Novel Isothiourea Derivatives as Potent Neuroprotectors and

Mar 10, 2009 - ... effects of 3 and cyclothiazide (CT) on transmembrane currents caused by kainic acid (KA) and glutamate in Purkinje neurons was perf...
0 downloads 0 Views 280KB Size
Download PDF
J. Med. Chem. 2009, 52, 1845–1852

1845

Novel Isothiourea Derivatives as Potent Neuroprotectors and Cognition Enhancers: Synthesis, Biological and Physicochemical Properties German L. Perlovich,*,†,‡ Alexey N. Proshin,‡ Tatyana V. Volkova,† Sergey V. Kurkov,† Vlaimir V. Grigoriev,‡ Ludmila N. Petrova,‡ and Sergey O. Bachurin‡ Institute of Solution Chemistry, Russian Academy of Sciences, 153045 IVanoVo, Russia, and Institute of Physiologically ActiVe Compounds, Russian Academy of Sciences, 142432, ChernogoloVka, Russia ReceiVed October 13, 2008

Various salts of 3-allyl-1,1-dibenzyl-2-ethyl-isothiourea, 1 (hydrochloride), 2 (hydrobromide), and 3 (hydroiodide), were synthesized. Ca-blocking properties of these salts were studied. Comparative analysis of the potentiating effects of 3 and cyclothiazide (CT) on transmembrane currents caused by kainic acid (KA) and glutamate in Purkinje neurons was performed. Analysis of the effects of 1 on N-methyl-D-aspartate (NMDA) receptors was performed on primary culture of heterogeneous neurons of rat cerebral cortex. Single crystals were grown and X-ray diffraction experiments solving the crystal structures of 1-3 were carried out. Analysis of conformations of the molecules in the crystal lattices was performed. The temperature dependencies of the solubility of 1-3 in water and n-octanol were obtained, and the thermodynamic parameters of solubility process were calculated. The effect of halogen atoms on the solubility was analyzed. The partitioning processes in the water-octanol system were studied at 25 °C. Chemical stability of tested salts in pH 7.4 phosphate buffer was determined at 25 °C. Introduction a

The N-methyl-D-aspartate (NMDA ) receptor belongs to a family of ionotropic glutamate receptors and performs important functions in the central nervous system. It is mainly involved in neuronal signaling processes, memory consolidation, and synaptic plasticity.1,2 The neurotoxicity that is induced by NMDA hyperactivation leads to a number of pathological conditions ranging from acute neurodegenerative disorders, such as stroke and trauma, to chronic forms of neurodegenerative diseases, i.e., Huntington’s disease, Parkinson’s disease, Alzheimer’s disease (AD), and lateral amyotrophic sclerosis.3-6 It is generally recognized that specific inhibition of calcium ion influx via hyperactivated glutamate(Glu)-receptor-channel complex (GluR), particularly the NMDA-subtype receptor, provides sufficient protection against a diverse group of neurological stresses related to the Glu excitotoxicity, such as brain ischemia, AD, etc.7 In the case of AD, it is considered that specific inhibitors of Glu-stimulated calcium uptake in nervous cells provides sufficient neuronal protection against the neurotoxic effect associated with β-amyloid peptide (Aβ) which is the major pathogen in AD, which is particularly realized by potentiating of excitotoxic effects of endogenous glutamate.8 On the other hand, it is assumed that glutamate receptor agonists (especially R-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype receptor agonists) exhibit strong cognition-enhancing effects due to the activation of glutamatergic neurotransmission.9 Such a dualism in properties of glutamatergic compounds explains the strong interest in these compounds as promising neuroprotectors and cognition enhancers. * To whom correspondence should be addressed. Phone: (+7) 4932 533784. Fax: (+7) 4932 336237. E-mail: [email protected]. † Institute of Solution Chemistry. ‡ Institute of Physiologically Active Compounds. a Abbreviations: NMDA, N-methyl-D-aspartate; AD, Alzheimer’s disease; GluR, glutamate(Glu)-receptor-channel complex; Glu, glutamate; Aβ, β-amyloid peptide; AMPA, R-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Glu-Ca-uptake, glutamate-stimulated calcium ions uptake; CNS, central nervous system; CT, cyclothiazide; KA, kainic acid.

The major goal of this study was focused on the properties of various 3-allyl-1,1-dibenzyl-2-ethylisothiourea salts 1 (hydrochloride, IP5051_Cl), 2 (hydrobromide, IP5051_Br), and 3 (hydroiodide, IP5051_I) (Figure 1) that we synthesized and developed.20 In the present study, we used a variety of in vitro and in vivo methods to perform the primary screening and selection of potential neuroprotectors and cognition enhancers in wide spectrum of GluR ligands. Particularly, we analyzed properties of these salts to inhibit the total and specific glutamatestimulated calcium ions uptake (Glu-Ca-uptake) in rat brain synaptosomes. Interaction of the drug molecule with target receptors is a key feature in the design drug molecules; however, drug delivery plays an equally important role. Therefore, screening parameters characterizing the solubility, partitioning, and passive transport processes are an essential part of the drug design. Hansch’s group10 suggested that the octanol-water partitioning (log Poct) is most commonly used method in QSAR studies. The index around 2 represents the optimal lipophilic nature of the tested molecule that is required for penetrating the CNS; however, this rule is not based on the permeability rates or equilibrium concentrations. Instead it only takes into account tests of biological activity. Two decades ago Ganellin’s group11 failed to predict the brain penetration of H2 antagonists. The log P data from the cyclohexane-water system (log Pchex) were likewise inadequate. In contrast, Young et al.11 reported that there is a significant correlation between the difference of log Poct and log Pchex data (∆ log P) as well as the logarithm of the brain/ blood equilibrium concentration ratios of H2 antagonists. They concluded that ∆ log P accounts for hydrogen bonding and reflects two different events. The log Pchex value quantifies the partitioning of the molecule into nonpolar regions of the brain, whereas the log Poct value describes protein interactions in blood. Thus, in order to optimize the brain penetration of a drug molecule, the overall H-bonding property of this molecule has to be minimized. Abraham12-14 and colleagues interpret the

