Molecules 2015, 20, 3821-3840; doi:10.

3390/molecules20033821
OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Synthesis, Central Nervous System Activity and

Structure-Activity Relationships of Novel 1-(1-Alkyl-4-aryl-4,5-
dihydro-1H-imidazo)-3-substituted Urea Derivatives
Elżbieta Szacoń 1,*, Marzena Rządkowska 1, Agnieszka A. Kaczor 1,2, Ewa Kędzierska 3,
Sylwia Fidecka 3 and Dariusz Matosiuk 1,*

1
Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer
Modeling Lab, Faculty of Pharmacy with Division of Medical Analytics,
Medical University of Lublin, 4a Chodźki St., Lublin PL-20093, Poland;
E-Mails: [email protected] (M.R.); [email protected] (A.A.K.)
2
School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, P.O. Box 1627,
Kuopio FI-70211, Finland
3
Department of Pharmacology and Pharmacodynamics, Faculty of Pharmacy with Division of
Medical Analytics, Medical University of Lublin, 4A Chodźki St., Lublin PL-20093, Poland;
E-Mails: [email protected] (E.K.); [email protected] (S.F.)

* Authors to whom correspondence should be addressed; E-Mails: [email protected] (E.S.);

[email protected] (D.M.); Tel.: +48-81448-7275 (E.S. & D.M.);
Fax: +48-81448-7272 (E.S. & D.M.).

Academic Editor: Derek J. McPhee

Received: 27 December 2014 / Accepted: 9 February 2015 / Published: 26 February 2015

Abstract: A series of 10 novel urea derivatives has been synthesized and evaluated for their
central nervous system activity. Compounds 3a–3h were prepared in the reaction between
the respective 1-alkyl-4-aryl-4,5-dihydro-1H-imidazol-2-amines 1a and 1b and appropriate
benzyl-, phenethyl-isocyanate or ethyl 4-isocyanatobenzoate and ethyl isocyanatoacetate 2
in dichloromethane. Derivatives 4c and 4g resulted from the conversion of 3c and 3g into
the respective amides due to action of an aqueous ammonia solution. The results obtained
in this study, based on literature data suggest a possible involvement of serotonin system
and/or the opioid system in the effects of tested compounds, and especially in the effect of
compound 3h. The best activity of compound 3h may be primarily attributed to its
favourable ADMET properties, i.e., higher lipophilicity (related to lower polar surface area
and greater molecular surface, volume and mass than for other compounds) and good
Molecules 2015, 20 3822

blood-brain permeation. This compound has also the greatest polarizability and ovality.
The HOMO and LUMO energies do not seem to be directly related to activity.

Keywords: central nervous system activity; opioid receptors; urea derivatives

1. Introduction

It is well known that many diseases are accompanied by inflammation and pain. Therefore, the
search for new antinociceptive compounds is an important focus of attention for chemists as well as for
pharmacologists [1,2]. Opioid receptors are key molecular targets for antinociceptive medications.
Most morphine-like analgesics possess similar structural features, i.e., the phenyl ring, tertiary nitrogen
atom and the two carbon fragment (e.g., as a part of the piperidine ring), which are required by the
receptor cavity [2–4]. These structural features are present in bezitramide, fentanyl and petidine, and
their analogues (Figure 1) [2].

H3C O
Ph
H 3C N N NC Ph N N N CH 3
N O
O O Ph
H3C

Bezitramide Fentanyl Petidine

Figure 1. Structural formulas of bezitramide, fentanyl and petidine. Pharmacophoric features

according to the Beckett’s model are shown in bold [2,5,6].

This “pharmacophore” model was elaborated by Beckett (with its subsequent modifications [7–9])
and was one of the first models used to explain the antinociceptive activity of morphine derivatives [2].
Later, non-classical pharmacophore models explaining opioid receptor activity were suggested, as
presented in Figure 2. These models consist of a base (B), a hydrophobic (H) and aromatic moiety (Ar)
or hydrogen bond acceptor (HA), hydrophobic (H), and aromatic groups (Ar) [7,10–12].

7.6 H 9.4 Å Ar
Å
B 6.7 Å H 7.0 Å

4.5 Ar 3.6 Å HA
Å

Figure 2. Non-classical opioid receptor pharmacophore models. B—base, Ar—aromatic;

H—hydrophobic region, HA—hydrogen bond acceptor [2,5,6].

Based on the non-classical pharmacophore models for opioid receptor activity we have previously
reported a few series of compounds with antinociceptive activity mediated through the opioid system
Molecules 2015, 20 3823

(series A–E [10–13]), partially mediated through opioid system (series F [2]) or with a different
mechanism of antinociceptive activity (series G–K [5,6], Figure 3). Some of these compounds also
exerted serotoninergic activity according to the pharmacophore model presented in Figure 4.

R1 R1 R1 R1

N N NH N N
N N N
NH O
N N N N O
O NH N
H R2
O O O
R2 R2

A B C D

R1 R1
R1
R1
( )n ( )n
N N OH N OH
N N N N
NH NH O
N NH
N N
R3 R4 N
O O O
O O O

E F G H

R1 R1 R1

N NH O N N N
NH O N
O
N N O N O
O NH2 N N
O O O NH2

I J K

n=0,1; R1, R2 = H, 2-Cl, 3-Cl, 2-CH3, 4-CH3, 2-OCH3, 4-OCH3; R3 = H, Ph, R4 = Bz, 2-ClBz

Figure 3. Previously reported antinociceptive and serotoninergic compounds. Series A–E

and partially F exert their antinociceptive activity through the opioid system. Series G–K
and some of compounds from series F have antinociceptive activity of unknown
mechanism [2,5,6,10–13].

Figure 4. Pharmacophore model for the 5HT2 receptor [2,5,6,12].

