Article pubs.acs.org/crystal
Solid State Investigation and Characterization of a Nepadutant Precursor: Polymorphic and Pseudopolymorphic Forms of MEN11282 Paola Paoli,*,† Patrizia Rossi,† Laura Chelazzi,‡ Maria Altamura,§ Valentina Fedi,§ and Danilo Giannotti∥ †
Department of Industrial Engineering, University of Florence, via S. Marta 3, 50139 Florence, Italy Centro di Cristallografia Strutturale, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy § Menarini Ricerche S.p.A., via Sette Santi 3, 50131 Florence, Italy ∥ Menarini Ricerche S.p.A., Via Livornese 897, 56122 Pisa, Italy ‡
S Supporting Information *
ABSTRACT: MEN11282 (1) is a precursor of nepadutant (MEN11420), a potent and selective antagonist at the human tachykinin NK-2 receptor (hNK-2), that has been evaluated in clinical trials for different therapeutic indications. Three crystalline forms of 1 were identified and characterized by both single crystal and powder Xray diffraction (SCXRD and XRPD): a monohydrate (1·H2O, SCXRD) and two different anhydrous forms, namely, 1_α and 1_β (XRPD). Because of the relevance that the solid form of a substance of pharmaceutical interest plays during the manufacturing process, variable temperature powder X-ray diffraction (VT-XRPD) in conjunction with differential scanning calorimetry were used to investigate the behavior of the different solid forms of 1. The rationale for the dehydration and hydration process involving 1·H2O and 1_α and the stability of 1_β toward water uptake is provided based on their crystal packings.
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INTRODUCTION Nepadutant (lab code: MEN11420, Scheme 1b) is a fully synthetic bicyclic glyco-hexapeptide, namely, {[Asn(β-DGlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2β-5β)}, that has been evaluated in clinical trials for different indications including infant colic.1 It acts as a potent and selective antagonist at the human tachykinin NK-2 receptor (hNK-2), the latter being a target for important chronic diseases at the respiratory, gastrointestinal, and genitourinary level.2 MEN11420, which is the glycosylated analogue of MEN10627 (Scheme 1c) or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo-(2β-5β) (also a potent antagonist at the tachykinin NK-2 receptor), was synthesized to circumvent the low bioavailability of the latter due to its extremely poor water solubility. The structures of MEN106273 and MEN114204 were determined by NMR spectroscopy (accompanied by in vacuo restrained molecular dynamics simulations, rMD, for MEN11420) in CD3CN and DMSO-d6 solution, respectively, and show, in agreement with the hypothesis behind the rational design of these conformationally constrained bicyclic hexapeptides, that in both cases the cyclic polypeptide adopts a single backbone conformation which contains a double β-turn arrangement, a key feature for binding the hNK-2 receptor.5 In addition, MEN10627 shows an almost identical conformation (see later) in the solid state as provided by single crystal X-ray diffraction (SCXRD).3 © 2016 American Chemical Society
As for the side chains departing from the hexapeptide backbone, special attention is deserved for those of the Trp-Phe sequence in view of the major role played by this dipeptide in the binding interactions with the NK-2 receptor.5 MD refinement of the NMR data (DMSO solution) suggests in MEN11420 the formation of a hydrophobic pocket with the indolyl and phenyl rings belonging to the Trp-Phe active sequence. As for MEN10627, in the solid state the two aromatic rings are perpendicular to each other, with the Trp3 side chain showing an eclipsed conformation, compared to the trans one observed in solution (NMR data). This different behavior is not surprising giving that side chains have in general a larger conformational freedom with respect to a cyclic backbone. In addition, their conformations can be biased by intermolecular interactions, as suggested by the authors for MEN10627, where, in the solid state, the Trp3εNH atom is involved in hydrogen bonds with a symmetry related molecule. In this context it appears interesting to gain further structural evidence about MEN11282 (cyclo(-Asp1-Asp2-Trp3-Phe4-Dap5Leu6-)cyclo(2β-5β); Dap = 2,3-diaminopropionic acid), (1 hereafter, Scheme 1a), the precursor and key synthesis Received: May 31, 2016 Revised: August 9, 2016 Published: August 15, 2016 5294
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Scheme 1. Schematic Drawings of 1 and of the Related Bicyclic Polypeptides
the amino acid side-chains in the right way to achieve affinity at the tachykinin NK2 receptor. Thus, while 1 is not a drug per se, it is in fact a molecule of pharmaceutical interest as the key precursor of a drug. During the solid state investigation of 1 a monohydrate (1· H2O) and two different anhydrous forms, namely, 1_α and 1_β, were identified and characterized by single crystal (1· H2O) and powder (1_α and 1_β) X-ray diffraction (XRD). The molecular and crystal structure of all the crystalline forms have been discussed and compared to the literature data cited above. In particular the three-dimensional (3D) arrangement of
