Selection of a Suitable Physical Form and Development of a

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Selection of a Suitable Physical Form and Development of a Crystallization Process for a PDE10A Inhibitor Exhibiting Enantiotropic Polymorphism Y.-H. Kiang, Eric A Bercot, Qiong Wu, Jodi Liu, Robert R Milburn, Dawn E Cohen, Christopher J Borths, Robert E Saw, Richard J Staples, Carl Davis, and Oliver R. Thiel Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00031 • Publication Date (Web): 31 Mar 2015 Downloaded from http://pubs.acs.org on April 3, 2015

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Organic Process Research & Development

Selection of a Suitable Physical Form and Development of a Crystallization Process for a PDE10A Inhibitor Exhibiting Enantiotropic Polymorphism

Y.-H. Kiang*,†, Eric A. Bercot†, Qiong Wu†, Jodi Liu†, Robert R. Milburn†, Dawn E. Cohen†, Christopher J. Borths†, Robert E. Saw†, Richard J. Staples†, Carl Davis‡, and Oliver R. Thiel†

†Process Development, ‡Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320

for Submission to


Revised March 18, 2015

*Author to whom correspondence should be addressed ([email protected])

ACS Paragon Plus Environment


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Abstract:

AMG 579 (1) is a potent and selective phosphodiesterase 10 (PDE10A) inhibitor selected for clinical development for the treatment of schizophrenia. Extensive polymorph and salt screening identified two free-base anhydrous polymorphs (Form 1 and Form 2) that are viable for further development. Crystal structures of these two polymorphs were determined by single-crystal Xray study. Form 1 and Form 2 are enantiotropically related with the transition temperature between 190 °C and 210 °C. After full characterization, quality attributes were evaluated and Form 2, the thermodynamically more stable form at room temperature, was selected for clinical development. A crystallization process for Form 2 was developed, and in situ Raman spectroscopy was used as a PAT tool to monitor and control the physical form. Use of this integrated control strategy allowed access to multi-kilogram quantities of AMG 579 in the desired form.


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Introduction: AMG 579 (1) is a potent, selective small molecule inhibitor of PDE10A in clinical development targeted for the treatment of schizophrenia.1 During lead optimization stage, a total of six lots of 1 were prepared. Purification of these six lots of 1 was achieved using flash chromatography. All six lots of chromatographically purified material were characterized as a crystalline solid of the same form, Form 1. During initial process development, however, a new polymorph, Form 2, was crystallized out adventitiously with slow cooling of the reaction solution. Subsequently a 96well high throughput polymorph screening study was conducted using a library containing 30 single and 66 binary solvent systems. Slurry, evaporation, anti-solvent precipitation, and cooling experiments were carried out in four 96-well plates, resulting in 258 crystalline samples out of 384 total conditions. No new phases were found beyond the two known polymorphs. The two polymorphs were fully characterized and their thermodynamic relationship thoroughly investigated. With unfavorable pKa values (2.5, 10.6) of 1, extensive salt screening did not result in any salt forms suitable for further development. Form 2 was selected for clinical study and long term development based on quality attributes such as crystallinity, drug substance stability, hygroscopicity, drug substance manufacture, and in vivo performance. A crystallization process was developed to manufacture the GMP batch of 1. In situ Raman spectroscopy was used as a Process Analytical Technology (PAT) tool to monitor and control the physical form in real time.



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Results and Discussion:

Polymorph Screen and Characterization. Two polymorphs, Form 1 and Form 2, were identified after extensive polymorph screening. X-ray diffraction patterns of Form 1 and Form 2 are characteristic of crystalline material, with distinct diffraction peaks from 3-40° 2-theta (Figure 1).

The DSC and TGA thermograms indicate that both Form 1 and Form 2 are

anhydrous (Figures 2 and 3, respectively).

In the DSC thermograms, sharp endothermic

transitions were observed for both polymorphs. As there is no weight loss in the TGA traces corresponding to the endothermic transitions, they are due to congruent melting of the solids. As such, thermal data obtained from the TGA and DSC measurements, namely the melting temperature and heat of fusion, can be used directly to infer thermodynamic stability of the two polymorphs. Both polymorphs are non-hygroscopic, taking up less than 0.2% of water at 95% relative humidity (RH) in the moisture sorption isotherms (Figure 4).

