Structural aspects of intermolecular interactions in the solid state of 1,4-dibenzylpiperazines bearing nitrile or amidine groups

X-ray diffraction analyses for new pentamidine analogs are presented: 1,4-bis(4-cyanobenzyl)piperazine (1) crystallizes in the triclinic space group () and 1,4-bis(4-amidinobenzyl)piperazine tetrahydrochloride tetrahydrate (2) in the monoclinic space group (P21/n) revealing a complex system of hydrogen bonds for (2).

The crystal structures of the title 1,4-bis(4-cyanobenzyl)piperazine (1) and 1,4-bis(4-amidinobenzyl)piperazine tetrahydrochloride tetrahydrate (2) are reported. Compound (1) crystallizes in the triclinic space group P " 1 1 and compound (2) in the monoclinic space group P2 1 /n. In both (1) and (2) the asymmetric unit contains one half of the molecule because the central piperazine rings were located across a symmetry center. The packing of both molecules was dominated by hydrogen bonds. The crystal lattice of (1) was formed by weak C-HÁ Á ÁN and C-HÁ Á Á interactions. The crystal structure of (2) was completely different, with cations as well as chloride anions and water molecules taking part in intermolecular interactions. Single-crystal X-ray diffraction studies combined with density functional theory (DFT) calculations allowed the characterization of the intermolecular interactions in those two systems having different types of very strong electrophilic groups: non-ionic nitrile and ionic amidine. Chemical shift data from 13 C CP/MAS (Cross Polarization Magic Angle Spinning) NMR spectra were analyzed using the different procedures for the theoretical computation of shielding constants.

Introduction
The increasing demands of the pharmaceutical industry for rapid molecular structure determination of pharmaceutical solids have prompted the development of joint analysis methods spanning X-ray diffraction, 13 C CP/MAS NMR and molecular modeling. The solid-state form of a drug can have a dramatic effect on its bioavailability and physical properties, and the regulatory approval for many drugs is granted only for the defined polymorph (Barrett et al., 2013;Geppi et al., 2008;Pereira Silva et al., 2011). Moreover, because the existence of solvates (called pseudopolymorphs) is an abiding problem in pharmaceutical chemistry (the solid-state form of a solvate can also be treated as the separate form of the drug), it is important to structurally characterize these. Solid-state structural studies of new substances which are designed as potential chemotherapeutic agents has also aroused great interest (Harris, 2007). Hydrogen bonds are crucial to the interactions between biomolecules, with the macromolecular target and their analysis contributing to expanding the information about biomolecular interactions.
The molecules analyzed in this investigation can be considered as pentamidine analogs and are of interest because of their potential as chemotherapeutic agents against pneumocystis pneumonia (PCP) caused by the fungus Pneumocystis jiroveci in patients with compromised immune systems (Ponce et al., 2010;Furrer et al., 1999;Maini et al., 2013) or as anticancer and antimicrobial agents (Barrett et al., 2013).
The screening of new pentamidine analogs led to the selection of less toxic and highly active molecules (with in vitro IC 50 values as low as 0.002 mM compared with 0.5 mM for pentamidine) which contain functional groups such as the piperazine ring that increase the rigidity of the molecule Huang et al., 2009;Cushion et al., 2004Cushion et al., , 2006Mitsuyama et al., 2008). The intermolecular interactions in the solid state of piperazine-type pentamidine analogs were not examined, and the results obtained here could be useful in the future explanation of their biological features.
In this investigation we analyzed and compared the solidstate structures of new pentamidine analogs containing the piperazine moiety (Fig. 1), i.e. 1,4-bis(4-cyanobenzyl)piperazine (1) and 1,4-bis(4-amidinobenzyl)piperazine tetrahydrochloride tetrahydrate (2) with particular attention paid to hydrogen bonding, using different methods: single-crystal X-ray diffraction analyses combined with molecular modeling and 13 C CP/MAS NMR spectroscopy. The bis-nitrile compound (1) is an intermediate for the synthesis of bisamidine (2) via the Pinner reaction (Pinner & Klein, 1877). Diffraction-quality single crystals of bis-amidines are difficult to grow, and the crystal structures of few bis-amidines related to pentamidine have been reported (Maciejewska et al., 2006;Lowe et al., 1989;Srikrishnan et al., 2004;Donkor et al., 1995). Some authors used the information obtained for the crystals of bis-nitriles to explain the properties of bis-amidines (Cui et al., 2003). (The authors designed the inhibitors of key enzymes in the coagulation cascade, and to experimentally check the geometry of the amidine inhibitors they determined their cyanosynthetic intermediates by X-ray diffraction.) In this work we show that such an approach to the problem is inappropriate due to the limited similarity between those two groups.