intermediate of MEN11420, bearing an Asp residue instead of the Asn(β-D-GlcNAc) moiety. In fact MEN11420 is usually produced by a multistep synthesis6 starting from amino acids through an iterative process of deprotection/coupling. The crucial steps in the synthesis are the two cyclizations that lead to MEN 11282, the non glycosylated bicyclic hexapeptide precursor. Even if it needs to be glycosylated to MEN11420 in order to optimize its pharmaceutical and pharmacological properties, 1 contains in its structure all the essential features for the affinity to the target: actually, the rigid, bicyclic structure can orientate 5295
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the Trp-Phe active sequence has been examined with respect to the intermolecular contacts found in 1·H2O, 1_α, and 1_β which can provide indirect hints about its ability to form intermolecular contacts. Additional information on the conformational space available to the Trp-Phe dipeptide was obtained from molecular dynamics simulations and quantum mechanical methods. Furthermore, the existence of different crystalline forms of 1 appears very important giving that in most cases molecules of pharmaceutical interest are handled as solids during the manufacturing process and their physical or chemical properties usually depend significantly on the solid form.7,8 In this context, the occurrence of polymorphs and pseudopolymorphs of 1 prompted us to investigate the environmental changes that promote solid−solid transformations. Hence variable temperature XRPD (VT-XRPD) and differential scanning calorimetry (DSC) experiments were used to study the thermal behavior of the different solid forms of 1 and the transitions between the pseudopolymorphs 1·H2O and 1_α. A mechanism of the dehydration−hydration process leading from 1·H2O to 1_α and backward has been proposed on the basis of the corresponding crystal lattices. At the same time, the packing motif of the anhydrous phase 1_β offers hints to account for the observed stability of this form toward hydration.
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Table 1. Crystallographic Data and Refinement Parameters for 1·H2O, 1_α and 1_β 1·H2O empirical formula formula weight T (K) crystal system, space group λ (Å) unit cell dimensions (Å, °)
volume (Å3) Z, dcalc (g/cm3) μ (mm−1) refinement method reflections collected/ unique data/ parameters/ restrains final R indices [I > 2σ(I)] R indices (all data) Rwp (%) χ2
EXPERIMENTAL SECTION
Material and Methods. Reagent grade solvents were used in the crystallization procedures. The synthesis of MEN11282 (1) has been reported elsewhere.4 1·H2O was obtained as a white polycrystalline powder by room temperature evaporation of a solution obtained by the following procedure. A total of 1.3 g of 1 was treated under stirring at room temperature with 15 mL of THF, and the resulting suspension was filtered and dried on the bench overnight. The solid was dissolved in 40 mL of methanol. The solution was filtered and left to evaporate slowly (1 month) to give crystals of 1·H2O suitable for single crystal X-ray diffraction analysis (SCXRD). The microcrystalline powder 1·H2O was completely dehydrated when held isothermally in oven at 383 K for 24 h affording the anhydrous form 1_α as polycrystalline material. Finally the treatment of a solution obtained by dissolving 4.2 g of 1 in 98 mL of methanol/water 9/1 v/v gave a second anhydrous form of 1, namely, 1_β, as a white polycrystalline powder. Single Crystal X-ray Diffraction (SCXRD). Intensity data from single crystal of MEN11282 monohydrate, 1·H2O, were collected at 100 K by using an Oxford Diffraction ExCalibur diffractometer equipped with a CCD area detector using the Cu Kα (λ = 1.54184 Å) radiation. Diffraction data were collected with the CrysAlis CCD9 program and reduced with the CrysAlis RED10 program. Absorption correction was performed with the program ABSPACK in CrysAlis RED. Structure was then solved using the SIR9711 program and refined by full-matrix least-squares against F2 using all data (SHELX201312). All the non-hydrogen atoms were refined anisotropically. The hydrogen atoms of the water molecule were found in the Fourier difference map and refined with an isotropic temperature factor riding the bound oxygen atom. All the other hydrogen atoms were set in calculated positions with their coordinates and temperature factors refined accordingly to the bound atoms. The correct absolute structure was chosen on the basis of the synthetic procedure. Geometrical calculations were performed by PARST97,13 and molecular plots were produced by the program ORTEP3,14 Mercury (v3.7),15 and Discovery Studio Visualizer (v2.5.5.9350).16 Crystallographic data and refinement parameters are reported in Table 1. Figure 1 shows an ORTEP3 view of 1 in 1·H2O. Structure Determination from XRPD Data and VT-XRPD Experiments. For crystal lattice parameters and structure determination of the two anhydrous forms of MEN11282, 1_α and 1_β, high-