Molecules 2015, 20 3824

In our continuous effort towards the discovery of novel antinociceptive compounds with additional
serotoninergic activity we have designed and synthesized a series of 10 N-substituted derivatives of
1-alkyl-4-ary(arylalkyl)imidazolidyn-2-ylideneureas 3a–3h, 4c, 4g (Scheme 1). The rationale of this
work can be summarized as follows: (1) the designed compounds follow the non-classical
pharmacophore model for opioid receptor activity as well as the pharmacophore model for the
serotoninergic activity; (2) the set of substituents in the aryl ring was selected on the basis of our
earlier experience with the substituent effect on the activity. Here we present the synthesis, drug-likeness
evaluation, ADMET prescreening, pharmacological studies for central nervous system activity and
structure-activity relationship analysis for 10 N-substituted derivatives of 1-alkyl-4-ary(arylalkyl)-
imidazolidyn-2-ylideneureas.

2. Results and Discussion

2.1. Chemistry

The synthetic route employed for the preparation of 1-(1-alkyl-4-aryl-4,5-dihydro-1H-imidazo)-3-

substituted urea derivatives is shown in Scheme 1. We have previously reported a few series of
1-aryl-4,5-dihydro-1H-imidazol-2-amine derivatives which do not possess a protonable nitrogen atom
but exhibit serotoninergic and antinociceptive activity mediated [2,10–13] or not [5,6] through the
opioid system. Encouraged by these results, we designed, synthesized and studied compounds 3a–3h,
4c, 4g (Scheme 1). Compounds 3a–3h were prepared in the reaction between the respective 1-alkyl-4-
aryl-4,5-dihydro-1H-imidazol-2-amines 1a and 1b and appropriate benzyl, phenethyl- isocyanate or
ethyl 4-isocyanatobenzoate, ethyl isocyanatoacetate 2 in dichloromethane. Derivatives 4c and 4g
resulted from the conversion of 3c and 3g into the respective amides due to the action of aqueous
ammonia solution.

CH 3 CH 3 CH 3
N N N
NH 2 NH NH
R1 N= C=O NH NH
N R1 N CON H 2
N
O O

R R R

1a, 1b 2 3a -3h 4c, 4g

Scheme 1. The synthesis scheme of the investigated compounds.

2.2. Estimation of Drug-Likeness

The descriptors applied for estimation of drug-likeness are presented in Table 1. Drug-likeness was
assessed using Lipinski’s rule as well as the placement of the investigated compounds in the chemical
space determined by the databases of the pharmacologically active compounds (CMC, Comprehensive
Medicinal Chemistry Database, containing about 7000 compounds and MDDR, MACCS-II Drug
Data Report, containing about 100,000 compounds) according to the methodology of PREADMET [14]
Molecules 2015, 20 3825

service as described previously [5,6]. Concerning Lipinski’s rule, all the compounds possess the molar
mass below 500, the number of hydrogen bond donors below five, the number of hydrogen bond
acceptors below 10, and the lipophilicity below 5. Optimal value of lipophilicity for drugs acting on
central nervous system is from 2 to 4 [15].
Regarding subsequent criteria of drug-likeness, most compounds collected in the CMC database has
lipophilicity from −0.4 to 5.6, molar refractivity in the range of 40–130, molar mass from 160 to 480,
and the number of atoms from 20 to 70 [5,6]. All the investigated compounds fulfill this criterion.
Concerning the compounds in MDDR database, the drug-like substances have the number of rings
equal or greater than 3, the number of rigid bonds equal or greater than 18, and the number of rotatable
bonds equal or greater than 6 [5,6]. Compounds 3c, 3g, 4c and 4g possess too few rings. Compound 3c
has in addition too few rotatable bonds. Compounds 3a, 3b, 3e, 3f, 4c and 4g have too low a number of
rotatable bonds which we will consider in the design of next series of compounds. Finally, molecule
drug-likeness score (fragment-based score) was calculated using Osiris Property Explorer [16]. According
to this score compounds 3a, 3b, 3c, 3f, 4c, 4g are more drug-like than the rest of compounds.
In summary, the investigated compounds may be termed drug-like, and it is justified to test them in the
in vivo experiments. Evaluation of drug-likeness has not allowed to exclude any compound from
in vivo experiments. In particular compound 3h has low drug-likeness score but it was selected for
in vivo studies as the validation of in silico approach.

Table 1. Parameters for drug-likeness estimation. HBD—a number of hydrogen bond

donors; HBA—a number of hydrogen bond acceptors.
Number
Molar Molar Rigid Rotatable Druglikeness
Comp. AlogP HBD HBA of Rings
Mass Refractivity Bonds Bonds Score
Atoms
3a 308.378 2.630 2 5 43 91.64 3 21 4 5.22
3b 322.404 2.951 2 5 46 96.24 3 19 5 5.74
3c 304.344 1.102 0 7 42 82.41 2 17 6 5.77
3d 366.414 2.827 2 7 49 102.91 3 23 6 1.30
3e 322.404 3.116 2 5 46 96.06 3 22 4 3.87
3f 336.431 3.437 2 5 49 100.67 3 22 5 4.39
3g 318.371 1.589 2 7 45 86.84 2 18 6 -1.23
3h 380.44 3.313 2 7 52 107.33 3 24 6 -0.14
4c 261.280 0.815 4 7 34 69.30 2 18 2 5.56
4g 275.306 1.301 4 7 37 73.72 2 19 2 4.16

2.3. Prediction of ADMET Properties

In order to facilitate the selection of compounds for animal studies, some ADMET parameters were
calculated (Table 2). The plot presented in Figure 5 confirms that most of the tested compounds possess
favorable ADMET properties. Comparing the plot in Figure 5 with lipophilicity values from Table 1
and polar surface areas from Table 3, it can be concluded that compounds from series 4c and 4g have
less favorable blood-brain permeation properties. All compounds are well absorbed (Figure 5), however
compounds 3f and 3h are not enough soluble in water as they have values of logS below −4 [16].
Moreover, compounds 4c and 4g have lower overall drug score which combines drug-likeness, cLogP,
Molecules 2015, 20 3826

LogS, molecular weight and toxicity risks in one convenient value than may be used to judge the
compound’s overall potential to qualify as a drug [16]. Importantly, compounds from series 3a–3h are
predicted to be non-toxic (all scores equal to 1.00 in Table 2) whereas compounds from series 4c and 4g
have middle risk (score 0.8) of mutagenic and tumorigenic properties and may have reproductive
effects. On the basis of calculation of ADMET parameters and our earlier experience on the effect of
substituents on the activity, we decided to test in vivo three compounds 3a, 3h and 4c.