10.1021/jm8012882 CCC: $40.75  2009 American Chemical Society Published on Web 03/10/2009

1846 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

PerloVich et al.

Figure 1. Structures of the compounds studied: (a) 1; (b) 2; (c) 3.

∆ log P value in a more complex way. They suggest not only that the hydrogen bond acidity is important but also that the solute hydrogen bond basicity and dipolarity/polarizability contribute and lead to a more positive ∆ log P value, thus favoring the penetration through the blood-brain barrier. In other words, the larger is the solute, the more negative the ∆ log P value becomes, thereby favoring penetration into the brain. In the second part of this study we determined the solubility of compounds that were selected in model solvents (water and n-octanol) within a wide temperature range and analyzed the impact of different types of anions (Cl-, Br-, and I-) on the outlined processes. Moreover, the partitioning processes of the compound in the model systems were described and the relationship between the partitioning coefficients and biological activity parameters was discussed. Results and Discussion Biological Experiments. Ca-Blocking Properties. The results of the tested compounds for their ability to inhibit GluCa-uptake are the following: 3, KI ) 6.7 ( 1.8; 2, KI ) 3.0 ( 1.2; 1, KI ) 4.2 ( 1.5. Comparative Study of 1-3 and Cyclothiazide Action on AMPA Receptors. 3 is the lead compound of the studied salts in our analysis. Both 3 and 2 salts were investigated. The goal of this stage was to determine the mechanism of the 1-3 effect. Our previous experiments revealed that the positive effect of this compound on learning and memory in animal models is probably linked to its ability to potentiate kainate-induced currents in brain neurons.15 Nowadays, cyclothiazide (CT) is one of the most effective positive modulators of AMPA receptors. The mechanism of its effect is determined via the dose-dependent inhibition of desensitization of AMPA receptors which is caused by their agonists such as AMPA agonists themselves, glutamate, and to a lesser extent kainate. Therefore, we performed a comparative study of CT and 1-3 effects on AMPA receptors on the transmembrane currents induced by the activation of these receptors with their agonists. The comparison of CT and 1-3 was useful in revealing the diversity and/or similarity of their interactions with AMPA receptors. Experiments were performed on Purkinje neurons from rat cerebellum that contained AMPA receptors. Since both glutamate and AMPA caused fast and significant desensitization of the currents, making it impossible to obtain the dose depended response, kainate was used as agonist in our experiments (Figure 2). The experiments demonstrated qualitatively similar effects of CT and 3 on kainate and glutamate currents (Figure 3). To find the quantitative difference between effects of these compounds, the dose-dependent responses for both kainate and kainate in the presence of CT and 3 were measured. As a result, our analysis revealed an inverse dependence of the potentiating effect of both of these compounds on the agonist dose; i.e., low

Figure 2. Transmembrane currents induced in Purkinje neurons by different doses of glutamate and KA. (a) Currents induced by different concentrations of glutamate: (1) 100 µΜ, (2) 500 µM, (3) 2000 µM. (b) Currents induced by different concentrations of kainate: (1) 50 µΜ, (2) 100 µΜ, (3) 500 µΜ, (4) 1000 µΜ, and (5) 2000 µΜ.

concentrations of the agonist led to more pronounced potentiation of the receptors. Examples of the potentiating effect of 3 on the transmembrane currents of various amplitudes that were caused by different concentrations of kainate are shown in Figure 4. The kainate dose-dependent responses that were obtained in the presence of 3 in its maximum soluble concentration (30 µM) were compared with those measured in the presence of the same concentration of CT (Figure 5). According to our results, both 3 and CT caused an unparallel shift of the kainate dosedependent response within wide range of concentration from 10 to 4000 µM. The response curve obtained in the presence of 3 is shifted more to the left and it is higher than the response obtained in the presence of CT, which demonstrates a higher efficacy of 3 in comparison to CT. In another series of experiments the concentrations of CT and 1 were varied within the 0.5-50 µM range whereas kainate dose remained constant (200 µM). The magnitude of this potentiation significantly depended on kainate concentrations. For low (20-50 µM) kainate doses the magnitude of potentia-

Isothiourea DeriVatiVes

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1847

Figure 3. Comparative potentiation of KA and Glu responses in Purkinje neurons by CT and 3. Transmembrane currents were induced by 0.5 mM kainic acid and 0.5 mM glutamate. Both CT and 3 were applied in 30 µM dose: (1) control, responses induced by 0.5 mM of corresponding agonist; (2) 30 µM CT; (3) 30 µM 3 action on kainic acid (a) and glutamate (b) responses.

