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Studies of the electronic structure and biological activity of chosen 1,4-benzodiazepines by 35Cl NQR spectroscopy and DFT calculations
Chemical Physics 330 (2006) 301–306 www.elsevier.com/locate/chemphys
Studies of the electronic structure and biological activity of chosen 1,4-benzodiazepines by 35Cl NQR spectroscopy and DFT calculations K. Bronisz a, M. Ostafin b
a,*
, O. Kh. Poleshchuk b, J. Mielcarek c, B. Nogaj
a
a Department of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland Department of Chemistry, Tomsk Pedagogical University, Komsomolskii 75, 634041 Tomsk, Russia c Faculty of Pharmacy, University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Received 9 June 2006; accepted 1 September 2006 Available online 6 September 2006
Abstract Selected derivatives of 1,4-benzodiazepine: lorazepam, lormetazepam, oxazepam and temazepam, used as active substances in anxiolytic drugs, have been studied by 35Cl NQR method in order to find the correlation between electronic structure and biological activity. The 35Cl NQR resonance frequencies (mQ) measured at 77 K have been correlated with the following parameters characterising their biological activity: biological half-life period (t0.5), affinity to benzodiazepine receptor (IC50) and mean dose equivalent. The results of experimental study of some benzodiazepine derivatives by nuclear quadrupole resonance of 35Cl nuclei are compared with theoretical results based on DFT calculations which were carried out by means of Gaussian’98 W software. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Benzodiazepines; Anxiolytic drugs; Nuclear quadrupole resonance (NQR) spectroscopy; DFT calculations
1. Introduction Recently, the clinical use of 1,4-benzodiazepines (BDZ) as anxiolytic, anticonvulsant, sedative-hypnotic and muscle relaxant agents has increased enormously [1–6]. After the first synthesis of the prototypical agents chlorodiazepoxide and diazepam, thousands of benzodiazepine congeners have been synthesised and studied at numerous laboratories [7–9]. However, the molecular mechanism of their activity remains to be clarified. The discovery of stereospecific, high affinity BDZ binding sites in mammal brains that are capable of being saturated, has rekindled new interest in this field. Notwithstanding, no clear hypothesis has so far been put forward as to the pharmacodynamic moiety of the BDZ molecule, a point which is of fundamental importance in understanding BDZ molecular interactions with the target sites in the brain [10,11]. *
Corresponding author. Tel.: +48 61 8295255. E-mail address: [email protected] (M. Ostafin).
0301-0104/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2006.09.001
So far many authors have tried to correlate the results of general biological tests on BZD with their molecular structure. The general formula of 1,4-benzodiazepine derivatives, with the types of substituents at the positions 1, 3, 7 and 2 0 , is presented in Fig. 1 along with Table 1. The profile of activity of a given derivative depends on the type of atom or atoms being substituents R1, R3, R7 and R2 0 in the benzodiazepine molecule. For example, the presence of a nitro group or a halogen atom at position R7 enhances the drug activity in the sequence F < Br < NO2. Introduction of a methyl group at R1 facilitates resorption but such an extension of the aliphatic chain is detrimental to the biological activity. The substituent at R3 determines the process of glucuronide, the absorbability and time of activity. Enhanced activity can be also achieved by introducing a halogen at the ortho position of the aromatic ring (R2 0 ). However, the presence of a halogen at the meta or para positions significantly reduces the therapeutic activity of the drug.
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Fig. 1. Chemical structures of 1,4-benzodiazepines.
Table 1 Four selected derivatives of 1,4-benzodiazepine Benzodiazepine derivative
Substituent
Lorazepam
R7 = Cl R3 = OH R7 = Cl R3 = OH R7 = Cl R3 = OH R7 = Cl R3 = OH
Lormetazepam Oxazepam Temazepam
R1 = H R2 0 = Cl R1 = CH3 R2 0 = Cl R1 = H R2 0 = H R1 = CH3 R2 0 = H
A quantitative structure-activity relationship (QSAR) study was carried out on over 50 BZD with a variety of substituents at positions 7 and 2 0 . Using CNDO/2 methodology and calculated values for dipole moment and net charge on the carbonyl oxygen, the data of several different in vivo tests have been analysed [12–15]. On the grounds of the crystal structure determinations and references, structure-activity relationships, pharmacokinetic and spectroscopic data, the presumable importance of the N1H–C2@O amidic moiety, which can bind to the receptor via a cooperative system of hydrogen bonds has been pointed out. A Free-Wilson analysis by Borea of in vivo data of several benzodiazepines has led to the following rank orders of contributions to the activity of substituents at position 7 and 2 0 thus showing the importance of electron withdrawing groups at these positions, e.g. the rank of electronegativities of the following substituents at position 7 can be represented as –CF3 > –Br > –NO2 and at position 2 0 –F > –Cl > –Br accordingly [16–18]. Position 7 is the most important location for enhancing BZR (benzodiazepine receptor) affinity. An increase in the substituent lipophilicity and electronic charge were found to be directly related to the increase in the receptor binding [19]. The optimal R7 substituents are: –CH2CF3, halogens and nitro, while the worst: –CH@CH–. The substituents at position 2 0 are of second most importance in positively influencing BZR affinity, and an increase in the polar nature of these substituents was shown to be beneficial, although this effect was diminished if the groups were bulky. Substitution at positions 3 and 8 results in reduced pharmacological activity of benzodiazepines. In addition, to corroborate the positive effect of the electron withdrawing moieties at positions 7 and 2 0 , the results of this analysis supported the importance of a carbonyl at position 2, the