Table 2. ADMET parameters of the studied compounds. S—solubility.

Toxicity Risk
Comp. LogS Drug Score
Mutagenic Tumorigenic Irritant Reproductive Effective
3a −3.454 1.00 1.00 1.00 1.00 0.85
3b −3.618 1.00 1.00 1.00 1.00 0.82
3c −2.118 1.00 1.00 1.00 1.00 0.93
3d −3.818 1.00 1.00 1.00 1.00 0.67
3e −3.923 1.00 1.00 1.00 1.00 0.80
3f −4.078 1.00 1.00 1.00 1.00 0.77
3g −2.594 1.00 1.00 1.00 1.00 0.55
3h −4.255 1.00 1.00 1.00 1.00 0.51
4c −2.004 0.80 0.80 1.00 0.80 0.48
4g −2.501 0.80 0.80 1.00 0.80 0.46

4
ADMET AlogP98

-2

-50 -25 0 25 50 75 100 125 150

ADMET PSA 2D

Figure 5. Evaluation of ADMET properties of the studied compounds.

Molecules 2015, 20 3827

Table 3. Structural and electronic parameters of investigated compounds. PSA—polar

surface area, HOMO—Highest Occupied Molecular Orbital, LUMO—Lowest Unoccupied
Molecular Orbital.
Surface PSA Volume HOMO LUMO Molecular
Comp. Ovality Polarizability
Å2 Å2 Å3 eV eV Weight
3a 586.1 61.2 288.6 1.652 −9.24 0.43 34.836 308.37
3b 573.1 65.0 304.7 1.706 −9.10 0.49 36.896 322.40
3c 589.8 107.7 278.8 1.702 −9.14 0.58 31.729 304.34
3d 660.9 104.3 328.4 1.734 −8.92 −0.65 39.549 366.41
3e 614.6 55.6 301.1 1.670 −9.20 0.42 36.854 322.40
3f 616.3 54.3 321.9 1.739 −9.07 0.53 38.789 336.43
3g 601.7 131.0 290.9 1.697 −9.26 0.38 33.862 318.37
3h 683.8 92.9 342.6 1.770 −9.00 −0.14 41.710 380.44
4c 482.5 170.8 225.8 1.556 −9.40 0.46 27.094 261.28
4g 523.3 175.1 245.2 1.630 −9.28 0.42 28.801 275.30

2.4. Pharmacological Activity

In this study we tested properties of three new 1-(1-alkyl-4-aryl-4,5-dihydro-1H-imidazo)-3-substituted

urea derivatives: 3a, 3h and 4c. Toxicity of tested compounds was 550 mg/kg ip for 3a, 1300 mg/kg ip
for 3h and 800 mg/kg ip for 4c, and therefore these ED50 values were adopted for further study. The
spontaneous activity and amphetamine hyperactivity were evaluated. The effect on body temperature
and behaviour of animals caused by administration of L-5-HTP, motor coordination as well as nociceptive
and anticonvulsant activity were also estimated. These allowed preliminary determination of the impact
of the new substances on the central nervous system (CNS) of experimental animals.
The antinociceptive properties were tested by performing “writhing test”. This test is one of the
most sensitive methods to determine the antinociceptive properties, and by its use it is possible to
detect even very weak antinociceptive agents. It is also considered as an experimental model closest to
the nature of clinical pain. It allows evaluation of analgesic action of both central and peripheral origin.
However, this method introduces some limitations: in this test it is difficult to determine the length
of antinociceptive activity, and the test is not specific as it can show an analgesic effect for many
substances [17,18]. Only substance 3h showed antinociceptive effect in this test and when
administered both in higher dose (0.1 ED50) and at half dose (0.05 ED50) caused very clear and
statistically significant (respectively p < 0.01 and p < 0.001) reduction in the number of writhing
episodes in mice (Figure 6). In order to more closely determine the mechanism of antinociceptive activity,
the writhing test was performed with the use of nonselective opioid antagonist—naloxone [19]. Naloxone
very clearly and statistically significant (p < 0.01), decreased antinociceptive activity of the substance
3h (Figure 7). This suggests the possibility of linking the activity of this compound with the opioid
system, and also makes this compound a good predictor of the structure in the search for a new group
of compounds acting through the opioid system.
Compound 3c significantly reduced the locomotor activity of animals only in a dose of 0.1 ED50
(p < 0.01), and the compound 3h—in the dose of 0.1 and 0.05 ED50 (p < 0.05) (Figure 8), while none
Molecules 2015, 20 3828

of the tested compounds did not change the hyperactivity caused by administration of amphetamine
(Figure 9).

25 control
0.1 ED50
20
0.05 ED50
writhing episodes

15 0.025 ED50
** ** p<0.01
10 *** *** p<0.001
vs control
5

0
3a 3h 4c

Figure 6. The antinociceptive effects of the tested compounds 3a, 3h and 4c, assessed
in the “writhing” test in mice. The results are expressed as mean ± SEM of a group of
8–10 mice. One-way ANOVA showed significant changes in the number of writhing
episodes of mice after the administration of the compound 3h (F5,47 = 5.734, p < 0.001).
Post hoc Dunnett’s test confirmed a significant reduction in the writhing episodes of mice
after the administration of the compound 3h in doses of 0.1 and 0.05 ED50 (p < 0.01 and
p < 0.001, respectively).
control
0.05 ED50
0.05 ED50 + Nx 5 mg/kg
30
*** p < 0.001 vs control
##
writhing episodes

## p < 0.01 vs 3h
20

***
10

0
3h

Figure 7. The influence of naloxone, 5 mg/kg s.c. on antinociceptive activity of compound

3h evaluated in “writhing” test in mice. The results are expressed as mean ± SEM of a group
of 6–14 mice. One-way ANOVA showed significant changes in the numer of writhing
episodes of mice after the administration of the compound 3h and coadministration of
compound 3h and naloxone (F2,25 = 11.42; p < 0.001). Post hoc Dunnett’s test confirmed
a significant reduction in the writhing episodes of mice after the administration of the
compound 3h in the 0.05 ED50 dose (p < 0.001). Pretreatment with naloxone increased the
numer of writhing episodes compared to compound 2 g group (p < 0.01).

