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1. WO2013155592 - CRYSTAL FORMS OF GOLOTIMOD AND PROCESS FOR ITS MANUFACTURING

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]
CRYSTAL FORMS OF GOLOTIMOD AND PROCESS FOR ITS MANUFACTURING

TECHNICAL FIELD

This invention relates to crystal forms of Golotimod and processes for their manufacturing.

BACKGROUND

The name Golotimod (CAS#: 229305-39-9) refers to a dipeptide H-D-Glu(L-Trp- OH)-OH with the IUPAC name (R)-2-amino-5-(((S)-1-carboxy-2-(1H-indol-3- yl)ethyl)amino)-5-oxopentanoic acid. It is also known as SCV-07 or gamma-D-glutamyl-L- tryptophan or bestim. It is disclosed in US5,916,878:


It is reported as useful for modulating the immune system of a patient (US

5,744,452), and useful for treating: lung cancer (WO 2009/025830A1 ), tuberculosis (WO 2003/013572 A1 ), genital viral infections (WO 2006/076169), melanoma (WO

2007/123847), hemorrhagic viral infections (WO 2006/047702), respiratory viral infections (WO 2005/112639), hepatitis C (WO 2010/017178), and injury or damage due to disease of mucosa (WO 2008/100458). SCV-07 is also reported as a vaccine enhancer (WO 2006/116053).

The preparation of a sodium salt of H-D-Glu(L-Trp-OH)-OH is described in example 1 D of US5.916,878. A solution of Cbz-D-Glu(L-Trp-OBzl)-O-Bzl in methanol is

hydrogenated over 10% palladium-on-charcoal (Pd/C) catalyst in the presence of one equivalent of sodium bicarbonate over a period of 4 hours. In this procedure, 2.5 g of 10% Pd/C is used per 5 g of Cbz-D-Glu(L-Trp-OBzl)-O-Bzl. The amount of 10% Pd/C is definitely not used in calalytic amount. In addition, a hydrogenation room and a

hydrogenator are required for large scale manufacturing in the plant. There is no

characterization data or purification procedure reported. The product (2.14 g) is obtained by triturating the methanol solution after hydrogenation and filtration with acetonitrile.

Since acetonitrile is also a toxic solvent and is the last solvent used in the production of this active pharmaceutical ingredient, this procedure is not amenable to the production of a pharmaceutically acceptable salt in kg amounts for the manufacturing of a finished dosage.

The synthesis of gamma-D-glutamyl-L-tryptophan is described in example 1A of US5.916,878. It is prepared by hydrogenation of a solution of N-benzyloxycarbonyl- gamma-D-glutamyl-L-tryptophan dibenzyl ester in methanol over Pd/C over 5 hours

(Scheme A).


Scheme A

In this synthesis: (a) 200 mg of Pd/C is used to convert 600 mg of N- benzyloxycarbonyl-gamma-D-glutamyl-L-tryptophan dibenzyl ester to 260 mg of gamma-D- glutamyl-L-tryptophan. The catalyst is removed by filtration across charcoal and the filtrate is evaporated to give gamma-D-glutamyl-L-tryptophan. The Pd/C is not used in a catalytic amount; (b) preparative HPLC is used to purify the crude gamma-D-glutamyl-L-tryptophan (260 mg) to pure gamma-D-glutamyl-L-tryptophan (190 mg), the eluant is a mixture of 0.1 % trifluoroacetic acid and acetonitrile; (c) the material is obtained by lyophilization. The yield is not reported, and the calculated yield of the transformation is about 61.5%.

In another approach (Suzuki et al., Journal of Biotechnology 2004, 111 , 291-295), H-D-Glu(L-Trp-OH)-OH is synthesized from D-glutamine and L-tryptophan through the transpeptidation reaction using Escherichia coli gamma-glutamyltranspeptidase (GGT) as a catalyst. The optimal condition reported involved the use of 50 mM D-glutamine and 50 mM L-tryptophan. The conversion did not go to completion. The reaction mixture was purified on a Dowex 1 x 8 column, washed with 150 ml of water, and then 5 N CH3COOH. The fractions which contained only gamma-D-glutamyl-L-tryptophan were saved and lyophilized to produce 126 mg of product.

Summary

This invention is based, at least in part, on crystal forms of Golotimod (H-D-Glu(L- Trp-OH)-OH) herein termed crystal form A and crystal form B and methods of preparing the crystal forms.

This invention is also based, at least in part, on methods for the preparation of pharmaceutical grade H-D-Glu(L-Trp-OH)-OH from H-D-Glu(L-Trp-O-T)-O-G wherein T is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or sec-butyl; G is benzyl, or C1-C3 alkyl.

In illustrative embodiments, there is provided, a crystalline form of golotimod characterized by an X-ray powder diffraction pattern comprising peaks, in degrees 2-theta + 0.2, at: 10.59, 11.40, and 23.25.

In illustrative embodiments, there is provided a crystalline form described herein wherein the X-ray powder diffraction pattern further comprises peaks, in degrees 2-theta + 2, at: 17.24, 18.04, 19.27, 19.49, 20.52, 21.36, 24.92, 25.33, 28.19, 29.74, and 32.68.

In illustrative embodiments, there is provided a crystalline form described herein further characterized by a solid state 13C NMR spectrum comprising peaks, in ppm at 25.7, 35.2, 59.1 , 1 16.1 , and 179.3.

In illustrative embodiments, there is provided a crystalline form described herein wherein the solid state 13 CNMR spectrum further comprises peaks, in ppm at: 24.7, 54.5, 114.0, 1 18.0, 120.9, 122.4, 125.9, 135.7, 173.7, and 176.8.

In illustrative embodiments, there is provided a crystalline form described herein wherein the X-ray powder diffraction pattern is substantially the same as shown in FIG. 2.

In illustrative embodiments, there is provided a crystalline form described herein further characterized by a solid state 13C NMR spectrum substantially the same as shown in FIG. 1.

In illustrative embodiments, there is provided a crystalline form described herein further characterized by an IR spectra having absorption maxima, in cm-1, at 742, 1094, 1231, 1455, 1536 1598, 1702, 3354, and 3405.

In illustrative embodiments, there is provided a pharmaceutical composition

comprising a crystalline form of golotimod described herein and a pharmaceutically acceptable exciprent.

In illustrative embodiments, there is provided a crystalline form of golotimod characterized by an X-ray powder diffraction pattern comprising peaks, in degrees 2-theta + 0.2, at 10.30, 12.22, and 26.68.

In illustrative embodiments, there is provided a crystalline form described herein wherein the X-ray powder diffraction pattern further comprises peaks, in degrees 2-theta + 2, at: 15.6, 17.53, 17.83, 18.10, 18.89, 19.68, 20.95, 22.17, 24.29, 24.88, 27.85 and 32.63.