Figure 5. Effect of CT (cyclothiazide) and 3 on dose-dependent response for the currents induced by kainic acid in Purkinje neurons. All responses are normalized to those induced by maximum (4 mM) concentration of kainate: (b) control; (O) in presence of 30 µM cyclothiazide; (0) in presence of 30 µM 3.

Figure 4. Concentration dependence of 3 effect on the currents induced by different kainate doses. Five doses of 3 were used: 1.0, 5.0, 10.0, 30.0, and 50 µM. Kainate was applied in 50 µM (b), 500 µM (O), and 1000 µM (0) doses. Magnitude of the kainate current in the absence of 3 was taken as 1 for each of these three doses.

tion was increased to 19-fold, whereas at high kainate doses (∼4000 µM) the increase was 1.5-fold relative to the maximum value. Thus, 1 was found to be more effective than CT (Figure 6). For example, for the responses that were caused by 1000 µM kainate exposure, the potentiation was 150% in the presence of 30 µM CT. In contrast, exposure at the same concentration of 1 resulted in a 3.9-fold increase, which was equal to 290%. In additional experiments we determined the potentiating effect of the maximum doses of CT and 1 (30 µM) on the receptor responses caused by the highest dose of kainate (∼4000 µM). The magnitudes of potentiation caused by CT and 1 were equal to 60% (n ) 4) and 132% (n ) 5), respectively (p < 0.05). Because of the high solubility of 1, we measured the effects of higher doses of this compound, compared these values with CT, and examined the differences between 1 and 3. Further experiments demonstrated that there was no difference between 1 and 3 at the 0.5-20 µM range. However, at 30 µM, 1 showed a 5-10% increase of potentiation in response to kainate relative to the potentiation of 3 at the same dose, which was probably due to low solubility of 3 at this concentration. More significant

Figure 6. Comparative effect of different doses of CT and 1 on currents, induced by the fixed concentration of KA. (a) Cyclothiazide effect: (1) current induced by 200 µM kainate application (control); (2-5) currents induced by 200 µM kainate application in presence of 1.0 µM (2), 10 µM (3), 30 µM (4), and 50 µM (5) of cyclothiazide. (b) 1 effect.

differences were detected between CT and 1 at 50 µM. For example, CT led to 5.55-fold increase of 500 µM kainateinduced response, whereas 1 resulted in a 12.2-fold increase at the same concentration of kainate. Both 1 and 3 potentiated the glutamate-induced currents in the same way as CT; however, their effects were more significant. Because of restrictions of compound applications on neurons, it was hard to determine the peak response caused by the glutamate and kainate. The glutamate application resulted

1848 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

PerloVich et al.

Table 1. Effect of 1 on NMDA-Induced Currents in Rat Cerebral Cortex currenta/responseb

control 0.5 µM 1 1 µM 1 2 µM 1 5 µM 1 10 µM 1 30 µM 1 a

50 µM NMDA

100 µM NMDA

5000 µM NMDA

10000 µM NMDA

325/100% 320/98% 220/68% 155/48% 110/34% 100/31% 35/11%

425/100% 420/99% 340/48% 235/55% 150/35% 100/23% 35/8%

710/100% 420/59% 250/35% 255/36% 145/20% 100/14% 30/4%

950/100% 425/45% 330/35% 245/26% 155/16% 100/10% 30/3%

Current in pA. b % of the responses in the control.

in a flat curve and did not depend on the agonist dose. The application of the studied compounds led to a significant potentiation of the glutamate responses, whereas this potentiation exceeded the kainate-induced responses. For example, 30 µM CT and 3 caused 7.0- and 26.9-fold increases of receptor responses after exposure to 500 µM glutamate, respectively. Some differences were observed in the initiation of the potentiating effect of CT and 3. The effect of CT evolves during several seconds and remains unchanged, whereas the effect of 3 becomes constant only after 30-40 s. The time required for recovery of the kainate-induced currents during washing of CT and 3 also differs for these compounds. The amplitude of these currents becomes normal after 1-3 min of washing of 30 µM CT, whereas stabilization of currents at 30 µM 3 takes about 9-11 min after washing. All these experiments led to the conclusion that CT and 3 showed similar effects on the responses caused by the activation of AMPA receptors in Purkinje neurons by different agonists. Therefore, 3 can potentiate AMPA responses due to inhibition of desensitization caused by agonists of these receptors. Effects of 1 on Activity of NMDA Receptors. The effects of 1 on NMDA receptors were analyzed on a heterogeneous population of a primary culture of neurons isolated from rat cerebral cortex. NMDA receptors (and more precisely receptors in cortex neurons) were divided into two groups according to their sensitivity to 1. In the first group, NMDA receptors were effectively inhibited by low concentrations of 1 (n ) 5). The efficacy of 1 inhibition of NMDA-induced currents depends on agonist concentration and amplitude of currents. Data are shown in Table 1. In general, exposure to 1 µM 1 caused 32-65% inhibition of NMDAinduced responses in neurons in the first group. The higher the amplitude of the current, the more pronounced is the effect detected. Exposure with 10 and 30 µM 1 resulted in 70-90% and 89-97% inhibition of the receptor, respectively. The inhibition effect of 1 was significantly decreased in the neurons in the second group (n ) 13). A decrease (about 10-30%) of NMDA-induced currents was observed for 10 µM 1 only. The magnitude of this effect was independent of the amplitude of the responses. However, the exposure of 30 µM 1 led to 50-70% inhibition of the responses in these neurons. Physicochemical Experiments. Crystal Structures of 1-3. In order to analyze the molecular conformations in crystals of 1-3, single crystals were grown from the acetone-hexane solutions and the complete crystal structures were resolved. Atomic numbering of the studied molecules and four conformationally independent fragments (Ph1, Ph2, R1, and R2) are shown in Figure 7. These salts create a hydrogen bond between the halogen atom and the N1-H1(N) fragment. The longest distance between N and the halogen atom corresponds to Br (3.716 Å), whereas

