Synthesis and structure-activity relationship studies of theophylline analogs on population responses in the rat hippocampus in vitro

Article abstract of DOI:10.1016/j.bmc.2008.07.045

We synthesized several theophylline analogs and tested the hypothesis that these compounds may be nootropic or cognitive enhancers by examining their effects on evoked population spikes recorded extracellularly in the CA1 region of the rat hippocampus. Whereas the length of the carbon chain on N7 had no effect, different size of the terminal lactam ring strongly influenced neuroactivity. Our results suggest that hexahydroazepin-2-one analogs have potential for further development as cognitive enhancers.

Full text of DOI:10.1016/j.bmc.2008.07.045

Bioorganic & Medicinal Chemistry 16 (2008) 8142–8150
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship studies of theophylline analogs
on population responses in the rat hippocampus in vitro
a
b
a
c
d
´
Kethireddy V. V. Ananthalakshmi , Tomáš Bartl , Mohammed H. Aziza , Ladislav Novotny , Radek Marek ,
b
a,
*
ˇ
Ludek Beneš , Samuel B. Kombian
a Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
b Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, Brno CZ-61242, Czech Republic
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
d National Center for Biomolecular Research, Faculty of Sciences, Masaryk University, Kamenice 5/A4, Brno CZ-62500, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized several theophylline analogs and tested the hypothesis that these compounds may be
nootropic or cognitive enhancers by examining their effects on evoked population spikes recorded extra-
cellularly in the CA1 region of the rat hippocampus. Whereas the length of the carbon chain on N7 had no
effect, different size of the terminal lactam ring strongly influenced neuroactivity. Our results suggest
that hexahydroazepin-2-one analogs have potential for further development as cognitive enhancers.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 21 June 2008
Revised 15 July 2008
Accepted 17 July 2008
Available online 23 July 2008
Keywords:
Hippocampus
Acetylcholine receptors
Dementia
Nootropic
Theophylline analogs
1. Introduction
inhibitors donepezil, revastigmine, and galantamine.2,15,16 How-
ever, therapy with these drugs provides only modest clinical relief
Dementia is a debilitating, chronic neurodegenerative disorder
characterized by mild to severe impairment in cognitive function
including memory, orientation, language, visual perception, and
executive function.1,2 These deficiencies affect the ability of suffer-
ers, who are not unconscious, to perform activities of everyday liv-
ing. Different types of dementia, with pathological and clinical
features that may overlap, are recognized including Alzheimer’s
dementia or disease (AD), vascular dementia, dementia with Lewy
bodies, and hippocampal sclerosis dementia.3–6 AD, the most com-
mon type of dementia, has a complex pathophysiology that in-
volves alteration of protein metabolism (b-amyloid and tau
protein) and inappropriate neurotransmission including those
mediated by glutamate, serotonin, dopamine, noradrenalin, and
acetylcholine.6,7 The most common neurochemical change found
in brains of AD patients is a decrease in acetylcholine in cortical
and subcortical regions such as the hippocampus, which are in-
volved in higher cognitive function.8–13 Currently, disease modify-
ing agents (e.g., amyloid modifying agents) and symptomatic
management agents6,7,14 are two main pharmacotherapeutic ap-
proaches. The latter approach represents an essential part of cur-
rent treatment of AD, and includes the anticholinesterase
of symptoms,1,17 and does not stop or reverse disease progression.
In addition, the long-term benefits of their use are unclear, and
their use is often accompanied by intolerable side or adverse ef-
fects.18–20 These drawbacks of the cholinesterase inhibitors have
triggered a search for drugs characterized by reduced side effects,
patient acceptance in long-term use and potential to inhibit the
neuronal loss associated with AD and other dementias.21 Recently,
memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist
with neuroprotective action and a potential to halt disease pro-
gression, has been approved for use in AD.16
In this study, neuronal responses to novel theophylline analogs
in the hippocampus, a structure involved in learning and memory,
have been investigated in order to identify possible candidates for
further development as cognitive enhancers. The compounds
tested were synthesized by chemically linking a purine-based
compound (theophylline) with lactam rings through a carbon
chain because both these chemical groups have each shown poten-
tial for cognitive enhancement. For example, methylxanthines that
are naturally present in some foods have CNS actions that may
influence memory.22,23 Similarly, some derivatives of purines are
currently undergoing clinical trials for use in AD patients because
of their nootropic or cognitive enhancement effects.24 As well, lac-
tam rings are the primary structure of the racetams, which are also
currently being used for memory enhancement.25 The rationale for
* Corresponding author. Tel.: +965 498 6916; fax: +965 534 2807.
E-mail address: kombian@hsc.edu.kw (S.B. Kombian).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.045

K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
8143
the synthesis of these compounds was therefore to see if a combi-
O
O
nation of a purine structure with a lactam ring would yield com-
pounds with enhanced nootropic potential. Here, we report that
some structural analogs of theophylline (a purine compound) syn-
thesized by linking different lactam rings to this primary structure
produce effects on hippocampal population responses that are con-
sistent with cognitive enhancement while others have either no ef-
fect or produce the exact opposite effects.
O
N
O
12
O
14
O
N
10
15
16
13
6
1
N
N
18
7
5
H3C
H3C
O
N
N9
N
N
N
8
O2
N
6
4
3CH3
CH3
11
7
8
O
O
O
O
O
N
N
N
2. Results
H3C
O
O
N
N
H3C
N
N
N
N
2.1. Chemistry
CH3
O
N
CH3
The target compounds were prepared in a two-step synthesis.
10
9
Briefly, in the first step, N-(x-chloroacyl)-lactams 1–6 were pre-
pared by reacting -chloroacyl chlorides with various lengths of
the chain (2-chloroacetyl chloride, 3-chloropropionyl chloride, 4-
chlorobutyryl chloride) and five- to seven-membered lactams
x
O
N
O
O
14
16
O
17
15 N
18
19
12
O
13
10
6
22
21
O
1
N
7
5
H3C
N
N9
(Scheme 1). N-(x-chloroacyl)-lactams were purified by vacuum
20
H3C
O
8
N
N
O2
N
N
distillation except for 4, which was unstable under heating.26 In
the second step, theophylline analogs 7–12 were obtained by treat-
4
3CH3
N
11
CH3
ment of N-(x-chloroacyl)-lactams with theophylline in N,N-
dimethylformamide (Scheme 1, Fig. 1), and were re-crystallized
12
11
from propan-2-ol.
Figure 1. Structures of theophylline analogs with atom numbering.
Structures of final compounds were determined by using MS, IR,
and 1H, 13C, and 15N NMR spectroscopy. 1H–13C gs-HSQC, 1H–13
C
tion, and bath perfused for 5–6 min. Bath application of 10 lM
GSQMBC, 1H–15N gs-HMBC, and 1H–15N GSQMBC experiments were
used to assign individual NMR resonances to the particular atoms.
Generally, purine compounds can be N-alkylated at N3, N7, or N9
nitrogens due to the tautomerism phenomenon.27 In this contribu-
tion, the position of N-alkylation of theophylline ring was unequiv-
ocally determined by using 1H–13C GSQMBC experiment.28 Three
bond correlation of H12 of acyl chain with C5 and C8 of purine ring
in compounds 7–12 unambiguously confirmed the position of sub-
stituent at N7. This conclusion is further supported by analysis of
compound 12 caused a consistent enhancement in the amplitude
of PS by 56.5 14.7% (p < 0.05, paired t-test, n = 7, Figs. 2 and 4).