For the thermodynamic stability of the two forms, the Heat-of-Fusion Rule proposed by Burger and Ramberger states: if the higher melting form has the lower heat of fusion, the two forms are usually enantiotropic; otherwise they are monotropic.2 In the case of 1, the higher melting form, Form 1, has a lower heat of fusion. Therefore, Form 1 and Form 2 should be enantiotropically related with Form 1 being the more stable form at higher temperature. To aid form selection and crystallization process development, it is critical to understand the temperature at which the thermodynamic stability between Form 1 and Form 2 switches, known as the transition temperature. In pharmaceutical industry, the most common practice to obtain the transition temperature of enantiotropic polymorphs is to plot the logarithm solubility versus inverse absolute temperature (1/T) for each phase (van’t Hoff plot).3

The solubility of the two

polymorphs in acetonitrile was measured at 28, 35, and 60 °C and used to construct the van’t Hoff plot (Figure 5). Solubility data used to construct Figure 5 are summarized in Table 1. In the van’t Hoff plot, the logarithm of solubility plotted against the inverse absolute temperature resulted in two apparent linear lines that cross at 357 K (84 °C), which is the transition temperature Tt, indicating that Form 2 is more stable below 84 °C.


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In addition to the Heat-of-Fusion Rule, DSC data can be used to estimate transition temperature with the following equation proposed by Yu:4

ܶ௧ =

∆ு೘,మ ି∆ு೘,భ ା൫஼೛,ಽ ି஼೛,భ ൯

೅೘,భ

×൫்೘,భ ି்೘,మ ൯

∆ಹ೘,మ ∆ಹ೘,భ ೅ ି ା൫஼೛,ಽ ି஼೛,భ ൯ ×௟௡൬ ೘,భ ൰ ೅೘,మ ೅೘,భ ೅೘,మ ೅೘,భ

(1)

In equation 1, Tt is the transition temperature between Form 1 and Form 2; Tm,1, Tm,2, ∆Hm,1, ∆Hm,2 are the melting temperature of Form 1, melting temperature of Form 2, heat of fusion of Form 1 and heat of fusion of Form 2, respectively; (Cp,L – Cp,1)Tm,1 is the heat capacity difference of the super-cooled liquid and solid Form 1 at Tm,1.

In the equation, melting temperature and heat of fusion for Form 1 and 2 can be obtained directly from DSC measurements; however, the heat capacity of super cooled liquid and of Form 1 at its melting temperature cannot be measured directly. One approach is to extrapolate (Cp,L – Cp,1)Tm,1 from the heat capacity data below and above melting using linear regression.5 In equation 1, let k = ((Cp,L – Cp,1)Tm,1 ÷ ∆Hm,1). Yu has reported that for crystalline solids, k is generally between 0.001 and 0.007 K-1 with an average 0.003 K-1.4 Using the average value of 0.003 K-1 reported in Yu’s paper for k, and the thermal data summarized in Table 2, the transition temperature Tt was estimated as 207 °C, 120 °C higher than that obtained from the van’t Hoff plot.

Given the importance of transition temperature for determining which form to develop, as well as for defining processing parameters during crystallization of the room temperature stable Form 2, it was necessary to determine whether the true transition temperature was more closely reflected in the result given by the van’t Hoff plot or the thermal data. Thus, mixtures of solid Form 1 and Form 2 were subjected to variable temperatures from 70 °C to 210 °C, and the crystalline phase was monitored by X-ray to further investigate the transition temperature. It was found that when starting with a 1:1 ratio mixture of Form 1 and Form 2, the resulting solids were



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of pure Form 2 up to 190 °C, and of pure Form 1 at 210 °C. This form conversion study placed the transition temperature between 190 °C and 210 °C, much closer to that estimated from the thermal data. This high transition temperature significantly reduced the risk of a phase transition of From 2 occurring at room temperature, such as during manufacturing and upon storage. It has been reported that large error was resulted in estimated transition temperature by extrapolating van’t Hoff plots.6,

7

Possible reasons that the van’t Hoff plot failed to predict transition

temperature correctly may include 1) solubility measurement error, particularly at 60 °C, due to difficulty in maintaining constant temperature during sample handling for analysis, as seen in the large standard deviation at 60 °C; 2) the validity of the assumption that the curves in the plots were linear.8

Consistent with Form 2 being the thermodynamically stable form at room

temperature, the water solubility measured at room temperature was 0.34 µg/mL for Form 2 and 0.73 µg/mL for Form 1.

Chemical stability of Form 1 and Form 2 was evaluated at stressed conditions. The powder samples of Form 1 and Form 2 were placed in open vials at 40 °C/75% RH and 60 °C, and analyzed by high performance liquid chromatography (HPLC) at 2, 4, and 8 weeks. No increase in chemical impurity was observed over the 8 week period as shown in Table 3. Both Form 1 and Form 2 are chemically stable under ICH stress conditions.

