Assessing the stable conformations of ibuprofen in solution by means of Residual Dipolar Couplings

Accepted Manuscript Assessing the stable conformations of ibuprofen in solution by means of Residual Dipolar Couplings

Maria Enrica Di Pietro, Giorgio Celebre, Christie Aroulanda, Denis Merlet, Giuseppina De Luca PII: DOI: Reference:

S0928-0987(17)30257-9 doi: 10.1016/j.ejps.2017.05.029 PHASCI 4043

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

21 January 2017 21 April 2017 13 May 2017

Please cite this article as: Maria Enrica Di Pietro, Giorgio Celebre, Christie Aroulanda, Denis Merlet, Giuseppina De Luca , Assessing the stable conformations of ibuprofen in solution by means of Residual Dipolar Couplings, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.05.029

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ACCEPTED MANUSCRIPT Assessing the stable conformations of Ibuprofen in solution by means of Residual Dipolar Couplings Maria Enrica Di Pietro,a * Giorgio Celebre,a Christie Aroulanda,b Denis Merlet,b and Giuseppina De Lucaa Lab. LXNMR_S.C.An., Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, via P.

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a.

Bucci, 87036, Arcavacata di Rende (CS), Italy, Fax: +39 0984493301; Tel: +39 0984493323. E-mail:

Equipe de RMN en milieu orienté, ICMMO, UMR 8182, Université Paris-Sud, Université Paris-Saclay, 15

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b.

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[email protected]

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rue Georges Clemenceau, 91405, Orsay, France.

Abstract

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Detailing the conformational equilibria between global and local minimum energy structures of anti-

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inflammatory α-arylpropionic acids directly in solution is of the utmost importance for a better understanding of the structure-activity relationships, hence providing valuable clues for rational structurebased drug design studies. Here the conformational preferences of the widely used pharmaceutical

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ibuprofen were investigated in solution by NMR spectroscopy in weakly ordering phases. A thorough

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theoretical treatment of the anisotropic interactions that are relevant for NMR spectra led to a conformational model characterized by six pairs of symmetry-related conformers, in particular four couples of gauche structures, with a total probability of 93%, and 2 couples of trans structures, counting for the remaining 7%.

Keywords Non-steroidal anti-inflammatory drugs – NMR spectroscopy – Alignment media – Conformational surface

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Chemical compounds studied in this article (S)-(+)-Ibuprofen (PubChem CID: 39912)

Introduction

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1.

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Ibuprofen (2-(4-(2-methylpropyl)phenyl)propanoic acid) is a poorly water-soluble, widespread non-

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prescriptive drug, frequently used for the treatment of painful and inflammatory conditions, such as rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. It is listed by the World Health Organisation

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as one of the essential drugs for a basic health-care system (WHO, 2015). Structurally, ibuprofen represents the parent compound of the 2-arylpropionic-type non-steroidal anti-

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inflammatory drugs (NSAIDs) commonly called “profens”, which inhibit the enzyme cyclooxygenase (COX) by interacting with its active site and thus competitively blocking it (Dannhardt and Kiefer, 2001; Hart et al.,

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1984).

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Although it was first synthetized in the ‘60s (Adams et al., 1967) and its three-dimensional crystal structure was reported earlier (Freer, 1993; McConnell, 1974; Shankland et al., 1996, 1997), there is currently a

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renewed interest in the investigation of the conformational space of ibuprofen (Betz et al., 2015; Fu et al., 2011; Khodov et al., 2014a; Klimovich and Mobley, 2010; Liu and Gao, 2012; Oparin et al., 2016; Paluch et

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al., 2011; Vueba et al., 2008). This can be related to the growing awareness of the key role that the 3D structure plays in driving the biological activity of pharmaceutical compounds. Indeed, it is now clear that a deeper knowledge of the conformational features of biologically active molecules, including NSAIDs, can help in understanding why and how they work, shedding more light on the structure-activity relationships underlying their biological effects (Llorens et al., 2002; Selinsky et al. 2002; Smeyers et al., 1985). In addition, the conformational preferences affect the drug’s transport property ( mbec and yubartse , 2013; Loverde, 2014) and its release from delivery systems (Geppi et al., 2005) and hence its bioavailability. However, the experimental determination of spatial structure and conformational state of pharmaceuticals 2

ACCEPTED MANUSCRIPT is still a serious challenge to researchers, especially in the presence of interconverting multiple conformations. Ibuprofen consists of a benzene ring bridging two para-substituents, an isobutyl and a propanoic acid group (Fig. 1), so that a rich conformational landscape can be expected. The conformational problem for ibuprofen was addressed by several quantum-chemical calculations (Fu et al., 2011; Jubert et al., 2006;