Maximal PS enhancement was observed after 6 min application,
and recovery was not significant after more than 20 min washout
of compound 12 as the effect was still significantly enhanced at
45.8 22.7% (p < 0.05, paired t-test, n = 7, Fig. 2). This effect of com-
pound 12 on PS amplitude was concentration-dependent with a
minimum effective concentration of 0.1
lM and a maximum con-
centration of 100
lM with a calculated EC50 value of ꢀ2
lM (Fig.
3JH8–C5 (4.7–5.0 Hz) and JH8–C4 coupling constants (12.7–13.1 Hz).
3
4A). Similar to the effects of compound 12, both compounds 10
and11 caused enhancement of PS amplitude (42.4 14.2% and
40.9 14.0%, respectively, p < 0.05, paired t-test, n = 4 each, Fig.
4B). In contrast to the above analogs, compound 9 caused a concen-
tration-dependent and reversible depression in the amplitude of PS
(À25.9 4.6%, p < 0.05, paired t-test, n = 5, Figs. 3 and 4). Recovery
from depression was almost complete (91.1 4.6%) and rapid
(within 10 min of washout). The threshold concentration was
3
3
The larger values of JH8–C4 (ꢀ 13 Hz) as compared to JH8–C5 are
characteristic of the N7-substituted purine derivatives.29
2.2. Electrophysiology
Theophylline analogs used in this study included compounds 7–
12. The results reported here were obtained from extracellular field
recordings of populations spikes (PS) in 90 hippocampal slices. The
PS recorded in the CA1 region was shown to be TTX-sensitive as
0.01
l
M with a peak concentration of 10
lM, and a calculated
EC50 value was 0.06
lM. Finally, the pyrrolidin-2-one derivatives
they were abolished by bath application of 1 lM TTX (Fig. 2)
7 and 8 did not cause significant changes in the PS amplitude
(6.4 4.4%, p > 0.05, n = 6/5, Fig. 4).
2.3. Effect of theophylline analogs on PS recorded in area CA1
of the hippocampus
2.4. Pharmacology of compound 12 effects on PS
Following the recording of stable PS for at least 15 min, all the
theophylline analogs were diluted to the final desired concentra-
To explore possible mechanisms by which the enhancers pro-
duced their effect on PS amplitude, we chose compound 12 as their
(CH2)y
(CH2)y
Et3N
(CH2)x
(CH2)x
Cl
N
+
Cl
Cl
HN
EtOAc
O
O
O
O
1-6
(CH2)y
O
(CH2)y
H
O
(CH2)x
H3C
O
N
N
KI, K2CO3
DMF
N
(CH2)x
N
H3C
O
N
N
N
+
Cl
N
O
O
O
N
O
N
CH3
CH3
7-12
Scheme 1. Synthesis of intermediates 1–6 and theophylline analogs 7–12; x = 1–3, y = 1–3.

8144
K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
Figure 2. The theophylline analog compound 12 enhances evoked PS amplitude
recorded in the cell body layer of area CA1 of the hippocampus. (A) A time-effect
plot in a representative cell showing that compound 12 (10 lM) enhances the
amplitude of TTX sensitive PS. Inserts are sample PS traces taken at the times
indicated by alphabets in the graph. In this and in all time-effect plots, each filled
circle represents the average response taken in a minute. The length of the solid line
below each drug represents the duration of application, and the broken lines
indicate baseline. (B) An average time-effect plot obtained from 8 slices showing
the consistent effect of compound 12 in enhancing PS amplitude. (C) A bar graph
Figure 3. Another theophylline analog compound 9 depresses evoked PS amplitude
recorded in the cell body layer of area CA1 of the hippocampus. (A) A time-effect
plot in a representative cell showing that compound 9 (10 lM) reversibly depresses
the amplitude of evoked PS. Inserts are sample PS traces taken at the times
indicated by alphabets in the graph. (B) An average time-effect plot obtained from 5
slices showing the consistent effect of compound 9 in depressing PS amplitude. (C)
A bar graph summarizing the effect of 10
amplitude.
lM compound 9 (n = 5) on evoked PS
summarizing the effect of 10 lM compound 12 (n = 8) and 1 lM TTX (n = 6) on
evoked PS amplitude (In this figure and in all other figures, Ãindicates significance
Further, we tested if compound 12 employed other mechanisms
(non-cholinergic) to produce the enhancement. In the hippocam-
pus and other brain regions, blockade of GABAB receptors results
in disinhibition and increase in PS.34,35 Therefore we tested if the
potent GABAB receptor antagonist, CGP 55845 (CGP;36), would
block or occlude the effect of compound 12 since it has been re-
ported to cause PS enhancement in the CA1 region.35 Bath applica-
compared to control in aCSF at p 6 0.05).
prototype and 10 lM as a suitable concentration because its effects
at this concentration were robust. We first tested if compound 12
employed cholinergic mechanism to enhance the PS amplitude be-
cause alteration in acetylcholine and cholinergic neurons/termi-
nals is implicated in the pathophysiology of dementias.2,6 We
tested for a role of the cholinergic system by using d-tubocurarine
(d-TC) and atropine, nicotinic (nAChR) and muscarinic (mAChR)
acetylcholine receptor antagonist, respectively, as both these
receptors are reported to be involved in AD, a common cause of
tion of 1 lM CGP predictably enhanced the PS amplitude by
55.4 15.5%, p < 0.05, paired t-test; n = 6 (Fig. 5C). Compound 12
was applied at the peak of this CGP effect, and it further enhanced
the PS amplitude by 32.7 6.9%, p < 0.05 compared to baseline in
the presence of CGP, paired t-test; n = 6 (Fig. 5C). Another neuro-
modulator in the CNS is adenosine37, and blockade of adenosine
A1 receptors also causes disinhibition and enhancement in synap-
tic responses. We therefore tested if blockade of A1 receptors
would occlude the compound 12 effect. The relatively selective
dementia.30–33 Bath application of atropine (100
lM) did not pro-
duce a significant effect on PS amplitude (8.2 15.3%, p > 0.05,
paired t-test; n = 5, Fig. 5A). In the presence of atropine, compound
12 (10 lM) was no longer able to cause an enhancement in PS
amplitude (0.2 2.8%, p > 0.05 compared to the effect in atropine
alone, paired t-test; n = 5, Fig. 5A). Similar to atropine, d-TC by it-
self did not change PS amplitude (1.8 6.4%, p > 0.05, paired t-test;
n = 6, Fig. 5B) but blocked the ability of compound 12 to enhance
the PS (À2.5 5.0%, p > 0.05 compared to the effect in d-TC alone,
paired t-test; n = 6, Fig. 5B).
A1 receptor antagonist, CPT (1 lM), enhanced PS amplitude by it-
self (76.5 18.3%, p < 0.05, paired t-test; n = 6, Fig. 5C). In the first
two slices tested, at the peak of this CPT effect, compound 12 pro-
duced very little enhancement in the PS amplitude. Since the CPT
effect was very big, we suppose that this may be a saturating effect

K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
8145
Figure 4. The effects of theophylline analogs on evoked PS are concentration-
dependent. (A) Concentration–response curves generated by applying different
concentrations of 3 different theophylline analogs (compounds 7, 9, and 12). Each
point on the graph has an n value of 3–8 slices (except compound 7, where n = 1).
(B) An integrated bar graph showing the effects of 10 lM of all TH-analogs tested.