Figure 7. Atom numeration of studied molecules and four conformationally independent fragments.

the shortest one corresponds to Cl (3.042 Å). This distance for 3 is 3.487 Å. For the mentioned compounds, the DsH · · · A angles have approximately the same value (within the experimental error) of ∼153°, and they essentially differ from the ideal geometric value of 180°. The following angles were chosen for the quantitative comparison of the molecular conformations (Table 2): ∠S1-C4-N2-C7, ∠N2-C7-C8-C9, ∠N2-C14C15-C16, ∠N2-C4-S1-C5, and ∠C4-N1-C3-C2. The projections of these molecules along the C4-N2 bond are shown in Figure 8 as a pictorial rendering to highlight the geometric differences. The bond between Ph1 and Ph2 fragments is positioned in the background of the picture with regard to the noted bond (dotted circles), whereas the R1 and R2 fragments are located in the foreground (full lines). It is obvious that the molecular conformations of the bromide and iodide salts are similar; however, they essentially differ from that of the chloride salt. As a result, these differences may account for the physicochemical properties of these compounds both in crystals and in solutions. For example, for 1 R1, R2 and Ph2 fragments are positioned above the imaginary plane passing through N1S1N2 atoms, whereas the Ph1 fragment is located below the mentioned plane. For 2 and 3, the R1 and Ph2 fragments are positioned above the plane, but R2 and Ph1 are below the plane. As a result of crystallization of these salts from the solutions, the organic part of the molecules takes on special conformation. Thus, we may assume that during solubilization of crystals and dissociation of salts on cations and anions, the organic part of the molecule can partly keep the conformational state (inherited from the crystal) in the solution. Hence, such a fact may explain the different molecular behavior during drug delivery as well as interactions with the biological receptor. Solubility and Partitioning Properties. We carried out solubility tests of salts in solvents modeling biological media. Initially, we used phosphate buffer at pH 7.4. However, the kinetic solubility experiments showed that the tested salts were not chemically stable in this buffer; therefore, water was chosen as the primary solvent (where the experimental values were strictly reproducible) imitating delivery pathways. In order to mimic the lipophilic medium (the model of transcellular delivery pathways), we used n-octanol. The thermodynamic parameters of solubility process were obtained on the basis of the temperature dependencies of the solubility data within the wide temperature range from 18 to 42 °C. Results of the solubility experiments in water and n-octanol as well as the thermody-

Isothiourea DeriVatiVes

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1849

Table 2. Angles (deg) Describing Molecular Conformations in the Crystal Lattices compd

∠S1-C4-N2-C7

∠N2-C7-C8-C9

∠N2-C14-C15-C16

∠N2-C4-S1-C5

∠C4-N1-C3-C2

1 2 3

26.8 29.5 34.8

-69.6 -58.7 162.4

62.9 117.8 113.3

55.1 50.2 40.2

119.9 -94.8 -79.1

Table 3. Temperature Dependencies of Solubility (X2 Mole Fraction) of 1-3 in Water and n-Octanol 1 T (K)/t (°C)

water, X2 × 102

2 n-octanol, X2 × 102

291.15/18 293.15/20 295.15/22 298.15/25 300.15/27 303.15/30 310.15/37 315.15/42

8.20 8.60 9.10 10.2 10.7 11.3

Aa Ba Rb σc

6.1 ( 0.5 2505 ( 153 0.9926 1.75 × 10-2

a

water, X2 × 104

3 n-octanol, X2 × 103

water, X2 × 105

n-octanol, X2 × 104

4.23

3.44

2.82

7.28

4.89

4.96

3.46

9.85

5.66 7.23 8.17

6.74 10.4 13.2

4.27 5.81 6.88

12.6 18.3 24.8

1.9 ( 0.3 2830 ( 86 0.9986 1.65 × 10-2

13.6 ( 0.5 5645 ( 139 0.9991 2.67 × 10-2

2.6 ( 0.2 3834 ( 62 0.9996 1.19 × 10-2

10.1 ( 0.4 5080 ( 121 0.9992 2.32 × 10-2

Parameters of the correlation equation ln X2 ) A - B/T. b R: pair correlation coefficient. c σ: standard deviation.

Table 4. Thermodynamic Functions of Solubility Process of 1-3 in Studied Solvents compd

X2 (mole fraction)

0 ∆Gsol (kJ · mol-1)

X a ∆Hsol (kJ · mol-1)

0 b ∆Hsol (kJ · mol-1)

0 T∆Ssol (kJ · mol-1)

0 ∆Ssol (J · mol-1 · K-1)

Water 1 1 · 4H2O 2 3

5.05 × 10-4 3.46 × 10-5

18.8 25.5

23.5 ( 0.7 31.9 ( 0.5

1 2 3

1.00 × 10-1 4.84 × 10-3 9.68 × 10-4

5.7 13.2 17.2

21 ( 1 47 ( 1 42 ( 1

19.1 ( 0.3 25.2 ( 0.3 4.7 6.4

16 ( 3 22 ( 2

15.3 33.8 24.8

51 ( 3 113 ( 4 83 ( 4

n-Octanol

a

Data obtained from solubility experiments. b Data obtained from calorimetric experiments. Enthalpy per mole of 1.

