Synthesis and evaluation of antiallodynic and anticonvulsant activity of novel amide and urea derivatives of valproic acid analogues

Article abstract of DOI:10.1021/jm901229s

Valproic acid (VPA, 1) is a major broad spectrum antiepileptic and central nervous system drug widely used to treat epilepsy, bipolar disorder, and migraine. VPA's clinical use is limited by two severe and lifethreatening side effects, teratogenicity and hepatotoxicity. A number of VPA analogues and their amide, N-methylamide and urea derivatives, were synthesized and evaluated in animal models of neuropathic pain and epilepsy. Among these, two amide and two urea derivatives of 1 showed the highest potency as antineuropathic pain compounds, with ED₅₀ values of 49 and 51 mg/kg for the amides (19 and 20) and 49 and 74 mg/kg for the urea derivatives (29 and 33), respectively. 19, 20, and 29 were equipotent to gabapentin, a leading drug for the treatment of neuropathic pain. These data indicate strong potential for the above-mentioned novel compounds as candidates for future drug development for the treatment of neuropathic pain. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901229s

7236 J. Med. Chem. 2009, 52, 7236–7248
DOI: 10.1021/jm901229s
Synthesis and Evaluation of Antiallodynic and Anticonvulsant Activity of Novel Amide and Urea
Derivatives of Valproic Acid Analogues
Dan Kaufmann,† Meir Bialer,†,‡ Jakob Avi Shimshoni,† Marshall Devor,§ and Boris Yagen*, ,‡
†
Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel, ‡David R. Bloom Center
for Pharmacy, The Hebrew University of Jerusalem, Israel, §Department of Cell and Developmental Biology, Institute of Life Sciences, Faculty of
Natural Sciences and Center for Research on Pain, The Hebrew University of Jerusalem, Israel, and Department of Medicinal Chemistry and
Natural Products, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
Received August 18, 2009
Valproic acid (VPA, 1) is a major broad spectrum antiepileptic and central nervous system drug widely
used to treat epilepsy, bipolar disorder, and migraine. VPA’s clinical use is limited by two severe and life-
threatening side effects, teratogenicity and hepatotoxicity. A number of VPA analogues and their
amide, N-methylamide and urea derivatives, were synthesized and evaluated in animal models of
neuropathic pain and epilepsy. Among these, two amide and two urea derivatives of 1 showed the
highest potency as antineuropathic pain compounds, with ED50 values of 49 and 51 mg/kg for the
amides (19 and 20) and 49 and 74 mg/kg for the urea derivatives (29 and 33), respectively. 19, 20, and 29
were equipotent to gabapentin, a leading drug for the treatment of neuropathic pain. These data indicate
strong potential for the above-mentioned novel compounds as candidates for future drug development
for the treatment of neuropathic pain.
Introduction
seizures, bipolar disorder, and migraine.9-11 It was also
shown to be effective for treating some forms of neuropathic
pain. However, its efficacy as a pain reliever, including as a
reliever of neuropathic allodynia (antiallodynic drug), is con-
troversial.12-15 VPA’s clinical use is limited by two severe
and life threatening side effects, teratogenicity and hepatoto-
xicity.16-19 Hepatotoxicity induced by 1 is caused by the
formation of metabolite(s) possessing a terminal double bond
(e.g., 4-ene-VPA). Teratogenicity induced by 1 is caused by
the parent compound.20-24 As 1 is a small branched fatty acid
having eight carbons in its structure and a wide range of CNS
activity, it is a good target for structure modification and
structure-activity relationship (SAR) studies.
Many studies have been conducted in an attempt to find
derivatives superior to 1, with a similarly broad spectrum of
activity and an improved side effects profile, by synthesiz-
ing analogues and derivatives of 1 and evaluating their
anticonvulsant activity.25-40 Studies conducted by our
group also evaluated the antiallodynic activity of such
derivatives in animal models for neuropathic pain. Among
the compounds tested for this activity are the corresponding
amide of 1 and its constitutional isomers and cyclopropyl
analogue.41,42,49 Valpromide (VPD, 2), the corresponding
amide of 1, and the constitutional isomers of 2 valnocta-
mide (VCD, 3), diisopropylacetamide (DID, 4), and pro-
pylisopropylacetamide (PID, 5) (Figure 1), were 10 times
more potent than 1 as anticonvulsants, and 4 times more
potent as antineuropathic pain compounds than 1. In
addition, they were also more potent than their correspond-
ing acids in animal models of both anticonvulsant and
antiallodynic activity.41,42
Neuropathic pain is a neurological disorder affecting be-
tween 3% and 8% of adults. It often has devastating effects on
patients’ quality of life, general mood, and occupational
functioning and is associated with a heavy financial burden
to family and to society.1,2 Many patients are resistant to
current therapy, and thus, there is a substantial need for
further development of novel medications for the treatment
of neuropathic pain.1,3,4 Among the pharmacotherapy cur-
rently used to treat neuropathic pain conditions are antiepi-
leptic drugs (AEDsa), e.g., carbamazepine, gabapentin, and
pregabalin. Indeed, these are among the first line treatment
options for many neuropathic pain conditions.2,5-7 It is
believed that epilepsy and neuropathic pain share aspects of
their underlying pathophysiology8 and that this is the reason
that some AEDs are useful for the treatment of neuropathic
pain.5
Valproic acid (VPA, 1, Figure 1) is a broad spectrum AED
widely used today for treating various types of epileptic
*To whom correspondence should be addressed. Address: Depart-
ment of Medicinal Chemistry and Natural Products, School of Phar-
macy, Faculty of Medicine, The Hebrew University of Jerusalem,
Jerusalem 91120, Israel. Phone: 972-2-6758606. Fax: 972-2-6757076.
E-mail: Yagen@cc.huji.ac.il.
aAbbreviations: VPA, valproic acid; AEDs, antiepileptic drugs;
CNS, central nervous system; VPD, valpromide; VCD, valnoctamide;
DID, diisopropylacetamide; PID, propylisopropylacetamide; SNL,
spinal nerve ligation; MES, maximal electroshock seizure; scMet, sub-
cutaneous pentylentetrazole; LDA, lithium diisopropylamine; NMR,
nuclear magnetic resonance; GC-MS, gas chromatography-mass
spectrometry; VFF, von Frey filaments; TLC, thin layer chromatogr-
apy; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone; TMCA,
2,2,3,3-tetramethylcyclopropylcarboxylic acid; TMCD, 2,2,3,3-tetra-
methylcyclopropylcarboxamide; MTMCD, N-methyl-2,2,3,3-tetrame-
thylcyclopropyl carboxamide.
In the present study, 17 new amide derivatives of analogues
of 1, including amide and uride derivatives, were synthesized
r
pubs.acs.org/jmc
Published on Web 10/30/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7237
Table 2. Anticonvulsant Activity and Toxicity (Tox) of Compounds
6-13 Administered Intraperitoneally to Mice
MESa
scMetb
Toxc
compd dose (mg/kg) 0.5 hd 4 hd 0.5 hd 4 hd 0.5 hd 4 hd
6
30
100
300
30
0/1
1/3
1/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
1/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/4
0/8
3/4
0/4
0/8
0/4
0/4
0/8
1/4
0/4
0/8
0/4
0/4
1/8
0/4
0/4
0/8
0/4
0/4
0/8
3/4
0/4
0/8
0/4
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
0/2
7
100
300
30
8
100
300
30
Figure 1. Chemical structure of valproic acid (VPA, 1), its amide
derivative (VPD, 2), and the amide derivatives of its constitutional
isomers.
