Preparation and Solid-State Characterization of Nonstoichiometric

Preparation and Solid-State Characterization of Nonstoichiometric Cocrystals of a Phosphodiesterase-IV Inhibitor and L-Tartaric Acid Variankaval,*,†

Wenslow,†

Murry,†

Hartman,†

Narayan Robert Jerry Robert Elizabeth Kwong,‡ Sophie-D. Clas,‡ Chad Dalton,‡ and Ivan Santos†

Roy

Helmy,†

CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 3 690-700

Merck Research Laboratories, Merck & Co., PO Box 2000, Rahway, New Jersey 07065, and Merck Frosst, Kirkland, Quebec, Canada ReceiVed September 9, 2005; ReVised Manuscript ReceiVed NoVember 28, 2005

ABSTRACT: Evidence for a series of nonstoichiometric, isostructural, cocrystalline complexes of L-883555, a phosphodiesteraseIV inhibitor, and L-tartaric acid with stoichiometries ranging from 0.3:1 to 0.9:1 is reported here. The free base form of this compound had insufficient bioavailability and, hence, could not be developed as a candidate for safety assessment studies. Several L-tartaric acid complexes were produced during an attempted salt-formation process, with the objective of increasing the bioavailability. It was found that the amount of L-tartaric acid incorporated in the cocrystalline complexes could be controlled by adjusting the acid: base ratio in the reaction mixture without accompanying proton transfer between acid and base. Spectroscopic techniques were employed to locate the site of intermolecular interaction between the acid and base as the N-oxide group in the base and the carboxylic acid of L-tartaric acid. Thermal and spectroscopic analysis of the degradation behavior for the various complexes showed the existence of at least two types of binding between the acid and base in those complexes with stoichiometries >0.5:1. The canonical hemitartrate complex was found to be more thermally stable than the other complexes, with acid:base stoichiometries lesser than or greater than 0.5:1 and was found to have much higher bioavailability than the free base in rhesus monkeys. This work shows the potential of designing suitable cocrystalline complexes driven by favorable interactions between an acid and base in cases where conventional proton transfer does not occur to form a true salt, offering a route toward increased bioavailability in poorly absorbed compounds. Introduction There are several advantages of preparing and developing the salt form of a drug. The principal among them is higher aqueous solubility, which usually translates to higher bioavailability. It is also possible that the dissolution rate of a salt in aqueous systems is much higher than that of the corresponding free acid or base, since the diffusion layer surrounding the dissolving salt has a pH different from that of the bulk. In situations where the free acids or bases are oily or low melting, salt formation can produce solids that have improved crystallinity and, hence, enhanced physicochemical stability. From a process development point of view the salt formation step could be used as an isolation method to obtain high-purity material. Less common but nevertheless useful drivers for salt formation include avoiding topical irritation, taste masking, and avoiding corrosivity of manufacturing equipment when the free acid or base is developed.1 Salt formation of a free base with an acid counterion is characterized by proton transfer from the acidic to the basic species. At present there exists no a priori prediction procedure to determine the feasibility of salt formation in a given acidbase pair. In those cases where salts are crystallized from solution, these salts are typically characterized by a fixed stoichiometry of acid to base. There are three possibilities when a mixture of acidic and basic species are concomitantly crystallized from solution: (i) a true salt can be formed with complete proton transfer from the acidic to the basic species, (ii) the molecules can cocrystallize in a single crystalline lattice, and (iii) the kinetics of the cocrystallization may be so prohibitive that a physical mixture of the two species is obtained. Which of these three processes actually occurs depends on the * To whom correspondence [email protected]. † Merck Research Laboratories. ‡ Merck Frosst.

should

be

addressed.

E-mail:

