Design, synthesis, computational and biological evaluation of new anxiolytics

Article abstract of DOI:10.1016/j.bmc.2004.09.016

New anxiolytics have been discovered by prediction of biological activity with computer programs pass and derek for a heterogeneous set of 5494 highly chemically diverse heterocyclic compounds (thiazoles, pyrazoles, isatins, a-fused imidazoles and others)

Full text of DOI:10.1016/j.bmc.2004.09.016

Bioorganic & Medicinal Chemistry 12 (2004) 6559–6568
Design, synthesis, computational and biological evaluation
of new anxiolytics
a,
Athina Geronikaki, Eugeni Babaev, John Dearden, Wim Dehaen,
b
c
d
*
e
f
f
Dmitrii Filimonov, Irina Galaeva, Valentina Krajneva, Alexey Lagunin,
e
g
Fliur Macaev, Guenadiy Molodavkin, Vladimir Poroikov, Serghei Pogrebnoi,
f
e
g
h
e
g
Victor Saloutin, Alla Stepanchikova, Eugenia Stingaci, Natalia Tkach,
b
g
Liudmila Vlad and Tatiana Voronina
f
a
School of Pharmacy, Department of Pharmaceutical Chemistry, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
b
Chemical Department of Moscow State University, Moscow 119899, Russia
School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool L3 3AF, UK
c
d
Leuven University, Leuven 3001, Belgium
Institute of Biomedical Chemistry of Russian Academy of Medicinal Science, 119121 Moscow, Russia
e
f
Institute of Pharmacology of Russian Academy of Medicinal Science, Moscow 125315, Russia
g
Institute of Chemistry of Moldova Academy of Science, Chisinau MD-2028, Moldova
Institute of Organic Chemistry of Urals Division of Russian Academy of Science, Ekaterinburg 620219, Russia
h
Received 13 July 2004; accepted 10 September 2004
Available online 2 October 2004
Abstract—New anxiolytics have been discovered by prediction of biological activity with computer programs PASS and DEREK for a
heterogeneous set of 5494 highly chemically diverse heterocyclic compounds (thiazoles, pyrazoles, isatins, a-fused imidazoles and
others). The majority of tested compounds exhibit the predicted anxiolytic effect. The most potent activity was found in 2-(4-nitro-
phenyl)-3-(4-phenylpiperazinomethyl)imidazo[1,2-a]pyridine 8, 1-[(4-bromophenyl)-2-oxoethyl]-3-(1,3-dioxolano)-2-indolinone 3, 5-
hydroxy-3-methoxycarbonyl-1-phenylpyrazole 5 and 2-(4-fluorophenyl)-3-(4-methylpiperazinomethyl)imidazo[1,2-a]pyridine 7. The
application of the computer-assisted approach significantly reduced the number of synthesized and tested compounds and increased
the chance of finding new chemical entities (NCEs).
ꢀ
2004 Elsevier Ltd. All rights reserved.
1
. Introduction
Computer-aided structure–activity relationship analysis
and molecular modelling are widely used now by pharma-
ceutical chemists to discover new lead compounds
Neurotic disturbances, anxiety, neurosis-like disorders
and stress-related illnesses are widespread, and many
types of pharmaceuticals have been developed for their
treatment. They influence different molecular targets
4
and optimize their structure and properties. However,
the majority of available approaches are focused on a sin-
gle macromolecular target, or on the only known phar-
macological/biochemical action, and/or compounds
from the same chemical series. Most (Q)SAR methods
are focused on a single biological activity, whereas in
reality each compound has both main and side pharma-
1
including GABA-A-benzodiazepine receptor complex,
1
2
glutamate and 5HT receptors. However, all currently
3
used anxiolytics demonstrate serious adverse effects.
Therefore, to reduce the probability of side reactions,
there is an urgent need to find new anxiolytics, prefer-
ably in chemical classes in which such activity has not
yet been observed.
5
,6
cological effects. Moreover, the number of existing tar-
gets is expected to increase from about 500 to about
7
5000–10,000 in a few years; thus, experimental evalua-
tion of potential biological activity is becoming more
complicated.
Keywords: Anxiolytics; Synthesis; Thiazoles; Pyrazoles; 2-Indolinones;
Fused imidazoles; PASS.
*
Corresponding author. Tel.: +30 2310997616; fax: +30 2310
97612; e-mail: geronik@pharm.auth.gr
An innovative approach to computer-aided prediction of
general biological activity spectra on the basis of chemical
9
0
968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.016

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A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
8
–11
32
structure of a compound has been developed
2–17
and
This approach is based on a ro-
2-phenyl-amino-4-phenyl-thiazolyl, 2-amino-4-methy-
33
l-thiazolyl and in general 2-(N-substituted or N,N-
1
is now widely used.
bust analysis of structure–activity relationships in a het-
erogeneous training set, including many thousands of
compounds from different chemical series. The approach
is the basis of the computer program PASS (Prediction of
Activity Spectra for Substances). PASS version 1.811 pre-
dicts 900 types of biological activity for a compound with
an average accuracy of about 85% according to the leave-
one-out cross-validation procedure (LOO CV).
3
4
disubstituted) acetamido derivatives have significant
local anaesthetic activity. Anti-inflammatory, analgesic
and antipyretic activities for some thiazolyl and benzo-
9
3
5,36
thiazolyl derivatives are also known.
Meloxicam,
for example, is a new NSAID possessing a thiazole
group. A number of thiazolyl-amino ketones as well as
thiazolyl amides have been found to be strong anti-
3
7,38
inflammatory agents.
Also, in a series of hydr-
azine-thiazoles and derivatives (and their Ôopen chainÕ
thiosemicarbazide analogues) inhibitory activity to
MAO rat liver mitochondria was found.