1_α
1_β
C37H46N8O10
C37H44N8O9
C37H44N8O9
762.82 100 monoclinic, P21
744.80 295 monoclinic, P21
1.54184 a = 9.0030(5)
1.54175 a = 9.0161(5)
744.80 295 orthorhombic, P212121 1.54175 a = 36.301(2)
b = 10.9243(5); β = 107.048(7) c = 19.661(2) 1848.7(2) 2, 1.370 0.842 full-matrix leastsquares on F2 6066/3989 (Rint = 0.0790)
b = 10.9561(5); β = 103.399(4) c = 18.838(2) 1810.2(2) 2, 1.367 0.822
c = 9.0801(2) 3637.4(2) 4, 1.360 0.822
3.81 1.58
4.91 2.31
b = 11.0350(3)
3989/503/4 R1 = 0.0667, wR2 = 0.1503 R1 = 0.1368, wR2 = 0.1725
quality XRPD data were recorded in a 0.5 mm capillary at room temperature by using a Bruker New D8 Da Vinci diffractometer equipped with a Bruker LYNXEYE-XE detector (Cu−Kα radiation, 40 kV × 40 mA, scanning range 2θ = 3−50°, 0.01° increments of 2θ and a counting time of 2 s/step). The 1_α sample was prepared by putting a 0.5 mm capillary containing 1·H2O in an oven at 383 K for 24 h, and then the capillary was rapidly closed in order to avoid the sample rehydration (see below). A volume of 1810.2(2) Å3 and 3637.4(2) Å3 was found respectively for 1_α and 1_β with the algorithm DICVOL.17 The two asymmetric units contain one molecule of MEN11282. Space group determination with Highscore plus resulted in space group P21 with Z = 2 (1_α) and P212121 with Z = 4 (1_β). The two structures were solved by simulated annealing that runs with structure fragments, performed with EXPO201318 using as a model structure for MEN11282 that found in 1·H2O from SCXRD. Ten runs for simulated annealing trial were set, and a cooling rate (defined as the ratio Tn/Tn−1) of 0.95 was used. Best solutions were chosen for Rietveld refinements, which were performed with the software TOPAS.19 Two shifted Chebyshev functions with 12 and 15 parameters for each form and a Pseudo-Voigt function were used to fit background and peak shape, respectively. Soft constraints were applied for bond distances and angles of the side arms departing from the cyclic polypeptide of the MEN11282 molecule. A common thermal parameter for all the non-H atoms was adopted. All the hydrogen atoms were fixed in calculated positions. Refinement details are reported in Table 1. Figure S1 shows the experimental, calculated, and difference diffraction patterns of the two forms. Variable-temperature experiments (performed in triplicates) were carried out in air in the range 298−590 K for 1·H2O and 298−610 K for 1_β20 by using an Anton Paar HTK 1200N hot chamber mounted on a Panalytical XPERT PRO diffractometer (Cu−Kα radiation, 40 kV x 40 mA), equipped with the PIX-CEL solid state fast detector. Scanning range 2θ = 3−40° with a 1 s/step counting time and 0.01° increments of 2θ. In all cases the temperature variation rate was 5 K/ 5296
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Figure 1. ORTEP3 view of 1 as found in 1·H2O. Ellipsoids are drawn at 20% probability.
Table 2. Intra- and Intermolecular Hydrogen Bonds in 1·H2O, 1_α and 1_β as Obtained from X-ray Analysis Intramolecular H-Bonds 1·H2O (1_α, 1_β) X−H···Y
X···Y (Å)
N(2)−H(2)···O(6) N(5)−H(5)···O(2) N(6)−H(6)···O(3) N(7)−H(7)···O(7)
3.14(1) 2.85(1) 3.61(1) 3.089(9)
H···Y (Å)
X−H···Y (deg)
2.31 2.01 2.82 2.26 Intermolecular H-Bonds
157 158 152 156
1·H2O/1_α/1_β X−H···Y
X···Y (Å)
O(1w)−H(1w)···O4 N(4)−H(4)···O(1w)a N(1)−H(1)···O(1)b N(8)−H(8)···O(8)c O(9)−H(9)···O(3)d
2.79(1)/−/− 2.86(1)/−/− 2.86(1)/2.8/3.0 3.00(1)/3.0/3.2 2.62(1)/2.8/2.6 Intermolecular Contacts
H···Y (Å)
X−H···Y (deg)
2.05(8)/−/− 1.99/−/− 2.00/1.9/2.1 2.16/2.1/2.3 1.84/2.0/1.8
143(6)/−/− 171/−/− 166/171/173 159/157/157 153/158/145
1·H2O/1_α/1_β X−H···Y e
O(1w)−H(1w)···CT N(4)−H(4)···CTe
X···Y (Å)
H···Y (Å)
X−H···Y (deg)
3.42(2)/−/− −/4.6/4.1
2.6(1)/−/− −/3.8/3.3
154(8)/−/− −/158/170
−x + 1, +y − 1/2, −z + 1 in 1·H2O. b−x + 2, +y + 1/2, −z + 2 in 1·H2O, −x, +y + 1/2, −z in 1_α, −x, + y + 1/2, −z + 1/2 + 1 in 1_β. cx − 1, +y, +z in 1·H2O, x + 1, +y, +z in 1_α, x, +y, +z − 1 in 1_β. d−x + 2, +y − 1/2, −z + 2 in 1·H2O, −x, +y − 1/2, −z in 1_α, −x, −1/2 + y, 3/2 − z in 1_β. eCT is the centroid of the six-membered ring C(23)−C(28) in 1·H2O, 1_α (−x + 1, +y − 1/2, −z + 1) and of the C(14)−C(19) ring in 1_β (−x + 1/2, −y, +z + 1/2). a
300−610 and 300−670 K ranges, for 1·H2O/1_α and 1_β, respectively. A linear heating rate of 10 K/min was used. Because MEN11282 appears to decompose near the melting point, experiments to determine the heats of fusion (ΔH) were carried on in the 310−720 K range with a linear heating rate of 100 K/min and 310− 750 K range (200 K/min), for 1_α and 1_β, respectively, in order to try to separate the melting effect by moving the decomposition reaction to higher temperatures. A TA Instruments Thermobalance model Q5000IR was employed for thermogravimetric measurements (which were performed in duplicates). The experiments were performed at a rate of 10 K/min, from 300 to 870 K under nitrogen flow (25 mL/min) as purging gas.