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Klimovich and Mobley, 2010; Liu and Gao, 2012; Okulik and Jubert, 2006; Oparin et al., 2016; Paluch et al., 2011; Shankland et al., 1998; Villa et al., 2001, 2004; Vueba et al., 2008), predicting a large number of low-

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energy conformers. As alternative to theoretical calculations, high-resolution broadband rotational

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spectroscopy was recently proposed as a technique to investigate experimentally the conformations of the

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isolated, unperturbed molecule of ibuprofen in the gas-phase (Betz et al., 2015). Being the liquid phase the physiological environment where ibuprofen acts, it would be nevertheless interesting to study the

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molecular conformational equilibrium directly in solution. NMR spectroscopy has been proven to be a sensitive tool in this respect. Indeed, in the fast exchange limit, the measured NMR spectral parameters are

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averaged over the torsional internal motions, allowing the investigation of molecular flexibility. More

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importantly, the NMR studies of bioactive molecules can be carried out directly in a liquid phase, which is closer to physiological conditions with respect to the gas or the solid states. Standard NMR parameters used over the years for structural and conformational investigations are J-couplings and nuclear

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Overhauser effect (nOe) measurements (Luy et al., 2008). 2D nOe spectroscopy was recently applied to

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determine the preferred conformations of ibuprofen in deuterochloroform (CDCl3) saturated solution (Khodov et al., 2014a). The aim was to get information on the conformational distribution in a saturated solution and thus shed light on the mechanism of formation of a certain crystal morphology within the nucleation process. Prominent differences in terms of relative population of the minimum energy conformers emerged from the comparison between the results obtained in saturated solution of CDCl3 (Khodov et al., 2014a) and those obtained from quantum chemical calculations and vibrational spectroscopy (Vueba et al., 2008). As such studies pointed out, a change in conformer distribution moving from gas phase/unsaturated solution to saturated solution is not astonishing and it was already observed in similar cases (Khodov et al. 2014b). In the present work, we aim at experimentally probing the 3

ACCEPTED MANUSCRIPT conformational space of ibuprofen in dilute organic solution by combining NMR spectroscopy with the use of anisotropic media. Unfortunately, standard NMR methods based on nOe measurements or J-couplings are limited to short-range connectivities and often do not provide enough unambiguous information for the structural and conformational elucidation of remote molecular fragments, particularly in the presence of multiple conformations in fast mutual exchange (Di Pietro et al., 2014). An appealing complementary

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strategy to access conformational information is applicable when the orientations of the solute molecules in the solution are not isotropically distributed, but rather statistically ordered (to a higher or lower extent)

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along a preferred direction, usually called “director”. This is what typically happens for NMR experiments in

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anisotropic solutions: the spectral parameters, called Residual Dipolar Couplings (RDCs), otherwise

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undetectable in isotropic phases due to their vanishing isotropic average, can then be observed and obtained from the NMR spectra. Physically speaking, the dipolar coupling constants

originate from the

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through-space interaction between two magnetically active nuclear spins and and, according to the laws of magnetic interactions, depend on both the distance

with respect to the external NMR magnetic field

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internuclear vector

between them and the angle

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molecules endowed with internal torsional degrees of freedom), both

where

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conformation, so that the following expression holds for the

and

of the

. For flexible solutes (i.e. depend on the molecular

RDC:

is the orientational order parameter and the symbol

represents the

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statistical average over the so-called “molecular tumbling” (the o erall molecular reorientational motion). The upper bar indicates an average, weighted over the complete conformational distribution of the flexible solute,

being the set of torsional angles defining the entire conformational space sampled by the

molecule (for more details, see ESI, eq. 1, and refs. therein). Due to their geometrical dependence, the knowledge of experimental RDCs enables, in principle, the determination of the relative spatial arrangements of distant atoms of a molecule (Burnell and De Lange, 2003; Emsley, 2007, 1985; Gil, 2011; Kummerlöwe and Luy, 2009; Thiele, 2008) and, consequently, it allows to acquire significant information about structural molecular properties such as constitution, configuration and conformation(s) (Aroulanda 4

ACCEPTED MANUSCRIPT et al., 2006; Celebre et al., 2006, 1992; De Luca et al. 2005; Emsley et al., 1994). The value of dipolar couplings to solve complex structure assignment problems is today recognized in pharmaceutical research (Liu et al., 2017). Their application also for the conformational analysis of small drugs sounds hence very appealing. The methodology was successfully applied to probe the conformational distributions of NSAIDs belonging to the family of salicylates and profens (Di Pietro et al., 2015, 2014). In an effort to gain a deeper

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insight on the conformational features of profens, we extend here the investigation to ibuprofen. The compound shows a stereogenic center at the α-carbon site connecting the carboxyl group and the aromatic

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ring (C2 in Fig. 1) and it can then exist as an R(-) or an S(+) enantiomer. Although only the S(+) form is

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pharmaceutically active (Evans, 2001; Geisslinger et al., 1989), ibuprofen is commercially available as a

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racemic mixture, since in vivo the COX-inactive R(-) ibuprofen undergoes a unidirectional chiral inversion into the active S(+) form (Baillie et al., 1989; Tracy and Hall, 1992). Here we studied the biologically active S-

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ibuprofen (SIBU, Fig. 1).

