Combined Virtual/Experimental Multicomponent Solid Forms Screening of Sildenafil: New Salts,

43 44 New multicomponent solid forms of sildenafil have been discovered 45 by means of a combined virtual/experimental cocrystal screening. Coformer 46 selection of candidates was conducted based on an in silico screening method from 47 a database of more than 2000 organic compounds, and the intensive experimental 48 screen produced 23 new solid forms. Since the 12 coformers chosen have a 49 combination of phenol and carboxylic acid groups, a variety of cocrystals, salts, and 50 hybrid salt-cocrystals were discovered and characterized. 51 52 53 54 55 56 57 58 59


INTRODUCTION 60 61
Cocrystals of active pharmaceutical ingredients (APIs) have received massive attention over the past 62 decade because they offer many opportunities to improve physicochemical properties of drugs.1,2 63 Indeed, solubility is one of the most important properties for a drug compound since it has a direct 64 impact on bioavailability, and the cocrystal approach is a versatile toolbox to tune this and another 65 important property3,4 such as stability5 because of the high number of available potential coformers. 66 On February 2018, the Food and Drug Administration (FDA) released a final guidance titled 67 "Regulatory Classification of Pharmaceutical Co-Crystals Guidance for Industry",6 providing applicants 68 planning to submit new drug applications with information on the regulatory classification of 69 pharmaceutical cocrystals, classifying them as a new form of the API, comparable in that respect to 70 polymorphs, hydrates, salts, etc. The FDA asks the applicants to provide evidence to demonstrate that 71 "both the API and coformers are present in the unit cell" and "the component API and coformer co-exist 72 in the cocrystal which interact nonionically". The FDA guidance suggests that the applicant consider the 73 difference of ΔpKa between the API and the coformer or to provide evidence that proton transfer has not 74 occurred in the lattice by means of spectroscopic tools or other orthogonal approaches. Thus, from a 75 regulatory point of view, it is very important to assess the proton transfer in a multicomponent 76 Sildenafil, the active principle of Viagra, is the first oral drug used for the medical treatment of erectile 77 dysfunction in elderly patients, and it was initially used as an antihypertensive drug8,9 but due to its 78 poor aqueous solubility and low bioavailability it is generally formulated as sildenafil citrate.10 This 79 solid form still exhibits moderate bioavailability, and this is the reason why some efforts have been 80 conducted to discover new salts and cocrystals with enhanced physicochemical properties. In this sense, 81 a sildenafil/acetylsalicylic cocrystal exhibiting enhanced intrinsic dissolution rate compared to sildenafil 82 citrate has been reported.11 Moreover, pharmacokinetics of salts and cocrystals of sildenafil with 83 dicarboxylic acids has been studied, and the glutarate salt was revealed to be a good candidate for 84 alternative formulation of the citrate salt.  Table S1). Stoichiometry has been 130 assessed based on NMR and thermogravimetric analysis (TGA) measurements when the crystal 131 structure is not available. In those cases where the crystal structure has not been solved, the definition of 132 the form as a salt or a cocrystal has been done based on the probability of proton transfer determined 133 with eq 3. 134 Twenty-three multicomponent forms of SIL (cocrystals, salts, and hybrid salt−cocrystal) have been 135 obtained through a cocrystal screening with 8 out of the 12 coformers used. Five cocrystal forms of SIL 136 have been obtained with three coformers: two forms with quercetin in a 1:1 stoichiometry (one as an 137 isopronanol solvate, SILQUE I, and one as a tetrahydrofuran solvate, SIL-QUE II); two with resorcinol 138 in two different stoichiometries (one in a 1:1 molar ratio, SIL-RES I, and one in a 1:2 molar ratio, SIL-139 RES II); one form with methyl gallate in a 1:1 stoichiometry, SIL-MEG. Fourteen salts of SIL have been 140 obtained with five coformers: one form with 3,4-dihydroxybenzoic acid in a 1:1 stoichiometry as an 141 isopropanol solvate, SIL-3,4-DHBA I; four forms with tartaric acid in two different stoichiometries: two 142 in a 1:1 molar ratio (an anhydrous form, SIL-TAR I, and an isopropanol solvate, SIL-TAR III); two in a 143 2:1 molar ratio (an anhydrous form, SIL-TAR II, and an isopropanol solvate, SIL-TAR IV); two forms 144 with caffeic acid in a 2:3 stoichiometry (one as an anhydrous form, SIL-CAF I, and one as a 145 monohydrate, SIL-CAF II); three forms with 3-hydroxybenzoic acid in a 1:1 stoichiometry (one as an 146 acetonitrile solvate, SIL-3-HBA I, one as a tetrahydrofuran solvate sesquihydrate, SIL-3-HBA II, and 147 one as an anhydrous form, SIL-3-HBA III); four forms with 4-hydroxybenzoic acid in a 1:1 148 stoichiometry (two as anhydrous forms, SIL-4-HBA I and SIL-4-HBA III, one as a hemiisopropanol 149 solvate, SIL-4-HBA II, and one as a tetrahydrofuran solvate, SIL-4-HBA IV). Four hybrid salt-cocrystal 150 forms of SIL have been obtained with two coformers: three forms with 3,4-dihydroxybenzoic acid in 151 two different stoichiometries (two of them in a 1:2 molar ratio as acetonitrile solvates, SIL-3,4-DHBA II 152 and SIL-3,4-DHBA III, and one in a 2:3 molar ratio as a dehydrate, SIL-3,4-DHBA IV) and finally one 153 form with 3-hydroxybenzoic acid in a 2:3 stoichiometry as a dehydrate, SIL-3-HBA IV. 154 2.2.5. X-ray Crystallographic Analysis. Single crystal X-ray diffraction intensity data of the different 155 crystal forms of sildenafil were collected using a D8 Venture system equipped with a multilayer 156 monochromator and a Mo microfocus (λ = 0.71073 Å). Frames were integrated with the Bruker SAINT 157 software package using a SAINT algorithm. Data were corrected for absorption effects using the 158 multiscan method (SADABS).20 The structures were solved and refined using the Bruker SHELXTL 159 Software Package, a computer program for automatic solution of crystal structures and refined by 160 fullmatrix least-squares method with ShelXle Version 4.8.0, a Qt graphical user interface for SHELXL 161 computer program.21 162 Powder X-ray diffraction patterns were obtained on a PANalytical X'Pert PRO MPD diffractometer in 163 transmission configuration using Cu Kα1 + 2 radiation (λ = 1.5406 Å) with a focusing elliptic mirror 164 and a PIXcel detector working at a maximum detector's active length of 3.347°. Configuration of 165 convergent beam with a focalizing mirror and a transmission geometry with flat sample sandwiched 166 between low absorbing films measuring from 2 to 40° in 2θ, with a step size of 0.026° or from 2 to 70° 167 in 2θ, with a step size of 0.013° with measuring times of 30 min to 4 h. The powder diffractograms were 168 indexed, and the lattice parameters were refined by means of LeBail fits by means of Dicvol04,22 and 169 the space groups were determined from the systematic absences. A summary of crystal data and relevant 170 refinement parameters are given in Tables 1 and 2 formation.23 This computational tool has been validated using experimental data extracted from the 199

