Myelography Iodinated Contrast Media. I ... - ACS Publications

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Myelography Iodinated Contrast Media. I. Unraveling the Atropisomerism Properties in Solution Luca Fontanive,†,▽ Nicola D’Amelio,*,‡,○ Attilio Cesàro,*,†,§ Amelia Gamini,∥ Letizia Tavagnacco,†,⊥ Marco Paolantoni,# John W. Brady,⊥ Alessandro Maiocchi,◆ and Fulvio Uggeri◆ †

Laboratory of Physical and Macromolecular Chemistry, Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy ‡ Bracco Imaging SpA-CRB Trieste, AREA Science Park, SS 14, Km 163,5, 34012 Basovizza (Trieste), Italy § Elettra Sincrotrone Trieste, Area Science Park, I-34149 Trieste, Italy ∥ Department of Life Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy ⊥ Department of Food Science, Cornell University, Ithaca, New York 14853, United States # Department of Chemistry, Biology, and Biotechnologies, University of Perugia, Via Elce di Sotto 8, I-06123 Perugia, Italy ◆ Centro Ricerche Bracco, Bracco Imaging SpA, Via Ribes 5, 10010 Colleretto Giacosa (TO), Italy S Supporting Information *

ABSTRACT: The present work reports a thorough conformational analysis of iodinated contrast media: iomeprol, iopamidol (the world’s most utilized contrast agent), and iopromide. Its main aim is the understanding of the complex structural features of these atropisomeric molecules, characterized by the presence of many conformers with hindered rotations, and of the role of atropisomerism in the physicochemical properties of their aqueous solutions. The problem was tackled by using an extensive analysis of 13C NMR data on the solutions of whole molecules and of simple precursors in addition to FT-IR investigation and molecular simulations. This analysis demonstrated that out of the many possible atropisomers, only a few are significantly populated, and their relative population is provided. The conformational analysis also indicated that the presence of a sterically hindered amidic bond, allowing a significant population of cis forms (E in iopromide and exo in iomeprol), may be the basis for an increased thermodynamic solubility of concentrated solutions of iomeprol. KEYWORDS: iodinated contrast media, atropisomerism, conformational analysis, NMR, IR, MM

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relatively low osmolality and viscosity.4−6 A high amount of iodine is required in a molecule providing an efficient radioopaque compound, as in these NICMs iodine constitutes about 50% of the weight. Furthermore, iodine covalently bound to organic molecules is more stable and nontoxic and has completely replaced the use of ionic iodide ions. However, although the presence of atropisomer chirality is of major concern in active drug research because it may affect properties such as pharmacokinetics and toxicity, the question may not be so relevant for NICMs. An extremely slow rate of atropisomer interconversion (of the order of years) means that the “formally chiral” compounds can be regarded as thermodynamically distinct species that can be isolated as

INTRODUCTION The phenomenon of atropisomerism has been very recently addressed as a largely overlooked alternative source of drug chirality with strong potential pharmaceutical implications.1 Atropisomerism has the distinct feature of creating molecular chirality as a result of hindered rotation about a bond axis and, therefore, has to be considered as a temperature dependent (noncovalent) molecular dissymmetry, with an energy barrier ranging from a few to almost a hundred kcal per mole. Iomeprol, iopamidol, and iopromide (Scheme 1) are injectable contrast agents belonging to the class of nonionic iodinated contrast media (NICM)2 characterized by an extensive atropisomer chirality. They are commonly used for diagnostic purposes in radiological analyses such as urography, angiography, and myelography.3 As the image quality depends on the iodine concentration, these drugs are commercialized as highly concentrated aqueous solutions, characterized, however, by © 2015 American Chemical Society

Received: Revised: Accepted: Published: 1939

November 11, 2014 May 3, 2015 May 6, 2015 May 6, 2015 DOI: 10.1021/mp5007486 Mol. Pharmaceutics 2015, 12, 1939−1950

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Molecular Pharmaceutics Scheme 1. Molecular Structures of Iopamidol, Iomeprol, and Iopromidea

a

Carbon atom numbering is given; for symmetrical side arms, the notation is referred to as “primed”.

molecular interactions potentially responsible for the soluble aggregates in concentrated solution. In the following sections, after a brief analysis of the atropisomer forms of NICMs and the assignment of NMR peaks of iomeprol, iopamidol, and iopromide, the presence of atropisomer forms is quantified by sorting out the NMR results for model molecules and, then, a molecular picture of the energetics of the molecular conformation is provided by MD simulations.

pure substances and stored as individual formulations for a suitable and/or required shelf life. However, NICMs are used exclusively as diagnostic molecules and lack any biological or therapeutic activity. Thus, a mixture of atropisomer forms can be used without danger at a concentration level above the solubility limit of each individual species. In this respect, the concentrated solution is not a metastable oversaturated solution because interconversion is prevented. A thorough physicochemical study of some properties of NICMs in aqueous solution and in solid state has been carried out in our laboratory in order to provide a clearer picture of their complex behavior and shed light on subtle differences occurring in analogous molecules. The present work aims at understanding the role of atropisomerism in the physicochemical properties of the aqueous solutions of NICMs, confining its attention mainly to three similar compounds reported in Scheme 1, namely, iopamidol, iomeprol, and iopromide. In particular, quantification of the atropisomer population and some relevant aspects of intramolecular interactions have been achieved in solutions of iopamidol, iomeprol, iopromide by 13C NMR and by molecular dynamics simulations. The task of assigning NMR peaks deriving from different conformers has been made possible by using simplified precursors. Indeed, the relevant result is that only a few of the many possible forms are found to be significant in solution. The whole set of conformational results provides an indication of the concentration dependence of the NMR experiments that are reported in an accompanying paper7 and an insight into the nature of the