To provide greater diversity, many potentially synthesiz-
able compounds from different chemical classes should
be investigated. In contrast to high-throughput screen-
ing programs carried out by pharmaceutical companies,
the academic community has very limited resources for
the execution of interdisciplinary projects for experi-
mental studies of new pharmaceutical agents. Using
PASS predictions, the number of ÔactivesÕ in the selected
compounds can be increased by up to 17 times. Thus,
PASS-based computer pre-screening of large databases of
diverse compounds can increase the probability of find-
ing of new anxiolytic agents, and reduce the number of
compounds that have to be synthesized and studied
experimentally in in vivo tests.
3
9
Heterocycles of the pyrazole class are widely used in
medicine. They are non-narcotic analgesics, PDE 5
40
4
1
42
inhibitors and factor Xa inhibitors. Pyrazole deriva-
4
tives also have antimicrobial and antitumour activity.
3
Although fused imidazo[1,2-a]pyridine-2-acetamides
(alpidem and zolpidem) possess anxiolytic activity, there
are no reports on such properties for 3-dialkylamino-
methyl-derivatives of imidazo[1,2-a]pyridines and
related a-fused imidazoles.
1
5
In spite of the lack of information about the anxiolytic ef-
fect of the above-mentioned classes of heterocyclic com-
pounds, such an effect was predicted with the computer
program PASS for some particular derivatives (see below).
2
. Results and discussion
2
.1. Compounds design
2.2. Compound selection
Potentially synthesizable compounds were designed by
five groups of organic chemists from different institu-
tions: Moscow State University (MSU), the Institute
of Organic Chemistry of Urals Division of the Russian
Academy of Science (IOC), the Institute of Chemistry
of the Moldova Academy of Science (IC), Leuven Uni-
versity (LU) and the Aristotelian University of Thessa-
loniki (AU). In total, a virtual set of 5494 structures
has been designed. The proposed compounds belonged
to numerous chemical classes, including substituted isa-
tins, pyrazoles, thiazoles and imidazoles fused with pyr-
idine, thiazole and benzothiazole rings.
Prediction of biological activity spectra was made for
5494 structures designed by the chemical synthesis teams.
The computer program PASS was used to predict the bio-
logical activity spectra of designed compounds, including
900 pharmacological effects and mechanisms of actions.
Since PASS is based on so-called ligand-based design ap-
proach, it cannot discover new targets and needs the
training set of ligands that were earlier tested experimen-
tally on anxiolytic action. The list of activities, which are
currently predicted by PASS, includes 40 kinds of biolog-
ical activity associated with the anxiolytic effect (Table 1).
On the basis of computer-aided predictions we selected
potential anxiolytics (virtual hits). The following criteria
were used for the hitsÕ selection:
Isatins (1H-indole-2,3-diones) are synthetically versatile
precursors of several other classes (e.g., indoles and quino-
lines), and are also intermediates for drug synthesis. It
has recently been reported that isatin possesses anticon-
1. Compounds were selected as hits if the value of prob-
ability (P ) of possessing anxiolytic activity exceeded
50%.
1
8
vulsant and proconvulsant activities along with other
pharmacological properties.
a
19,20
3-Hydroxy-3-substituted
oxindoles derived from isatin, 3-(4-thiazolidone-2-hydraz-
21
2. If, among the compounds designed by a certain
chemical team, too many similar compounds satisfied
criterion 1, then only several representative structures
were selected.
2
2
ono)-isatin, 1-morpholino-methyl-3-(aryloxy-arylthio-
2
3
acetyl hydrazone)-isatin and isatin based spiroazetidi-
2
4
nones have been reported to possess anticonvulsant
activity. Hydrazones and Schiff and Mannich bases of isa-
3. If none of the designed compounds appeared as hits,
then the cutoff value P was decreased for the com-
2
5
tin also exhibit significant anticonvulsant activity. Isatin
derivatives are also reported to show antibacterial, anti-
a
pounds designed by a given chemical team.
4. If, for a compound selected as a hit, any adverse and/
or toxic effects were predicted, then this compound
was excluded from the subset of hits.
2
6–28
fungal, cytotoxic and anti-HIV activities.
The thiazole group is of great importance in biological
systems. It has been found that alkyl/aryl-aminoacetyl
2
9
derivatives of 2-amino-4-phenylthiazolyl,
2-amino-
31
benzothiazolyl, 2-amino (substituted) benzothiazolyl,
The structures of eight potential anxiolytics from differ-
ent chemical series, selected on the basis of these criteria,
3
0

A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
Table 1. Biological activities associated with anxiolytic effect in PASS
6561
a
b
MPA , %
No.
Number
Activities
1
2
3
4
5
6
7
8
9
1462
390
232
126
131
60
82
93
92
91
94
89
90
85
79
86
83
94
93
86
87
97
92
92
91
83
91
83
94
84
91
81
76
95
83
91
97
84
95
89
95
94
89
92
90
94
Anxiolytic
5 Hydroxytryptamine 1 agonist
5 Hydroxytryptamine 1A agonist
5 Hydroxytryptamine 1A antagonist
5 Hydroxytryptamine 1D agonist
5 Hydroxytryptamine 1D antagonist
5 Hydroxytryptamine 2A antagonist
5 Hydroxytryptamine 2B antagonist
5 Hydroxytryptamine 2C agonist
5 Hydroxytryptamine 2C antagonist
5 Hydroxytryptamine 3 agonist
5 Hydroxytryptamine 3 antagonist
5 Hydroxytryptamine 4 antagonist
5 Hydroxytryptamine antagonist
5 Hydroxytryptamine uptake inhibitor
Adenosine A1 receptor agonist
AMPA receptor antagonist
131
24
16
60
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
242
51
986
219
30
147
146
16
Benzodiazepine agonist
Benzodiazepine inverse agonist
Benzodiazepine omega receptor agonist
Beta adrenoreceptor antagonist
Chloride channel agonist
5
325
40
88
Cholecystokinin B antagonist
7
10
Corticotropin releasing factor 1 receptor antagonist
DOPA decarboxylase inhibitor
GABA A receptor agonist
GABA A receptor antagonist
GABA B receptor antagonist
GABA receptor agonist
58
11
12
209
104
14
Glutamate receptor agonist
Histamine H3 receptor agonist
MAO A inhibitor
42
38
Melatonin agonist
Neurokinin 1 antagonist
47
79
Neurokinin 2 antagonist
Neurokinin antagonist
161
414
41
NMDA receptor antagonist
NMDA receptor glycine site antagonist
Sigma receptor antagonist
Substance P antagonist
101
255
a
Number, is the number of compounds from the PASS training set exhibiting a particular activity.