min, and after the target temperature was reached the sample was kept for 10 min at that temperature before proceeding with data collection. Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). DSC experiments were carried out using a Mettler Toledo DSC1 Excellence system. Measures were run in aluminum pans with a pierced lid (mass samples range from 0.7 to 5 mg). Temperature and enthalpy calibration were done using indium as a standard. All the experiments were carried out in air. DSC peaks were analyzed using the STARe software.21 The melting data reported were the average of two measurements; standard errors were ±0.1 K for temperature and ±0.2 kJ/mol for enthalpy. Dehydration temperature range and dehydration heat for 1·H2O, as well as melting points for 1_α and 1_β, were determined by measurements in the 5297
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Table 3. Most Important Torsional Angles (deg) Defining the Molecular Shape of MEN11282 as Derived from the X-ray Structure of 1·H2O ϕ ψ ω χ1 χ2,1 χ2,2
Asp1
Asp2
Trp3
Phe4
Dap5
Leu6
60(1) 31(1) 169.9(9) −75(1) 172(1) −9(1)
−156(1) −174(1) −177(1) 79(1) −80(1) 102(1)
−63(1) −37(1) −172.6(9) −48(1) −89(1) 92(1)
−130(1) 34(1) −167.0(9) −66(1) 89(1) −90(1)
−165.1(9) 174.3(8) 161.3(9) 66(1) −94(1) 177.0(8)
−63(1) 129(1) 178.3(9) 178.2(9) −178.1(9) 58(1)
shows ϕ and ψ dihedral angles typical of positions 2 and 3 of a type II β-turn. As expected, the overall shape of the MEN11282 molecule in 1_α and 1_β is almost identical with respect to that observed in 1·H2O (Figure S2). In fact, in all the MEN11282 samples (1· H2O, 1_α, and 1_β) the four side chains definitely point away from the 18-membered ring.31 The Asp1, Trp3, and Phe4 side chains adopt a gauche (+) conformation, while that adopted by the Asp2 and Dap5 ones is the gauche (−), and finally the side chain of Leu6 takes a trans conformation (see Table 3). As expected, the conformation of the cyclic polypeptide as well as the two β-turn motifs compare well with those observed in the strictly related MEN114204 and MEN10627.3 In particular, as for the β-turn, a type I β-turn in the region Asp-Trp-Phe-Dap in both the MEN11420 and MEN10627 molecules and a type II β-turn in the region Dap-Leu-Met-Asp (MEN10627) and Dap-Leu-Asn-Asp for MEN11420 were found. In addition, the fact that MEN10698 [cyclo(Met-AspTrp-Phe-Dap-Leu)cyclo-(2β-5β), Scheme 1e], a pseudosymmetrical analogue of MEN10627, contains both type I and II βturns (SCXRD, NMR, and rMD data)32 is further evidence that this type of bicyclic structure, notwithstanding the amino acid composition, can be used as a scaffold to build bioactive molecules featuring β-turned structures. On the other hand we have already shown that it is possible to retain the β-turn feature of one of the cycles considered responsible for the binding to the NK-2 receptor5 even in structures of reduced size and complexity such as the monocyclic MEN13365, i.e., N[5(S),8(R)-dibenzyl-2(S)-(1H-indol-3-ylmethyl)-3,6,11,14-tetraoxo-1,4,7,10-tetraaza-cyclotetradec-12(R)-yl]-2-(4-sulfamoylpiperazin-1-yl)-acetamide (Scheme 1d), as provided by SCXRD analysis.33 As for the Trp and Phe side chains, in all the abovementioned cyclic polypeptides (Scheme 1), two different overall arrangements for the aromatic rings can be identified, as illustrated in Figure 2: (i) that found in the monohydrate (1· H2O) and anhydrous (1_α and 1_β) forms of MEN11282, in MEN13365 and in MEN10698 featuring the Trp ring almost perpendicular with respect to the 18-membered cycle, with the Phe arm parallel (labeled as I); (ii) that with the heterocycle parallel (eclipsed conformation for χ2,1), while the phenyl ring is almost perpendicular to the polypeptide cycle (labeled as II), as found in MEN10627 and MEN10698. It is to be noted here that MEN 10698 contains two Trp-Phe sequences and shows both the I and II arrangements. The first family can be further divided into two subgroups depending on the sign of the χ2,1 dihedral angle about the C(11)−C(12) bond (see Figure 1 for atom labeling): negative in 1·H2O, 1_α, and 1_β (hence the I− conformational isomer) and positive in MEN13365 and MEN10698 (hence the I+ conformational isomer). Results from geometry optimization (DFT level) of the three conformational isomers of 1, namely, 1_I−, 1_I+, and 1_II
