Crystal structure and conformational analysis of doxorubicin nitrate

The conformations of the two free doxorubicin (DoxH+) cations present in the crystal structure of the title compound and (Dox) bound to proteins and DNA are compared.


Chemical context
Since its discovery and isolation by genetic mutation of Streptomyces peucetius in 1969 (Arcamone et al., 1969), the anthracycline antibiotic doxorubicin [(Dox); trade name adriamycin] has become one of the most potent and widely used drugs in cancer chemotherapy (Denel-Bobrowska & Marczak, 2017;Cagel et al., 2017;Cappetta et al., 2018). Extensive studies of the anticancer activities of doxorubicin (Weiss, 1992;Shafei et al., 2017) have led to FDA approval for the treatment of cancer forms, such as breast (Shafei et al., 2017), ovarian (Duggan & Keating, 2011) and small-cell lung cancer (Ló pez-Gonzá lez et al., 2013). The anticancer action of doxorubicin is a consequence of its intercalation into base pairs of double-stranded DNA and subsequent inhibition of human DNA topoisomerase II (Arcamone, 1981;Liu, 1989;Chaires, 1998;Yang & Wang, 1999;Jung & Reszka, 2001). Although a few crystal structures of doxorubicin bound to DNA, enzymes and proteins have been reported, to the best of our knowledge, there is no crystal structure determination of doxorubicin itself in the literature. A Cambridge Structural Database (CSD version 5.38; Groom et al., 2016) search for the doxorubicin skeleton structure gave only two hits for hydrochloride salts of N-and O-substituted variants [CSD entries ADRMVL (Eckle & Stezowski, 1980) and BUJZIP (Eckle & Stezowski, 1983)]. Even for daunorubicin (also known as daunomycin), a closely related anthracycline antibiotic, only the crystal structures of its hydrochloride solvates have been reported (Neidle & Taylor, 1977;Courseille et al., 1979). In the absence of a high-resolution crystal structure, researchers have so far relied on computational and solution studies to ascertain the preferred conformational geometry of (Dox) (Zhu et al., 2010; Agrawal et al., 2009;Barthwal et al., 2008). ISSN 2056-9890 In order to probe and improve the activity of (Dox), several derivatives have been studied (Post et al., 2005). Metal complexation to doxorubicin is known to alter its pharmaceutical activity and several Fe, Mn, Pt and Sn derivatives of the anthracycline have been studied with regard to their anticancer activities (Ming, 2003). (DoxH) + -functionalized iron oxide nanoparticles have been studied as cancer theranostics (Yu et al., 2008). With a similar objective, we have attempted to coordinate Ag + to (Dox). However, mixing stoichiometric amounts of (DoxH)Cl and AgNO 3 in the presence of Et 3 N yielded only the nitrate derivative of (Dox) as (DoxH)NO 3 in crystalline form. In this article, we report the 0.80 Å resolution crystal structure determination of doxorubicin nitrate and analyze and compare conformational details.

Structural commentary
The title compound crystallizes in the chiral monoclinic P2 1 space group with two protonated doxorubicin cations (DoxH + ) and two nitrate anions in the asymmetric unit. The (DoxH) + cations consist of an aglycone, containing three approximately planar fused rings (the root-mean-square deviations of the six rings in the asymmetric unit are between 0.009 and 0.027 Å , B-D; the atom-numbering scheme and ring labels are shown in the scheme), and a sugar moiety in a chair conformation attached to ring A. Two nitrate ions hold pairs of cations with their fused rings at an approximately right angle to each other [86.4 (4) between C1-C20 and N62(O64-O66)]. The two cations present in the asymmetric unit are rather similar, exhibiting insignificant differences (Fig. 1).
In 2010, Zhu and co-workers published a detailed conformational analysis of anthracycline antibiotics, including doxorubicin, based on previously published (Dox)-protein and (Dox)-DNA complexes as well as DFT calculations (Zhu et al., 2010). The analysis identified three important doxorubicin conformational domains: (1) the aromatic ring system, (2) the functional group at C9 and (3) at C7 relating to the aminal linkage: (1) The aromatic anthracycline ring system does not vary significantly in any of the DNA-bound (Dox) structures and in the structure in this study. A somewhat more pronounced variation is encountered in protein-bound-(Dox), such as the one in 4dx7 or 4mra (vide infra). Based on the B3LYP level of theory, Zhu et al. have proposed four types of stable conformational isomers, with type I tautomer -forming two hydrogen bonds between C5-O and C6-OH and between C12-O and C11-OH -being the preferred one. The crystal structure in the present report confirms this prediction.
(2) The C8 carbon can either be above or below the anthracycline planes; in this structure, C8 is above the plane and the C19-C20-C7-C8 torsion angles are 16.6 (6) and 17.5 (7) ( Table 1). This is in the expected range for an intercalating (Dox), but significantly deviates from that found in a protein-bound (Dox). The conformation at C9 is similar to that at C8, as C9 is almost coplanar the anthracycline plane [C20-C19-C10-C9 torsion angles are 18.9 (6) and 19.2 (6) ]. More dramatic variations between the conformations of C8 and C9 are observed in the protein-bound (Dox) (5mra), where their torsion angles are 47.75 and À49.70 , respectively. According to a study based on resonant molecular dynamic calculations and NMR experiments, the conformation with a C7-O7-C1*-C2* torsion angle of 142-143 was found to be biologically relevant (Barthwal et al., 2008;Agrawal et al., 2009). However, this seems to be only applicable to DNA-intercalated (Dox). Protein-bound (Dox) have a wider range of torsion angles, for example, 88.43 in sorcin-bound (Dox), to 150.82 in AcrB-bound (Dox). The (Dox) structure in the present study has torsion angles of 161.6 (5) and 162.6 (4) .
(3) The C7-connected daunosamine is the most flexible conformational entity in (Dox). The N3*-O7(C5) distance (2.74-8.50 Å ) determines the conformational diversity. In the Molecular structure of (DoxH) + showing the atom-labeling scheme. Only one of the molecules present in the asymmetric unit is shown, with displacement ellipsoids drawn at the 40% probability level. H atoms are not presented for clarity. Inset: Molecular overlay of the two crystallographically independent (DoxH) + moieties present in the asymmetric unit. The molecular overlay was performed using the function available within the Discovery Studio Visualizer Suite. The target chosen was one of the (Dox) units from the crystal structure, with the H atoms ignored. present structure, the corresponding distances are 7.947 (1) and 8.042 (2) Å , which are on the longer end of the spectrum. The presence of the nitrate ions between the two (Dox) fragments of the asymmetric unit influences this distance greatly.