MPA, is the minimal prediction accuracy (calculated by leave-one-out procedure) for every type of activity from the PASS training set.
b
are presented in Figure 1: 1 (from IOC), 2 and 3 (from
IC), 4 (from AU); 5 (from LU), 6–8 (from MSU).
fluorinated b-oxo-ester (in refluxing ethanol) or with di-
methyl acetylenedicarboxylate (Scheme 1).
As additional criteria, prediction of toxicities with the
Isatin derivatives 2 and 3 were prepared by phenacyl-
ation of isatin ketal 9 with x-bromoacetophenones at
room temperature (Scheme 2).
4
4
expert system DEREK was performed for the eight se-
lected compounds. For the most of them, carcinogeni-
city, mutagenicity and skin sensitization were estimated
as being plausible. This level of probability of possessing
these undesirable effects is not an obstacle for investiga-
tion of compounds at the stage of lead finding; however,
these adverse effects should be tested for at the next
stages of preclinical study. Thus, all selected hits were
synthesized and experimentally tested for their anxio-
lytic effect.
The thiazole derivative 4 was obtained by a modified
Mannich reaction (Scheme 3) described earlier.
33
Fused dialkylaminomethylimidazoles 6, 7 and 8 were
obtained by adapting the Mannich reaction (rarely ap-
4
5
plied to this class ) to known parent imidazoles 10–
4
6–48
12
(Scheme 4).
2
.3. Chemistry
2.4. Pharmacology
All selected compounds predicted to be anxiolytics were
successfully synthesized. Pyrazole derivatives 1 and 5
were obtained from phenylhydrazine by reaction with
Anxiolytic activity in selected compounds was evaluated
by the conflict situation test (Table 2). This showed that
the majority of tested compounds produced a significant

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A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
O
F
F
O
O
HCl.N
O
F
O
OH
O
N
O
O
N
N
N
N
F
F
Br
S
O
O
1
2
3
4
S
O
N
N
N
N
N
N
N
O
N
N
N
O
N
N
N
+
N
O
OH
F
F
O
5
6
7
8
Figure 1. Structures of potential anxiolytics selected on the basis of PASS prediction from 5494 virtually designed compounds.
F
F
OMe
COOMe
O
F
F
OH
OH
F
O
O
MeOOC
Ph-NHNH2
H
F
F
MeO
N
N
N
N
F
5
1
Scheme 1. The synthesis of pyrazoles
phenylpyrazole).
1
(5-hydroxy-3-(1,1,2,2-tetrafluoroethyl)-1-phenylpyrazole) and
5
(5-hydroxy-3-methoxycarbonyl-1-
S
S
O
O
O
CH =O
2
HO(CH ) OH
O
ArCOCH Br
O
2
2
2
N
N
N
N
O
O
O
+
H , C H
K CO , DMF
O
NH
6
6
2
3
N
N
H
N
H
Ar
O
N
1
0
9
6
2
3
Ar = p-FPh,
Ar =p-BrPh
O
F
F
Scheme 2. The synthesis of compounds 2 (1-[(4-fluorophenyl)-2-
oxoethyl]-3-(1,3-dioxolano)-2-indolinone) and 3 (1-[2-(4-bromophenyl)-
CH =O
2
2-oxoethyl]-3-(1,3-dioxolano)-2-indolinone).
N
N
N
N
R"
N
NH
O
O
R"
N
N
O
N
11,12
S
Ph
O
N
HCl
HCl.N
7,8
7
,11: R' = F, R" = Me
N
R'
R'
N
HCl(g), DME
8,12: R' = NO
2
, R" = Ph
HCI
Ph
S
O
4
Scheme 4. The synthesis of compounds 6 (3-(morpholinomethyl)-2-(4-
fluorophenyl)imidazo[2,1-b][1,3]thiazole), 7 (2-(4-fluorophenyl)-3-(4-meth-
ylpiperazinomethyl)imidazo[1,2-a]pyridine) and 8 (2-(4-nitrophenyl)-
Scheme 3. The synthesis of compound 4 (1-(4-methyl-2-phenyl-1,3-
thiazol-5-yl)-3-morpholin-4-yl-propan-1-one hydrochloride).
3
-(4-phenylpiperazinomethyl)imidazo[1,2-a]pyridine).
The average number of punished water licks caused by
administration of compound 1 is 243; that is, less than
for medazepam but more than found for the control.
ÔBenzodiazepine 1 agonistÕ is predicted as the most prob-
able (36.0%) mechanism of anxiolytic action for com-
pound 1. Compound 2 caused on average 308
punished water licks, which is comparable to both the
control and compound 1 but less than for medazepam.
ÔBenzodiazepine 1 agonistÕ (46.6%) and Ô5HT2A antago-
nistÕ (33.8%) are predicted as the most probable mecha-
nisms of anxiolytic action for compound 2.
anxiolytic action displayed as a considerably increased
number of punished water licks (Fig. 2).