detrimental effect of groups larger than –H or –CH3 at positions 3, and the negative impact of substituents at position 4 0 . The latter observation suggests that the pendant phenyl ring occupies a hydrophobic pocket in the receptor binding site and that the depth of the pocket cannot accommodate a para substituent [20,21]. Conformational and electronic properties of 21 benzodiazepines were calculated by using empirical energy and semiempirical molecular orbital methods [22–24]. Compounds that were either highly active or very weakly active in BZR binding (e.g. diazepam and medazepam) were found to have very similar low energy conformations, thus indicating that conformational factors are not important for receptor recognition. However, mapping the electrostatic potential of the benzodiazepine molecule led to the postulate that the interactions between the electron withdrawing substituents R7, the carbonyl oxygen at position 2 and the N4 imine nitrogen with three different cationic receptor sites are required for high affinity analogs [ibid.]. The NQR spectroscopy provides parameters characterising the electronic structure (35Cl NQR frequency) of substances which can be related to their biological activities [25,26]. This paper reports such a study for 1,4-benzodiazepine derivatives. The aim of the paper was to determine the resonance frequencies 35Cl NQR at the chlorine nuclei at position 7 of the benzodiazepine ring and to determine the correlations with the parameters describing the biological activity of a compound studied. 2. Materials Benzodiazepine derivatives: lormetazepam, oxazepam and temazepam were purchased in a hermetically sealed containers from Tarchomin Pharmaceutical Works ‘‘Polfa’’ Joint Stock Company. Lorazepam: 7-chloro-5-(2chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin2-one. Lormetazepam:(R,S)-7-chloro-5-(2-chlorophenyl)1,3-dihydro-3-hydroxy-1-methyl-2H-1,4-benzodiazepin-2one. Oxazepam:7-chloro-1,3-dihydro-3-hydroxy-5-phenyl2H-1,4-benzodiazepin-2-one. Temazepam: 7-chloro-1,3dihydro-3-hydroxy-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one. 3. Experimental 35
Cl NQR frequencies were measured in liquid nitrogen (77 K) on a pulsed Fourier transform NQR spectrometer type NQS-300 (0.5–300 MHz) from MBC Elektronics (Warsaw, Poland) constructed on the basis of a laboratory made NQR spectrometer. The 35Cl NQR spectra of the compounds studied were recorded by a standard method of Fourier transform of a FID signal observed after a p/2 pulse and the pulse duration was 7 ls. Portions of about 3 g of each substance were placed in glass vials of 5 cm in length and 1 cm diameter, capped with a plastic cork. The samples were placed in a cylindrical coil of 3 cm in
K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
length and 1.3 cm in diameter. The samples were cooled to 77 K by immersing in a liquid nitrogen bath. 4. Computational details The molecular geometry was fully optimised with Gaussian’98W program [27], using the hybrid method B3PW91 [28] with the 6-31G(d,p) basis set. This basis set presents a high-level performance theoretical study (polarisation functions) of molecules. The calculations were carried out on a personal computer Pentium 4. The values of the NQR frequencies and quadrupole coupling constants were calculated from the principal components of the EFG tensor at the quadrupolar nuclei of chlorine and nitrogen. It is known that in general two parameters can be obtained from NQR experiment: the quadrupole coupling constant, v, and asymmetry parameter of the EFG tensor g: v ¼ e2 Qqzz =h;
g ¼ ðqxx qyy Þ=qzz
ð1Þ
where eqii are components of the EFG tensor at the quadrupolar nucleus (defined in the EFG principal axes system), Q the nuclear quadrupole moment, e the proton charge and h is the Planck’s constant. We used qii from the results of the Gaussian program and then calculated v and g using Eq. (1). The Q values were taken from [29]. The electronic structure of the studied compounds was analysed by means of the natural bond orbitals (NBO) partitioning scheme approach [30]. The natural population analysis (NPA) scheme developed by Weinhold has been used to obtain charges on atoms in molecules. Proft et al. [31] have found that the B3PW91 functional provide comparatively good results.
303
Table 2 The 35Cl NQR frequencies determined at 77 K and parameters describing the biological activity of the compounds studied: biological half-lifetime (t0.5), affinity to the benzodiazepine receptor (IC50) and mean dose equivalent Benzodiazepine derivative
mQ at 77 K (MHz)
t0.5 (h)
Lorazepam
34. 787 35.406* 35.549 35.571*
14
4
0.75
Lormetazepam
35.232?
12
4
0.75
*
IC50 (nmol/l)
Mean dose equivalent (mg)
Oxazepam
34.798 34.945*
11.5
18
15.0
Temazepam
35.128* 35.244* 35.302*
11
16
7.5
* The values marked by asterisk refer to the chlorine atoms at position 7 in benzodiazepines. ? Unambiguous assignment of this frequency to the chlorine atoms at position 7 or 2 0 was not possible.
Exemplary spectra of temazepam and lorazepam are shown in Fig. 2. The full-width half-maximum (FWHM) values of the resonance lines were within 8–16 kHz. It is also interesting to analyse the multiplicity of the spectra against the
5. Results and discussion The resonance frequencies at the chlorine atoms at position 7 of the benzodiazepine (R7) and at the ortho positions of the phenyl ring (R2 0 ), measured at 77 K are given in Table 2. The same table presents the pharmacological and pharmacokinetic parameters of the drugs: biological half-life period (t0.5), affinity to benzodiazepine receptor (IC50) and mean dose equivalent. Biological half-life time (t0.5) is the time in which the concentration of the active substance decreases by half in a given form of drug. The affinity to benzodiazepine receptor (IC50) is expressed as the number of moles of a given compound that can be bonded to the receptor considered. The mean dose equivalent is the mean amount of the active therapeutic substance administered per 24 h. To analyse the intra- and inter-molecular interactions of particular chlorine atoms in the lorazepam molecule, the 35 Cl NQR resonance frequencies have been assigned to each of them. The probe was the chlorine atom at position 7 of benzodiazepine ring to which the resonance frequencies labelled as (*) in Table 2 were assigned [32–34].
Fig. 2. The 77 K.
35
Cl NQR spectra of (a) temazepam (b) lorazepam taken at
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Table 3 The results of the calculation at B3PW91/6-31G(d,p) level Molecule
mCl (MHz)
e2Qqzz14N (MHz)
qCl
qN
qcarb: O
qhydr: O
LPOcarb./LPhydr.