The n values of each compound are shown below the bar.
on the response rather than pharmacological antagonism. To
examine the possibility that CPT actually blocked the effect of com-
pound 12, in other four slices, at the peak of the CPT effect, the
stimulus strength was decreased to produce responses close to
the initial baseline response recorded in normal aCSF. Once this
new baseline stabilized, subsequent application of compound 12
produced an enhancement of 47.9 22.7% (n = 4; Fig. 5C) in the
PS amplitude, an effect similar to that observed for compound 12
in aCSF alone (p > 0.05, unpaired t-test).
2.5. Pharmacology of compound 9 effects on PS
Figure 5. Compound 12 effects on PS amplitude are blocked by atropine and d-
tubocurarine but not by CGP55845 or CPT. (A) Bar graph summarizing the lack of
effect of compound 12 (n = 5) on PS amplitude when applied after pretreatment of
To investigate the mechanism by which compound 9 produced
the observed depression in PS amplitude, we tested if it employed
mechanisms known to modulate synaptic responses in the CNS. In
the hippocampus, GABA, acting on presynaptic GABAB receptors, is
known to cause depression in synaptic and cellular responses.38,39
To test if compound 9 employed GABAB receptors or GABAergic
mechanism to cause PS suppression, we pretreated slices with
slices with atropine (ATR; 100
tubocurarine (d-TC; 10 M) also prevented compound 12 from enhancing the PS
amplitude (n = 5). (C) In contrast to the effect of ATR and d-TC shown in A and B,
pretreatment of slices with both CGP (1 M; n = 6) and CPT (1 M; n = 4) enhanced
lM). (B) Similar to A, pretreatment of slices with d-
l
l
l
the PS amplitude but did not prevent compound 12 from further enhancing the PS
amplitude. (ÃÃindicates significance at p 6 0.05 compared to the response in the
presence of CGP).
1 l
M CGP,36 a potent GABAB receptor antagonist. Bath application
of CGP for 8 min caused an enhancement in the PS amplitude
(53.8 15.5%, p < 0.05; n = 7; Fig. 6A and C). At the peak of this
3. Discussion
CGP effect, concurrent application of 10
lM compound 9 no longer
produced significant depression in the PS amplitude
a
We report here the results of a study on the effects of different
derivatives of theophylline on neuronal responses in the rat hippo-
campus, a region involved in memory processing and implicated in
the pathophysiology of dementias. These compounds were synthe-
sized for use as possible nootropic agents or cognitive enhancers in
patients with dementias such as AD. Our results reveal that termi-
nal ring substitution of theophylline at position 7 determined the
type of effect on hippocampal PS recorded in vitro. All chain length
substituents with hexahydroazepin-2-one (compounds 10–12)
caused similar enhancements in population spike amplitude
regardless of the carbon chain length (x = 1–3) between the ring
and the theophylline skeleton. In contrast, piperidin-2-one ring
(10.6 11.5%, p > 0.05 compared to baseline in the presence of
CGP; n = 7; Fig. 6A and C). To determine if the action of compound
9 was mediated by adenosine acting as an intermediate to suppress
the PS amplitude, we blocked adenosine A1 receptors with 1 lM
CPT, and then tested compound 9 to determine if the PS amplitude
was suppressed in the presence of this antagonist. Similar to CGP
above, CPT also caused an enhancement in PS amplitude
(51.6 26.7%, p < 0.05; n = 5; Fig. 6B and C) but did not block the
depressing effect of 10 lM compound 9 on PS amplitude
(À27.6 6.5, p < 0.05 compared to baseline in the presence of
CPT; n = 5; Fig. 6B and C).

8146
K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
ring on the substituent at nitrogen 7 in the theophylline structure
since compounds 10–12 all of which have the hexahydroazepin-2-
one ring produced statistically indistinguishable enhancement in
PS amplitude at 10
lM; ꢀ56%; ꢀ42%; and ꢀ41% enhancement,
respectively. The only structural difference among these deriva-
tives being the length of the carbon chain (x) between the nitrogen
at position 7 of the theophylline ring and the nitrogen at position 1
of the hexahydroazepin-2-one, a 7-membered ring (y = 3) common
to compound 12: x = 3; compound 11: x = 2; and compound 10:
x = 1. Thus, the carbon chain length is not an essential requirement
for enhancement in hippocampal neuronal activity. Furthermore,
the same carbon chain length (x = 3) with a different terminal ring
structure (6-membered ring; y = 2), such as in compound 9, pro-
duced the opposite effect on hippocampal neuronal activity, while
another derivative compound 7 with the same chain length (x = 1)
as contained in compound 10 but with a different terminal ring (5-
membered ring; y = 1) had no detectable neuroactivity. Finally,
compound 8 with a bridging carbon chain length of x = 3, but with
the 5-membered pyrrolidin-2-one ring (y = 1) as in compound 7
(x = 1), also had no effect on hippocampal neuronal activity. To
summarize our findings, the above data indicate that the length
of the bridging carbon chain is not essential for neuroactivity in
this series of theophylline derivatives. Furthermore, the data sug-
gest that the pharmacophore for enhancing PS amplitude resides
in the hexahydroazepin-2-one ring. The nitrogen in this 7-mem-
bered ring (y = 3) may possess the ideal configuration to be proton-
ated in solution to generate a quaternary compound such as that
contained in the natural transmitter ACh, possibly endowing the
analogs with this ring substitution with an ability to interact at
the acetylcholine binding site on cholinergic receptors.
3.2. Mechanism(s) of action of enhancers to increase PS
amplitude
As indicated above, all of the enhancers produce almost identi-
cal increases in PS amplitude at 10 lM. Because the compound 12
effect appeared to be more robust, compound 12 was chosen as a
prototype of the enhancers to investigate the possible mecha-
nism(s) by which they produced enhancement in PS amplitude. Be-
cause of the well-known role of the cholinergic system in the
pathophysiology of dementias,1,6,32,33,40 we tested to see if com-
pound 12 employed this system in the hippocampus to cause PS
enhancement. Both nAChRs and mAChRs are present in the
CNS41–43 and are reported to be involved in cognitive processes,
and drugs are being developed that target both these receptors.33,40
In these studies, we showed that the enhancing effect of compound
12 on PS amplitude was blocked by atropine, a mAChR antago-
nist.44,45 Furthermore, they were also blocked by d-tubocurarine,
a nAChR antagonist.46
The above findings indicate that compound 12 and possibly the
other PS enhancers employ cholinergic mechanisms to enhance PS
amplitude. The likely site of action is either at the muscarinic or
nicotinic receptors or at both sites. Presynaptic muscarinic recep-
tor activation is reported to depress excitatory synaptic transmis-
sion in the hippocampus.47–49 Since acetylcholine is reported to
produce a tonic effect in the hippocampus and other regions50–52
blockade of mAChRs would be expected to reverse a muscarinic to-
nic inhibition leading to excitation. Interestingly, although we did
not unmask a tonic mAChR-mediated effect with atropine, the lat-
ter nonetheless blocked the effect of compound 12. This suggests
that cholinesterase activity in our preparation is so efficient that
released ACh has no chance of activating its receptors. This would
tend to rule out blockade of mAChR as the mechanism by which
compound 12 is enhancing the PS. On the other hand, nAChRs
are reported to cause excitation by increasing glutamate re-
lease53,54 and activation of these receptors by compound 12 and
Figure 6. The effect of compound 9 on PS amplitude is blocked by CGP but not by
CPT. (A) An average time-effect plot showing that pre-treatment of slices with CGP
(1
(n = 7). (B) Another average time-effect plot showing that pre-treatment of slices
with CPT (1 M) also enhances PS amplitude, but in contrast to the effect of CGP
lM) enhances PS amplitude and blocks the ability of compound 9 to depress PS
l
above does not block the ability of compound 9 to depress PS amplitude (n = 5). (C)
Summary bar graphs showing that the compound 9-induced PS amplitude
depression is blocked in the presence of CGP, but not CPT. Note that the effect of
compound 9 alone is from Fig. 3 for comparison only. (ÃÃindicates significance at
p 6 0.05 compared to the response in the presence of CPT).
substitution (compound 9) caused a suppression of PS amplitude
while pyrrolidin-2-one substituted compounds (compounds 7
and 8) produced no detected neuroactivity in the hippocampus.