9
100
300
30
10
11
12
13
Table 1. Antiallodynic Activity of Compounds 6-34 30-240 min after
Intraperitoneal Administration to Rats at 80 mg/kg
100
300
30
SNLa
100
300
30
compd
30 minb
60 minb
120 minb
180 minb
240 minb
6
1/9
0/9
1/9
1/8
0/9
0/8
0/9
1/9
1/9
0/8
2/10
1/8
1/10
6/9
3/9
4/9
5/9
0/10
0/10
0/10
0/8
0/9
2/7
5/8
2/9
0/9
0/9
1/9
0/9
3/9
0/9
1/9
1/8
0/9
0/8
0/9
1/9
3/9
1/8
1/10
1/8
2/10
7/9
7/9
6/9
6/9
2/10
2/10
1/10
2/8
0/9
2/7
6/8
2/9
0/9
0/9
3/9
1/9
4/9
1/9
2/9
3/8
2/9
0/8
0/9
1/9
3/9
1/8
2/10
1/8
2/10
8/9
4/9
4/9
7/9
2/10
6/10
1/10
1/8
0/9
2/7
6/8
3/9
0/9
0/9
2/9
1/9
4/9
1/9
2/9
3/8
2/9
2/8
0/9
1/9
4/9
3/8
2/10
1/8
2/10
2/9
4/9
3/9
6/9
2/10
5/10
1/10
2/8
0/9
1/7
4/8
3/9
0/9
0/9
4/9
1/9
2/9
1/9
2/9
1/8
1/9
1/8
0/9
0/9
2/9
2/8
0/10
0/8
0/10
2/9
1/9
1/9
5/9
2/10
5/10
1/10
2/8
0/9
1/7
3/8
3/9
2/9
0/9
4/9
0/9
7
100
300
30
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
100
300
a Maximal electroshock test (number of animals protected/number of
animals tested). b Subcutanous metrazol test (number of animals pro-
tected/number of animals tested). c Neurotoxicity evaluated as motor
impariment or sedation (number of animals protected/number of ani-
mals tested). d Time after drug administration.
model of neuropathic pain (Table 1). Antiallodynic acti-
vity of these compounds was compared to their antiepi-
leptic activity in the maximal electroshock (MES) and
subcutaneous pentylenetetrazol (scMet) seizure tests
(Tables 2-4).
Chemistry
The general synthesis for the analogues of 1 and their
corresponding amides, N-methylamides and urea derivatives,
is depicted in Figure 3. Stated briefly, the starting material for
compounds 6, 7, 25, 28, 31 was valeric acid; for compounds
8, 9, 24, 30 isovaleric acid; for compounds 19, 20, 23, 29
3-methylvaleric acid; for compounds 15, 17, 18 4-methylva-
leric acid;forcompounds 12, 16, 21, 22, 27, 33dimethylbutyric
acid; for compounds 13, 34 pivaloylacetic acid. In order to
produce the corresponding branched acids, the above-men-
tioned acids were converted to the corresponding enolates by
the use of lithium diisopropylamine (LDA), followed by
substitution of a hydrogen atom on the carbon R to the
carboxyl with the appropriate alkyl by using specific alkyl
iodides.43 The carboxylic acids were treated with thionyl
chloride in order to produce the corresponding acyl chloride
followed by treatment with 28-30% ammonium hydroxide
or 41% methylamine solution in water at 0 °C for 2 h to yield
the corresponding amide or N-methylamide derivative, re-
spectively. The urea derivatives of the corresponding acids
were synthesized by coupling the acyl chloride moiety with
urea according to a previously published procedure.34,44 The
chemical structures of the synthesized amides, N-methyla-
mides, and urea derivatives were identified by 1H NMR
and GC-MS, while purity was established using elemental
analysis.
a Spinal nerve ligation model of neuropathic pain (number of animals
protected/total number of animals tested). b Time after drug adminis-
tration.
by modifying the alkyl side chain(s) of the carboxyl moiety
of the appropriate acids. We aimed to determine the SAR
of these derivatives in an attempt to develop additional
novel and more potent compounds for the treatment of
neuropathic pain. We further synthesized 12 N-methyl and
urea derivatives by replacing the amino group in those amides
that yielded the strongest antiallodynic effect among the ones
evaluated in this study. The aim was to determine the effect on
the antiallodynic and anticonvulsant potencies of substitution
of the NH2 (in the amide moiety) with N-methyl and urea
substituents.
The antiallodynic activity for all 29 compounds mentioned
above was evaluated utilizing the spinal nerve ligation (SNL)

7238 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Kaufmann et al.
Table 3. Anticonvulsant Activity and Toxicity of Compounds 14-22
Administered Intraperitoneally to Mice
Table 4. Anticonvulsant Activity and Toxicity of Compounds 23-34
Administered Intraperitoneally to Mice
MESa
scMetb
Toxc
MESa
scMetb
Toxc
compd dose (mg/kg) 0.5 hd 4 hd 0.5 hd 4 hd 0.5 hd 4 hd
compd dose (mg/kg) 0.5 hd 4 hd 0.5 hd 4 hd 0.5 hd 4 hd
14e
15g
16
17
18
19
20
21
22
30
100
300
30
0/1
3/3
NTf
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
3/3
1/1
0/1
2/3
1/1
0/1
2/3
1/1
0/1
2/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
1/3
1/1
0/1
0/1
NTf
0/1
0/3
0/0
0/1
0/1
1/1
0/1
0/1
1/1
0/1
0/1
1/1
0/1
1/1
1/1
0/1
0/1
1/1
0/1
1/1
1/1
0/1
0/1
1/1
0/1
0/1
NTf
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/4
0/8
4/4
0/4
0/8
4/4
0/4
0/8
2/4
0/4
0/8
2/4
0/4
0/8
4/4
0/4
8/8
4/4
0/4
0/8
3/4
0/4
2/8
4/4
0/4
6/8
3/4
0/2
0/4
1/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
0/2
0/2
0/4
2/2
0/2
0/4
1/2
23
30
100
300
30
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
2/3
1/1
0/1
0/3
0/1
0/1
3/3
1/1
0/1
3/3
1/1
0/1
2/3
1/1
0/1
0/3
0/1
0/1
1/3
1/1
0/1
0/3
1/1
NAf
NAf
NAf
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
1/3
1/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
NAf
NAf
NAf
0/1
0/1
1/1
0/1
0/1
4/5
0/1
0/1
1/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
1/1
1/1
0/1
1/1
1/1
0/1
1/1
1/1
0/1
1/1
1/1
0/1
0/1
1/1
0/1
4/5
1/1
NAf
NAf
NAf
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
NAf
NAf
NAf
0/4
1/8
3/4
0/4
1/8
2/4
0/4
0/8
4/4
0/4
0/8
4/4
0/4
0/8
0/4
1/4
6/8
3/4
1/4
8/8
4/4
1/4
6/8
4/4
1/4
6/8
3/4
0/4
0/8
4/4
0/4
3/8
4/4
NAf
NAf
NAf
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
1/2
0/4
1/2
0/2
0/4
2/2
0/2
0/4
2/2
1/2
1/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
NAf
NAf
NAf
24
100
300
30
100
300
30
25
100
300
30
100
300
30
26
100
300
30
100
300
30
27
100
300
30
100
300
30
28e
29e
30e
31e
32
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
100
300
30
a Maximal electroshock test (number of animals protected/number of
animals tested). b Subcutanous metrazol test (number of animals pro-
tected/number of animals tested). c Neurotoxicity evaluated as motor
impariment or sedation (number of animals protected/number of ani-
mals tested). d Time after drug administration. e Data taken from ref 27.
f NT: not tested. g Data taken from ref 28.
100
300
30
33g
34g
100
300
30
100
300
Biology
In Vivo. The surgical procedure used to produce allodynia
was previously described by Kim and Chung.45Briefly, rats
were anesthetized using intraperitoneal (ip) administration
of 85 mg/kg ketamine and 15 mg/kg xylazine. With the rats in
prone position the paraspinal muscles on the left were care-
fully separated from the L4 to the S2 transverse processes
followed by removal of the L6 transverse process in order to
visualize the L5-L6 spinal nerves. These were tightly ligated
with a 5-0 silk thread and cut just distal to the ligature. The
paraspinal muscles were closed with sutures, and then
the skin was closed with Michel clips. A bacteriostatic
powder was applied topically followed by intramuscular
administration of ampicillin. We allowed at least 5 days
for recovery after surgery before commencement of the
behavioral tests.
