Search for anticonvulsant and analgesic active derivatives of dihydrofuran-2(3H)-one

Article abstract of DOI:10.1016/j.bmc.2012.08.037

A series of derivatives of dihydrofuran-2(3H)-one (γ-butyrolactone, GBL) was synthesized and tested for anticonvulsant, neurotoxic and analgesic activity. In the anticonvulsant screening 10 lactones were effective in the maximal electroshock test (MES) at the highest doses (300 and 100 mg/kg, 0.5 h, ip, mice). Statistical analysis showed correlation between the anticonvulsant activity and relative lipophilicity parameters determined by experimental and computational methods (R_M0, C log P and M log P). Preliminary antinociceptive evaluation of selected derivatives revealed strong analgesic activity. The majority of the tested compounds showed high efficacy in animal models of acute pain (hot plate and writhing tests) and strong local anesthetic activity (modified tail immersion test). The obtained ED₅₀ values were comparable with such analgesics as acetylsalicylic acid and morphine.

Full text of DOI:10.1016/j.bmc.2012.08.037

Bioorganic & Medicinal Chemistry 20 (2012) 6533–6544
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Search for anticonvulsant and analgesic active derivatives
of dihydrofuran-2(3H)-one
Krzysztof Wie˛ckowski a, Kinga Sałat b, Justyna Bytnar a, Marek Bajda a, Barbara Filipek b, James P. Stables c,
Barbara Malawska a,
⇑
a Department of Physicochemical Drug Analysis, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b Department of Pharmacodynamics, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
c Consultant Potomac, MD 20854, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of derivatives of dihydrofuran-2(3H)-one (c-butyrolactone, GBL) was synthesized and tested for
Received 24 May 2012
Revised 25 July 2012
Accepted 8 August 2012
Available online 31 August 2012
anticonvulsant, neurotoxic and analgesic activity. In the anticonvulsant screening 10 lactones were effec-
tive in the maximal electroshock test (MES) at the highest doses (300 and 100 mg/kg, 0.5 h, ip, mice). Sta-
tistical analysis showed correlation between the anticonvulsant activity and relative lipophilicity
parameters determined by experimental and computational methods (RM0, ClogP and MlogP). Prelimin-
ary antinociceptive evaluation of selected derivatives revealed strong analgesic activity. The majority of
the tested compounds showed high efficacy in animal models of acute pain (hot plate and writhing tests)
and strong local anesthetic activity (modified tail immersion test). The obtained ED50 values were com-
parable with such analgesics as acetylsalicylic acid and morphine.
Keywords:
Dihydrofuran-2(3H)-one
c
-Butyrolactone
Phenylpiperazines
Analgesic activity
Anticonvulsant activity
Lipophilicity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
mechanism-driven designs.7 Therefore, the identification of anti-
convulsants is usually conducted through multiple in vivo screen-
Epilepsy is a serious neurological condition affecting up to 1% of
the world’s population.1,2 This disorder is defined by recurrent sei-
zures caused by synchronized discharges of neurons in the brain
leading to an altered feeling or behavior of the affected patient.
Worldwide, over 40 antiepileptic drugs (AEDs) are in clinical use,
however these drugs are only effective in about one third of pa-
tients, the remainder being resistant to the available first-line
AEDs. Many of the clinically used drugs have not been definitively
linked with a specific site of the brain nor have the exact mecha-
nisms of action been fully elucidated.3–6 Most AEDs possess more
than one mechanism of action which may account for their efficacy
in different seizure types, patterns and individual patients. Due to
the lack of understanding and complexity of epileptogenic pro-
cesses drug candidates are typically being discovered through con-
ventional screening and structural modifications rather than by
ing tests that are frequently based on seizure type or non-specific
mechanisms rather than well-defined etiology.
Due to the shared pathomechanism of many central and periph-
eral nervous system disorders, several AEDs have found application
in the treatment of non-epileptic conditions associated with neu-
ronal irregularities. Among them chronic pain of peripheral or cen-
tral origin and various psychiatric disorders (anxiety, bipolar
affective disorder) are the most important. AEDs such as valproic
acid, carbamazepine, gabapentin, pregabalin or lamotrigine are
clinically used in neuropathic pain as the first-line treatment.8–10
Numerous studies have also demonstrated analgesic activity of
other anticonvulsant agents in animal models of pain and some
of them are being currently evaluated in clinical studies (Fig. 1).11
O
O
O
NH2
O
HN
Abbreviations: AEDs, antiepileptic drugs; ASP, Anticonvulsant Screening Pro-
N
N
H
gram; CNS, central nervous system; GBL,
c-butyrolactone, dihydrofuran-2(3H)-one;
GHB, -hydroxybutyric acid, 4-hydroxybutyric acid; DCM, dichloromethane; DMF,
c
O
dimethylformamide; HP, hot plate test; ip, intraperitioneal; MIT, modified tail
immersion test; po, per os; RP-TLC, reversed-phase thin-layer chromatography;
TBAB, tetrabutylammonium bromide; WT, writhing test.
BRIVARACETAM
LACOSAMIDE
⇑
Corresponding author.
E-mail address: mfmalaws@cyf-kr.edu.pl (B. Malawska).
Figure 1. Examples of analgesic-active anticonvulsants.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.037

6534
K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
In the recent years these facts have directed the search for novel
antiepileptic compounds towards identification and development
of potent agents endowed with both anticonvulsant and analgesic
activity. In a highly saturated epilepsy market this approach has
proven successful by providing expanded financial and marketing
incentives that do not independently exist in the field of epilepsy.
Presented work is a part of our search for potential anticonvul-
F
N
2
O
N
O
F
F
N
N
O
O
1
sant agents among derivatives of 4-hydroxybutyric acid
(c-
hydroxybutyric acid, GHB). In this paper we describe studies in a
group of derivatives of dihydrofuran-2(3H)-one, (oxolan-2-one,
Figure 3. Analgesic-active 3-substituted GBLs.
c-butyrolactone, GBL), a cyclic analogue and precursor of GHB.
GBL-derived structures and the lactone itself are well known for
their central nervous system (CNS) pharmacological potential
and were found to possess anticonvulsant activity. GBL ring is pres-
ent in the structures of different GABAA receptor ligands: antago-
nists (e.g., bicuculline, picrotoxin) as well as positive allosteric
modulators bearing anticonvulsant properties (I-III) (Fig. 2).12–16
Our pilot screen of a small group of 3-substituted GBL deriva-
tives revealed not only anticonvulsant but also potent analgesic
and local anesthetic properties in several tests in mice.17 In that
study two compounds (1, 2, Fig. 3) demonstrated high analgesic
activity, comparable with or even higher than reference drugs: ace-
tylsalicylic acid, morphine, lidocaine and mepivacaine.
Pharmacological evaluation of the most potent compound 2
was further developed and showed high efficacy in other models
of pain, anticonvulsant activity in mice and antioxidant activity
in vitro.18 These results encouraged us to follow the GBL path of
the project and to synthesize more analogs thereof and for selected
compounds to extend the pharmacological screening to include
analgesic activity tests.
The obtained derivatives were evaluated for their anticonvul-
sant activity and neurotoxicity within the Anticonvulsant Screen-
ing Program at the National Institute of Neurological Disorders
and Stroke, National Institutes of Health, USA formally directed
by J. Stables. Selected compounds were then tested in rodent mod-
els of pain. For the purpose of properties–activity relationship anal-
ysis, lipophilicity parameters were determined by experimental
and computational methods.