Our data further indicate that the enhancers of PS employ cholin-
ergic mechanisms to do this and hence supports the hypothesis
that hexahydroazepin-2-one derivatives may be useful as a cogni-
tive enhancer in conditions such as AD where neuronal communi-
cation in the hippocampus and some cortical regions is diminished
due to a decline in cholinergic inputs or activity or both.1,6,32,33,40
Our data also indicate that piperidin-2-one and pyrrolidin-2-one
derivatives are not good candidates for use as cognitive enhancers.
However, piperidin-2-one derivatives may have potential utility in
other CNS disorders characterized by excessive excitation such as
epilepsy.
3.1. Structural requirements for PS enhancement
The results of this study reveal that the main requirement for
enhancing PS amplitude in the hippocampus is the nature of the

K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
8147
the other enhancers can thus lead to the observed effects in this
study. Another possible nAChR-dependent mechanism through
which compound 12 may cause the enhancement will be as an
allosteric potentiating ligand at the nAChRs such as that reported
for galantamine.55,56
and a Bruker Avance 300 spectrometer operating at frequencies
of 300.13 MHz (1H), 75.48 MHz (13C), and 30.41 MHz (15N). NMR
samples were prepared by dissolving individual compounds in
CDCl3 (550 l
L). 1H and 13C chemical shifts (d) were referenced rel-
ative to the signal of solvent [d = 7.27 ppm for 1H (residual CHCl3);
d = 77.23 ppm for 13C (CDCl3)]. 15N NMR chemical shifts deter-
mined by 1H–15N correlation experiments are reported relative to
liquid ammonia.59,60 The 1H–13C GSQMBC NMR experiment was
adjusted for a long-range coupling of 7.5 Hz. 1H–15N GSQMBC
and 1H–15N gs-HMBC NMR experiments were adjusted for cou-
plings ranging from 9 to 11 Hz. Chemical shifts are reported in
ppm, and indirect spin–spin coupling constants (J) are reported
in hertz (Hz).
Since our evidence indicates the involvement of both choliner-
gic receptors in the action of compound 12 to enhance PS ampli-
tude, a more plausible mechanism of action of these enhancers
may be to alter the levels of endogenous acetylcholine, which then
acts non-selectively to produce the observed effect. If this were the
case, the enhancing effect may reflect a predominant nAChR activ-
ity in normal conditions so that excitation is observed. Similar dual
effects of ACh have been reported in the nucleus accumbens
whereby the effect of nAChR was only unmasked after blockade
of mAChR.51 Such an indirect action to enhance ACh levels could
explain why both mAChR and nAChR receptor blockades are both
effective in preventing the effects of the enhancers. This prelimin-
ary study is unable to dissect out the exact mechanism or what
contribution each receptor subtype makes to the final effect. The
evidence, however, clearly rules out the involvement of other
well-known neuromodulators, such as adenosine57 and GABA58
in the action of compound 12 to enhance the PS amplitude.
The PS depression caused by compound 9 on the other hand
was blocked by CGP, a GABAB receptor antagonist but not by CPT,
an adenosine A1 receptor antagonist, indicating that the compound
9-induced PS depression was either direct through activation of
GABAB receptors or indirect through increasing the levels of endog-
enous extracellular GABA. Further studies are required to deter-
mine if this analog is a GABAB receptor agonist, a GABA reuptake
inhibitor, or a GABA degradatory enzyme inhibitor, all of which
can cause depression in excitatory synaptic transmission. Thus,
the piperidin-2-one analogs may have a role in CNS disorders char-
acterized by excessive excitation.
5.1.1. General procedure for N-(
The solution of a proper lactame (107 mmol) in ethyl acetate
(10 mL), -chloroacyl chloride (90 mmol) was added, and the mix-
x-chloroacyl)-lactames
x
ture was stirred at 30 °C for 20 min. The solution of triethylamine
(11.2 mL, 80 mmol) in ethyl acetate (40 mL) was added dropwise
in the course of 1.5 h, and the reaction mixture was heated at
50 °C for 1 h. The solid precipitate was filtered off, and the filtrate
was treated with water and 3% solution of sodium bicarbonate. Or-
ganic layer was dried over sodium sulfate, filtered and evaporated
to dryness under vacuum.
5.1.2. N-(2-Chloroacetyl)-pyrrolidin-2-one (1)
The title compound was prepared by a reaction of pyrrolidin-2-
one and 2-chloroacetyl chloride. Crude product was suspended in
diisopropyl ether (100 mL), and the mixture was refluxed for
5 min, filtered, and the filtrate was let to crystallize at À15 °C.
White crystals were washed with diisopropyl ether, and were dried
at ambient condition. Yield: 8.99 g, 66.2%; mp 42–45 °C; 1H NMR
and 13C NMR spectra were identical with those published
previously.61
4. Conclusion
5.1.3. N-(4-Chlorobutyryl)-pyrrolidin-2-one (2)
The title compound was prepared by a reaction of pyrrolidin-2-
one and 4-chlorobutyryl chloride. Crude product obtained was dis-
tilled under vacuum (88.0–89.5 °C, 33 Pa) to provide colorless li-
The results of this study indicate that the hexahydroazepin-2-
one analogs of theophylline have actions on hippocampal neuronal
activity that are consistent with cognitive enhancement, and hence
have the greatest potential for use as cognitive enhancers or noo-
tropic agents, whereas the piperidin-2-one and pyrrolidin-2-one
derivatives have no potential in the management of memory and
cognitive impairment associated with dementias. Indeed, the
piperidin-2-one analogs may exacerbate memory and cognitive
deficits in dementia patients but may be useful in other CNS disor-
ders such as epilepsy. Our results also show that structural modi-
fications of methylxanthines can yield products with different
pharmacological actions from those commonly associated with
methylxanthine analogs, for example, adenosine receptor
antagonism.
quid. Yield: 13.01 g, 76.5%; 1H NMR (200 MHz)
d 3.64 (t,
J = 6.9 Hz, 2H, NCH2 pyrrolidin-2-one), 3.08 (t, J = 7.0 Hz, 2H,
NCH2), 2.53 (t, J = 6.9 Hz, 2H, CH2CO pyrrolidin-2-one), 2.37 (t,
J = 7.0 Hz, 2H, CH2CO), 1.96–2.21 (m, 4H, CH2CH2CH2 pyrrolidin-
2-one, CH2CH2CH2); 13C NMR (50 MHz) d 175.32, 173.06, 45.33,
44.16, 33.93, 33.55, 27.08, 17.21.