pKa difference between the acid and base, the crystallization conditions, and the relative steric and electrostatic properties of the two molecules. According to a general rule of thumb, the probability of formation for a true salt is high when the pKa difference between acid and base is at least greater than 2 units in aqueous solutions.2 When this is not the case, one of the possibilities that can occur is the cocrystallization of the two species in a single crystalline lattice. This has been observed in such systems as the bidentate hydrogen-bonded units of 2-amino 6-methylpyridine with carboxylic acids,3 in inclusion crystals of cholic acid and cholamide with alcohols,4 in cocrystals of bases with carboxylic acids,5-7 and in fats and lipids.8-10 The packing motif of these crystals is undergirded by the presence of complementary hydrogen-bonding donor and acceptor groups in the pairs of molecules involved in the supramolecular complex formation. All of these complexes are characterized by well-defined stoichiometric ratios of the molecules bearing the hydrogen-bonding donor and acceptor groups. Nonstoichiometric cocrystalline complexes of acidic and basic species in the solid state are, however, quite rare. The reports available in the literature on nonstoichiometric, organic, solid-state complexes have been restricted to channel hydrates or solvates of drugs,11-13 gas hydrates of the kind observed in oil drilling,14-16 and polymer-solvent complexes, such as polyelectrolytesurfactant.17 Each type of nonstoichiometric compound has its own unique features. A singular characteristic of most channel hydrates and solvates of drug molecules is the ability for facile removal of the solvent from the crystal lattice and the isomorphous nature of the crystals compared to the corresponding anhydrous materials. In such systems the crystal lattice is punctuated by channels, the size of which determines the maximum extent and type of solvent molecules that can be included.18,19 The gas hydrates are unique in the sense that it is the gas molecules that are captured in an icelike network structure of water molecules containing voids large enough for gas incorporation. The nonstoichiometric complexes of poly-

10.1021/cg050462u CCC: $33.50 © 2006 American Chemical Society Published on Web 01/21/2006

Cocrystals of L-883555 and L-Tartaric Acid

electrolyte-surfactants are types of ionomer in which the polyelectrolyte chain units form a Coulombic bond with the surfactant molecules. There have not been any reports on the formation of nonstoichiometric cocrystalline complexes in organic systems to our knowledge. Compound I is a potent phosphodiesterase-IV (PDE(IV)) inhibitor with indications for asthma and/or chronic obstructive pulmonary disease. In every drug development program the first steps after the identification of a molecule as a potential drug candidate is the development of the drug in a stable solid form for dosing in animals. These investigations shed light on potential toxic effects and the levels at which such toxicity is observed in animals, so that doses for humans can be suitably established in clinical trials. The primary requirement for the solid form of the drug is that it is bioavailable in the plasma in sufficient amounts to enable identification of toxicity. Compound I is a neutral molecule which is a stable crystalline solid amenable to simple formulation; however, it has very poor pharmacokinetic properties in rats and rhesus monkeys. These studies are referred to as safety assessment studies. As a result, it would be very difficult to develop this compound utilizing standard formulations for oral delivery. The compound does not have any acidic or basic sites that are amenable for salt formation, with the exception of the pyridine N-oxide functionality. Salts of pyridine N-oxides have been formed utilizing strong mineral acids. Unfortunately, all attempts to form distinct salts of I with strong acids were not fruitful. We next turned our attention to the possibility of producing a cocrystalline complex that may be amenable to a simple oral formulation without dissociation and exhibit improved pharmacokinetic properties. In this paper, we report the preparation of stable cocrystalline complexes of compound I with L-tartaric acid wherein the primary interaction between the acid and base in the crystal lattice is hypothesized to be intermolecular hydrogen bonding as opposed to protonation of the base. The unique nature of these complexes arises from the fact that the acid:base ratio in the complex can be tuned, depending on the amount of acid charged to the reacting system. Furthermore, the crystal lattices of these complexes do not seem to undergo significant perturbation upon changing the acid:base ratio and, as such, are isomorphous crystal forms. Even though several complexes with specific acid:base ratios are quoted in this work, the possibility of a continuous series of such cocrystals is apparent, and hence the use of the term nonstoichiometric may be appropriate. These L-tartaric acid complexes demonstrated much higher bioavailability in rhesus monkeys compared to the free base form of compound I. Specifically, the hemitartrate complex was found to have better physical properties and, hence, was chosen as a viable solid form for safety assessment studies. Materials and Methods Process for Making Tartrate Complexes. The free base (compound I) was slurried in 60 volumes of 15% aqueous ethanol. This mixture was heated to reflux until a clear solution was obtained. L-Tartaric acid (variable equivalents) was then added to this mixture, and the resulting solution was slowly cooled to room temperature over 3 h. The slurries obtained at room temperature were filtered and dried overnight ∼60 °C under vacuum with an N2 sweep. 13 C and 15N NMR. All SS-NMR spectra were obtained on the Bruker DSX-400 NMR spectrometer (9.4 T magnetic field strength) using a Bruker double-resonance CPMAS probe and a standard CPMAS pulse sequence. For 1H/13C CPMAS, the 13C and 1H resonance frequencies are 100.63 and 400.14 MHz, respectively, at this magnetic field strength. 1 H/13C CPMAS NMR experiments were performed using a standard CPMAS pulse sequence with 2.0 ms contact times; 4K of data points