2. Chemistry
The majority of the derivatives were prepared by N- or
O-alkylation of appropriate reagent by 3-bromodihydrofuran-
2(3H)-one (1–7, 10–13, 15–17, 21–24) or 3-bromo-5-methyl-
dihydrofuran-2(3H)-one (8, 14) (Fig. 4, Table 1). The reactions were
carried out in dichloromethane (DCM) or acetonitrile (MeCN) in the
presence of anhydrous potassium carbonate and in some cases
tetrabutylammonium bromide (TBAB).17,23–26 N-alkylation of 2,
6-xylidine (21) required maintenance of relatively low basic pH
(around 8) which was achieved by slow addition of potassium car-
bonate in aqueous solution over the course of the reaction.27 The
phthalimide derivative 17 was obtained by heating dimethylform-
Considering the above, we designed a series of c-butyrolactone
derivatives, synthesized and tested them for anticonvulsant and
analgesic activity. This study was focused on exploration of the
substitution at position 3 by introduction of various alkyl- and
arylakyl-substituents linked by nitrogen atom, oxygen atom or
aminomethylene group. As a natural consequence of the results
from the previous studies several modifications of the phenylpip-
erazine moiety were examined (3–7, 10). This conception is also
supported by the fact that arylpiperazines are present in various
CNS-active compounds and their anticonvulsant and analgesic
activities have been reported in numerous publications, including
our studies.17–20 In order to verify the pharmacological significance
amide (DMF) solution of
a-bromo-GBL with phthalimide in the
form of potassium salt, thus did not required addition of a base.28,29
The 4-substituted dihydrofuran-2(3H)-one (9) was prepared by
Michael addition of amines to 5-H-furan-2-one (c-crotonolactone)
(Fig. 5, Table 1).16,30
Derivatives of 1-amino ethylidene dihydrofuran-2(3H)-one
(18–20) were obtained by condensation of 3-acetyl-dihydrofuran-
2(3H)-one with primary amines (Fig. 6, Table 1).31–33 The reaction
was accomplished by refluxing toluene solution of the reagents
of the size and aromaticity of the substituent at the a-position dif-
ferent substituents were introduced (11–13, 15–24), among them
1,2,3,4-tetrahydroisoquinolines which have been also reported to
possess anticonvulsant properties.21,22 As a preliminary attempt
to investigate the substitution at other positions of GBL ring 5-
methyl analogs of 1 and 11 (8 and 14) and a positional isomer of
compound 2, 4-phenylpiperazine-GBL (9) were also synthesized.
O
O
A or B or C
N(O)
Br
O
O
N(O)
H/K +
H(Me)
H(Me)
NH
R
O
N
NH
R
O
O
O
H
O
O
O
O
O
R
O
N(O)
H/K :
n
NH2
N
K
H
R
NH
II
NH
I
N
O
O
NH2
NH2
OH
O
R
O
O
NH
O
O
Br
NH2
NH
III
BICUCULLINE
Figure 2. Structures of bicuculline and anticonvulsant-active derivatives of
c-
butyrolactone.
Figure 4. Synthesis of 3- and 3,5-substituted derivatives of GBL.

K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
6535
DCM
3h
O
O
N
NH
+
N
N
O
O
Figure 5. Synthesis of 4-substituted derivative of GBL.
obtained from two programs: ChemOffice (ClogP) and Marvin
(MlogP). Relative lipophilicity and the partition coefficients
calculated for all the compounds are presented in Figure 7 and in
Supplementary data.
R
R
O
n
NH
O
toluene
O
n
+
O
Δ
, 5h
NH2
O
The lipophilicity parameters determined by RP-TLC and ex-
pressed as RM0 values were in the 0.47–2.40 range which matches
the requirements for CNS-active compounds and augurs well for
the penetration to the brain.34,35 Good correlation was found be-
tween relative lipophilicity RM0 and its partition coefficients calcu-
lated using ChemOffice and Marvin software, proving their
usefulness for the prediction of the lipophilicity at early stages of
studies. Linear relationship of RM0 values with ClogP and MlogP
was characterized by correlation coefficients (R2) of 0.7712 and
0.7233, respectively.
Figure 6. Synthesis of 3-arylalkylaminoetylidene derivatives of GBL.
with azeotropic removal of water by the use of Dean–Stark appara-
tus. All the obtained compounds were tested as racemates or mix-
tures of diastereoisomers (8 and 14).
3. Lipophilicity determination
Physicochemical properties of bioactive compounds influence
both, their pharmacological activity and pharmacokinetic profile.
Lipophilicity is one of the crucial physicochemical properties that
comprise influence of different molecular parameters and has a
strong impact on biological activity of compounds, especially con-
sidering the absorption and penetration to CNS.34,35 The lipophilic-
ity of the obtained dihydrofuran-2(3H)-one derivatives was
determined using experimental and computational methods. Rela-
tive lipophilicity (RM0) was estimated by reversed-phase thin-layer
chromatography (RP-TLC) with mixtures of MeCN and Tris buffer
(pH 7.4) as the mobile phase. Theoretical parameters were
4. Pharmacology
4.1. Anticonvulsant activity
Preliminary anticonvulsant evaluation (Test 1 ASP) of all syn-
thesized compounds was performed according to procedures de-
scribed before.36–38 These initial studies involved three in vivo
tests: maximal electroshock seizure (MES), subcutaneous metraz-
ole (scMet), and minimal locomotor impairment as an assessment
of neurotoxicity (TOX). These initial screens are qualitative assays
involving a relatively small number of animals (16 male albino
Figure 7. Relative lipophilicity of compounds 1–24: RM0 and calculated logP values.
MES 300
MES 100
TOX 300
TOX 100
1
2
3
4
5
6
7
8
9
10 16 17 18 19 20 21 24
Compound
Figure 8. Test 1 ASP scores for active compounds (mice, 100 or 300 mg/kg, ip, 0.25–4 h).

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K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
mice of comparable age and weight). The tested compound is
administered to one, three and one additional animal at three
respective doses (30, 100, and 300 mg/kg ip), 0.5, 1, 2 and 4 h prior
to the test. Identical dosage and timing is used in MES, scMet and
toxicity testing. Compounds found to be effective in these seizure
challenges are regarded as potentially useful candidates for new
anticonvulsants. The MES test is a proven method of inducing gen-
eralized tonic-clonic seizures and identifying clinical candidates
that prevent partial and generalized seizure while the scMet-active
compounds are effective in the treatment of absence seizures.
Neurotoxicity of the tested compounds was determined using the
rotarod test that measures locomotor impairment.
0.75
0.50
0.25
0.00
-0.25
-0.50
MES rat
TOX rat
0.25
0.5
1
2
4
Time [h]
Compounds selected on the basis of the data obtained from
intraperitoneal (ip) screening are rolled into advanced evaluation
including MES test in Sprague dawley rats and 6-Hz test.37,38 The
minimal clonic seizure test, otherwise known as the 6-Hz model,
is used in much the same way as the MES test to assess a com-
pound’s efficacy against electrically-induced seizures. It is a ran-
dom screening test for therapy-resistant epilepsy and can help
identify novel anti-seizure agents.39,40 Classical sodium channel
antiepileptic drugs like phenytoin and carbamazepine are not ac-
tive in this model at non-toxic doses. Conversely, certain newer
anticonvulsant agents such as levetiracetam, which is not tradi-
tionally recognized to be effective in the conventional MES test, in-
deed does show protection in the minimal clonic seizure (6-Hz)
model. The 6-Hz model was found to be highly sensitive to the
antiepileptic drugs that positively modulate GABAA receptors and
thus may be valuable as a tool for the identification of such
compounds.
Figure 9. ASP test results for compound 2 (rats, 30 mg/kg, po).
Figure 10. 6-Hz test in mice for compound 23 (mice, 100 mg/kg, ip).
4.1.1. Test 1 ASP
Among 24 compounds subjected to the ASP screen, 10 lactones
showed protection against MES seizures at doses of 100 and
300 mg/kg 0.5 h following administration, however only two of
them did not produce neurotoxic effects (4, 19). Only lactones 6
and 18 showed anticonvulsant activity 15 min. after the adminis-
tration and only lactone 2 showed longer protection, being active
after 4 h. Isolated neurotoxic effects at doses 100 and 300 mg/kg
were observed in seven compounds (5, 7, 9, 16, 17, 21, 24). The re-
sults of the ASP screen are presented in Figure 8 in a form of scores
gained in the Test 1 ASP in mice, at doses of 100 or 300 mg/kg. In
the graph the columns above axis represent fraction of animals
that was protected in the MES test while the columns below the
axis correspond to the fraction of animals that failed in the TOX
test (100 and 300 mg/kg, 0.25–4 h). Detailed results of these tests
are given in Supplementary data.