Single crystal structure of Form 1 was solved in a monoclinic cell with the space group P21/c (Figure 6); that of Form 2 was solved in an orthorhombic cell with the space group Pna21 (Figure 7). In addition to the difference in crystal packing, the conformation of 1 is significantly different in the two polymorphs (Figure 8). As the powder X-ray diffraction pattern of a material can be calculated from a single crystal structure, having the single crystal structures of both polymorphs allowed determination of powder X-ray diffraction patterns of the pure phases, which can be fully utilized for phase identification and phase purity determination for compound 1. Comparison of the powder diffractions of the experimental and calculated using the single crystal structure was illustrated in Figures 9 and 10, for representative batches of Form 1 and Form 2, respectively. In these two Figures, the excellent agreement between the experimental


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and the calculated X-ray diffraction patterns indicates phase purity of the representative bulk materials. Salt Screen. Solid-state characterization of the free base polymorphism confirmed enantiotropy with a transition temperature of the two polymorphs between 190 °C and 210 °C. Although the transition temperature is high, and Form 2 is the thermodynamically stable form in the practical temperature range, it was desirable to identify a salt form that does not exhibit enantiotropic polymorphism.

In addition to form control, a viable crystalline salt form may potentially

improve in vivo performance. Compound 1 is a very weak acid/base, with pKa values of 2.5 and 10.6, as derived from fitting the pH solubility profile of Form 2. A manual salt screening was conducted9 using methanesulfonic acid, HCl, H2SO4, p-toluenesulfonic acid, potassium methoxide, and sodium methoxide: from this screening, crystalline mesylate and sulfate salts were identified. DSC and TGA analyses showed melting/decomposition at about 157 °C for the mesylate salt and about 223 °C for the sulfate salt. However, further characterization revealed that the mesylate salt was highly hygroscopic, taking up 40% of water at 80% RH and turning amorphous, whereas the sulfate salt disproportionated in water rapidly and converted to the free base. Given that each crystalline salt of 1 suffered a serious physical liability, neither salt was deemed suitable for further development.

Initial Crystallization Process Development. With the freebase the only viable form for clinical development a process capable of delivering kilogram quantities of the desired polymorph needed to be identified. Based on the complex polymorph behavior of 1, it was decided to decouple synthesis and form setting step. As such crude 1 was obtained through a convergent coupling of two major fragments. The crude drug substance was of relatively high purity (> 97 LCAP, > 96 wt%, no residual solvents > 5000 ppm), and it was subjected to a separate form-setting recrystallization.10 Selected room temperature solubility data of 1 (Form 1) in organic solvents is shown in Table 4. The compound is sparingly soluble in most solvents. The highest solubility is observed in THF and the polar aprotic solvents DMSO, DMF and NMP. Out of these solvents DMSO was selected as the process solvent due to the least concerns from a toxicity perspective (ICH class III). The goal was to design a process that would allow



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dissolution of the compound at elevated temperature in a reasonable amount of solvent (< 30 L/kg), followed by a hot polish filtration, and crystallization of the desired polymorph of freebase by addition of water. Early experiments focused on identification of a process that was able to deliver gram quantities of both forms of 1. This would allow for adequate

material to conduct studies (characterization,

pharmacokinetics) aimed at selecting a form for development. These early experiments were also geared towards an assessment of the relative manufacturability of the two polymorphs. The method for isolation of Form 1, which is the kinetically favored form at relevant processing conditions, needed to rely on a nucleation driven anti-solvent crystallization. Consequently isolation of Form 1 could be achieved by an inverse addition of a DMSO solution into water. The crude freebase could be dissolved in hot DMSO (20 L/kg) at 65 °C. The material could then be crystallized by addition of this solution to a slurry of Form 1 seed (1 wt%) in water (10 L/kg). Isolation using this method produced material of high purity, however with a very small particle size (< 10 µm), as judged by optical microscopy, that resulted in a rather slow filtration. Under these isolation conditions, Form 1 was stable with regard to conversion to Form 2. Addition of Form 2 (2 wt%) at the endpoint of the crystallization did not lead to turnover of Form 1 to Form 2. Performing this inverse addition based crystallization process in the presence of Form 2 seed (2 wt% in water) led to isolation of Form 2. This experimental result indicated that this process for isolation of Form 1, could incur a significant risk upon scale-up. Small contamination of equipment or seed with Form 2, could result in isolation of Form 2. Therefore it was prudent to consider isolation of the thermodynamically favored polymorph (Form 2).

Isolation of Form 2 (thermodynamic form) could be achieved by employing a more controlled growth based seeded crystallization, in which the anti-solvent (water) was added to the DMSO solution of 1 (vide infra for details).

Form Selection. In order to determine which physical form should be recommended for phase I clinical trial and/or long term development, the following quality attributes were compared:


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crystallinity, drug substance stability, hygroscopicity, drug substance manufacture, and in vivo performance. Both forms are crystalline with high melting point, non-hygroscopic, and chemically stable under stressed conditions. The differentiating attributes are therefore in manufacturability and in vivo performance.