In illustrative embodiments, there is provided a crystalline form described herein further characterized by a solid state 13C NMR spectrum comprising peaks, in ppm, at 30.3, 112.6, and 129.3.

In illustrative embodiments, there is provided a crystalline form described herein wherein the solid state 13CNMR spectrum further comprises peaks, in ppm, at: 24.0, 54.8, 111.4, 118.6, 120.8, 134.6, 172.3, 176.0, and 177.2.

In illustrative embodiments, there is provided a crystalline form described herein wherein the X-ray powder diffraction pattern is substantially the same as shown in FIG. 8.

In illustrative embodiments, there is provided a crystalline form described herein further characterized a solid state 13C NMR spectrum substantially the same as shown in FIG. 7.

In illustrative embodiments, there is provided a crystalline form described herein further characterized by an IR spectra having absorption maxima, in cm-1, at 744, 860, 1029, 1087, 1226, 1258, 1455, 1531 , 1609, 1655, 1700, 3374, 3401 , and 3418.

In illustrative embodiments, there is provided a pharmaceutical composition comprising a crystalline form of golotimod described herein and a pharmaceutically acceptable excipient.

In illustrative embodiments, there is provided a process for the preparation of golotimod comprising: (a) reacting a solution of H-D-Glu(L-Trp-OMe)-O-Bzl.HCl salt in an alcohol with 3N NaOH at about 0°C, thereby forming a solution; (b) removing insoluble particles from the solution by: (b-i) (b-i-a) washing the solution with ethyl acetate, thereby forming an aqueous layer; and (b-i-b) separating and filtering the aqueous layer, thereby forming an aqueous solution; or (b-ii): (b-ii-a) filtering the solution, thereby forming a filtrate; (b-ii-b) adjusting a pH of the filtrate with 6N HCl to a pH of from about 10 to about 11, thereby forming a basic solution; (b-ii-c) evaporating the basic solution to about 50% of the original volume of the basic solution, thereby forming an evaporated basic solution; (b- ii-d) extracting the evaporated basic solution with ethyl acetate, thereby forming an aqueous layer; and (b-ii-e) separating the aqueous layer, thereby forming an aqueous solution; or (b-iii) (b-iii-a) filtering the solution, thereby forming a filtrate; (b-iii-b) adjusting a pH of the filtrate with 6N HCI to a pH of about 7.0, thereby forming a neutral solution; (b-iii- c) evaporating the neutral solution to about 50% of the original volume of the neutral solution, thereby forming an aqueous solution; (c) acidifying the aqueous solution to a pH of from about 2.6 to about 3.1 with 6N HCl solution at about 0°C with stirring, thereby forming an acidic solution; (d) isolating a solid from the acidic solution; and (e) drying the solid by drying the solid in air followed by drying in a vacuum oven at about a pressure of about 0.2 inch Hg and at a at temperature of about 42°C for a period of time of from about 10 hours to about 12 hours.

In illustrative embodiments, there is provided a process described herein wherein the isolating the solid from the acidic solution comprises filtering the acidic solution.

In illustrative embodiments, there is provided a process described herein wherein the isolating the solid from the acidic solution further comprises suspending the solid in water at a temperature of about 48°C by stirring for at least about 30 minutes followed by cooling to a temperature of from about 20°C to about 24°C, followed by filtering.

In illustrative embodiments, there is provided a process described herein wherein the isolating the solid from the acidic solution further comprises washing the solid with de-ionized water followed by washing the solid with acetone.

In illustrative embodiments, there is provided a process described herein wherein the alcohol is methanol or isopropanol.

In illustrative embodiments, there is provided a process described herein wherein the acidifying the aqueous solution comprises stirring for a period of time of from about 8 hours to about 16 hours.

In illustrative embodiments, there is provided a process described herein wherein the drying the solid in air comprises drying for a period of time of from about 2 hours to about 2 days.

In illustrative embodiments, there is provided a process described herein wherein the drying the solid in air comprises drying for a period of time of from about 2 hours to about 4 hours.

In illustrative embodiments, there is provided a process for the preparation of golotimod from H-D-Glu(L-Trp-OMe)-OBzl.HCl salt comprising: (a) reacting a solution of H-D-Glu(L-Trp-OMe)-O-Bzl.HCl salt in isopropanol with 3N NaOH at a temperature of about 0°C, thereby forming a solution; (b) washing the solution with ethyl acetate, thereby forming an aqueous layer; (c) separating and filtering the aqueous layer thereby forming a filtrate; (d) acidifying, with HCOOH solution at a temperature of about 0°C, the filtrate to a pH of from about 2.6 to about 3.1 , thereby forming a material; (e) stirring the material for a period of time of from about 8 hours to about 16 hours and filtering, thereby forming a solid; (f) washing the solid with de-ionized water, thereby forming a washed solid; (g) drying the washed solid in air for a period of time of about 4 hours, followed by drying the washed solid in a vacuum oven at a pressure of about 0.2 inch Hg and at a temperature of about 42°C for a period of time of from about 10hours to about 12 hours.

In illustrative embodiments, there is provided a process for the crystallization of golotimod comprising: (a) dissolving H-D-Glu(L-Trp-OH)-OH in water by heating a suspension of golotimod in water, thereby forming a solution; (b) filtering the solution, thereby forming a filtrate; (c) adding isopropanol to the filtrate at a ratio of about 1 :2 to the volume of the filtrate, thereby forming a crystallization solution; (d) leaving the

crystallization solution to crystallize.

In illustrative embodiments, there is provided a process for the crystallization of golotimod comprising: (a) dissolving H-D-Glu(L-Trp-OH)-OH in water by heating a mixture of H-D-Glu(L-Trp-OH)-OH in water to a temperature of about 80°C, thereby forming a material; (b) filtering the material, thereby forming a solution; (c) cooling the solution, thereby crystallizing the H-D-Glu(L-Trp-OH)-OH in a crystallized solution; (d) filtering the crystalized solution, thereby forming a solid; and (e) drying the solid in air for a period of time of from about 2 hours to about 4 hours followed by drying the solid under vacuum at a temperature of from about 40°C to about 45°C.

In illustrative embodiments, there is provided a process for the crystallization of golotimod comprising: (a) adjusting, with sodium hyyroxide solution, a pH of a solution of

H-D-Glu(L-Trp-OH)-OH in water to a pH of from about 10 to about 11 , thereby fomning a basic solution; (b) extracting the basic solution with ethyl acetate thereby forming an aqueous solution; (c) filtering the aqueous solution, thereby forming a filtrate; (d) precipitating crystalline H-D-Glu(L-Trp-OH)-OH by adjusting a pH of the filtrate to pH of from about 2.6 to about 3.1 , thereby forming a precipitated solution; (e) filtering the precipitated solution, thereby forming a solid; (f) washing the solid thereby forming a washed solid; and (g) drying the washed solid in air for a period of time of from about 2 hours to about 4 hours followed by drying the washed solid under vacuum at a temperature of about 40°C to about 45°C.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

Brief Description of the Drawings

In drawings which illustrate embodiments of the invention,

Figure 1 shows the solid state 13C NMR spectrum for Form A of H-D-Glu(L-Trp-OH)- OH.