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STRUCTURE AND HINDERED ROTATIONS IN NICMS The generic molecular structure of NICMs consists of a fully substituted aromatic ring (Figure 1) with three iodine atoms covalently linked in positions 2, 4, and 6. Positions 1, 3, and 5 are substituted with three hydrophilic side chains attached to the benzene ring by amidic bonds via the amino group (position 1) or the carbonyl group (positions 3 and 5). The main role of the side chains is to (i) increase the water solubility (relevant for image quality),3,8 (ii) increase their biocompatibility, and (iii) aid their clearance.5 Given the presence of two neighboring iodine atoms, the rotation about the bond of these positions can be greatly hindered (see rotations φ1, φ3, φ5, in Figure 1). Rotamers about φ1 can have high interconversion barriers (atropisomers) only in the methylated compounds (tertiary amide). Thus, the restricted rotations shown in Figure 1 give rise to the following independent atropisomeric forms (where capital letters SYN/ANTI refer to the orientation of the three groups, 1940

DOI: 10.1021/mp5007486 Mol. Pharmaceutics 2015, 12, 1939−1950

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Molecular Pharmaceutics

in their recent perspective,1 with a significant temperature dependence for their interconversion. Literature data on iopamidol and analogues in solution at room temperature have shown that the syn and anti conformers are populated in similar amounts and that their splitting into endo/exo populations exists only when the anilide moiety is a tertiary amide,9 but no reference was made in this particular work to SYN/ANTI forms. Furthermore, other studies on the physicochemical properties of NICMs do not deal with molecular conformation in aqueous solution, but rather refer to the question of the association equilibria as a function of concentration, studied by osmolality, calorimetry,11 and densitometry,10,12−14 in order to correlate the free energy, enthalpy, and volume properties of NICM solutions. In particular, a preliminary report on the differences in the thermodynamic properties of the compounds iopamidol, iomeprol, and iopromide in aqueous solution has already been reported.11

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RESULTS AND DISCUSSION 1. NMR Assignment of Iomeprol, Iopamidol, and Iopromide. Inspection of the molecular structures of iomeprol, iopamidol, and iopromide (shown in Scheme 1) reveals that many distinct conformations are accessible due to hindered rotations and to Z,E equilibria regarding the amidic bonds. In addition to dissymmetry due to atropisomer forms, at least one stereocenter is present in each molecule (one in position 11 on iopamidol and two in positions 9 and 9′ on iomeprol and iopromide). 1 H NMR spectra only partially reflect this complexity (see for example iomeprol in Figure 2, top) showing mainly one peak for each resonance (with the exception of methyl 13 and methylene 12 in iomeprol, due to exo/endo conformations in the anilido branch). On the other hand, 13C spectra (Figure 2, bottom) are certainly more informative due to enhanced resolution but are also more difficult to interpret. Nonetheless, a full assignment of the main forms was carried out according to the procedure described in the following section. 2. Assignment of Main Atropisomeric Forms. The complexity of 13C NMR spectra, such as the one reported in Figure 2, is due to the fact that virtually all the different conformations are visible but difficult to distinguish. For this reason, assignment was accomplished by comparing the 13C NMR spectra of NICMs with those of molecules with a similar structure but lower complexity. The molecular species analyzed in this work are mainly characterized by the absence of substituents in one or more arms, as shown in Scheme 2. (The 13 C NMR spectra of the compounds listed in Scheme 2 are reported in Figures S1−S7 of the Supporting Information, and the assignments of the three NICMs are in Tables S1−S3.) Figure 3 shows an enlargement of the spectral region of aromatic carbon atoms in positions 3 and 5, a region that is sensitive to conformational changes but is also well resolved (similar effects are displayed by C5 and also by C1, but are not discussed here). Compound A (named Iodoftal) was chosen as the reference compound for its greater simplicity (Figure 3), because atropisomeric forms are absent in this molecule. Atropisomeric Forms: Syn/Anti and E/Z. A comparison of the 13C spectrum of Iodoftal (compound A) with that of compound C is able to unravel the presence of atropisomeric syn/anti and E/Z forms in the disubstituted molecule C. In particular, the singlet belonging to carbons 3 and 5 is split into two signals with similar intensity (red arrow in Figure 3C), with an additional splitting generating two extra peaks with very

Figure 1. Generic molecular structure of NICMs with the identification of the hindered rotations φ1, ψ1, φ3, φ5, (top). The nomenclature identifying different atropisomers (bottom) is specified by the vertical arrows indicating the orientation of carbonyl groups (top, rotations φ3 and φ5) and of the anilido group (middle, rotation φ1) with respect to the aromatic plane. The arrows parallel to the ring (bottom, rotation ψ1) indicate whether the carbonyl moiety of the anilido group points toward the interior or the exterior of the ring.

while small syn/anti is used for the orientation of the two carbonyl groups, when the N-methyl substitution is absent): • syn/anti: generated by the two possible relative orientations of the two carbonyls in positions 3 and 5 with respect to the aromatic ring plane; • SYN/ANTI: generated by the two possible orientations of the anilido carbonyl with respect to the other two carbonyl groups when in syn form (this atropisomerism is not defined in the anti conformer); • endo/exo: generated by the cis−trans forms in the anilido moiety (endo with the carbonyl group pointing inward and exo pointing outward); • an additional Z/E interconversion at the level of the amidic bonds may be present in the branches in positions 3 and 5. The atropisomerism phenomenon has already been investigated by NMR for iopamidol9 and by HPLC for iomeprol,10 revealing the presence of distinct rotamers with hampered interconversion rating their energetic barriers as high as 20 kcal/mol.3,10 These molecules, therefore, fall in the lower limit of group 2 within the classification used by LaPlante et al. 1941


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Figure 2. 1H (top) and 13C (bottom) NMR spectra of 1 M iomeprol in H2O (10% D2O) at 600 MHz. The concentration reproduces the standard preparation of the X-ray contrast agent.