The amount of sample in each TG measurement varied between 2 and 3 mg. Theoretical Calculations. Geometry optimizations (MM) and molecular dynamics (MD) simulations were performed on the conformational isomers of 1, namely, 1_I−, 1_I+, and 1_II, as representatives of the different disposition of the side chains of the Trp-Phe sequence as found in the solid state structures of MEN11282 (1_I−), the latter both monohydrate (1·H2O) and anhydrous (1_α and 1_β), MEN13365 and MEN10698 (1_I+, see molecular drawings in Scheme 1) and finally in MEN10627 (1_II) (details in the Results and Discussion). All calculations were made by using the CHARMm Force Field.22 MM calculations were performed on each species by using the Smart Minimizer energy minimization procedure implemented in Accelrys Discovery Studio 2.116 and before starting the MD simulations, the geometry of each compound was further optimized using the steepest descent and conjugate gradient algorithms. MD simulations were carried out at 300 K in vacuum. In the molecular dynamics simulations, the time step was 1 fs for all runs, equilibration time = 500 ps and production time = 5000 ps, snapshot conformations were sampled every 10 ps. The programs used for the MD and the energy minimization were the Standard Dynamics Cascade and Minimization protocols, and trajectories were analyzed by the Analyze Trajectory protocol, all implemented in Discovery Studio 2.1. Gaussian09 (rev. c01)23 was used for quantum chemical calculations using the following functionals: B3LYP24,25 and B97D.26 The basis set was 6-31+G(d,p).27 The Berny algorithm was used.28 The reliability of the stationary points was assessed by the evaluation of the vibrational frequencies. Geometry optimizations were performed on the three conformational isomers 1_I−, 1_I+, and 1_II.
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RESULTS AND DISCUSSION Molecular Structures from X-ray Diffraction and Modeling Studies. The molecular structures of 1·H2O, 1_α, and 1_β were investigated by single crystal (1·H2O) and powder (1_α and 1_β) X-ray diffraction techniques. In all cases in the asymmetric unit there is an independent molecule of 1 together with a water molecule in 1·H2O. Accordingly to the rationale beyond the design of the polypeptide bicycle, in 1·H2O all the peptide bonds show a trans conformation, with the Cα atoms of Asp1, Trp3, Phe4, and Leu6 occupying the corners of the rectangle outlined by the backbone atoms (see Figure 1). This conformation is stabilized by two weak intraturn hydrogen bonds involving the oxygen atom O(3) of the carbonyl group of Asp2 and the hydrogen atom of the Dap5 NH group [(N(6)−H(6)], and the carbonyl oxygen atom O(6) of Dap5 and the amidic hydrogen atom of Asp2 [(N(2)−H(2)], respectively (see Table 2). In addition the bridging moiety is involved in two stronger H-bonds interactions (see Table 2): the βCO group of Asp2 is a Hbond acceptor to the NH group of Phe4, and the β amidic group of Dap5 is a H-bond donor to the carbonyl oxygen atom of Leu6. The Trp3-Phe4 peptide sequence shows ϕ and ψ dihedral angles typical of those in positions 2 and 3 of a type I β-turn29,30 (see Table 3), while the Leu6-Asp1 peptide segment 5298
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the bulk material was checked by comparing calculated and measured (298 K) powder diffraction patterns (Figure S4). In the crystal all the amide groupings of the bicyclic ring, except those featuring O(2)/N(7)H(7) and O(5)/N(6)H(6), together with the carboxylic group of Asp1 are involved in intermolecular H-bonds (see Table 2) with four different symmetry related MEN11282 molecules. The cocrystallized water molecule acts as a bridge between two symmetry related MEN11282 molecules via strong H-bonds (see Table 2). In fact it works both as H-bond donor to the carbonyl oxygen atom O(4) of Trp3 and as H-bond acceptor with the NH moiety [N(4)−H(4)] of the Trp3 of a symmetry related molecule. Finally an OH···π (Phe4) interaction further contributes to hold in place the water molecule (see Table 2). The water molecules give rise to a zigzag motif which propagates along the b axis direction and hold together chains of MEN11282 molecules (Figures 3a and S5). Finally, the cocrystallized water molecules do not interact with each other, and, accordingly, they are lodged in isolated sites37,38 which are not interconnected by a channel (Figure S6). The lack of channels should make the departure of the water molecules from the crystal lattice quite difficult, and, as a consequence, the host framework is unlikely to survive unaffected the water removal. Thermogravimetric analysis shows that 1·H2O completely lost the water molecules (a mass loss of 2.4% was observed, corresponding to one water molecule per 1·H2O) at around 333 K (Figure S7). DSC measurements (Figure S8a) indicate that the process is associated with a rather broad peak (peak: 347.7 K; extrapolated peak: 348.5 K; enthalpy: 64.0 