized products
were purified by crystallization. 1H NMR spectra of the
synthesized compounds were measured in CDCl3 using TMS
as the internal standard. Elemental analyses were performed for
all the synthesized compounds.
MES and scMet tests, both in mice and in rats.23,24,28,31-33 In
mice, the protective index (PI) values of these compounds were
of the same magnitude as that of 1. However, in the rat, the PI
values improved significantly and were much wider than those
of 1, especially for compound 8.28 All the CNS-active amide
derivatives of 4 reported in the literature to date represented
substitution of the hydroxyl group in the carboxyl moiety of 4
by different amine or amide groups, without modification of
the tetramethylcyclopropane moiety.23,24,28,31-33,37
Results and Discussion
The present study explored the anticonvulsant activity of
TMCA derivatives that are halogenated or methylated at the
R-position to the carboxyl or carboxamide moiety (compounds
10-19, Figure 3).
The positive preliminary results obtained for 11 in the rat
scMet anticonvulsant model led us to synthesize additional
TMCA derivatives (12-19, Figure 3) and explore SAR of these
compounds.
By utilizing the modified procedure for the synthesis of
R-fluoro-VPA (20, Figure 4),17 we were able to obtain R-fluo-
rotetramethylcyclopropanecarboxylic acid (R-F-TMCA, 10) in
high yields. Chlorination and bromination of 4 resulted in very
low yields of the halogenated products R-Cl-TMCA (16) and
R-Br-TMCA (18) using common literature procedures, such as
the Hell-Volhard-Zelinski reaction frequently used for R-h-
alogenations of carboxylic acids.38,39 Therefore, syntheses of
18 was performed by a two-step reaction as described in Scheme
In spite of a large number of existing AEDs, about 30% of
epileptic patients are still not seizure-free.1-3 Because of
multiple mechanisms of action, discovery of new potent an-
alogues and derivatives of 1 is based on a structure-activity
relationship (SAR) approach utilizing structural modification of
the molecule (1) and comparative evaluation of the potency and
safety margin of the new synthesized compunds in established
anticonvulsant animal models. Compound 4, a cyclic analogue
of 1, does not possess anticonvulsant activity in the MES test
model.30 Recently a number of amide derivatives of 4 were
synthesized by our research group23,24,28,31-33 and their anti-
convulsant potency was evaluated in the MES and in the scMet
tests in mice and in rats. These models predict the efficacy of
a drug to treat human generalized tonic-clonic seizures (MES)
and myoclonic seizures (scMet).36 Compounds 5-9 (Figure 2)
showed improved anticonvulsant activities relative to 1 in the

2236 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Pessah et al.
Table 1. Anticonvulsant Activity and Toxicity of Compounds 10-19 Administered Intraperitoneally to Mice
a Maximal electroshock test (number of animals protected/number of animals tested). b Subcutaneous metrazol test (number of animals protected/number
of animals tested). c Neurotoxicity (number of animals exhibiting neurotoxicity/number of animals tested). d Time after drug administration.
2.40,41 In the first step a 1,1-dibromo-2,2,3,3-tetramethylcyclo-
propane was obtained by reaction of 2,3-dimethyl-2-butene with
a dibromocarbene, produced by the reaction of bromoform with
potassium tert-butoxide.40 In the second step, a metallodeha-
logenation reaction was carried out using butyllithium (BuLi)
followed by passing carbon dioxide gas through the reaction
mixture to yield the desired compound 18. We utilized the
above-described procedure for the synthesis of 16 by the reaction
of dichloro carbene with 2,3-dimethyl-2-butene, followed by
metallodehalogenation and carboxylation reaction as described
above to yield compound 16.
The remaining compounds presented in Table 1 were inactive
in the MES and scMet models tests.
With the exception of 17, none of the tested compounds
presented in Table 2 showed anticonvulsant activity in the rat
MES test. Compounds 18 and 19 were not tested in this model
because of a very low activity obtained by their screening in
mice (Table 1). In the scMet model test in rats (Table 3)
compound 11 exhibited excellent activity with no toxicity at
50 mg/kg. Compounds 10 and 15 showed marginal anticon-
vulsant activity, and 17 showed partial anticonvulsant activity
at this dose. Compound 13 showed partial anticonvulsant activity
at 30 mg/kg but was toxic and caused myoclonic jerks in some
of the tested animals and therefore was not further investigated.
In general, the R-halogenated and R-methyl free acids 10, 14,
16, and 18 were inactive in the anticonvulsant scMet and MES
tests (Tables 1-3). R-F-MTMCD (12) and R-F-TMCU (13)
The anticonvulsant activity and toxicity profiles of the
synthesized R-halogenated TMCA and their amide derivatives
(compounds 10-19, Figure 3) are presented in Tables 1-6.