Chemistry
All chemicals were purchased from major chemical suppliers as high or the highest purity grade and were used without any further purification. The solvents, K 2 CO 3 , HCl, NaOH and ammonia, were obtained from POCH (Gliwice, Poland). The substrates 4-cyanobenzyl bromide and piperazine were obtained from Alfa AESAR (Karlsruhe, Germany). The scheme below presents the synthetic route to bis-amidine (2) via bis-nitrile (1). In the first step 1,4-bis(4cyanobenzyl)piperazine (1) was prepared by a modification of the procedure given by Gruenenthal (2008). The Pinner reaction of (1) to form the bis-amidine (2) was conducted for 2 weeks due to the poor solubility of (1) in ethanolic HCl.
Compound (1) was mentioned in the paper by Spychała (1999), but the detailed synthetic and spectral information was not given and therefore it is described in x2.1.1. Melting points were determined with an Electrothermal 9001 digital melting point apparatus. Elemental analyses were performed on a Vario EL III CHNS element analyzer, and were averaged from two independent determinations. Chemical shifts (p.p.m.) in CDCl 3 (1) or DMSO-d 6 (2) were referenced to tetramethylsilane (TMS) .

Crystallography
Crystals of (1) suitable for X-ray analysis were grown by slow evaporation from acetone, and crystals of (2) were grown by slow evaporation from 96% ethanol and a few drops of water (which were added to the ethanol mixture until its homogeneity was achieved). Diffraction data were collected on an Oxford Diffraction KM4 CCD diffractometer using Mo K radiation at room temperature for (1) and on an Oxford Diffraction SuperNova CCD diffractometer using Cu K radiation at 130 K for (2). Data reduction was carried out using CrysAlis Pro (Agilent, 2011(Agilent, , 2012 for (1) and (2), respectively.
The unit-cell parameters were determined by least-squares treatment of the angles of the highest-intensity reflections chosen from all the experiments. The structures were solved by direct methods using SHELXS97 and refined on F 2 by fullmatrix least-squares with SHELXL97 (Sheldrick, 2008). The function For (1) an empirical extinction correction was also applied according to the formula F 0 c ¼ kF c ½1 þ ð0:001F 2 c 3 = sin 2Þ À1=4 , and the extinction coefficient was equal to 0.08 (1).
All non-H atoms were refined with anisotropic displacement parameters. The coordinates of the H atoms of (1) and the majority of hydrogen positions of (2) were generated geometrically. In (2) the hydrogen at N1 and the H atoms of water molecules were found on the difference map. Then the H atom involved in hydrogen bonds in (1) and all the H atoms in (2) were refined isotropically. The other atoms were refined as a riding model with U iso (H) = 1.2U eq (carrier atom). All the  Table 1 Crystal data, data collection and structure refinement for (1) and (2). (1) (2)