namic functions of the solubility processes are summarized in Tables 3 and 4. 1 is very soluble in water, and it creates crystallohydrate(s) in the bottom phase. Therefore, it is not possible to obtain reproducible solubility measurements. In contrast, the results for the remaining salts were reproducible in water and n-octanol

Figure 8. Projection of the studied molecules along the C4-N2 bond: (a) 1; (b) 2; (c) 3. Dotted line corresponds to molecular fragment located behind the C4-N2 bond (second plane), whereas solid line corresponds to molecular fragment located in front of the bond.

solutions, since there was no formation of any crystallohydrates/ crystallosolvates in the bottom phases. On the basis of the thermogravimetric experiments, the stoichiometry of 1 crystallohydrate was derived: [1 · 4H2O]. The chloride salt is the most soluble both in water and in n-octanol, whereas the iodide one is less soluble in these solvents (Table 3). The impact of the halogen atom on the solubility data at 25 °C can be roughly estimated. The bromide salt is 14 times more soluble in water in comparison with the iodide one. In turn, the bromide salt is 5 times more soluble in n-octanol compared to iodide salt, whereas the chloride salt is 21 times more soluble in contrast with the bromide salt. The entropies of the solubility processes both in water and in n-octanol have positive signs, and this fact testifies that the studied molecules are distributed in a more disordered state in the solutions than in the crystals. It is worth noting that the octanolic solutions are more disordered than the aqueous ones. In order to estimate the difference of crystal lattice energies of the crystallohydrate [1 · 4H2O] and the unhydrated phase (in other words, the stabilization of crystal lattice energy by solvent (water) molecules), we used our previously described method.16 The standard values of solution enthalpies (per mole of 1) for the hydrated and for the unhydrated phases were determined in the same solvent (water) by the calorimetric method. The results are presented in Table 4. As a result, the crystal lattice energy of the crystallohydrate is 6.1 ( 0.6 kJ · mol-1 higher than energy of the unhydrated phase. The partition coefficients were studied within the wide concentration range of n-octanol phase solutions at 25 °C. It is

1850 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

PerloVich et al.

conformations in this buffer, which they “inherit” from the crystal lattices during the solubility process. Conclusions As a result of our studies, we determined Ca-blocking properties of 1-3 and arranged them in the following manner: (KI(3) ) 6.7 ( 1.8) > (KI(1) ) 4.2 ( 1.5) > (KI(2) ) 3.0 ( 1.2).

Figure 9. Kinetic dependencies of the decomposition processes of 1-3 in phosphate buffer with pH 7.4 at 25 °C.

worth noting that we did not detect any concentration dependencies of partition coefficients, which indicates that the associative processes are negligible and activity coefficients are equal to 1. The partitioning coefficients have the following values: P(1) ) 2.1, P(2) ) 4.4, and P(3) ) 0.42. Thus, the iodide salt is mainly distributed in the aqueous phase, in contrast to the chloride and bromide salts. In other words, the iodide salt “prefers” water enriched pathways (paracellular pathways) for the delivery process. Probably the chloride and bromide salts undertake the lipophilic (transcellular) delivery pathways. Stability of 1-3 in Phosphate Buffer. Chemical stability of the tested salts was studied in phosphate buffer (pH 7.4) in the following way. Three test tubes were filled with the same mass of each salt and diluted with the same volume of buffer followed by intensive stirring by mechanical stirrer at 25 °C. The analyzed probes were collected (2 mL) from the initial test tube mixtures at a definite time interval and mixed with 1 mL of deuterated chloroform (CDCl3). The obtained mixture was stirred in a thermostat for 30 min, and finally the chloroform phase was separated for NMR analysis. It is worth noting that 30 min after the beginning of stirring, the tested salts dissociated to the organic phase and the halogen anion (no peak was detected corresponding to the quaternary proton on nitrogen at ∼10.2 ppm), whereupon the organic part of the molecule started to decay and resulted in decomposition products. One of these products was a dibenzylamine fragment (peaks, ∼4.6-4.9 ppm). The amount of the decay product was estimated by integral values of the proton peaks. The kinetic dynamics of the decomposition process of the studied compounds are shown in Figure 9 where the organic parts of these molecules (obtained from different salts) have various chemical stabilities in the phosphate buffer. In other words, the organic phase that is derived from the iodide salt is the most stable to the decay process, whereas the organic phase from the chloride salt demonstrates less stability to the decomposition. Interestingly, the Ca-blocking properties (a parameter describing biological activity) of tested salts correlate with their chemical stability (i.e., more stable molecules have higher KI): (KI(3) ) 6.7 ( 1.8) > (KI(1) ) 4.2 ( 1.5) > (KI(2) ) 3.0 ( 1.2). Since the organic phases of these molecules in phosphate buffer have similar chemical composition, the above-mentioned differences in the chemical stability can be linked to various molecular