As in previous studies (Di Pietro et al., 2015, 2014), the solvent used was the well-known weakly ordering

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chiral liquid crystal obtained by dissolving the synthetic homopolypeptide poly-γ-benzyl-L-glutamate (PBLG)

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in the organic CDCl3 co-solvent (Samulski and Tobolski, 1968). Similar polymer - organic solvent mixtures were already applied in several NMR studies of small molecules (Aroulanda et al., 2003, 2001; Courtieu et al., 2002; Lesot et al., 2015, 2003; Rivard et al., 2003; Thiele and Berger, 2003) and also for the

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stereochemical assignment of ibuprofen enantiomers using RDCs combined with theoretical modelling

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(Berger et al., 2012; Marathias et al., 2007). One of their main features is that the degree of orientational order experienced by a solute in such media is usually weak enough to give mainly high-resolution first order NMR spectra. Moreover, SIBU is poorly soluble in water, but it dissolves readily in such anisotropic phase. In the present study, the conformational distribution of SIBU in solution will be investigated starting from the experimental RDCs measured in a PBLG/CDCl3 phase via the application of the robust theoretical approach known as the AP-DPD model (Celebre et al., 2003; Emsley et al., 1982). Results will be discussed also in the light of literature data obtained by molecular modelling calculations and other experimental methods.

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= H2C2C4C5,

= C11C10C7C6 and

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Fig. 1. Topological structure, carbon atoms labelling and torsional angles

= H11C11C10C7 of S-ibuprofen (SIBU). Protons nuclei are numbered after the carbon they are bound to.

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The (a,b,c) axes of the molecular reference frame adopted for the molecules are also shown. The c axis,

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perpendicular to the (ab) plane of the aromatic ring, points behind the plane, defining a right-handed

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Cartesian system.

Experimental and Theoretical Tools

2.1

Measurement of Residual Dipolar Couplings

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2.

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The conformational study addressed here is based on the experimentally observed RDCs,

, between

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the -th and -th magnetically active nuclei of the molecule, measured from the NMR spectra of partially oriented S-ibuprofen. RDCs are null in isotropic media, whereas they do affect NMR spectra of partially aligned solutes. Experimentally speaking, what is directly measured from the anisotropic spectra is the total coupling constant,

. Due to the dominant first-order features of the observed spectra and neglecting,

as usual, the anisotropy of indirect coupling (Diehl, 2007; Vaara et al., 2002), (Canet, 1996):

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can be defined as follows

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, one has to measure the set of scalar couplings

spectra of the isotropic sample (SIBU/CDCl3) and the set of total couplings

from the NMR

from the spectra of the

anisotropic sample (SIBU/PBLG/CDCl3). To describe accurately the torsional distribution around the three dihedral angles

,

and

for SIBU, the needed set of data has to be large and informative, i.e.

containing conformationally dependent inter-fragment

. For this reason, the experimental set of two

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anisotropic samples at different PBLG concentration (11.7% and 9.0%) were extracted and mixed in the

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conformational analysis. Details on sample preparation are reported in the ESI.

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On both the isotropic and anisotropic samples we performed routine 1D NMR 1H, 13C and 13C-{1H} spectra as well as COSY, HSQC and HMBC correlation experiments for the spectral assignment of the isotropic sample.

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1D 13C and 2D 1H-13C J-resolved spectra together with more elaborated 2D 1H-1H SERF (Fäcke and Berger, 1995; Farjon et al., 2002) and 1H-13C HETSERF (Bax and Freeman, 1982; Farjon et al., 2004) experiments

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were needed for the measurement of extended sets of non-trivial 1H-1H and 1H-13C

and

couplings.

All spectra were recorded at room temperature on a high-resolution Bruker Avance 500MHz spectrometer

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(11.74 T) equipped with a 5 mm TBO probe and a standard variable-temperature unit BVT-3000.