Tests were also carried out to evaluate the effect of the new urea derivatives on the head-twitch
responses (HTR) in mice caused by administration of a serotonin precursor, L-5-HTP, which may
indicate the involvement of the serotonergic system in the observed effects. The head twitch response
evoked in mice occurs as a result of increased activity of central 5-hydroxytryptamine (5-HT) neuronal
systems [20]. This behavior appears to be mediated by 5-HT2 receptors. Several studies have established
Molecules 2015, 20 3829

that direct and indirect 5-HT agonists induce HTR in rodents [20–27]. Furthermore, 5-HT2 receptor
antagonists selectively block HTR [26–30], and their potency is highly correlated with the antagonist’s
affinity for 5-HT2 receptors [27,31]. However, this test is not very specific because, other compounds
such as adrenergic ligands, can change the HTR [32].

control
400 0.1 ED50
0.05 ED50
0.025 ED50
300
* p<0.05
motility

** p< 0.01
200 * * vs control
**
100

0
3a 3h 4c

Figure 8. The influence of the tested compounds 3a, 3h, and 4c on the spontaneous locomotor
activity of mice. The results are expressed as mean ± SEM of a group of 6–14 mice.
One-way ANOVA showed significant changes in locomotor activity of mice after the
administration of compounds 3a and 3h (F6,47 = 6,204, p < 0.0001). Post hoc Dunnett’s
test confirmed a significant reduction in motility of mice after the administration of the
compound 3a in the dose of 0.1 ED50 (p < 0.01) and 3h—0.1 and 0.05 ED50 (p < 0.05).
control
amph 5 mg/kg
1500 0.1 ED50
** + amph 5 mg/kg
** p<0.01 vs control
1000
motility

500

0
amph 3a 3h 4c

Figure 9. The influence of the tested compounds 3a, 3h, and 4c on amphetamine-induced
hyperactivity in mice. The results are expressed as mean ± SEM of a group of 7–8 mice.
One-way ANOVA revealed significant changes in locomotor activity of mice after the
administration of amphetamine (F4,31 = 3.885; p < 0.05). Simultaneous injection of each of
the tested compounds with amphetamine did not change the activity of mice compared to
amphetamine group.

HTR to 5-HTP were significantly decreased by all the substances investigated (p < 0.01), from
a mean of 12.2 ± 3.12 to 4.0 ± 0.86 by 3c, 4.4 ± 2.2 by 3h and 3.0 ± 0.89 by 4c (Figure 10). The result
seems to point out some connection with the 5-HT system.
Molecules 2015, 20 3830

15

head-twitch responses (HTR)

control
0.1 ED50

10 ** p < 0.01
vs control

5 ** ** **

0
3a 3h 4c

Figure 10. The influence of the tested compounds 3a, 3h and 4c on the head-twitch responses
(HTR) evoked by L-5-HTP (230 mg·kg−1). The results are expressed as mean ± SEM of
a group of 8–10 mice. One-way ANOVA showed significant changes in the number of
HTR (F3,30 = 6.902, p < 0.01). The post hoc Dunnett’s test confirmed a significant decrease
in the numer of HTR after the administration of all of the tested compounds: 3a, 3h and 4c
in the dose of 0.1 ED50 (p < 0.01).

Only the substance 3h had a significant effect on body temperature in mice: given in a dose
corresponding to 0.1 ED50 caused a very clear, statistically significant and prolonged decrease in body
temperature in mice (p < 0.001 from 30 to 90, and in 180 min, and p < 0.01 from 120 to 150 min).
Moreover, administration of a lower dose of this substance (0.05 ED50) resulted in statistically
significant reduction of the observed parameter, but this effect was somewhat lower and short-lived
(p < 0.001 at 30 min only, and p < 0.01 from 60 to 90 min) (Figure 11). Administration of other
compounds practically did not affect the body temperature of animals.
control
0.1 ED50 3h
0.05 ED50 3h
1

0
** *
-1 * ***
*** *** *
Δ°C

***
-2
***
-3
30 60 90 120 150 180
Fi
-4

Figure 11. The influence of compound 3h (used in dose of 0.1 and 0.05 ED50) on the body
temperature of mice. Each point represents the mean for a group of 8–10 mice. Two-way
ANOVA revealed significant effects for both dose [F(2.153) = 73.38; p < 0.0001] and time
[(F(5.153) = 9.29; p < 0.0001], as well as a statically significant dose × time [F(10.153) = 3.22;
p < 0.001]. Post hoc Bonferroni test confirmed a significant decrease of the body temperature
of mice after the administration of compound 3h at the dose of 0.1 ED50 in 30, 60, 90, 180 min
(p < 0.001), and from 120 to 150 min (p < 0.05) and at the dose of 0.05 ED50 in 30 min
(p < 0.001), 60 min (p < 0.01) and 90 min (p < 0.05). * p < 0.05; ** p < 0.01; *** p < 0.001.
Molecules 2015, 20 3831

Serotonin has been reported to play an important role in central regulation of body temperature [33–35].
The MAO (monoamine oxidase) type A inhibitors appear to be crucially involved in hypothermia [36].
As a result of MAO-inhibition, 5-HT levels in the body are increased and may precipitate a serotonin
syndrome. Hypothermia in rodents has been reported for MAO type A enzyme inhibitors (antidepressant
drugs) such as clorgyline [37] and harman (1-methyl-β-carboline) [38]. In the pentetrazol seizure test,
none of the tested compounds clearly reduced the severity of clonic or tonic seizures, or protected the
animals from dying (data not presented).
It should be noted that new compounds, used at a dose of 0.1 ED50, caused no coordination
disorders, as they did not change the behaviour of mice in either assay—the chimney, as well as the
rota-rod test (Figures 12 and 13). If present, impairment of motor coordination can express not only
neurotoxicity, but can also influence on the results of the other tests, e.g., on the reaction to nociceptive
stimuli of laboratory animals. Based on performed experiments, we cannot exclude any acute side
effects of these compounds. Motility (after administration of compound 3a and 3h (only at the dose of
0.1 ED50) as well as body temperature of normothermic mice (after compound 3h—at the dose of 0.1
and 0.05 ED50) were decreased, indicating some depressant action on the CNS.
80
control
0.1 ED50
time on rota-rod (s)

60

40

20

0
3a 3h 4c

Figure 12. The influence of the tested compounds 3a, 3h and 4c on motor coordination in
the rota-rod test. The results are expressed as mean ± SEM of a group of 10 mice. One-way
ANOVA did not show any significant changes in time spent on rota-rod [F3,36 = 0.61;
p = 0.6129].