Figure 2 shows the powder X-ray diffraction pattern of Form A of H-D-Glu(L-Trp- OH)-OH.

Figure 3 shows the FTIR of Form A of H-D-Glu(L-Trp-OH)-OH as a KBr pellet.

Figure 4 shows the single crystal structure of Form AH-D-Glu(L-Trp-OH)-OH.

Figure 5 shows the single crystal structure of From A of H-D-Glu(L-Trp-OH)-OH in the crystal lattice and its relationship with other molecules of H-D-Glu(L-Trp-OH)-OH.

Figure 6 shows a XRPD pattern of Form A of golotimod simulated from a single-crystal X-ray diffraction crystal structure obtained from a representative single crystal of Form A.

Figure 7 shows the solid state 13C NMR spectrum for Form B of H-D-Glu(L-Trp-OH)- OH.

Figure 8 shows the powder X-ray diffraction of Form B of H-D-Glu(L-Trp-OH)-OH. Figure 9 shows the FTIR of Form B of H-D-Glu(L-Trp-OH)-OH as a KBr pellet Figure 10 shows the single crystal structure of Form B of H-D-Glu(L-Trp-OH)-OH.

Figure 11 shows the single crystal structure of Form B of H-D-Glu(L-Trp-OH)-OH in the crystal lattice and its relationship with other molecules of H-D-Glu(L-Trp-OH)-OH.

Figure 12 shows a XRPD pattern of Form B of H-D-Glu(L-Trp-OH)-OH simulated from a single-crystal X-ray diffraction crystal structure obtained from a representative single crystal of Form B.

Figure 13 shows a comparison of the XRPD pattern of Form A and Form B. The XRPD patterns of Form B (top) are compared to Form A (bottom) of H-D-Glu(L-Trp-OH)- OH.

Figure 14 shows the speciation plot of H-D-Glu(L-Trp-OH)-OH.

Figure 15 shows the FTIR of Apo816DL-Form A recorded with The PerkinElmer Spectrum Two FTIR instrument equipped with Attenuated total reflectance (ATR) as the sampling technique. The data were analyzed by Spectrum Two software (Application Version: 10.03.09.0139). The FTIR shown refers to Apo816DL-Form A with ATR

correction.

Figure 16 shows the FTIR of Apo816DL-Form B recorded with The PerkinElmer Spectrum Two FTIR instrument equipped with Attenuated total reflectance (ATR) as the sampling technique. The data were analyzed by Spectrum Two software (Application Version: 10.03.09.0139). The FTIR shown refers to Apo816DL-Form B with ATR

correction.

Detailed Description

As used herein, the term "golotimod" is a nonproprietary name adopted by the

USAN council for the chemical D-gamma-Glutamyl-L-Tryptophan with the lUPAC name (2R )-2-amino-5-[[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino]-5-oxopentanoic acid, having the chemical structure:


"Golotimod" is also termed herein "Apo816DL".

As used herein, the term "substantially" when used with respect to an X-ray powder diffraction pattern or part thereof, means that any spectrum exhibiting the same sequence of peaks and minima, showing the same Theta and d interlattice interval position (X-ray) all within a standard deviation of + 0.5% complies with the subject X-Ray powder diffraction pattern and may be considered as an X-ray diffraction pattern of the same material.

A crystalline form of Golotimod designed crystal Form A is disclosed herein and may be characterized by at least one of the following characteristics:

(i) a solid state 13C NMR spectrum comprising peaks at 24.8, 25.8, 35.2, 54.5, 59.1 , 109.7, 1 14.1 , 1 16.2, 118.2, 121.0, 122.6, 126.1 , 135.7, 173.66, 176.8, and 179.3 ppm and the NMR spectra was referenced with respect to tetramethylsilane (δ(13C) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm;

(ii) an X-ray powder diffraction pattern comprising at least three approximate positions (°2θ + 0.2), when measured using CuKa radiation, selected from the group consisting of 10.77, 1 1.59, 17.42, 18.21 , 19.41, 19.65, 20.72, 21.96, 23.42, 25.03, 25.47, 28.35, 29.87, and 32.84.

A crystalline form of Golotimod designated crystal Form A is disclosed and may be characterized by a solid state 13C NMR spectrum comprising peaks substantially in agreement with the solid state 13C NMR pattern as set out in Figure 1.

A crystalline form of Golotimod designated crystal Form A is disclosed and may be characterized by an X-ray powder diffraction pattern substantially in agreement with the X-ray powder diffraction pattern as set out in Figure 2.

A crystalline form of Golotimod designated crystal Form A is disclosed and may be characterized by an FTIR spectrum comprising peaks substantially in agreement with the FTIR pattern provided in Figure 3. IR absorption maxima are observed at, 742, 1094, 1230, 1455, 1536 1598, 3454, and 3405 cm-1.

A single crystal structure of Form A was obtained from representative Form A crystals which is illustrated in Figures 4 and 5.

A composition comprising a crystalline form of Golotimod designed crystal Form A is also disclosed.

A crystalline form of Golotimod designed crystal Form B is also disclosed and may be characterized by at least one of the following characteristics:

(i) a solid state 13C NMR spectrum comprising peaks at 24.0, 30.3, 54.8, 111.4, 112.6, 118.6, 120.8, 129.3, 134.6, 172.3, 176.0, and 177.2 ppm and the NMR spectra was referenced with respect to tetramethylsilane (δ(13C) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm;

(ii) an X-ray powder diffraction pattern comprising at least three

approximate positions (°2θ ± 0.2), when measured using CuKα radiation, selected from the group consisting of 10.30, 12.22, 15.6, 17.53, 17.83, 18.10, 18.33, 18.89, 19.68, 20.95, 22.17, 24.29, 24.88, 26.68, 27.85, and 32.63.

A crystalline form of Golotimod designated crystal Form B is disclosed and may be characterized by a solid state 13C NMR spectrum comprising peaks substantially in agreement with the solid state 13C NMR pattern as set out in Figure 7.

A crystalline form of Golotimod designated crystal Form B is disclosed and may be characterized by an X-ray powder diffraction pattern substantially in agreement with the X- ray powder diffraction pattern as set out in Figure 8.

A crystalline form of Golotimod designated crystal Form B is disclosed and may be characterized by an FTIR spectrum comprising peaks substantially in agreement with the FTIR pattern as set out in Figure 9. IR absorption maxima are observed at 747, 860, 1029, 1087, 1226, 1258, 1455, 1531, 3374, 3418, and 3401 cm-1.