Lorazepam
35.49(0.06)*
4.37
0.020 0.032
0.403
0.598
0.743
1.841/1.930
36.01(0.11) Lormetazepam
35.22(0.06)* 35.54(0.09)
4.46
0.012 0.017
0.446
0.584
0.757
1.852/1.943
Oxazepam
34.18(0.08)
4.20
0.046
0.436
0.598
0.740
1.840/1.932
Temazepam
35.13(0.06)
4.27
0.010
0.476
0.586
0.757
1.852/1.944
*
The values marked by asterisk refer to the chlorine atoms at position 7 in benzodiazepines.
chemical structure of the molecules. It appears that for some samples the number of resonance lines is not the same as that of chlorine atoms. For example the lorazepam molecule has two chlorine atoms and its 35Cl NQR spectrum shows four resonance lines three of which are shown in Fig. 2b. This observation confirms that the chlorine atoms in the lorazepam molecule show both chemical and crystallographic inequivalence. On the other hand, for lormetazepam with two chlorine atoms in its molecule only one NQR line has been detected despite a very careful scanning of the entire frequency range of interest. Since chemical equivalence of these two chlorines is rather unlikely as confirmed by DFT results which show chemical inequivalence of more than 300 kHz for chlorine atoms in positions 7 and 2 0 (see Table 3) we cannot provide convincing explanation of this fact as yet. Obviously this ambiguity did not enable us to assign the NQR frequency marked by (?) in Table 2 to one of the two chlorine atoms in lormetazepam molecule.
The 35Cl NQR spectra has been recorded off-resonance so the relative intensities of NQR lines, as Fig. 2b shows, do not reflect the relative number of molecules occupying inequivalent positions in the crystalline unit cell. For oxazepam with only one chlorine atom only one 35 Cl NQR line was recorded, which suggests that all the molecules take equivalent positions in the elementary cell [35]. The NQR spectrum of temazepam, also having one chlorine atom, shows three resonance lines indicating the presence of at least three molecules of inequivalent positions in the elementary cell [36]. The 35Cl NQR resonance frequencies determined at 77 K were correlated with the parameters of biological activity of the compounds, see Fig. 3. Assuming linear correlation model the correlation coefficients determined for data shown in Fig. 3a–c are R = 0.715, 0.768 and 0.890, respectively. Although these values of R are too small to corroborate the true linearity of the correlation given the
Fig. 3. The mean 35Cl NQR frequency in the spectra taken at 77 K versus (a) biological half-lifetime, (b) affinity to the benzodiazepine receptor and (c) mean dose equivalent.
K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
four data points only available a positive correlation of data shown in Fig. 3a and a negative one for data in Fig. 3b and c is clearly visible. As follows from Fig. 3a, the mean resonance frequencies increase with increasing half-lifetime of the compounds studied. This correlation suggests that benzodiazepines characterised by the lowest resonance frequencies are more easily metabolised. We also found a negative correlation between the affinity to the benzodiazepine receptor and the resonance 35Cl NQR frequency, so as the electron density at Cl increases, the affinity to this receptor increases. Therefore, it can be concluded that the compounds characterised by lower resonance frequencies better bind to the benzodiazepine receptor. Moreover, as shown in Fig. 3c, as the mean 35 Cl NQR resonance frequency on the chlorine atoms in benzodiazepines decreases, the mean dose equivalent increases. This indicates that the compounds characterised by higher resonance frequencies are administered in lower doses. 6. DFT calculations The B3PW91/6-31G(d,p) optimised structure by one of the molecules studied is displayed in Fig. 4. Its bond lengths were typical for the organic molecules. A comparison of the geometrical parameters with the experimental data [34–36] for benzodiazepine derivatives showed that the optimised C–C, C–N, C–O and C–Cl bond lengths indicated the high reliability of our calculations. Analysis of the obtained results leads to the following correlation between the calculated and experimental bond lengths for the compounds studied (Fig. 5): ˚ ¼ 0:01 þ 0:99½Rðexp :Þ=A ˚ ½Rðcal:Þ=A ðr ¼ 0:995; s ¼ 0:01; n ¼ 79Þ
ð2Þ
(where r is the correlation coefficient, s the standard deviation and n is the number of the compounds). The factor
Fig. 4. Molecular structure of lorazepam, optimised at B3PW91/631G(d,p) level.
305
Fig. 5. The dependence between experimental and calculated bond lengths of benzodiazepines.
preceding R(exp.) is close to unity, which indicates the high reliability of the calculations at the B3PW91 level of the theory. These results encouraged us to perform the calculations of v for the chlorine and nitrogen atoms for some benzodiazepines (Table 3). Note that a fairly good correlation exists between the experimental and calculated 35Cl NQR frequencies, as demonstrated in Fig. 6 where straight correlation line was drawn according to the equation: mCl ðexp:Þ ¼ 20:5 þ 0:4mCl ðcal:Þ ðr ¼ 0:985; s ¼ 0:05; n ¼ 4Þ ð3Þ A similar dependence was obtained earlier in paper [37] for many compounds containing chlorine–carbon and chlorine–nitrogen bonds. The obtained dependences between NQR frequencies and biological activity parameters: biological half-life, affinity to the receptor, and mean dose equivalent allow hypothesising, that these parameters roughly at least should depend on the atomic charges of chlorine. However, in a series of compounds presented here the calculated by NBO approach chlorine effective charges are virtually identical (Table 3) what implies the lack of dependence between effective charges on chlorine atoms and the biological activity of the molecules. We have tried to estimate charges on other atoms in molecules, which would correlate sufficiently with IC50 values. The following
Fig. 6. The dependence between experimental and calculated frequencies of benzodiazepines.
35
Cl NQR
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References
Fig. 7. The dependence between calculated nitrogen quadrupole coupling constant and IC50 of some benzodiazepines.
correlations between IC50 values and charges on nitrogen and oxygen atoms were obtained: IC50 ¼ 616 359qN þ 1020qhydr: O
ðr ¼ 0:993; s ¼ 1:1; n ¼ 4Þ ð4Þ
for the diazepine nitrogen 4 and hydroxyl oxygen, respectively, and IC50 ¼ 892 370qN 1250qcarb: O
ðr ¼ 0:983; s ¼ 1:7; n ¼ 4Þ ð5Þ
for the diazepine nitrogen 4 and carboxyl oxygen. Indirect confirmation to that the reactive part in molecules contains nitrogen and oxygen atoms is considerably better correlation of IC50 on the calculated NQR parameters of nitrogen rather than chlorine atoms (Fig. 7). Like for the correlations shown in Fig. 3a–c linear model was chosen for simplicity sake only and the same remarks apply here regarding the only four data points available. Besides, from our calculation it is evident, that the population of lone electron pair (LP) of carboxylic oxygen is much lower, than that of hydroxyl oxygen (Table 3). This may indicate that the series of 1,4-benzodiazepine molecules including carboxylic atoms, play a key role in interacting with GABA receptor. 7. Conclusions The above-discussed results of the study lead to the following conclusions: 1. There is a relationship between the 35Cl NQR frequency and the parameters characterising biological activity of 1,4-benzodiazepine derivatives. It can be supposed that the distribution of electron density in a given compound permits prediction of its biological activity. 2. The information provided by the 35Cl NQR frequency may help adjust a proper dose of a given drug. 3. The analysis of 35Cl NQR frequencies shows distinctive crystallographic and chemical inequivalence of chlorine atoms in some 1,4-benzodiazepine derivatives.