Molecular modeling
Structural optimizations were performed at the density functional theory (DFT) level with B3LYP/6-311(d,p) hybrid functional, and the locations of the true minima were confirmed by vibrational analysis using GAUSSIAN09 (Frisch et al., 2009). The crystal atomic coordinates were used as the starting point for DFT computations. To investigate the intermolecular interactions in the solid state (Gholivand et al., 2013), the optimization of H atoms positions was performed for the cluster C1 built up from three molecules of (1). The target molecule of (1) was surrounded by two neighboring molecules from one layer which were connected to each other via two C3-H3AÁ Á ÁN2 hydrogen bonds with a donoracceptor distance of 3.629 (2) Å (Fig. 2). For compound (2) cluster C2 was built up from one cation of molecule (2) surrounded by 14 chloride anions and ten water molecules (Fig. 2), because compound (2) did not form hydrogen bonds directly with another molecule of (2). All hydrogen-bond parameters are given in Table 2. The positions of the H atoms were optimized, while the other atoms were kept fixed during the optimizations. This approach allowed us to perform the analyses of the central molecule when the neighboring molecules were present. The hydrogen-bonding energies were calculated for compound (1) using the equation: E Hbond = 1 2 (E cluster À E two À E one ), taking into account the two hydrogen bonds C3-H3AÁ Á ÁN2 formed in the layer. E two is composed of two neighboring molecules at the left-hand side of Fig. 2, and E one is the remaining part of C1. For compound (2) the average energy of hydrogen bonds was calculated based on the energy difference between the hydrogen-bonded cluster and its fragments as represented by the equation: E Hbond = 1/28(E cluster À E anions+water À E one ).
The isotropic 13 C shielding constants (p.p.m.) for (1) and (2) in the solid state were computed with the GIAO (gauge including the atomic orbital) method using GAUSSIAN09 (Frisch et al., 2009) at the DFT level with B3LYP/6-311(d, p) hybrid functional. For the assignment of 13 C CP/MAS NMR resonances, the structures obtained by X-ray diffraction were optimized prior to chemical shielding calculations using four different procedures: (i) the positions of all atoms in (1) and (2) were fully optimized; (ii) the positions of the non-H atoms were fixed, but the H atoms were allowed to move in (1) and (2); (iii) the positions of all atoms in the clusters C1 and C2 were optimized, and the shielding constants were analyzed for the target molecules (1) and (2); (iv) the positions of the non-H atoms were fixed in clusters C1 and C2, and the shielding constants were analyzed for the target molecules (1) and (2).
The results show that the compounds 1,4-bis(4-cyanobenzyl)piperazine (1) and 1,4-bis(4-amidinobenzyl)piperazine tetrahydrochloride tetrahydrate (2), the piperazine-derived analogs of pentamidine, crystallize in the triclinic space group P " 1 1 and monoclinic space group P2 1 /n, respectively. In both (1) and (2), the asymmetric units contain one half of the molecule because the central piperazine rings are located across symmetry centers according to the symmetry operators (Àx, 2 À y, 1 À z) in (1) and (Àx, Ày, Àz) in (2). Additionally, in the asymmetric unit of (2) there are two chloride ions and two molecules of water. As a consequence, we found the tetrachloride salt of this derivative in the lattice. The structure of (1) consists of two cyanobenzyl groups which are joined by the piperazine ring which adopts the expected chair conformation. The cyanobenzyl moiety is almost planar with a maximum deviation of 0.045 (1) Å for C8. The orientation of the piperazine ring with respect to the cyanobenzyl fragment is char-acterized by C1-C8-N1-C9 and C6-C1-C8-N1 torsion angles of À168.6 (1) and 26.0 (2) , respectively.
In (2) the benzyl group is essentially planar with a maximum deviation of 0.011 (1) Å for C2. The C7 atom from the amidine group was found to be coplanar with the above fragment and the N2 and N3 atoms of this group were displaced by 0.556 (2) and À0.653 (2) Å , respectively. The location of the amidine group with respect to the benzene ring can also be characterized by the C3-C4-C7-N2 and C3-C4-C7-N3 torsion angles of À31.6 (2) and 147.1 (1) , respectively. The central piperazine ring adopts a chair conformation and its orientation with respect to the benzyl fragment moiety is characterized by C1-C8-N1-C9 and C6-C1-C8-N1 torsion angles of 66.5 (1) and 100.0 (1) , respectively.
The packing of both molecules is dominated by hydrogen bonds (Table 2). In the crystal of (1), the nitrile groups take part in intermolecular C3-H3AÁ Á ÁN2 (Àx + 2, Ày + 1, Àz + 2) hydrogen bonds which link the molecules into extended chains. The molecules are further organized into layers parallel to the (011) plane via weak C9-H9AÁ Á Á(x À 1, y, z) contacts (Figs. 3 and 4). We also found such a participation of the nitrile substituents in the hydrogen bonds for other analogs of pentamidine (Ż abiń ski et al., 2007Maciejewska et al., 2008).
The crystal structure of (2) differs in that the packing involves cations, chloride anions and water molecules. Each cation is surrounded by chloride anions and molecules of water and they are linked by N-HÁ Á ÁO, N-HÁ Á ÁCl, C-HÁ Á ÁO and C-HÁ Á ÁCl hydrogen bonds (see Table 2   The chains of the molecules of (1) within a layer.