Marginal activity for compounds 11, 12, and 17 was obtained
in the scMet test in mice using a 100 mg/kg dose (Table 1).

Carboxamide AnticonVulsant
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2237
Table 2. Anticonvulsant [Anti-MES] Activity and Toxicity of Compounds 10-17 Administered Orally to Ratsa
a Symbols are as follows: ++++, 100% of the animals were protected; +++, 75% of the animals were protected; ++, 50% of the animals were
protected; +, 25% of the animals were protected; -, no protection. In the case of toxicity: ++++, 100% of the animals exhibited neurotoxicity; +++, 75%
of the animals exhibited neurotoxicity; ++, 50% of the animals exhibited neurotoxicity; +, 25% of the animals exhibited neurotoxicity; -, no neurotoxicity.
b Neurotoxicity. c Not tested.
possess significantly reduced anticonvulsant activity in the MES
and scMet models in mice (Table 1), compared to their parent
compounds 6 and 8.23,24,28,31 Compound 12 was inactive at 50
mg/kg in the scMet model test in rats (Table 3). Compound 13
was slightly active in scMet (Table 3) and inactive in the MES
test (Table 2). We assume that 12 and 13, the fluorinated
derivatives of 6 and 8, activate one or more of their presumed
targets with lower affinity than their nonfluorinated counter-
partners.
R-Cl-TMCD (17) was active in the MES and scMet tests in
rats at 50 mg/kg and showed no toxicity at this dose (Tables 2
and 3). The insertion of a chlorine atom (compound 17) instead
of the fluorine atom (compound 11) at the R-position to the
carboxamide 5 has a critical influence on the anticonvulsant
activity in the rat MES test. Compound 19 was inactive in the
MES and scMet tests in mice and was toxic at 450 mg/kg (Table
1). Furthermore, compound 15 showed reduced anticonvulsant
activities compared to 5 (Tables 1 and 2), although R-methyl-
VPA (21, Figure 4) was found to be more potent relative to
1.14 Compound 11 was more potent than 5 in the scMet model
test both in mice and in rats (Tables 4 and 6). In a similar
manner, R-fluoro-VPD (22, Figure 4) was more potent in the
scMet test in mice than VPD (23),29,34 exhibiting the same
positive effect on anticonvulsant potency of an R-fluoro sub-
stituent in this compound. However, compound 20 (Figure 4)
was less potent than 1 in the scMet anticonvulsant model test
in mice.17 At a dose of 50 mg/kg, 14 was inactive in the MES
and in the scMet model tests in rats (Tables 2 and 3), and
compound 15 was also inactive at the same dose (Tables 2 and
3) compared to 5 (Table 6).
The excellent potency of 11 in the rat scMet test (Table 2)
led us to further investigation and quantification of its pharma-
cological properties in the following additional animal models
for anticonvulsant activity: the 6 Hz psychomotor seizure model
(Table 5), the rat-kindling model, and the pilocarpine-induced
status model which is used to discover active compounds for
the treatment of status epilepticus (SE).42 SE is one of the most
severe epileptic seizure conditions in which seizure duration
lasts for 30 min or more and consciousness is not always
regained.43,44
In the mice scMet model 11 was ∼7 times more potent than
1 and it was also more potent than compounds 23 and 5 (Table
4). In the 6 Hz model tests in mice at the currents of 32 and 44
mA, 11 was also more potent than 1, 23, and 5 and had relatively
high PI (PI ) TD50/ED50) values (Table 5). The high potency
of the new AED levetiracetam (LEV) in the 6 Hz psychomotor

2238 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Pessah et al.
Table 3. Anticonvulsant [Anti-scMet] Activity and Toxicity of Compounds 10-17 Administered Orally to Ratsa
a Symbols are as follows: ++++, 100% of the animals were protected; +++, 75% of the animals were protected; ++, 50% of the animals were
protected; +, 25% of the animals were protected; -, no protection. In the case of toxicity: ++++, 100% of the animals exhibited neurotoxicity; +++, 75%
of the animals exhibited neurotoxicity; ++, 50% of the animals exhibited neurotoxicity; +, 25% of the animals exhibited neurotoxicity; -, no neurotoxicity.
b Neurotoxicity.