In the scMet test neither of the compounds showed protective
effect at the indicated doses and time points (data not shown),
whereas lactone 15 displayed proconvulsive properties at all doses
(30–300 mg/kg), at 0.5 and 4 h. It is interesting to note that com-
pound 15 is a derivative of 1,2,3,4-tetrahydroisoquinoline (TIQ), a
moiety present in various anticonvulsant and neuroprotective
compounds.21,22 On the other hand, TIQ derivatives are also known
for their neurotoxicity, and some of them are considered to be
endogenous neurotoxins.41
properties. The results proved that the compound 2 was absorbed
after po administration and penetrated to CNS. In our extended
study of compound 2, its ED50 value in the MES test in mice was
determined to be 112 mg/kg.18
Two compounds (22 and 23) were selected for the evaluation of
anticonvulsant activity in the 6-Hz test in mice (Fig. 10). Com-
pound 23 administered ip at a dose of 100 mg/kg showed moder-
ate protection after 1 and 2 h. Compound 22 was inactive in this
model at the dose and times.
4.1.3. Structure–anticonvulsant activity relationship
The presented results show that the CNS activity of these
derivatives of dihydrofuran-2(3H)-one hinges upon the presence
of a phenylpiperazine moiety. Among eight phenylpiperazine-
containing structures, seven compounds were active in the MES
test and exhibited TOX effects, indicating their penetration to CNS
and inhibitory influence thereon. Using unsubstituted derivative
of N-phenylpiperazine (1) as a reference compound, neurotoxicity
was found to exceed anticonvulsant activity (100 vs 300 mg/kg).
Introduction of a methoxy substituent at the position 2 of the phe-
nyl ring (3) decreased the toxicity while further introduction of a
chlorine atom at the position 5 (6) intensified both effects. Substi-
tution at the position 3 of the phenyl ring seems to give a beneficial
effect. The MES activity of the 3-methoxy derivative (4) was slightly
improved while the presence of an electron-attracting trifluoro-
methyl group (2) further increased anticonvulsant activity and
prolonged duration of action to 4 h. Derivatives substituted at posi-
tion 4 of the phenyl ring, 4-methoxy (5) and 4-fluoro (7) produced
only neurotoxic effects. Substituting the N-phenylpiperazine frag-
ment with a 4-benzylpiperidine group (10) or introducing a methyl
substituent at position 5 of dihydrofuran-2(3H)-one (8) did not im-
prove the activity profile. Transfer of the phenylpiperazine moiety
to position 4 of GBL yielded inactive and more neurotoxic com-
pounds (9) when compared with reference-structure 1. Apart from
4.1.2. Advanced ASP studies
According to ASP procedures37,38 basing on the data obtained
from the ip screening compounds 2 and 18 were selected for fur-
ther evaluation of their anticonvulsant (MES) and neurotoxic
(TOX) properties in rats after oral administration at dose of
30 mg/kg (Fig. 9). Compound 18 was inactive while lactone 2
was effective in this model showing also some neurotoxicity, how-
ever its protective activity in MES increased while neurotoxicity
decreased as a function of time. Even more important outcome
from this test concerns the bioavailability and pharmacokinetic

K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
6537
the activity and neurotoxicity of the derivatives of dihydrofuran-
2(3H)-ones. 4-Phelypiperazin-1-yl fragment, along with it’s 30-
trifloromethyl analogue, are preferable in terms of activity (1–9),
however others substituents are able to elicit the activity as well.
Anticonvulsant effect in the MES test was also observed for
structures with a phenyl substituent connected to dihydrofuran-
2(3H)-one by a suitable alkylamino linker (compound 10 but not
11), or an aminoethylidene spacer (18–20).
3
2
1
0
4.1.4. Lipophilicity–anticonvulsant activity relationship
In search for relations between molecular properties and phar-
macological activity of the presented set of dihydrofuran-2(3H)-
one derivatives an interesting relationship has been found.
Statistical analysis revealed significant differences between mean
values of lipophilicity descriptors (RM0 as well as ClogP) and
activity in the MES and TOX (at any doses) tests. More specifically,
active compounds (doses 30, 100 or 300 mg/kg) displayed higher
RM0 values than inactive compounds (1.90 vs 1.33, Fig. 11, Table 2).
A similar tendency was observed for calculated log P values (ClogP
and MlogP) but in this case mean logP values for active com-
pounds were two times higher than for the inactive. Table 2 pres-
ent the results of this correlation study for ClogP values. Obtained
results show that inactive compounds are characterized by lower
log P values. These findings can be helpful in elimination of
potentially inactive compounds at early stages of the project.
Figure 11. Correlation between lipophilicity parameters RM0 and activity in MES
and TOX tests.
these examples only the 1-amino ethylidene derivatives of dihydro-
furan-2(3H)-one (18–20) showed some protective effect in MES and
low or no neurotoxicity. The N-phenyl derivative (18) displayed
moderate activity in the MES test (100 mg/kg), along with TOX
properties (300 mg/kg). Increasing the distance between the
aromatic ring and the nitrogen atom by exchanging phenylamine
for a benzylamine fragment (19) yielded decrease in anticonvulsant
and TOX activity. Moreover, introducing a homoveratrylamine
fragment at the same position (20) resulted in detectable activity
in both tests (300 mg/kg). Compounds without aromatic substitu-
ents (23, 24) were inactive in the MES test and TOX effects were
observed only for the morpholine derivative 24. Introduction of
benzylamine substituent, its rigid or amide analogs (compounds
11–17), as well as arylamino- or aryloxy- substituents (21, 22) gave
derivatives inactive in ASP tests.
4.2. Analgesic activity
Twelve compounds (1–6, 11, 15–17, 22, 24) were selected for
further pharmacological evaluation focused on their antinocicep-
tive activity in acute pain and local anesthesia tests in mice. The
selection was based on the anticonvulsant activity, low toxicity
and diversity of the structures.
Summing up, results of the preliminary anticonvulsant screen-
ing suggests that the substituent at position 3 determines both
Figure 12. Analgesic activity of the selected compounds and morphine in the hot plate test in mice [ED50].
Figure 13. Analgesic activity of selected compounds, and reference drugs in the writhing test in mice [ED50].

6538
K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
Figure 14. Local anesthetic activity of the tested compounds and the reference drugs: latency of reaction in the MTI test in mice (% of difference in comparison with control
group).
Table 1
Table 1 (continued)
Structures of the synthesized compounds
Symbol
Structure
R
Symbol
Structure
R
O
NH
1
2
H
3-CF3
R
19
20
O
3
4
5
6
7
O
O
2-MeO
3-MeO
4-MeO
2-MeO-5-Cl
4-F
N
O
O
N
O
O
NH
O
O
O
N
N
H
8
N
N
N
O
21
22
O
O
H2N
O
9
O
O
O
O
O
H
N
10
N
O
O
23
24
x HCl
x HCl
11
12
13
x HCl
4-MeO x HCl
2-CF3
O
R
R
H
O
N
O
O
N
O
O
H
N
14
O
O
4.2.1. Hot plate test
15
16
H
The hot plate assay (HP) is a rodent model of acute pain. In this
test pain is induced by perceptible but not harmful thermal stim-
uli. The characteristic response that occurs (jumping, licking of
paws) is of central origin and it is thought that drugs with antino-
ciceptive properties in the hot plate test act primarily in the spinal
medulla and/or higher layers of the central nervous system.42,43
Since peripherally acting analgesic drugs are generally inactive in
this pain model,42 morphine was chosen as a reference drug.