Crystal Growth & Design, Vol. 6, No. 3, 2006 691 were acquired in 60 ms and then zero-filled to 8K before transformation using 40.0 Hz of line broadening. A total of 1K of scans were acquired with a recycle delay of 3.0 s. The rotor frequency was 15.0 kHz. All 13 C spectra were referenced to TMS using the carbonyl carbon of glycine (176.03 ppm) as a secondary reference. 1H T1 experiments were performed with a standard inversion recovery pulse sequence. For 1H/15N CPMAS, the 15N and 1H resonance frequencies are 40.56 and 400.14 MHz, respectively, at this magnetic field strength. 1H/15N CPMAS NMR experiments were performed using a standard CPMAS pulse sequence; 1K of data points were acquired in 20 ms and then zero-filled to 2K before transformation using 100.0 Hz of line broadening. A total of 12K scans were acquired with a recycle delay of 3.0 s. The rotor frequency was 5.0 kHz. All 15N spectra were referenced to liquid CH3NO2 using the nitrogen in natural-abundance glycine (-342 ppm) as a secondary reference. Attenuated Total Reflectance (ATR) Fourier Transform Infrared Spectroscopy. A Nicolet Nexus-670 FT-IR (Nicolet Instrument Co., Madison, WI) equipped with a DTGS detector was employed for all experiments. A Golden Gate Diamond ATR sampling accessory (Specac Inc., Smyrna, GA) was employed for ATR-FT-IR experiments. Each sample was placed on the ATR sampling device and aligned according to the manufacturer’s recommendation. In all experiments, a torque of ∼20 cNm was applied by the Golden Gate Diamond ATR sampling device. Each spectrum represents 32 coadded scans measured at a spectral resolution of 2 cm-1 in the 4000-600 cm-1 range with an aperture of 36. Spectral data were acquired with Omnic ESP software, version 5.1 (Nicolet Instrument Co.). Bioavailability Studies. Four rhesus monkeys were used for these studies. The studies were performed at ITR Laboratories Canada Inc. They were housed in a CAALAC-accredited facility in accordance with the guidelines. The doses were given either as 5 or 10 mL/kg volume. The pharmacokinetic parameters were calculated using WinNonlin version 3.0. The concentrations of compound I in the plasma of animals were analyzed by HPLC. The following conditions were used: HPLC system, Hewlett-Packard 1090 HPLC system Ace3 C-18, 150 × 4.6 mm, 3 mm with a mobile phase of 0.1% trifluoroacetic acid in water (67%) and 0.1% trifluoroacetic acid in acetonitrile (33%). The flow rate was 1.0 mL/min. The oven temperature was 40 °C. A UV detector at 250 nm was used. Other parameters include an injection volume of 25 mL and a stop time of 6 min. Standard solutions of the compound with concentrations of 0.01-5 mg/mL were prepared by serial dilution of the stock solution with acetonitrile. To individual 0.1 mL aliquots of plasma blanks was added an equal volume of the standard solution. The mixture was vortexmixed for 15 min and centrifuged at 14 000 rpm for 15 min. The clear supernatant was transferred to a microvial and injected into the HPLC column.

Results and Discussion Compound I (mol wt 456.5), N-cyclopropyl-1-{3-[6-(1hydroxy-1-methylethyl)-1-oxidopyridin-3-yl]phenyl}-1,4-dihydro[1,8]naphthyridin-4-one 3-carboxamide (cf. Figure 1), is a phosphodiesterase IV (PDE-IV) inhibitor.20 It is a crystalline solid with a melting point >200 °C.21 The predicted pKa (ACD/ pKa DB, ACD Labs, Toronto, Canada) of relevant groups in the molecule are also shown in Figure 1. The aqueous solubility of the free base is 7.5 ( 0.3 µg/mL,22 and it was found to produce unacceptable exposures in animals (see Table 4). Hence, a salt form of compound I was highly desirable for conducting safety assessment studies. Attempts at salt formation with conventional anions yielded a few stable salt forms, including succinate, tartrate, and citrate. The only feasible counterion that produced a thermally stable crystalline solid with compound I was L-tartaric acid. Characterization of Solids Produced from the Crystallization Experiments. In conventional salt formation, a single acid:base stoichiometry or a series of discrete stoichiometries is usually observed, depending upon the degree of acidity/ basicity of the free base and acid, respectively, the pKa difference

692 Crystal Growth & Design, Vol. 6, No. 3, 2006

Variankaval et al.