Drug substance manufacture:

Form 2 is isolated by a controlled anti-solvent and cooling

crystallization, which can be seeded with Form 2. To date, all crystallizations conducted in this manner have resulted in isolation of Form 2. Additionally, mixed slurries of Form 1 and Form 2 in a variety of solvents at relevant processing conditions (< 100 °C) result in the isolation of Form 2. Form 1 is more difficult to control and manufacture. Form 1 can only be produced with a reverse addition anti-solvent driven precipitation; however, during isolation, Form 1 is at risk of converting to Form 2 in the presence of small amounts of Form 2. The reverse addition process employed to access Form 1 also leads to the formation of very small particles, creating additional issues during filtration and drying.

In vivo performance: In a crossover dog pharmacokinetics study, Form 1 and Form 2 suspensions were compared against a solution formulation, at a dose 10-fold higher than the projected clinical efficacious dose. This dose was selected to reflect the highest possible FIH ending dose, provided no adverse effect is observed in the single ascending dose study. The area under the plasma concentration-time curve from time zero to 24 hours (AUC0-t) for Form 1 was 32% of that for solution, and Form 2 was 14% of that for solution (Table 5). Based on these pharmacokinetics data, an enabling formulation for Form 2, such as an amorphous solid dispersion or nano-crystals, would likely be required to achieve a sufficient dose dependent increase in exposure should the actual target dose prove to be many fold higher than the projected efficacious dose.



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Based on the analysis of the quality attributes, Form 2, being the thermodynamically stable form in the practical temperature range, was selected for long-term development to mitigate and control drug substance manufacturing risk.

Scale-up Crystallization Process Development. Based on the selection of Form 2, additional process development was performed in order to establish a suitable process for production of kilogram quantities. For this purpose the crystallization design was based on solubility curves in different DMSO/water at different temperatures (Figure 11).

A Raman method was developed for PAT on-line monitoring of the polymorph during the crystallization process. Raman spectroscopy was capable of distinguishing between polymorphs in the crystallizing mixture through use of an immersion probe. Although DMSO features dominated the spectrum, the region between 1110 and 1120 cm-1 was free of DMSO interference and displayed distinct features of the two crystal forms. Integration of a Form 2 peak located at 1143 cm-1 and a Form 1 peak located at 1150 cm-1 yielded kinetic information about polymorph growth dynamics. While the limit of detection of Form 1 in a Form 2 slurry was between 10 and 25% at the concentrations used in the isolation procedure, this technique still provided valuable information and was used during the GMP production run of 1. Raman was employed as an online monitoring technique to track polymorph dynamics during the crystallization process. Online data was confirmed by off-line X-ray powder diffraction measurements. In situ Raman showed the formation of between 25 and 50% Form 1 during the water addition after seeding in both the use test and production run of 1 (Figure 12). It also indicated that after aging 2 hours at 60 °C, Form 1 had diminished to less than 25% (Figure 13). The conversion rate of Form 1 to Form 2 was comparable between scales.

The scale-up process for crystallization of 1 was carried out on a kilogram scale.10 The material was dissolved in DMSO (28 L/kg) at 60 °C and polish filtered. Water (2 L/kg) was added at


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temperature, followed by 1 wt% seed (Form 2) in water (1 L/kg). After holding the batch at 60 °C for 15 min, during which time the seed held, the remaining water (3 L/kg) was added over 1 hour. At this point, in situ Raman monitoring indicated the presence of between 25 and 50% of Form 1. The batch was aged at 60 °C for 17 hours; Raman did not detect Form 1, X-ray powder diffraction did not detect Form 1, and microscopy indicated particles greater than 50 micron. The batch was cooled to 20 °C over 4 hours and held at that temperature for 1 hour. The solids were isolated by filtration and rinsed with water. Drying under vacuum with a nitrogen bleed yielded a total of 1.61 kg of 1 (91 % recovery, 99.77 wt%, 99.9 LCAP, 2100 ppm DMSO, KF 620 ppm)).

The above described Form 2 crystallization from DMSO/water was fit-for-purpose for a first delivery of material for clinical studies. The process suffered from two major liabilities that would be desirable to improve upon prior to production of additional material: the widepoint of the process was 35 L/kg of 1, which would limit throughput on large-scale production, and the detection of Form 1 during anti-solvent addition was concerning potential risk to long term process robustness, although the turn-over to Form 2 was shown to be facile in preceding batches. Ultimately it was desirable to use a process that would minimize formation and detection of Form 1 during any stage of the crystallization. Therefore additional solvents were considered and, based on the solubility curves, acetic acid and water emerged as an attractive alternative system for the isolation of 1 (Figure 14). A relatively high solubility (> 140 mg/mL) could be achieved in acetic acid/water mixtures (9:1), thereby allowing for a significantly more volume-efficient polish filtration. In situ Raman was used to evaluate the presence of Form 1 and did not detect Form 1 in crystallizations utilizing the acetic acid and water solvent system.