The rat’s foot withdrawal in response to a tactile stimulus
was used to detect tactile allodynia using a set of nine nylon
von Frey filaments (VFF). The VFF used produced an initial
bending force of 5.8, 13.7, 19.6, 39.2, 58.7, 78.3, 97.9, 146.9,
and 254.5 mN, equivalent to a mass of 0.6, 1.4, 2, 4, 6, 8, 10,
15, and 26 g. The same set was calibrated and used in all
experiments. All compounds were administered ip at 7, 14,
and 21 days after surgery using a Latin square design
protocol where the experimenter who performed the beha-
vioral tests was not aware of the dose or substance given to
the animals tested. Compounds containing one or two chiral
centers were evaluated as their racemates or diastereomeric
a Maximal electroshock test (number of animals protected/number of
animals tested). b Subcutanous metrazol test (number of animals pro-
tected/number of animals tested). c Neurotoxicity (number of animals
protected/number of animals tested). d Time after drug administration.
e Data taken from ref 34. f NA: not available. g Data taken from ref 58.
mixtures. The VFF were applied briefly just before and 30,
60, 120, 180, 240 min after injection, at 1-2 s intervals, to the
mid-plantar skin of the hind paw. Stimulation began with the
0.6 g VFF, using a perpendicular force to the skin that was
just sufficient to bend the monofilament. If the animal failed
to respond with a brief paw withdrawal to at least three out of
five stimuli, the next monofilament was tested using an
ascending staircase protocol. The response threshold was
set as the average of the minimal force required to obtain a
criterion response on the two repeats. Rats were considered
“protected” from allodynia if they failed to respond to the
15 g VFF. All compounds were administered at 80 mg/kg
ip, a dose previously shown to produce full protection
from allodynia for compounds 2-5. Compounds displaying
significant antiallodynic activity at 80 mg/kg ip had their
ED50 value and 95% confidence interval (CI) calculated
utilizing two additional doses with a minimum of 8 rats per
dose.
The antiepileptic potential of the tested compounds was
established using the MES, scMet seizure, and pilocarpine
tests. In the MES test, 60 Hz 50 mA of alternating current

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7239
was delivered through corneal electrodes for 0.2 s. During
the time of administration of the test substance a drop of
0.5% tetracaine in saline is applied to the eyes of the animals.
Animals were restrained by hand during administration of
the electrical stimulus and then released for observation of
the seizure throughout its entire course. A test substance/
dose that is able to abolish the hind limb tonic extensor
component of the seizure indicates prevention of the MES
induced seizure spread and thus is considered “active”. Tonic
extension was considered abolished if the hind limbs were not
fully extended at 180° to the plane of the body.
In the scMet seizure test, a convulsive dose of pentylenete-
trazole was injected subcutaneously (85 mg/kg in mice) at
the time of peak effect of the test substance, followed by
observation of seizure occurrence. Absence of seizures
indicates that the tested compound/dose can elevate the
pentylenetetrazole seizure induction threshold.
Table 5. Comparison of ED50 Values in Models of Epilepsy (MES) and
Neuropahtic Pain (SNL)a
ED50, mg/kg (95 % confidence interval)
compd
SNLb
MESc
MES/SNL
1
269 (227-310)d
61 (44-77)d
52 (9-78)d
58 (18-97)d
42 (14-52)e
50 (35-61)
49 (9-61)
485 (324-677)d
32 (22-42)d
29 (19-38)d
51 (34-66)d
22 (15-28)e
NA
1.8
2
0.52
0.56
0.88
0.52
3
4
5
19
20
29
33
NA
24 (16-35) f
49 (16-59)
74 (31-84)
0.49
0.86
64 (55-74) f
a ED50 values and 95% confidence intervals of the log transforms
were calculated by the probit analysis. b Spinal nerve ligation test.
c Maximal electroshock test. d Data taken from ref 42. e Data taken from
ref 49. f Data taken from ref 34.
Systemic administration of pilocarpine, a cholinergic ago-
nist, has been used to induce status epilepticus. This seizure
state is clinically defined as continuous seizure activity or
multiple seizures without regaining consciousness, lasting
more than 30 min. To determine if a test substance can
prevent acute pilocarpine induced status, candidate drugs
were administered to male Sprague-Dawley rats via the ip
route, followed by administration of a challenge dose of
pilocarpine immediately (0 min) and 30 min after treatment
with a candidate drug. The outcome measures were “protec-
tion” or “no protection” from epileptic seizures. In addition,
morbidity was also determined 24 h after each test was
completed. Quantitative determination of the protective
effect was undertaken for compounds found to possess
significant protection. This included calculations of the peak
time response as well as determination of ED50 and 95%
confidence limits.
In the present study we evaluated the antiallodynic activity
of amide derivatives of 1 (6-22) and several of their N-methyl
(23-27) and urea derivatives (28-34) (Figure 2) in the SNL
model for neuropathic pain. All tested compounds were given
at a dose of 80 mg/kg (ip). The dose was chosen as the highest
tolerated dose that induced complete reversal of tactile allo-
dynia in all rats for compounds 2-5.41,49 We also evaluated
the correlation between the antiallodynic and anticonvulsant
activities of the synthesized compounds in the MES, scMet,
and pilocarpine models for epilepsy.
Figure 2 lists the synthetic amide derivatives of the analo-
gues of 1 that were tested: (1) Compounds 6-13 possessed five
to seven carbons in the carboxylic moiety. (2) Constitutional
isomers of VPD had eight carbons in their carboxylic moiety
(compounds 14-16), and (3) compounds 17-22 had nine
carbons in their carboxylic moiety (Figure 2).
We also synthesized several N-methylamide derivatives
(compounds 23-27) and urea derivatives (compounds
28-34) of those acids that yielded the strongest antiallodynic
effect as amide derivatives.
Results and Discussion
Neuropathic pain and epilepsy share similar underlying
pathophysiology. This is presumed to be the reason that
AEDs have become a mainstay of treatment for various
neuropathic pain syndromes.2,3,7,8,46 Both epilepsy and
neuropathic pain have a debilitating effect on patient’s daily
performance, mood, and quality of life. Current therapy
options are often insufficient and limited by severe side
effects.1,4,47,48 Relatively few SAR studies have been per-
formed in order to develop new potent compounds for
neuropathic pain.41,49
In this study we designed and synthesized a new series of
amide, N-methylamide and urea derivatives of closely related
analogues of 1, and examined their antiallodynic activity in
the SNL (Chung) model of neuropathic pain. We also eval-
uated their anticonvulsant activity in the MES, scMet, and
pilocarpine seizure models. Previously we evaluated the effi-
cacy of compounds 2-5 (Figure 1) and their corresponding
acids in the SNL model of neuropathic pain.41,49 Compounds
2-5 (Figure 1) were 4-6 times more potent in the neuropathic
pain assay and 10-20 times more potent in the anticonvulsant
assay than 1. On the basis of their ED50 values (Table 5), they
are equipotent to gabapentin (ED50 = 32 mg/kg, p > 0.05),
one the most widely used drugs today in the treatment of
neuropathic pain.5,50-52 The corresponding acids of com-
pounds 2-5 were synthesized and evaluated as well.41 These
were inactive in antiallodynic and anticonvulsant animal
models.
The results obtained for antiallodynic effect evaluated in
the rat SNL model of neuropathic pain are presented in
Table 1. The results for anticonvulsant activity in the MES
and scMet seizure tests in mice are presented in Tables 2-4,
and the results obtained in the rat pilocarpine model of status
epilepticus are presented in Tables 6 and 7.
Amide Derivatives of the Analogues of 1 with Five to Seven
Carbons. Amides 6-13, having five to seven carbons in
their carboxylic moiety, showed a decrease in antiallodynic
activity compared to the amides 2-5 which had eight
carbons. Compound 6 had the highest antiallodynic activity
in this group of compounds. By use of this compound,
four out of nine rats tested were considered protected after
120 and 180 min (Table 1). Compounds 6-13 evaluated in
the anticonvulsant tests on mice were either inactive or
showed no therapeutic window between activity and toxicity
(Table 2). ClogP values are an important indicator of the
balance between the lipophilic and hydrophilic moieties in a
given molecule. It was previously shown that there is
a correlation between anticonvulsant activity and ClogP
values, where an increase in ClogP is correlated with
increased anticon vulsant properties.40,53,54 Relatively favor-
able anticonvulsant activity was indicated in one study for
compounds displaying ClogP values between 1.84 and
2.64.40 Since the ClogP values of 6-13 (0.123-1.311) were
the lowest among the compounds presented in Figure 2 this
might explain the weaker anticonvulsant effect of this group

7240 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Kaufmann et al.