J/g) and that the heat effect is about 20% higher than the enthalpy of vaporization of water at the same temperature (49 kJ per mole of water ripped out from the crystal lattice vs 41 kJ/mol, respectively). Finally, no further significant thermal events are present in the DSC curve until, at a temperature of about 573 K, the compound started to melt and during melting decomposed (melting temperature: 586 K,39 peak 583.6 K, extrapolated peak 583.4 K; melting enthalpy: 64.6 J/g, 48.1 kJ/ mol (see Figure S8). Then VT-XRPD analyses were carried out in the range 298−590 K (Figure S9). The XRPD patterns indicate that a phase change takes place between 333 and 353 K (Figure S10), which, consistent with TGA and DSC data, has been assigned to a dehydration process which leads to a crystalline anhydrous phase. The anhydrous form, identified as the 1_α one from the comparison of the corresponding diffraction patterns (Figure S11), retains (vide inf ra) most of the structural features present in the parent hydrate phase (1· H2O), and, accordingly, their XRPD patterns are quite similar (Figure 4). In other words, notwithstanding the lack of channels interconnecting the cavities that host the water molecules (at least in the static structure, see Figure S6), the latter fully escape without causing a detectable (in the XRPD patterns, Figure S9) collapse of the crystal network, at least in the experimental conditions adopted. Thus, while the water molecules leave the crystal, a reorganization of the host molecules can be postulated. As for the latter, in the anhydrous form their relative position remains almost unchanged within each chain (Figure 3b). In fact, as in the parent hydrate form, each MEN11282 molecule interacts with four different symmetry related molecules through the carboxylic group of Asp1 and all the amide groupings of the bicyclic ring, except those featuring O(2)/N(7)H(7) and O(5)/N(6)H(6) (see Table 2).
Figure 2. Superimposition of the molecular structures of MEN11282, as found in 1·H2O (ball and stick, red), MEN13365 (fuchsia), MEN10627 (yellow), and MEN10698 (two independent molecules in black and green).
(see Figure S3 top), as representatives of the different conformations of the side chains of the Trp-Phe sequence above-discussed, suggest that the arrangement found in 1·H2O, 1_α, and 1_β for the Trp side chain (1_I−) is the preferred one irrespective of the model (B3LYP and B97D) used.34 In addition most of the MD trajectory snapshots show the heterocycle perpendicularly arranged with respect to the 18membered ring (i.e., the I families), with the NH grouping oriented either outside (as found in 1·H2O, 1_α, 1_β, MEN13365 and MEN10698 solid state structures where the NH group is involved in intermolecular contacts, vide inf ra) and inside with respect to the Phe4 ring (Figure S3 bottom). The latter is in most cases perpendicularly arranged (as in the II family and in the majority of the solid state structures deposited in the Cambridge Structural Database35 featuring a 18membered cyclohexapeptide) and involved in weak contacts of NH···π type with the close Trp side arm. Finally the eclipsed conformation is never adopted by the Trp side arm during the MD simulations (in vacuum at 300 K),36 thus further suggesting that such arrangement, observed in the solid state of MEN10627 and MEN10698, is the result of the intermolecular interactions involving the Trp3εNH atom as already suggested by Pavone.3 Finally several conformations are also sampled with the indolyl and phenyl rings of the Trp-Phe sequence facing each other and forming a hydrophobic pocket as already reported (Figure S3 bottom).4 In summary, solid state and modeling results are consistent in suggesting that the Trp3-Phe4 sequence is able to change its overall arrangement in response to the local environment, as evidenced by X-ray (crystal environment) and DFT and MD (vacuum) data, thus further confirming3,4,32 the flexibility of the Trp-Phe dipeptide side chains which is essential for the optimal fit into the receptor. Crystal Structures from X-ray Diffraction and Thermal Analyses with Differential Scanning Calorimetry. First of all the correspondence between the crystal structure of MEN11282, as determined by SCXRD (1·H2O), and that of 5299
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Figure 3. Crystal packings of (a) 1·H2O view along the a axis highlighting the zigzag pattern described by the cocrystallized water molecules; (b) 1_α view along the a axis highlighting the strict similarity with the pattern described by the MEN11282 molecules in the parent hydrate form; (c) 1_β view along the c axis.