Scheme 2. Molecular Species Studied in This Worka

a

Compounds A, B, C, and D are precursors of compounds E, F, G, which are iopamidol, iomeprol, and iopromide, respectively.

weak intensity (the right flanking each higher peak). The first splitting was indirectly attributed to the syn/anti forms, for to the following reasons: (i) E/Z conformers are rarely energetically equivalent, thus unlikely to show peaks with similar intensities; (ii) IR spectra performed on an iopamidol solution (a close analogue) indicate a strong prevalence of the Z conformation with respect to the E one. In particular, Figure 4a,b show IR profiles of iopamidol solutions obtained at different concentrations in the 1200− 1700 cm−1 and 2600−3800 cm−1 spectral regions, where signals typically attributed to amide vibrations (i.e., Amide I, II, III) can be observed.15,16 The Amide I band, which is mainly due to a CO stretching vibration,17,18 is found at ∼1630 cm−1 while the bands at ∼1510 cm−1 and ∼1560 cm−1 can be attributed to the Amide II modes (combination of C−N stretching and N−H bending17,18) of distinct secondary amide groups in the trans conformation. In fact, cis secondary amides should present intense signals at around 1440−1450 cm−1, arising from basically uncoupled N−H deformations, while no Amide II modes above 1500 cm−1 are expected for these forms.15,16 It

is interesting to note that the presence of two distinct bands assigned to the nonequivalent amide groups (namely, the anilido moiety and the two equivalent amide groups in positions 3 and 5) is in agreement with the NMR study of Longo et al.,19 who reported a different rate of proton exchange for the two types of N−H groups. Coupling between a C−N stretching and an N−H bending in trans amides is also at the origin of the characteristic Amide III vibration commonly found at 1200−1310 cm−115,16 and assigned to the band at ∼1281 cm−1 in the spectrum of Figure 4a. Its counterpart for cis amides (mostly a C−N stretching) is normally found at higher frequencies, in the 1310−1350 cm−1 range.16 On the other hand, the band at ∼1355 cm−1 (Figure 4a) can be attributed mainly to the aryl C−N stretching vibration and the overall interpretation is confirmed by the presence of a band at ∼3060 cm−1 in Figure 4b assigned to the overtone of the Amide II band of trans secondary amides.16 Figure 4b also shows that, in spite of the low signal-to-noise ratio above ca. 3100 cm−1 connected with the strong water contribution in this region, a broad feature 1942


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the Internet at http://pubs.acs.org.) An analysis of the temperature behavior, as described in Supporting Information, provides the two values for the direct and reverse first order rate constants, k1 and k−1, and for the respective free energies of activation: k1 = 9.5s−1; ΔG‡ A = 18.1 kcal/mol k1 = 4.2s−1; ΔG‡ B = 18.6 kcal/mol

suggesting that, although hampered (see also section 4), the interconversion also occurs at room temperature. Other Atropisomeric Forms: Endo/Exo and SYN/ANTI. The existence of exo/endo and SYN/ANTI forms generates two further splittings. Three groups of signals relating to carbons C1, C3, and C5 are observed in the spectrum of compound D in Figure S4, where C3 and C5 are no longer equivalent. To investigate the endo/exo equilibrium, the anisotropy of the aromatic ring and of carbonyl moiety of anilido branch were considered. The aromatic ring is the static part of the molecule and of its shielding cloud too (see Figure S11). The rotation of the amidic bond of anilido branch results in mobility of the shielding cloud of the carbonyl moiety. Exploiting these anisotropic properties on protons of C12 the endo/exo equilibrium was studied. The larger splitting leading to peaks with different intensities (Figure 3D showing C3) is due to the exo/endo forms. The assignment was verified in two ways: (i) A splitting with similar population is also found in the 1H spectrum and well evident for signals of the methyl in position 13. A 2D-1H,13C HOESY heteronuclear NOE cross peak between the methyl protons in position 13 and the methylene carbon in position 12 was found only in the minor form, which is compatible with the endo conformer (see Figure S9 and S10 for compound F, iomeprol). (ii) The value of the chemical shift of methylene 12 of compound F (iomeprol) is expected to be significantly different in the exo and endo conformations. In the exo form, these protons are placed on top of the shielding cloud of the aromatic ring, while in the endo form, they point in the opposite direction (Figure S11). Accordingly, the two forms are found at very different chemical shifts (exo at 3.82 and endo at 4.53 ppm), whose intensity is larger for the exo form than for endo. Figure S11 also shows how the anisotropy of the carbonyl cannot significantly affect the chemical shift of protons in position 12 but does influence the shifts of aromatic carbons. In particular, as expected, C1 is deshielded, while C3 and C5 are shielded in the less-populated endo form. Figure 3B shows the 13C spectrum of compound B in which only the exo/endo forms are present. The figure shows only one peak for each carbon, demonstrating that either only one form is favored or the two forms are in fast exchange on the NMR time scale. The remaining splitting in Figure 3F is consequently attributed to the SYN/ANTI equilibrium, generating forms with similar intensities. Indeed, an insight into the stability of SYN/ANTI forms is provided by the comparison of the 13C spectrum of compound A with that of compound D and that of iomeprol (compound F). There are two protons of C8 in the iomeprol side chain attached to C3 (or C5). These protons are present in two similarly populated forms, each displaying an NOE of the opposite anilido branch either with the methyl 13, as expected