20 control
0.1 ED50
time in chimney (s)

15

10

0
3a 3h 4c

Figure 13. The influence of the tested compounds 3a, 3h and 4c on motor coordination
in the chimney test. The results are expressed as mean ± SEM of a group of 8–10 mice.
None of the investigated compounds caused motor deficits in the chimney test. All animals
were able to leave the chimney within less than 60 s.
Molecules 2015, 20 3832

The results of the pharmacological investigation showed that compounds tested exert significant
influence on the CNS of laboratory animals. The observed effects seem to be connected primarily with
serotonergic and/or opioid system. This involvement, however, is unclear and requires further study.

2.5. Structure-Activity Relationship

HOMO and LUMO orbitals for selected compounds are shown in Figure 14. Molecular structures
and electrostatic potential distribution of selected compounds are presented in Figure 15.

Figure 14. HOMO (A, C) and LUMO (B, D) for 3h (A, B) and 4c (C, D).

Figure 15. Molecular structures of 3h (A) and 4c (B). The map of the electrostatic
potential (ESP) onto a surface of the electron density for 3h (C) and 4c (D).
Molecules 2015, 20 3833

The best activity of compounds 3h may be primarily attributed to its favourable ADMET properties,
i.e., higher lipophilicity (related to lower polar surface area and greater molecular surface, volume
and mass than for other compounds, Table 3) and good blood-brain permeation. Ths compound has
also the greatest polarizability and ovality. The HOMO and LUMO energies do not seem to be directly
related to activity (Table 3).

3. Experimental Section

3.1. Chemistry

All commercial reagents and solvents were purchased from Sigma-Aldrich (Spruce, St. Louis, MO,
USA) and used without purification. Reactions were routinely monitored by thin-layer
chromatography (TLC) in silica gel (60 F254 plates Merck, Darmstadt, Germany) and the products were
visualized with ultraviolet light of 254 nm wavelength. All NMR spectra were acquired on a Bruker
AVANCE III 300 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with BBO Z-gradient
probe. Spectra were recorded at 25 °C using DMSO as a solvent with a non-spinning sample in 5 mm
NMR-tubes. MS spectra were recorded on Bruker microTOF-Q II and processed using Compass Data
Analysis software. The elementary analysis was performed with the application of Perkin-Elmer
analyzer (940 Winter St., Waltham, MA, USA). Melting points were determined with a Boetius
apparatus (Jena, Germany).

3.1.1. General Procedure for the Synthesis of Compounds 3a–3h

Benzyl/phenethylisocyanate or ethyl 4-isocyanatobenzoate, ethyl isocyanatoacetate 2 were dissolved

in dichloromethane (25 mL) under atmosphere of dry nitrogen and added to a solution of the free
base of 1-alkyl-4-aryl-4,5-dihydro-1H-imidazol-2-amines 1a, 1e (0.01 mol) dissolved in
dichloromethane (100 mL). The mixture was shaken for 24 h at room temperature. Solvent was
removed by distillation and the rubber-like residue was treated with warm propan-2-ol. The solid product
was filtrated off and recrystallized from propan-2-ol.

1-(1-Methyl-4-phenyl-4,5-dihydro-1H-imidazo)-3-benzylurea (3a). From 1a (1.75 g) 3a (1.51 g, 49%

yield) was obtained as a white crystalline solid, mp 145–146 °C; 1H-NMR (DMSO-d6): δ = 8.63 (s,
1H, NH); 8.01 (s, 1H, NH); 7.08–7.42 (m, 10H, H-Ar); 4.01–4.17 (d, 2H, C4, J = 7.6 Hz); 3.61 (s, 1H,
C5), 3.48 (s, 2H, CH2benzyl); 2.24 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ = 17.1 (CH3); 38.2 (CH2);
48.5 C5 (CH2); 68.9 C4 (CH); 161.0 (C=N); 171.0 (C=O); 119.1, 120.3, 120.7, 122.9, 124.2, 127.6 128.4,
129.1, 129.8 (C-Ar); EIMS m/z 309.1 [M+H]+. HREIMS (m/z): 308.1340 [M+]; (Calcd for C18H20N4O
308.3900); Anal. Calcd for: C18H20N4O; Anal. Found C, 70.35; H, 6.59; N, 17.17; Calcd C, 70.10; H,
6.35; N, 18.16.

1-(1-Methyl-4-phenyl-4,5-dihydro-1H-imidazo)-3-phenethylurea (3b). From 1a (1.75 g) 3b (1.06 g,

33% yield), was obtained as a white crystalline solid, mp 120–122 °C; 1H-NMR (DMSO-d6): δ = 8.56
(s, 1H, NH); 7.29 (s, 1H, NH); 6.89–7.63 (m, 10H, H-Ar); 4.00–4.10 (d, 2H, C4, J = 7.5 Hz); 3.45 (s,
1H, C5); 3.78–3.85 (m, 2H, CH2phenethyl); 3.17–3.24 (m, 2H, CH2phenethyl); 2.06 (s, 3H, CH3); 13C-NMR
(DMSO-d6): δ = 18.4 (CH3); 41.9 (CH2); 45.2 (CH2); 49.96 C5 (CH2); 67.8 C4 (CH); 161.3 (C=N);
Molecules 2015, 20 3834

171.6 (C=O); 114.2, 120.4, 120.8, 126.5, 127.1, 128.2, 129.1, 131.4,131.8, (C-Ar); EIMS m/z 323.5
[M+H]+. HREIMS (m/z): 322.1921 [M+] (Calcd for C19H22N4O 322.4170); Anal. Calcd for:
C19H22N4O: Anal. Found Found C, 70.65; H, 6.79; N, 17.49; Calcd C, 70.78; H, 6.87; N, 17.37.

