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

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 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.


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][3][4]. These structural features are present in bezitramide, fentanyl and petidine, and their analogues ( Figure 1) [2].  [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 (series A-E [10][11][12][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.

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][11][12][13].  [2,5,6,12]. 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.

Estimation of Drug-Likeness
The descriptors applied for estimation of drug-likeness are presented in Table 1 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.  21 4 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, 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.

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 of the tested compounds did not change the hyperactivity caused by administration of amphetamine ( Figure 9).  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 that direct and indirect 5-HT agonists induce HTR in rodents [20][21][22][23][24][25][26][27]. Furthermore, 5-HT2 receptor antagonists selectively block HTR [26][27][28][29][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].  . 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. 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.  Serotonin has been reported to play an important role in central regulation of body temperature [33][34][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.  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.

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.  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).

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).

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.

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).

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 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].

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.