Figure 3. Spectra of aromatic carbon atoms in position 3 for compounds coded as in Scheme 2 (C = 0.3 M). Splitting due to atropisomeric forms is shown with colored arrows (code: syn/anti red ↔; SYN/ANTI ↔; endo/exo blue ↔; E/Z green ↔). Compounds A, B, C, and D are precursors; compounds E, F, and G are iopamidol, iomeprol, and iopromide, respectively.

at ca. 3210−3250 cm−1 is clearly discernible. In line with the previous discussion, this contribution is assigned to the N−H stretching mode of trans amide groups.16 In terms of the overall band shape and relative intensity, negligible spectral variations are observed at different concentrations in Figure 4a, and only slight frequency shifts of Amide peak positions were detected. Any effect arising from the modulations of solute−solute and solute−solvent interactions, related to any possible aggregation, would not influence the proposed conformational picture. Thus, from the spectrum of compound C reported in Figure 3 the conclusion can be drawn that the syn and anti atropisomers are energetically similar and therefore exclude significant interaction between the branches that could favor one form with respect to the other. Moreover, the further doubling observed in each peak is assigned to the presence of E/Z forms with the Z conformer largely predominating over the E one. Before concluding this part of assigning the atropisomeric forms syn/anti, it is worth mentioning an additional result regarding the temperature dependence of the C3 (or C5) peak position of iopamidol (compound E in Figure 3). At room temperature, the rotation around the C−CO bond is slow and the signals of the two nonequivalent groups (syn/anti) are sharp lines at 149.1 and 149.2 ppm, corresponding to a frequency difference of Δν = 9.46 Hz at 600 MHz. When the temperature is increased, the two lines broaden and merge into a single broad line at 70 ± 5 °C. Above this coalescence temperature, the single line gets sharper and sharper until the natural line width is reached. The process is fully reversible upon cooling the solution back to room temperature. (The spectra are reported in Figure S8, available free of charge via 1943


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Figure 4. Infrared spectra of iopamidol aqueous solutions at three concentrations in the region of fundamental amide modes (a) and in the high wavenumber region (b).

in the ANTI form, or the methylene 12, as expected in the SYN form. A confirmation of the assignment is obtained by analyzing the NOE connection in the Figures S9a,S9b, in which the protons of C8 are connected with protons of C12 or C13. Figure S10 shows the interaction between these protons, while the similar intensity of the peaks assigned to the SYN and ANTI forms suggests poor interaction between the branches. 3. Population of Atropisomeric Forms of Iopamidol, Iomeprol, and Iopromide in Solution. Iopamidol. The present study on precursor B shows that the secondary amide is predominantly in the trans (endo) conformation, as are the remaining amides (see compound C). Moreover, Bradamante and Vittadini demonstrated that there is “free rotation” along the C1−N bond.9 In this respect, iopamidol displays only the syn/anti atropisomerism of compound C; therefore, two peaks are expected for each aromatic carbon due to the presence of the syn/anti forms. Furthermore, the spectrum in Figure 3 shows that in iopamidol one form is preferred (about 70%

instead of the 50% found in compound C). This effect is probably due to the different geometries of the branches with respect to compound C. Steric hindrance considerations would suggest a larger population for the anti form, as confirmed by preliminary quanto-mechanical energy calculations carried out to simulate Raman spectra (manuscript in preparation). This assignment has been reported in Table 1. A somewhat different Table 1. Relative Populations of the Conformers in Concentrated Solution of Iopamidol, Iomeprol, and Iopromide (ca. 0.3 M)a atropisomer forms

SYN/ANTI

syn/anti

endo/exo

E/Z

not present 50:50 not present

31:69 67:33 38:62a

not present 23:77 not present

≈ 0:100 ≈ 0:100 27:73

NICM iopamidol iomeprol iopromide a

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Tentative values based on assignment not confirmed. DOI: 10.1021/mp5007486 Mol. Pharmaceutics 2015, 12, 1939−1950

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depicted in Figure 1, the hindered rotations, φ1, ψ1, φ3, and φ5, identify the presence of atropisomer forms through the orientation, with respect to the aromatic plane, of carbonyl groups linked to C3 and C5 (rotations φ3 and φ5, respectively) and of the anilido group linked to C1 (rotation φ1). The populations calculated by NMR and reported in Table 1 refer to the actual noninterconvertible conformers found in commercial solutions (1 M) and do not provide any information about the molecular freedom or the energetic barriers between rotamer forms. In addition, some reported populations may reflect an “equilibrium” reached at higher than ambient temperatures (due to their thermal history during the industrial process). It should be clear that while the kinetics of the interconversion depends on the energetic barriers, the putative population (in the absence of the barriers) is solely a function of the conformer energy, according to Boltzmann statistics. Thus, the investigation of the conformational energetics of transitions primarily refers not only to the rotations φ1, φ3, and φ5 but also to the conformational freedom of the hydrophilic side chains. As a corollary, because the hydrophilic character of iomeprol appears to be different from that of iopamidol and iopromide,11 the hydration properties of the three molecules will be treated in more detail elsewhere.7 Hindered Conformation at the Ring-Linked Bonds. Conventional MD simulations are usually limited to time scales in the 10−100 ns range and thus cannot achieve equilibrium between states separated by barriers on the order of 20 kcal/mol. Therefore, it is good practice to carry out simulations over the conformational wells by sampling all the accessible regions. This problem was tackled by exploring first the energetic profile for rotations φ3 or φ5 about the C−C bond through a preliminary molecular mechanics calculation on the energetic barriers arising from the substituents on neighboring aromatic carbons (i.e., the iodine atoms; Figure 5). Two large energetic