Figure 2 (continued on the next page)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7241
Figure 2. Structures of valproic acid (VPA) and the amide, N-methylamide, and urea derivatives of VPA analogues.
of compounds and their correspondingly weaker antiallody-
nic activity.40,53
moderately active at all time points compared to 15-16,
compounds that were almost inactive. When administered at
a higher dose, 100 mg/kg ip, 14 remained partially active as
an antiallodynic, but only half of the animals reached the
criterion for protection (data not shown). An even higher
dose, 120 mg/kg, caused sedation in the rats, and thus, 14 was
excluded from further testing. 14 was also previously eval-
uated in the anticonvulsant test in mice and was found to
have ED50 values of 78 and 103 mg/kg in the MES and scMet
Constitutional Isomers of 2. Consistent with previously
synthesized constitutional isomers of 2,35,37,42,55 three amide
derivatives 14-16 (Figure 2), containing eight carbons in
their carboxylic moiety, were synthesized and evaluated for
antiallodynic and anticonvulsant activities. They showed
lower activity as antiallodynic compounds at 80 mg/kg ip
than their constitutional isomers 2-5. Compound 14 was

7242 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Kaufmann et al.
Table 6. Anticonvulsant Activity of Compounds in the Pilocarpine Test
of Status Epilepticus after po Administration to Ratsa
compd
dose (mg/kg)
time (h)
pilocarpineb
deaths
9
450
600
100
200
400
65
0.0
0.5
0.5
0.0
0.5
0.0
0.5
0.0
0.5
0.0
0.0
0.0
0.5
0.5
0.0
0.5
8/8
0/7
1/8
8/8
7/7
8/8
8/8
7/7
0/8
0/8
6/8
8/8
0/8
10/13
8/8
2/6
0
0
1
0
1
0
0
0
0
8
0
0
0
3
0
1
14
16
20
22
130
65
130
450
37.5
75
24
29
31
32
33
200
300
100
200
Figure 3. Synthesis of VPA analogues, and their amide (2-22),
N-methylamide (23-27), and urea (28-34) derivatives. Reagents
and conditions are as follows: (i) LDA, THF, -15 °C, 20 min; (ii)
propyl iodide, isopropyl iodide, ethyl iodide, or methyl iodide,
THF, 0 °C, 30 min; (iii) SOCl2, CH2Cl2, 25 °C,12 h; (iv) 28-30%
NH4OH, acetonitrile, 0 °C, 2 h; (v) 41% methylamine, acetonitrile,
0 °C, 2 h; (vi) urea, acetonitrile, 80 °C, 2 h.
a A challenge dose of pilocarpine is given 0 and 30 min following ip
administration of a candidate drug to male Spragu-Dawley rats.
b Pilocarpine test (number of animal protected/total number of rats
tested).
Table 7. ED50 in the Pilocarpine Test of Status Epilepticusa
was observed at 2 h, the time of the peak antiallodynic effect
of 19, 20, and 22. Compounds 19, 20, and 22 were further
tested at two additional doses (60 and 40 mg/kg ip) in order
to determine their ED50 values in the SNL model. Com-
pound 21, the least potent among the four active compounds
19-22, was not tested further. The ED50 values of com-
pounds 19 and 20 were 50 and 49 mg/kg, respectively
(Table 5). 22, although active at 80, 60, and 40 mg/kg (data
not shown), showed a nonlinear dose-response curve, and
thus, its ED50 value could not be calculated. On the basis of
ED50 values, 19 and 20 are equipotent to gabapentin (p >
0.05),50-52 indicating the potential for these compounds
to become candidates for further investigation as analge-
sic drugs. Although 19-22 were active in the mouse MES
and scMet seizure tests (Table 3), they did not show separa-
tion between activity and toxicity, and thus, these com-
pounds were not evaluated further. 20 was shown to be
active in the pilocarpine status model, having an ED50 value
of 84 mg/kg. It was thus the most potent compound in
both epilepsy and neuropathic pain models among the
group with nine carbon atoms in the carboxylic moiety.
Interestingly 19-22 showed a correlation between antiallo-
dynic and anticonvulsant activity, but neurotoxicity was
evident starting at 100 mg/kg (Table 3). Neurotoxicity was
not seen in compounds 6-13 having five to seven carbons
in their carboxylic moiety. The decreased safety margin
between activity and toxicity for the acids containing more
than eight carbons in their structures was already noted
in the literature for analogues of 1 (2-propylhexanoic acid,
2-pentylheptanoic acid, and 2-hexyloctanoic acid) having 9,
12, and 14 carbons in their carboxylic moiety.56 For this
reason amide derivatives of analogues of 1 having more than
9 carbons in their carboxylic moiety were not synthesized
and evaluated.
ED50, mg/kg
(95% CI)
ED97, mg/kg
(95% CI)
compd
time (h)
16
20
29
32
0.5
0.5
0.0
0.5
257 (192-317)
84 (62-103)
23 (4-33)
451 (345-1269)
149 (114-393)
NT
247 (207-295)
439
a Analysis of statistical significance was done by means of probit
analysis. ED50 and ED97 values and 95% confidence intervals of the log
of the transforms were calculated by the probit analysis.
tests, respectively, 2-3 times more potent than 1.27 This
compound was not evaluated in rats as an anticonvulsant. 15
and 16 were considered inactive at 80 mg/kg ip, as only a
small fraction of animals were protected at this dose com-
pared to full protection provided by 2-5 in this model
(Table 1). 15 was previously reported to have only partial
activity in the anticonvulsant tests in mice,28 and thus, it was
not evaluated further. 16, however, was active as an antic-
onvulsant at the highest dose tested (300 mg/kg), although a
mild sedative effect was observed after 0.5 h (Table 3). It is
worth mentioning that at these doses compound 16, unlike
other compounds in this group, was active in the pilocarpine
test for status epilepticus having an ED50 value of 256 mg/kg.
Overall, compounds 14-16 were less potent as antiallodynic
and anticonvulsant compounds than their structural iso-
mers, compounds 2-5, despite the fact that they had similar
ClogP values. The exception was activity in the pilocarpine
model for status epilepticus which was an attribute of
compound 16 alone (Tables 6 and 7).
Amide Derivatives of the Analogues of 1 with Nine Carbons.
In order to increase the lipophilicity of the analogues of 1 and
thus increase their cell membrane penetration and brain-to-
plasma ratio, amide derivatives having nine carbons in their
carboxylic moieties were synthesized (compounds 17-22)
(Figure 2). Four of them (19-22) proved to be the most
active of the compounds tested in the SNL model (Table 1).
However, compounds 19-22 were found to be mildly seda-
tive at the highest dose tested in the SNL model, 80 mg/kg ip
(at 0.5 h), more so than compounds with five to eight carbons
in their carboxylic moiety (data not shown). The sedative
effect started to dissipate 1 h after dosing, and no sedation
2,2,3,3-Tetramethylcyclopropanecarboxylic acid (TMCA)
is a cyclic analogue of 1, and its amide (TMCD) and
N-methylamide derivatives (MTMCD) (Figure 4) were eval-
uated previously in our laboratory as antiallodynic and anti-
convulsant compounds.49 TMCD was much more potent
than its corresponding acid. However, its N-methylamide
derivative, MTMCD, was even more potent than TMCD in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7243
Table 8. ClogP Values of Valproic Acid and the Amide and Urea
Derivatives of Its Analoguesa
compd
ClogP
compd
ClogP
1
2.58
1.4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
2.239
2.109
2.239
1.979
2.109
1.746
1.616
1.746
1.876
0.778
1.92
2
3
1.71
1.58
4
5
1.71
6
1.311
0.782
1.181
0.652
0.782
0.253
0.742
0.123
1.84
7
8
9
10
11
12
13
14
15
16
17
Figure 4. Cyclic analogue of 1 (TMCA), its amide derivative
(TMCD), and its N-methyl derivative (MTMCD).
1.79
1.66
1.79
1.92
both epilepsy and neuropathic pain models.41 Therefore,
in order to evaluate the effect of substitution of NH2 in the
amide with the N-methyl group, we synthesized five
N-methylamides corresponding to compounds 23-27
(Figure 1) and evaluated their antiallodynic and anticonvul-
sant activities (Tables 1 and 4).