Figure 1. Molecular structures of compound I and L-tartaric acid. The predicted pKa values of basic groups in compound I are as follows: N(1), too weak; N(2), 4.21(0.70); N(3), -0.69(0.53); N(4), -3.14(0.20); OH, 12.48(0.29). Table 1. Residual Solvent and L-Tartaric Acid Content in the Complexes mole ratio acid:base

amt of acid added (equiv)

residual ethanol (wt %)

residual water (wt %)

0.3:1 0.4:1 0.5:1 0.7:1 0.9:1

1 2 4 10 20

1.58 1.4 0.8 0.7 0.4

NDa 1.2 0.7 0.7 0.6

a

ND ) not determined.

between acid and base, the solubility of the components in solution, and the pH of the solution. On the basis of Figure 1 it is clear that the pKa difference between L-tartaric acid (pKa ) 3.02 [23]) and the pyridinyl N, N(2) (pKa ) 4.21), may be insufficient for proton transfer. The other basic groups in compound I also appear at first glance to be unsuitable for salt formation. In agreement with these expectations, the results of the aqueous titration (cf. Table 1) show that the amount of L-tartaric acid incorporated in the solid seems to scale with the equivalents of L-tartaric acid charged to the reaction vessel. The titration accounts for all the L-tartaric acid present in the solids and is not discriminating against various types of binding should they existsfor example, deprotonated acid, hydrogen-bonded acid, and free L-tartaric acid. To decipher the nature of the acid: base binding, further studies were undertaken. Gas chromatography and Karl Fischer titration results show that ethanol and water are also present in the solids.24 The residual solvent content did not undergo any change upon drying in a vacuum oven at 60 °C overnight under a N2 sweep, indicating strong binding of these solvents in the solids. In general, reduced amounts of ethanol and water are observed at higher stoichiometries.25 Two general conclusions can be drawn from this observation: (i) the inclusion of solvents in the solids points to some type of imperfection in the crystalline lattice and (ii) to some extent, increased incorporation of L-tartaric acid seems to displace residual solvents from the solid. Structural Investigations of the Compound I-L-Tartaric Acid Complexes. The morphologies of the solids obtained from the crystallization experiments are shown in Figure 2. The particles of all complexes are needle-shaped, with a few agglomerates that were easily broken up upon disturbance. Neither the birefringence nor the habit of the crystals seemed to be affected by incorporation of varying amounts of L-tartaric acid. Solid-state structural investigations were carried out using X-ray diffraction and SS-NMR.

The powder patterns of the complexes are different from those of the free base and acid but are isostructural with each other, with no peak shifting, providing evidence of formation of a series of new solid phases which are identical with each other from a crystallographic viewpoint but different only in the amount of incorporated L-tartaric acid. These observations from polarized microscopy and X-ray diffraction point to a rather remarkable trend in the complexes. Incorporation of varying amounts of L-tartaric acid in the solid does not produce any major change in the crystalline structure or morphology of the complexes. This behavior is akin to that of the channel solvates alluded to earlier, because one of the easiest ways in which a second component can fit in a lattice without perturbing the unit cell significantly is when this component forms a channel in the crystal structure. Increased incorporation of the acid leads to a gradual filling up of the channel in the crystal. When this is the case, the dimensions of the channel need not change significantly to accommodate more acid. This also provides support to the hypothesis that increased incorporation of L-tartaric acid in a sense displaces the ethanol and water present in the solids. DSC traces of free base, L-tartaric acid, and the solids from the crystallization experiments are shown in Figure 4. The free base shows a single melting peak with onset at 266 °C, while L-tartaric acid has a melting onset at 170.5 °C. A single melting endotherm (with accompanying decomposition) is observed for complexes with a stoichiometry close to the canonical 0.5:1 acid: base ratio. Additional thermal events are observed in complexes with a deficiency or excess of L-tartaric acid in comparison to 0.5:1. The melting endotherm develops a shoulder prior to the main peak in the complexes with stoichiometries 0.5:1, additional lowtemperature endotherms were observed. Thus, even though the complexes appear to be similar crystallographically, they display differences upon heating and are quite different from the free base and acid. To delineate the origins of these thermal events, thermogravimetric analysis (TGA) was carried out. The total weight loss is plotted as a function of stoichiometry in Figure 5. The data show that the 0.5:1 complex exhibits the lowest weight loss in the same temperature range compared to the other complexes. To further understand the high-temperature behavior of the complexes, TG-FTIR was carried out. TG-FTIR data were also collected for the free base and L-tartaric acid. The FTIR spectra of evolved gases from the free base did not show any signal above the background up to decomposition (see the Supporting Information). The onset of CO2 evolution in L-tartaric acid was 175 °C. The TG-FTIR spectra of the gases evolved from four complexess0.4:1, 0.5:1, 0.7:1, and 0.9:1 were analyzed as a function of temperature. The evolution of CO2 is observed at temperatures at least 15 °C lower in the other complexes compared to that for the 0.5:1 complex (cf. Figure 6). In all the complexes CO2 evolution commences at a higher temperature than in pure L-tartaric acid. This provides some evidence that free or unbound L-tartaric acid may not be present in the complexes. 1H/13C CPMAS and 1H T SS-NMR experiments were 1 performed on complexes with varying L-tartaric acid contents to determine structural perturbations on compound I caused by incorporated L-tartaric acid. Figure 7 displays the 13C spectra of L-tartaric acid and compound I, as well as samples with increasing equivalents of incorporated L-tartaric acid. The 13C spectra of complexes display significantly shifted 13C peak positions compared to the free base sample. This is evidence