The crude freebase was dissolved in acetic acid/water (9:1, 8 L/kg) at 70 °C and polish filtered. The mixture was then driven into supersaturation by addition of water (1 L/kg) and cooling to 60 °C. The batch was then seeded (1 wt% Form 2), additional water added (5.8 L/kg), and the batch cooled to room temperature. This procedure was performed on 5.44 kg scale and afforded 1 of high quality (92 % recovery, 99.4 LCAP, 99.1 wt%). It is readily apparent that this process is



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significantly more volumetrically efficient (15 L/kg) as compared to the original process (35 L/kg). More significant, however, is the absolute absence of Form 1 at the end of anti-solvent addition, as indicated by Raman monitoring during both the use test and production run of 1 (Figure 15). This difference between the acetic acid and DMSO system may likely be affected by the different solubility of 1 at the seed point, thereby allowing a better turnover to the more thermodynamically stable Form 2. The solubility at the beginning of the antisolvent addition is ~ 80 mg/mL in the acetic acid system, as compared to ~ 25 mg/mL in the DMSO system.


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Conclusions A polymorph and salt screen was conducted on 1. Two polymorphs of the free base, as well as a sulfate salt and a mesylate salt, were identified as crystalline solids. After further characterization, only the free base forms appeared to be viable for development. Both forms are non-hygroscopic and chemically stable under stressed conditions. Thermodynamically these two forms are enantiotropically related with a high transition temperature between 190 °C and 210 °C. The more stable form at room temperature, Form 2, was selected for long term development based on evaluation of quality attributes. A robust process for isolation of Form 2 was developed. The design of the crystallization was guided by employing in-situ Raman monitoring.



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Experimental Section General Procedure. Unless otherwise indicated, all commercially available reagents were purchased from Aldrich Chemical Company, Inc and used without further purification. Analytical grade solvents were obtained from commercial suppliers (Aldrich, Fisher Scientific, and Mallinckrodt).

Powder X-ray diffraction (pXRD) patterns were collected with a PANalytical X-ray powder diffractometer (X’Pert PRO) equipped with a real-time-multiple-strip detector, using Ni filtered Cu Kα radiation at 45 kV, 40 mA. The diffractometer employs a divergence slit of 1/4o, an antiscatter slit of 1/2o, and soller slits of 0.04 radian at both the incident and the diffracted sides. Each sample was scanned between 3-40o 2θ.

Moisture sorption isotherms were obtained on an SGA-100 vapor sorption analyzer. Between 5 and 10 mg of sample were dried at 40 °C until the weight change was less than 0.02% for 10 min or a maximum equilibration time of 120 minutes had been reached. Subsequent to drying the sample was cooled to 25 °C and subjected to relative humidity (RH) in the range of 5 to 95% in increments of 5% RH. A point isotherm was recorded either when the equilibrium criterion of less than 0.01% weight change for 10 minutes was achieved, or at every 90 min.

Karl Fisher titrations for water content determination were performed using a Mettler Toledo C20 Coulometric KF titrator. An Aquastar Combi Coulomat Methanol Solution was used as the solvent. Between 10-20 mg of sample was used for each analysis.

Thermal properties of the samples were characterized by differential scanning calorimetry (DSC Q2000 and Q200, TA Instruments) and thermal gravimetric analysis (TGA Q500, TA Instruments). Heating rate of 10 °C/min was employed over a temperature range of 25–225 °C unless otherwise indicated. Standard aluminum pans were used.


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Analysis was performed by HPLC-UV on an Agilent 1100 series, equipped with a quaternary pump (G1311A), diode-array detector (G1315A) set at 220 nm, autosampler (G1329A) and a 150 mm×4.6 mm Phenomenex Luna C18 column. A gradient method from 20 to 95% acetonitrile in water and 0.1% trifluoroacetic acid at 1 mL/min for 15 min was used. Standards were prepared in 50% acetonitrile at 0.05 mg/mL and injected at 1, 5, 10, 25 and 50 µL.

Single crystal data were collected using a Bruker CCD (charge coupled device) based diffractometer equipped with an Oxford Cryostream low-temperature apparatus operating at 173 K. Data were measured using omega and phi scans of 0.5° per frame for 10 (Form 2) and 160 (Form 1) seconds. The total number of images was based on results from the program COSMO where redundancy was expected to be 4.0 and completeness to 100% out to 0.83 Å. Cell parameters were retrieved using APEX II software and refined using SAINT on all observed reflections. Data reduction was performed using the SAINT software which corrects for Lp. Scaling and absorption corrections were applied using SADABS multi-scan technique, supplied by George Sheldrick. The structures are solved by the direct method using the SHELXS-97 program and refined by least squares method on F2, SHELXL- 97, which are incorporated in SHELXTL-PC V 6.10. All non-hydrogen atoms are refined anisotropically. Hydrogens were calculated by geometrical methods and refined as a riding model. A summary of parameters for the X-ray data collection and subsequent refinement is given in Table 6. The crystal used for the diffraction study showed no decomposition during data collection. All drawings are done at 50% ellipsoids.