A comparative study of the potentiating effect of 3 and cyclothiazide (CT) as a well-known positive modulator of AMPA receptors transmembrane currents induced by kainic acid (KA) and glutamate in Purkinje neurons was performed. In order to determine the quantitative differences between effects of these compounds, we measured the dose-dependent responses for kainate alone and for kainate in the presence of CT and 3. Analysis of these results revealed an inverse correlation of the potentiating effect for both of these compounds on the agonist, i.e., low concentration of the agonist led to more pronounced potentiation. As a result of these experiments, we concluded that effects of CT and 3 on responses induced by activation of AMPA receptors in Purkinje neurons by different agonists are similar. Therefore, 3 potentiates AMPA responses because of inhibition of desensitization caused by agonists of these receptors. The effects of 1 on NMDA receptors were analyzed on heterogeneous population of primary culture of neurons isolated from rat cerebral cortex. NMDA receptors were divided into two groups according to their sensitivity to 1. Single crystals were grown and X-ray diffraction experiments were performed to solve the crystal structures of 1-3. The molecular conformations of the bromide and iodide salts are similar, whereas they essentially differ from that of the chloride salt. The temperature dependencies of 1-3 solubility in water and n-octanol were obtained, and thermodynamic parameters of solubility process were calculated. The chloride salt is mostly soluble in both water and n-octanol, whereas the iodide salt is less soluble in these solvents. The bromide salt is 14 times more soluble in water compared to the iodide salt. In turn, the bromide salt is 5 times more soluble in n-octanol compared to iodide salt, whereas the chloride salt is 21 times more soluble in contrast with the bromide salt. The partition coefficients were determined within a wide concentration range of the n-octanol phase solutions at 25 °C. The partition coefficients have the following values: P(1) ) 2.1; P(2) ) 4.4; P(3) ) 0.42. Thus, the iodide salt is mainly distributed in the aqueous phase in contrast to the chloride and bromide salts. 1 is very soluble in water, and it creates crystallohydrate in the bottom phase of the solubility experiments. On the basis of the thermogravimetric experiments, we determined the stoichiometry of 1 crystallohydrate: [1 · 4H2O]. The crystal lattice energy of the crystallohydrate is 6.1 ( 0.6 kJ · mol-1 higher than the unhydrated phase. Chemical stabilities of tested salts in phosphate buffer (pH 7.4) were studied at 25 °C. The salts dissociated into pure organic phase and the halogen anion 30 min after the beginning of stirring. The organic phase, which is obtained from the iodide salt, is mostly stable to the decay process, whereas the organic phase obtained from the chloride salt demonstrates less stability to the decomposition. Interestingly, the Ca-blocking properties

Isothiourea DeriVatiVes

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1851

Scheme 1

of the discussed salts correlate with the chemical stability: more stable molecules have higher KI. Experimental Section Chemical Procedures. The standard and accessible methods of the synthesis of tetra-substituted isothioureas were developed by the Institute of Physiologically Active Compounds of Russian Academy of Science.20 Synthetic approaches to novel isothioureas were carried out according to Scheme 1. S-Ethyl-N-allyl-N′,N′-dibenzylisothiourea Hydrochloride (1). S-Ethyl-N-allyl-N′,N′-dibenzylisothiourea hydroiodide (4.52 g, 0.01 mol) was added to a stirred mixture of 10% aqueous solution NaOH (50 mL) and benzene (50 mL). The mixture was stirred at 0-5 °C until the salt was completely dissolved. After that, the benzene layer was separated and washed out with water (2 × 50 mL). The solution was dried (Na2SO4), and the solvent was evaporated. S-Ethyl-Nallyl-N′,N′-dibenzylisothiourea acted as base which was quickly used in the next step because of its lability. 1H NMR [200 MHz, DMSO-d6] δ: 1.20 (t, 3H, CH3), 2.30 (q, 2H, SCH2), 4.15 (m, 2H, NCH2), 4.60 (s, 4H, CH2Ph), 5.25 (m, 2H, dCH2), 5.80 (m, 1H, -CHd), 7.20-7.40 (m, 10H, ArH). S-Ethyl-N-allyl-N′,N′-dibenzylisothiourea (1.6 g, 0.005 mol) was dissolved in isopropanol (30 mL) followed by addition (by drops) of isopropanolic HCl solution (0.006 M, 20 mL) to the stirred solution. The reaction mixture was kept under stirring at 20 °C for 8 h. The grown crystals were filtered, and the yield was 1.2 g (66.6%). Mp 143-145 °C. Anal. (C20H25ClN2S) C, H, N, S. S-Ethyl-N-allyl-N′,N′-dibenzylisothiourea Hydrobromide (2). Bromoethane (1.31 g, 0.012 mol) was added to a solution of N-allylN′,N′-dibenzylthiourea (2.96 g, 0.01 mol) in acetone (50 mL). The reaction mixture was kept for 72 h at 40 °C. The grown crystals were filtered and recrystallized from isopropanol (30 mL) and yielded 3.1 g (76.5%). Mp 156-158 °C. Anal. (C20H25BrN2S) C, H, N, S. S-Ethyl-N-allyl-N′,N′-dibenzylisothiourea Hydroiodide (3). Iodoethane (1.87 g, 0.012 mol) was added to a solution of N-allylN′,N′-dibenzylthiourea (2.96 g, 0.01 mol) in acetone (50 mL). The reaction mixture was kept for 48 h at 40 °C. The grown crystals were filtered and recrystallized from isopropanol (30 mL) and yielded 3.5 g (77.3%). The melting point was 150-152 °C. Anal. (C20H25IN2S) C, H, N, S. Materials and Solvents. 1-Octanol (n-octanol, CH3(CH2)7OH, MW ) 130.2, lot no. 11K3688), ARG, was from Sigma Chemical