However, because of the missed water molecules O(4)/ N(5)H(5) does not participate in any H-bond contact, while the NH hydrogen-bond donor of Trp3 is now involved in a contact of NH···π(Phe4) type with a symmetry related molecule of the adjacent chain (Table 2, Figure S12). In particular the release of the water molecules makes the host molecules smoothly rearrange in a more compact structure as provided by a comparison of the volume of the voids in the crystal lattice of 1_α (i.e., 1.1% of the unit cell volume, ca. 19 Å3, probe radius 1.2 Å,15 see Figure S13) and those virtually generated by deleting the water molecules in 1·H2O (i.e., 2.4% of the unit cell volume, ca. 45 Å3, probe radius 1.2 Å, Figure S6). In particular adjacent chains move closer on passing from
1·H2O to 1_α and make the empty volume left by the water escape almost disappear as also quantified by the reduction of the distance separating the (Trp3)NH···centroid(Phe4) of MEN11282 molecules belonging to adjacent chains (5.2 vs 3.8 Å, Figures S5 and S12). As a result, the anhydrous form 1_α is well-packed as well as the starting parent hydrated species 1· H2O, K.P.I.40,41 = 0.69. Moreover the dehydration process leading from 1·H2O to 1_α is reversible as evidenced by a second series of XRPD measurements carried out in the 298−353−298 K range (see Figure 4) showing that the anhydrous form (1_α) spontaneously reconverts into 1·H2O at the end of the cooling cycle. The fact that the dehydrated form retains the 3D order of the 5300
DOI: 10.1021/acs.cgd.6b00826 Cryst. Growth Des. 2016, 16, 5294−5304
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Figure 4. Changes in the experimental XRPD pattern of 1·H2O with temperature, together with the calculated patterns for 1·H2O and 1_α. The broad signal at 7° (2θ) is due to the Kapton polymer window of the Anton-Paar hot chamber.
Figure 5. Superimposition of the XRPD patterns of 1_β collected at 298, 323, 353, 413, 453, 513, 553, and 583 K. The broad signal at 7° (2θ) is due to the Kapton polymer window of the Anton-Paar hot chamber.
the hydrated crystalline form (1·H2O). In fact we reasoning that the (Trp3)NH···π(Phe4) interactions can act as a switch triggered by the humidity/temperature conditions: they lengthen allowing the uptake of the guest molecules (which H-bind adjacent chains of host molecules) during the rehydration process, while they shorten upon water release (dehydration process) bridging the adjacent chains.
original crystal, as defined by space group symmetry and lattice parameters, suggests that the dehydration process of the monohydrate phase 1·H2O leads to the formation of an isomorphic desolvate (1_α), that is a crystalline phase that retains the molecular packing of the parent hydrate after dehydration.42,43 Finally, the lack of strong interactions between the adjacent chains in the crystal lattice of 1_α could account for its ability to reincorporate water giving back 5301
DOI: 10.1021/acs.cgd.6b00826 Cryst. Growth Des. 2016, 16, 5294−5304
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Figure 6. XRPD patterns of 1·H2O, 1_α, and 1_β. The broad signal at 7° (2θ) is due to the Kapton polymer window of the Anton-Paar hot chamber.
conditions than 1_α (see above) appears quite surprising. However, the comparative inspection of the crystal lattice of the two anhydrous forms offers some hints to try to rationalize their different behavior. The region of interest is that connecting two adjacent chains of the host molecules which in both the lattices are held together by a NH···π type interaction involving the NH group provided by the Trp3 residue and the phenyl ring belonging to Phe4 and Trp3 in 1_α and 1_β, respectively (see Table 2). As a result of these interactions, in 1_β, the Trp rings describe a zigzag pattern, and each void is separated from the adjacent ones (Figure 7) by the hydrophobic groups belonging to four MEN11282 molecules (i.e., the aromatic rings of the Trp and Phe residues) which
In summary, from a molecular point of view, it appears that chains of adjacent MEN11282 molecules are joined by strong H-bonds in the hydrate through the water molecules, the latter are replaced by the significantly weaker (Trp3)NH···π(Phe4) contacts in the isomorphic dehydrate, hence the instability of 1_α toward rehydration. On the contrary, the anhydrous phase 1_β is stable when left in air at room temperature as well as when kept at r.t. in a closed chamber maintained at constant relative humidity (ca. 75%) by using a saturated solution of NaCl. In addition no phase changes are observed from r.t. to 583 K as provided by the VT-XRPD patterns collected in the 298−610 K range in air (see Figure 5). Consistently no thermal events were observed in the DSC curve until the melting/decomposition events occur (Figure S14, melting temperature: 603 K).44,45 As in 1_α each independent hexapeptide molecule of 1_β is H-bonded to four different symmetry related molecules through the carboxylic group of Asp1 and all the amide groupings of the bicyclic ring (Table 2), except O(2)/N(7)H(7) and O(5)/N(6)H(6), together with O(3)/N(3)H(3). In addition in this case also the O(4)/N(5)H(5) is not involved in intermolecular contacts, and the NH hydrogen-bond donor of Trp3 is involved in a NH···π interaction, now with the phenyl ring of Trp3 provided by a symmetry related molecule belonging to the adjacent chain. Finally, the arrangement of MEN11282 molecules within each chain is very similar to that already observed in 1·H2O and 1_α (Figures 3c and 6). A map of the cavities, obtained by using a probe of radius 1.2 Å, shows no channels in the crystal lattice and gives 1.4% of empty volume in the unit cell (ca. 52 Å3) compared to 1.1% of the 1_α form. The packing efficiency is the same as 1·H2O and 1_α (i.e., K.P.I. = 0.69). In other words the two anhydrous forms appear quite similar in terms of number, strength, and type of H-bond interactions, voids and packing efficiency. On these grounds the stability toward hydration observed for the 1_β form even in more severe