A single crystal structure of Form B was obtained from representative Form B crystals which is illustrated in Figures 10 and 11.

There is also provided a process, termed herein Process A, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) reacting a solution of H-D-Glu(L-Trp-OMe)-O-Bzl.HCl salt in isopropanol with 3N NaOH at about 0°C;

(b) washing of the solution from step (a) with ethyl acetate;

(c) separating the aqueous layer from step (b) and filtering the aqueous layer to obtain the filtrate;

(d) acidifying the filtrate to pH 2.9 to 3.12.6 to 3.1 with 6N HCl solution at about

0°C;

(e) filtering the solid after stirring the material from step (d) for 8 to 16 hours;

(f) washing the solid with de-ionized water; and

(g) drying the solid in air for 4 hours, followed by drying in a vacuum oven at about 0.2 inch Hg at about 42°C for 10-12 hours;

There is also provided a process, termed herein Process B, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) reacting a solution of H-D-Glu(L-Trp-OMe)-0-Bzl.HCl salt in methanol with 3N NaOH at about 0°C;

(b) filtering the solution from step (a) from insoluble particles;

(c) adjusting the pH of the filtrate from step (b) with 6N HCl to pH 10 to 11 ;

(d) evaporating the solution from step (c) to about 50% of its original volume;

(e) extraction of the solution from step (d) with ethyl acetate;

(f) separating the aqueous layer from step (e);

(g) acidifying the filtrate from step (f) to pH 2.6 to 3.1 with 6N HCl solution at about 0°C;

(h) filtering the solid from step (g);

(i) washing the solid from step (h) with de-ionized water;

(j) suspending the solid from step (i) in water at 48°C and stirring it for 30 min. and then cooling the reaction mixture to 20-24°C;

(k) filtering the solid from step (j); and

(I) drying the solid in air for 2 days, followed by drying in a vacuum oven at about 0.2 inch Hg at about 41 °C for 10-12 hours;

There is also provided a process, termed herein Process C, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) reacting a solution of H-D-Glu(L-Trp-OBzl)-OMe.HCl salt in methanol with 3N NaOH at about 0°C;

(b) filtering the solution from step (a) from insoluble particles;

(c) adjusting the pH of the filtrate from step (b) with 6N HCl to pH 6.0 to 6.5;

(d) evaporating the solution from step (c) to about 50% of its original volume;

(e) acidifying the filtrate from step (d) to pH 2.6 to 3.1 with 6N HCl solution at about 0°C;

(f) filtering the solid from step (e);

(g) washing the solid from step (f) with de-ionized water;

(h) washing the solid from step (g) with ether;

(i) suspending the solid from step (h) in water at 20-24°C and stirring it for 10 min, followed by stirring it for 30 min at 48°C; and then cooling the reaction mixture to 20- 24°C;

(j) filtering the solid from step (i); and

(k) drying the solid from step (j) in air for 4 hours, followed by drying in a vacuum oven at about 0.2 inch Hg at about 40°C for 10-12 hours.

The material produce by process A, B and/or C above, when characterized by XRPD and solid state 13C NMR, typically exhibits XRPD and solid state 13C NMR characteristics of golotimod crystal Form A.

When Process A or B or C is used to produce crystal form A, the use of seed crystals of Form A may be advantageous to provide greater control of the production of form A, particularly on large scale.

There is also provided a process, termed herein Process D, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) heating a mixture of H-D-Glu(L-Trp-OH)-OH in water to about 80°C to effect dissolution;

(b) filtration of the material from step (a) to remove insoluble particulates;

(c) cooling the solution from step (b) to crystallize the H-D-Glu(L-Trp-OH)-OH;

(d) filtration of the insoluble solid from step (c); and

(e) drying the solid in air for 2 to 4 hours and further drying the solid under vacuum at 40°C to 45°C;

There is also provided a process, termed herein Process E, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) adjusting the pH of a solution of H-D-Glu(L-Trp-OH)-OH in water to pH 10-11 with sodium hydroxide solution;

(b) extracting the solution with ethyl acetate and retaining the aqueous solution

(a);

(c) filtering the aqueous solution from step (b);

(d) adjusting the pH of the filtrate from step (c) to pH 2.6 to 3.1 to precipitate the crystalline form of H-D-Glu(L-Trp-OH)-OH;

(e) filtering the resulting solid and washing the resulting solid with water; and

(f) drying the solid in air for 2 to 4 hours and further drying the solid under vacuum at 40°C to 45°C;

There is also provided a process, termed herein Process F, for manufacturing golotimod, which process does not require chromatographic purification and comprises:

(a) reacting a solution of H-D-Glu(L-Trp-OMe)-O-Bzl.HCl salt in isopropanol with 3N NaOH at about 0°C;

(b) washing of the solution from step (a) with ethyl acetate;

(c) separating the aqueous layer from step (b) and filtering the aqueous layer to obtain the filtrate;

(d) acidifying the filtrate to pH 2.6 to 3.1 with HCOOH solution at about 0°C;

(e) filtering the solid after stirring the material from step (d) for 8 to 16 hours;

(f) washing the solid with de-ionized water; and

(g) drying the solid in air for 4 hours, followed by drying in a vacuum oven at about 0.2 inch Hg at about 42°C for 10-12 hours.

The material produce by process D, E and/or F above, when characterized by XRPD and solid state 13C NMR, typically exhibits XRPD and solid state 13C NMR

characteristics of golotimod crystal Form B.

In one embodiment, the present invention provides a method for manufacturing pharmaceutical grade golotimod, without chromatographic purifications. As an illustrative example for large scale manufacturing, the compound H-D-Glu(L-Trp-O-T)-O-G.HCl wherein G is benzyl and T is methyl, namely H-D-Glu(L-Trp-OCH3)-O-BzI.HCl is often used herein to describe an illustrative process.

In some embodiments, there is provided a method for manufacturing H-D-Glu(L-Trp-OH)-OH without chromatography purification which method comprises process A or process B or process C as shown in Table 8. The material isolated in these three processes typically match the characterization of crystal form A of golotimod.

Depending on the pH of the solution, H-D-Glu(L-Trp-OH)-OH can exist in different protonated forms, namely H3L, H2L, HL, L. The definition of these species is set out in Scheme 2 below:


Scheme 2

The speciation distribution of H3L, H2L, HL, L depends on the pH of the solution and can be computed using the experimental pKas of the compound with the HySS 2009 software. The speciation plot of H-D-Glu(L-Trp-OH)-OH is shown in Figure 13.

The computed speciation distribution results are shown in Table 1 below. At pH 2.5 to 3.0, the majority of the product in the mixture is H2L, which has the chemical structure H-D-Glu(L-Trp-OH)-OH.