conclusion was reached by Bradamante and Vittadini,9 who assigned an equal probability of cis/trans (E/Z) and syn/anti forms. This interpretation seems to be unlikely in view of the different energies usually ascribed to cis/trans conformers in the amidic bonds.20,21 Iomeprol. This molecule is rather more complex than iopamidol, mixing the atropisomer forms of compound C (syn/ anti and E/Z) with those of compound D (SYN/ANTI and endo/exo). However, for the reasons reported above, only the E form is present in the branches in positions 3 and 5 of iomeprol, while endo/exo splitting (Figure 3) is very evident in the spectrum (and similar to that observed in compound D). The relative populations of the endo and exo forms is about 1:3. Having solved the complexity due to the amidic bonds, further subdivisions of the peaks are expected due to the SYN/ ANTI and syn/anti forms. In particular, three main conformers should be present: SYNsyn, ANTIsyn and anti (the hypothetical SYNanti and ANTIanti forms are equivalent molecules). Because the SIN/ANTI equilibrium only affects the syn form, two of the three peaks observed in Figure 3F (at 150.2 ppm) are likely to belong to this form. The sum of the areas of two peaks (syn) in comparison with the remaining (anti), reveals that the syn is more populated, independently of the assignment of two of the three peaks to a specific atropisomer. If this tentative interpretation is correct, the prevalence of syn form with respect to the anti, constitute a major difference with Iopamidol. The relative population of atropisomer is more difficult to interpret in the less populated endo form (peaks at 149.7 ppm), although the number of peaks is the same. Iopromide. Iopromide is definitely the most complex molecule in terms of the NMR spectrum, as the three different branches create many different signals, each complicated by the presence of many conformations. Among the three different amidic bonds, only one (in the branch linked to C3) is expected to display both E and Z forms in sizable populations. Due to their similarity with the anilido branch of iopamidol, the syn/anti forms (but not the SYN/ANTI) are expected to be present. The aromatic region of the 13C spectrum (Figure 3G) clearly shows a splitting in the region of C3, most likely due to the E/Z forms introduced by the methylated amide. The assignment of the forms was based on the 1H shift of the methyl in position 14, which in the E form lies in the shielding cone of the aromatic ring and appears at higher chemical shifts. A ratio of about 1:3 has been estimated for the E and Z conformers, respectively. A further evident splitting is displayed in the 1H spectrum for the E form, which might be due to the remaining syn/anti equilibrium. In this case, however, a close inspection of the spectra (both 1H and 13C) reveals many other splittings making the complexity virtually unsolvable. The effect is probably due to the reduced symmetry of the molecule, which is able to emphasize the effect of the stereochemistry of the two chiral branches that are present in racemic forms. Summary of the Relative Population of Atropisomers of Iopamidol, Iomeprol, and Iopromide. The above-reported results and interpretations have provided a reasonable estimate of the relative populations of the conformers of iomeprol, iopamidol, and iopromide, as reported in Table 1. Because the accuracy of the values depends on the concentration of the NICMs, the data refer to populations calculated for 0.3 M solutions. 4. Side Chain Conformation by Computer Simulations. According to the molecular structure of the NICMs

Figure 5. Energetic profile for the rotation about the dihedral angle C(x − 1)−C(x)−C(O)−N C−C (rotations φ3 or φ5 in Figure 1) through molecular mechanics calculation on iopamidol; here the energetic barriers arise from the neighboring iodine atoms.

barriers are located at about 170° and 350°, because the angle definition C(x − 1)−C(x)−C(O)−N implies that both the positions at about 0° and 180° are strongly prohibited because of the side iodine atoms. The energetic barriers for the complete rotation are only slightly less than 20 kcal/mol (ca. 18.5 and 17 kcal/mol for the clockwise and counterclockwise 1945


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Figure 6. Distributions of dihedral angles φ1, φ3, and φ5 of iomeprol side chains in anti (either C3 or C5 group is on the same side of the C1 group).

linked to C5. The interactions between the former groups at C1 and C3 makes the conformation state, with φ3 at about −120°, slightly less favored than that at −60°, while no effect is seen on the population of the other arm linked to C5. It is noteworthy to mention, however, that the overall probability for C3 (or C5) above or below the aromatic ring is 50:50, and therefore, the global effect maintains an overall symmetry of the system with respect to the side arm orientations. Conformation of Iomeprol Side Arm. Figure 7 shows a molecular representation of the side arm of iomeprol with the

rotations, respectively), in reasonable agreement with the coalescence temperature findings (see section 2) and of other literature values.9,10 Assuming this value would imply a transformation rate (interconversion between atropisomer forms) of the order of seconds at ambient temperature, a time that is too long to be observed in the statistics of conventional MD simulations. On the other hand, the details of the two minima at about 50° and 250° should be taken as approximate and only indicative for the subsequent analysis. The actual population of the conformational angles in the accessible range between the two maxima at 170° and 350° has been evaluated by MD simulation. An initial simulation was carried out by using the atomic coordinates of the crystalline anhydrous iopamidol.22 Indeed, this molecule is in the syn form for the two symmetric arms, showing a conformation different from that (anti) recently found in the pentahydrate-iopamidol crystal structure.23 Notwithstanding this difference, the starting conformation of iopamidol can explore all the rotations of the anilido group linked to C1 (rotation φ1), while the freedom of the other two side arms is clearly hampered and limited to regions designated here as g+ and g−. A second simulation was made on the iomeprol molecule. Here the rotation φ1 is hindered by the methyl substitution, and the anilido group can be either SYN or ANTI, only when the other two equal arms are syn. However, when the symmetric arms are in the anti conformation (and there are two mirror images with respect to the position of C3, C5, and C1), the position of the anilido arm discriminates between the two arms C3 and C5, because only one the these arms is on the same side of the aromatic ring. This breaking of degeneracy is clearly shown in the distribution of the accessible dihedral angles φ3 and φ5 reported in Figure 6. Each probability curve for rotations φ3 and φ5 presents two maxima located within the g+ and g− regions, reflecting that the carbonyl group is not confined in the position orthogonal to the aromatic ring but flips between two tilted positions, with angle φ3 at about −60° and −120° and φ5 at about +60° and +120°, respectively (neither of the positions at −90° and +90° are favored). The anilido group in this chosen conformation is on the same side of the arm linked to C3 and on the opposite side of the arm

