Influence of Solvent Selection on the Crystallizability and Polymorphic Selectivity Associated with the Formation of the “Disappeared” Form I Polymorph of Ritonavir

The comparative crystallizability and polymorphic selectivity of ritonavir, a novel protease inhibitor for the treatment of acquired immune-deficiency syndrome, as a function of solvent selection are examined through an integrated and self-consistent experimental and computational molecular modeling study. Recrystallization at high supersaturation by rapid cooling at 283.15 K is found to produce the metastable “disappeared” polymorphic form I from acetone, ethyl acetate, acetonitrile, and toluene solutions in contrast to ethanol which produces the stable form II. Concomitant crystallization of the other known solid forms is not found under these conditions. Isothermal crystallization studies using turbidometric detection based upon classical nucleation theory reveal that, for an equal induction time, the required driving force needed to initiate solution nucleation decreases with solubility in the order of ethanol, acetone, acetonitrile, ethyl acetate, and toluene consistent with the expected desolvation behavior predicted from the calculated solute solvation free energies. Molecular dynamics simulations of the molecular and intermolecular chemistry reveal the presence of conformational interplay between intramolecular and intermolecular interactions within the solution phase. These encompass the solvent-dependent formation of intramolecular O–H...O hydrogen bonding between the hydroxyl and carbamate groups coupled with differing conformations of the hydroxyl’s shielding phenyl groups. These conformational preferences and their relative interaction propensities, as a function of solvent selection, may play a rate-limiting role in the crystallization behavior by not only inhibiting to different degrees the nucleation process but also restricting the assembly of the optimal intermolecular hydrogen bonding network needed for the formation of the stable form II polymorph.


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The supplementary material supports the main manuscript by providing further details of the following: Section S1 provides further details on induction time analysis using classical nucleation theory.Figure S1 shows the DSC patterns of ritonavir nucleation in different solvents.Figure S2 presents the exponential fitting of nucleation rates in toluene with supersaturation for estimating the nucleation rate at S = 5.Table S1 lists the melting points, enthalpies of fusion, and full widths at half maximum (FWHM) for toluene, acetonitrile, ethyl acetate and acetone based on the DSC data.Table S2 presents the comparisons of the relevant nucleation properties of ritonavir form I in different solvents and other compounds (butyl paraben 1 , form PABA [2][3][4] , methyl stearate 5 , TFA form II 6 ).

S1. Induction Time Analysis Using Classical Nucleation Theory
Measured induction times were analysed using classical nucleation theory (CNT) to calculate the nucleation rate (J) through the Arrhenius relationship as follows: where  is the pre-exponential kinetic factor,  is the thermodynamic parameter, ∆  is the free energy assuming a spherical nucleus growth to critical size,  is the supersaturation (S = C/C*; C = concentration in solution (g/g solvent), C* = solubility at the crystallisation temperature),  is the Boltzmann constant,  is the nucleation absolute temperature,  is the effective interfacial energy, and  is the molecule volume.
The pre-exponential kinetic factor, A, associating with the molecular kinetics of the nucleation process, can be determined 7 by Eq. ( 3): where C 0 is the concentration of nucleation sites, * is the attachment frequency of building units to a nucleus and can be impacted by changes of solvent, supersaturation and molecular conformation of the solute, z is the Zeldovich factor which can be derived and calculated 8,9 by: Considering the dependence of Zeldovich factor and attachment frequency on supersaturation, and the higher concentration leading to higher attachment frequency, the pre-exponential kinetic factor, A, and the classical nucleation expression can be re-written as Eqs.( 5) and ( 6), respectively: where the pre-exponential kinetic factor constant, A 0 , and the thermodynamic parameter  can be derived from the intercept and slope, respectively, by plotting the linear function of The attachment rate, f* C 0 , can be estimated by combining equations ( 3) -( 5): From the CNT, the critical size,   , for the nucleation cluster was calculated from:

S4. Comparisons of Nucleation Properties of Different Compounds
Table S2 compares the relevant nucleation properties of ritonavir form I in different solvents and other compounds (butyl paraben 1 , -form PABA [2][3][4] , methyl stearate 5 , TFA form II 6 ) from literature.The nucleation rate, critical nuclei size, number of nuclei and effective surface energy obtained from this study are generally compatible with these compounds from literature with two compounds (methyl stearate and TFA form II) having much higher nucleation rates obtained using the KBHR method 10,11 with progressive nucleation mechanism.The supersaturation values for nucleation studies of ritonavir form I in various solvents in the current study are serval times higher than the other compounds from literature with the nucleation driving force being also higher than butyl paraben, and the average induction time being in the similar order of magnitude to -form PABA but about 10 times longer than butyl paraben.
Table S2.Nucleation parameters and the associated properties of different compounds in various solvents.Note that the grey filled area indicates the corresponding solvent not being studied in the literature, the symbol (-) means the value of the property not being available and the grey area presents the solvent not being studied in the paper.

Figure S1 .
Figure S1.The DSC patterns of ritonavir nucleation in different solvents.

Figure S2 .
Figure S2.Exponential fitting of nucleation rates in toluene with supersaturation for estimating the nucleation rate of 11.51 m -3 s -1 at S = 5.

Table S1 .
Melting points, enthalpies of fusion, and FWHMs for toluene, acetonitrile, ethyl acetate and acetone based on the DSC data.Note that unfortunately, the original DSC data for ethyl acetate is no longer available due to an instrument software upgrade.