Table 4. Quantitative Anticonvulsant Data (Anti-MES and Anti-scMet)
in Mice Dosed Intraperitoneally with Compound 11 Compared to 1, 5,
22, and 23
Table 5. Quantitative Anticonvulsant Data in the 6 Hz Psychomotor
Test in Mice Dosed Intraperitoneally with Compound 11 Compared to
1, 5, and 23
g
g
e,g
MESa ED50
(mg/kg)
scMetc ED50
(mg/kg)
TD50
(mg/kg)
6 Hz test at 32 mA,
ED50 (mg/kg)
6 Hz test at 44 mA,
ED50 (mg/kg)
d
d
compd
PIb
PId
compd
PIa
PIb
1f
263 (237-282) 1.5 220 (177-268) 1.8 398 (356-445)
1c
126 (95-152)
57 (47-65)
57f (32-85)
72 (46-97)
3.2
1.4
2.1
1.4
310 (258-335)
66 (29-87)
59g (42-75)
>200
1.3
1.2
2
23h
22i
11
5m
56 (51-64)
NT
1.4 55 (45-63)
NT 23
1.5 81 (74-91)
3.8 0.46
23e
11
5h
>200j
>120
38k (26-54)
57 (39-76)
3.2 120l (108-135)
1.7 99 (85-109)
a Protective index (TD50/ED50 ratio) in the 32 mA current 6 Hz test.
b Protective index (TD50/ED50 ratio) in the 44 mA current 6 Hz test. c Data
taken from ref 36. d The interval in parentheses stands for 95% confidence
interval. e Data taken from ref 53. f The ratio of protected-to-tested rats
was 1/8 (25 mg/kg), 4/8 (70 mg/kg), and 8/8 (130 mg/kg). g The ratio of
protected-to-tested rats was 0/8 (25 mg/kg), 3/8 (50 mg/kg), 6/8 (75 mg/
kg), and 7/8 (100 mg/kg). h Data taken from ref 23.
a Maximal electroshock test. b Protective index (PI ) TD50/ED50) in the
MES test. c Subcutaneous metrazol test. d Protective index (PI ) TD50/
ED50) in the scMet test. e Neurotoxicity. f Data taken from ref 36. g The
interval in parentheses stands for the 95% confidence interval. h Data taken
from ref 53. i Data taken from refs 29 and 34. j The ratio of protected-to-
tested mice was 0/8 (60 and 150 mg/kg), 1/4 (200 mg/kg), 4/4 (400 mg/kg).
k The ratio of protected-to-tested mice was 1/8 (25 mg/kg) and 8/8 (50 and
100 mg/kg). l The ratio of neurotoxic-to-tested mice was 0/8 (60 mg/kg),
1/8 (100 mg/kg), 3/8 (120 mg/kg), and 8/8 (150 mg/kg). m Data taken from
ref 23.
32mA current (ED50 ) 57 mg/kg) and at the 44 mA current
(ED50 ) 59 mg/kg) with similar potency (ED50) (Table 5).
Compound 11’s potency at 44 mA is by far better than that of
LEV.36 The unique pharmacological profile of this compound
in the 6 Hz seizure test suggests it as a potential candidate for
treatment of partial seizures and secondary generalized seizures
in refractory epileptic patients, in analogy to LEV.36,46
The ED50 of 11 in the rat scMet test was 6 mg/kg (Table 6),
being 120 and 10 times more potent than 1 and 23, respectively.
Compound 11 possesses an excellent safety margin with a PI
seizure test and its lack of anticonvulsant activity in the MES
and scMet tests certify that LEV has anticonvulsant activity in
seizures with different neuronal mechanisms than those induced
by the MES and scMet models.36 In the 6 Hz seizure test at 32
mA, LEV ED50 ) 19 mg/kg, and at 44 mA its potency decreases
significantly (ED50 ) 1089 mg/kg).36,45 We have shown that
11 is efficacious in the 6 Hz psychomotor model both at the

Carboxamide AnticonVulsant
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2239
Table 6. Quantitative Anticonvulsant Data in Rats Dosed Orally with
Compounds 11 and 17 Compared to Compounds 1, 5, 6, 8, and 23
respectively. Therefore, a safety margin of at least 6 times is
still maintained after administration of 11 at the 336 mg/kg dose.