Raman data was obtained using a RamanRXN1 at lab scale, and a RamanRXN3 at pilot scale (Kaiser Optical Systems, Ann Arbor, Michigan, USA). Both instruments were equipped with 785cm-1 excitation lasers (Invictus NIR laser 785nm, 500mW) and CCD camera detectors. Prior to data collection, an exposure time was selected to achieve optimal detector saturation. Exposure times were typically below 10 seconds. iC Raman software (Metter-Toledo Autochem Inc., Columbia, Maryland, USA) was employed for data collection.



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Preparation of Form 1 single crystals: 1 (50 mg, 0.11 mmol) was added to a 20 mL glass vial containing 15 mL of acetone. This container was placed in a sonication bath and sonicated for 15 min at 25 °C. The thin suspension after sonication was filtered through a 0.45 µm polytetrafluoroethylene filter. The filtrate was collected into two 20-mL glass vials in equal amount. Slow evaporation afforded colorless block single crystals suitable for X-ray data collection.

Preparation of Form 2 single crystals: 1 (600 mg, 1.36 mmol) was added to a 20 mL glass vial containing 4 mL of N-methyl-2-pyrrolidone. This vial was heated to 60 °C for 10 min. The suspension was filtered through a 0.45 µm polytetrafluoroethylene filter. The filtrate was collected into two 4-mL glass vials in equal amount. The 4-mL vial containing the filtrate was placed into a 20-mL scintillation vial containing 5 mL of water. Vapor diffusion afforded block single crystals suitable for X-ray data collection.

Form conversion study: Form conversion experiments were performed at 70 °C, 90 °C, 135 °C, 150 °C, 190 °C, 200 °C and 210 °C. Except for 150 °C, a minimal amount of solvent (20-300 µL) was added to a 4 mL vial containing a magnetic stir bar and 200 mg of a 1:1 (w/w) mixture of Form 1 and Form 2 to form a thick suspension. The mixture was heated to the set temperature on a stir plate while being stirred. The solvents used were 15% water in DMSO for 70 °C and 90 °C, m-xylene for 135 °C, and hexamethylphosphoramide for 190 °C, 200 °C and 210 °C. No solvent was used for the experiments performed at 150 °C.

High throughput polymorph screening: High-throughput polymorph screen was conducted using a library of 30 single and 66 binary solvent systems in 96-well plates. The solvents used were heptane, toluene, isopropyl acetate, methylethyl ketone, THF, methanol, ethanol, DMF, acetonitrile, ethyl acetate, benzonitrile, propylene glycol, anisole, cyclohexane, dichloromethane, nitromethane, 2-methoxyethanol, diisopropyl ether, methyl chloroform, n-butyl acetate, methyl isobutyl ketone, 2-methyltetrahydrofuran, n-butanol, dioxane, 2-propanol, 2,2,2-trifluroethanol, propanol, acetone, dimethoxyethane, trifluorotoluene, and water. Ten milligrams of Form 1 solid (99.37% purity) was dispensed into each well of a source plate and 960 μL of solvent


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system was added. After 30 minutes of sonication at room temperature and 60 minutes of heating at 80 °C, aliquots of each mixture were filtered and transferred to evaporation, antisolvent and cooling plates. The source plate was stored open at 55 °C for 8 hours. The evaporation plate was stored open at ambient temperature for 24 hours. The cooling plate was sealed and cooled cubically from 60 °C to 5 °C over 8 hours then stored for an additional 8 hours at 5 °C. An equal volume of antisolvent (water or n-butyl ether) was added to each filtrate in the anti-solvent plate, which was then sealed and cooled linearly from 25 °C to 5 °C over 8 hours. Samples were analyzed by pXRD to determine polymorphism.

Solubility measurements: Solubility of Forms 1 and 2 in acetonitrile was determined at 28°C, 35°C and 60°C by placing an excess amount of solid into a 8 mL glass vial containing 4 mL acetonitrile. The sample vial was rotated on a shaker. The sample suspension was withdrawn at regular time intervals and passed through a 0.45 µm polytetrafluoroethylene membrane filter with the first portion discarded to ensure saturation of the filter. An aliquot of the filtrate was then diluted and analyzed by HPLC.