The H2L species is only sparingly soluble in the mixture and the compound will precipitate out of the aqueous solution at pH of about 2.6-3.2, substantially free or free of all impurities. The optimal pH range for the precipitation of H2L species appears to be between pH 2.7 to 2.9 wherein the H2L species is present in about 70%. Thus, this method eliminates prep HPLC purification requirements for the manufacture of H-D-Glu(L-Trp-OH)- OH. Further, no hydrogenation reaction is required because the crude H-D-Glu(L-Trp-OH)- OH is prepared from the base hydrolysis of H-D-Glu(L-Trp-OCH3)-O-Bzl. Individual Kas of the compound H-D-Glu(L-Trp-OH)-OH, as set out below, may be used to compute the speciation composition of the mixture at different pHs, specifically from pH 1 to 10.


In some embodiments, a solution of H-D-Glu(L-Trp-OH)-OH is adjusted to pH 10-11 with sodium hydroxide, and filtered to remove insoluble particulates. The pH of the solution is adjusted to between 2.6 to 3.2. The crystal form obtained depends on the solvent composition of the solution of H-D-Glu(L-Trp-OH)-OH in sodium hydroxide and also on the acid used in the neutralization to the pH range as desired. With process A or B or C, crystal form A may be isolated. Other variations of the processes parameters may result in designated form B.

In some embodiments, the predominant species H2L precipitates out of the solution and is filtered to give pharmaceutical grade pure H-D-Glu(L-Trp-OH)-OH.

When 1 g of H-D-Glu(L-Trp-OH)-OH produced from process A or B or C is dissolved in 40 ml of hot water at about 80°C, a material that is suitable for single crystal structure determination may be obtained. The single crystal structure shown in Figures 10 and 11 has the following unit cell parameters when measured at approximately 150K: a =

10.1575(3) Å; b = 5.2975(1 ) Å; c = 14.5631 (4) Å; a= 90°; b= 94.7680(14)°; g = 90°; V = 780.92(3) A3; Z = 2; calculated density is 1.417 Mg/m3; and the space group is P2V The computed XRPD using the single crystal structure data does not match with XRPD of crystal form A. This crystal form golotimod crystal form B.

Form B may be produced from the recrystallization of golotimod in water according to a representative scheme shown as process D in Table 9. Table 9 also shows two more processes useful for preparing crystal form B, processes E and F.

Golotimod has limited solubility in water. Process E may be used to prepare form B. In process E, sodium hydroxide solution is added to a mixture of golotimod in water to adjust the pH to between 10 and 11. The solution is extracted with ethyl acetate to remove organic impurities and the aqueous layer is filtered to remove any insoluble particulates. The pH of the solution is then adjusted with hydrochloric acid solution to between 2.7 and 3 and the insoluble solid is filtered. Upon drying, the solid obtained has the characteristics of Form B, as characterized by its XRPD (Figure 7), solid state C13 NMR (Figure 8), FTIR (Figure 9), and a single crystal structure (Figures 10 and 11). It should be noted that there is no co-solvent used in Process E. Water is the only solvent.

In process D and E, golotimod may be used as a starting material to prepare crystalline Form B of golotimod.

Process F is another method that may be used for preparation of crystal Form B of golotimod. In process F, a solution of the diester H-D-Glu(L-Trp-OMe)-O-Bzl is hydrolyzed with sodium hydroxide. The material is extracted with ethyl acetate to remove organic impurities. The aqueous filtrate is filtered and then acidified with formic acid to pH between 2.6 to 3.2 at about 0°C. The insoluble solid is filtered, washed with water and dried in a vacuum oven at 40-45°C. This solid is golotimod crystal form B.

In some embodiments, Form A of golotimod may be characterized by a solid state 13C NMR spectrum comprising peaks at 24.8, 25.8, 35.2, 54.5, 59.1 , 109.7, 114.1 , 116.2, 1 18.2, 121.0, 122.6, 126.1 , 135.7, 173.66, 176.8, and 179.3 ppm and the NMR spectra was referenced with respect to tetramethylsilane (δ(13C) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm.

In some embodiments, Form A of golotimod exhibits a solid state 13C NMR spectrum substantially similar to the spectrum set out in Figure 1.

In some embodiments, Form A of golotimod is characterized by XRPD peaks located at two or more of the following positions: about 10.59, about 11.40, about 17.24, about 18.04, about 19.27, about 19.49, about 19.71 , about 20.52, about 21.38, about 21.80, about 23.25, about 24.92, about 25.32, about 28.19, about 29.74, about 32.68, about 34.47, about 36.21 degree 2θ. It is understood by a person of skill in the art when solvents and/or water are added or removed from lattice, the lattice may slightly expand or contract, resulting in minor shifts in the position of the XRPD peaks.

In some embodiments of the present invention, Form A of golotimod is provided which may be characterized by an XRPD pattern substantially similar to the pattern as set out in Figure 2.

In some embodiments, Form A of golotimod exhibits an XRPD pattern substantially similar to the pattern set out in Figure 2 but may exhibit small shifts in peak positions resulting from the presence or absence of specific solvents or water in the crystal lattice.

The single crystal of Form A is prepared from the recrystallization from isopropanol and water. A single-crystal X-ray diffraction (XRD) crystal structure was obtained from a representative single crystal of Form A of golotimod. Using XRD data collected at about 150 K, the following unit cell parameters were obtained: a = 5.1386(1) Å; b = 9.7099(2) Å; c = 15.2429(4) Å; \α = 90°; β = 95.002(1 )°; γ = 90°; V = 757.65(3) Å 3; Z = 2; calculated density is 1.461 Mg/m3; and the space group is P2i.

A crystal packing diagram from the single-crystal XRD structure of Form A, is set out in Figure 5. A simulated XRPD pattern was generated for Cu radiation using Platon (Platon for Windows Taskbar V1.081 , 2005) and the atomic coordinates, space group, and unit cell parameters from the single crystal data. Such a simulated XRPD pattern of Form A of golotimod is set out in Figure 6.

In some embodiments, Form B of golotimod is characterized by a solid state 13C NMR spectrum comprising peaks at 24.0, 30.3, 54.8, 1 11.4, 1 12.6, 118.6, 120.8, 129.3, 134.6, 172.3, 176.0, and 177.2 ppm and the NMR spectra was referenced with respect to tetramethylsilane (δ(13C) = 0.0 ppm) by setting the high-frequency resonance of

adamantane to 38.56 ppm.

In some embodiments, Form B of golotimod may exhibit a solid state 13C NMR spectrum substantially similar to the spectrum as set out in Figure 7.

In some embodiments, Form B of golotimod may be characterized by XRPD peaks located at two or more of the following positions: about 10.30, about 12.22, about 15.6, about 17.53, about 17.83, about 18.10, about 18.33, about 18.89, about 19.68, about 20.95, about 22.17, about 24.29, about 24.88, about 26.68, about 27.85, about 32.63 degree 2θ. It is understood by a person of skill in the art when solvents and/or water are added or removed from lattice, the lattice may slightly expand or contract, resulting in minor shifts in the position of the XRPD peaks.