As shown in Figure 12 almost all the tested compounds showed
analgesic activity in the HP test (p <0.001 or 0.01 compared to
vehicle-treated mice, Student’s test). The lowest ED50 values were
assigned for compounds 3, 4, 11 and 24 along with the previously
published 1 and 2 and were comparable with morphine (MOR).17
As it was already published, derivative 22 was inactive in this
test.17
6,7-(MeO)2
N
O
O
O
17
18
N
O
O
O
NH
O

K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
6539
4.2.2. Writhing test
properties of the salicylamide moiety present in the structure. In
the MTI test the influence of the substibution at the phenyl ring
on the activity was similar to that observed for the activity in acute
pain tests. No correlation between the analgesic and anticonvul-
sant activity or lipophilicity has been found.
The writhing test (WT) is an inflammatory pain model widely
used for detection of compounds that exhibit peripheral antinoci-
ceptive activity.44 In this assay the phenylbenzoquinone-induced
pain phenomenon is associated with the synthesis and release of
arachidonic acid metabolites via cyclooxygenase and lipooxygen-
ase pathways.
5. Conclusion
Each of the tested compounds showed strong analgesic activity
in this test reducing the mean number of writhing responses in a
dosage-dependent and statistically significant manner (p <0.02
compared to the control group, Student’s test). All the ED50 values
were lower than for the reference drug, acetylsalicylic acid (ASA)
and in many cases comparable with morphine (Fig. 13).
As a part of our search for potential antiepileptic drugs, a series
of 3-, 4- or 5-substituted derivatives of dihydrofuran-2(3H)-one
was synthesized and tested in maximal electroshock, subcutane-
ous metrazole and neurotoxicity screens. Ten lactones were found
to be effective in the MES screen at a dose of 300 mg/kg and four of
them showed also protective effect at doses of 100 mg/g 0.5 h after
the administration. The results have shown that the CNS activity of
these derivatives of dihydrofuran-2(3H)-one hinges upon the pres-
ence of a phenylpiperazine moiety. Among eight phenylpiperazine-
containing structures, seven compounds were active in the MES
test and exhibited TOX effects, indicating their penetration to
CNS and inhibitory influence thereon.
Comparison of relative lipophilicity parameters determined by
chromatographic or computational methods with anticonvulsant
activity in MES test showed statistically significant differences be-
tween the mean values of RM0 and ClogP for active and inactive
compounds. These results along with strong correlation between
RM0 and ClogP parameters suggest that both computational meth-
ods may be used for prediction the lipophilicity of target com-
pounds, rendering useful assistance at early stages of design of
new potential anticonvulsant derivatives of 3-substituted dihydro-
furan-2(3H)-one or closely related structures.
4.2.3. Local anesthetic activity (modification of tail immersion
test, MTI)
The tail immersion test is an assay evaluating systemic analge-
sic activity, although with slight modifications can be used to
determine local anesthetic properties as well. In this test the la-
tency of animal’s response to thermal stimuli was measured for
two concentrations of the investigated compound (1% and 2%).45
The MTI test confirmed local anesthetic potency of five selected
compounds, however among them only compound 3 showed the
efficacy similar to the previous results (1 and 2) and the reference
compounds, mepivacaine (MEP) or lidocaine (LID). Compounds 4, 5
and 11 were inactive in the lower concentration. The differences in
latency of the response in comparison to the vehicle-treated mice
is presented in Figure 14.
4.2.4. Structure–analgesic activity relationship
The antinociceptive screening revealed mostly high analgesic
activity of the investigated compounds. In the group of phenylpip-
erazine derivatives, comparing with unsubstituted 1, HP activity
improves with introduction of a methoxy group at position 2 giv-
ing compound 3, the most active in this test but lowers slightly
with transfer thereof to the meta-position (4), and clearly drops
down with the para-substitution (5). Introduction of a chlorine
atom at position 50 of compound 3 gave also distinct decrease in
the activity (6). Benzylamino-GBL 11 showed stronger analgesic
properties than its rigid or amido- analogs 15–17. Similarly
to the HP, in the WT test the methoxy substitution at the
phenypiperazine gave the highest efficacy for the ortho-position
yielding the lowest ED50 value among the tested compounds while
for meta- and para-isomers and 5-chloro analogue gradual
decrease in the activity was observed. The most potent non-
phenylpiperazine derivative was lactone 11 (as in HP) with ED50
value lower than for morphine. No significant differences between
ED50 values were observed in this group, proving that aromatic
substituent is not necessary for the activity in this test. Interest-
ingly, compound 22 was inactive in HP test implies that the
presence of the lactone ring is insufficient to achieve the analgesic
activity. On the other hand 22 showed definite activity in the WT
test. This fact may be explained by anticipated anti-inflammatory
Due to proven efficacy of various anticonvulsant drugs in the
treatment of certain kinds of pain the pharmacological screening
was extended to antinociceptive activity evaluation in mice. Our
studies revealed strong analgesic activity of the tested derivatives,
comparable or even higher than of the reference compounds, mor-
phine, acetylsalicylic acid or mepivacaine. Derivative 3 was the
most potent among the tested lactones with ED50 of 0.66 mg/kg
in the HP and 0.28 mg/kg in the WT tests. Low ED50 values ob-
tained in these screens in combination with the lack of neurotoxic
effect at low doses (TOX test 1 ASP in mice, 30 mg/kg) have posi-
tively verified the initial concept to broaden our studies giving
good prospects for the development of this path of the project.
The specific mechanism which accounts for the pharmacologi-
cal activity of the presented compounds remains unknown. Some
conjectures concerning the activity of the most studied compound
2 have already been presented18 although, further investigation is
certainly necessary.
6. Experimental
6.1. Synthesis
The methods used for the synthesis of the desired compounds
are shown in Figures 4–6 and Table 1. Melting points were deter-
mined on an Electrothermal 9300 Melting-Point Apparatus and
are uncorrected. Reactions were monitored by TLC on silica gel
plates (5 ꢀ 8 cm, 0.25 mm; Kiselgel 60 F254, Merck) using the fol-
lowing solvent systems: S1 chloroform/acetone (1:1, v/v) and S2
CHCl3/MeOH/AcOH (60:10:5, v/v/v) with visualization of spots un-
der UV light. All reagents were purchased from Sigma–Aldrich Co.
1H NMR spectra were recorded with Varian Mercury 300 spec-
trometer at 300 MHz with tetramethylsilane as an internal stan-
dard. The following abbreviation are used: Ph, phenyl ring; 2-, 3-,
4-, 5- or 6-Ph, indicating position of the proton at the phenyl ring;
Table 2
Correlation between calculated lipophilicity parameter (ClogP) and anticonvulsant
activity (MES) and neurotoxicity (TOX)—statistical analysis
ASP test
MES
Activity
Mean
SEM
p
RM0
+
1.90
1.33
1.67
1.40
1.97
0.95
1.54
1.09
0.08
0.13
0.12
0.16
0.14
0.20
0.21
0.26
0002
ꢁ
TOX
MES
TOX
+
0.203
0.001
0.198
ꢁ
+
ClogP
ꢁ
+
ꢁ
PIP, piperazine ring; a-, b- or c-GBL, indicating 3-, 4- or 5-position

6540
K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
in the lactone ring; s, singlet; d, doublet; t, triplet; m, multiplet.
Elemental analyses of C, H, and N were carried out on a Varian
EL III Model Elemental Analyzer. Mass spectra were recorded on
MDX SCILEX API 2000 (Concord, ON, Canada) using the ESI method.
The synthesis of several compounds has already been published
in our publications (1,23 2,17 10,24 11,25 15,26 19,31 22,17 2446) or
elsewhere (17,28,29 18,32 2032).
(t, J = 5.0 Hz, 4H, CH2-3,5-PIP), 2.99 (m, 2H, CH2-2,6-PIP), 2.72 (m,
2H, CH2-2,6-PIP), 2.35 (m, 2H, CH2-b). ESI-MS (m/z) 277.5 [M+H]+.