g
g
e,g
MESa ED50
(mg/kg)
scMetc ED50
(mg/kg)
TD50c
compd
PIb
PId
(mg/kg)
Conclusions
1f
485 (324-677) 1.6
646 (466-869) 1.2
784 (503-1176)
87 (68-107)
381 (355-418)
163 (138-179)
538 (437-664)
117m (71-170)
23h
5i
32 (22-42)
2.7
59 (44-47)
52 (42-163)
45 (31-55)
1.5
7.3
3.6
5.9
20
The engine that has driven AED discovery is screening in
animal models. Although there is a large number of models,
the MES and scMet remain the “gold standard” in early stages
of testing, since compounds active in the MES and scMet tests
have generally been efficacious in clinical trials.50,51 Among
the corresponding R-halo-TMCD derivatives emerging from this
study compound 11 was the most active compound. Its
anticonvulsant potency, 120 times greater in the rat scMet test
(Table 6) than that of the most widely used AED 1, and its
high potency in the 6 Hz psychomotor seizure test in mice, in
the hippocampal-kindled rat model and in the pilocarpine
induced status model in rats make it a good candidate for
development as a new CNS drug. Compound 17 was found to
be potent in the rat scMet test (ED50 ) 27 mg/kg) and, unlike
11, showed anticonvulsant activity also in the rat MES test (ED50
) 97 mg/kg, Table 6) and is also 4 times more potent than 1 in
the rat MES test.36 None of the R-halo- and R-methyltetram-
ethylcyclopropanecarboxylic acids were found to be active in
the MES and scMet tests both in mice and in rats (Tables 1-3).
In addition, compounds 12, 13, 15, and 19 exhibited no
anticonvulsant activity in the mice MES and scMet tests
(Table 1).
Compound 11, a potent CNS-active derivative of 4, possesses
two quaternary carbons in the ꢀ-position to the carboxamide
group and therefore cannot be converted to metabolites with a
terminal double bond, analogues to hepatotoxic compound 2.12,15
It is also not teratogenic and is a much more potent compound
possessing a much wider safety margin than 1.52 Since existing
AEDs fail to control seizures in about 30% of epileptic patients
and since all frontline AEDs exhibit teratogenic effects in
humans, 11 could be a promising candidate for further develop-
ment as a new, potent, and safe antiepileptic and CNS drug,
taking into account its high potency also in the 6 Hz psycho-
motor seizure test, the hippocampal-kindled rat model, and the
pilocarpine-induced status model in rats.
>250
6i
82 (64-102)
29 (18-47)
>100k
2.0
8j
18.5 92 (50-151)
11
17
6.4l (3.5-9.5)
97n (43-244)
>2.1 27o (16-39)
>7.8 >200p
a Maximal electroshock test. b Protective index (TD50/ED50 ratio) in the
MES test. c Subcutaneous metrazol test. d Protective index (TD50/ED50 ratio)
in the scMet test. e Neurotoxicity. f Data from ref 36. g The interval in
parentheses stands for 95% confidence interval. h Data from ref 25. i Data
from ref 23. j Data from ref 28. k The ratio of protected-to-tested rats was
0/8 (100 mg/kg). l The ratio of protected-to-tested rats was 0/8 (2 mg/kg),
5/8 (6 mg/kg), 6/8 (12.5 mg/kg), 8/8 (25 mg/kg). m The ratio of neurotoxic-
to-tested rats was 1/8 (50 mg/kg), 3/8 (100 mg/kg), 6/8 (175 mg/kg), and
7/8 (250 mg/kg). n The ratio of protected-to-tested rats was 1/8 (25 mg/
kg), 2/8 (50 mg/kg), 6/8 (100 mg/kg), 6/8 (150 mg/kg), and 4/8 (200 mg/kg).
o The ratio of protected-to-tested rats was 1/8 (10 mg/kg), 3/8 (25 mg/kg),
4/7 (40 mg/kg), 8/8 (50 mg/kg). p The ratio of neurotoxic-to-tested rats was
0/8 (200 mg/kg).
value of 20 that is much wider than those of 1 (PI ) 1.2) and
23 (PI ) 1.5). Compound 11 was further tested in the
hippocampal kindled rat model and was active with an ED50 of
30 mg/kg. It was also active in the rat pilocarpine-induced status
model.47 The main feature of the model consists of a large
number of spontaneous recurrent seizures of both acute induced
SE and chronic spontaneous seizures. At the time of SE
induction by pilocarpine injection, 11 significantly reduced the
development of seizures in the tested rats (status at time 0 test)
and exhibited an ED50 of 23 mg/kg. It also showed protection
against pilocarpine-induced SE 30 min after status induction
(after first stage III seizure54) and exhibited an ED50 of 80 mg/
kg.
The ability of 11 to prevent and to reduce seizure threshold
in several types of seizures in a broad range of animal models
of epilepsy suggests that this compound may have a multiple
mechanism of action and, like 1, it may exhibit a broad spectrum
of action for epilepsy treatment.48 The relatively good potency
of this compound in the 6 Hz model at both 32 and 44 mA
currents (Table 5) and in the rat kindling model may also imply
that in analogy to LEV it can be useful for the treatment of
therapy-resistant epileptic patients.