literature. 200
The difference between the calculated energy of the cocrystal and the pure components was used to rank 201 potential cofomers.24 This approach uses surface site interaction points (SSIPs) calculated from the ab 202 initio molecular electrostatic potential surface (MEPS) of the isolated molecule in the gas phase.19,25 203 The interaction of a molecule with its environment is described by a discrete set of SSIPs pairing energies of the 1:1 cocrystal and the pure components was calculated for each 226 sildenafil/coformer combination using a coformer database which contains more than 2000 organic 227 compounds (including 860 products from the GRAS list). The coformers were ranked in order of 228 decreasing ΔE, and only 12 coformers were chosen from the top 100 compounds according to toxicity 229 criteria and probability of success in a cocrystallization experiment (Table 3). This theoretical approach 230 defines an energy threshold of 11kJ/mol where the probability of cocrystal formation is higher than 231 50%. Thus, only coformers with ΔE > 11 kJ/mol were chosen for experimental screening 232 Since sildenafil has a strong basic group, the formation of salts with strong carboxylic acids is expected. 233 In fact, the formation of a salt or a cocrystal can be assessed based on the "rule of thumb"31 which 234 states that salts are formed when ΔpKa [pKa(base) − pKa(acid)] ≥ 3, and a cocrystal is expected when 235 this value is ≤0, the combinations with a value 0 ≤ [pKa(base) − pKa(acid)] ≤ 3 being much less reliable 236 and falling around a "salt-cocrystal continuum" region.32 This uncertainty motivated the analysis and 237 correlation by Cruz-Cabeza33 of a big set of experimental cocrystal/salt data in order to develop a more 238 reliable equation to predict the salt/cocrystal outcome. According to this statistical analysis, eq 3 allows 239 prediction of the probability of proton transfer around the region of ΔpKa values between −1 and 4. 240 241 P(%) = 17ΔpKa + 28 (3) 242