The reference anxiolytic agent medazepam was also
tested in the same series of experiments. Administration
of medazepam in rats increased the number of punished
water licks from 167 to 392. Medazepam is a benzo-
diazepine-like compound acting on the GABA-A-ben-
zodiazepine receptor complex. Biological activities of
medazepam agreed well with those predicted by PASS:
calculated probabilities (P ) equal to 89%, 78%, 73%
a
for ÔanxiolyticÕ, ÔBenzodiazepine 1 agonistÕ and ÔGABA
receptor agonistÕ activities, respectively.
Compound 3 was administrated in DMSO because it is
unstable in aqueous solution. In general, DMSO is

A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
Table 2. The results of PASS prediction and experimental testing of selected compounds
6563
Name
Structure
P
a
P
i
Predicted activity
Number of punished water licks
Control
167 ± 32
398 ± 52
N
Medazepam
0.891
0.006
0.002
0.005
Anxiolytic
Benzodiazepine 1 agonist
GABA receptor agonist
N
0
0
.783
.734
Cl
F
F
F
OH
*
1
N
N
0.434
.359
0.051
0.132
Anxiolytic
Benzodiazepine 1 agonist
243 ± 71
F
0
O
O
O
*
2
N
0.318
0.088
0.030
0.020
Anxiolytic
Benzodiazepine 1 agonist
5 HT 2A antagonist
308 ± 53
F
0
0
.466
.338
O
DMSO
172 ± 52
O
O
O
*
3
4
(in DMSO)
N
0.394
.450
0.062
0.038
Anxiolytic
Benzodiazepine 1 agonist
785 ± 38
Br
0
O
O
HCl.N
O
*
N
0.372
.397
0.068
0.022
Anxiolytic
GABA A agonist
463 ± 100
0
S
O
N
*
N
5
6
O
0.714
0.008
0.017
0.023
Anxiolytic
Benzodiazepine 1 agonist
GABA A agonist
655 ± 78
0
0
.506
.392
OH
S
N
N
*
0.694
0.688
0.354
0.010
0.005
0.007
Anxiolytic
GABA A agonist
Benzodiazepine agonist
475 ± 59
O
N
F
F
*
7
N
0.579
0.023
0.001
0.009
0.010
0.007
Anxiolytic
588 ± 66
0
0
0
0
.641
.515
.469
.425
Benzodiazepine omega agonist
GABA A agonist
5HT2C agonist
N
N
N
5HT3 agonist
O
+
O
N
N
*
8
0.655
0.014
0.001
0.018
0.007
0.045
Anxiolytic
800 ± 128
N
0
0
.670
.424
Benzodiazepine omega agonist
GABA A receptor agonist
5HT3 agonist
N
N
0.415
.368
0
5HT2C agonist
*
Difference from control group is determined by P < 0.05.

6564
A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
Figure 2. Anxiolytic activity of tested compounds.
widely used in biological screening as a universal sol-
vent. However, to exclude the influence of DMSO on
the anxiolytic effect shown by compound 3, we deter-
mined the anxiolytic activity of DMSO separately. No
significant anxiolytic activity of DMSO was observed.
The average number of punished water licks caused by
compound 3 was 785. This value is almost twice of that
for medazepam. Agonistic action on benzodiazepine 1
receptors is predicted as the most probable (45%) mech-
anism of anxiolytic action for compound 3.
nisms of anxiolytic action for compound 8 are
ÔBenzodiazepine omega agonistÕ (67%), ÔGABA A agon-
istÕ (42.4%), Ô5HT3 agonistÕ (41.5%) and Ô5HT2C agon-
istÕ (36.8%).
Thus, we found the following order of average anxio-
lytic potency for tested compounds: 8 > 3 > 5 >
7 > 6 > 4 > medazepam > 2 > 1 >DMSO > control. This
does not completely correspond to the order of calcu-
lated probabilities for their anxiolytic effect P : 65.6%
a
(compd 8), 39.4% (compd 3), 71.4% (compd 5), 57.9%
Administration of compound 4 increased the average
number of punished water licks to 463. However, its
anxiolytic activity does not differ significantly from that
of medazepam. ÔGABA A agonistÕ (39.7%) is predicted
as the most probable mechanism of anxiolytic action
of compound 4.
(compd 7), 69.4% (compd 6), 37.2% (compd 4), 89.1%
(medazepam), 31.8% (compd 2), 43.4% (compd 1). Since
P values calculated by PASS reflect the probabilities of
compoundsÕ belonging to the class of ÔactivesÕ rather
than the values of their potency, the absence of such cor-
respondence is not surprising.
a
The average number of punished water licks caused by
compound 5 was 655. This value is less than that for
compound 3, but is significantly more than the number
of punished water licks observed for medazepam. ÔBenzo-
diazepine 1 agonistÕ (50.6%) and ÔGABA A agonistÕ
In the majority of tested compounds the probability of
anxiolytic effect (P ) is less than 70%, hence, one may
a
suggest that they may appear to be new chemical entities
9
,10
(NCEs).
To check this suggestion, we performed a di-
rect estimation of their similarity to known pharmaco-
logical agents.
(39.2%) are predicted as the most probable mechanisms
of anxiolytic action for compound 5.
2
.5. Similarity assessment
Administration of compound 6 increased the average
number of punished water licks to 475, which is approxi-
mately the same as the value for medazepam. The most
probable mechanisms of anxiolytic action predicted by
PASS for compound 6 are ÔGABA A agonistÕ (68.8%)
and ÔBenzodiazepine agonistÕ (35.4%).
We compared the selected compounds with those from
4
9
the MDDR database. The ÔsimilarityÕ procedure of
4
9
ISIS/Base 2.1.1 was used in the search for similar com-
pounds. It is considered that compounds exhibit similar
biological activity if their similarity is more than 70%.
Administration of compound 7 increased the number of
punished water licks to 588. This value exceeds the value
for compound 6 and medazepam but it is less than that
for compounds 5 and 3. The most probable predicted
mechanisms of anxiolytic action are ÔBenzodiazepine
omega agonistÕ (64.1%), ÔGABA A agonistÕ (51.5%),
Ô5HT2C agonistÕ (46.9%) and Ô5HT3 agonistÕ (42.5%).