Anal. calcd for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14. Found: C,
64.80; H, 7.69; N, 10.15.
6.1.4. 3-[4-(4-Methoxyphenyl)-piperazin-1-yl]-dihydrofuran-
2(3H)-one (5)
Procedure P1.
Reagents: 1-(4-methoxyphenyl)piperazine
6.1.1. General procedures for the synthesis of derivatives of
dihydrofuran-2(3H)-one
(4.81 g, 25 mmol), 3-bromo-dihydrofuran-2(3H)-one (4.12 g,
25 mmol), K2CO3 (3.46 g, 25 mmol), MeCN (80 ml); reaction time
5 h; crystallization from i-PrOH; yield: 4.6 g (67 %); mp 115–
116 °C; TLC: Rf (S1) = 0.63, Rf (S2) = 0.84; 1H NMR (CDCl3, d ppm):
6.1.1.1. Procedure P1 (3–9, 13, 16).
A mixture of the appro-
priate amine (1 equiv) or its hydrochloride salt and anhydrous
K2CO3 (1–1.2 equiv) in dry solvent (MeCN, DCM or toluene) was
6.87 (m, 4H, Ph), 4.40 - 4.22 (m, 2H, CH2-c), 3.76 (s, 3H, CH3O),
stirred at room temperature for 30 min.
A
solution of 3-
3.59 (t, J = 9.5 Hz, 1H, CH- ), 3.14 (t, J = 4.9 Hz, 4H, CH2-3,5-PIP),
a
bromo-dihydrofuran-2(3H)-one (1 equiv) in dry solvent (MeCN,
DCM) was then added dropwise over 15 min and stirring continued
for 5–48 h at ambient temperature. The mixture was then filtered
and the filtrate was evaporated to obtain a crude oily residue
which was purified by recrystallization from 2-propanol (i-PrOH)
or EtOAc.
3.00 (m, 2H, CH2-2,6-PIP), 2.73 (m, 2H, CH2-2,6-PIP), 2.35 (m, 2H,
CH2-b). ESI-MS (m/z) 277.5 [M+H]+. Anal. calcd for C15H20N2O3: C,
65.20; H, 7.30; N, 10.14. Found: C, 65.44; H, 7.54; N, 10.09.
6.1.5. 3-[4-(5-Chloro-2-methoxyphenyl)-piperazin-1-yl]-
dihydrofuran-2(3H)-one (6)
Procedure
P1.
Reagents:
1-(5-chloro-2-methoxyphenyl)
6.1.1.2. Procedure P2 (12, 14, 23, 24).
Starting with a solu-
piperazine (2.27 g, 10 mmol), 3-bromo-dihydrofuran-2(3H)-one
(1.65 g, 10 mmol), K2CO3 (1.38 g, 10 mmol), DCM (30 ml); reaction
time 7 h; crystallization from i-PrOH; yield: 2.07 g (67 %); mp 91–
92 °C; TLC: Rf (S1) = 0.72, Rf (S2) = 0.87; 1H NMR (CDCl3, d ppm):
6.91 (dd, J = 2.5, 8.6 Hz, 1H, 3-Ph), 6.89 (d, J = 2.5 Hz, 1H, 4-Ph),
tion of the appropriate amine (2.5 equiv) in dry MeCN or DCM,
anhydrous K2CO3 (1 or 2 equiv) and tetrabutylammonium bromide
(TBAB, 0.1 equiv) were added and the reaction mixture was stirred
at room temp for 30 min. Subsequently, the reaction mixture was
cooled down to 0 °C and a solution of 3-bromo-dihydrofuran-
2(3H)-one (2.5 equiv) in dry solvent (MeCN or DCM) was added.
Stirring was continued at 0 °C for 1 h and then at room temp for
3–50 h. The resultant mixture was filtered and the solvent evapo-
rated. The oily residue was dissolved in solution of EtOH or MeOH
with Et2O and acidified to pH 2–3 with saturated HCl solution in
EtOH. After 2–7 days of at 5 °C a hydrochloride salt precipitated.
The salt was then purified by recrystallization from the appropriate
solvent (EtOH or MeOH).
6.79 (d, J = 8.6 Hz, 1H, 6-Ph), 4.40 - 4.28 (m, 2H, CH2-
c), 3.89 (s,
3H, CH3O), 3.62 (t, J = 9.5 Hz, 1H, CH- ), 3.21–3.06 (m, 4H, CH2-
a
3,5-PIP), 3.01 (m, 2H, CH2-2,6-PIP), 2.76 (m, 2H, CH2-2,6-PIP),
2.46–2.28 (m, 2H, CH2-b). ESI-MS (m/z) 311.5 [M+H]+. Anal. calcd
for C15H19N2O3Cl: C, 57.97; H, 6.16; N, 9.01. Found: C, 57.41; H,
6.49; N, 8.91.
6.1.6. 3-[4-(4-Fluorophenyl)-piperazin-1-yl]-dihydrofuran-
2(3H)-one (7)
Procedure
P1.
Reagents:
1-(4-fluorophenyl)piperazine
6.1.1.3. Procedure P3 (18, 20).
To a solution of 3-acetyl-
(7.21 g, 40 mmol), 3-bromo-dihydrofuran-2(3H)-one (6.60 g,
40 mmol), K2CO3 (6.91 g, 50 mmol), MeCN (65 ml); reaction time
5 h; crystallization from i-PrOH; yield: 7.97 g (75 %); mp 95–
97 °C; TLC: Rf (S1) = 0.56, Rf (S2) = 0.75; 1H NMR (CDCl3, d ppm):
6.95 (m, 2H, 3,5-Ph), 6.87 (m, 2H, 2,6-Ph), 4.41 - 4.22 (m, 2H,
dihydrofuran-2(3H)-one (1 equiv) in toluene, an appropriate amine
(1 equiv) was added. The mixture was refluxed and the water
azeotropically removed (Dean–Stark apparatus). The solvent was
evaporated and a solid residue was recrystallized from i-PrOH.
CH2-c), 3.59 (t, J = 9.5 Hz, 1H, CH-a), 3.16 (m, 4H, CH2-3,5-PIP),
6.1.2. 3-[4-(2-Methoxyphenyl)-piperazin-1-yl]-dihydrofuran-
2(3H)-one (3)
3.00 (m, 2H, CH2-2,6-PIP), 2.73 (m, 2H, CH2-2,6-PIP), 2.40–2.29
(m, 2H, CH2-b). ESI-MS (m/z) 265.6 [M+H]+. Anal. calcd for
Procedure P1.
Reagents: 1-(2-methoxyphenyl)piperazine
C14H17N2OF: C, 63.62; H, 6.48; N, 10.60. Found: C, 63.30; H, 6.33;
(1.92 g, 10 mmol), 3-bromo-dihydrofuran-2(3H)-one (1.65 g,
10 mmol), K2CO3 (1.66 g, 12 mmol), toluene (30 ml); reaction time
7 h; crystallization from i-PrOH; yield: 1.25 g (45 %); mp 127–
128 °C; TLC: Rf (S1) = 0.63, Rf (S2) = 0.85; 1H NMR (CDCl3, d ppm):
N, 10.47.
6.1.7. 5-Methyl-3-(4-phenylpiperazin-1-yl]-dihydrofuran-
2(3H)-one (8)
7.05–6.82 (m, 4H, Ph), 4.45–4.27 (m, 2H, CH2-
c), 3.84 (s, 3H,
Procedure
P1.