1-(1-Methyl-4-phenyl-4,5-dihydro-1H-imidazo)-3-(ethoxycarbonylmethyl)urea (3c). From 1a (1.75 g)

3c (1.49 g, 48% yield) was obtained as a white crystalline solid, mp 159–161 °C; 1H-NMR (DMSO-d6):
δ = 8.60 (s, 1H, NH); 8.27 (s, 1H, NH); 7.11–7.61 (m, 5H, H-Ar); 3.76 (s, 1H, C5), 4.02–4.15 (d, 2H,
C4, J = 7.4 Hz); 3.98–4.02 (m, 2H, CH2); 3.71–3.82 (m, 2H, CH2); 2.30 (s, 3H, CH3); 1.26–1.31 (t, 3H,
CH3, J = 7.1 Hz) 13C-NMR (DMSO-d6): δ = 17.9 (CH3); 19.3 (CH3); 41.3 (CH2); 43.6 (CH2); 49.6 C5
(CH2); 68.1 C4 (CH); 160.9 (C=N); 171.3 (C=O); 119.1, 120.4, 120.9, 123.5, 127.4, 129.1, 129.8,
132.5, 132.8, 133.5 (C-Ar); EIMS m/z 305.3 [M+H]+. HREIMS (m/z): 304.16730 [M+] (Calcd for
C15H20N4O3 304.3570); Anal. Calcd for: C15H20N4O3; Anal. Found C, 59.35; H, 6.29; N, 18.38; Calcd
C, 59.20; H, 6.22; N, 18.41.

1-(1-Methyl-4-phenyl-4,5-dihydro-1H-imidazo)-3-(4-ethoxycarbonylphenyl)urea (3d). From 1a (1.75 g)

3d (2.82 g, 77% yield) was obtained as a white crystalline solid, mp 170–172 °C; 1H-NMR (DMSO-d6):
δ = 9.05 (s, 1H, NH); 8.16 (s, 1H, NH); 7.05–7.68 (m, 9H, H-Ar); 4.01–4.18 (d, 2H, C4, J = 7.6 Hz);
3.29 (s, 1H, C5); 2.73–2.90 (m, 2H, CH2); 1.33 (s, 3H, CH3); 1.15–1.28 (t, 3H, CH3, J = 7.0 Hz);
13
C-NMR (DMSO-d6): δ = 18.3 (CH3); 21.3 (CH3); 41.3 CH2; 41.5 C5 (CH2); 69.3 C4 (CH); 161.2
(C=N); 171.7 (C=O); 170.9 (C=O); 117.6, 119.7, 128.3, 129.7, 131.3, 131.6, 133.5, 134.9, 136.1,
136.7 (C-Ar); EIMS m/z 367.1 [M+H]+. HREIMS (m/z): 366.1120 [M+] (Calcd for C20H22N4O3
366.4280); Anal. Calcd for: C20H22N4O3; Anal. Found C, 65.45; H, 6.19; N, 15.38; Calcd C, 65.56; H,
6.05; N, 15.29.

1-[1-Methyl-4-(methylphenyl)-4,5-dihydro-1H-imidazo]-3-benzylurea (3e). From 1b (1.89 g) 3e (1.96 g,

61% yield) was obtained as a white crystalline solid, mp 132–134 °C; 1H-NMR (DMSO-d6): δ = 8.73 (s,
1H, NH); 8.04 (s, 1H, NH); 7.03–7.51 (m, 9H, H-Ar); 3.63 (s, 1H, C5), 4.02–4.18 (d, 2H, C4, J = 7.6 Hz);
3.85 (s, 2H, CH2benzyl); 2.29 (s, 3H, CH3); 1.63 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ = 18.8 (CH3);
20.8 (CH3); 41.3 (CH2); 43.6 (CH2); 49.6 C4 (CH2); 68.4 C5 (CH); 162.5 (C=N); 172.0 (C=O); 119.2,
121.4, 121.9, 123.5, 128.2, 129.5, 130.8, 133.5, 133.8, 135.3 (C-Ar); EIMS m/z 319.1 [M+H]+.
HREIMS (m/z): 318.2630 [M+] (Calcd for C19H22N4O 322.7050); Anal. Calcd for: C19H22N4O; Anal.
Found C, 70.65; H, 6.69; N, 17.42; Calcd C, 70.71; H, 6.87; N, 17.38.

1-[1-Methyl-4-(4-methylphenyl)-4,5-dihydro-1H-imidazo)-3-phenethylurea (3f). From 1b (1.89 g) 3f

(2.05 g, 61% yield) was obtained as a white crystalline solid, mp 116–117 °C; 1H-NMR (DMSO-d6):
δ = 8.81 (s, 1H, NH); 8.54 (s, 1H, NH); 7.19–7.61 (m, 9H, H-Ar); 3.81 (s, 1H, C5), 4.09–4.19 (d, 2H,
C4, J = 7.5 Hz); 3.61–3.76 (m, 2H, CH2phenethyl); 3.11–3.37 (m, 2H, CH2phenethyl); 2.30 (s, 3H, CH3);
13
C-NMR (DMSO-d6): δ = 17.6 (CH3); 21.8 (CH3); 41.2 (CH2); 43.6 (CH2); 49.6 C4 (CH2); 69.2 C5
(CH); 160.8 (C=N); 172.8 (C=O); 120.0, 127.3, 120.5, 122.7, 127.2, 129.1, 129.4, 131.0, 131.9, 133.5
(C-Ar); EIMS m/z 337.3 [M+H]+. HREIMS (m/z): 336.3210 [M+] (Calcd for C20H24N4O 336.4440);
Anal. Calcd for: C20H24N4O; Anal. Found C, 71.55; H, 7.32; N, 16.68; Calcd C, 71.40; H, 7.19;
N, 16.65.
Molecules 2015, 20 3835