[1] M.C. Gravielle, R. Faris, S.J. Russek, S.H. Farb, J. Biol. Chem. 280 (2005) 20954. [2] S.I. Wikinski, G.B. Acosta, M.C. Gravielle, C.D. Bonavita, V. Bisagno, S. Fiszer-Plazas, M.C. Rubio, Pharmacol. Biochem. Behav. 68 (2001) 721. [3] D.J. Greenblatt, J.S. Harmatz, R.I. Shader, Clin. Pharmacokinet. 21 (1991) 165. [4] B.G. Katzung, Basic and Clinical Pharmacology, Medical Publishing Division, London, 2004. [5] M. Fukinaga, K. Ishizawa, K.C. Kamei, Pharmacology 57 (1998) 233. [6] W.B. Mendelson, Psychiatr. Dev. 2 (1984) 161. [7] J.F. Liegeois, L. Eyrolles, B.A. Ellenbroek, C. Lejeune, P. Carato, J. Bruhwyler, J. Geczy, J. Damas, J. Delarge, J. Med. Chem. 45 (2002) 5136. [8] P. Giusti, R. Arban, Pharmacol. Res. 27 (1993) 201. [9] P. Giusti, G. Guidetti, E. Costa, A. Guidotti, J. Pharmacol. Exp. Ther. 257 (1991) 1062. [10] L.G. Aguayo, R.W. Peoples, H.H. Yeh, G.E. Yevenes, Curr. Top. Med. Chem. 2 (2002) 869. [11] P. Ducrot, C.R. Andrianjara, R. Wrigglesworth, J. Comput. Aided. Mol. Des. 15 (2001) 767. [12] A. Ravi-Keerti, B. Ashok-Kumar, T. Parthasarathy, V. Uma, Bioorg. Med. Chem. 13 (2005) 1873. [13] A. Lu, J. Zhou, J. Chem. Inf. Comput. Sci. 44 (2004) 1130. [14] J. Kawakami, K. Hoshi, A. Ishiyama, S. Miyagishima, K. Sato, Chem. Pharm. Bull. 52 (2004) 751. [15] S.P. Gupta, Curr. Pharm. Des. 8 (2002) 111. [16] P.A. Borea, G. Gilli, V. Bertolasi, V. Ferretti, Mol. Pharmacol. 31 (1987) 334. [17] P.A. Borea, G. Gilli, Arzneim.-Forsch. 34 (1984) 649. [18] R. Kumar, J.W. Lown, Org. Biomol. Chem. 7 (2003) 3327. [19] P.A. Borea, A. Bonora, Biochem. Pharmacol. 32 (1983) 603. [20] P.A. Borea, Bol. Soc. Ital. Bio. Sper. 57 (1981) 628. [21] S.P. Gupta, R.N. Saha, V. Mulchandani, J. Mol. Recognit. 5 (1992) 75. [22] D. Berezhnoy, R. Baur, A. Gonthier, B. Foucaud, M. Goeldner, E. Sigel, J. Neurochem. 92 (2005) 859. [23] L.T. Schove, J.J. Perez, G.H. Loew, Bioorg. Med. Chem. 2 (1994) 1029. [24] G.H. Loew, J.R. Nienow, M. Poulsen, Mol. Pharmacol. 26 (1984) 199. [25] L.J. Latosinska, J. Pharm. Biomed. Anal. 38 (2005) 577. [26] E. Balchin, D.J. Malcolme-Lawes, I.J.F. Poplett, M.D. Rowe, J.A.S. Smith, G.E.S. Pearce, S.A.C. Wren, Anal. Chem. 77 (2005) 3925. [27] M.J. Frisch, G.W. Trucks, H.B. Schlegel, P.M.W. Gill, B.G. Johnson, M.A. Robb, J.R. Cheeseman, T. Keith, G.A. Petersson, J.A. Montgomery, K. Raghavachari, M.A. Al-Laham, V. Zakrzewski, J.V. Ortiz, J.B. Foresman, J. Cioslowski, B.B. Stefanov, A. Nanayakkara, M. Challacombe, C.Y. Peng, P.Y. Ayala, W. Chen, M.W. Wong, J.L. Andres, E.S. Replogle, R. Gomperts, R.L. Martin, D.J. Fox, J.S. Binkley, D.J. Defrees, J. Baker, J.P. Stewart, M. HeadGordon, C. Gonzales, J.A. Pople, Gaussian 98, Revision A, Gaussian, Inc, Pittsburg, PA, 1998. [28] J.P. Perdew, K. Burke, Y. Wang, Phys. Rev. B54 (1996) 16533. [29] P. Pyykko¨, Mol. Phys. 99 (2001) 1617. [30] E.D. Glendening, A.E. Reed, J.E. Carpenter, F. Weinhold, NBO Version 3.1.7, 1997. [31] F. De Proft, J.M.L. Martin, P. Geerlings, Chem. Phys. Lett. 256 (1996) 400. [32] B. Nogaj, A. Glaser, J. Spychala, Magn. Reson. Chem. 34 (1996) 995. [33] B. Nogaj, G. Schroeder, Magn. Reson. Chem. 25 (1987) 565. [34] G. Gilli, V. Bertolasi, M. Sacerdoti, P.A. Borea, Acta. Crystallogr. B33 (1977) 2664. [35] G. Gilli, V. Bertolasi, M. Sacerdoti, P.A. Borea, Acta Crystallogr. B34 (1978) 2826. [36] Z. Gałdecki, M.L. Gło´wka, Acta Crystallogr. B36 (1980) 3044. [37] O. Kh. Poleshchuk, K. Makiej, M. Ostafin, B. Nogaj, Magn. Reson. Chem. 39 (2001) 329.