1.71
1.58
0.822
0.203
2.109
a ClogP was calculated by utilizing the ChemDraw Ultra software,
version 8.
N-Methyl Derivatives of the Analogues of 1. 23-25 are the
N-methyl derivatives of the acids, synthesized by replacing
the NH2 group in the amides 3-5, respectively. The anti-
allodynic data indicated that the N-methyl derivatives were
either less active or inactive as analgesic compounds com-
pared to their amide counterparts. Compound 25 was active
in the rat MES model; its ED50 value was 63 mg/kg. Thus, 25
is less active as an anticonvulsant than 5, the amide derivative
of the same acid (ED50 = 22 mg/kg), but it is 8 times more
potent than 1 in the rat MES test. 25 was not active at all in
the SNL model of neuropathic pain, compared to its amide
analogue 5 which was previously found to be highly active
and equipotent to gabapentin.42 24, was the most active as an
antiallodynic among the N-methyl derivatives 23-27
(Table 1). However, at 80 mg/kg ip it produced unfavorable
side effects such as sedation and severe wet dog shake
reminiscent of the “serotonin syndrome”,57 unlike its amide
counterpart 4. For this reason it was not evaluated further.
26 and 27 were inactive in the SNL model (Table 1), but 26
was active in both the MES and scMet models in mice with
ED50 values of 134 and 155 mg/kg for the MES and scMet
tests, respectively. Compound 27, having only six carbons in
its carboxylic moiety, was not active in either the antic-
onvulsant or the antiallodynic animal models, and thus no
other N-methylamide derivative of acids with side chains
shorter than seven carbons were synthesized or evaluated.
23-25 had ClogP values similar to those of their amide
counterparts 3-5. However, they were less active in both
antiallodynic and anticonvulsant tests, indicating that other
requirements, such as spatial arrangement of the com-
pounds, dictate the antiallodynic and anticonvulsant activ-
ities in addition to ClogP values. These results indicate that
the N-methylamide moiety in 23-27 decreased or abolished
potency in both antiallodynic and anticonvulsant animal
models compared to the amide derivatives 3-5, 12, and 14 of
their corresponding acids (Tables 1 and 4, respectively). The
results obtained with the derivatives of the branched alipha-
tic carboxylic acids contrasted with those seen with TMCD
and MTMCD, the amide and N-methylamide derivatives of
TMCA, the cyclopropyl analogue of 1.49
viously been synthesized in our lab and were found to be very
potent as anticonvulsant compounds in the MES and
scMet tests, as well as in other models of epilepsy.34-36,58 It
is worth mentioning that the urea moiety is found as a
fused ring in three leading antiepileptic drugs: carbamaze-
pine, phenytoin, and phenobarbital. We decided to synthe-
size seven urea derivatives of acids with five, six, and mainly
eight carbons in their structures (28-34) and to evaluate
their antiallodynic activity (Figure 2). 29, a urea derivative of
2-ethyl-3-methylpentanoic acid, was the most active com-
pound with an ED50 value of 49 mg/kg in the SNL model
of neuropathic pain. Although mild sedation was witnessed
at 80 mg/kg, the highest dose tested, the two lower doses,
60 and 40 mg/kg, did not produce sedation or motor
impairment. Compared to 29, 1-(3,3-dimethylbutanoyl)
urea (33), a urea derivative of butyric acid containing two
methyl groups at the β-position to the carbonyl, was found to
be the second most active compound in the urea group. 33
was evaluated at two high doses, 100 and 110 mg/kg, and its
ED50 value was found to be 74 mg/kg. This indicates
significantly less potency than gabapentin (p < 0.05). At a
higher dose, 120 mg/kg, 33 was sedative, and thus, it was not
evaluated further. These results indicate that substitution of
NH2 by urea in compounds 2-5 and 12-14 (Figure 2)
decreases antiallodynic activity. Only 29, the urea derivative
of 2-ethyl-3-methylpentanoic acid, retained the antiallodynic
activity seen in compound 3, the corresponding amide of this
acid (Table 5). The ClogP values of these compounds were
1.71 and 1.79 for 3 and 29, respectively (Table 8). Although
33 was found to have moderate antiallodynic activity
(Table 1 and 5), it was highly active as an anticonvulsant
compound (Table 5). The amide (12) and N-methylamide
(27) derivatives of 3,3-dimethylbutanoic acid, having similar
ClogP values (around 0.8, Table 8), were not active as
antiallodynic or anticonvulsant compounds (Tables 1 and
4, respectively). One of the most remarkable observations
stemming from this research was the marked effect of the
location of a single methyl group in the acyl moiety of the
constitutional isomers of amides 17-22, and the constitu-
tional isomers of the urea derivatives 28-34, on the anti-
allodynic activity of the tested compounds (Figure 2,
Table 1).
Urea Derivatives of the Analogues of 1. Urea derivatives of
1 and its constitutional isomers and analogues have pre-

7244 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Kaufmann et al.
Conclusion
A gas chromatography-mass spectrometry assay was pre-
formed on a HP5890 series II GC equipped with a Hewlett-
Packard MS engine (HP5989A) single quadrupole mass spectro-
meter, HP7673 autosampler, HP MS-DOS Chemstation, and
HP-5MS capillary column (0.25 μm ꢀ 15 m ꢀ 0.25 mm). The
temperature program was as follows: injector temperature,
180 °C; initial temperature, 40 °C for 3 min; gradient of 3 °C/
min until 140 °C; gradient of 20 °C/min until 190 °C; hold time of
3 min. The MS parameters were set as follows: source tempera-
ture 180 °C; transfer line temperature 280 °C; positive ion
monitoring; EI-MS (70 eV). The molecular ion and the five
most pronounced ions are provided. Elemental analyses were
preformed on a 2400-2 Perkin-Elmer C, H, N analyzer. C, H, N
analyses of all newly synthesized compounds were within (0.4
of theoretical values and thus were considered satisfactory.
General Procedure for the Synthesis of Compounds 6-9 and
15-22. Anhydrous THF (70 mL) and 160 mmol of diisopropy-
lamine were added to a round-bottomed flask cooled to -15 °C
under nitrogen (N2) atmosphere, followed by dropwise addition
of 160 mmol n-butyllithium in order to prepare 160 mmol of
lithium diisopropylamine (LDA). The reaction mixture was
stirred for 30 min, and a 1:1 mixture of 10 mL of dry THF
and 72 mmol of valeric acid (for the synthesis of compounds 6
and 7), isovaleric acid (for the synthesis of compounds 8 and 9),
3-methylvaleric acid (for the synthesis of compounds 19 and 20),
4-methylvaleric acid (for the synthesis of compounds 15, 17, and
18), or tertbutylacetic acid (for the synthesis of compounds 16,
21, and 22) was added and allowed to stir for an additional 15
min below 0 °C. Then 72 mmol of DMPU was added dropwise
after maintaining a temperature of 5 °C and allowed to stir for
additional 30 min followed by a slow dropwise addition of a 1:1
solution of 160 mmol of the corresponding alkyl iodide (either
methyl iodide, ethyl iodide, propyl iodide, or isopropyl iodide)
in 10 mL of anhydrous THF. The reaction mixture was allowed
to stir at room temperature for 2 h. THF was distilled from the
reaction mixture at 60-80 °C at normal pressure, and the oily
product was dispersed in petroleum ether. A 10% HCl solution
was added until pH 1 was reached, and the organic phase was
separated from the aqueous phase and washed three times with
brine. The aqueous phase was combined and extracted with
petroleum ether (3 ꢀ 50 mL). The petroleum ether extracts were
combined, dried over MgSO4, filtered, and evaporated to yield
97% oily product. The oily product was further distilled under
reduced pressure to yield the pure corresponding acid. The free
carboxylic acids produced were chlorinated with thionyl chlor-
ide according to a previously published method.59 The acyl
chloride (44 mmol) obtained was dissolved in 20 mL of dry ACN
and was added dropwise to a 50 mL ammonium hydroxide
solution (28-30% in water) at 0 °C and was left to stir for 2 h.