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In contrast to what measured in the isotropic sample, RDCs between the three dynamically equivalent protons within each methyl group (H3, H12 and H13) are observed in the NMR spectrum of the PBLG/CDCl3

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anisotropic samples. For instance, the signal of H3 at 1.6 ppm in the 1D 1H spectrum of SIBU in PBLG(11.7%)/CDCl3 (Fig. 2a, red spectrum) consists of a triplet of doublets, since each methyl proton is

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coupled both to the vicinal proton H2 and to the other two geminal equivalent methyl protons. For the sample SIBU/PBLG(9.0%)/CDCl3 the signal appears as a pseudoquadruplet in the 1H 1D spectrum (Fig. 2a, black spectrum), but the multiplet structure becomes clear in the 2D SERF experiment of Fig 2b. Selective refocusing 1H-1H and 13C-1H NMR experiments, SERF and HETSERF, turned out to be particularly useful for the measurement of long-range couplings between spins belonging to different molecular moieties (i.e. isobutyl chain, phenyl ring and propionic fragment). SERF and HETSERF experiments recorded on the sample SIBU/PBLG(11.7wt%)/CDCl3 are shown as examples in Fig. S1 and 3 (see figure captions for more details). Thanks to such homonuclear and heteronuclear selective spectra, we managed to edit long-

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ACCEPTED MANUSCRIPT range couplings, like 8TH3H10 and 4TC6H2, otherwise hardly measurable in the 1H and proton-coupled 13C 1D spectra (Fig. 2a and 3a, respectively). The complete set of measured

and

as well as the chemical shifts are listed in Tables S1-S2 of ESI.

Note that second-order effects as well as the presence of diastereotopic sites, namely the hydrogen nuclei H10a and H10b and the methyl groups labelled 12 and 13, complicated the spectral analysis. Magnitude and were calculated from the experimentally measured

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sign of the target

and

couplings,

, 48 for the sample with PBLG at 11.7% and 49 for the sample with PBLG at 9.0%,

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The resulting sets of

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according to eqs. (2) and (3), as previously reported (Di Pietro et al., 2014, 2015; Emsley et al., 2010, 2007).

are reported in Table 1. Note that no data sensitive to the torsions about the OC1C2C4 and HOC1O dihedral

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angles were collected, thus preventing us from investigating the flexibility of the carboxylic group.

Fig. 2. (a) 1D 1H spectra recorded at 300 K for the samples SIBU/PBLG/CDCl3 with PBLG at 11.7wt% (in red) and 9.0wt% (in black). (b) 2D 1H-1H SERF tilted spectrum of the sample SIBU/PBLG(9.0wt%)/CDCl3 where the offset of the excitation selective pulse was set at υ3 and the offsets of the refocusing selective pulse at υ2 and υ3. During t1 only the H3H3 and H2H3 couplings evolve, so that they can be easily measured on the indirect F1 dimension. The SERF spectrum was recorded using a data matrix of 1024 (t2) x 120 (t1) with 40 8

ACCEPTED MANUSCRIPT scans per t1 increment. The relaxation delay was 1 s. Data were processed using a sine filter in the F1 dimension prior to be zero-filled to 256 points and Fourier transformed. The durations of the selective REBURP refocusing and the selective E-BURP excitation pulses were 30.0 ms (frequency width of 165 Hz) at υ3

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frequency and 28.3 ms (frequency width of 175 Hz) at υ2 frequency.

Fig. 3. (a) 1D proton-coupled 13C spectrum of S-ibuprofen in PBLG(11.7wt%)/CDCl3 and enlargements of signals corresponding to the aromatic carbons. The spectrum was recorded at 300 K by using 32768 points and 8192 scans and Fourier-transformed without any preliminary apodisation. (b) 2D

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C-1H HETSERF

spectrum of the same sample where the offset of the selective refocusing pulse on the 1H channel was set at υ2. The HETSERF spectrum was recorded using a data matrix of 1024 (t2) x 248 (t1) with 160 scans per t1 increment. The relaxation delay was 1.7 s. Data were processed using zero-filling on t1 up to 512 points and

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ACCEPTED MANUSCRIPT qsine filter in F1 dimension. The duration of the RE-BURP refocusing pulse was 16.5 ms, corresponding to a frequency width of 300 Hz. An inset with the enlarged signal of C6, C8 is also shown.

Table 1 obtained by the NMR analysis of SIBU dissolved in PBLG/CDCl3. [a]

[a]

(Hz)

PBLG 11.7% PBLG 9.0%

PBLG 11.7% PBLG 9.0%

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1.8 ± 0.4 H2-H3 H2-H5 = H2-H9 29.6 ± 0.3 H2-H6= H2-H8 H2-H10 [b] -0.5 ± 0.3 H2-H11 6.9 ± 0.4 H2-H12 ; H2-H13 [b] -8.7 ± 0.5 H3-H3 -1.3 ± 0.4 H3-H5 = H3-H9 -0.5 ± 0.3 H3-H6 = H3-H8 -0.6 ± 0.5 H3-H10 [b] H3-H11 -0.9 ± 0.4 H3-H12 ; H3-H13 [b] -1.2 ± 0.5 H5-H6 = H8-H9 -1.6 ± 0.4 H5-H9 -4.4 ± 0.4 H5-H10 = H9-H10 [b] H5-H11 = H9-H11 H5-H12 = H9-H12 ; H5-H13 = H9-H13 [b] H6-H8 -4.5 ± 0.5 H6-H10 = H8-H10 [b] -2.2 ± 0.4 H6-H11 = H8-H11 H6-H12 = H8-H12 ; H6-H13 = H8-H13 [b] -1.0 ± 0.4 H10a-H10b -1.4 ± 0.4 H10-H11 [b] -0.5 ± 0.3 H12-H12 6.2 ± 0.3 H13-H13 28.1 ± 0.3 -1.2 ± 0.4 14.1 ± 0.4 -1.6 ± 0.6 -0.3 ± 0.6 5.0 ± 0.5 0.3 ± 0.5