5.1.4. N-(4-Chlorobutyryl)-piperidin-2-one (3)
The title compound was prepared by a reaction of piperidin-2-
one and 4-chlorobutyryl chloride. Crude product obtained was dis-
tilled under vacuum (87.5–90.5 °C, 20 Pa) to provide colorless li-
quid. Yield: 12.92 g, 70.5%; 1H NMR (200 MHz) d 3.50–3.70 (m,
2H, J = 6.9 Hz, NCH2 piperidin-2-one), 3.26 (t, J = 7.0 Hz, 2H,
NCH2), 2.50 (t, J = 6.9 Hz, 2H, CH2CO), 2.30-2.50 (m, 2H, J = 7.0 Hz,
CH2CO piperidin-2-one), 2.30-2.50 (m, 6H, CH2CH2CH2CH2 piperi-
5. Experimental
5.1. Synthesis of theophylline analogs
din-2-on, CH2CH2CH2); 13C NMR (50 MHz)
d 177.40, 174.49,
Starting materials were obtained from the commercial suppli-
ers, and were used without further purification. Reactions were
monitored by thin-layer chromatography on 0.25 mm silica gel
plates (Merck 60 F 254), and were visualized with UV light or io-
dine vapor. ESI-MS spectra were determined on an Agilent LC-
MSD 1100 VL Series. IR spectra were recorded on a Nicolet Impact
410 FT spectrometer using KBr discs, UV spectra were obtained on
a HP 8453 spectrophotometer. Melting points were determined on
an electrothermal melting point apparatus, and are uncorrected.
NMR spectra were obtained on a Varian Gemini spectrometer
operating at frequencies of 199.98 MHz (1H), 50.29 MHz (13C),
44.45, 43.97, 39.09, 33.51, 28.76, 23.20, 21.86.
5.1.5. N-(2-Chloroacetyl)-hexahydroazepin-2-one (4)
The title compound was prepared by a reaction of hexa-
hydroazepin-2-one and 2-chloroacetyl chloride. Yield: 16.51 g,
81.4% of brown liquid; 1H NMR and 13C NMR spectra were identical
with the literature data.26
5.1.6. N-(3-Chloropropionyl)-hexahydroazepin-2-one (5)
The title compound was prepared by a reaction of hexa-
hydroazepin-2-one and 3-chloropropionyl chloride. Crude product

8148
K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
obtained was distilled under vacuum (86.0–89.0 °C, 13 Pa) to pro-
vide colorless liquid. Yield: 10.51 g, 58.0%; 1H NMR (200 MHz) d
3.85–4.00 (m, 2H, NCH2 hexahydroazepin-2-one), 3.80 (t,
J = 6.8 Hz, 2H, CH2Cl), 3.36 (t, J = 6.8 Hz, 2H, ClCH2CH2), 2.60–2.75
(m, 2H, COCH2 hexahydroazepin-2-one), 1.50-1.90 (m, 6H,
CH2CH2CH2CH2CH2 hexahydroazepin-2-one); 13C NMR (50 MHz)
d 177.69, 172.73, 43.27, 42.11, 39.54, 39.46, 29.05, 28.46, 23.58.
5.1.11. 1,3-Dimethyl-7-(4-oxo-4-(2-oxopiperidine-1-yl)-butyl)-
3,7-dihydro-1H-purine-2,6-dione (9)
The title compound was prepared by a reaction of theophylline
and 3. Yield: 1.04 g, 27.2%; mp 124–126 °C; 1H NMR (300 MHz) d
7.54 (s, 1H, aromatic H), 4.62 (t, J = 6.4 Hz, 2H, NCH2), 3.69–3.74
(m, 2H, NCH2 piperidin-2-one), 3.59 (s, 3H, N3CH3), 3.40 (s, 3H,
N1CH3), 2.94 (t, J = 6.3 Hz, 2H, CH2CO), 2.47–2.60 (m, 2H, COCH2
piperidin-2-one), 2.23 (qi, J = 7.2 Hz, 2H, CH2CH2CH2), 1.76–1.86
(m, 4H, CH2CH2CH2CH2 piperidin-2-one); 13C NMR (75 MHz) d
175.36 (C15), 173.27 (C17), 155.05 (C6), 151.69 (C2), 148.96 (C4),
140.95 (C8), 107.02 (C5), 46.34 (C12), 43.93 (C21), 35.86 (C14),
34.76 (C18), 29.65 (C11), 27.86 (C10), 26.18 (C13), 22.34 (C20),
20.14 (C19). 15N NMR (30 MHz) d 230.5 (N9), 170.5 (N16), 168.2
(N7), 151.6 (N1), 114.7 (N3); IR (cmÀ1) 3116, 2956, 1706, 1704,
1666, 1548, 1475, 1359, 1291, 1201, 1162; MS m/z 370.3 (MÀNa+);
UV kmax (methanol) 205 nm, 274 nm.
5.1.7. N-(4-Chlorobutyryl)-hexahydroazepin-2-one (6)
The title compound was prepared by a reaction of hexa-
hydroazepin-2-one and 4-chlorobutyryl chloride. Crude product
obtained was distilled under vacuum (88.0–95.0 °C, 35 Pa) to pro-
vide colorless liquid. Yield: 12.18 g, 76.6%; 1H NMR (200 MHz) d
3.80–4.00 (m, 2H, NCH2 hexahydroazepin-2-one), 3.61 (t,
J = 7.0 Hz, 2H, NCH2), 3.05 (t, J = 7.0 Hz, 2H, CH2CO), 2.65–2.70
(m, 2H, CH2CO hexahydroazepin-2-one), 2.12 (qi J = 7.0 Hz, 2H,
CH2CH2CH2), 1.50-1.90 (m, 6H, CH2CH2CH2CH2CH2 azepan-2-
one); 13C NMR (50 MHz) d 178.48, 175.00, 43.32, 43.27, 39.75,
36.28, 29.08, 28.52, 28.01, 23.69.
5.1.12. 1,3-Dimethyl-7-(2-oxo-2-(2-oxoazepan-1-yl)-ethyl)-3,7-
dihydro-1H-purine-2,6-dione (10)
The title compound was prepared by a reaction of theophylline
and 4. The reaction mixture was heated at 50 °C for 4 h. Yield:
1.34 g, 36.2%; mp 148–151 °C; 1H NMR (300 MHz) d 7.57 (s, 1H,
aromatic H), 5.51 (s, 2H, NCH2), 3.80–4.00 (m, 2H, NCH2 hexa-
hydroazepin-2-one), 3.61 (s, 3H, N3CH3), 3.36 (s, 3H, N1CH3),
2.72–2.84 (m, 2H, COCH2 hexahydroazepin-2-one), 1.65–2.05 (m,
6H, CH2CH2CH2CH2 hexahydroazepin-2-one); 13C NMR (75 MHz)
d 178.18 (C15), 169.68 (C13), 155.24 (C6), 151.76 (C2), 148.64
(C4), 142.05 (C8), 107.28 (C5), 52.78 (C12), 44.07 (C20), 39.21
(C16), 29.71 (C19), 29.13 (C11), 28.15 (C10), 27.77 (C17), 23.52
(C18); 15N NMR (30 MHz) d 231.8 (N9), 174.5 (N14), 158.8 (N7),
151.7 (N1), 114.7 (N3); IR (cmÀ1) 3113, 2943, 1704, 1659, 1550,
1475, 1376, 1253, 1192, 1158; MS m/z 356.2 (MÀNa+); UV kmax
(methanol) 206 nm, 274 nm.