1-[1-Methyl-4-(4-methylphenyl)-4,5-dihydro-1H-imidazo]-3-(ethoxycarbonylmethyl)urea (3g). From

1b (1.89 g) 3g (1.56 g, 49% yield) was obtained as a white crystalline solid, mp 183–185 °C; 1H-NMR
(DMSO-d6): δ = 9.04 (s, 1H, NH); 8.33 (s, 1H, NH); 7.02–7.64 (m, 4H, H-Ar); 3.49 (s, 1H, C5),
4.04–4.20 (d, 2H, C4, J = 7.5 Hz); 3.98–4.02 (m 2H, CH2); 3.51–3.62 (m 2H, CH2); 2.45 (s, 3H, CH3);
2.01 (s, 3H, CH3); 1.28–1.39 (t, 3H, CH3, J = 7.2) 13C-NMR (DMSO-d6): δ = 17.9 (CH3); 20.5 (CH3);
22.9 (CH3); 41.0 (CH2); 43.6 (CH2); 48.6 C5 (CH2); 67.1 C4 (CH); 161.5 (C=N); 171.9 (C=O); 112.3,
112.3, 112.8, 119.5, 120.9, 121.1, 128.7, 130.5, 131.8, 132.0 (C-Ar); EIMS m/z 319.1 [M+H]+.
HREIMS (m/z): 318.1560 [M+] (Calcd for C16H22N4O3 318.3840); Anal. Calcd for: C16H22N4O3; Anal.
Found C, 60.45; H, 6.59; N, 17.38; Calcd C, 60.36; H, 6.97; N, 17.60.

1-[1-Methyl-4-(4-methylphenyl)-4,5-dihydro-1H-imidazo]-3-(4-ethoxycarbonylphenyl)urea (3h). From

1b (1.89 g) 3h (2.51 g, 66% yield) was obtained as a white crystalline solid, mp 154–156 °C; 1H-NMR
(DMSO-d6): δ = 9.18 (s, 1H, NH); 8.44 (s, 1H, NH); 7.18–7.60 (m, 8H, H-Ar); 4.09–4.20 (d, 2H, C4,
J = 7.4 Hz); 3.56 (s, 1H, C5); 2.79–2.81 (m, 2H, CH2); 1.97 (s, 3H, CH3); 1.33 (s, 3H, CH3); 1.12–1.25
(t, 3H, CH3); 13C-NMR (DMSO-d6): δ = 18.2 (CH3); 21.1 (CH3); 22.0 (CH3); 40.2 (CH2); 49.6 C5
(CH2); 68.0 C4 (CH); 162.1 (C=N); 171.5 (C=O); 119.9, 120.2, 121.6, 122.5, 123.4, 128.1, 122.8,
127.1, 127.8, 129.0 (C-Ar); EIMS m/z 381.4 [M+H]+. HREIMS (m/z): 380.3510 [M+] (Calcd for
C21H24N4O3 380.4550); Anal. Calcd for: C21H24N4O3; Anal. Found C, 66.45; H, 6.59; N, 14.81; Calcd
C, 66.30; H, 6.36; N, 14.73.

3.1.2. General Procedure to Obtain Compounds 4c, 4g

1-[1-Methyl-4-(alkyl)phenyl-4,5-dihydro-1H-imidazo]-3-(ethoxycarbonylmethyl)ureas 3c, 3g
(0.01 mol) were dissolved in methanol (50 mL) and added to 20% aqueous ammonia solution. The
mixture was shaken for 6 h at room temperature. The solid product was filtrated off and recrystallized
from propan-2-ol.

1-(1-Methyl-4-phenyl-4,5-dihydro-1H-imidazo)-3-aminocarbonylmethylurea (4c). From 3c (3.04 g) 4c

(1.65 g, 60% yield) was obtained as a white crystalline solid, mp 142–144 °C; 1H-NMR (DMSO-d6):
δ = 9.62 (s, 1H, NH); 8.46 (s, 1H, NH); 8.11–8.16 (d, 2H, NH2); 7.40–7.69 (m, 5H, H-Ar); 3.89 (s, 1H,
C4), 4.03–4.17 (d, 2H, C5, J = 7.5 Hz); 3.98–4.02 (m 2H, CH2); 2.30 (s, 3H, CH3); 13C-NMR
(DMSO-d6): δ = 23.8 (CH3); 41.3 (CH2); 49.9 C5 (CH2); 69.8 C4 (CH); 160.3 (C=N); 171.3 (C=O);
172.5 (C=O); 119.3, 120.3, 121.6, 125.5, 125.9, 128.1, 129.7, 129.9 (C-Ar); EIMS m/z 276.3 [M+H]+.
HREIMS (m/z): 275.3210 [M+] (Calcd for C13H17N5O4 275.3190); Anal. Calcd for: C13H17N5O4; Anal.
Found C, 56.45; H, 6.39; N, 25.38; Calcd C, 56.71; H, 6.22; N, 25.43.

1-[1-Methyl-4-(4-methylphenyl)-4,5-dihydro-1H-imidazo]-3-aminocarbonylmethylurea (4g). From 3g

(3.18 g) 4g (1.33 g, 46% yield) was obtained as a white crystalline solid, mp 128–130 °C; 1H-NMR
(DMSO-d6): δ = 9.23 (s, 1H, NH); 8.61 (s, 1H, NH); 8.05–8.11 (d, 2H, NH2); 6.76–7.38 (m, 4H,
H-Ar); 3.73 (s, 1H, C5), 4.03–4.18 (d, 2H, C4, J = 7.6 Hz); 3.98–4.02 (m 2H, CH2); 2.30 (s, 3H, CH3);
1.93 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ = 17.5 (CH3); 23.4 (CH3); 40.2 (CH2); 45.0 C5 (CH2); 68.5
C4 (CH); 160.7 (C=N); 172.1 (C=O); 169.8 (C=O); 118.3, 118.4, 120.1, 127.0, 127.9, 128.1, 129.7,
131.1 (C-Ar); EIMS m/z 290.5 [M+H]+. HREIMS (m/z): 289.1330 [M+] (Calcd for C14H19N5O2
Molecules 2015, 20 3836

289.3460); Anal. Calcd for: C14H19N5O2; Anal. Found C, 58.23; H, 6.52; N, 24.38; Calcd C, 58.11; H,
6.61; N, 24.21.