Figure 7. Simple model used for the side arm of iomeprol: definition is given for the rotational angles ω1-ω6 in a conformation close to a putative minimum energy (carbon C at the left end is either C3 or C5 of the ring).

definition of the dihedral angles ω1, ω2, ω3, ω4, ω5, ω6, in a conformation close to a putative minimum energy. The molecular structure was simplified by the substitution of a methyl group (label C) at the left end, instead of the ring with the other two side chains. Molecular Mechanics calculations were carried out to explore the conformational freedom of the side arm (the S-enantiomeric form was simulated here). In the first approach, the conformational energy space was explored using the CHARMM force field “in vacuo”. Sections of the energy contours at fixed dihedral angle ω2 were calculated for the model side chain with a terminal methyl group on the carbonyl, instead of the aromatic ring, and keeping the angle ω1 fixed at 180°. The partial Ramachandran maps for six 1946


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Figure 8. Ramachandran maps of model molecule used for the side arm of iomeprol in the planes ω3-ω4 for six sections of ω2 (0, 60, 120, 180, 240 and 300°). Energy contour lines are shown from black (0 kcal/mol) to white (>5 kcal/mol) in steps of 1 kcal/mol.

Figure 9. Three side arm conformations of iomeprol selected among those with the lowest energy.

orientation in the position orthogonal to the CO−NH plane, which is at about +90° or +270°. Some of these angle rotations make the H-bond possible between the carbonyl oxygen (C7) and the hydroxyl group on C9. Angle ω3 preferentially remains in the trans conformation (ca. 180°) with some minor presence in the gauche positions (either +60° or +300°). Finally, angle ω4 occupies the trans (180°) and gauche (+60) conformers with almost equal probability, as this bond would offer higher energy for the g− with two close interactions between the primary hydroxyl group (terminal) and C8 and O9. In conclusion, the side chain preferentially elongates away, with an overall geometry that is essentially trans planar (zigzag) and orthogonal to the aromatic ring for half of the arm, but still statistically exploring a large domain space. The second half of the arm is still trans zigzag shaped, but its orientation is tilted by 90°. Thus, in aqueous solutions, the long side arms fluctuate between conformational states that ensure flexibility and hydration at the same time, with a geometry that is at least

sections are plotted in Figure 8a−f, showing the energy contour lines in the planes ω3-ω4 with the existence of quite a few well-defined minima, the most relevant ones in the range ω2 = 180−240−300°, ω3 = 60 or −60°, and ω4 = 60° or 180° (see the bottom panels) Similar conclusions were drawn from the analysis of the whole iomeprol molecule using the AMBER force field and charge set using the semiempirical method CNDO. The six dihedral angles, ω1 to ω6, were randomly varied to generate 10 000 conformations, and the energy of each conformation was evaluated. Indeed, many conformers lie in the energy range within 1 kcal/mol above the minimum energy conformation, most of them differing mainly in the orientation of the two dihedral angles ω5 and ω6 of the terminal hydroxyl groups. Figure 9 shows some side arm conformation of iomeprol selected among those with the lowest energy values. Among the main angles, ω1 oscillates around the value of 180° (large oscillations are forbidden), while ω2 has a preferential 1947


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sterically hindered amidic bond, allowing a significant population of cis forms (E in iopromide and exo in iomeprol), may be the basis of the known higher stability of concentrated solutions of iomeprol. As a perspective, the determination of the conformational isomers and their populations can be used to understand the self-association and the formation of soluble aggregates in concentrated solutions (at the concentration used for clinical purposes), with their potential evolution into structured aggregates that may nucleate the crystalline forms. These aspects are analyzed in the accompanying paper and in other works in preparation. As a final comment, the authors are indebted to an anonymous reviewer of the present paper for providing insightful comments and suggesting direction for useful corrections and ideas for future work. Without the supportive work of the anonymous reviewers, this paper would not have been properly presented.

partially maintained in the crystalline state, providing the intermolecular hydrogen bonding pattern.22−24 5. Is Atropisomerism the Efficiency Key for Concentrated NICMs? Although an attempt has been made to rationalize the complexity step-by-step through a factorization of the various contributions, the picture emerging for the solution properties of the NICMs is indeed still quite complex. First of all, the different time scales of equilibration for the three molecules considered here (namely, iopamidol, iomeprol, and iopromide) is not a differentiating factor for the NMR measurements. This means that NMR experiments see all the different conformations arising from rotations φ1, φ3, and φ5 separately (see Figure 3), except for iopamidol, which lacks any distinction for its orientation about φ1. However, some differences in the energy barriers make the time scale change from one species to another (energy varies from 18 to 40 kcal/mol). A direct dynamic NMR experimental evaluation of these energy barriers in all relevant compounds needs to be performed, as the computational studies suffer from the use of approximations. Although the aim of this work has been the study of the accessible conformation in solution, no special attention has been given to the influence of concentration. Indeed, the effect of the concentration on both the conformer populations and on the dynamics of transformation may be a relevant issue. Some visible effects have been detected in 13C spectra performed as a function of concentration, most probably due to intermolecular interactions in the aggregates, and reported in a related paper.7 Of even greater interest is the comparison between the solution conformation and the molecular conformation in the solid crystalline polymorphs. This aspect is relevant from many viewpoints: it could clarify whether the packing symmetry in the crystals is able to select some specific conformations and how the population in solution is modified upon crystallization (i.e., the nature of the competition between crystallization kinetics and atropisomerism kinetics). Experimental results on the crystal structures of pentahydrate iopamidol23 and of anhydrous iomeprol24 show that only the anti form is present in both cases, while the syn form occurs in the anhydrous iopamidol polymorph.22 In addition, the iomeprol crystal presents only the exo conformer, which was found to be more abundant in our solution study. This means that the concentration of these species may be the determining thermodynamic factor for their solubility curve. Still, the solution conformational variety with a moderate interconversion rate should be considered the relevant key to ensuring stability of apparently oversaturated solutions, which may appear as a thermodynamic mixture of enantiomeric compounds.