Studies of the electronic structure and biological activity of chosen 1,4-benzodiazepines by 35Cl NQR spectroscopy and DFT calculations K. Bronisz a, M. Ostafin b
a,*
, O. Kh. Poleshchuk b, J. Mielcarek c, B. Nogaj
a
a Department of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland Department of Chemistry, Tomsk Pedagogical University, Komsomolskii 75, 634041 Tomsk, Russia c Faculty of Pharmacy, University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Received 9 June 2006; accepted 1 September 2006 Available online 6 September 2006
Abstract Selected derivatives of 1,4-benzodiazepine: lorazepam, lormetazepam, oxazepam and temazepam, used as active substances in anxiolytic drugs, have been studied by 35Cl NQR method in order to find the correlation between electronic structure and biological activity. The 35Cl NQR resonance frequencies (mQ) measured at 77 K have been correlated with the following parameters characterising their biological activity: biological half-life period (t0.5), affinity to benzodiazepine receptor (IC50) and mean dose equivalent. The results of experimental study of some benzodiazepine derivatives by nuclear quadrupole resonance of 35Cl nuclei are compared with theoretical results based on DFT calculations which were carried out by means of Gaussian’98 W software. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Benzodiazepines; Anxiolytic drugs; Nuclear quadrupole resonance (NQR) spectroscopy; DFT calculations
1. Introduction Recently, the clinical use of 1,4-benzodiazepines (BDZ) as anxiolytic, anticonvulsant, sedative-hypnotic and muscle relaxant agents has increased enormously [1–6]. After the first synthesis of the prototypical agents chlorodiazepoxide and diazepam, thousands of benzodiazepine congeners have been synthesised and studied at numerous laboratories [7–9]. However, the molecular mechanism of their activity remains to be clarified. The discovery of stereospecific, high affinity BDZ binding sites in mammal brains that are capable of being saturated, has rekindled new interest in this field. Notwithstanding, no clear hypothesis has so far been put forward as to the pharmacodynamic moiety of the BDZ molecule, a point which is of fundamental importance in understanding BDZ molecular interactions with the target sites in the brain [10,11]. *
Corresponding author. Tel.: +48 61 8295255. E-mail address: [email protected] (M. Ostafin).
0301-0104/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2006.09.001
So far many authors have tried to correlate the results of general biological tests on BZD with their molecular structure. The general formula of 1,4-benzodiazepine derivatives, with the types of substituents at the positions 1, 3, 7 and 2 0 , is presented in Fig. 1 along with Table 1. The profile of activity of a given derivative depends on the type of atom or atoms being substituents R1, R3, R7 and R2 0 in the benzodiazepine molecule. For example, the presence of a nitro group or a halogen atom at position R7 enhances the drug activity in the sequence F < Br < NO2. Introduction of a methyl group at R1 facilitates resorption but such an extension of the aliphatic chain is detrimental to the biological activity. The substituent at R3 determines the process of glucuronide, the absorbability and time of activity. Enhanced activity can be also achieved by introducing a halogen at the ortho position of the aromatic ring (R2 0 ). However, the presence of a halogen at the meta or para positions significantly reduces the therapeutic activity of the drug.
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K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
Fig. 1. Chemical structures of 1,4-benzodiazepines.
Table 1 Four selected derivatives of 1,4-benzodiazepine Benzodiazepine derivative
Substituent
Lorazepam
R7 = Cl R3 = OH R7 = Cl R3 = OH R7 = Cl R3 = OH R7 = Cl R3 = OH
Lormetazepam Oxazepam Temazepam
R1 = H R2 0 = Cl R1 = CH3 R2 0 = Cl R1 = H R2 0 = H R1 = CH3 R2 0 = H
A quantitative structure-activity relationship (QSAR) study was carried out on over 50 BZD with a variety of substituents at positions 7 and 2 0 . Using CNDO/2 methodology and calculated values for dipole moment and net charge on the carbonyl oxygen, the data of several different in vivo tests have been analysed [12–15]. On the grounds of the crystal structure determinations and references, structure-activity relationships, pharmacokinetic and spectroscopic data, the presumable importance of the N1H–C2@O amidic moiety, which can bind to the receptor via a cooperative system of hydrogen bonds has been pointed out. A Free-Wilson analysis by Borea of in vivo data of several benzodiazepines has led to the following rank orders of contributions to the activity of substituents at position 7 and 2 0 thus showing the importance of electron withdrawing groups at these positions, e.g. the rank of electronegativities of the following substituents at position 7 can be represented as –CF3 > –Br > –NO2 and at position 2 0 –F > –Cl > –Br accordingly [16–18]. Position 7 is the most important location for enhancing BZR (benzodiazepine receptor) affinity. An increase in the substituent lipophilicity and electronic charge were found to be directly related to the increase in the receptor binding [19]. The optimal R7 substituents are: –CH2CF3, halogens and nitro, while the worst: –CH@CH–. The substituents at position 2 0 are of second most importance in positively influencing BZR affinity, and an increase in the polar nature of these substituents was shown to be beneficial, although this effect was diminished if the groups were bulky. Substitution at positions 3 and 8 results in reduced pharmacological activity of benzodiazepines. In addition, to corroborate the positive effect of the electron withdrawing moieties at positions 7 and 2 0 , the results of this analysis supported the importance of a carbonyl at position 2, the