No compound had a similarity with compound 1 greater
than 65%. Forty-one compounds had about 60% simi-
larity to compound 1. However, most of these com-
pounds
possess
antifungal
(20
compounds),
antihypertensive (11 compounds) and antiarthritic (6
compounds) effects, but none has an anxiolytic effect.
The most similar anxiolytics (19 compounds) from the
MDDR database have about 50% similarity to com-
pound 1. Compound 2 had about 70% similarity with
nine compounds from MDDR database, but none of
them is known to have anxiolytic activity. Most of them
(seven compounds) have an antidiabetic effect and
Compound 8 revealed the most potent anxiolytic effect
in comparison with the other tested substances. The
number of observed punished water licks for this com-
pound was 800. The most probable predicted mecha-

A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
6565
aldose reductase inhibiting activity. The most similar
anxiolytics (five compounds) had about 65% similarity
to compound 2. Similar results were obtained for com-
pound 3: 10 compounds from MDDR database with
antidiabetic effect and aldose reductase inhibiting activ-
ity had about 70% similarity. The most similar anxioly-
tics (three compounds) had about 65% similarity to
compound 3. Eight compounds from MDDR database
had about 70% similarity to compound 4. All of them
are antipsychotics; seven are dopamine D4 antagonists
and one is a sigma antagonist. One anxiolytic from the
MDDR database had 65% similarity to compound 4.
There are six compounds in MDDR database whose
similarity to compound 5 exceeded 70%. All of them
are neuronal injury inhibitors; five compounds are lipid
peroxidation inhibitors and one compound is an anti-
oxidant. Ten anxiolytics from the MDDR database
had about 60% similarity to compound 5. Thirteen com-
pounds with 80% similarity to compound 6 were found
in the MDDR database. Most of them are anxiolytics
tor of two. Although an anxiolytic effect of some of the
tested compounds might be predicted based on their
structural similarity to known anxiolytics, the finding
of an anxiolytic effect in compounds 3, 5 and 4 can be
considered as the discovery of NCEs. Thus, computer
prediction provides the possibility of (1) finding of new
potent anxiolytic agents and (2) significantly decreas-
ing the number of synthesized and tested compounds.
4. Experimental
4.1. PASS method
PASS software (Prediction of Activity Spectra for Sub-
stances) was used for prediction of anxiolytic effect for
5494 compounds designed in this study. PASS version
1.811 predicts 900 types of biological activity with a mean
accuracy of 85%. The general list of activity types pre-
dicted by the current version of PASS is given on the
9
(
nine compounds), anticonvulsants (nine compounds)
Website. For representation of the structural formula
of a compound, PASS uses MNA (multilevel neighbour-
and cognition enhancers (four compounds). Ten com-
pounds from the MDDR database had 80% similarity
to compound 7. They are antipsychotics (six com-
pounds), dopamine D4 antagonists (five compounds),
anxiolytics (four compounds), anticonvulsants (three
compounds) and three compounds with sedative/hyp-
notic effect. Three compounds from the MDDR data-
base had about 80% similarity to compound 8. All of
them are antipsychotics and two are dopamine D4
antagonists. Sixteen anxiolytics were similar to com-
pound 8, with 70% similarity.
5
0
hoods of atoms) descriptors. The PASS training set
contains 45,660 substances, which are represented by
41644 different MNA descriptors. The calculation of
biological activity spectrum is based on structure–
activity relationships that are stored in a SAR
knowledgebase.
A list of predicted activities, including anxiolytic effect
and molecular mechanisms of action, is given in Table
1. In most cases the prediction accuracy, calculated by
leave-one-out cross-validation, is better formolecular
mechanisms than for anxiolytic effect. Mean prediction
accuracy is about 82% for anxiolytic effect, whereas
for various mechanisms of anxiolytic action it varies
from 83% to 97%. Only three mechanisms have slightly
less prediction accuracy than that of anxiolytic effect (5
Hydroxytryptamine 2C agonist, GABA A receptor
antagonist and GABA A receptor agonist).
Thus, only 3-(Morpholinomethyl)-2-(4-fluorophenyl)-
imidazo[2,1-b][1,3]thiazole 6, 2-(4-fluorophenyl)-3-(4-
methylpiperazinomethyl)imidazo[1,2-a]pyridine 7 and
2
[
-(4-nitrophenyl)-3-(4-phenylpiperazinomethyl)imidazo-
1,2-a]pyridine 8 from the tested compounds had consid-
erable similarity to known anxiolytics from the MDDR
database, and could have been discovered by similarity
search at the 70% threshold. All other compounds can
be considered as quite new anxiolytic agents, presuma-
bly NCEs.
PASS uses MOL- or SDF-files as input of structural for-
mula(s), and PASS output is presented as a list of activity
names and probability values for the compound to be
either active (P ) or inactive (P ), respectively. Interpret-
a
i
3. Conclusions
ation of prediction results is based on consideration of
P values.
a
To discover new anxiolytics, an innovative computer-as-
sisted approach based on the PASS predictions has been
applied. The probability of finding new chemical entities
was increased by virtual combinatorial design of highly
diverse chemical compounds including different types of
heterocycles (thiazoles, pyrazoles, isatins, a-fused imi-
dazoles). Eight prospective hits from 5494 structures
presented in the initial database, selected on the basis
of computer prediction, were synthesized and tested as
potential anxiolytics. Six tested compounds 3, 4, 5, 6,
1. P > 0.7: the chance of finding activity experimentally
a
is high; in many cases the compound may be a close
analogue of known pharmaceutical agents.
2. 0.5 < P < 0.7: the chance of finding activity experi-
a
mentally is less; the compound is not so similar to
known pharmaceutical agents.