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C1-H3 2.7 ± 0.3 C1-H5 = C1-H9 -0.7 ± 0.4 C2-H2 61.3 ± 0.3 C2-H3 4.7 ± 0.6 C2-H5 = C2-H9 C3-H2 12.5 ± 0.3 C3-H3 -16.4 ± 0.6 C3-H5 = C3-H9 -0.7 ± 0.2 C4-H2 -0.9 ± 0.4 C4-H3 -1.1 ± 0.6 C4-H11 -1.2 ± 0.5 C5-H2 = C9-H2 -2.0 ± 0.4 C5-H3 = C9-H3 -1.3 ± 0.6 C5-H5 = C9-H9 15.3 ± 0.5 C5-H6 = C9-H8 C5-H9 = C9-H5 1.2 ± 0.5 C5-H11= C9-H11 -1.3 ± 0.5 C6-H2 = C8-H2 -0.6 ± 0.4 C6-H5 = C8-H9 C6-H6 = C8-H8 15.2 ± 0.4 C6-H8= C8-H6 1.1 ± 0.4 [b] C6-H10 = C8-H10 C6-H11 = C8-H11 -2.5 ± 0.5 C10-H6 = C10-H8 -0.3 ± 0.2 C10-H10a 17.2 ± 0.2 C10-H10b 50.2 ± 0.2 C11-H6= C11-H8 -1.0 ± 0.3 C11-H11 30.6 ± 0.3 C11-H12 -2.5 ± 0.7 C11-H13 -0.3 ± 0.7 C12-H12 7.2 ± 0.4 C13-H13 1.1 ± 0.4 [a] obtained from eqs. (2) and (3) [b]

(Hz)

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Experimental dipolar couplings

19.0 ± 0.2 10.5 ± 0.2 -11.0 ± 0.2 -7.5 ± 0.3 -2.9 ± 0.4 -1.5 ± 0.3 -0.8 ± 0.2 -0.6 ± 0.2 -1.4 ± 0.4 -0.8 ± 0.4 -0.8 ± 0.1 -25.2 ± 0.1 -13.0 ± 0.1 -3.0 ± 0.4 -1.5 ± 0.4 -1.3 ± 0.3 -0.9 ± 0.3 -1.3 ± 0.3 -0.9 ± 0.3 -1.1 ± 0.4 -0.6 ± 0.3 -26.3 ± 0.2 -15.3 ± 0.2 3.5 ± 0.2 1.3 ± 0.2 -2.3 ± 0.3 -1.3 ± 0.2 -1.6 ± 0.2 -1.1 ± 0.2 3.5 ± 0.2 1.3 ± 0.2 -5.5 ± 0.3 -3.4 ± 0.2 -10.0 ± 0.2 -4.8 ± 0.2 -2.2 ± 0.2 -35.3 ± 0.1 -15.9 ± 0.1 9.2 ± 0.3 7.9 ± 0.2 11.4 ± 0.1 6.9 ± 0.1 2.3 ± 0.1 0.8 ± 0.1

only averaged values were observed for these theoretically different RDCs involving diastereotopic sites

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ACCEPTED MANUSCRIPT 2.2

Conformational Analysis by means of RDCs

What makes the RDCs a unique tool for conformational studies of flexible molecules is that they can be related to the probability distribution function of the solute,

, that is a function of the

conformations the molecule assumes. This term describes the conformational surface of the studied molecule and hence it represents the real target of the present work. In the case of S-ibuprofen, the

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conformational flexibility is due to the internal rotations of the propionic acid fragment and of the isobutyl

and

can be then rewritten as

, with

= H2C2C4C5,

= C11C10C7C6

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torsional distribution

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group that can be described through three dihedral angles about the C2-C4, C7-C10, and C10-C11 bonds. The

= H11C11C10C7. A relevant point for the following discussion is that the rotations of the propionic acid

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fragment and of the isobutyl chain are non-cooperative motions (Khodov et al., 2014a; Vueba et al., 2008), due to the limited interactions between the two fragments, whereas the torsions about the C7-C10 and the

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C10-C11 bonds are coupled. Therefore, it is admissible and advantageous to treat the non-coupled torsions separately, considering two sub-fragments of the molecule, the S-(+)-2-phenylpropanoic acid and the

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isobutylbenzene.

conformational distribution

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It is clear that, in order to exploit the set of experimental RDCs values to obtain a physically meaningful , it is necessary to adopt a proper theoretical model. Here we

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chose to apply a combination of the Additive Potential (Emsley et al., 1982) and Direct Probability Description (Celebre et al., 2003) models, the so-called AP-DPD approach (Celebre et al., 2010, 2012), which

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is implemented in an in-house software called AnCon (Pileio, 2005). The interested reader is referred to the ESI for a detailed description of the theoretical procedure.