Reagents:
1-phenylpiperazine
(1.62 g,
CH3O), 3.66 (m, 1H, CH- ), 3.06 (t, J = 4.5 Hz, 4H, CH2-3,5-PIP),
a
10 mmol), 3-bromo-5-methyl-dihydrofuran-2(3H)-one (1.79 g,
10 mmol), K2CO3 (1.38 g, 10 mmol), MeCN (50 ml); reaction time
5 h; crystallization from i-PrOH; yield: 1.3 g (50 %); mp 105–
107 °C; TLC: Rf (S1) = 0.75, Rf (S2) = 0.89; 1H NMR (CDCl3, d ppm):
7.27 (m, 2H, 3,5-Ph), 7.00–6.79 (m, 3H, 2,4,6-Ph), 4.55–4.41 (m,
3.05 (m, 2H, CH2-2,6-PIP), 2.82–2.71 (m, 2H, CH2-2,6-PIP), 2.47–
2.26 (m, 2H, CH2-b). ESI-MS (m/z) 277.4 [M+H]+. Anal. calcd for
C15H20N2O3: C, 65.20; H, 7.30; N, 10.14. Found: C, 64.91; H, 7.32;
N, 9.94.
1H, CH-c), 3.72 (m, 1H, CH-a), 3.29 (m, Hz, 2H, CH2-3,5-PIP),
6.1.3. 3-[4-(3-Methoxyphenyl)-piperazin-1-yl]-dihydrofuran-
2(3H)-one (4)
3.08 (m, 2H, CH2-3,5-PP), 2.81 (m, 4H, CH2-2,6-PIP), 2.50 (m, 1H,
CH2-b), 1.97 (m, 1H, CH2-b), 1.52 (m, 3H, CH3). ESI-MS (m/z)
261.5 [M+H]+. Anal. calcd for C15H20N2O2: C, 69.20; H, 7.74;
N,10.76. Found: C, 69.53; H, 8.12; N, 10.92.
Procedure P1.
Reagents: 1-(3-methoxyphenyl)piperazine
(1.92 g, 10 mmol), 3-bromo-dihydrofuran-2(3H)-one (1.65 g,
10 mmol), K2CO3 (1.66 g, 12 mmol), MeCN (30 ml); reaction time
5 h; crystallization from i-PrOH; yield: 1.62 g (59 %); mp 79–
81 °C; TLC: Rf (S1) = 0.68, Rf (S2) = 0.85; 1H NMR (CDCl3, d ppm):
7.17 (t, J = 8.1 Hz, 1H, 5-Ph), 6.54–6.39 (m, 3H, 2,4,6-Ph), 4.40 -
6.1.8. 4-(4-Phenylpiperazin-1-yl]-dihydrofuran-2(3H)-one (9)
Procedure.
To a solution of 1.43 g (17 mmol) of 5-H-furan-2-
one in 2 ml of DCM a solution of 2.76 g (17 mmol) of 1-phenylpip-
erazine in 2 ml of DCM was added dropwise over 30 minutes. The
4.22 (m, 2H, CH2-c), 3.79 (s, 3H, CH3O), 3.59 (m, 1H, CH-a), 3.24

K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
6541
mixture was then stirred for 3 h. Evaporation of the solvent and
6.1.13. 2-(2-Oxotetrahydrofuran-3-yl)-isoindolin-1,3,dion (17)
recrystallization from EtOAc yielded 1.87 g (45 %) of compound
28; mp 127–129 °C; TLC: Rf (S1) = 0.55, Rf (S2) = 0.88; 1H NMR
(CDCl3, d ppm): 7.35–7.20 (m, 2H, 3,5-Ph), 6.99–6.81 (m, 3H,
Procedure.
A mixture of 4.08 g (22 mmol) of potassium
phthalimide, 3.63 g (22 mmol) of 3-bromo-dihydrofuran-2(3H)-
one and 30 ml of DMF was stirred and heated at 100 °C for 8 h.
The mixture was then poured on crumbled ice (100 g). The precip-
itated product was filtered off, washed with water and recrystal-
lized from i-PrOH; yield 4.42 g (87 %); mp 167–169 °C; TLC: Rf
(S1) = 0.76, Rf (S2) = 0.85; 1H NMR (CDCl3, d ppm): 7.93–7.83 (m,
2H, 4,7-PHT), 7.79–7.73 (m, 2H, 5,6-PHT), 5.10 (m, 1H, CH-
4.66 (m, 1H, CH2- ), 4.40 (m, 1H, CH2- ), 2.79 (m, 1H, CH2-b),
2.60 (m, 1H, CH2-b). ESI-MS (m/z) 232.4 [M+H]+. Anal. calcd for
2,4,6-Ph), 4.46 (dd, J = 7.0, 9.2 Hz, 1H, CH2-
c), 4.24 (dd, J = 6.5,
9.2 Hz, 1H, CH2- ), 3.38 (t, J = 6.8 Hz, 1H, CH-b), 3.21 (m, 4H,
c
CH2-3,5-PIP), 2.72–2.51 (m, 6H, CH2-2,6-PIP, CH2-a). ESI-MS (m/z)
247.5 [M+H]+. Anal. calcd for C14H18N2O2: C, 68.27; H, 7.37;
N,11.37. Found: C, 67.91; H, 7.41; N, 11.27.
a -),
c
c
6.1.9. 3-(4-Methoxybenzylamine)-dihydrofuran-2(3H)-one x
HCl (12)
Procedure P2.
C12H9NO4: C, 62.34; H, 3.92; N, 6.06. Found: C, 61.69; H, 4.06; N,
Reagents: 4-methoxybenzylamine (3.43 g,
5.88. According to29 mp 173–176 °C.
25 mmol), 3-bromo-dihydrofuran-2(3H)-one (4.12 g, 25 mmol),
K2CO3 (6.91 g, 50 mmol), MeCN (70 ml); reaction time 50 h; crys-
tallization from EtOH; yield: 5.4 g (84 %); mp 192–195 °C; TLC: Rf
(S1) = 0.63, Rf (S2) = 0.59; 1H NMR (CDCl3, d ppm): 10.13 (s, 2H,
NH2+), 7.48 (d, J = 8.8 Hz, 2H, 2,6-Ph), 6.97 (d, J = 8.8 Hz, 2H, 3,5-
6.1.14. 3-[1-(Phenylamino)-ethylidene]-dihydrofuran-2(3H)-
one (18)
Procedure P3.
Reagents: phenylamine (1.16 g, 12.5 mmol), 3-
acetyl-dihydrofuran-2(3H)-one (1.60 g, 12.5 mmol), toluene
(30 ml); reaction time 1 h; crystallization from i-PrOH; yield:
1.5 g (59 %); mp 85–87 °C; TLC: Rf (S1) = 0.84, Rf (S2) = 0.90; 1H
NMR (CDCl3, d ppm) 9.98 (s, 1H, NH), 7.32 (t, J = 7.8 Hz, 2H, 3,5-
Ph), 7.10 (t, J = 7.4 Hz, 1H, 4-Ph), 7.09 (d, J = 7.4 Hz, 2H, 2,6-Ph),
Ph), 4.46 (m, 1H, CH-a), 4.37–4.09 (m, 4H, CH2-c, CH2-BA), 3.75
(s, 3H, CH3O), 2.64–2.33 (m, 2H, CH2-b). -b). ESI-MS (m/z) 222.3
[M+H]+. Anal. calcd for C12H16NO3Cl: C, 55.93; H, 6.26; N, 5.43.
Found: C, 55.74; H, 6.44; N, 5.41.
4.35 (m, 2H, CH2-c), 2.91 (t, J = 7.9 Hz, 2H, CH2-b), 2.02 (s, 3H,
6.1.10. 3-[2-Trifluomethylbenzylamine)-dihydrofuran-2(3H)-
one (13)
CH3). ESI-MS (m/z) 204.3 [M+H]+. Anal. calcd for C12H13NO2: C,
70.92; H, 6.45; N, 6.89. Found: C, 70.50; H, 6.60; N, 6.77. According
to32 mp 75–77 °C.
Procedure P1.