5.1.8. General procedure for 1,3-dimethyl-7-(x-oxo-x-(1-
azacycloalkan-2-one-1-yl)- alkyl)-3,7-dihydro-1H-purine-2,6-
diones
The suspension of theophylline (2.00 g, 11 mmol), proper N-(x-
chloroacyl)-lactame (12 mmol), potassium carbonate (2.30 g,
17 mmol) and potassium iodide (0.37 g, 2 mmol) in N,N-dimethyl-
formamide (10.0 mL) was heated at 80 °C for 4 h. Reaction mixture
was diluted with water (50.0 mL), and aqueous solution was ex-
tracted with chloroform. Chloroform solution was treated with
1M sodium hydroxide, dried over sodium sulfate, and evaporated
to dryness under vacuum. Acetone (3 mL) was added to residuum,
and the solution was let to crystallize at À15 °C. Crystals were fil-
tered, and were re-crystallized from propan-2-ol.
5.1.9. 1,3-Dimethyl-7-(2-oxo-2-(2-oxopyrrolidine-1-yl)-ethyl)-
3, 7-dihydro-1H-purine-2,6-dione (7)
5.1.13. 1,3-Dimethyl-7-(3-oxo-3-(2-oxoazepan-1-yl)-propyl)-
3,7-dihydro-1H-purine-2,6-dione (11)
The title compound was prepared by a reaction of theophylline
and 1. Yield: 1.35 g, 40.1%; mp 209–212 °C; 1H NMR (300 MHz) d
7.55 (s, 1H, aromatic H), 5.61 (s, 2H, NCH2), 3.87 (t, J = 7.1 Hz, 2H,
NCH2 pyrrolidin-2-one), 3.61 (s, 3H, N3CH3), 3.37 (s, 3H, N1CH3),
2.68 (t, J = 8.1 Hz, 2H, COCH2 pyrrolidin-2-one), 2.16 (qi,
J = 7.2 Hz, 2H, CH2CH2CH2 pyrrolidin-2-one); 13C NMR (75 MHz) d
176.16 (C15), 167.15 (C13), 155.29 (C6), 151.69 (C2), 148.59 (C4),
141.94 (C8), 107.39 (C5), 50.94 (C12), 45.22 (C18), 32.91 (C16),
29.76 (C11), 27.81 (C10), 17.62 (C17). 15N NMR (30 MHz) d 232.7
The title compound was prepared by a reaction of theophylline
and 5. Yield: 3.31 g, 85.8%; mp 146–147 °C; 1H NMR (300 MHz) d
7.70 (s, 1H, aromatic H), 4.62 (t, J = 6.8 Hz, 2H, NCH2), 3.80–3.95
(m, 2H, NCH2 hexahydroazepin-2-one), 3.49 (t, J = 6.9 Hz, 2H,
CH2CO), 3.59 (s, 3H, N3CH3), 3.40 (s, 3H, N1CH3), 2.62–2.78 (m,
2H, COCH2 hexahydroazepin-2-one), 1.55–1.95 (m, 6H,
CH2CH2CH2CH2CH2 hexahydroazepin-2-one); 13C NMR (75 MHz)
d 177.50 (C16), 172.97 (C14), 155.11 (C6), 151.75 (C2), 148.96
(C4), 142.15 (C8), 106.86 (C5), 43.30 (C12), 42.81 (C21), 40.39
(C13), 39.56 (C17), 29.69 (C11), 29.01 (C20), 27.91 (C10), 27.92
(C18), 23.63 (C19); 15N NMR (30 MHz) d 230.4 (N9), 176.5 (N15),
166.3 (N7), 151.5 (N1), 114.6 (N3); IR (cmÀ1) 3123, 2935, 2857,
1700, 1668, 1547, 1475, 1387, 1260, 1185, 1151; MS m/z 370.2
(MÀNa+); UV kmax (methanol) 206 nm, 274 nm.
(N9), 168.6 (N14), 155.2 (N7), 151.8 (N1), 114.8 (N3); IR (cmÀ1
)
3116, 2963, 1744, 1700, 1673, 1550, 1456, 1407, 1369, 1294,
1274, 1236, 1192; MS m/z 328.2 (MÀNa+); UV kmax (methanol)
208 nm, 274 nm.
5.1.10. 1,3-Dimethyl-7-(4-oxo-4-(2-oxopyrrolidine-1-yl)-
butyl)-3, 7-dihydro-1H-purine-2,6-dione (8)
5.1.14. 1,3-Dimethyl-7-(4-oxo-4-(2-oxoazepan-1-yl)-butyl)-3,7-
dihydro-1H-purine-2,6-dione (12)
The title compound was prepared by a reaction of theophylline
and 2. Yield: 1.61 g, 44.0%; mp 136–137 °C; 1H NMR (300 MHz) d
7.57 (s, 1H, aromatic H), 4.38 (t, J = 7.3 Hz, 2H, NCH2), 3.81 (t,
J = 7.0 Hz, 2H, NCH2 pyrrolidin-2-one), 3.59 (s, 3H, N3CH3), 3.41
(s, 3H, N1CH3), 2.90 (t, J = 7.0 Hz, 2H, CH2CO pyrrolidin-2-one),
2.60 (t, J = 8.0 Hz, 2H, CH2CO), 2.24 (m, 2H, CH2CH2CH2), 2.06 (qi,
J = 7.1 Hz, 2H CH2CH2CH2 pyrrolidin-2-one); 13C NMR (75 MHz) d
175.34 (C17), 172.76 (C15), 154.98 (C6), 151.72 (C2), 149.04 (C4),
140.96 (C8), 106.94 (C5), 46.27 (C12), 45.39 (C20), 33.49 (C14),
33.15 (C18), 29.69 (C11), 27.89 (C10), 25.35 (C13), 17.21 (C19);
15N NMR (30 MHz) d 230.6 (N9), 170.9 (N16), 168.0 (N7), 151.6
(N1), 114.6 (N3); IR (cmÀ1) 3109, 2956, 1741, 1707, 1666, 1549,
1475, 1403, 1362, 1294, 1222, 1191; MS m/z 356.2 (MÀNa+); UV
kmax (methanol) 207 nm, 274 nm.
The title compound was prepared by a reaction of theophylline
and 6. Yield: 1.54 g, 38.4%; mp 108–110 °C; 1H NMR (300 MHz) d
7.58 (s, 1H, aromatic H), 4.38 (t, J = 6.7 Hz, 2H, NCH2), 3.85–3.95
(m, 2H, NCH2 hexahydroazepin-2-one), 3.59 (s, 3H, N3CH3), 3.41
(s, 3H, N1CH3), 2.91 (t, J = 6.9 Hz, 2H, CH2CO), 2.65–2.75 (m, 2H,
COCH2 hexahydroazepin-2-one), 2.24 (qi, J = 6.8 Hz, 2H
CH2CH2CH2), 1.55–1.95 (m, 6H, CH2CH2CH2CH2 hexahydroazepin-
2-one); 13C NMR (75 MHz) d 177.56 (C17), 174.65 (C15), 155.05
(C6), 151.70 (C2), 148.97 (C4), 140.97 (C8), 107.03 (C5), 43.34
(C12), 43.29 (C22), 39.60 (C18), 35.53 (C14), 29.65 (C11), 29.02
(C21), 28.44 (C19), 27.86 (C10), 26.63 (C20), 26.30 (C13); 15N
NMR (30 MHz) d 230.6 (N9), 176.7 (N16), 168.5 (N7), 151.5 (N1),
114.6 (N3); IR (cmÀ1) 3099, 2943, 2853, 1707, 1656, 1550, 1478,

K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
8149
1383, 1332, 1185, 1151; MS m/z 384.3 (MÀNa+); UV gkmax (meth-
plots and bar graphs. Graphical representations were done using
GraphPad PrismÒ (GraphPad Software, San Diego, CA, USA), Sigma-
PlotÒ (Systat Software Inc., San Jose, CA, USA), and CorelDrawÒ
(Corel Corp., Ottawa, ON, Canada) softwares.
anol) 205 nm, 274 nm.