Figure 4
The packing arrangement of (1) showing layers parallel to the (011) plane.

Table 3
Selected bond lengths (Å ), valence angles ( ) and torsion angles ( ) for (1) and (2). (1) (2) interesting to note that there are no direct hydrogen bonds between neighboring molecules of (2). In contrast, the pentamidine analog with three O atoms in the linker forms direct hydrogen bonds using these atoms as proton acceptors (Maciejewska et al., 2006;Lowe et al., 1989;Donkor et al., 1995) even in the presence of water molecules. The protonated N atoms of the piperazine ring in (2) cannot be involved in intermolecular interactions as proton acceptors, and water molecules serve as both proton donors and proton acceptors, providing the main intermolecular links. So far, X-ray studies of the structurally related bis-amidines did not present detailed analysis of the intermolecular hydrogen bonding (Lowe et al., 1989) or showed only a few intermolecular hydrogen bonds involving the anions or water molecules (Donkor et al., 1995;Maciejewska et al., 2006;Lowe et al., 1989;Srikrishnan et al., 2004).

Parameters of hydrogen bonding in the clusters
The theoretical hydrogen-bond parameters for the target molecules (1) and (2) in clusters C1 and C2 are presented in Table 4. The donoracceptor distances for hydrogen bonds in model clusters are equal to the experimental values since the optimizations were performed only for the H-atom positions. The N-H, C-H and O-H bonds are longer by 0.11-0.16 Å than those obtained from the crystal structure determinations, and as a result the hydrogen-acceptor distances HÁ Á ÁA are shorter, suggesting stronger intermolecular interactions. Theoretical N-HÁ Á ÁO, O-HÁ Á ÁCl and N-HÁ Á ÁCl angles are more linear than the crystallographic values.
The computed hydrogen-bonding energy in the C1 model cluster between the molecules (1) was equal to À8.8 kJ mol À1 , which is characteristic of very weak interactions. In the first approximation (the hydrogen bonds presented in Table 4), the average hydrogen-bonding energy in the C2 model cluster between the molecule of (2) and the surrounding water and chloride anions was calculated as À406.8 kJ mol À1 , a very high value. In the second approximation, we considered the 48 interactions with d(HÁ Á ÁA) distances below 3.6 Å (thereby incorporating solvation of the cations by water molecules), and the average intermolecular interaction energy of compound (2) with all surrounding chloride anions and water molecules was À237.3 kJ mol À1 . The intermolecular bonds connecting bisnitriles are much weaker than the intermolecular bonds formed by bis-amidines. The short contacts in the bis-nitrile crystal (1) are due to weak, nonpolar interactions, whereas in the bis-amidine crystal (2) they are dominated by hydrogen bonds between water molecules and chloride anions.
3.3. Solid-state 13 C CP/MAS NMR spectra analysis of (1) and (2) Solid-state 13 C CP/MAS NMR spectra of 1,4-bis(4-cyanobenzyl)piperazine (1) and 1,4-bis(4-amidinobenzyl)piperazine (2) are presented in Fig. 6. The crystals for the 13 C CP/MAS NMR experiments were collected in the same manner as for the single-crystal X-ray diffraction. In neither spectrum were multiplets observed, i.e. we observed a single resonance for each pair of chemically equivalent C atoms. Only for the piperazine ring was an additional signal detected, in accordance with the crystallographic results. Preliminary assignments were carried out on the basis of solution chemical shifts and on the basis of the computed shielding constants obtained for the fully optimized structures of (1) and (2) Table 4 Theoretical hydrogen-bonding parameters for (1) and (2) calculated in clusters C1 and C2, respectively (see Fig. 2).
coefficients were also obtained for the shielding constants calculated by procedures (ii) and (iii). The computations correctly predicted higher shielding constants for C9/C9 0 than for C10/C10 0 in the piperazine ring. As can be seen from Table  5, significant differences between the isotropic chemical shifts measured in solution and in the solid state were found for C7/ C7 0 and C3/C3 0 which are engaged in hydrogen bonding and for the piperazine C atoms. Surprisingly, the calculations on cluster C1 performed using procedure (iv) produced the worst correlation, although the correlation coefficient was still high (R 2 = 0.991). Apparently, the strength of specific solid-state effects is weak, and a simple comparison between the solution and solid-state resonances, and the simple calculation for the