3. P < 0.5: the chance of finding activity experimentally
a
is even less; the compound has only a low similarity
to the compounds from the training set.
7
and 8 have equivalent or higher anxiolytic effect than
the reference anxiolytic medazepam. Compounds 8 (2-
4-nitrophenyl)-3-(4-phenylpiperazinomethyl)imidazo[1,-
-a]pyridine) and 3 (1-[(4-bromophenyl)-2-oxoethyl]-3-
1,3-dioxolano)-2-indolinone) have the most potent
anxiolytic effect, exceeding that of medazepam by a fac-
4.2. DEREK method
(
2
(
To estimate the toxic effects of compounds, their struc-
4
4
tures were run through DEREK, an expert system for
prediction of toxicity, developed by Lhasa Ltd (Leeds,

6566
A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
UK). DEREK predicts six categories, namely certain,
probable, plausible, implausible, improbable or impossi-
ble, for carcinogenicity, mutagenicity and skin
sensitization.
(1.92g, 0.01mol) for 1h. The mixture was stirred for
1h, and poured into water (250mL). The white precipi-
tate was filtered, washed with water and dried over
P O . Pure product 2 was prepared by crystallization
2
5
from ethanol. Yield 2.56g (78.2%), mp 190–191ꢂC.
4
.3. Chemical methods
C H FNO 327.30.
1
8
14
4
1
H NMR spectra were recorded on Aspect 3000, Bruker
AC400 (400MHz), Bruker AC-80 (80MHz), Bruker-
AW-80 spectrometers, and the chemical shifts are re-
ported in parts per million (d) downfield from
tetramethylsilane as internal standard. Electron ioniza-
tion mass spectra were recorded on a VG-250 spectro-
meter (VG Labs., Tritech England) with ionization
energy maintained at 70eV. The IR spectra were re-
corded with Perkin–Elmer 597 Specord M-80 spectrom-
eter (The Perkin–Elmer Corporation Ltd, Beaconsfield,
Bucks, England). Elemental analyses were obtained with
an acceptable range (±0.4%) using a Perkin–Elmer
Calculated (%): C 66.04; H 4.31; F 5.81; N 4.30. Found
(%): C 66.28; H 4.50; F 5.63; N 4.18. H NMR (DMSO-
1
d6): 4.35–4.55 m (4H, dioxolane ring); 4.95 s (2H,
CH CO); 7.15–7.43 m (4H, isatin ring); 7.93–8.04 m
(4H, aromatic ring).
2
4.3.4. 1-[2-(4-Bromophenyl)-2-oxoethyl]-3-(1,3-dioxolano)-
2-indolinone (3). 1-[2-(4-Bromophenyl)-2-oxoethyl]-3-
(1,3-dioxolano)-2-indolinone 3 was prepared by the
same method as for 2, using the following amounts: 3-
(1,3-dioxolano)-2-indolinone 9 (1.91g, 0.01mol), potas-
sium carbonate (2.07g, 0.015mol), 2-bromo-1-(4-
bromophenyl)-1-ethanone (2.95g, 0.01mol), DMF
(20mL). Yield 3.21g (82.7%), mp 155–156ꢂC (from
EtOH). C H FNO 327.30. Calculated (%): C 55.68;
2
400B CHN analyzer. Thin-layer chromatography
TLC) was performed on silica gel analytical TLC plates
(
(
(
(
ꢁ
60 F254, Merck, Darmstadt, Germany) and Silufol
Silpearl on aluminium foil Czechia). Melting points
uncorrected) were determined on a Boetius apparatus.
18
14
4
H 3.63; Br 20.58; N 3.62. Found (%): C 55.52; H 3.84;
Br 20.76; N 3.47. H NMR (DMSO-d ): 4.37–4.54 m
1
6
(4H, dioxalane ring); 4.93 s (2H, CH CO); 7.14–7.32
m (4H, isatin ring); 7.43–7.63 m (4H, Ar).
2
4.3.1. 5-Hydroxy-3-(1,1,2,2-tetrafluoroethyl)-1-phenylpyr-
azole (1). A mixture of ethyl-3-oxo-4,4,5,5-tetrafluoro-
pentanoate (20.14g, 0.1mol) and phenylhydrazine
(
4
4
4
.3.5. 1-(4-Methyl-2-phenyl-1,3-thiazol-5-yl)-3-morpholin-
51
-yl-propan-1-one hydrochloride (4) . The distilled ace-
10.5g, 0.1mol) in ethanol (25mL) was refluxed for
h. The solvent was removed under reduced pressure.
The resulting residue was recrystallized from hexane to
0
tylchloride (0.15mol) was added to the 4,4 -methylen-
edimorpholine (0.15mol) in dimethoxyethane (100mL).
The product obtained was allowed to stand at room
temperature for 2h. Then 2-phenyl-4-methyl-5-acetyl-
thiazole (0.1mol) was added, hydrogen chloride
was passed for 15min and the mixture was refluxed
for 2h.
give pyrazole 1 as a white powder. Yield 13.8g (53%),
mp 156–157ꢂC. C H F N O 260.1. Calculated (%): C
1
1
8
4
2
5
N 10.58. IR spectrum (cm ): 2200 (OH); 1600, 1515
0.77; H 3.08; N 10.77. Found (%): C 50.87; H 3.78;
ꢀ
1
1
(
CH), 6.44 tt (1H, H(CF ) , J = 56.6, 5.2Hz), 7.5 ws
C@C, C@N). H NMR (acetone-d ): d 5.91 s (1H,
6
2
2
(
1H, OH), 7.83 m (5H, C H ).
6 5
The solvent was evaporated and the residue was recrys-
tallized from CH OH/CH COOC H to give the final
product 4 (79%) as a white powder, mp 216–216.5.