5.2. Animal experiments
All animals used in this study were male Sprague–Dawley rats
supplied by Kuwait University Animal Resource Centre. All exper-
iments were done in accordance with guidelines on humane han-
dling of experimental animals as established by the Canadian
Council on Animal Care. The procedures were designed to mini-
mize animal suffering, and the smallest number of animals neces-
sary to produce the required results was used.
5.6. Chemicals and drugs
All the theophylline analogs were synthesized in-house, and
were dissolved in dimethyl sulfoxide. Stock solutions of 10 mM
were prepared, aliquoted, and stored at À20 °C and were used
within 2 weeks. Tetrodotoxin, atropine, 8-cyclopentyl-theophyl-
line, and d-tubocurarine and all salts used in the preparation of
aCSF and recoding solutions were obtained from Sigma Company
(Steinheim, Germany). CGP55845 was obtained from Tocris (Bris-
tol, UK). All drugs and chemicals were diluted by at least 1000-fold
with aCSF to the desired concentration, and were applied by bath
perfusion.
5.3. Slice preparation
Extracellular electrophysiological experiments were performed
in coronal hippocampal slices generated from rats (75–150 g)
using previously published techniques and methods.62,63 Briefly,
rats were deeply anesthetized with halothane, and were killed by
quick decapitation. The brains were quickly removed and placed
in ice cold (4 °C) artificial cerebrospinal fluid (aCSF) bubbled con-
tinuously with 95% O2 and 5% CO2 (Carbogen). The composition
of the aCSF used for dissection, storage, and PS recording was (in
mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.23 NaHPO4, 25 NaH-
Acknowledgments
Work in Dr. Kombian’s laboratory is supported by research
grants from Kuwait University. Chemical synthesis work was sup-
ported by Grant No 25/2006/FaF awarded by University of Veteri-
nary and Pharmaceutical Sciences. NMR part of the project was
supported by the Ministry of Education of the Czech Republic
(MSM0021622413 and LC06030 to R.M.). We thank Dr. O.A. Phil-
lips for discussions on chemical structures and nomenclature, Dr.
Emil Švajdlenka for measurement of mass spectra, and Prof. M.A.
Oriowo for generous gift of d-tubocurarine.
CO3, and 10 D-glucose. 400 lm thick coronal slices containing the
hippocampus were cut from a block of brain tissue in ice-cold aCSF
using Leica VT 1000S (Leica Microsystems, Wetzlar, Germany) or
OTS-4000 (Electron Microscopy Sciences, Hatfield PA, USA) tissue
slicers. Prior to recording, slices were incubated for 1 h in aCSF,
which was continuously bubbled with carbogen at room tempera-
ture. Slices were trimmed and suspended on nylon mesh in a
Supplementary data
500 lL capacity recording chamber. Bath temperature was tightly
maintained at 29–31 °C to ensure that changes in responses were
not due to variation in temperature.64 Slices were perfused at a
flow rate of 2–3 mL/min with aCSF that was bubbled with
carbogen.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.07.045.
References and notes
5.4. Population spike recordings
1. Birks, J.; Harvey, R. J. Cochrane Database Syst. Rev. 2006, CD001190.
2. Birks, J. Cochrane Database Syst. Rev. 2006, CD005593.
3. Chui, H. C.; Mack, W.; Jackson, J. E.; Mungas, D.; Reed, B. R.; Tinklenberg, J.;
Chang, F. L.; Skinner, K.; Tasaki, C.; Jagust, W. J. Arch. Neurol. 2000, 57,
191–196.
4. Beach, T. G.; Sue, L.; Scott, S.; Layne, K.; Newell, A.; Walker, D.; Baker, M.;
Sahara, N.; Yen, S. H.; Hutton, M.; Caselli, R.; Adler, C.; Connor, D.; Sabbagh, M.
Brain Pathol. 2003, 13, 263–278.
An extracellular field recording glass electrode (3–15 MX) filled
with 3 M NaCl was placed in the stratum pyramidale of area CA1,
and bipolar stimulating electrodes were placed in the stratum rad-
iatum near area CA1 to activate Schaffer collateral/commissural fi-
bers. A PS was recorded by stimulating these afferents, and by
moving the field recording electrode around until an optimal PS
could be recorded. These responses were monitored for at least
30 min and were shown to be stable prior to the application of
any drug.
5. McKeith, I. Int. Psychogeriatr. 2004, 16, 123–127.
6. Caselli, R. J.; Beach, T. G.; Yaari, R.; Reiman, E. M. J. Clin. Psychiatry 2006, 67,
1784–1800.
7. Doraiswamy, P. M. CNS Drugs 2002, 16, 811–824.
8. Nagy, Z.; Hindley, N. J.; Braak, H.; Braak, E.; Yilmazer-Hanke, D. M.; Schultz, C.;
Barnetson, L.; King, E. M.; Jobst, K. A.; Smith, A. D. Dement. Geriatr. Cogn. Disord.
1999, 10, 115–120.
9. Beach, T. G.; Kuo, Y. M.; Spiegel, K.; Emmerling, M. R.; Sue, L. I.; Kokjohn, K.;
Roher, A. E. J. Neuropathol. Exp. Neurol. 2000, 59, 308–313.
10. Ikonomovic, M. D.; Mufson, E. J.; Wuu, J.; Cochran, E. J.; Bennett, D. A.; DeKosky,
S. T. J. Alzheimers Dis. 2003, 5, 39–48.
11. Pennanen, C.; Kivipelto, M.; Tuomainen, S.; Hartikainen, P.; Hanninen, T.;
Laakso, M. P.; Hallikainen, M.; Vanhanen, M.; Nissinen, A.; Helkala, E. L.; Vainio,
P.; Vanninen, R.; Partanen, K.; Soininen, H. Neurobiol. Aging 2004, 25, 303–310.
12. Scheff, S. W.; Price, D. A. J. Alzheimers Dis. 2006, 9, 101–115.
13. Scheff, S. W.; Price, D. A.; Schmitt, F. A.; DeKosky, S. T.; Mufson, E. J. Neurology
2007, 68, 1501–1508.
14. Hull, M.; Berger, M.; Heneka, M. Drugs 2006, 66, 2075–2093.
15. Doody, R. S. J. Clin. Psychiatry 2003, 64(Suppl. 9), 11–17.
16. Cosman, K. M.; Boyle, L. L.; Porsteinsson, A. P. Expert Opin. Pharmacother. 2007,
8, 203–214.
17. Birks, J.; Flicker, L. Expert Opin. Investig. Drugs 2007, 16, 647–658.
18. Cummings, J. L. J. Geriatr. Psychiatry Neurol. 2004, 17, 112–119.
19. Doody, R. S.; Geldmacher, D. S.; Gordon, B.; Perdomo, C. A.; Pratt, R. D. Arch.
Neurol. 2001, 58, 427–433.