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MATERIALS AND METHODS NMR Experiments. All NMR measurements were performed at 300 K on a Bruker Avance III Ultra Shield Plus 600 MHz spectrometer provided with a two-channel BBI probe. Assignment of NMICM and precursors was accomplished via a series of 1D and 2D spectra, namely: 2D-1H,13CHSQC, 2D- 1 H, 13 C-TOCSY-HSQC, 2D- 1 H, 13 C-HMBC, 2D-1H,1H−COSY, and 2D-1H,1H-NOESY. NOESY spectra of the mixture of iopamidol and iomeprol was recorded with a mixing time of 300 ms. The NMR assignment of the main species in iopamidol, iomeprol, and iopromide is reported in Tables S1−S3 of the Supporting Information. The assignment was based on spectra (as shown in Figure 3) at a concentration of NICMs of 0.3 M, to eliminate the complexities introduced by aggregation (as discussed in the accompanying paper7). Unless otherwise stated, all other experimental data and their discussion refer to spectra recorded at concentrations of about 1 M. Iomeprol and iopamidol were provided by Bracco Imaging SpA either as powders or as aqueous solutions, while iopromide was purchased as Ultravist by Schering at concentrations of 400 mgI/mL and 370 mgI/mL. Solutions at lower concentrations were prepared by dilution. All samples were prepared in D2O or in water with 10% D2O. TSP (sodium 3-(trimethylsilyl)propionate-2,2,3,3,d4) was used as the internal standard for the chemical shift reference: its concentration in every sample in the test tube was 1:100 of the NICMs in each solution. Several precursors of iomeprol were provided by Bracco Imaging SpA as anhydrous powders and listed in Scheme 2 (A, B, C, and D). Compounds A, B, and C were solubilized by adjusting the pH. FTIR Experiments. Infrared experiments in Attenuated Total Reflectance (ATR) configuration were performed using an FTIR Bruker ISF28 spectrometer equipped with a Deuterium Triglycine Sulfate (DTGS) detector. A Specac Gateway multireflection ATR system with a 45° horizontal ZnSe crystal was employed. Each spectrum was the average of 50 scans recorded with a resolution of 2 cm−1. ATR spectra of iopamidol aqueous solutions were recorded as a function of concentration at room temperature. A concentrated iopamidol sample (0.9 M) was provided by Bracco Imaging SpA and diluted solutions were prepared from the original formulation by using deionized water. Measured ATR spectra were converted to “absorbance” ones by considering the simple correction implemented in the Opus 5.5 (Bruker Optics)

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CONCLUSIONS A conformational analysis of iodinated contrast media iomeprol, iopamidol, and iopromide is a prerequisite for understanding the complexity of the structural features of these molecules. However, the presence of many possible conformers, generated by rotations hindered to a greater or lesser degree, has prevented a straightforward interpretation of the experimental data. This problem was tackled using an extensive analysis of NMR data on the whole molecules and simple precursors in addition to exploratory molecular simulations. This work has demonstrated that out of the many possible atropisomers, only few are significantly populated and quantification of the population fractions has been provided. The conformational analysis also indicated that the presence of a 1948


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Shield Plus, and CBM (Consorzio per il Centro di Biomedicina Molecolare S.c.r.l., AREA Science Park, Basovizza, Trieste, Italy) for hosting the instrumentation are also acknowledged.

software, accounting for the wavelength dependence of the penetration depth. Depicted spectra were also corrected by subtracting the spectrum of pure water from that of the solutions after normalization to the water combination band at ca. 2100 cm−1. Computational Procedures. Molecular dynamics (MD) simulations were performed on water/iopamidol solutions at 0.043, 0.5, and 1 m in the isothermal−isobaric (N,P,T) ensemble, where the number of particles N, the pressure P, and the temperature T are constant. Iopamidol and water molecules were randomly oriented and uniformly placed in a cubic simulation box according to standard procedures. The CHARMM22 force field25 was used to simulate iopamidol (and iomeprol) molecules, and water molecules were represented by the modified TIP3P model.26,27 All simulations were performed using the CHARMM program.28 The length of covalent bonds involving hydrogen atoms was kept fixed using the SHAKE algorithm,29 and a time step of 1 fs was used to integrate the Newton equations of motion. Electrostatic interactions were treated using the Ewald summation method30 with a real space cutoff of 11.5 Å, κ = 0.4, and a Kmax2 value of 27, while van der Waals interactions were smoothly truncated on an atom-by-atom basis using switching functions from 10.5 to 11.5 Å. Temperatures were maintained constant using a Hoover thermostat31 with a coupling constant of 1000 kcal·mol−1·ps−2 and pressures by a pressure piston32 of a mass of 1000 amu. The simulations were run for 4 ns and configurations were saved every 0.2 ps.