Co. Phosphate buffer solution (pH 7.4) was prepared by mixing of solutions of appropriate sodium and potassium salts of phosphoric acid. All chemicals were AR grade. The pH values were measured with a Toledo MP 220 pH meter (Mettler) standardized with pH 1.68 and 9.22 solutions. Solubility Determination. Solubilities of these compounds were determined within a wide temperature range (18-42) ( 0.1 °C by the isothermal saturation technique. The solid phase precipitated upon centrifugation, the surfactant was filtered (Acrodisc CR syringe filter, PTFE, 0.2 µm pore size), and the bulk solution was measured spectrophotometrically by UV-2401 PC spectrophotometer (Shimadzu, Japan) according to a previously described protocol.17 The solubility measurements are reported as averages of at least three replicated experiments with statistical errors within 2.5%. Determination of Partition Coefficients. The method of isothermal saturation was used for the determination of partition coefficients P (D7.4). We prepared n-octanol solutions with fixed concentrations, varying the concentrations between one-tenth and the maximum solubility value. The respective solution was left in a thermostated ampule, with the same volume of added water. The equilibration was carried out for 24 h under continuous stirring. The initial concentration and final concentration of the tested compound in the octanol solutions were determined by spectrophotometry before and after the equilibration. All experiments were carried out at 25 °C. The partition coefficient was calculated using the following formula:

P ) Cow /Cwo

(1)

where Cow and Cwo are the molar concentrations of the solute in the mutually saturated phases of octanol and water. The accuracy of the P-value was verified by checking the mass balance of the starting amount of the compound i compared to the total amount of the compound partitioned between the two phases,

mi ) mow + mwo

(2)

where mi ) CiVi is the starting mass (in moles) of the compound, mow ) CowVow is the mass of the substance dissolved in the watersaturated octanol phase, and mwo ) CwoVwo is the mass of the substance dissolved in the octanol-saturated water phase. Each experiment was performed at least three times.

1852 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7

PerloVich et al.

TG Experiments. Thermogravimetric studies were carried out within the temperature range 25-200 °C with a heating rate of 10 °C/min and a flow rate of dry argon gas at 5.4 L/h using NETZSCH TG 209 F1 (Germany). The calibration of the TGA cell was performed using a sample of calcium oxalate monohydrate in an atmosphere of flowing dry argon. The measurements were repeated three times. The determinations were off by 3%. X-ray Diffraction Experiments. Single-crystal X-ray measurements were carried out using a Nonius CAD-4 diffractometer with graphite-monochromated Mo KR radiation (λ ) 0.710 69 Å). The intensity data were collected at 25 °C by means of a ω-2θ scanning procedure. The crystal structures were solved using direct methods and refined by means of a full-matrix least-squares procedure. CAD-4 Software (1989)18 was applied for data collection, data reduction, and cell refinement. Programs SHELXS-97 and SHELXL9719 were used to solve and to refine structures, respectively. NMR Experiments. NMR data were obtained by standard methods using Bruker AMX-200. Biological Methods. Calcium-Blocking Property Experiments. Interaction between the compounds and glutamate-dependent calcium uptake system was studied on newborn (8-11 days old) rat brain synaptosomal P2-fraction isolated according to the following method: synaptosomes were suspended in the incubation buffer A (132 mM NaCl, 5 mM KCl, 5 mM HEPES), pH 7.4, and stored at 0 °C during all experiments. Aliquots of synaptosomes (50 µL) were transposed to buffer A containing the testing compound and 45Ca samples. Calcium ion uptake was stimulated by introducing 200 µM glutamate solution. After 5 min of incubation at 37 °C, the process was terminated by filtration on GF/B filters. The sample was washed three times with cold buffer B (145 mΜ KCl, 10 mΜ Tris, 5 mΜ Trilon B) followed by measurements of radioactivity with scintillator counter SL-4000 Intertechnic. In preliminary experiments, all compounds were tested at 100 µM for their ability to inhibit the glutamate stimulated Ca uptake. If inhibition of the Glu-Ca-uptake was 50% or more, then further studies were carried out to determine the concentration dependence of its inhibition and the corresponding value of KI was measured according to the following equation:

KI ) K(43/21) ) [(Ca4 - Ca3)/(Ca2 - Ca1)] × 100 (3) 2+

where Ca1 is the Ca influx in blank experiment (without glutamate and tested compounds). Ca2 is the Ca2+ influx in the presence of glutamate only (Glu-Ca-uptake), Ca3 is the Ca2+ influx in the presence of tested compound (without glutamate), and Ca4 is the Ca2+ influx in the presence of both glutamate and tested compound. Electrophysiological Experiments. The AMPA-ligands activity studies were carried out on isolated Purkinje neurons that contain a broad population of AMPA-receptors. The main series of experiments were performed with kainic acid (KA) as receptor agonist because glutamate and especially AMPA induce fast and strong desensitization of receptors, which makes the study of dose-response relationships very difficult. Electrophysiological experiments were carried out on freshly isolated neurons from different areas of the brain of 14-18 days old rats using the patchclamp technique. The preparation of isolated cells was performed as follows: a selected region of the brain was cut into slices of 0.4-0.6 mm width followed by the incubation in saline (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4) for 1 h. The slices were transferred to fresh saline solution with 2 mg/mL Pronase (Serva) and 1 mg/mL collagenase (Sigma). Slices were incubated at 34 °C and pregassed with 100% O2. Finally, the slices were mechanically dissociated into individual cells by means of Pasteur pipettes. The composition of extracellular saline was 150 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.32. The composition of the intracellular saline was 140 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl2, 1 mM ATP. The transmembrane currents were registered in the configuration of “whole cell”. Compounds were exposed to neurons by the method of fast perfusion.