5.5. Data acquisition, analysis, and statistics
All recordings were made using an Axopatch 1D amplifier and
pClamp software (Clampex 8, Axon instruments) in current clamp
mode at sampling rates of 50 kHz, filtered at 1 kHz, digitized and
stored for off-line analysis. Each stored PS trace was an average
of five successively triggered responses elicited at 10 s intervals.
The amplitude of the PS was measured from the peak of the posi-
tive going wave to the tip of the negative going wave. Drugs were
perfused between 5 and 20 min depending on the type of experi-
ment. All data are expressed as mean standard error (SE). Statis-
tical significance of all measures was determined using Student’s t-
test (paired or unpaired where appropriate) and was considered
significant at p 6 0.05 using SigmaStatÒ (Systat Software Inc., San
Jose, CA, USA). PS amplitudes were normalized by taking the mean
of 4–5 responses prior to drug application, and by dividing the rest
of the values by this mean. These values were used for average
20. Winblad, B.; Jelic, V. Alzheimer Dis. Assoc. Disord. 2004, 18(Suppl. 1), S2–S8.
21. Ringman, J. M.; Cummings, J. L. Behav. Neurol. 2006, 17, 5–16.
22. Nehlig, A.; Daval, J. L.; Debry, G. Brain Res. Brain Res. Rev 1992, 17, 139–170.
23. Ribeiro, J. A.; Sebastiao, A. M.; de, M. A. Prog. Neurobiol. 2002, 68, 377–392.

8150
K. V. V. Ananthalakshmi et al. / Bioorg. Med. Chem. 16 (2008) 8142–8150
24. Grundman, M.; Capparelli, E.; Kim, H. T.; Morris, J. C.; Farlow, M.; Rubin, E. H.;
Heidebrink, J.; Hake, A.; Ho, G.; Schultz, A. N.; Schaffer, A. N.; Houston, W.;
Thomas, R.; Thal, L. J. Life Sci. 2003, 73, 539–553.
46. Mulle, C.; Changeux, J. P. J. Neurosci. 1990, 10, 169–175.
47. Fernandez de, S. D.; Cabezas, C.; de Prada, A. N.; Sanchez-Jimenez, A.; Buno, W.
J. Physiol. 2002, 545, 51–63.
25. Narahashi, T.; Marszalec, W.; Moriguchi, T.; Yeh, J. Z.; Zhao, X. Life Sci. 2003, 74,
281–291.
48. Fernandez de, S. D.; Buno, W. Eur. J. Neurosci. 2003, 17, 555–558.
49. Buno, W.; Cabezas, C.; Fernandez de, S. D. J. Mol. Neurosci. 2006, 30, 161–164.
50. Alreja, M.; Wu, M.; Liu, W.; Atkins, J. B.; Leranth, C.; Shanabrough, M. J.
Neurosci. 2000, 20, 8103–8110.
51. Zhang, L.; Warren, R. A. J. Neurophysiol. 2002, 88, 3315–3330.
52. Wu, Y.; Wang, W.; Richerson, G. B. J. Neurophysiol. 2003, 89, 2021–2034.
53. Gray, R.; Rajan, A. S.; Radcliffe, K. A.; Yakehiro, M.; Dani, J. A. Nature 1996, 383,
713–716.
54. Alkondon, M.; Pereira, E. F.; Albuquerque, E. X. J. Neurophysiol. 2003, 90, 1613–
1625.
55. Schrattenholz, A.; Pereira, E. F.; Roth, U.; Weber, K. H.; Albuquerque, E. X.;
Maelicke, A. Mol. Pharmacol. 1996, 49, 1–6.
26. Mathias, L. J.; Moore, D. R. J. Am. Chem. Soc. 1985, 107, 5817–5818.
27. Pullman, B.; Pullman, A. Adv. Heterocycl. Chem. 1971, 13, 77–159.
28. Marek, R.; Kralik, L.; Sklenar, V. Tetrahedron Lett. 1997, 38, 665–668.
29. Marek, R.; Sklenar, V. Annu. Rep. NMR Spectrosc. 2005, 54, 201–242.
30. Nordberg, A. Biol. Psychiatry 2001, 49, 200–210.
31. Woodruff-Pak, D. S.; Gould, T. J. Behav. Cogn. Neurosci. Rev. 2002, 1, 5–20.
32. Piggott, M. A.; Owens, J.; O’Brien, J.; Colloby, S.; Fenwick, J.; Wyper, D.; Jaros, E.;
Johnson, M.; Perry, R. H.; Perry, E. K. J. Chem. Neuroanat. 2003, 25, 161–173.
33. Clader, J. W.; Wang, Y. Curr. Pharm. Des. 2005, 11, 3353–3361.
34. Mott, D. D.; Lewis, D. V. Epilepsy Res. 1992, 7(Suppl.), 119–134.
35. Ananthalakshmi, K. V.; Edafiogho, I. O.; Kombian, S. B. Epilepsy Res. 2007, 76,
85–92.
56. Samochocki, M.; Hoffle, A.; Fehrenbacher, A.; Jostock, R.; Ludwig, J.;
Christner, C.; Radina, M.; Zerlin, M.; Ullmer, C.; Pereira, E. F.; Lubbert,
H.; Albuquerque, E. X.; Maelicke, A. J. Pharmacol. Exp. Ther. 2003, 305,
1024–1036.
36. Davies, C. H.; Pozza, M. F.; Collingridge, G. L. Neuropharmacology 1993, 32,
1071–1073.
37. Phillis, J. W.; Wu, P. U. Prog. Neurobiol. 1981, 17, 141–184.
38. Dutar, P.; Nicoll, R. A. Nature 1988, 332, 156–158.
39. Davies, C. H.; Collingridge, G. L. J. Physiol. 1996, 496, 451–470.
40. Geerts, H.; Finkel, L.; Carr, R.; Spiros, A. J. Neural Transm. 2002(Suppl), 203–216.
41. Swanson, L. W.; Simmons, D. M.; Whiting, P. J.; Lindstrom, J. J. Neurosci. 1987, 7,
3334–3342.
42. Volpicelli, L. A.; Levey, A. I. Prog. Brain Res. 2004, 145, 59–66.
43. Champtiaux, N.; Changeux, J. P. Prog. Brain Res. 2004, 145, 235–251.
44. Stolerman, I. P. Pharmacol. Biochem. Behav. 1988, 30, 235–242.
45. Renner, U. D.; Oertel, R.; Kirch, W. Ther. Drug Monit. 2005, 27, 655–665.
57. Wu, L. G.; Saggau, P. Neuron 1994, 12, 1139–1148.
58. Otis, T. S.; De, K. Y.; Mody, I. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7698–7702.
ˇ
59. Marek, R.; Lycka, A. Curr. Org. Chem. 2002, 6, 35–66.
ˇ
60. Marek, R.; Lycka, A.; Kolehmainen, E.; Sievänen, E.; Toušek, J. Curr. Org. Chem.
2007, 11, 1154–1205.
61. Moore, D. R.; Mathias, L. J. Macromolecules 1986, 19, 1530–1536.
62. Otis, T. S.; De, K. Y.; Mody, I. J. Physiol. 1993, 463, 391–407.
63. Klapstein, G. J.; Colmers, W. F. J. Neurophysiol. 1997, 78, 1651–1661.
64. Motamedi, G. K.; Salazar, P.; Smith, E. L.; Lesser, R. P.; Webber, W. R.; Ortinski,
P. I.; Vicini, S.; Rogawski, M. A. Epilepsy Res. 2006, 70, 200–210.