243
Sildenafil has a basic functional group (piperazine) with a pKa value of 6.78,34 and we have applied this 244 statistical approach to the coformers with acidic groups selected from the virtual cocrystal screening to 245 assess the probability of salt formation (Table 4)  Although 184 structures showed total proton transfer between donor and acceptor, 63 of them revealed 262 that cocrystals or mixed salt-cocrystals were formed. This encouraged us to test the carboxylic acids 263 previously chosen in the virtual cocrystal prediction. Table 5   In addition, one molecule of water is also present acting as a bridge between carboxylates ( Figure 6). 293 Nonionized 3-hydroxybenzoic acid molecules are located in channels establishing strong hydrogen 294 bonds with other 3-hydroxybenzoate molecules (Figure 7). 295

3-Hydroxybenzoic Acid Salt THF Hemisolvate Sesquihydrate (SIL-3-HBA II). The salt formed 296
by 3-hydroxybenzoic acid and sildenafil crystallizes with one molecule of sildenafil cation, one 297 molecule of the carboxylate, half disordered molecule of THF, and 1.5 molecules of water in the 298 asymmetric unit. In spite of the different degree of proton transfer, this solid form is isostructural to the 299 hybrid 3-hydroxybenzoic acid saltcocrystal, and the same interactions between sildenafil and 3-300 hydroxybenzoate molecules are established. Moreover, identical channels are formed but filled by 301 disordered tetrahydrofuran and water molecules instead of molecules of 3-hydroxybenzoic acid. Only 302 small differences between both structures are present like, for instance, centroid−centroid distances 303 measured betwee pyrimidinone rings and torsion angles of propyl groups (Figure 8). However, the antiparallel dipole−dipole interactions between stacked pyrimidinone rings are not 319 observed in this form. This is the only structure of this family of hybrid salts−cocrystals where water is 320 not present, and this produces a different architecture of the coformer self-assembling, which consists of 321 layers of alternate carboxylic/carboxylate interactions ( Figure 11). 322 3.4. Dissolution Study. The dissolution studies were carried out at pH 1.2, pH 6.5, and FaSSIF (pH 323 6.5), which represent the average pH values of the fast state stomach and intestine, respectively. SIL has 324 pH dependent solubility which decreases with an increase in pH. One of the major challenges in the 325 dissolution study of multicomponent entities is continuous change in the solution composition due to 326 precipitation of either of the component over the dissolution testing period. The solubility data generated 327 may be erroneous due to limitations of the analytical method; for example, estimations carried out by 328 UV spectrophotometry are subject to the overlap in the absorption spectra of the two components. We 329 have used the HPLC method to quantify the amount of SIL dissolved; hence we see some difference in On the other hand D1h for SIL-RES II > SIL-QUE I and SIL-QUE II. As expected, the amount 337 dissolved at pH 6.5 was at least 10 times lower than the amount dissolved at pH 1.2. But in both 338 conditions, the amount of SIL dissolved was significantly higher for salts than for cocrystals and hybrid 339 salt−cocrystals. Most of the cocrystals and hybrid salt−cocrystals did not provide any release of SIL at 340 pH 6.5 or even in FaSSIF, which contains an additive like lecithin included in the dissolution media.
Thus, although an exhaustive solubility study of the new forms was not the main objective of this 342 research, our data suggest that SILTAR could be a potential alternative (in terms of bioavailability) to 343 the commercial citrate salt of sildenafil.