In some embodiments of the present invention, Form B of golotimod is provided which may be characterized by an XRPD pattern substantially similar to the pattern set out in Figure 8.

In some embodiments, Form B of golotimod exhibits an XRPD pattern substantially similar to the pattern set out in Figure 8 but may exhibit small shifts in peak positions resulting from the presence or absence of specific solvents or water in the crystal lattice.

A single-crystal X-ray diffraction (XRD) crystal structure was obtained from a representative single crystal of Form B of golotimod. Using XRD data collected at about 150 K, the following unit cell parameters were obtained: a = 10.1575(3) Å; b = 5.2975(1) Å; c = 14.5631(4) Å; a = 90°; β = 94.7680(14)°; γ = 90°; V = 780.92(3) Å3. A crystal packing diagram from the single-crystal XRD structure of Form B, is set out in Figure 11. A simulated XRPD pattern was generated for Cu radiation using Platon (Platon for Windows Taskbar V1.081, 2005) and the atomic coordinates, space group, and unit cell parameters from the single crystal data. Such a simulated XRPD pattern of Form B of golottmod is set out in Figure 12.

In certain embodiments of the invention, recrystallization of Form A in hot water resulted in the formation of Form B. One such example is the use of process D described herein to prepare Form B.

In some embodiments, Form A is treated with sodium hydroxide solution to produce a solution of golotimod at a pH of 10 to 11. Readjustment of the pH to 2.6-3.1 with hydrochloric acid results in the formation of Form B which may be precipitated out of solution. One such example is the use of process E described herein to prepare Form B.

Thus, depending on the conditions of crystallization and the crystallization time and stirring time, an unequal mixture of Form A and Form B may be obtained in process A or B or C because pure Form B is readily formed from recrystallization from water. In a

laboratory that is free of a history of preparation of Form B, only Form A is obtained when process A or B or C is executed.

The present invention also provides pharmaceutical compositions comprising the designated crystal Form A or designated crystal Form B, in combination with one or more pharmaceutically acceptable carriers or excipients.

In illustrative embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active pharmaceutical ingredient (API) in admixture with pharmaceutically acceptable excipients wherein the API comprises a detectable amount of the designated crystal Form A or designated Form B of the present invention.

In illustrative embodiments, the pharmaceutical composition comprises

therapeutically effective amount of the API in admixture with pharmaceutically acceptable excipients wherein the API comprises from about 5% to about 100% by weight of the designated crystal Form A or designated crystal Form B of the present invention.

In illustrative embodiments, the API in such compositions comprises from about 10% to about 100% by weight of the designated crystal Form A or designated crystal Form B.

In illustrative embodiments, the API in such compositions comprises from about 25% to about 100% by weight of the designated crystal Form A or designated crystal Form B.

In illustrative embodiments, the API in such compositions comprises from about 50% to about 100% by weight of the designated crystal Form A or designated crystal Form B.

In illustrative embodiments, the API in such compositions comprises from about 75% to about 100% by weight of the designated crystal Form A or designated crystal Form B.

In illustrative embodiments, substantially all of the API is the crystalline golotimod designated crystal Form A or designated crystal Form B.

The compositions in accordance with the invention are suitably in unit dosage forms such as tablets, pills, capsules, powders, granules or solutions. The compositions are intended for oral administration or for administration by injection. Formulation of the compositions according to the invention can conveniently be effected by methods known from the art, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., 1995.

For instance, for oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose,

magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral API can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate,

carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

As an example of a pharmaceutical composition, the designated crystal Form A was formulated into a capsule formulation as follows: A 30 mg potency capsule was composed of 30 mg of the API, 57 mg of microcrystalline cellulose; and about 28 mg gelatin as in #0 white opaque gelatin capsule. The API and microcrystalline cellulose were first blended, and the mixture was then encapsulated in gelatin capsules.

Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day.

Form A (solubility: 5.3 mg/mL) is about two times more soluble than Form B

(solubility: 2.6 mg/mL) in water. The saturated solution of both Form A and Form B has a pH of about 2.5. If the aqueous solution of Form A is left for about 1 week at room

temperature under normal light, the solution will turn a slight pinkish color. However, under similar conditions, the aqueous solution of Form B will remain colorless for over a week. Thus, Form B may be more suited for solution formulation, for example for oral use or injection purposes. In either case, the aqueous solution can be adjusted with sodium hydroxide or carbonate to give a solution at around pH 7. For pharmaceutical composition that are designed for solid dose tablets containing the active pharmaceutical ingredient golotimod using wet granulation with other excipients, Form B may be the more preferred than Form A because the observed coloration of the aqueous solution. Form A can easily be converted to Form B using the conditions of Process E.

The stability of crystalline Apo816DL Form A is investigated by stirring a suspension of crystalline Form A in (a) deionized water; (b) dichloromethane; (c) tetrahydrofuran; (d) methanol : water (1 : 1 ) for about 16 h. The recovered solid material after filtration and drying shows identical XRPD to the starting material Apo816 Form A. As a result, crystalline Form A does not change when it is stirred as a suspension in the above solvents.

The same set of experiments was repeated with Apo816DL Form B. The recovered solid material after filtration and drying shows identical XRPD to the starting material Apo816 Form B. Thus, crystalline Form B does not change when it is stirred as a suspension in the above solvents.

Apo816 crystalline Form A and Form B retain their respective crystalline polymorph form when they are stirred as a suspension in a variety of solvents for 16 h.

The intrinsic dissolution rates (IDR) of both forms are determineddin 0.1 N HCI with the set up as shown in USP (<1087> Fig. 1 and 2). The IDR for Apo816DL Form A and Form B are 2.834 mg/cm2/min (r2 = 0.99) and 1.806 mg/cm2/min (r2 = 0.99) in 0.1 N HCI dissolution medium respectively. The trend is parallel to the water solubility of Form A and Form B. Thus Apo816DL Form B is an alternative to Apo816 Form A in formulation studies because this crystalline form is also stable as a suspension in water, and in a mixture of water and methanol.

The storage stability of both Form A and Form B are evaluated at relative humidity (RH) at 11%, 57% and 86% for 13 days. The results are analyzed by FTIR. There is virtually no change in the FTIR for both forms after 13 days of RH storage studies.

Therefore, both Form A and Form B are stable at the conditions studied.

Examples

The following examples are illustrative of some of the embodiments of the invention described herein. These examples do not limit the spirit or scope of the invention in any way.

