Structures of the hydrate and dihydrate forms of the DNA-binding radioprotector methylproamine

The dihydrate and hydrate forms of the DNA-binding bis-benzimidazole radioprotector methylproamine are reported. These are the first single-crystal structures of bis-benzimidazoles related to Hoechst 33342 to be reported.


Chemical context
Methylproamine (1) is a bibenzimidazole derivative which binds in the minor groove of DNA in adenine-thymine-rich regions of four or more consecutive AT pairs (Martin et al., 2004) and is related to the Hoechst family of DNA-binding bibenzimidazoles (Pjura et al., 1987). Although the structure of methylproamine with the DNA dodecamer d(CGCGAATTCGCG) 2 has been determined and reported by us, the structure of the free ligand has not yet been published as it is very difficult to obtain good quality crystals for these types of compounds. In order to examine the conformational and tautomeric differences between the uncomplexed ligand and that which is bound to DNA, the structures of both the dihydrate (1)Á2H 2 O and the monohydrate (1)ÁH 2 O, which were grown from water in the presence of -cyclodextrin, are reported. ISSN 2056-9890 2. Structural commentary Displacement ellipsoid plots for (1)Á2H 2 O and (1)ÁH 2 O are presented in Figs. 1 and 2, respectively. The two structures represent two different conformations of (1); (1)Á2H 2 O exists in an extended conformation as determined by the C9-C10-C14-N4 torsion angle which is 173.54 (14) with an N1Á Á ÁN6 distance of 17.251 (2) Å while (1)ÁH 2 O adopts a crescent shape with a C9-C10-C14-N4 torsion angle of À19.8 (2) and an N1Á Á ÁN6 distance of 16.859 (2) Å . In addition, they represent different tautomeric forms of (1); (1)Á2H 2 O can be described as the N2, N4 tautomer whereas (1)ÁH 2 O exists in the crystal as the N2, N5 tautomer as defined by the numbering scheme used in Figs. 1 and 2. The tautomeric form adopted in each case is implied not only by the N-H hydrogen atoms, which were located in difference maps and refined satisfactorily without restraint, but also by the C-N bond distances of the two benzimidazole moieties within the structures (Tables 1  and 2). The tautomeric form assigned in each case is also supported by the intermolecular hydrogen bonds that these N-H groups participate in. It is the intermolecular hydrogen-bonded interactions involving these N-H groups which no doubt play a major role in which tautomer is adopted in each case in the solid state.
In both structures the ortho-methyl substituent in ring A lies on the opposite side of the structure to the N-H hydrogen atom of benzimidazole ring B, this is very likely for steric reasons; the dihedral angles between the two rings as defined by C2-C1-C7-N3 in (1)Á2H 2 O and by C2-C1-C7-N2 in (1)ÁH 2 O, which are À30.0 (2) and À23.6 (2) , respectively, reflect a balance between electronic effects which prefer coplanarity between the two aromatic rings and steric effects whereby the ortho-methyl group would be unreasonably close to the benzimidazole nitrogen of ring B. The dihedral angles between the two benzimidazole rings (rings B and C) are À5.7 (2) and À19.8 (2) , respectively.
The geometry of the para-dimethylamino substituent on ring A differs between the two structures; the mean C-N1-C angles are 116.4 and 119.7 , respectively, for (1)Á2H 2 O and (1)ÁH 2 O, suggesting that the former is more pyramidalized, consistent with this are the significant differences in the C4-N1 bond distances which are 1.3923 (18) and 1.374 (2) Å for (1)Á2H 2 O and (1)ÁH 2 O, respectively.
It is interesting to compare the conformation of (1) in these two structures with that adopted by (1) when bound in the minor groove of the palindromic DNA dodecamer [d(CGCGAATTCGCG) 2 ; Martin et al., 2004]. The ligand must adopt the 2-H, 4-H tautomeric form with a crescent shape similar to that adopted by (1)ÁH 2 O so that it can direct the necessary N-H hydrogen-bond donors into the minor groove, in addition the ortho-methyl substituent on ring A must be facing away from the crescent. A superposition of the two structures with that of (1) bound to DNA is shown in Fig. 3 Displacement ellipsoid plot of the asymmetric unit for hydrate (1)ÁH 2 O.

Figure 1
Displacement ellipsoid plot of the asymmetric unit for dihydrate (1)Á2H 2 O.

Figure 3
Overlay for the structures of A; (1)Á2H 2 O and B; (1)ÁH 2 O with DNAbound (1). In the LH-structure the DNA-bond ligand is indicated by capped sticks, while in the RH structure it is ball and stick.

Figure 5
The water tetramer with additional hydrogen-bonded interactions with (1).

Figure 4
The water tetramer with diagonally hydrogen-bonded molecules of (1).
The structure of the hydrate (1)ÁH 2 O is also characterized by extensive hydrogen-bonding interactions, both directly between the benzimidazole moieties of (1), and via the water  Hydrogen bonding between (1) and the water molecule in (1)ÁH 2 O.

Figure 10
Two-dimensional hydrogen-bonded network in (1)ÁH 2 O. molecule. The water molecule participates in two O-HÁ Á ÁN hydrogen bonds as donor and one N-HÁ Á ÁO hydrogen bond as acceptor to form a cluster of three molecules of (1) ( Fig. 8 and Table 4). This cluster is then further hydrogen bonded via N-HÁ Á ÁN interactions between the remaining benzimidazolebased hydrogen-bond donors and acceptors ( Fig. 9 and Table 4), to form two-dimensional hydrogen-bonded sheets lying in the (101) plane (Fig. 10).

Database survey
A search of the CSD (version 1.23; Groom et al., 2016) for structures related to (1) uncovered no hits.

Synthesis and crystallization
The synthesis of methylproamine (1) has been previously reported (Martin et al., 2004) but previous attempts to obtain crystals of the free ligand of suitable quality for X-ray analysis were not successful. In this study, crystals were serendipidously obtained during an attempt to obtain crystals of (1) complexed to -cyclodextrin. Thus a solution of (1) (6.8mg) in 1 ml of water saturated with -cyclodextrin was left in a vapour diffusion tank with acetone allowed to diffuse into the solution. It is worth noting that (1) has very low solubility in water in the absence of -cyclodextrin. After 12 h, brown plates of (1) as its dihydrate developed, which were then harvested for X-ray analysis. The resulting solution when left to evaporate over a period of several months gave further needle-like crystals in a viscous matrix of -cyclodextrin that were shown to be the monohydrate (1)ÁH 2 O.

Refinement
Crystal data, data collection and structure refinement details for (1)Á2H 2 O and (1)ÁH 2 O are summarized in Table 5. In both structures, carbon-bound H atoms were placed in calculated positions and refined using a riding model, with methyl C-H = 0.96 Å and aromatic C-H = 0.93 Å and U iso (H) =1.5U eq (C) for methyl and 1.2U eq (C) for aromatic C-H. Hydrogen atoms attached to N and O were located in difference maps and allowed to refine with isotropic displacement parameters. In the structure of (1)ÁH 2 O there are solvent-accessible voids of 154 Å 3 per unit cell; however, there was no significant difference electron density associated with these voids. The largest difference electron density of 0.5 e Å 3 was associated with the piperazine group. Application of the SQUEEZE procedure (Spek, 2015) found eight electrons associated with the voids.

Funding information
Funding for this research was provided by: Sirtex Medical.  (Westrip, 2010). where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.001 Δρ max = 0.21 e Å −3 Δρ min = −0.24 e Å −3 Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.