Crystal Growth & Design, Vol. 6, No. 3, 2006 693

Figure 2. Micrographs of (a) free base and (b)-(f) L-tartaric acid complexes. The magnification is 20×, and the scale bar is 50 µm in all images.

that the samples are L-tartaric acid-compound I complexes and not merely a physical mixture of compound I and L-tartaric acid. This was also obviously evident from the unique nature of the X-ray patterns of the various complexes. In the complexes, the acid is most likely incorporated near the carbon whose peaks are perturbed in comparison to free base. A closer inspection of Figure 7 shows that the main spectral perturbations appear in the unsaturated region of the 13C spectrum (100-165 ppm), pegging this as the most probable region in which binding occurs. However, no attempt was made to correlate each 13C peak to the carbon atoms in the compound I molecule, due to the number of different 13C sites in this region and lack of adequate peak resolution in the spectrum. The 13C peaks representing L-tartaric acid (74 and 171 ppm in particular) are clearly present in the complexes and increase in relative intensity as the acid content is increased. When the acid peaks in the complexes are compared to those of free L-tartaric acid, only a minor perturbation of chemical shift is witnessed. This suggests only minor structural changes to the L-tartaric acid as it is complexed with compound I.

Table 2.

1H

T1 Values for L-Tartaric Acid, Free Base, and Tartrate Complexes

peak (ppm)

L-tartaric

176 171 74 72

27.4 21.1 21.6 23.3

acid

free base 1.3 1.3

tartrate complex 0.34:1

0.42:1

0.51:1

0.67:1

0.88:1

1.6 1.6 1.3 1.5

1.5 1.5 2 2.3

1.6 1.8 1.3 2.5

2.3 3.2 2.1 1.9

2.1 4.8 2.9 3.2

Additional evidence of complex formation and insight into the nature of the complexes is seen in the 1H T1 values corresponding to specific peaks in the 13C spectrum (cf. Table 2). From Table 2, it is clear that L-tartaric acid has appreciably longer 1H T1 values (∼25 s) compared to those of free base (∼1.3 s). If free L-tartaric acid were present in the solids, a significant overall 1H T1 increase for L-tartaric acid 13C peaks relative to the amount of L-tartaric acid in the sample should be expected. However, as seen in Table 2, no appreciable gain in 1H T1 is evidenced until the tartrate content is above 0.5:1. Once the acid content is above this value, 1H T1 values for all


Variankaval et al.

Figure 3. XRPD of L-tartaric acid complexes. The acid:base stoichiometry is indicated in the figure.

Figure 4. DSC traces of L-tartaric acid, free base, and tartrate complexes. The onset temperatures of the endotherms for all samples are marked in the figure. The stoichiometries of the complexes are indicated next to the DSC curves. The onset temperatures of the largest peak are shown for the 0.3:1 and 0.4:1 complexes.

acid 13C peaks do not display a uniform T1 increase. An unchanged T1 value from free base to the 0.5:1 complex suggests one type of acid binding to compound I. As the acid:base stoichiometry is raised above 0.5:1, an additional type of L-tartaric acid binding is witnessed, resulting in a nonuniform increase in 1H T1 value. Specifically, in samples with stoichiometries greater than 0.5:1, the 1H T1 value of the L-tartaric acid carbonyl peak at 171 ppm increases at a faster rate than the carbonyl peak at 176 ppm. This trend suggests different types of binding for each L-tartaric acid carbonyl carbon as the stoichiometry exceeds 0.5:1. To refute the possible theory that the noncanonical complexes (stoichiometry not equal to 0.5:1)

could be physical mixtures of the 0.5:1 complex and free L-tartaric acid, the T1 values of L-tartaric acid and the 0.5:1 complex were used to generate theoretical estimates of T1 for the other solids using the simple relation