Reaction mixture was extracted with ethyl acetate (3 ꢀ 30 mL),
the organic phase was washed with 2 N NaOH, dried over
MgSO4, and evaporated, and the oily product was recrystallized
with ethyl acetate petroleum ether (3:1) to obtain the desired
product.
We have reported in this study a novel class of amide,
N-methylamide and urea derivatives of several new analogues
of 1. It appears that amide derivatives of the analogues that
have eight carbons in their carboxylic moiety are both active
as antiallodynic and anticonvulsant agents and have a rela-
tively wide safety margin between activity and toxicity. Amide
derivatives of analogues of 1 with fewer than eight carbons in
their carboxylic moiety were the least potent of the com-
pounds evaluated, in both antiallodynic and anticonvulsant
animal models. The amide derivatives from the analogues of 1
that contained nine carbons in their carboxylic moiety were
morepotent than those containingeight carbons, but theyhad
a narrower safety margin between antiallodynic activity and
undesirable motor impairment or sedation. Compounds 19
(ED50 = 50 mg/kg), 20 (ED50 = 49 mg/kg), and 29 (ED50
=
49 mg/kg) were the most promising compounds for neuro-
pathic pain evaluated in this study, with potencies equal to
that of gabapentin. 3-Methyl-2-propylpentanamide (20) was
also active in the pilocarpine model of epilepsy, thus making it
a superior candidate for future drug development as both an
antiallodynic and an anticonvulsant drug. Interestingly 20 is a
close analogue of 3, previously evaluated and found to be
active in both models for epilepsy and neuropathic pain. 20
differs from 3 by having a propyl rather than an ethyl side
chain (Figure 2). Substitution of hydrogen by a methyl
group on the amide nitrogen (23-27) reduced the antiallo-
dynic and the anticonvulsant activity in all compounds
tested in this study. 1-(2-Ethyl-3-methylpentanoyl)urea
(29) was the most active compound among the group
28-34 (the urea derivatives of carboxylic acids with five,
six, and mainly eight carbons in their structures) in both the
antiallodynic and the anticonvulsant tests. However, all of
these induced mild sedation at the highest dose tested.
We conclude that 20 and 29 offer the best combination of
safety and efficacy among the active compounds evaluated
in this study. Thus, they are potential candidates for fur-
ther development as both anticonvulsant and analgesic
compounds.
Experimental Section
Chemicals. All chemicals were purchased from Sigma-Al-
drich. Tetrahydrofuran (THF), acetonitrile (ACN), thionyl
chloride, dichloromethane (DCM), petroleum ether, and ethyl
acetate were purchased from Frutarom Israel. Ammonium
hydroxide, 28-30% in water, methylamine, 41% in water,
and urea were purchased from Acros Organics Company Inc.
Dry dichloromethane, tetrahydrofuran, acetonitrile, and
DMPU were obtained by refluxing over CaH2 for 2 h and
distillation, fresh prior to use. DMPU was refluxed over CaH2
2-Ethylpentanamide (6). White powder. Mp 104-106 °C. MS-
EI, m/z (%): 128 (Mþ - 1, 0.17), 100 (19), 87 (47), 72 (100), 55
(17). 1H NMR (300 MHz, CDCl3, δ TMS): 0.91 (t, J = 7, 6H),
1.26-1.45 (m, 3H) 1.46-1.66 (m, 3H), 2.0-2.11 (m, 1H),
5.5-5.8 (br d, J = 60, 2H). Anal. (C7H15NO) C, H, N. Yield
40%.
2-Methylpentanamide (7). Silver powder. Mp 82-84 °C. MS-
EI, m/z (%): 100 (Mþ - 15, 4.5), 86 (18), 73 (100), 72 (21), 55
(13). 1H NMR (300 MHz, CDCl3, δ TMS): 0.89 (t, J = 7.2, 3H),
1.14 (d, J = 6.9 3H) 1.28-1.42 (m, 3H), 1.56-1.68 (m, 1H),
2.2-2.34 (m, 1H), 5.5-5.7 (br s, 1H), 5.8-6.0 (br s, 1H). Anal.
(C6H13NO) C, H, N. Yield 55%.
˚
for 2 h, distilled under reduced pressure, and stored over 4 A
molecular sieves (8-12 mesh) under nitrogen atmosphere.
Compounds 6-34 were prepared according to a method de-
scribed further in this section.
Materials and Methods. Product formation follow-up was
performed by means of 1H NMR and TLC. TLC analyses were
performed on precoated silica gel on aluminun sheets (Kieselgel
1
60 F254, Merck). H NMR spectra were recorded on a Varian
Mercury series NMR 300 spectrometer. Chemical shifts (δ scale)
are reported in parts per million (ppm) relative to the indicated
reference. Coupling constants are given in Hz.
Chemical structure and purity of the compounds newly
synthesized were assessed by TLC, 1H NMR, GC/MS, and
elemental analysis. Melting point was determined on a
1002-230 VAC Mel-Temp capillary melting point apparatus.
2-Ethyl-3-methylbutanamide (8). White “cotton-like” pow-
der. Mp 138-140 °C. MS-EI, m/z (%): 114 (Mþ - 15), 1.7),
100 (18), 87 (68), 72 (100), 55 (16). 1H NMR (300 MHz, CDCl3,
δ TMS): 0.88-1.0 (m, 9H), 1.52-1.64 (m, 2H), 1.5-1.86 (m,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7245
2H), 5.38-5.5 (br s, 1H), 5.6-5.8 (br s, 1H). Anal. (C7H15NO)
C, H, N. Yield 65%.
2-Ethylbutanamide (10). White crystals. Mp 113-115 °C.
MS-EI, m/z (%): 100 (Mþ - 15, 7), 87 (85), 86 (44), 72 (100),
55 (25). 1H NMR (300 MHz, CDCl3, δ TMS): 0.88-1.0 (t, J =
9, 6H), 1.4-1.68 (br m, 4H), 1.9-2.4 (br m, 1H), 5.4-5.6 (br s,
1H), 5.8-6.0 (br s, 1H). Anal. (C6H13NO) C, H, N. Yield 60%.
2-Methylbutanamide (11). Silver crystals. Mp 112-114 °C.
MS-EI, m/z (%): 86 (Mþ - 15, 26), 73 (100), 72 (17), 57 (30), 55
(12). 1H NMR (300 MHz, CDCl3, δ TMS): 0.9-1.0 (t, J = 9,
3H), 1.14-1.22 (d, J = 6.3, 3H), 1.38-1.54 (m, 1H), 1.6-1.78
(m, 1H), 2.12-2.28 (m, 1H), 5.4-5.8 (br s, 2H). Anal.
(C5H11NO) C, H, N. Yield 65%.
3,3-Dimethylbutanamide (12). Silver crystals. Mp 135-
137 °C. MS-EI, m/z (%): 115 (Mþ, 0.43), 59 (100), 58 (8), 57
(33), 55 (4). 1H NMR (300 MHz, CDCl3, δ TMS): 1.0 (s, 9H),
2.04 (s, 2H), 1.9-2.4 (br m, 1H), 5.4-5.6 (br s, 1H), 5.8-6.0 (br
s, 1H). Anal. (C6H13NO) C, H, N. Yield 60%.
2,3-Dimethylbutanamide (9). Silver powder. Mp 131-133 °C.
MS-EI, m/z (%): 115 (Mþ, 0.35), 100 (15), 73 (100), 72 (24), 55
(14). 1H NMR (300 MHz, CDCl3, δ TMS): 0.95 (dt, J = 6.6, 8.1
6H), 1.12 (d, J = 6.9 3H), 1.76-1.9 (m, 1H), 1.94-2.04 (m, 1H),
5.4-5.7 (br s, 1H), 5.7-6.0 (br s, 1H). Anal. (C6H13NO) C, H,
N. Yield 62%.
2-Ethyl-4-methylpentanamide (15). White needles. Mp 86-
88 °C. MS-EI, m/z (%): 128 (Mþ - 15, 2.5), 100 (19), 87 (51), 72
(100), 57 (27). 1H NMR (300 MHz, CDCl3, δ TMS): 0.86-1.0
(m, 9H), 1.18-1.3 (m, 1H), 1.4-1.66 (br m, 4H), 2.1-2.2 (m,
1H), 5.38-5.62 (br d, J = 36, 2H). Anal. (C8H17NO) C, H, N.