2.3

DFT Calculations

Key elements in the conformational analysis via AnCon are the a priori knowledge (assumed or obtained from experimental techniques and/or theoretical calculations) of the geometries of the rigid molecular fragments involved in the -dependent molecular torsions and a tentative estimate of the potential energy surface (PES). To get such information we run theoretical calculations in vacuo, individuating approximately 11

ACCEPTED MANUSCRIPT the same minimum energy structures reported in the literature. Density Functional Theory (DFT) calculations were performed through the Gaussian03 software package (Frisch et al., 2003), with the widely employed hybrid method functional B3LYP and the basis set 6-31++G**, since previously they gave reliable results for similar molecules (Di Pietro et al., 2015) and for ibuprofen itself (Liu and Gao, 2012). Assuming the independence of the rotations of the two para-substituents, rigid and relaxed PES scans (i.e. not

= H2C2C4C5, for the propanoic acid fragment, o er the 0°−360° range with

a 3°-step sampling, and for the torsion angles

= C11C10C7C6 and

= H11C11C10C7, for the isobutyl chain,

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separately for the torsion angle

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allowing or allowing, respectively, for bond lengths and angles relaxation of the structure) were performed

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o er the 0°−360° range with a 20°-step sampling (Fig. S2 of ESI). For the sake of comparison with the

probability distributions

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experimental probability distributions, the PES were trivially converted into the normalized theoretical and

. Molecular geometries of the global and local minima

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found in the PES relaxed scans were then fully optimized and confirmed as real minima by the absence of imaginary harmonic frequencies (Table S3 of ESI). A detailed theoretical description is out of the aim of this

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work. Therefore, more results are given in the ESI, whereas in the following we have restricted the

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discussion to a comparison with the experimental NMR findings.

Conformational Results and Discussion

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3.

S-ibuprofen is a three-rotor molecule, whose conformational surface can be described according to the

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three dihedral angles displayed in Fig.1. As mentioned, it is advisable to treat the torsional distribution about the dihedral angle and

as separate from the flexibility of the isopropyl chain, described by the angles

.

Rotation of the propionic group. The rotation of the propanoic acid fragment, P, with respect to the phenyl ring, R, is described by the dihedral angle

. The two subsets of Table 1 corresponding to the

phenylpropanoic acid moiety of the molecule in the two samples (namely 24 independent

for each

sample, see Table S4 of ESI for details) were used as input in the AnCon iterative process. The experimental dipolar couplings were reproduced with a good agreement and a satisfactory RMS error of 0.37 Hz (Fig. 4a). 12

ACCEPTED MANUSCRIPT The corresponding torsional distribution

(Fig. 4b) is characterized by two equally populated maxima

at 13°± 1° (conformation I) and 193°± 1° (conformation II), indicating the quasi-planarity of the hydrogen atom directly bound to the stereogenic carbon atom (α-hydrogen). The distribution is reasonably in agreement with the DFT-calculated

of Fig. 4b and similar values were also found for ibuprofen by

optical vibrational spectroscopy (Vueba et al., 2008) and theoretical calculations (Liu and Gao, 2012;

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Smeyers et al., 1985; Villa et al., 2001, 2004) as well as for other profens like naproxen and flurbiprofen by NMR in ordered phases (Di Pietro et al., 2015). Moreo er, analogous preferred conformations with the α-

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hydrogen lying approximately in the aromatic plane were predicted via ab initio calculations also in

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aqueous solution, with only small variations in the conformational angles (Villa et al., 2004). Indeed, this

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preferred conformation of the α-hydrogen is a common feature of arylacetate (Di Pietro et al., 2015; Smeyers et al., 1998), since it allows to minimize steric hindrance due to the bul iest α-methyl and carboxyl

angle

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substituent groups, by placing them above and below the plane of the phenyl ring. Note that the dihedral is strongly dependent on the intermolecular interactions and/or packing, so that much larger values

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up to 40° were found in the solid state (Goossens et al., 2007).

Fig. 4. (a) NMR-observed versus AP-DPD-calculated dipolar couplings for the S-(+)-2-phenylpropanoic acid fragment. (b) Experimental (black solid line) and theoretical (dashed grey line) torsional probability distributions as a function of

= H2C2C4C5, obtained for S-ibuprofen by varying

a 3°-step sampling.