detrimental effect of groups larger than –H or –CH3 at positions 3, and the negative impact of substituents at position 4 0 . The latter observation suggests that the pendant phenyl ring occupies a hydrophobic pocket in the receptor binding site and that the depth of the pocket cannot accommodate a para substituent [20,21]. Conformational and electronic properties of 21 benzodiazepines were calculated by using empirical energy and semiempirical molecular orbital methods [22–24]. Compounds that were either highly active or very weakly active in BZR binding (e.g. diazepam and medazepam) were found to have very similar low energy conformations, thus indicating that conformational factors are not important for receptor recognition. However, mapping the electrostatic potential of the benzodiazepine molecule led to the postulate that the interactions between the electron withdrawing substituents R7, the carbonyl oxygen at position 2 and the N4 imine nitrogen with three different cationic receptor sites are required for high affinity analogs [ibid.]. The NQR spectroscopy provides parameters characterising the electronic structure (35Cl NQR frequency) of substances which can be related to their biological activities [25,26]. This paper reports such a study for 1,4-benzodiazepine derivatives. The aim of the paper was to determine the resonance frequencies 35Cl NQR at the chlorine nuclei at position 7 of the benzodiazepine ring and to determine the correlations with the parameters describing the biological activity of a compound studied. 2. Materials Benzodiazepine derivatives: lormetazepam, oxazepam and temazepam were purchased in a hermetically sealed containers from Tarchomin Pharmaceutical Works ‘‘Polfa’’ Joint Stock Company. Lorazepam: 7-chloro-5-(2chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin2-one. Lormetazepam:(R,S)-7-chloro-5-(2-chlorophenyl)1,3-dihydro-3-hydroxy-1-methyl-2H-1,4-benzodiazepin-2one. Oxazepam:7-chloro-1,3-dihydro-3-hydroxy-5-phenyl2H-1,4-benzodiazepin-2-one. Temazepam: 7-chloro-1,3dihydro-3-hydroxy-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one. 3. Experimental 35
Cl NQR frequencies were measured in liquid nitrogen (77 K) on a pulsed Fourier transform NQR spectrometer type NQS-300 (0.5–300 MHz) from MBC Elektronics (Warsaw, Poland) constructed on the basis of a laboratory made NQR spectrometer. The 35Cl NQR spectra of the compounds studied were recorded by a standard method of Fourier transform of a FID signal observed after a p/2 pulse and the pulse duration was 7 ls. Portions of about 3 g of each substance were placed in glass vials of 5 cm in length and 1 cm diameter, capped with a plastic cork. The samples were placed in a cylindrical coil of 3 cm in
K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
length and 1.3 cm in diameter. The samples were cooled to 77 K by immersing in a liquid nitrogen bath. 4. Computational details The molecular geometry was fully optimised with Gaussian’98W program [27], using the hybrid method B3PW91 [28] with the 6-31G(d,p) basis set. This basis set presents a high-level performance theoretical study (polarisation functions) of molecules. The calculations were carried out on a personal computer Pentium 4. The values of the NQR frequencies and quadrupole coupling constants were calculated from the principal components of the EFG tensor at the quadrupolar nuclei of chlorine and nitrogen. It is known that in general two parameters can be obtained from NQR experiment: the quadrupole coupling constant, v, and asymmetry parameter of the EFG tensor g: v ¼ e2 Qqzz =h;
g ¼ ðqxx qyy Þ=qzz
ð1Þ
where eqii are components of the EFG tensor at the quadrupolar nucleus (defined in the EFG principal axes system), Q the nuclear quadrupole moment, e the proton charge and h is the Planck’s constant. We used qii from the results of the Gaussian program and then calculated v and g using Eq. (1). The Q values were taken from [29]. The electronic structure of the studied compounds was analysed by means of the natural bond orbitals (NBO) partitioning scheme approach [30]. The natural population analysis (NPA) scheme developed by Weinhold has been used to obtain charges on atoms in molecules. Proft et al. [31] have found that the B3PW91 functional provide comparatively good results.
303
Table 2 The 35Cl NQR frequencies determined at 77 K and parameters describing the biological activity of the compounds studied: biological half-lifetime (t0.5), affinity to the benzodiazepine receptor (IC50) and mean dose equivalent Benzodiazepine derivative
mQ at 77 K (MHz)
t0.5 (h)
Lorazepam
34. 787 35.406* 35.549 35.571*
14
4
0.75
Lormetazepam
35.232?
12
4
0.75
*
IC50 (nmol/l)
Mean dose equivalent (mg)
Oxazepam
34.798 34.945*
11.5
18
15.0
Temazepam
35.128* 35.244* 35.302*
11
16
7.5
* The values marked by asterisk refer to the chlorine atoms at position 7 in benzodiazepines. ? Unambiguous assignment of this frequency to the chlorine atoms at position 7 or 2 0 was not possible.
Exemplary spectra of temazepam and lorazepam are shown in Fig. 2. The full-width half-maximum (FWHM) values of the resonance lines were within 8–16 kHz. It is also interesting to analyse the multiplicity of the spectra against the
5. Results and discussion The resonance frequencies at the chlorine atoms at position 7 of the benzodiazepine (R7) and at the ortho positions of the phenyl ring (R2 0 ), measured at 77 K are given in Table 2. The same table presents the pharmacological and pharmacokinetic parameters of the drugs: biological half-life period (t0.5), affinity to benzodiazepine receptor (IC50) and mean dose equivalent. Biological half-life time (t0.5) is the time in which the concentration of the active substance decreases by half in a given form of drug. The affinity to benzodiazepine receptor (IC50) is expressed as the number of moles of a given compound that can be bonded to the receptor considered. The mean dose equivalent is the mean amount of the active therapeutic substance administered per 24 h. To analyse the intra- and inter-molecular interactions of particular chlorine atoms in the lorazepam molecule, the 35 Cl NQR resonance frequencies have been assigned to each of them. The probe was the chlorine atom at position 7 of benzodiazepine ring to which the resonance frequencies labelled as (*) in Table 2 were assigned [32–34].
Fig. 2. The 77 K.
35
Cl NQR spectra of (a) temazepam (b) lorazepam taken at
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K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
Table 3 The results of the calculation at B3PW91/6-31G(d,p) level Molecule
mCl (MHz)
e2Qqzz14N (MHz)
qCl
qN
qcarb: O
qhydr: O
LPOcarb./LPhydr.