3
3
2
5
4
2
.3.2. 3-(1,3-Dioxolano)-2-indolinone (9) (for compounds
and 3). A mixture of isatin (14.7g, 0.1mol), of ethylene
ꢀ4
UV: 8.49 · 10 mol/mL in abs C H OH gave:
2
5
glycol 18.62g, 0.3mol) and KY-2-8 (2.0g) (in acidic
form) in benzene (400mL) was refluxed for 8–10h with
water separator. The reaction was controlled by TLC
k 340nm, k 227nm, k 203nm. IR: Hydrochloride salt
1
2
3
4
gion 1720–1740cm
(mN –H). Analysis of base: C H N SO 316.40 Calcu-
lated (%): C 64.54; H 6.37; N 8.85. Found (%): C 64.31;
in liquid paraffin showed strong absorption in the re-
(mC@O) and 2700–2650cm
ꢀ1
ꢀ1
(
Silufol, UV 254, chloroform/acetone = 10:1, iodine va-
pours for peak detection). On complete reaction, KY-
-8 was filtered, the solvent was evaporated in vacuum
+
1
7
20
2
2
2
H 6.44; N 8.89.
to 100mL and the residue was cooled. The product
was filtered to give white crystals of 9. Additional
amounts of product 9 were obtained from the mother
liquor.
1
H NMR of base in CDCl : 2.75 (s, 3H, 3-thiazole CH ).
3
3
3
7
.2–3.8 (m, 4H, CH C@O), 7.1–6.8 d (m, 2H, Ar), 8.05–
2
.8 (m, 2H, Ar).
Yield 14.95g (78.3%), mp 134ꢂC (from Benzene).
C H NO 191.18. Calculated (%): C 62.82; H 4.74; N
7
4.3.6. 5-Hydroxy-3-methoxycarbonyl-1-phenylpyrazole
(5). A solution of phenylhydrazine (5.41g, 50mmol) in
methanol (100mL) was treated with dimethyl acetylene
dicarboxylate (7.11g, 50mmol). The solution was al-
lowed to stand at ambient temperature for 24h. After
evaporation of the solvent, xylenes (100mL) were added
and the mixture was heated under reflux for 2h. After
removal of the xylenes, the hydroxypyrazole 5 (71%)
was crystallized from toluene, mp 192–194ꢂC, IR
1
0
9
3
.32. Found (%): C 62.79; H 4.78; N 7.48. IR spectrum
ꢀ
1
(
O– of dioxolane ring).
cm ): 3280 (N–H); 1740 (C@O); 1200 and 1600 (–C–
4
2
.3.3. 1-[(4-Fluorophenyl)-2-oxoethyl]-3-(1,3-dioxolano)-
-indolinone (2). 2-Bromo-1-(4-fluorophenyl)-1-etha-
none (2.17g, 0.01mol) was added by small portions to
stirred mixture of DMF (20mL), potassium carbonate
ꢀ
(KBr) 1735cm ; H NMR (400MHz, DMSO-d ): 3.9
6
1 1
(
2.07g, 0.015mol) and 3-(1,3-dioxolano)-2-indolinone 9

A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
6567
(
1
s, 3H, OCH ), 6.3 (s, 1H, CH-4), 7.2–7.8 (m, 5H, Ph),
3
uation consisted of three parts: the experimental cham-
ber, the electronic block and the counter.
1
3
2.2 (br, 1H, OH);
C NMR (100MHz): 51.5
(
OCH ), 89.2 (CH-4), 122.1, 127.0, 129.0 (CHPh),
3
1
1
38.1 (CPh), 141.9 (C pyrazole), 153.4 (C pyrazole),
3
The experimental chamber, of size 275 · 275 · 450mm,
was made of an organic glass. It was based on a stand-
5
+
62.3 (CO); MS (EI, m/z, %) 218 (M , 100), 187
Å
53
(
MꢀOCH , 26), 77 (Ph, 71).
ard electrode floor made from stainless steel bars of
4
3
mm diameter with a distance between them of 8–
4.3.7. Precursors of compounds 6, 7 and 8. The following
starting materials were obtained according to published
10mm. A drinking bowl was attached to a lateral wall
of each chamber, comprising a glass vessel with a nipple
made of stainless steel. Nipples protruded on 2cm into
the chamber at a height of 5cm from the floor. In con-
trast to many other devices, in our device the drinking
bowl was in the chamber space, and not in the blacked
out compartment. This is because animals in a new sit-
uation instinctively try to hide in a dark compartment.
Thus, they are able to find a drinking bowl not due to
the deliberate search for satisfaction of motivation, but
accidentally. The electrode floor and nipples of the
drinking bowl were connected to an electronic block.
methods and further used to prepare the compounds 6–
8
: 2-(4-fluorophenyl)-benzo[d]imidazo[2,1-b]thiazole 10,
4
0
mp 151ꢂC, 2-(4-fluorophenyl)-imidazo[1,2-a]pyridine
4
1
1
1, mp 158–160ꢂC, 2-(4-nitrophenyl)-imidazo[1,2-a]-
42
pyridine 12, mp 265–267ꢂC.
4
.3.8. Synthesis of compounds 6, 7 and 8 (general
procedure). A solution of fused imidazole A (2mmol),
paraform (2.5mmol) and secondary amine (2.5mmol)
in 50mL of acetic acid was stirred at 60ꢂC for 4–5h.
The reaction mixture was poured into water, neutralized
and the precipitate filtered and, if necessary, additionally
purified by column chromatography (silica gel, chloro-
form). The following compounds have been obtained
by this method.
The electronic block contained current stabilizers (one
on each channel that provided an opportunity for inde-
pendent adjustment of their current), drive circuits of
standard signals for the counting device and delay gen-
erators for delivering punishing current to the drinking
bowls on the day of experiment. The device allowed
the registration of nonpunishable water licks during
acquisition of the skill of water licks (training, without
delivering the current to drinking bowls), and also pun-
ishing current and signals of punishable water licks dur-
ing experiments.