Experimental Section
Chemicals. All reagents were purchased from Sigma-Aldrich.
The NFSI was purchased from Fluorochem U.K. Compounds
10-19 were prepared according to the methods described further
in this section.
The teratogenicity and embryotoxicity of 11 was tested in
the SWV mouse model at three different doses, and the results
were compared to those obtained for 1 at similar doses (Table
7). The teratogenicity of 1 is one of its major side effects limiting
its use in women of childbearing age.10,49 Compound 1 induces
high percentages of severe malformations in animal models8,10,13,49
and in humans.8,10,49 Evaluation of the teratogenic potential of
derivatives and analogues of compound 1 is highly important
for their development as new improved AEDs. Unlike 1,
compound 11 caused no malformations and was found to be
nonteratogenic in the three doses administered: 671, 502, and
336 mg/kg. This compound, however, was toxic at 671 and 502
mg/kg both to the pregnant dams and to the embryos (Table
7). At the lower dose, 336 mg/kg, however, no toxic effect was
observed. The doses of 11 presented in Table 7 are similar to
the doses used for establishing the teratogenic effect of 1.23 It
is important to emphasize that in the case of compound 11 the
three tested doses are 18, 14, and 9 times higher than its ED50
values in the mice scMet test (Table 4) and 12, 9, and 6 times
higher than its ED50 values in the 6 Hz model (Table 5),
Materials and Methods. Product formation follow-up was
performed by means of GC/MS and TLC techniques. TLC analyses
were performed on precoated silica gel on aluminum sheets
(Kieselgel 60 F254, Merck). Gas chromatography-mass spectros-
copy assays were performed on an HP5890 series II GC instrument
equipped with a Hewlett-Packard MS engine (HP5989A) single
quadrupole MS spectrometer, HP7673 autosampler, HP MS-DOS
Chemstation, and HP-5MS capillary column (0.25 µm × 15m ×
0.25 mm). The temperature program was as follows: injector
temperature, 180 °C; initial temperature, 40 °C for 6 min; gradient
of 20 °C/min until 140 °C; gradient of 10 °C until 200 °C; hold
time, 3 min. The MS parameters were set as follows: source
temperature, 180 °C (140 °C for compounds 18 and 19); transfer
line, 280 °C; positive ion monitoring, EI-MS (70 eV).
The chemical structure and purity of the synthesized compounds
were assessed by GC/MS, NMR, and combustion elemental
analysis. Melting points were determined on a Buchi 530 capillary
1
melting point apparatus and are uncorrected. H NMR spectra, in
CDCl3 using TMS as internal standard, were recorded on a Varian
Mercury series NMR 300 spectrometer. Chemical shifts (δ scale)
are reported in parts per million (ppm) relative to TMS. Coupling

2240 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Pessah et al.
Table 7. Teratogenic Effect of 11 Compared to 1 in the SWV Mouse Modela
dose, mg/kg
(mmol/kg)
no. of
resorptions (%)
no. of live
fetuses (%)
no. of dead
fetuses (%)
no. of fetuses with
NTDb (%)
compd
no. of litters
no. of implants
control
0
11
13
13
12
4
128
131
160
154
55
0
128
0
0
1c
1
600 (3.6)
452 (2.7)
301 (1.8)
671 (4.2)
502 (3.2)
336 (2.1)
10 (7.6)
18 (11.3)
17 (11.0)
43 (78.2)d,e
34 (26.8)d,e
11 (9.8)
107 (81.7)
141 (88.1)
133 (86.4)
12 (21.8)
93 (73.2)
101 (90.2)
14 (10.7)d
57 (53.3)
41 (29.1)
1 (0.6)
1
4 (2.6)d
2 (1.5)
11
11
11
0d
0
0d
0d
0
10
8
127
112
0
a All dams received the drugs intraperitoneally on the morning of day 8 of gestation. b Neural tube defects. c Compound 1 was administered in the form
of its sodium salt. d Significantly different when compared to the control group: p < 0.05, Fishers exact test. e Significantly different when compared to group
treated with similar dose of 1: p < 0.05, Fishers exact test.
constants (J) are given in hertz (Hz). Elemental analyses were
performed on a 2400-2 Perkin-Elmer C, H, N, F, Br, Cl analyzer.
Analyses of all newly synthesized compounds had satisfactory
results indicating g98% purity.