Lorazepam
35.49(0.06)*
4.37
0.020 0.032
0.403
0.598
0.743
1.841/1.930
36.01(0.11) Lormetazepam
35.22(0.06)* 35.54(0.09)
4.46
0.012 0.017
0.446
0.584
0.757
1.852/1.943
Oxazepam
34.18(0.08)
4.20
0.046
0.436
0.598
0.740
1.840/1.932
Temazepam
35.13(0.06)
4.27
0.010
0.476
0.586
0.757
1.852/1.944
*
The values marked by asterisk refer to the chlorine atoms at position 7 in benzodiazepines.
chemical structure of the molecules. It appears that for some samples the number of resonance lines is not the same as that of chlorine atoms. For example the lorazepam molecule has two chlorine atoms and its 35Cl NQR spectrum shows four resonance lines three of which are shown in Fig. 2b. This observation confirms that the chlorine atoms in the lorazepam molecule show both chemical and crystallographic inequivalence. On the other hand, for lormetazepam with two chlorine atoms in its molecule only one NQR line has been detected despite a very careful scanning of the entire frequency range of interest. Since chemical equivalence of these two chlorines is rather unlikely as confirmed by DFT results which show chemical inequivalence of more than 300 kHz for chlorine atoms in positions 7 and 2 0 (see Table 3) we cannot provide convincing explanation of this fact as yet. Obviously this ambiguity did not enable us to assign the NQR frequency marked by (?) in Table 2 to one of the two chlorine atoms in lormetazepam molecule.
The 35Cl NQR spectra has been recorded off-resonance so the relative intensities of NQR lines, as Fig. 2b shows, do not reflect the relative number of molecules occupying inequivalent positions in the crystalline unit cell. For oxazepam with only one chlorine atom only one 35 Cl NQR line was recorded, which suggests that all the molecules take equivalent positions in the elementary cell [35]. The NQR spectrum of temazepam, also having one chlorine atom, shows three resonance lines indicating the presence of at least three molecules of inequivalent positions in the elementary cell [36]. The 35Cl NQR resonance frequencies determined at 77 K were correlated with the parameters of biological activity of the compounds, see Fig. 3. Assuming linear correlation model the correlation coefficients determined for data shown in Fig. 3a–c are R = 0.715, 0.768 and 0.890, respectively. Although these values of R are too small to corroborate the true linearity of the correlation given the
Fig. 3. The mean 35Cl NQR frequency in the spectra taken at 77 K versus (a) biological half-lifetime, (b) affinity to the benzodiazepine receptor and (c) mean dose equivalent.
K. Bronisz et al. / Chemical Physics 330 (2006) 301–306
four data points only available a positive correlation of data shown in Fig. 3a and a negative one for data in Fig. 3b and c is clearly visible. As follows from Fig. 3a, the mean resonance frequencies increase with increasing half-lifetime of the compounds studied. This correlation suggests that benzodiazepines characterised by the lowest resonance frequencies are more easily metabolised. We also found a negative correlation between the affinity to the benzodiazepine receptor and the resonance 35Cl NQR frequency, so as the electron density at Cl increases, the affinity to this receptor increases. Therefore, it can be concluded that the compounds characterised by lower resonance frequencies better bind to the benzodiazepine receptor. Moreover, as shown in Fig. 3c, as the mean 35 Cl NQR resonance frequency on the chlorine atoms in benzodiazepines decreases, the mean dose equivalent increases. This indicates that the compounds characterised by higher resonance frequencies are administered in lower doses. 6. DFT calculations The B3PW91/6-31G(d,p) optimised structure by one of the molecules studied is displayed in Fig. 4. Its bond lengths were typical for the organic molecules. A comparison of the geometrical parameters with the experimental data [34–36] for benzodiazepine derivatives showed that the optimised C–C, C–N, C–O and C–Cl bond lengths indicated the high reliability of our calculations. Analysis of the obtained results leads to the following correlation between the calculated and experimental bond lengths for the compounds studied (Fig. 5): ˚ ¼ 0:01 þ 0:99½Rðexp :Þ=A ˚ ½Rðcal:Þ=A ðr ¼ 0:995; s ¼ 0:01; n ¼ 79Þ
ð2Þ
(where r is the correlation coefficient, s the standard deviation and n is the number of the compounds). The factor
Fig. 4. Molecular structure of lorazepam, optimised at B3PW91/631G(d,p) level.
305
Fig. 5. The dependence between experimental and calculated bond lengths of benzodiazepines.
preceding R(exp.) is close to unity, which indicates the high reliability of the calculations at the B3PW91 level of the theory. These results encouraged us to perform the calculations of v for the chlorine and nitrogen atoms for some benzodiazepines (Table 3). Note that a fairly good correlation exists between the experimental and calculated 35Cl NQR frequencies, as demonstrated in Fig. 6 where straight correlation line was drawn according to the equation: mCl ðexp:Þ ¼ 20:5 þ 0:4mCl ðcal:Þ ðr ¼ 0:985; s ¼ 0:05; n ¼ 4Þ ð3Þ A similar dependence was obtained earlier in paper [37] for many compounds containing chlorine–carbon and chlorine–nitrogen bonds. The obtained dependences between NQR frequencies and biological activity parameters: biological half-life, affinity to the receptor, and mean dose equivalent allow hypothesising, that these parameters roughly at least should depend on the atomic charges of chlorine. However, in a series of compounds presented here the calculated by NBO approach chlorine effective charges are virtually identical (Table 3) what implies the lack of dependence between effective charges on chlorine atoms and the biological activity of the molecules. We have tried to estimate charges on other atoms in molecules, which would correlate sufficiently with IC50 values. The following
Fig. 6. The dependence between experimental and calculated frequencies of benzodiazepines.
35
Cl NQR
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References
Fig. 7. The dependence between calculated nitrogen quadrupole coupling constant and IC50 of some benzodiazepines.
correlations between IC50 values and charges on nitrogen and oxygen atoms were obtained: IC50 ¼ 616 359qN þ 1020qhydr: O
ðr ¼ 0:993; s ¼ 1:1; n ¼ 4Þ ð4Þ
for the diazepine nitrogen 4 and hydroxyl oxygen, respectively, and IC50 ¼ 892 370qN 1250qcarb: O
ðr ¼ 0:983; s ¼ 1:7; n ¼ 4Þ ð5Þ
for the diazepine nitrogen 4 and carboxyl oxygen. Indirect confirmation to that the reactive part in molecules contains nitrogen and oxygen atoms is considerably better correlation of IC50 on the calculated NQR parameters of nitrogen rather than chlorine atoms (Fig. 7). Like for the correlations shown in Fig. 3a–c linear model was chosen for simplicity sake only and the same remarks apply here regarding the only four data points available. Besides, from our calculation it is evident, that the population of lone electron pair (LP) of carboxylic oxygen is much lower, than that of hydroxyl oxygen (Table 3). This may indicate that the series of 1,4-benzodiazepine molecules including carboxylic atoms, play a key role in interacting with GABA receptor. 7. Conclusions The above-discussed results of the study lead to the following conclusions: 1. There is a relationship between the 35Cl NQR frequency and the parameters characterising biological activity of 1,4-benzodiazepine derivatives. It can be supposed that the distribution of electron density in a given compound permits prediction of its biological activity. 2. The information provided by the 35Cl NQR frequency may help adjust a proper dose of a given drug. 3. The analysis of 35Cl NQR frequencies shows distinctive crystallographic and chemical inequivalence of chlorine atoms in some 1,4-benzodiazepine derivatives.