T1(solid) ) [w(0.5:1)][T1(0.5:1)] + [w(L-tartaric acid)][T1(L-tartaric acid)] (1) where w is the weight fraction of the components. This relation assumes that the difference in T1 values between the two types of bound L-tartaric acid is not significant compared to that between free and bound L-tartaric acid. Given this assumption,


Figure 5. Weight loss observed in TGA as a function of the acid: base ratio of complex. The temperature range for the weight loss computation was from ambient temperature to the onset of melting for each sample.

Figure 6. Lowest temperature at which CO2 is first observed in the gas phase in the TG-FTIR as a function of acid:base ratio.

Figure 8 shows that the estimated values of T1 for the physical mixtures are significantly different from the observed T1 values for the complexes. This is additional evidence that true nonstoichiometric complexes are formed.

Figure 7.

13


These observations, coupled with the X-ray diffraction data, additional features in the DSC, and CO2 evolution in TG-FTIR of the noncanonical complexes, support two conclusions. (i) All the L-tartaric acid present in the crystals is bound. (ii) There are at least two types of binding of the L-tartaric acid to the free base: a strong interaction, such as observed in the 0.5:1 complex, and other weaker binding interactions, as evidenced in the complexes with acid:base ratios less than or greater than 0.5:1. Determining the Binding Site. 13C NMR provided evidence that L-tartaric acid is incorporated into the crystal lattice containing compound I. However, due to the complexity of the 13C spectral region that is perturbed by L-tartaric acid, it is quite difficult to determine the binding site. To determine the site of the compound I-L-tartaric acid interaction, 15N CPMAS experiments were performed. Figure 9 shows 15N CPMAS spectra for complexes with varying stoichiometries. From Figure 9 it is quite clear that, as the acid content is increased, the N(3) site transforms from a singlet at -90 ppm in the free base to a triplet (-90, -94, -101 ppm) in samples g0.5:1. Samples with stoichiometries 0.5:1 samples heated above the second endotherm are compared to a 0.5:1 sample in Figure 12 and Table 3, respectively. From Figure 12, it is clearly evident that L-tartaric acid 13C peaks (particularly 74 and 171 ppm) in >0.5:1 samples return to intensity levels similar to those of the 0.5:1 sample after they are heated past the endotherm. Additionally, from Table 3, 1H T1 values return to levels similar to those for the 0.5:1 sample. These results provide strong evidence supporting elimination of the second tartrate-binding site from these samples. Figure 13 shows 15N CPMAS spectra for samples >0.5:1 that have been heated past the second endotherm compared to 0.5: 1. The relative intensities of N(3) sites for >0.5:1 are not

Figure 9. 15N CPMAS spectra for (a) 0.9:1, (b) 0.7:1, (c) 0.5:1, (d) 0.4:1, and (e) 0.3:1 complexes and (f) free base. Asterisks represent spinning sidebands.


Figure 10. 15N NMR of compound I free base at contact times of (a) 10 ms and (b) 0.5 ms.


Figure 13. 15N CPMAS spectra for (a) 0.9:1 complex heated past the endotherm, (b) 0.7:1 complex heated past the endotherm, and (c) 0.5:1 complex. Table 4. Plasma Levels in Rhesus Monkey Given Orally in Methocel (n ) 4) compd mg/kgd)

0.5:1 complex (3 compound I (3 mg/kg)

AUC (µg/mL × h)a,b

Cmax (µg/mL)b,c

5.5 ( 4.3 0.24 ( 0.9

0.44 ( 0.21 0.03 ( 0.009

a AUC ) area under curve. b The uncertainties are expressed in standard deviations. c Cmax ) maximum concentration in plasma. d Dose based on free base.

Figure 11. 15N NMR of the 0.7:1 tartrate complex at contact times of (a) 10 ms and (b) 0.5 ms.