General Procedure for the Synthesis of Compounds 10
and 14. Compound 4 was refluxed for 48 h with ethyl alcohol
in a ratio of 1:15 in the presence of a catalytic amount of sulfuric
acid. The excess of alcohol was removed under reduced pressure
and the residue dissolved in 100 mL of hexane, washed with
saturated sodium bicarbonate solution following by brine, dried over
magnesium sulfate (MgSO4), and filtered, and the hexane was
evaporated. The ester, a colorless liquid, was further purified by
vacuum distillation.
A solution of lithium diisopropylamine (LDA, 0.032 mol) was
prepared by dissolving diisopropylamine in THF distilled over
calcium hydride. The reaction mixture kept under nitrogen was
cooled to -15 °C, and BuLi (1.6 M in hexanes, 0.032 mol) was
added slowly while stirring and maintaining the temperature at -15
°C. The resulting mixture was stirred for additional 10 min, allowed
to warm up to 0 °C, following by stirring for an additional 10 min.
The reaction mixture cooled to -15 °C, TMCA ethyl ester (0.03
mol) dissolved in 5 mL of dry THF was added dropwise, and the
mixture was stirred for 40 min at -15 °C.
The temperature was elevated to -8 °C, and an amount of 1.5
equiv of the R-substituiting reagent (NFSI for the fluorination and
methyl iodide for the methylation) dissolved in dry THF was added
to the reaction mixture. The reaction mixture was stirred for an
additional 30 min while the formation of R-fluoro or R-methyl
TMCA ethyl esters was monitored by GC-MS. After the reaction
was completed the organic solvent was removed under reduced
pressure and the oily residue dispersed in ethyl acetate and filtered
by using a Buchner funnel and a vacuum pump. The ethyl acetate
solution was washed with water, 1 N HCl, and brine, dried over
MgSO4, and filtered. The solvent was evaporated to yield R-fluoro
or R-methyl TMCA ethyl ester as yellow oils.
The obtained ethyl esters were hydrolyzed to the corresponding
acids using potassium hydroxide (0.045 mol) in a water/ethanol
mixture (1:1, v/v), while monitoring the hydrolysis progress by
GC-MS. Once the reaction was completed, the ethanol was
evaporated and the remaining aqueous solution washed with hexane.
The aqueous solution was cooled and slowly acidified to pH 1 using
1 N HCl and thereafter extracted three times with ethyl acetate.
The organic fractions were combined and extracted three times with
5% sodium bicarbonate solution. The combined sodium bicarbonate
extracts were acidified to pH 1 and extracted three times with ethyl
acetate. The organic fraction was dried over MgSO4 and filtered
and the solvent was evaporated under vacuum to yield R-substituted
TMCA typically as a white powder. Compounds 10 and 14 were
purified by crystallization from ethyl acetate.
General Procedure for the Synthesis of Compounds 16
and 18. Chloroform or bromoform (0.053 mol) in the presence of
potassium tert-butoxide solution in tert-butanol (0.064 mol) was
reacted with 2,3-dimethyl-2-butene (0.053 mol) at 0 °C. The
reaction mixture was allowed to warm up and stirred overnight at
room temperature. The solvent was evaporated under reduced
pressure and the residue dissolved in petroleum ether, washed three
times with water and brine, dried over MgSO4, and filtered. The
solvent was evaporated under reduced pressure to yield the 1,1-
dihalo-2,2,3,3-tetramethylcyclopropane as a white solid.
Under nitrogen atmosphere, 1,1-dihalo-2,2,3,3-tetramethylcy-
clopropane (0.02 mol) was dissolved in 30 mL of THF (freshly
distilled over lithium aluminum hydride) and cooled to -78 °C.
BuLi (1.6 M in hexane, 0.025 mol) diluted by dry THF (1:1, v:v)
was slowly added dropwise and the reaction mixture stirred for 30
min at -78 °C following by passing carbon dioxide gas for 2 h
through the reaction mixture. The reaction mixture was allowed to
warm up to room temperature. The organic solvent was evaporated
under reduced pressure and the residue dissolved in petroleum ether
and extracted three times with 5% sodium bicarbonate in water.
The water extracts were combined, cooled in an ice bath, acidified
by 1 N HCl to pH 1, and extracted three times with dichlo-
romethane. The organic extracts were combined, dried over MgSO4,
and filtered and the solvent was evaporated to yield 16 or 18 as
white solids. They were further purified by crystallization from ethyl
acetate.