Crystallization of Form 1: A 100 ml round-bottom flask was charged with crude 1 (1.00 g, 2.27 mmol) and DMSO (20 mL). The mixture was heated to 65 °C and complete dissolution of the solids was observed. A separate 100 ml round-bottom flask was charged with water (10 mL) and seeds of 1 (Form 1) (10 mg, 0.023 mmol). The DMSO solution was added at room temperature over 90 minutes. Immediate crystallization was observed at the point of addition. The mixture was aged for 2 h. The solids were isolated by filtration, rinsed with 2 x 3 ml water and then dried on the filter for 72 h, providing 1 as a white crystalline solid (Form 1, 916 mg, 92 % yield). Crystallization of Form 2 (DMSO process): A clean, dry reactor under nitrogen was charged with crude 1 (1.76 kg) and DMSO (50.6 L). The reaction mixture was warmed to 60 °C, and polish filtered (5 µm in line filter) into a second clean, dry reactor. At 60 °C water (3.4 L) was charged to the reaction followed by seed (12 g in 1.7L of water). The reaction mixture was held at 60 °C for 15 min then water (5.4L) was charged over 60 minutes. The reaction mixture was agitated at 60 °C for 14 h. The conversion to desired crystal form was confirmed by Raman



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spectroscopy and pXRD. The batch was cooled to ambient temperature over 4 hours and aged an additional 30 minutes at ambient. The solids were collected by filtration (25 µm Teflon filter cloth) and the reactor and cake washed with water (2 X 8.5L and 2 x 5.5L). The solids were dried under vacuum with a nitrogen bleed at ambient temperature providing 1 as a white crystalline solid (1.61 kg, 99.9 LCAP, 99.8 wt %, KF 0.1 wt%, DMSO 2100 ppm, 92 % yield). Crystallization of Form 2 (acetic acid process): A clean, dry reactor under nitrogen was charged with crude 1 (5.40 kg), acetic acid and water (9:1, 43.2 L). The reaction mixture was warmed to 75 °C and polish filtered (5 µm in line filter) into a second clean, dry reactor. The filter was rinsed with a mixture of acetic acid and water (9:1, 2.70 L). Water (5.40 L) was charged to the reactor and the temperature was lowered to 60 °C. Seed (54.4 g slurried in 540 mL of acetic acid/water 1:1) was added. The reaction mixture was held at 60 °C for 30 min, then water (31.3 L) was charged over 120 minutes. The reaction mixture was agitated at 60 °C for 30 min. The batch was cooled to ambient temperature over 2 hours and aged an additional 15 minutes at ambient. Presence of the desired crystal form was confirmed by Raman spectroscopy and pXRD. The solids were collected by filtration (25 µm Teflon filter cloth) and the reactor and cake washed with acetic acid/water (1:1, 16.2 L) and water (2 x 16.2 L). The solids were dried under vacuum with a nitrogen bleed at ambient temperature providing 1 as a white crystalline solid (5.04 kg, 99.4 LCAP, 99.1 wt%, KF 0.1 wt%, acetic acid 1397 ppm, 92 % yield).


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Associated Content Supporting Information. Further experimental data and Crystallographic Information Files for single crystal structures. This material is available free of charge via the Internet at http://pubs.acs.org.



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Author Information Corresponding Author *Y.-H. Kiang ([email protected]) Present Addresses Eric A. Bercot: Suterra LLC, 20950 Talus Place, Bend OR 97701 Jodi Liu: Pardee RAND Graduate School, 1776 Main Street, Santa Monica, CA 90407 Robert R. Milburn: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404


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Acknowledgements The authors thank Dr. Tian Wu for valuable discussion. In vivo support was provided by Mr. Marcus Soto, with bioanalytical support by Ms. Savannah Kerr and Dr. Sarah Wilson. High throughput polymorph screening was conducted by Mr. Helming Tan and Ms. Van Luu. This work is supported by the Amgen Small Molecule Process & Product Development.



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Abbreviations GMP, good manufacturing practice; DSC, differential scanning calorimetry; TGA, thermogravimetric analysis; ICH, International Conference on Harmonisation; DMSO, dimethyl sulfoxide; DMF, dimethylformamide; NMP, N-methylpyrrolidone; THF, tetrahydrofuran; FIH, first in human; LCAP, liquid chromatography area percent; KF, Karl Fisher titration; HPMC, hydroxypropyl methylcellulose


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References 1.

Hu, E.; Chen, N.; Bourbeau, M. P.; Harrington, P. E.; Biswas, K.; Kunz, R. K.; Andrews,

K. L.; Chmait, S.; Zhao, X.; Davis, C.; Ma, J.; Shi, J.; Lester-Zeiner, D.; Danao, J.; Able, J.; Cueva, M.; Talreja, S.; Kornecook, T.; Chen, H.; Porter, A.; Hungate, R.; Treanor, J.; Allen, J. R. J. Med. Chem. 2014, 57, 6632-41.

2.

Burger, A.; Ramberger, R. Mikrochimica. Acta [Wien] II 1979, 252-271.

3.

Higuchi, W. I.; Lau, P. K.; Higuchi, T.; Shell, J. W. J. Pharm. Sci. 1963, 52, 150-3.

4.

Yu, L. J. Pharm. Sci. 1995, 84, 966-74.

5.

Li, H.; Kiang, Y. H.; Jona, J. Eur. J. Pharm. Sci. 2009, 38, 426-32.

6.