4
.3.8.1. 3-(Morpholinomethyl)-2-(4-fluorophenyl)imid-
azo[2,1-b][1,3]thiazole (6). Yield 75%, mp 187–188ꢂC.
C H FN OS 367.45. Calculated (%): N 11.4. Found
(
2
0
18
3
1
%): N 11.3. H NMR (DMSO-d ): 3.19 (morpholine,
6
4
H, m), 3.86 (morpholine, 4H, m), 4.06 (3-CH , 3H,
2
s), 7.3 (fluorophenyl, 2H, m), 7.43 (H-6, 1H, m), 7.57
H-7, 1H, m), 7.78 (fluorophenyl, 2H, m), 8.02 (H-8,
H, d, J = 7.8Hz), 8.13 (H-5, 1H, d, J = 8.2Hz).
(
1
The counter provided registration of nonpunishable
water licks during training and punishable water licks
during the experiments. The program recorded duration
of freezing after placing the animal into the device
chamber, the latent period of the first approach to a
drinking bowl, locomotor activity and the number of
water licks. The data saved up in these files were sub-
jected to statistical processing by means of the statistical
package ÔStatisticaÕ.
4
.3.8.2. 2-(4-Fluorophenyl)-3-(4-methylpiperazinomet-
hyl)-imidazo[1,2-a]pyridine (7). Yield 72%, mp 150–
52ꢂC. C H FN 324.40. Calculated (%): N 17.3.
1
1
9
21
4
1
Found (%): N 17.4. H NMR (DMSO-d ): 2.72 (3H,
6
N–CH , s), 3.1 (8H, piperazine, br m), 4.07 (2H, 3-
3
CH , s), 6.37 (H-8, 1H, d, J = 9.6Hz), 7.01 (H-6, 1H,
2
m), 7.89 (fluorophenyl, 2H, m), 7.3 (fluorophenyl, H-7,
3
H, m), 8.58 (H-5, 1H, d, J = 6.3Hz).
Each experiment was carried out for 3days. On the first
day the animals were completely deprived of water. Next
day, that is, after 24-h deprivation, the training (acquisi-
tion of skill of water licking from a drinking bowl) was
performed. For this purpose animals were placed in
experimental chambers for 5min. The animals explored
the chamber, and after a while found a drinking bowl
and started to drink. That day a weak current
(0.05mA) not felt by the rats was delivered to the drink-
ing bowl and the floor of the chamber, therefore water
licks were nonpunishable and their number character-
ized the intensity of drinking motivation. The third
day the animals were placed again into the experimental
chambers for 10min, but this time a direct current of
0.25mA was delivered to dummy drinking bowls and
the electrode floor of the chambers after the first water
lick for 10s. Hence, each water lick became punishable
and for satisfaction of thirst the rats had to overcome
the fear developed as a result of punishment. The in-
crease in the number of punishable water licks in ani-
mals of the treated group in comparison with the
4
.3.8.3. 2-(4-Nitrophenyl)-3-(4-phenylpiperazinometh-
yl)imidazo[1,2-a]pyridine (8). Yield 69%, mp 176–178ꢂC.
C H N O 413.48. Calculated (%): N 16.9. Found (%):
2
4
23
5
2
1
N 16.9. H NMR (DMSO-d ): 3.1–3.2 (piperazine, 8H,
6
m), 4.15 (3-CH , 2H, s), 6.7–7.0 and 7.2–7.3 (H-6, H-
2
, nitrophenyl, phenyl, 9H, m), 7.65 (H-7, 1H, d,
J = 7.9Hz), 8.3 (nitrophenyl, 2H, m), 8.43 (H-5, 1H, d,
J = 7.1Hz).
7
4
.4. Anxiolytic test
5
2
The conflict situation test for the experimental evalua-
tion of anxiolytic effect was used. Experiments were car-
ried out on 235 male rats of Wag/Rij strain in the weight
range 300–350g. Substances at dose 10mg/kg were in-
jected intraperitoneally in suspension in Tween-80, ex-
cept for compound 3, which was administrated in
dimethyl sulfoxide (DMSO). The device for carrying
out experiments using a method involving a conflict sit-

6568
A. Geronikaki et al. / Bioorg. Med. Chem. 12 (2004) 6559–6568
control group served as a measure of the effect of strain
and/or of drug injected.
18. Bhattacharya, S. K.; Chakrabarti, S. Indian J. Exp. Biol.
998, 36, 118.
1
9. Bhattacharya, S. K.; Mitra, S. K.; Acharya, S. B. J.
1
2
2
2
Psychopharmacol. 1991, 5, 202.
0. Hota, D.; Acharya, S. B. Indian J. Exp. Biol. 1994, 32,
The rats were divided into groups with equal numbers of
individuals in each one. One group served as a control
7
10.
1. Pajaoshesh, H.; Parson, R.; Popp, F. D. J. Pharm. Sci.
983, 72, 318.
2. Karali, K.; Gursoy, A. Il Farmaco 1994, 49, 819.
(
animals of this group were injected with a physiological
solution with several drops of the Tween-80), whilst the
animals in the other group were administered a drug. In
the statistical estimation of results, average and confi-
dence intervals at P < 0.05 were calculated. Analysis of
variance was used for an estimation of reliability of dif-
ferentiation from the control group.
1
23. Gursoy, A.; Karali, N.; Buyuktimkin, S.; Demirayak, S.;
Ekinic, A. C.; Ozer, H. Il Farmaco 1996, 51, 432.
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5. Sridhar, S. K.; Pandeya, S. N.; Stables, J. P.; Ramesh, A.
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Acknowledgements
2
2
The authors are grateful to INTAS for supporting this
work (Grant no. 00-711) and to MDL Information Sys-
tems for providing licenses at the ISIS and MDDR
database.
2
3
9. Srivastava, P. N.; Rai, S. K. Eur. J. Med. Chem. 1980, 15,
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