General Procedure for the Synthesis of Compounds 11,
12, 15, 17, and 19. Thionyl chloride was added dropwise to the
R-substituted TMC acids (10, 14, 16, 18) dissolved in dry
dichloromethane (DCM) and kept at 0 °C. The reaction mixture
was stirred for 10 h, and the solvent and excess of thionyl chloride
were removed by distillation. The appropriate acyl chlorides were
used without further purification. The corresponding acyl chlorides
(0.01 mol), dissolved in dry dichloromehane (4 mL), were slowly
added at 0 °C to a stirred solution of 25% NH4OH in water (10-20
mL) and dichloromethane (8 mL) or to a solution of methylamine
(10 mL, 2 M in THF). The mixtures were kept at 0 °C for 60 min
and then extracted three times with 25 mL of ethyl acetate. The
extracts were combined and washed with 25 mL of water, 25 mL
of HCl (1 N), and 25 mL of brine. The organic layer was dried
over MgSO4, filtered, and evaporated. The obtained amides were
purified by crystallization from ethyl acetate.
1-Fluoro-2,2,3,3-tetramethylcyclopropanelcarboxylic Acid (10).
Yield 54%; white powder; mp 121-123 °C; MS-EI, m/z (%) 160
(M+, 0.46), 145 (69), 115 (100), 73 (38), 61 (70); Rf ) 0.24 (DCM/
MeOH, 97:3); 1H NMR (300 MHz, CDCl3 δ TMS) 1.20-1.26 (dd,
J ) 2.1, 15.5 Hz, 18H); Anal. (C8H13FO2) C, H, F.
1-Fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide (11).
Yield 76%; white needles; mp 99 °C; MS-EI, m/z (%) 159 (M+,
0.5), 144 (100), 124 (20), 81 (29), 61 (26); Rf) 0.3 (DCM/MeOH,
97:3); 1H NMR (300 MHz, CDCl3 δ TMS) 1.27-1.16 (bd, J ) 33
Hz, 18H), 5.43 (1H) 6.3 (1H). Anal. (C8H14FNO) C, H, N, F.
N-Methyl-1-fluoro-2,2,3,3-tetramethylcyclopropanecarbox-
amide (12). Yield 94%; yellow oil; MS-EI, m/z (%) 173 (M+, 0.01)
158 (100), 138 (24), 81 (40), 58 (69); Rf ) 0.15 (DCM/MeOH,
1
98:2); H NMR (300 MHz, CDCl3 δ TMS) 1.13-1.32 (ddd, J )
42 Hz, 11, 6, 18H), 2.8 (d, J ) 9 Hz, 3H) 6.43 (s, 1H). Anal.
(C9H16FNO) C, H, N, F.
Synthesis of 1-Fluoro-2,2,3,3-tetramethylcyclopropanecarbo-
nyl Urea (13). Urea (2.7 g, 0.045 mol), in dry acetonitrile (40 mL),
was refluxed for 1 h, followed by dropwise addition of R-fluoro-
TMC-acyl chloride (3.2 g, 0.018 mol). The reaction mixture was
stirred under reflux for additional 2 h and cooled to room
temperature, and the solvent was evaporated under reduced pressure.
Chloroform (40 mL) was added to the remaining oily residue, and

Carboxamide AnticonVulsant
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2241
the solids were filtered by vacuum, dissolved in ethyl acetate, and
washed with water. The organic extracts were dried over magnesium
sulfate, filtered, and evaporated to yield the pure R-F-TMCU. Yield
62%; white crystals; mp 234 °C; MS-EI, m/z (%) 202 (M+, 4),
187 (46), 144 (100), 81 (51), 61 (56); Rf ) 0.5 (DCM/MeOH, 96:
4); 1H NMR (300 MHz, CDCl3 δ TMS) 1.206-1.271 (d, J ) 19.5
Hz, 18H), 5.179 (1H), 8.068-8.293 (bd, J ) 67.5 Hz, 2H). Anal.
(C9H15FN2O2) C, H, N, F.
To determine if the test substance can prevent acute pilocarpine-
induced status, the compound was given ip to male albino
Sprague-Dawley rats (150-180 g). Then a challenge dose of
pilocarpine was administered and the treatment effects of the
candidate drug were observed. The outcome measures were
“protection” or “no protection”. The seizure severity was determined
by using the established Racine scale.54 Compounds found to
possess significant protection at time zero (time from the first stage
III seizure) were proceeded to further evaluation in a sustained
seizure model where the drug candidate was given 30 min after
pilocarpine status induction (or after first stage III seizure). To
calculate 11’s ED50 at time 0, eight rats per dose were utilized at
the following doses: 12, 25, 50, and 100 mg/kg. To calculate 11’s
ED50 at 30 min, 50, 75, and 100 mg/kg doses were utilized and the
number of rats per dose was 7, 6, and 7, respectively.