Figure 12. 13C CPMAS spectra for (a) 0.9:1 complex heated past the endotherm, (b) 0.7:1 complex heated past the endotherm, and (c) 0.5:1 complex.

affected by heating above the endotherm. This supports the theory that the L-tartaric acid incorporated in excess of 0.5:1 is bound in a different site and that this binding is more labile compared to the first type of binding. Heating past the endotherm releases this species of L-tartaric acid but does not affect the remaining acid strongly bound to N(3). This is clearly

evident from Figure 13, where no change is seen in the N(3) region of the spectrum compared to 0.5:1. The perturbations to the crystal lattice upon heating were investigated by variable-temperature X-ray powder diffraction. The 0.67:1 complex was chosen as an example. Plotted in Figure 14 are the d spacings calculated from peak positions of four peaks in the X-ray pattern of the complex. As the temperature is increased, anisotropic thermal expansion is observed as an increase in the lattice spacing. However, prior to melting (and well above the melting point of free L-tartaric acid and above the onset temperature of the low temperature endotherm seen in samples with stoichiometries >0.5:1), an abrupt lattice contraction is observed. This contraction is due to the release of the weakly bound L-tartaric acid, ethanol, and water from the crystal lattice. Supporting evidence for this is provided by the evolution of CO2, observed in the same temperature range in the TG-FTIR studies. That the solid retains its crystalline state is evident from the intense peaks observed at these temperatures; the only change is a shift of the peaks to higher 2θ positions. Finally, variable-temperature ATR-FTIR was carried out on the free base and complexes. The FTIR spectra of the free base do not show any changes whatsoever with increasing temperature (cf. Figure 15), consistent with thermal analysis of the material. Significant changes are observed with increasing temperature for three complexes in the N+-O- and C-OH regions (cf. Figure 16), providing additional evidence of intermolecular interactions at these moieties. However, no apparent differences between the complexes in the extent of interactions are discernible by FTIR. The objective of the complex formation was to enhance the bioavailability of compound I. The aqueous solubility obtained by slurrying the 0.5:1 tartrate complex in water was 23.7 µg/ mL. Increased exposure was indeed found in animals for the 0.5:1 complex compared to compound I (cf. Table 4). On the


Variankaval et al.

Figure 14. d spacings in the crystal lattice of the 0.7:1 complex for four peaks. The legend indicates the peak positions at 25 °C. The wavelength of the X-ray radiation used was 1.547 18 Å.

Figure 15. Variable-temperature ATR-FTIR spectra of compound I free base. Temperatures were 34, 100, 150, 160, 175, and 190 °C.

basis of the thermal properties of the hemitartrate and the accompanying bioavailability data, this form was chosen for further development. Conclusions Evidence for a series of nonstoichiometric, acid:base, cocrystalline complexes of compound I with L-tartaric acid has been presented. X-ray powder diffraction revealed the isomorphous nature of the complexes over a wide range of stoichiometries. The incorporation of increasing amounts of L-tartaric acid in the solid does not seem to have a significant effect on the unit

cell dimensions, which leads to the hypothesis that the acid is probably occupying channels within the crystal which progressively get filled upon addition of the acid. Variable contact time 15N SS-NMR studies clearly indicate the absence of proton transfer to the free base. This inability of the acidic and basic moieties to form a true salt is related to the low pKa difference between the acid and the base. The canonical 0.5:1 complex is found to be more thermally stable than the other complexes, on the basis of CO2 evolution studies. Using high-temperature X-ray, SS-NMR, and thermal analyses, the presence of multiple binding modes of acid to the base was established in complexes with a stoichiometry either lesser than or greater than 0.5:1.



Figure 16. The N-O, C-OH, and carboxylic acid stretching regions in the ATR-FTIR spectra for the tartrate complexes at various temperatures. 0.4:1 complex: 25, 50, 80, 100, 120, 140, 160, 164, 170, 176, 180, 190 °C. 0.5:1 complex: 25, 75, 100, 120, 140, 155, 158, 160, 166, 168, 172C, 180, 185 °C. 0.7:1 complex: 25, 80, 100, 120, 140, 150, 154, 158, 160, 164, 168, 172, 176, 180 °C.

The 0.5:1 complex seems to be the most homogeneous with respect to the binding of the L-tartaric acid to the free base and the most thermally stable. The fact that different acid:base compositions are found in the solid phase is indicative of a solid solution which requires the mutual miscibility of compound I and L-tartaric acid over the composition range. The miscibility that is indeed observed (and verified by the X-ray diffraction patterns) is driven by the strong intermolecular interactions between the carboxylic acid group in L-tartaric acid and N+O- moiety in the free base. Hence, the designed synthesis of such cocrystals can be incorporated into any salt-screening strategy and can be especially useful in those cases where the pKa difference between acid and base molecules is not sufficient to form a true salt. The critical factor is to identify appropriate acid or

base molecules such that the complementarity of functional groups drives the formation of stable cocrystals. In this particular case the synthesis of supramolecular hydrogen-bonded cocrystalline complex of compound I and L-tartaric acid resulted in increased bioavailability, which enabled the selection of this crystalline form for further development. Acknowledgment. We wish to acknowledge Rick Sidler for discovering the first tartrate complex of compound I. Supporting Information Available: Text and figures giving gas chromatography, titration, microscopy, X-ray powder diffraction and thermal analysis methods, Gram-Schmidt plot of evolved gases in TGIR for compound I, TG-MS data for tartrate complexes, and a plot of CO2 evolution for the complexes. This material is available free of charge via the Internet at http://pubs.acs.org.