Example 1 :

Preparation of H-D-Glu(L-Trp-OH)-OH - Form A (Apo816DL - Form A) in isopropanol


A clear solution of H-D-Glu(L-Trp-OMe)-OBzl hydrochloride salt (30.0 g, 0.063 mol) in isopropanol (120 mL) was cooled in a dry ice-water bath to about -0.7 °C. Then, a 3N NaOH solution (84 mL, 0.252 mol) was added dropwise over 30 min while the internal temperature was maintained at below 4.5°C. The reaction was completed after 70 min. The reaction mixture was poured into a separatory funnel, and then mixed with ethyl acetate (70mL). The separated aqueous layer was collected, washed with ethyl acetate (40mL) and filtered to remove residual small amount of grey solid. The filtrate was acidified to pH about 3 with dropwise addition of a 6N HCl solution (29 mL) under cooling in an ice- water bath. The resulting suspension was stirred at RT for overnight. The solid was collected by suction filtration, and washed with de-ionized water (1 x 25mL and 6 x 20mL), and the effluent from the last wash was shown to be chloride free by the silver nitrate test. The solid was air-dried for 4h, and then dried under vacuum at 42 °C for overnight to afford 15.4 g (72.8% yield) of the title compound. HPLC purity (peak percent area) = 99.8% (HPLC conditions: Column: Waters, Symmetry C18, δμm, 3.9 x 150 mm, part #

WAT046980; Mobile phase: A = Aqueous phase: 0.05% TFA in water; B = Organic phase; ACN, Flow rate: 1mL/min; Injection volume: 5μί; Temperature: room temperature; Method - Gradient method: min-B%: 0-0, 10-100, 11-100, 15-0; Retention time of title compound : 4.7 min). 1H NMR (D2O, 400MHz): δ (ppm) 7.62 (d, J = 8.1 Hz, 1 H), 7.44 (d, J = 8.1 Hz, 1H), 7.14 - 7.28 (m, 2H), 7.05 - 7.14 (m, 1 H), 4.64 - 4.70 (m, 1 H), 3.47 (t, J = 6.6 Hz, 1H), 3.34 (dd, J = 14.7, 5.6 Hz, 1 H), 3.10 - 3.21 (m, 1 H), 2.20 - 2.35 (m, 2H), 1.77 - 1.98 (m, 2H); MS-ESI (m/z): 334.2 [M+1]\ 205.2, 188.1(100%); Anal. Calcd. for C16H 19N3O5: C, 57.65; H, 5.75; N, 12.61 ; Found: C, 57.43; H, 5.80; N, 12.70. The water content of this material was 0.75% as determined by the Karl-Fischer test. The solid state 13C NMR

spectrum is displayed in Figure 1 , the XRPD pattern in Figure 2 and the FT-IR spectrum in KBr in Figure 3.

Example 2:

Preparation of H-D-Glu(L-Trp-OH)-OH- Form A (Apo816DL - Form A) in MeOH


Procedure 1 : A clear solution of H-D-Glu-(L-Trp-OMe)-OBzl hydrochloride salt (30.0 g, 0.063 mol) in methanol (120 mL ) was cooled in a water-dry ice bath to about -1.3°C. Then, a 3N NaOH solution (84 mL, 0.252 mol) was added dropwise over 40 min while the internal temperature was maintained at below 2°C. After 1h the reaction was completed. The reaction mixture was filtered to remove small grey particulates, and the filtrate was acidified under cooling in an ice-water bath to pH about 10-11 with the dropwise addition of a 6N HCl solution (10.5 mL ). The resulting solution was concentrated in vacuo using a rotary evaporator to a volume of about 100 mL. The remaining solution was poured into a separator/ funnel, and washed with ethyl acetate (2 x 35 mL). The aqueous layer was collected, cooled in an ice bath, and a 6N HCl solution was added (20.5 mL) until the pH reached about 3. The precipitated solid was collected by suction filtration, and washed with de-ionized water (4 x 50mL). The solid was placed back in a round bottom flask, and suspended in de-ionized water (80 mL). The mixture was stirred at 48°C for 30min, then allowed to cool to room temperature. The solid was collected by filtration, and washed with de-ionized water (3 x 25 mL). The effluent of the last wash was shown to be chloride free by the silver nitrate test. The solid was air-dried over the weekend, and then dried under vacuum at 41 °C for overnight to afford 14.9 g (yield = 70.4%) of the title compound. HPLC purity (peak percent area) is 98.3%. 1H NMR (D2O, 400MHz): δ (ppm) 7.61 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.14 - 7.23 (m, 2H), 7.06 - 7.14 (m, 1H), 4.64 - 4.70 (m, 1 H), 3.47 (t, J = 6.1 Hz, 1 H), 3.34 (dd, J = 14.7, 5.6 Hz, 1 H), 3.14 (dd, J = 14.7, 8.6 Hz, 1 H), 2.19 - 2.36 (m, 2H), 1.74 - 2.00 (m, 2H); MS-ESI (m/z): 334.2 [M+1]+, 205.2, 188.1(100%); Anal. Calcd. for C 16H 19N3O5: C, 57.65; H, 5.75; N, 12.61 ; Found: C, 57.42; H, 5.77; N, 12.73. The water content of this material was 0.51 % as determined by the Karl-Fischer test. The solid state 13C NMR spectrum, the XRPD pattern and the FT-IR spectrum in KBr are very similar to those as described in Example 1 above.

Procedure 2:

A clear solution of H-D-Glu-(L-Trp-OMe)-OBzl hydrochloride salt (30.0 g, 0.063 mol) in methanol (120 mL) was cooled in a water-dry ice bath to about 2.7°C. Then, a 3N NaOH solution (84 mL, 0.252 mol) was added dropwise over 25 min while the internal

temperature was maintained at below 7°C. After 1 h, the reaction mixture was filtered and the filtrate was acidified under cooling in an ice-water bath to pH about 7 with the dropwise addition of a 6N HCI solution. The resulting suspension was concentrated under reduced pressure using a rotary evaporator. The residue was acidified using a 6N HCl solution under cooling in an ice-water bath until the pH of the mixture reached about 3, and the resulting suspension was left stirred for overnight. The solid was collected via suction filtration, and washed with de-ionized water until the effluent of the wash was shown to be chloride free by the silver nitrate test. The solid was then rinsed with acetone, air-dried for 6h, and then further dried under vacuum at 40°C for overnight to afford 15.9 g (75.4% yield) of the title compound. The solution 1H NMR data (400 MHz) of this compound in D2O was similar to that described in Example 2, Procedure 1 above. The water content of this material was 0.47% as determined by the Kari-Fischer test. The XRPD pattern and the FT-IR spectrum in KBr are very similar to those as described in Example 1 above.