Threlfall, T. L. Org. Process Res. Dev. 2009, 13, 1224-1230.

7.

Veesler, S.; Lafferrère, L.; Garcia, E.; Hoff, C. Org. Process Res. Dev. 2003, 7, 983-989.

8.

Grant, D. J. W.; Mehdizadeh, M.; Chow, A. H. L.; Fairbrother, J. E. Int. J. Pharm. 1984,

18, 25-38.

9.

Due to the low pKa value of 1, only strong acids were chosen from the first tier acid list:

hydrochloric acid, sufuric acid, p-toluenesulfonic acid, methanesulfonic acid, benzensulfonic acid, maleic acid, phosporic acid, L-tartaric acid, fumaric acid, citric acid, and succinic acid.

10.

The process to manufacture 1 will be disclosed in a separate manuscript.



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Figure Captions Figure 1. X-ray diffraction patterns of a) Form 1, and b) Form 2 of 1.

Figure 2. DSC (solid line) and TGA (dash line) of 1 Form 1, showing a melting endotherm at 222.9 °C.

Figure 3. DSC (solid line) and TGA (dash line) of 1 Form 2, showing a melting endotherm at 221.7 °C.

Figure 4. Moisture sorption isotherm at 25 °C for 1 a) Form 1 and b) Form 2. Adsorption data are in closed circles and solid line; desorption data are in open circles and dashed line.

Figure 5. The van’t Hoff plot of 1 Form 1 (closed circle) and Form 2 (open circle), showing two linear regression lines crossing at 480 K.

Figure 6. Viewing down the b axis of 1 Form 1 single crystal structure. Carbon is represented as small sphere, nitrogen as medium sphere, and oxygen as large sphere. (Hydrogen atoms are removed for clarity.)

Figure 7. Viewing down the b axis of 1 Form 2 single crystal structure. Carbon is represented as small sphere, nitrogen as medium sphere, and oxygen as large sphere. (Hydrogen atoms are removed for clarity.)

Figure 8. Overlay of 1 molecule in Form 1 (black) and Form 2 (grey) showing the difference in conformation of 1 in these two polymorphs.

Figure 9. (a) Observed, and (b) calculated X-ray powder diffraction patterns of 1 Form 1.

Figure 10. (a) Observed, and (b) calculated X-ray powder diffraction patterns of 1 Form 2.


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Figure 11. Solubility profile of 1 Form 2 in DMSO/water at different temperatures.

Figure 12. In situ Raman spectra after anti-solvent addition a) during use test and b) scale-up for the DMSO process, showing 25-50% of Form 1. Raman spectra of Form 1 and Form 2 are plotted for comparison.

Figure 13. In situ Raman spectra after aging 2 hours at 60 °C a) during use test and b) scale-up for the acetic acid process, showing less than 25% of Form 1. Raman spectra of Form 1 and Form 2 are plotted for comparison.

Figure 14. Solubility profile of 1 Form 2 in acetic acid/water at different temperatures.

Figure 15. In situ Raman spectra after anti-solvent addition a) during use test and b) scale-up for the acetic acid process, showing absence of Form 1. Raman spectra of Form 1 and Form 2 are plotted for comparison.



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Table 1. Solubility of 1 Form 1 and Form 2 in Acetonitirle Form 1 T(ºC)

28

35

60

Solubility (mg/mL) 0.61 0.63 0.63 0.95 0.95 0.96 1.53 1.97 1.91

Form 2 Average (mg/mL) 0.62±0.01

0.95±0.01

1.80±0.24

Solubility (mg/mL) 0.28 0.29 0.29 0.46 0.46 0.48 1.28 1.26 1.44


Average (mg/mL) 0.29±0.01

0.46±0.01

1.33±0.10

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1 2 3 4 Average

Table 2. Thermal Data of 1 Form 1 and Form 2 Form 1 Form 2 m.p (ºC) heat of fusion m.p (ºC) heat of fusion (J/g) (J/g) 222.86 114.6 221.52 121.2 222.69 108.9 221.73 119.1 222.96 112.7 221.21 119.1 222.81 113.0 222.28 122.2 222.83±0.11 112.3±2.4 221.69±0.45 120.4±1.5



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week 2 4 8

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Table 3. Chemical Stability of 1 40 °C/75% RH 60 °C Form 1 Form 2 Form 1 Form 2 % % % % % % % % weight peak area weight peak area weight peak area weight peak area 98.9 99.8 100.2 99.5 98.8 99.8 100.8 99.5 99.1 99.8 101.4 99.5 99.3 99.7 100.0 99.5 100.9 99.8 100.9 99.5 99.9 99.8 101.2 99.5


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Table 4. Solubility of 1 Form 1 in Selected Organic Solvents at Room Temperature Solvent Solubility (mg/mL) ICH class Water

Selection of a Suitable Physical Form and Development of a

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