Evaluation of Teratogenicity. The teratogenicity of the com-
pounds was evaluated in the highly inbred SWV mice strain highly
susceptible to VPA-induced neural tube defects (NTDs) according
to a published procedure.23 On day 8.5 of gestation, each dam
received a single ip injection of the tested compounds in a range
of 1.8-4.2 mmol/kg or the control (25% water solution of
Cremophor EL, Fluka Biochemica, Germany). On day 18.5 of
gestation, the dams were sacrificed by carbon dioxide asphyxiation,
the location of all viable fetuses and resorption sites were recorded,
and the fetuses were examined for the presence of exencephaly or
other gross congenital abnormalities.
1-Methyl-2,2,3,3-tetramethylcyclopropanelcarboxylic Acid (r-
Methyl-TMCA, 14). Yield 57%; white crystals; mp 86 °C; MS-
EI, m/z (%) 156 (M+, 0.3), 141 (100), 123 (30), 95 (55), 69 (23);
1
Rf ) 0.33 (DCM/MeOH, 98:2); H NMR (300 MHz, CDCl3 δ
TMS) 1.05 (S, 6H), 1.2 (S, 6H), 1.27 (3H). Anal. (C9H16O2) C, H.
1-Methyl-2,2,3,3 tetramethylcyclopropanecarboxamide (r-
Methyl-TMCD, 15). Yield 30%; white crystals; mp 111-112 °C;
MS-EI, m/z (%) 156 (M+, 0.4), 141 (100), 123 (28), 95 (53). 69
(26); Rf ) 0.29 (DCM/MeOH, 98:2); 1H NMR (300 MHz, CDCl3
δ TMS) 1.02 (6H), 1.15 (6H) 1.3 (3H) 5.28 (bd, J ) 30 Hz, 2H).
Anal. (C9H17NO) C, H, N.
1-Chloro-2,2,3,3-tetramethylcyclopropanelcarboxylic Acid (16).
Yield 28%; white crystals; mp 128 °C; MS-EI, m/z (%) 176 (M+,
1.1), 161 (100), 83 (85), 67 (33), 59 (70); Rf ) 0.27 (DCM/MeOH,
1
97:3); H NMR (300 MHz, CDCl3 δ TMS) 1.24-1.28 (d, J ) 11
Hz, 12H). Anal. (C8H13ClO2) C, H, Cl.
1-Chloro-2,2,3,3-tetramethylcyclopropanecarboxamide (17).
Yield 86%; white crystals; mp 128 °C; MS-EI, m/z (%) 159 (M -
15, 100), 143 (28), 124 (23), 81 (24), 58 (54); Rf ) 0.67 (DCM/
1
Acknowledgment. This work is abstracted from the Ph.D.
thesis of Neta Pessah in partial fulfillment of the Ph.D. degree
requirements for The Hebrew University of Jerusalem. The
authors thank James P. Stables, Director of the NIH-NINDS-
Anticonvulsant Screening Program (ASP), for the testing of the
compounds in the ASP.
MeOH, 97:3); H NMR (300 MHz, CDCl3 δ TMS) 1.22-1.3 (d,
J ) 25 Hz, 12H), 5.43 (1H) 6.23 (1H). Anal. (C8H14ClNO) C, H,
N, Cl.
1-Bromo-2,2,3,3-tetramethylcyclopropanecarboxylic Acid (18).
Yield 33%; white crystals; mp 161-162 °C; MS-EI, m/z (%) 205,
207 (M - 15, 19,18), 123, 125 (30,34), 83 (100), 67 (40), 59 (59);
1
Rf ) 0.19 (PE/ether, 85:15); H NMR (300 MHz, CDCl3 δ TMS)
1.24-1.26 (d, J ) 4 Hz, 12H). Anal. (C8H13BrO2) C, H, Br.
1-Bromo-2,2,3,3-tetramethylcyclopropanecarboxamide (r-Br-
TMCD, 19). Yield 87%; white crystals; mp 157 °C; MS-EI, m/z
(%) 204, 206 (M - 15, 53, 51), 124 (21), 81 (28), 67 (20), 58
(100); Rf ) 0.75 (DCM/MeOH, 96:4); 1H NMR (300 MHz, CDCl3
δ TMS) 1.25-1.26 (d, J ) 2.7, 12H), 5.47-5.7 (bd, J ) 69, 2H).
Anal. (C8H14BrNO) C, H, N, Br.
Supporting Information Available: Purity determination of the
synthesized compounds by combustion analysis. Description of the
protocols of the animal models used for the screening of investi-
gational AED are available free of charge via the Internet at http://
pubs.acs.org.
References
Biological Testing. The evaluation of anticonvulsant activities
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