13

over the 0° - 360° range by

ACCEPTED MANUSCRIPT Rotation of the isobutyl chain. The flexibility of the isobutyl chain is due to two coupled torsions about the dihedral angles

and

phenyl ring R, while

. In particular,

describes the rotation of the -CH2- fragment with respect to the

describes the rotation of the -CH(CH3)2 fragment with respect to the -CH2- group.

The experimental input data in the AnCon calculations were the two subsets of Table 1 corresponding to the isobutylbenzene portion of the molecule in the two samples, namely 26 observed

for the sample with PBLG at 9.0% (for details see Table S5 of ESI). Among

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with PBLG at 11.7% and 29

for the sample

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the conformational models we tested, a good fitting of the experimental data could be achieved considering an equilibrium between 4 predominant gauche conformers (23.25% each in terms of relative

SC

population) and two barely populated trans structures (3.5% each). The corresponding torsional probability is reported in Figure 5a. Besides the low RMS error obtained (0.45 Hz), the

satisfactory reproduction of the

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distribution

can be also judged by considering Fig. 5c and Table S5.

103° ± 1° and

= 77° ± 1° and

= 257° ± 1° and

= 291° ± 1° (g1*),

= 283° ± 1° and

=

= 69° ± 1°

= 291° ± 1° (g2*). A simple conformational equilibrium of only gauche structures

D

(g2),

= 69° ± 1° (g1),

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The two symmetry-related couples of gauche conformers, named g1 / g1* and g2 / g2*, are located at:

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was not compatible with the experimental data. Indeed, despite their low relative percentage, two more t and t* conformers, located respectively at

= 90° ± 2° and

= 180° and

= 270° ± 2° and

= 180°,

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were needed in order to fit satisfactorily the two RDC sets. This emphasizes even more the accuracy of the conformational information achieved by NMR in liquid crystalline partially ordering media. The complete

AC

set of six conformers is displayed in Fig. 5d. The obtained conformational surface shows a significant qualitative agreement with the theoretical probability distribution

we calculated from the potential energy values obtained by DFT

calculations (Fig. 5b) as well as theoretical data found in the literature (Liu and Gao, 2012), although an upper shift of about 10°-15° can be noticed by comparing the

value for gauche conformers.

It is noteworthy that a good correspondence can be observed between the gauche and trans conformers of Fig. 5d and the structures identified by an optical vibrational study coupled with DFT calculations (Vueba et al., 2008). As extensively discussed by Vueba et al., such conformational preferences are the result of a

14

ACCEPTED MANUSCRIPT compromise between steric hindrance minimization, electrostatic interactions and hyperconjugative effects. Among them, particularly relevant is the contribution coming from concurrent steric repulsions between the two methyl groups of the isobutyl moiety and the phenyl ring, which hinders the trans arrangement. Interestingly, by roughly adding the room temperature populations found for the gauche and trans conformations in the mentioned study, a Gibbs population ratio of 94:6 was estimated, that is very

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close to our findings. Four energetically close structures were also recently identified in the gas phase via rotational spectra,

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which qualitatively match the gauche conformers of Fig. 5d (Betz et al., 2015). In the cited work, however,

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even though theoretically calculated, the trans conformers were not found in the gas phase and hence they

NU

were assumed not to be populated under the working conditions.

Note that for diastereotopic sites different RDCs values are theoretically measurable, as done for the -

,

-

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groups of couplings

,

-

and

However, for some long-range couplings only mean values could be extracted (see for instance -

. -

). Even in that case, we chose not to force the iterative procedure to average

D

or

-

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such theoretically-different values. Therefore, two calculated values are reported in Table S5 of ESI. Despite an averaged value would have allowed us to obtain an even better fitting and lower RMS, we used this additional parameter to further check the reliability and goodness of the iterative calculations. This is clear : the rough average of the two

CE

if one looks at

AC

experimentally measured value.

15

ends up being very close to the single

D

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RI

PT

ACCEPTED MANUSCRIPT

and

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Fig. 5. Experimental (a) and theoretical (b) torsional probability distributions as a function of = H11C11C10C7 obtained for S-ibuprofen by varying

and

= C11C10C7C6

over the 0° - 360° range by a 20°-step

CE

sampling. (c) NMR-observed versus AP-DPD-calculated dipolar couplings for the isobutylbenzene fragment. Averaged measured RDCs involving diastereotopic sites were excluded from the graph. (d) Structures of

AC

minimum energy conformers.