isolated molecule is sufficient for structural analysis. The resonances of C3/C3 0 and C5/C5 0 overlap at 131.8 p.p.m., although the calculation showed higher shielding for C5/C5 0 . This can be caused by the impact of intramolecular motions of the benzene rings in the solid state which were not considered in the calculations. The resonances of the ortho pairs C2/C2 0 and C6/C6 0 to the piperazine linker were clearly separated. This is a well known phenomenon dependent on intermolecular interactions and the nature of the substituent present at the neighboring C atom. The separation of signals indicated that methylene groups are slightly twisted relative to the benzene ring plane. The resonances of C7/C7 0 proximal to N2/N2 0 are broadened and split into unequal doublets due to a residual coupling to the quadrupolar 14 N atom (Olivieri et al., 1987). For other C atoms proximal to N atoms only some broadening was observed. The 13 C CP/MAS spectrum of molecule (2) had the same characteristics as the spectrum of (1) in that no multiplets were observed and the C7/C7 0 atoms were broadened by a dipolar coupling to the quadrupolar 14 N nucleus. After preliminary assignment based on the computed shielding constants obtained for the fully optimized structure as described in x2.3 point (i), the shielding constants obtained using three different procedures (ii), (iii) and (iv) were compared to the experimental chemical shifts. The correlation coefficients R 2 for the linear correlations = f() were within the range 0.973-0.996. The highest correlation coefficient was obtained for procedure (ii) -see the supporting information for more details. Procedure (iv), which considered cluster C2, produced the worst results as well as for cluster C1. This observation agreed with our earlier findings for bis-nitriles that the shielding constant computation based on the single molecule structure can be informative for the analysis of solidstate structure based on NMR spectra (Maciejewska et al., 2008). In the spectrum of (2) separated resonances for all aromatic C atoms were observed. Both C atoms ortho and both C atoms meta to the amidine group are shielded in different ways. The lack of coplanarity of amidine groups with the benzene ring clearly affects the shielding of these aromatic C atoms. Next we compared the 13 C resonances in the solid state, solid , and in solution, solution (Table 5). As can be seen, the highest negative differences between the their chemical shifts were found for C2/C2 0 , C3/C3 0 , C7/C7 0 and C10/C10 0 . This was attributed to the engagement of these atoms in the intermolecular interactions with water molecules and chloride anions, which are much stronger than in DMSO solution.
Linear pentamidine analogs are molecules of pharmaceutical interest which are rather poorly represented in the crystallographic database: chemical shifts from NMR solid-state spectra of the analyzed compounds have provided valuable information on the solid-state structure to complement the crystallographic data, allowing their hydrogen-bonding patterns to be determined.  Table 5 Differences (p.p.m.) between selected 13 C chemical shifts in the solid state and in solution for (1) and (2): (Á = solution À solid ). (1)

Conclusions
The structures of 1,4-bis(4-cyanobenzyl)piperazine (1) and 1,4-bis(4-amidinobenzyl)piperazine tetrahydrochloride (2) at 293 and 130 K were solved using single-crystal X-ray diffraction. Compound (1) crystallizes in the triclinic P " 1 1 space group, and compound (2) in the monoclinic space group P2 1 /n with four chloride anions and four H 2 O molecules. The crystal lattice of (1) is formed by weak C-HÁ Á ÁN and C-HÁ Á Á interactions. The intermolecular interaction energy (evaluated using the equation: E Hbond = 1 2 (E cluster À E two À E one ) for the former was À8.8 kJ mol À1 . The crystal lattice formed by (2) is dominated by water molecules and chloride anions, and the average intermolecular interaction energy of compound (2), taking account of all the surrounding chloride anions and water molecules was À237.3 kJ mol À1 . It is interesting to note that no direct hydrogen bonds exist between neighboring molecules of (2). Our result clearly indicated that structural information and intermolecular interactions in bis-nitriles are not transferable to the structural analysis of bis-amidines. The computation of shielding constants for isolated molecules together with the solid-state spectrum are of considerable value in understanding the solid-state structures of pentamidine analogs.