Figure 3
Representation highlighting theinteractions between C and D rings of the aglycone moieties. Turquoise spheres indicate centroids.
following observations: (1) The most important functional groups are, understandably, the amino group of the daunosamine moiety and the hydroxyl group of the glycolic site; (2) (Dox) binds in the DNA minor groove and (3) while the crystal structure reported here is by and large quite similar to the one of (Dox) bound in DNA, significant conformational differences are prominent in comparison to protein-bound (Dox), mainly because of differences in hydrogen-bond donors present in proteins.
Binding of (Dox) to sorcin, a calcium-binding protein that causes multidrug resistance (MDR) in human tumors, impairs cell death. Sorcin is overexpressed in human tumors and MDR cancers. Two sites, designated as pocket 1 and pocket 2, were found to bind (Dox), which was modeled satisfactorily at pocket 1, but not at pocket 2. The molecular overlay in Fig. 4a shows the significant differences in the conformation: methoxy and the glycolic units are significantly rotated from their native state. On the contrary, DNA-bound (intercalated) (Dox) and (Dox) in this study do not differ significantly in their conformations, as shown in (Dox)-1p20 in Fig. 4 below.
In another study, three molecules of (Dox) were found to bind the AcrB protein (PDB accession 4dx7; Eicher et al., 2012). Although the conformational differences are not as stark as they were in sorcin, the rotation now being about the bond between glycol-O carbon and the A ring [(Dox)-4dx7 in Fig. 5]. In the neurotoxin BoNT/B-(Dox) complex, O13 and O14 of the aglycone interact with the toxin and is stacked between Trp1261 and His1240 (Eswaramoorthy et al., 2001). All the O and H atoms of the structure are hydrogen-bonded with various amino acid residues of the neurotoxin. The conformational changes in this complex are minimal, similar to DNA-bound (Dox).

Figure 4
Representative molecular overlays of doxorubicin from this study (purple) and the ones from the literature.
then dissolved in EtOH. After filtration, the filtrate was layered with Et 2 O. Red sheet-like crystals of (DoxH)NO 3 were obtained in two weeks.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 4. All H atoms were positioned geometrically and refined using a riding model: O-H = 0.82, N-H = 0.89 and C-H = 0.93-0.98 Å with U iso (H) = 1.2 or 1.5U eq (parent atom). The structure was refined as a twocomponent twin (matrix to transform one domain into the other: (1 0 0 0 1 0 1 0 1); BASF = 0.3072). Atoms marked with a star correspond to the pyranose ring, following a numbering convention previously described for (Dox) (Eswaramoorthy et al., 2001).  Data collection: APEX3 (Bruker, 2016); cell refinement: SAINT (Bruker, 2016); data reduction: SAINT (Bruker, 2016); program(s) used to solve structure: SHELXT (Sheldrick, 2015b); program(s) used to refine structure: SHELXL (Sheldrick, 2015a); molecular graphics: OLEX2 (Dolomanov et al., 2009) and CrystalMaker (Palmer, 2017); software used to prepare material for publication: OLEX2 (Dolomanov et al., 2009). 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. Refinement. Refined as a 2-component twin.