References (1) Stahl, P. H.; Wermuth, C. G. Handbook of Pharmaceutical Salts: Properties, Selection and Use; VHCA: Zurich, Switzerland, and Wiley-VCH: Weinheim, Germany, 2002; p 5. (2) Tong, W. Q.; Whitesell, G. Pharm. DeV. Technol. 1998, 3, 215. (3) Fan, E.; Vincent, C.; Geib, S. J.; Hamilton, A. D. Chem. Mater. 1994, 6, 1113. (4) Sada, K.; Kondo, T.; Miyata, M. Chem. Mater. 1994, 6, 1103. (5) Aakeroy, A. A.; Beatty, A. M.; Nieuwenhuyzen, M.; Zhou, M. Tetrahedron 2000, 56, 6693. (6) Remenar, J. F.; Morissette, S. L.; Peterson, M. L.; Moulton, B.; MacPhee, J. M.; Guzman, H. R.; Almarsson, O. J. Am. Chem. Soc. 2003, 125, 8456. (7) Lynch, D. E.; Mistry, N.; Smith, G.; Byriel, K. A.; Kennard, C. H. L. Aust. J. Chem. 1998, 51(9), 813-818. (8) Koyano, T.; Hachiya, I.; Sato, K. J. Phys. Chem. 1992, 96, 1051410520. (9) Sato, K. Chem. Eng. Sci. 2001, 56, 2255-2265. (10) Takeuchi, M.; Ueno, S.; Sato, K. Food Res. Int. 2002, 35, 919-925. (11) Steiner, T.; Koellner, G.; Ali, S.; Zakim, D.; Saenger, W. Biochem. Biophys. Res. Commun. 1992, 188, 1060. (12) Steiner, T.; Koellner. G. J. Am. Chem. Soc. 1994, 116, 5122. (13) Ripmeester, J. A. Supramol. Chem. 1993, 2, 89. (14) Clausen, W. F. J. Chem. Phys. 1951, 19, 259. (15) von Stackelberg, M.; Muller, H. R. Z. Electrochem. 1954, 58, 215. (16) Raj Bishnoi, P.; Natarajan, V. Fluid Phase Equilib. 1996, 117, 168.

Variankaval et al. (17) Bakeev, K. N.; Shu, Y. M.; MacKnight, W. J.; Zezin, A. B.; Kabanov, V. A. Macromolecules 1994, 27, 300, (18) Morris, K. R. Structural Aspects of Hydrates and Solvates. In Polymorphism in Pharmaceutical Solids; Brittain, H. G., Ed.; Marcel Dekker: New York, 1999; pp 145, 146. (19) Brittain, H. G.; Bugay, D. E.; Bogdanovich, S. J.; DeVincentis, J. Drug DeV. Ind. Pharm. 1988, 14, 2029. (20) Albaneze-Walker, J.; Murry, J. A.; Soheili, A.; Ceglia, S.; Springfield, S. A.; Bazaral, C.; Hughes, D. L. Submitted for publication in J. Am. Chem. Soc. (21) See the Supporting Information. (22) The equilibrium solubility was evaluated by stirring excess solid in water for ca. 16 h in the absence of light at 25 °C. The supernatant was analyzed using a HP8453 UV spectrophotometer. (23) http://www.petrik.com/PUBLIC/library/misc/acid_base_pk.htm, accessed 29 Dec 2003. (24) Routine SS-NMR could not clearly locate ethanol in the crystal because of peak overlap with the free base and the complicated nature of the spectrum. (25) It was not possible to describe the exact nature of the residual solventsssurface-bound versus incorporated in the crystal lattices with the current data. However, analysis of TG-MS data indicates that ethanol and water are both released from the solids only at higher temperatures, with ethanol being somewhat more tightly bound than water. See the Supporting Information for details.

CG050462U

Preparation and Solid-State Characterization of Nonstoichiometric

Recommend Documents