[1] M.C. Gravielle, R. Faris, S.J. Russek, S.H. Farb, J. Biol. Chem. 280 (2005) 20954. [2] S.I. Wikinski, G.B. Acosta, M.C. Gravielle, C.D. Bonavita, V. Bisagno, S. Fiszer-Plazas, M.C. Rubio, Pharmacol. Biochem. Behav. 68 (2001) 721. [3] D.J. Greenblatt, J.S. Harmatz, R.I. Shader, Clin. Pharmacokinet. 21 (1991) 165. [4] B.G. Katzung, Basic and Clinical Pharmacology, Medical Publishing Division, London, 2004. [5] M. Fukinaga, K. Ishizawa, K.C. Kamei, Pharmacology 57 (1998) 233. [6] W.B. Mendelson, Psychiatr. Dev. 2 (1984) 161. [7] J.F. Liegeois, L. Eyrolles, B.A. Ellenbroek, C. Lejeune, P. Carato, J. Bruhwyler, J. Geczy, J. Damas, J. Delarge, J. Med. Chem. 45 (2002) 5136. [8] P. Giusti, R. Arban, Pharmacol. Res. 27 (1993) 201. [9] P. Giusti, G. Guidetti, E. Costa, A. Guidotti, J. Pharmacol. Exp. Ther. 257 (1991) 1062. [10] L.G. Aguayo, R.W. Peoples, H.H. Yeh, G.E. Yevenes, Curr. Top. Med. Chem. 2 (2002) 869. [11] P. Ducrot, C.R. Andrianjara, R. Wrigglesworth, J. Comput. Aided. Mol. Des. 15 (2001) 767. [12] A. Ravi-Keerti, B. Ashok-Kumar, T. Parthasarathy, V. Uma, Bioorg. Med. Chem. 13 (2005) 1873. [13] A. Lu, J. Zhou, J. Chem. Inf. Comput. Sci. 44 (2004) 1130. [14] J. Kawakami, K. Hoshi, A. Ishiyama, S. Miyagishima, K. Sato, Chem. Pharm. Bull. 52 (2004) 751. [15] S.P. Gupta, Curr. Pharm. Des. 8 (2002) 111. [16] P.A. Borea, G. Gilli, V. Bertolasi, V. Ferretti, Mol. Pharmacol. 31 (1987) 334. [17] P.A. Borea, G. Gilli, Arzneim.-Forsch. 34 (1984) 649. [18] R. Kumar, J.W. Lown, Org. Biomol. Chem. 7 (2003) 3327. [19] P.A. Borea, A. Bonora, Biochem. Pharmacol. 32 (1983) 603. [20] P.A. Borea, Bol. Soc. Ital. Bio. Sper. 57 (1981) 628. [21] S.P. Gupta, R.N. Saha, V. Mulchandani, J. Mol. Recognit. 5 (1992) 75. [22] D. Berezhnoy, R. Baur, A. Gonthier, B. Foucaud, M. Goeldner, E. Sigel, J. Neurochem. 92 (2005) 859. [23] L.T. Schove, J.J. Perez, G.H. Loew, Bioorg. Med. Chem. 2 (1994) 1029. [24] G.H. Loew, J.R. Nienow, M. Poulsen, Mol. Pharmacol. 26 (1984) 199. [25] L.J. Latosinska, J. Pharm. Biomed. Anal. 38 (2005) 577. [26] E. Balchin, D.J. Malcolme-Lawes, I.J.F. Poplett, M.D. Rowe, J.A.S. Smith, G.E.S. Pearce, S.A.C. Wren, Anal. Chem. 77 (2005) 3925. [27] M.J. Frisch, G.W. Trucks, H.B. Schlegel, P.M.W. Gill, B.G. Johnson, M.A. Robb, J.R. Cheeseman, T. Keith, G.A. Petersson, J.A. Montgomery, K. Raghavachari, M.A. Al-Laham, V. Zakrzewski, J.V. Ortiz, J.B. Foresman, J. Cioslowski, B.B. Stefanov, A. Nanayakkara, M. Challacombe, C.Y. Peng, P.Y. Ayala, W. Chen, M.W. Wong, J.L. Andres, E.S. Replogle, R. Gomperts, R.L. Martin, D.J. Fox, J.S. Binkley, D.J. Defrees, J. Baker, J.P. Stewart, M. HeadGordon, C. Gonzales, J.A. Pople, Gaussian 98, Revision A, Gaussian, Inc, Pittsburg, PA, 1998. [28] J.P. Perdew, K. Burke, Y. Wang, Phys. Rev. B54 (1996) 16533. [29] P. Pyykko¨, Mol. Phys. 99 (2001) 1617. [30] E.D. Glendening, A.E. Reed, J.E. Carpenter, F. Weinhold, NBO Version 3.1.7, 1997. [31] F. De Proft, J.M.L. Martin, P. Geerlings, Chem. Phys. Lett. 256 (1996) 400. [32] B. Nogaj, A. Glaser, J. Spychala, Magn. Reson. Chem. 34 (1996) 995. [33] B. Nogaj, G. Schroeder, Magn. Reson. Chem. 25 (1987) 565. [34] G. Gilli, V. Bertolasi, M. Sacerdoti, P.A. Borea, Acta. Crystallogr. B33 (1977) 2664. [35] G. Gilli, V. Bertolasi, M. Sacerdoti, P.A. Borea, Acta Crystallogr. B34 (1978) 2826. [36] Z. Gałdecki, M.L. Gło´wka, Acta Crystallogr. B36 (1980) 3044. [37] O. Kh. Poleshchuk, K. Makiej, M. Ostafin, B. Nogaj, Magn. Reson. Chem. 39 (2001) 329.