Reagents: 2-trifluomethylbenzylamine (5.25 g,
30 mmol), 3-bromo-dihydrofuran-2(3H)-one (4.95 g, 30 mmol),
K2CO3 (4.15 g, 30 mmol), MeCN (30 ml); reaction time 40 h; crys-
tallization from isopropanol; yield: 3.3 g (42 %); mp 51–52 °C;
TLC: Rf (S1) = 0.80, Rf (S2) = 0.89; 1H NMR (CDCl3, d ppm): 7.66
(m, 2H, 4,6-Ph), 7.59 (t, J = 7.5 Hz, 1H, 5-Ph), 7.42 (t, J = 7.6 Hz,
6.1.15. 3-[1-[2-(3,4-Dimethoxyphenyl)ethylamino]-ethylidene]-
dihydrofuran-2(3H)-one (20)
Procedure P3.
Reagents: 3,4-dimethoxyphenylethylamine
(3.62 g, 20 mmol), 3-acetyl-dihydrofuran-2(3H)-one (2.56 g, 20
mmol), toluene (30 ml); reaction time 1 h; crystallization from i-
PrOH; yield: 3.5 g (60 %); mp 114–115 °C; TLC: Rf (S1) = 0.77, Rf
(S2) = 0.92; 1H NMR (CDCl3, d ppm) 8.34 (s, 1H, NH), 6.88–6.66
1H, 3-Ph), 4.41 (m, 1H, CH2-
CH2-Bz), 3.60 (m, 1H, CH- ), 2.49–2.21 (m, 2H, CH2-b), NH proton
not detected. ESI-MS (m/z) 260.4 [M+H]+. Anal. calcd for
12H12NO2F3: C, 55.60; H, 4.67; N, 5.40. Found: C, 55.70; H, 4.88;
c), 4.25 (m, 1H, CH2-c), 4.07 (m, 2H,
a
C
(m, 3H, Ph), 4.29–4.21 (m, 2H, CH2-c), 3.96 (s, 3H, CH3O), 3.92 (s,
N, 5.50.
3H, CH3O), 3.42 (m, 2H, CH2-1-PEA), 2.87–2.70 (m, 4H, CH2-2-
PEA, CH2-b), 1.85 (s, 3H, CH3).). ESI-MS (m/z) 292.6 [M+H]+. Anal.
calcd for C16H21NO4: C, 65.96; H, 7.27; N, 4.81. Found: C, 65.85;
H, 7.43; N, 4.80. According to32 mp 112–113 °C.
6.1.11. 3-Benzylamine-5-methyl- dihydrofuran-2(3H)-one x HCl
(14)
Procedure P2.
bromo-5-methyl-dihydrofuran-2(3H)-one
Reagents: benzylamine (1.07 g, 10 mmol), 3-
(1.79 g, 10 mmol),
6.1.16. 3-(2,6-Dimethylphenylamine)-dihydrofuran-2(3H)-one
K2CO3 (1.38 g, 10 mmol), MeCN (35 ml); reaction time 20 h; crys-
tallization from acetone; yield: 1.43 g (59 %); mp 215–217 °C;
TLC: Rf (S1) = 0.74, Rf (S2) = 0.85; 1H NMR (CDCl3, d ppm): 10.31
(s, 2H, NH+), 7.58 (d, J = 4.1 Hz, 2H, 3,5-Ph), 7.44 (d, J = 4.8 Hz,
(21)
Procedure.
of 2,6-dimethylphenylamine and 4.12 (25 mmol) of 3-bromo-dihy-
drofuran-2(3H)-one, heated to 80 °C, solution of 1.73 g
To a solution of 8.5 ml toluene, 3.03 g (25 mmol)
a
3H, 2,4,6-Ph), 4.65 (m, 1H, CH2-
CH- ), 2.82 - 2.57 (m, 1H, CH2-b), 2.21–2.01 (m, 1H, CH2-b),
1.36 (m, 3H, CH3). ESI-MS (m/z) 206.2 [M+H]+. Anal. calcd for
12H16NO2Cl: C, 59.63; H, 6.67; N,5.79. Found: C, 59.33; H, 6.75;
c
), 4.57–4.14 (m, 3H, CH2-Bz,
(12.5 mmol) of K2CO3 in 5 ml of water was added dropwise over
5 h. After cooling, the mixture was washed with 5% solution of
KHCO3 (10 ml) and water (3 ꢀ 25 ml). The organic layer was dried
over Na2SO4, filtered off and the solvent removed in vacuo. The oily
residue was dissolved in Et2O which crystallized slowly after a few
days at 5 °C; yield 3.2 g (62 %); mp 83–84 °C; TLC: Rf (S1) = 0.79, Rf
(S2) = 0.94; 1H NMR (CDCl3, d ppm) 7.02 (d, J = 7.4 Hz, 2H, 3,5-Ph),
a
C
N, 5.76.
6.1.12. 3-(6,7-Dimethoxy-3,4,dihydro-1H-isoquinolin-2-yl)-
dihydrofuran-2(3H)-one (16)
6.93 (m, 1H, 4-Ph), 4.46–4.23 (m, 2H, CH2-c), 4.01 (m, 1H, CH-a),
Procedure P1.
Reagents: 6,7-dimethoxy-1,2,3,4-tetrahydro-
2.68 (m, 2H, CH2-b), 2.40 (m, 6H, 2CH3, CH2-b). ESI-MS (m/z)
206.0 [M+H]+. Anal. calcd for C12H15NO2: C, 70.22; H, 7.37; N,
6.82. Found: C, 69.69; H, 7.71; N, 6.84. According to48 mp 85–86 °C.
isoquinoline (2.71 g, 14 mmol), 3-bromo-dihydrofuran-2(3H)-one
(2.47 g, 15 mmol), K2CO3 (6.22 g, 45 mmol), MeCN (160 ml); reac-
tion time 5 h; crystallization from i-PrOH; yield: 0.88 g (23 %); mp
100–101 °C; TLC: Rf (S1) = 0.65, Rf (S2) = 0.87; 1H NMR (CDCl3, d
ppm): 6.59 (s, 1H, 8-TQ), 6.51 (s, 1H, 5-TQ), 4.42 - 4.24 (m, 2H,
6.1.17. 3-Cyclohexylamine-dihydrofuran-2(3H)-one x HCl (23)
Procedure: P2.
Reagents: cyclohexylamine (2.48 g, 25 mmol),
CH2-
c
), 4.01 (d, J = 14.1 Hz, 1H, CH2-1-TQ), 3.85 (s, 3H, CH3O),
3-bromo-dihydrofuran-2(3H)-one (4.13 g, 25 mmol), K2CO3
(3.45 g, 25 mmol), DCM (40 ml); reaction time 20 h; crystallization
from EtOH; yield: 1.65 g (30 %); mp 244–245 °C; TLC: Rf (S1) = 0.69,
Rf (S2) = 0.73; 1H NMR (CDCl3, d ppm) 10.05 (s, 1H, NH2+), 9.49 (s,
3.81 (s, 3H, CH3O), 3.80–3.66 (m, 2H, CH2-1-TQ , CH-
a), 3.16–3.03
(m, 1H, CH2-3-TQ), 2.92–2.79 (m, 3H, CH2-3-THIQ, CH2-4-TQ),
2.50–2.30 (m, 2H, CH2-b). ESI-MS (m/z) 278.4 [M+H]+. Anal. calcd
for C15H19NO4: C, 64.97; H, 6.91; N, 5.05. Found: C, 64.84; H,
7.22; N, 5.09.
1H, NH2+), 4.66–4.43 (m, 2H, CH-
CH2-
a
, CH2-
c), 4.36–4.17 (m, 1H,
c
), 3.20 (t, J = 10.8 Hz, 1H, CH-1-CYC), 2.63–2.45 (m, 2H,

6542
K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
CH2-b), 2.16–1.94 (m, 2H, CH2-2,6-CYC), 1.77 (m, 2H, CH2-2,6-CYC),
1.66–0.97 (m, 6H, CH2-3,4,5-CYC). ESI-MS (m/z) 184.6 [M+H]+.
Anal. calcd for C10H18NO2Cl: C, 54.67; H, 8.26; N, 6.37. Found: C,
54.03; H, 8.63; N, 6.34. Compound 20 has been described as a free
base.47
current of 50 mA intensity (5–7 times the levels necessary to elicit
minimal electroshock seizures) delivered over 0.2s via corneal
electrodes. A drop of 0.5% tetracaine hydrochloride in 0.9% saline
was instilled in the eye prior to application of the electrodes.