Yield 65%.
2-Ethyl-3,3,-dimethylbutanamide (16). White powder. Mp
136-138 °C. MS-EI, m/z (%): 128 (Mþ - 15, 1), 87 (71), 72
1
(100), 57 (23), 55 (11). H NMR (300 MHz, CDCl3, δ TMS):
Pivalamide (13). Silver crystals. Mp 156-158 °C. MS-EI, m/z
(%): 101 (Mþ, 65), 100 (84), 86 (52), 85 (60), 68 (100). 1H NMR
(300 MHz, CDCl3, δ TMS): 1.0 (s, 9H), 5.4-5.8 (br s, 2H). Anal.
(C5H11NO) C, H, N. Yield 65%.
0.91 (t, J = 7.2, 3H), 0.98 (s, 9H), 1.46-1.7 (br m, 2H),
1.72-1.79 (dd, J = 97.5, 3, 1H), 5.3-5.6 (br d, J = 36, 1H).
Anal. (C8H17NO) C, H, N. Yield 56%.
2-Isopropyl-4-methylpentanamide (17). White powder. Mp
108-110 °C. MS-EI, m/z (%): 142 (Mþ - 15, 2), 115 (21), 101
2-Ethylhexanamide (14). White crystals. Mp 79-80 °C. MS-
EI, m/z (%): 143 (Mþ, 0.2), 115 (11), 87 (58), 72 (100), 57 (25). 1H
NMR (300 MHz, CDCl3, δ TMS): 0.8-0.98 (m, 5H), 1.2-1.38
(m, 4H), 1.4-1.68 (m, 4H), 1.96-2.8 (m, 1H), 5.36-5.7 (ds, 2H).
Anal. (C8H17NO) C, H, N. Yield 95%.
1
(28), 86 (67), 72 (100). H NMR (300 MHz, CDCl3, δ TMS):
0.89 (t, J = 6.6, 6H), 0.94-0.98 (dd, J = 6.6, 1.2 6H), 1.18-1.3
(m, 1H), 1.5-1.64 (br m, 2H), 1.7-1.82 (m, 1H), 1.88-1.96 (m,
1H), 5.38-5.45 (br s, 1H), 5.6-5.7 (br s, 1H). Anal. (C9H19NO)
C, H, N. Yield 50%.
General Procedure for the Synthesis of Compounds 23-27.
The corresponding acids were prepared according to the above-
mentioned procedure for compounds 2-5 and 15-22. The
corresponding acids (propyl isopropylacetic acid, diisopropyla-
cetic acid, valnoctic acid, ethyl butylacetic acid, and tert-buty-
lacetic acid, corresponding to compounds 23-27, respectively)
were chlorinated with thionyl chloride according to a previously
published method.59 The obtained acyl chloride (33 mmol) was
dissolved in 10 mL of dry DCM and 5 mL of a 41% methylamine
solution in water, and was left to stir for 2 h. The reaction
mixture was extracted with ethyl acetate (3 ꢀ 30 mL), the
organic phase was washed with 2 N HCl, dried over MgSO4,
and evaporated, and the product was recrystallized with ethyl
acetate/petroleum ether (3:1) to obtain the desired product.
2-Ethyl-N,3-dimethylpentanamide (23). White powder. Mp
69-72 °C. MS-EI, m/z (%): 157 (Mþ, 0.33), 101 (72), 86 (100),
58 (42), 57 (36). 1H NMR (300 MHz, CDCl3, δ TMS): 0.76-0.94
(m, 9H), 1.0-1.2 (m, 2H), 1.4-1.62 (br m, 3H), 1.7-1.8 (m, 1H),
2.79 (d, J = 4.8, 3H), 5.6-5.8 (br s, 1H). Anal. (C9H19NO) C, H,
N. Yield 75%.
4-Methyl-2-propylpentanamide (18). White “needle-like” powder.
Mp 120-121 °C. MS-EI, m/z (%): 142 (Mþ - 15, 1), 114 (12), 101
1
(17), 72 (100), 55 (13). H NMR (300 MHz, CDCl3, δ TMS):
0.8-0.96 (m, 9H), 1.16-1.42 (br m, 4H), 1.5-1.68 (br m, 3H),
2.18-2.28 (m, 1H), 5.4-5.6 (br s, 1H), 5.7-5.9 (br s, 1H). Anal.
(C9H19NO) C, H, N. Yield 67%.
2-Isopropyl-3-methylpentanamide (19). White “cotton-like”
crystals. Mp 105-107 °C. MS-EI, m/z (%): 128 (Mþ - 29,
0.4), 115 (8), 101 (24), 86 (100), 69 (11). H NMR (300 MHz,
1
CDCl3, δ TMS): 0.84-1.0 (m, 12H), 1.00-1.3 (br m, 1H),
1.36-1.62 (br dm, 1H), 1.66-1.82 (m, 2H), 1.94-2.1 (br m,
1H), 5.4-5.6 (br s, 1H), 5.7-5.9 (br s, 1H). Anal. (C9H19NO) C,
H, N. Yield 35%.
3-Methyl-2-propylpentanamide (20). White “needle-like” pow-
der. Mp 120-121 °C. MS-EI, m/z (%): 142 (Mþ - 15, 0.35), 101
1
(35), 86 (32), 72 (100), 55 (16). H NMR (300 MHz, CDCl3, δ
TMS): 0.8-0.96 (m, 9H), 1.06-1.3 (br m, 2H), 1.3-1.64 (br m,
5H), 0.9-2.0 (m, 1H), 5.4-5.54 (br s, 1H), 5.7-5.9 (br s, 1H).
Anal. (C9H19NO) C, H, N. Yield 45%.
2-Isopropyl-N,3-dimethylbutanamide (24). White needles. Mp
2-Isopropyl-3,3-dimethylbutanamide (21). White powder. Mp
128-130 °C. MS-EI, m/z (%): 142 (Mþ - 15, 0.68), 101 (36), 100
102-104 °C. MS-EI, m/z (%): 157 (Mþ, 0.14), 115 (39), 100
1
(100), 58 (24), 57 (25). H NMR (300 MHz, CDCl3, δ TMS):
1
(30), 86 (100), 57 (22). H NMR (300 MHz, CDCl3, δ TMS):
0.86-0.96 (dd, J = 6.75, 12H), 1.549 (t, J = 15, 1H), 1.9-2.6
(m, 2H), 2.77 (d, J = 4.8, 3H), 5.6 (br s, 1H). Anal. (C9H19NO)
C, H, N. Yield 73%.
0.9-1.1 (m, 15H), 1.7 (d, J = 6, 1H) 2.0-2.1 (m, 1H), 5.3-5.5
(br s, 1H). Anal. (C9H19NO) C, H, N. Yield 45%.
2-tert-Butylpentanamide (22). White powder. Mp 118-
119 °C. MS-EI, m/z (%): 142 (Mþ - 15, 0.88), 101 (35), 100 (9),
72 (100), 57 (21). 1H NMR (300 MHz, CDCl3, δ TMS): 0.86-0.94
(t, 3H), 0.94-1.0 (s, 9H), 1.1-1.22 (m, 1H), 1.3-1.44 (m, 2H),
1.56-1.78 (m, 1H), 1.8-1.86 (dd, J = 12, 1H), 5.4-5.6 (br s,
1H), 5.8-6.0 (br s, 1H). Anal. (C9H19NO) C, H, N. Yield 40%.
General Procedure for the Syntheis of Compounds 10-14. The
corresponding acid (ethylbutyric acid, methylbutyic acid, tert-
butylacetic acid, and pivalic acid corresponding to compounds
6-9, respectively) was chlorinated with thionyl chloride accord-
ing to a previously published method.59 The acyl chloride
(44 mmol) obtained was dissolved in 20 mL of dry ACN
and was added dropwise to a 50 mL ammonium hydroxide
solution (28-30% in water) at 0 °C and then left to stir for 2 h.
Reaction mixture was extracted with ethyl acetate (3 ꢀ 30 mL),
the organic phase was washed with 2 N NaOH, dried over
MgSO4, and evaporated, and the oily product was recrystallized
with ethyl acetate petroleum ether (3:1) to obtain the desired
product.