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(1) (a) LaPlante, S. R.; Fader, L. D.; Fandrick, K. R.; Fandrick, D. R.; Hucke, O.; Kemper, R.; Miller, S. P. F.; Edwards, P. J. Assessing atropisomer axial chirality in drug discovery and development. J. Med. Chem. 2011, 54, 7005−7022. (b) Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R. The challenge of atropisomerism in drug discovery. Angew. Chem. 2009, 48, 6398−6401. (c) LaPlante, S. R.; Edwards, P. J.; Fader, L. D.; Jakalian, A.; Hucke, O. Revealing atropisomer axial chirality in drug discovery. ChemMedChem 2011, 6 (3), 505−513. (d) Clayden, J. Atropisomers and near-atropisomers: achieving stereoselectivity by exploiting the conformational preferences of aromatic amides. Chem. Commun. 2004, 2, 127−135. (2) Thomsen, H. S.; Morcos, S. K. Radiographic contrast media. BJU Int. 2000, 86.s1, 1−10. (3) Krause, W.; Schneider, P. W. Chemistry of X-Ray Contrast Agents. In Contrast Agents II: Optical, Ultrasound, X-Ray and Radiopharmaceutical Imaging; Krause, W., Ed.; Springer: Berlin, 2002; pp 107−150. (4) Bonnemain, B.; Meyer, D.; Schaefer, M.; Dugast-Zrihen, M.; Legreneur, S.; Doucet, D. New iodinated, low-osmolar contrast media. A revised concept of hydrophilicity. Invest Radiol. 1990, 25 (Suppl 1), S104−S106. (5) Krasuski, R. A.; Sketch, M. H., Jr; Harrison, J. K. Contrast Agents for Cardiac Angiography: Osmolality and Contrast Complications. Curr. Interv. Cardiol. Rep. 2000, 2, 258−266. (6) Yamauchi, T.; Furui, S.; Harasawa, A.; Ishimura, M.; Imai, T.; Hayashi, T. Optimum iodine concentration of contrast material through microcatheters: hydrodynamic analysis of experimental results. Phys. Med. Biol. 2002, 47, 2511−2523. (7) Gamini, A.; D’Amelio, N.; Fontanive, L.; Brady, J. W.; Cesàro, A.; Maiocchi, A.; Uggeri, F.; Myelography iodinated contrast media. II. Understanding the molecular mechanisms of aggregation (to be submitted). (8) Anelli, P. L.; Brocchetta, M.; Maffezzoni, C.; Paoli, P.; Rossi, P.; Uggeri, F.; Visigalli, M. Syntheses and X-ray crystal structures of derivatives of 2,2′,4,4′,6,6′-hexaiodobiphenyl. J. Chem. Soc., Perkin Trans. 1 2001, 1175−1181. (9) Bradamante, S.; Vittadini, G. Non-Ionic Contrast Agents: Detection of Ambient Temperature Isomers of Congested Hexasubstituted Benzenes with Multipositional Hindered Rotations. Magn. Reson. Chem. 1987, 25, 283−292. (10) Gallotti, A.; Uggeri, F.; Favilla, A.; Cabrini, M.; de Haën, C. The chemistry of iomeprol and physico-chemical properties of its aqueous solutions and pharmaceutical formulations. Eur. J. Radiol. 1994, 18 (Suppl 1), S1−S12. (11) Giannini, G.; Cuppo, F.; Fontanive, L.; D’Amelio, N.; Cesàro, A.; Maiocchi, A.; Uggeri, F. Interactions in iodinated contrast media solutions: Part 1. A thermodynamic study. J. Thermal Anal. Calorim. 2011, 103, 89−94. (12) Felder, E.; Grandi, M.; Pitrè, D.; Vittadini, G. Iopamidol. In Analytical profiles of drug substances. Florey, K., Ed.; Academic Press: San Diego, 1988; pp 115−154. (13) Miklauts, H.; Fichte, K.; Wegscheider, K. Osmolality of nonionic contrast media. Fortschr Geb Rontgenstrahlen Nuklearmed Erganzungsbd 1989, 128, 16−20. (14) Dooley, M.; Jarvis, B. Iomeprol: a review of its use as a contrast medium. Drugs 2000, 59, 1169−1186. (15) Dollish, F. G; Fately, W. G.; Bentley, F. F. In Characteristic Raman frequencies of organic compounds; John Wiley & Sons: New York, 1974. (16) Socrates, G. Infrared and Raman Characteristic Group Frequencies: Tables and Charts., 3rd ed., John Wiley & Sons: New York, 2004.

ASSOCIATED CONTENT

S Supporting Information *

13 C NMR spectra of compounds A−G with assignments of 1H and 13C chemical shifts of compounds E−G, and bidimensional spectra of compound F; data for the evaluation of energetic value of the rotational barriers of compound E. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/mp5007486.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Phone:+39-040-5583684. Fax: +39-040-5583691. *Phone: +34-91-2998878. Fax: +34-91-5854506. Present Addresses ▽

(L.F.) DUCSM, Piazza Ospedale 1 34100 Trieste. (N.D’A.) National Biotechnology Center/IMDEA Nanoscience, Calle Faraday 9, Cantoblanco, 28049 Madrid, Spain. ○

Notes

The authors declare the following competing financial interest(s): Alessandro Maiocchi and Fulvio Uggeri are employees of Bracco Imaging SpA, Italy. This paper is part of a series on Iodinated Contrast Media; see also refs 7, 11, 23, and 24. The work described has been taken in part from the Ph.D. thesis of L.F. at the University of Trieste, School of Nanotechnology.

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ACKNOWLEDGMENTS Research carried out in the framework of a collaboration between the University of Trieste with the Bracco Research Center under the Project “Physical Chemistry of Iodinated Contrast Media”. The authors acknowledge A. Lerbrert and P. Mason for running preliminary MD simulations and G. Giannini for fruitful discussions, Bruker Biospin s.r.l. for providing a 600 MHz spectrometer Bruker Avance III Ultra 1949


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