Complete set of conformers. The non-coupled rotations discussed above were finally combined together, to obtain a homogeneous conformational description for the whole molecule. Hence, both sets of independent

of Table 1 were fitted, including also 1H-1H RDC between nuclei very far apart in the

molecular structure (see Table 1), which were added for the sake of completeness in the calculations run for the whole molecular structure. Because of the huge computational burden, dihedral angles

16

,

and

ACCEPTED MANUSCRIPT were varied over the 0° - 360° range only by a 30°-step sampling, reaching an acceptable fit between experimental and calculated values (Tables S6-S7 and Fig.S3 of the ESI). The complete set of minimum energy conformers for S-ibuprofen is summed up in Table 2. As a consequence of the symmetry of the whole molecular system, the twelve conformers obtained combining the spatial arrangement for the propionic fragment (I and II) and for the isobutyl chain (g1, g1*, g2, g2*, t

PT

and t*) end up overlapping, so that only six independent minimum-energy structures are needed to describe exhaustively the conformational flexibility of the SIBU molecule.

RI

The proposed strategy allows therefore the accurate description of the conformational behavior of

SC

ibuprofen in an organic-based medium. Analogous experimental studies in aqueous solutions, closer to the

NU

physiological conditions, are unfortunately hindered by the drug’s poor water solubility. Howe er, it is worth noting that the lowest energy structures identified for ibuprofen using a PCM solvent model via

MA

thorough ab initio QM calculations (Fu et al., 2011) nicely match the minimum-energy conformers of Table

AC

CE

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D

2, hence adding value to the conformational information experimentally gathered in the present study.

17

ACCEPTED MANUSCRIPT Table 2 Structures, torsion angles ,

,

and relative abundance

of the six pairs of lowest minimum energy

conformers obtained for SIBU from the AP-DPD treatment of NMR data in PBLG/CDCl3 phase. = H2C2C4C5 (degree)

Conformers

= H11C11C10C7 (degree)

(%)

13

103

69

11.6

II-g2

193

283

69

11.6

I-g1*

13

257

291

11.6

II-g2*

193

77

291

11.6

I-g2

13

283

69

11.6

II-g1

193

103

69

11.6

I-g2*

13

77

291

11.6

193

257

291

11.6

13

90

180

1.8

II-t*

193

270

180

1.8

I-t*

13

270

180

1.8

193

90

180

1.8

CE

RI SC

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II-t

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I-t

PT

I-g1

II-g1*

4.

= C11C10C7C6 (degree)

Conclusions

NMR in a weakly ordering liquid crystal composed of PBLG and CDCl3 was applied to assess experimentally the conformational features of S-ibuprofen, a widespread anti-inflammatory drug with three cooperative internal rotations. NMR data from two samples at different PBLG concentration (11.7% and 9.0%) were collected and analyzed through the AP-DPD theoretical approach. Six pairs of symmetry-related conformers were found: four 18

ACCEPTED MANUSCRIPT couples of gauche structures (I-g1 / II-g2, I-g1* / II-g2*, I-g2 / II-g1, I-g2* / II-g1*), with a total probability of 93%, and two couples of trans structures (I-t / II-t*, I-t* / II-t), counting for the remaining 7%. A multipleconformation model was required to fulfil unambiguously the large amount of experimental RDCs used in the conformational analysis, whereas a single-state model or an equilibrium between a reduced number of only gauche structures were not compatible with the experimental data. Indeed, a thorough comparison

PT

with theoretical calculations and previous literature data confirm the existence of multiple conformations for the ibuprofen molecule.

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Following the successful conformational description of other profens, naproxen and flurbiprofen, the

SC

present study on ibuprofen confirms the reliability and usefulness of NMR spectroscopy in weakly ordering

NU

PB G phases for the precise drug’s conformational elucidation directly in solution. This kind of conformational analysis yields relevant information in the context of a rational structure-based drug design.

MA

Indeed, a detailed knowledge of the populated molecular structures in solution can be extremely helpful to clarify the structure-acti ity relationships (SAR) ruling the pharmacological characteristics of substituted α-

D

arylpropionic acids. Moreover, it is recognized that studies of drugs’ bioacti e conformations cannot ignore

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any more the complicated equilibria between interconverting low-energy structures and, to this end, the strategy applied here definitely stands out as a promising root for the conformational elucidation of small

AC

Acknowledgements

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to medium-size flexible pharmaceutics.

The present work has been supported by the “ ’Oréal Italia per le Donne e la Scienza 2014” program through the fellowship of M. E. Di Pietro. G. Celebre, G. De Luca and M. E. Di Pietro thank University of Calabria for financial support. D. Merlet and C. Aroulanda thank the CNRS and the French Ministry of Higher Education and Research for recurrent funding. Moreover, all authors warmly acknowledge Prof. Attilio Golemme for his critical reading of the manuscript.

19

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Model

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WHO,

Lists

of

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www.who.int/medicines/publications/essentialmedicines/en/ (accessed 17.03.17).

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Medicines.

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Graphical Abstract

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