Effects of 1-3 on NMDA receptors were analyzed on a heterogeneous population of a primary culture of neurons isolated from rat cerebral cortex.

Acknowledgment. The present research was funded as a part of the basic research program established by the Presidium of Russian Academy of Sciences “Fundamental Sciences for Medicine”. Supporting Information Available: Elemental analysis data, X-ray data, and NMR spectra of compounds 1-3. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Parsons, C. G.; Danysz, W.; Quack, G. Glutamate in CNS disorders as a target for drug development: an update. Drug News Perspect. 1998, 11, 523–579. (2) Nakanishi, N. S. Molecular diversity of glutamate receptors and implications for brain function. Science 1992, 258, 31–37. (3) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. F. The glutamate receptor ion channels. Pharmacol. ReV. 1999, 51, 7–61. (4) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. Ø.; Madsen, U.; Krogsgaard-Larsen, P. Ligands for glutamate receptors: design and therapeutic prospects. J. Med. Chem. 2000, 43, 2609–2645. (5) Petrov, V. I.; Piotrovsky, L. B.; Grigoriev, I. A. Exitatory Amino Acids; Volgograd Medical Academy: Volgograd, Russia, 1997; p 168. (6) Zefirova, O. N.; Zefirov, N. S. Physiologically active compounds acting on glutamate receptors. Zh. Org. Khim. 2000, 36, 1273–1300. (7) Khachaturian, Z. S. Calcium hypothesis of Alzheimer’s disease and brain aging. Ann. N.Y. Acad. Sci. 1994, 747, 1–11. (8) Mattson, M. P.; Cheng, B.; Davis, D.; Bryant, K.; Lieberburg, I.; Rydel, R. E. β-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J. Neurosci. 1992, 12, 376–389. (9) Bleakman, D.; Lodge, D. Neuropharmacology of AMPA and kainate receptors. Neuropharmacology 1998, 37, 1187–1204. (10) Hansch, C.; Bjorkroth, J. P.; Leo, A. Hydrophobicity and central nervous system agents: on the principle of minimal hydrophobicity in drug design. J. Pharm. Sci. 1987, 76, 663–686. (11) Young, R. C.; Mitchell, R. C.; Brown, T. H.; Ganellin, C. R.; Griffiths, R.; Jones, M.; Rana, K. K.; Saunders, D.; Smith, I. R.; Sore, N. E.; Wilks, T. J. Development of a new physicochemical model for brain penetration and its application to the design of centrally acting H2 receptor histamine antagonists. J. Med. Chem. 1988, 31, 656–671. (12) Abraham, M. H.; Chadha, H. S.; Mitchell, R. C. Hydrogen bonding. 33. Factors that influence the distribution of solutes between blood and brain. J. Pharm. Sci. 1994, 83, 1257–1268. (13) Abraham, M. H.; Chadha, H. S.; Mitchell, R. C. Hydrogen bonding. Part 36. Determination of blood brain distribution using octanol-water partition coefficients. Drug Des. DiscoVery 1995, 13, 123–131. (14) Abraham, M. H.; Chadha, H. S. Applications of a Solvation Equation to Drug Transport Properties. In Lipophilicity in Drug Action and Toxicology; Pliska, V., Testa, B., van de Waterbeemd, H., Eds.; Methods and Principles in Medicinal Chemistry, Vol. 4; VCH Publishers: Weinheim, Germany, 1995. (15) Bachurin, S. O. Medicinal chemistry approaches for the treatment and prevention of Alzheimer’s disease. Med. Res. ReV. 2003, 13 (1), 48–88. (16) Perlovich, G. L.; Hansen, L. K.; Volkova, T. V.; Mirza, S.; Manin, A. N.; Bauer-Brandl, A. Thermodynamic and structural aspects of hydrated and unhydrated phases of 4-hydroxy-benzamide. Cryst. Growth Des. 2007, 7 (12), 2643–2648. (17) Perlovich, G. L.; Volkova, T. V.; Bauer-Brandl, A. Towards an understanding of the molecular mechanism of solvation of drug molecules: a thermodynamic approach by crystal lattice energy, sublimation and solubility exemplified by hydroxybenzoic acids. J. Pharm. Sci. 2006, 95 (7), 1448–1458. (18) CAD-4 Software, version 5.0; Enraf-Nonius: Delft, The Netherlands, 1989. (19) Sheldrick, G. M. SHELXL97 and SHELXS97; University of Go¨ttingen: Go¨ttingen, Germany, 1997. (20) Tkachenko, S. E.; Proshin, A. N.; Bachurin, S. O.; Petrova, L. N.; Zefirov, N. S. Derivatives of N,S-Substituted N′-1-[(Hetero)arylalkyl]N′-[(hetero)aryl]-methylisothioureas, Method of Synthesis of Their Bases and Salts, Physiologically Active N′-1-[(hetero)arylalkyl]-N′[(hetero)aryl]-methylisothioureas, Pharmaceutical Formulation and Method of Treatment. Russian Patent 2223952, February 20, 2004 (according to the Patent Application 2001114922 of June 4, 2001).

JM8012882