Figure 7. Crystal packing of 1_β view along the b axis highlighting the NH···π interaction involving the Trp3 residues of adjacent chains and the voids mapped in the crystal lattice. 5302
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interactions, and isolated voids are located between adjacent chains. In addition the polycrystalline powder of 1_α easily rehydrates and gets back the 1·H2O form. We speculate that the replacement of the strong H-bonds in the hydrate form with the significantly weaker (Trp3)NH···π(Phe4) contacts in the isomorphic dehydrate could account for the instability of the latter toward rehydration. By contrast the anhydrous form 1_β is stable toward hydration. In this case also there are isolated voids in the region between adjacent chains (the latter joined by (Trp3)NH···π(Trp3) contacts); however at variance with 1_α, they are completely enclosed by the hydrophobic rings of the Trp and Phe residues provided by four symmetry related molecules, thus preventing the access of the water molecules. Finally, the molecular structure of the bicyclic hexapeptide MEN11282 was discussed and compared to the literature data focusing the attention on the double β-turn motif and on the arrangement of the Trp-Phe side chains, due to their key role in binding to the hNK-2 receptor. Modeling data suggest that the Trp3-Phe4 sequence is quite flexible and able to reorient in response to the local environment, which is essential for the optimal fit into the receptor.
could prevent the access of the water molecules to the crystal lattice. By contrast in 1_α, though voids are not connected by a channel, they are not completely screened from the outside by the side arms of the MEN11282 molecules (Figure 8).
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Figure 8. Crystal packing of 1_ α view along the a axis highlighting the NH···π interaction involving the Phe4 residues of adjacent chains and the voids mapped in the crystal lattice.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b00826. Additional XRPD patterns, DSC and TGA curves; molecular views of different conformational isomers as found from modeling studies; superimposition of the MEN11282 molecule as found in the X-ray structures of 1·H2O, 1_α, and 1_β; crystal packing of 1·H2O (PDF)
In summary, the isomorphic dehydration and hydration process involving 1·H2O and 1_α and the stability of 1_β toward water uptake was rationalized from their crystal structures. In 1·H2O the isolated water molecules are Hbonded to MEN11282 molecules belonging to adjacent chains and, once released upon heating, are replaced by isolated voids and weak (Trp3)NH···π(Phe4) contacts giving 1_α. In the latter species we postulate that the (Trp3)NH···π(Phe4) interactions, which join adjacent chains, lengthen or shorten in response to the environment changes allowing the water to escape. In 1_β MEN11282 molecules originate a very similar packing motif: isolated voids are present between adjacent chains of molecules, which are held together by (Trp3)NH···π(Trp3). However, at variance with 1_α, in the stable anhydrate 1_β, each void, being enveloped in a hydrophobic pocket, is completely screened from the environment.
Accession Codes
CCDC 1482792−1482794 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
CONCLUSION In this paper we have presented the solid state characterization of 1 (MEN11282), a precursor and key intermediate of MEN11420, the latter currently in phase II clinical trials for the treatment of infant colic. A combination of experimental (XRD by both single crystal and microcrystalline powder and DSC) and modeling (MM, MD, and DFT calculations) techniques were used to investigate the behavior of 1. During the solidstate study, a monohydrate (1·H2O) and two different anhydrous forms, namely, 1_α and 1_β, were identified. The stable monohydrate crystalline phase of MEN11282, 1· H2O, loses water upon heating giving the anhydrous phase 1_α without major changes in the crystal structure as provided by SCXRD (1·H2O) and XRPD (1_α) structure determinations. In fact in both the solids the MEN11282 molecules are arranged almost identically within chains propagating along the b axis. However, in 1·H2O adjacent chains are held together by the water molecules (located in isolated voids) through strong H-bonds which describe a zigzag motif, while in 1_α chains of MEN11282 molecules are connected by NH···π(Phe4)
*E-mail: paola.paoli@unifi.it. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Authors would like to thank the Centro di Cristallografia Strutturale (CRIST) of the University of Firenze for the X-ray diffraction facilities and Dr. Samuele Ciattini for his valuable technical assistance. In addition, authors would like to thank Dr. Andrea Ienco (ICCOM-CNR, Firenze) and Prof. Lucia Maini (Dip. Di Chimica “G. Ciamician”, University of Bologna) for their technical help. This work was supported in part by Regione Toscana [POR CREO FESR 2007-2013 Linea d’intervento 1.1.C].
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