2-Isopropyl-N-methylpentanamide (25). White powder. Mp
97-99 °C. MS-EI, m/z (%): 157 (Mþ, 0.40), 115 (56), 100 (53),
86 (100), 58 (45). 1H NMR (300 MHz, CDCl3, δ TMS): 0.8-0.96
(m, 9H), 1.06-1.26 (br m, 2H), 1.4-1.6 (br m, 2H), 1.6-1.82 (br
m, 2H), 2.8 (d, J = 4.5, 3H), 5.44-5.6 (br s, 1H). Anal.
(C9H19NO) C, H, N. Yield 70%.
2-Ethyl-N-methylhexanamide (26). White powder. Mp 73-
76 °C. MS-EI, m/z (%): 157 (Mþ, 0.74), 101 (59), 86 (100), 58
(53), 57 (37). 1H NMR (300 MHz, CDCl3, δ TMS): 0.86-0.96
(dd, J = 6.75, 12H), 1.549 (t, J = 15, 1H), 1.9-2.6 (m, 2H), 2.77
(d, J = 4.8, 3H), 5.6 (br s, 1H). Anal. (C9H19NO) C, H, N. Yield
70%.
N,3,3-Trimethylbutanamide (27). White needles. Mp 73-
75 °C. MS-EI, m/z (%): 129 (Mþ, 0.33), 114 (2.2), 73 (100), 58
(42), 57 (42). 1H NMR (300 MHz, CDCl3, δ TMS): 1.0 (s,
9H), 2.04 (s, 2H), 2.8 (d, J = 4.5, 3H), 5.4 (br s, 1H). Anal.
(C7H16NO) C, H, N. Yield 70%.
General Procedure for the Synthesis of Compounds 28-34.
The chemical synthesis for compounds 28-34 was identical to

7246 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Kaufmann et al.
that previously published by Shimshoni et al. in 2008. Briefly,
the corresponding acyl chlorides were prepared as previously
described for compounds 6-22. They were dissolved in dry
acetonitrile (50 mL) and slowly added to a boiling acetonitrile
solution (100 mL) of 0.14 mol of urea and refluxed for 2 h. The
organic solvent was evaporated under reduced pressure, fol-
lowed by dissolving the products in 100 mL of ethyl acetate. The
products were washed three times with 20 mL of distilled water.
The organic fraction was dried over magnesium sulfate and
evaporated, and products were purified by crystallization from
ethyl acetate.
1-(2-Propylpentanoyl)urea (28). White crystals. Mp 217-
220 °C. MS-EI, m/z (%): 157 (2), 144 (36), 115 (100), 72 (30),
61 (21). 1H NMR (300 MHz, CDCl3, δ TMS): 0.86-0.74 (t, J =
15, 6H), 1.2-1.74 (m, 8H), 2.24-2.4 (m, 1H), 5.62 (s, 1H), 8.4 (s,
1H), 9.42 (s, 1H). Anal. (C9H18N2O2) C, H, N. Yield 83%.
1-(2-Ethyl-3-methylpentanoyl)urea (29). White crystals. Mp
147-148 °C. MS-EI, m/z (%): 157 (6), 130 (100), 115 (69), 72
(32), 61 (36). 1H NMR (300 MHz, CDCl3, δ TMS): 0.86-0.96
(m, 9H), 1.08-1.24 (m, 1H), 1.38-1.74 (m, 4H), 1.96-2.06 (m,
1H), 5.35 (s, 1H), 8.37 (s, 1H), 8.7 (s, 1H). Anal. (C9H18N2O2) C,
H, N. Yield 61%.
1-(2-Isopropyl-3-methylbutanoyl)urea (30). White crystals.
Mp 199-200 °C. MS-EI, m/z (%): 144 (59), 129 (100), 86 (27),
69 (34), 61 (25). 1H NMR (300 MHz, CDCl3, δ TMS): 0.91-0.98
(t, J = 6, 12H), 1.78-1.84 (t, J = 6, 1H), 1.98-2.1 (m, 2H), 5.49
(s, 1H), 8.41 (s, 1H), 8.9 (s, 1H). Anal. (C9H18N2O2) C, H, N.
Yield 51%.
1-(2-Isopropylpentanoyl)urea (31). White crystals. Mp 213 °C.
MS-EI, m/z (%): 144 (81), 129 (65), 115 (100), 72 (35), 61 (33). 1H
NMR (300 MHz, DMSO-d6): 0.78-0.88 (m, 9), 1.06-1.22 (m,
2H), 1.28-1.54 (m, 2H), 1.62-1.74 (m, 1H), 2.1-2.2 (m, 1H),
7.21 (s, 1H), 7.86 (s, 1H), 10.14 (s, 1H). Anal. (C9H18N2O2) C, H,
N. Yield 71%.
dose was prepared according to a previously described standard
procedure.
Determination of the Median Effective Dose (ED50). In order
to determine the ED50 for the respective antiallodynic and
anticonvulsant protocols, doses were varied until three to four
points were established between the dose levels producing 0%
protection and 100% protection in both protocols. The data
were subjected to FORTRAN probit analysis,60 resulting in a
calculated ED50 and 95% confidence interval.
Evaluation of Rat Motor Dysfunction or Sedation. Com-
pounds 19-22, 29, and 33, which were found active in the
antiallodynic protocol, were evaluated in the accelerating rotor-
od apparatus using the highest dose administered in the anti-
allodynic sensory test (80 mg/kg). Naive male albino rats (n = 5)
(Sprague-Dawley, 200-250 g) were injected with 19-22, 29, or
33 and were placed on the rotorod after 60 and 120 min in
accordance with the time to peak antiallodynic effect previously
measured. The time before falling off the apparatus was mea-
sured, with a maximum cutoff time spent of 120 s.
Calculation of ClogP. ClogP was calculated by means of
ChemDraw Ultra software, version 8.
Statistical Analysis. The results are presented as either the
ED50 or 95% confidence intervals. A Student’s t test was used
for calculating significant difference between ED50 values. A p
value of <0.05 was considered significant.
Acknowledgment. This work was a part of the Ph.D. thesis
of Dan Kaufmann in partial fulfillment of the Ph.D. require-
ments for The Hebrew University of Jerusalem, Israel. The
authors thank James P. Stables, Director of the NIH-NINDS
Anticonvulsant Screening Program (ASP), for testing the
compounds in the ASP. The authors also thank Anne Minert
from the Department of Cell and Developmental Biology,
Institute of Life Sciences, The Hebrew University of Jerusa-
lem, Israel, for her help and support with all of the behavioral
antiallodynic experiments.
1-(2-Ethylhexanoyl)urea (32). White powder. Mp 164-
166 °C. MS-EI, m/z (%): 158 (Mþ - 28, 7.7), 130 (67), 115
1
(70), 72 (100), 57 (63). H NMR (300 MHz, CDCl3, δ TMS):
0.8-0.93 (m, 6H), 1.22-1.3 (m, 4H), 1.3-1.7 (br m, 4H), 2.17
(m, 1H), 5.5 (br s, 1H), 8.39 (br s, 1H), 9.15 (br s, 1H). Anal.
(C9H18N2O2) C, H, N. Yield 73%.
1-(3,3-Dimethylbutanoyl)urea (33). White crystals. Mp
175 °C. MS-EI, m/z (%): 143 (Mþ - 15, 0.9), 102 (100), 83
(18), 59 (79), 57 (45). 1H NMR (300 MHz, CDCl3, δ TMS): 1.0
(s, 9H), 2.18 (s, 2H), 5.62 (br s, 1H), 8.4 (br s, 1H), 9.55 (br s, 1H).
Anal. (C7H14N2O2) C, H, N. Yield 70%.
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
the investigational anticonvulsant and antiallodynic com-
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
1-(Pivaloyl)urea (34). White crystals. Mp 151-153 °C. MS-
EI, m/z (%): 144 (3), 129 (14), 89 (22), 88(16), 57 (100). 1H NMR
(300 MHz, CDCl3, δ TMS): 1.25 (s, 9H), 5.6 (br s, 1H), 8.3 (br s,
2H). Anal. (C6H12N2O2) C, H, N. Yield 70%.
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