Protective endpoints were defined as abolition of the hind limb to-
nic extension component of the seizure. Results are expressed as a
ratio of the number of animals protected/number of animals
tested.
6.2. Determination of lipophilicity
6.2.1. Reversed-phase thin-layer chromatography
Silica gel RP-18 F254s plates, 10 ꢀ 10 cm (Merck, Darmstadt,
Germany), were used for measurement by planar chromatography.
Mixtures of MeCN (Merck) and Tris buffer (pH 7.4) containing 10–
90% (v/v) of organic modifier decreasing in 5% (v/v) steps were
used as mobile phases. The Tris buffer was prepared by dissolving
24.35 g of Tris (Merck) in 250 mL distilled water and adjusting this
solution to pH 7.4 with 0.1 mol L-1 hydrochloric acid (POCH, Gliw-
ice, Poland). Analytical-grade chemicals were used unless indi-
cated otherwise. Solutions of the tested compounds in MeOH
6.3.1.3. Pentetrazole inducted seizure (scMet).
A dose of
85 mg/kg of pentetrazole which produces seizures in more than
95% of mice, was administered as a 0.5% solution subcutaneously
in the posterior midline. Animals were observed for 30 minutes.
Failure to observe even a threshold seizure (a single episode of clo-
nic spasms with a minimum duration of 5 seconds) was defined as
protection and the results were expressed as the number of ani-
mals protected/number of animals tested.
were prepared at concentrations of 1 mg/mL. 10 lL of each sample
6.3.1.4. Neurotoxicity (TOX)50,51
.
The rotorod test was used
was applied onto plates which were then placed in TLC chambers
(5 ꢀ 15 ꢀ 15), previously saturated with the solvents for 2 h. Fol-
lowing development, the plates were dried and the spots observed
under UV light at k = 254 nm. All measurements were conducted at
ambient temperature. Mean retention factor (Rf) values were cal-
culated for three independent determinations and converted to
retention parameters (RM) using the following equation:
to evaluate neurotoxicity in mice. Animals were placed on a 1-inch
diameter knurled plastic rod rotating at 6 rpm. Non-toxic (normal)
mice can remain on a rod rotating at this speed almost indefinitely.
Neurological toxicity is defined as the failure of the animal to re-
main on the rod for 1 min and is expressed as the number of ani-
mals exhibiting toxicity/number of animals tested. Animals are
considered toxic if they fail this test on three successive attempts.
Rat toxicity was determined using overt evidence of ataxia, abnor-
mal gait or the positional sense test.
RM ¼ logð1=Rf ꢁ 1Þ
ð1Þ
The RM values for each compound, obtained with different sol-
vent systems, were then extrapolated to zero concentration of
MeCN in the mobile phase to estimate the relative retention
parameters (RM0) according to the equation:
6.3.1.5. Minimal clonic seizure (6-Hz) model37–39
test is used to assess a compound’s efficacy against electrically-
induced seizures but uses lower frequency (6 Hz) and
.
The 6-Hz
a
onger duration of stimulation (3 s). Test compounds are pre-
administered to mice via ip injection. Corneal stimulation
(0.2 ms monopolar rectangular pulses at 6 Hz over 3 s) was
delivered by a constant-current device. During the stimulation,
mice were manually restrained and released into the obser-
vation cage immediately following the application of the current.
Untreated mice will display seizures characterized by a minimal
clonic phase followed by stereotyped, automatistic behaviors de-
scribed originally as being similar to the aura of human patients
with partial seizures. The duration of the seizure activity ranged
from 60 to 120 s in untreated animals. Upon resolution of seizures
animals resumed their normal exploratory behavior. The experi-
mental endpoint was protection against seizure. The animal was
considered to be protected if it resumed its normal exploratory
behavior within 10 s of stimulation.
RM ¼ RM0 þ aC
ð2Þ
where C is the concentration of MeCN in the mobile phase (% v/v).
6.2.2. Calculation of partition coefficients
Theoretical partition coefficients of the tested compounds were
calculated using two computer programs: ChemOffice Ver. 8,0;
1985–2003 CambridgeSoft Corporation, (ClogP) and MarvinView
5.2.0 (MlogP).
6.3. Pharmacology
6.3.1. ASP - anticonvulsant assays
Preliminary anticonvulsant evaluation (Test I) of all the synthe-
sized lactones was performed by applying test protocols49 de-
signed and sponsored by the National Institute of Neurological
Disorders and Stroke, NIH, Rockville, MD, USA, for the Anticonvul-
sant Screening Program (ASP). Test I evaluations consisted of three
tests performed in mice (ip): maximal electroshock, subcutaneous
pentetrazole, and rotarod test for neurological toxicity. Advanced
studies included MES and TOX tests in rats (po) and 6-Hz test in
mice.
6.3.2. Analgesic activity tests
Analgesic activity studies were carried out in collaboration with
the Department of Pharmacodynamics, Jagiellonian University,
Medical College. Preliminary antinociceptive activity evaluation
consisted of three tests performed in mice: hot plate test, writhing
test and modified tail immersion test.
6.3.1.1. Animals.
Male albino, CF No. 1 mice (18–25 g, Charles
6.3.2.1. Chemicals.
Except for the local anesthetic activity
River, Willimington, MA, USA) and male albino, Sprague-Dawley
rats (100–150 g, Charles River, Willimington, MA, USA) were used
in the experiment. Compounds were either dissolved in saline or
suspended in 0.5% methylcellulose. The compound was adminis-
tered to mice by ip injection at three dosage levels (30, 100 and
300 mg/kg) with anticonvulsant activity and neurotoxicity noted
15, 30, 60 and 240 minutes after administration.
test, the investigated compounds were administered intraperito-
neally (ip) in the form of a suspension in 0.5% methylcellulose
(MC) (Loba Chemie) solution 30 min before each assay. Phen-
ylbenzoquinone (INC Pharmaceuticals, Inc. N.Y.) was prepared as
a 0.02% solution. Morphine (Morphinum hydrochloricum, Polfa
Kutno), acetylsalicylic acid (Polpharma), mepivacaine (Meaverin
2%, Rhone-Poulenc Rorer) and lidocaine (Lignocainum hydrochlor-
icum 2%, WZF) were used as reference drugs. Control animals were
given appropriate amounts of vehicle (MC).
6.3.1.2. Maximal electroshock seizure (MES) test49
mal electroshock seizures were elicited with a 60-cycle alternating
.
Maxi-

K. Wie˛ckowski et al. / Bioorg. Med. Chem. 20 (2012) 6533–6544
6543
6.3.2.2. Animals.
For the behavioral experiments, adult male
Supplementary data
Albino Swiss (CD-1) mice weighing 18–30 g were used. The ani-
mals were kept in groups of 15 mice in cages at a temperature of
22 2 °C, under a light/dark (12/12) cycle and had free access to
food and water before the experiments. Each experimental group
consisted of 6–8 animals/dose and all the animals were used only
once. In all experiments the mice were habituated to the vivarium
for a minimum of 72 h prior to experimentation. The experiments
were performed between 08:00–15:00. The procedures were ap-
proved by the Local Ethics Committee of the Jagiellonian University
in Cracow (ZI/329/2006).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.037.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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Acknowledgements
The authors deeply appreciate the assistance of the Anticonvul-
sant Screening Program, National Institute of Neurological Disor-
ders and Stroke, NIH, USA, and would also like to express
´
personal thanks to Prof. dr Katarzyna Kiec Kononowicz from the
Department of Technology and Biotechnology of Drugs the Faculty
of Pharmacy, Jagiellonian University, Kraków Poland. This work
was supported by the Polish Ministry of Science and Higher Educa-
tion grant no. N N405 163339 and by the Jagiellonian University
Collegium Medicum grant no. K/ZDS/001918.
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