3.2. Pharmacology

The experiments were performed on male Albino Swiss mice (18–30 g). The animals were kept
8–10 to a cage, at room temp. of 20 ± 1 °C, on a 12:12 h dark-light cycle. Standard food (LSM, Motycz,
Poland) and water were available ad libitum. All experiments were performed between 9:00 a.m.
and 4:00 p.m. The experiments were performed in accordance with the opinion of Local Ethics
Committee for Animal Experimentation.
The investigated substances, 3c, 3h and 4c, were administered intraperitoneally (i.p.) or subcutaneously
(s.c.) in volume of 10 mL·kg−1 as suspensions in aqueous solution of 0.5% methylcellulose (tylose).
The compounds were injected 60 min before the tests. The controls received the equivalent volume of
the solvent. All tests performed, suggested by Vogel and Vogel [18], are generally accepted as basic in
investigation of the central activity by behavioral methods. The acute toxicity of the compound was
assessed in mice according to Litchfield and Wilcoxon method [39], as the ED50 calculated as “the lost
of righting reflex” within 48 h. The compounds were injected in doses equivalent to 0.1, 0.05 and
0.025 ED50.
In addition, the activity of compounds was assessed in the following test: (1) locomotor activity
was measured in photoresistor actometers for single mice for 30 min as spontaneous activity and
amphetamine-induced hyperactivity: mice received subcutaneusly (s.c.) 5 mg/kg of amphetamine 30 min
before the test; (2) nociceptive reactions were studied in the acetic acid (0.6%) induced writhing test [40].
The number of writhing episodes was measured for 10 min starting 5 min after i.p. administration of
acid solution; (3) motor coordination was evaluated in rota rod test: [41] motor impairments, defined
as the inability to remain on the rotating rod for 1 min were measured and the mean time spent on the
rota-rod was counted for each mouse) and chimney test: [42] motor impairments were indicated by the
inability to perform the test within 1 min); (4) body temperature in normothermic mice was measured
in the rectum by thermistor thermometer; (5) pentylenetetrazole (110 mg/kg, s.c.)-induced convulsions
were evaluated as the number of mice with clonic seizures, tonic convulsions and dead animals;
(6) head twitch responses (HTR) after 5-hydroxytryptophan (L-5-HTP), were estimated acc. to
Corne et al. [43]. Mice received L-5-HTP (230 mg/kg, i.p.) and the number of HTR was recorded in
6 two-minutes intervals (4–6, 14–16, 24–26, 34–36, 44–46, 54–56 min).
The obtained data were calculated by Fisher exact test (pentylenetetrazole-induced seizures),
two-way analysis of variance (ANOVA) and followed by a post hoc confirmation with the Bonferroni
test (body temperature), and one-way ANOVA followed by Dunnett’s post hoc test (other tests).

3.3. Molecular Modeling

The compounds investigated were modeled using the LigPrep protocol from the Schrödinger Suite [44].
In order to sample different protonation states of ligands in physiological pH, Epik module was used [45].
Parameters to evaluate drug-likeness were calculated using VegaZZ v. 3.0.1 [46] (number of atoms),
Discovery Studio v. 3.1. [47] (molar mass, number of rings, lipophilicity, number of rotatable bonds),
ACDLabs (molar refractivity, number of hydrogen bond donors and acceptors), and the Schrödinger
Molecules 2015, 20 3837

Suite (a number of rigid bonds) as described previously [2,12,13]. Drug-likeness was also evaluated
with Osiris Property Explorer [16]. This approach is based on a list of about 5300 distinct substructure
fragments with associated drug-likeness scores. The drug-likeness is calculated summing up score
values of those fragments that are present in the molecule under investigation. ADMET parameters
were calculated with Discovery Studio 3.1 (solubility, blood-brain permeation) or Osiris Property
Explorer [16] (toxicity risks). The prediction of toxicity by this tool relies on a precomputed set of
structural fragment that give rise to toxicity alerts in case they are encountered in the investigated
structure. For structure–activity relationship studies, HOMO and LUMO energies and polarizabilty
were calculated with Discovery Studio 3.1 and molar surface, volume, ovality and polar surface area
were calculated with VegaZZ as reported earlier [2,12,13]. The maps of the electrostatic potential
(ESP) onto a surface of the electron density were visualized with ArgusLab [48].

4. Conclusions

In conclusion, we have designed and studied 10 compounds and one of them exhibited antinociceptive
properties probably connected with the opioid system accompanied by serotoninergic properties. Further
studies are necessary to investigate the molecular mechanism for this compound which will enable to
apply structure-based design methods to obtain more favorable modifications.

Acknowledgments

The paper was developed using the equipment purchased within the project “The equipment of
innovative laboratories doing research on new medicines used in the therapy of civilization and
neoplastic diseases” within the Operational Program Development of Eastern Poland 2007–2013,
Priority Axis I modern Economy, operations I.3 Innovation promotion. The research was partially
performed during the postdoctoral fellowship of Agnieszka A. Kaczor at University of Eastern Finland,
Kuopio, Finland under a Marie Curie fellowship. Part of the calculations was performed under
a computational grant by Interdisciplinary Center for Mathematical and Computational Modelling
(ICM), Warsaw, Poland, grant number G30-18 and under resources and licenses by CSC, Finland.

Author Contributions

E.S., M.R., A.A.K. designed research, performed synthesis of title derivatives, analyzed data,
participated in the discussion of the obtained results and contributed to writing of this manuscript,
E.K. performed research, analyzed the data, contributed to writing of this manuscript. S.F. and D.M.
contributed to discussion of results. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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