Example 3:

Preparation of H-D-Glu(L-Trp-OH)-OH - Form B (Apo816DL - Form B)


To a cooled suspension of Apo816DL - Form A obtained as described in Example 2, Procedure 1 above (6.0 g, 18.0 mmol) in 12 mL of de-ionized water was added 6N NaOH (5mL, 30mmol) dropwise. The resulting solution was washed with EtOAc, then subjected to clarification filtration. The pH of the filtrate was adjusted to 2-3 using a 6N HCl solution. The resulting suspension was stirred for 2h, and the solid was collected via suction filtration. The solid was washed with de-ionized water (10 mL x7) and acetone (10 mL). The solid was air-dried for overnight, and then under vacuum at 40°C for 4h to give

Apo816DL Form B (5.5 g). The solid was shown to be chloride free by the silver nitrate test. The solution 1H NMR data (400 MHz) in D2O is similar to that described in Example 1 above. The water content of this material was 0.75% as determined by the Karl-Fischer test. The solid state 13C NMR spectrum is displayed in Figure 7, the XRPD pattern in Figure 8, and the FT-IR spectrum in KBr in Figure 9.

Example 4:

Preparation of H-D-Glu(L-Trp-OH)-OH - Form B (Apo816DL - Form B) in isopropanol / Acidification with Formic Acid


To a suspension of H-D-Glu-(L-Trp-OMe)-OBzl.HCI (6 g, 12.66 mmol) in isopropanol (24 mL) was added a 3N NaOH solution (16.9 mL, 50.64 mmol) dropwise while the internal temperature was maintained at below 9°C using an ice-water bath. The resulting mixture was stirred for 1 h and 45 min, then poured into a separatory funnel, and then mixed with ethyl acetate (25mL). The separated aqueous layer was washed with ethyl acetate (25mL) one more time. The aqueous layer was collected and subjected to clarification filtration. The filtrate was acidified to pH 3.0-3.5 with the dropwise addition of formic acid under cooling in an ice-water bath. The resulting suspension was stirred at RT for overnight. The

solid was collected by suction filtration, and washed with de-ionized water until the filtrate was shown to be chloride free by the silver nitrate test. The solid product was then rinsed with acetone, air-dried and then further dried under vacuum at 40 °C for overnight to afford 2.75 g (yield = 65.1 %) of Apo816DL The solution 1H NMR data (400 MHz) in D2O is similar to that described in Example 1 above. The water content of this material was 0.47% as determined by the Karl-Fischer test. The XRPD pattern and the FT-IR spectrum in KBr are very similar to those as described in Example 3 above.

Example 5:

Preparation of H-D-Glu(L-Trp-OH)-OH - Form A (Apo816DL - Form A)

A suspension of compound from Example 1 (1 g) in de-ionized water (43 mL ) was heated at an 80°C water bath temperature until a clear solution was obtained. After clarification filtration, 6 mL of the filtrate was mixed with 3 mL of isopropanol and the resulting solution was left at RT for 4 days. The product crystallized out of the solution in colorless plates. A suitable crystal was collected and used for single crystal X-ray structure determination. The single crystal structure is displayed in Figures 4 and 5. Crystal data information is given in Table 4 and selected geometric parameters are given in Table 5.

Example 6:

Preparation of H-D-Glu(L-Trp-OH)-OH - Form B (Apo816DL - Form B)

A sample of compound from Example 1 (1.026g) in deionized water (43 mL) was slowly warmed to 80°C in a water bath until a clear light purple solution was obtained.

After clarification filtration, 25 mL of the solution was left aside at RT. The product crystallized out of the solution as colorless needles. A suitable crystal was collected and used for single crystal X-ray structure determination (see Example 7 below). The single crystal structure is displayed in Figures 10 and 11. Crystal data information is given in Table 6 and selected geometric parameters are given in Table 7.

The rest of the solid was collected by suction filtration and dried. The water content of this material was 1.62% as determined by the Karl-Fischer test. The solid state 13C NMR spectrum, the XRPD pattem and the FT-IR spectrum in KBr are similar to those as described in Example 3 above.

Example 7: Analytical Methods

A. Solid State 13C NMR

Solid-state 13C cross-polarization magic-angle spinning (CP MAS) NMR experiments were performed on a Varian Infinity Plus 400 NMR spectrometer utilizing a Varian triple- resonance 4.0 mm HXY magic-angle spinning NMR probe. For each sample, 1100 scans were acquired using a spinning rate of 13.0 kHz, a 9 s recycle delay, a 3 ms contact time, a 50 kHz sweep width, and a 40.96 ms acquisition time. In addition, 1 H decoupling was achieved using TPPM decoupling with a 15-degree tip angle and 76.9 kHz decoupling field was applied during acquisition and the amplitude of the 13C channel pulse was increased during the contact time (variable-amplitude CP). The FIDs were processed using 2 zero- fills and the NMR spectra were referenced with respect to tetramethylsilane (6(13C) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm.

As illustrative examples, the solid state 13C NMR spectrum of the Apo816DL - Form A of Example 1 is shown in Figure 1 , and that of Apo816DL - Form B of Example 3 is shown in Figure 7.

B. Powder X-Ray Diffraction (XRPD)

The XRPD patterns were measured on a PANalytical X'Pert Pro Multi-Purpose X- ray Diffraction (MPD) system with X'Pert Data Collector Ver. 2.2d using Cu Ka irradiation. The X-ray tube was operated at a voltage of 45 kV and a current of 40 mA. Each sample was scanned between 4° and 40° in two-theta (2θ) angles with step size of 0.017° and a counting time of 10.3s per step. The sample was loaded into Zero-background sample holder after grinding.

As a first illustrative example, the XRPD pattern showing the crystalline nature of Apo816DL - Form A of Example 1 is displayed in Figure 2. The respective peak

information such as two-theta (2θ) angles, d-values and relative intensities are given in Table 2.

Table 2: Two-theta (2)) angles, d-values and relative intensities of Apo816DL - Form A of

Example 1


As a second illustrative example, the XRPD pattern showing the crystalline nature of Apo816DL - Form B of Example 3 is displayed in Figure 8. The respective peak information such as two-theta (2Θ) angles, d-values and relative intensities are given in

Table 3.


C. Fourier Transform Infra-Red (FT-IR)

The FT-IR spectra were obtained using a Perkin Elmer Paragon 1000PC Fourier Transform-IR spectrophotometer at a resolution of 4 cm-1 over a range of 4400 cm-1 to 600 cm-1. The sample was mixed with KBr by gently grinding with an agate pestle and mortar, and then the mixture was pressed into a disc.

As illustrative examples, the FT-IR (KBr) spectrum of Apo816DL - Form A of Example 1 is shown in Figure 3, and that of Apo816DL - Form B of Example 3 is shown in Figure 9.

Example 8:

Single Crystal X-Ray Structure Determination

Suitable crystals of Apo816DL - Form A (described in Example 5) and Form B (described in Example 6) were collected and used for single crystal X-ray structure determinations.

Data were collected on a Nonius Kappa-CCD diffractometer using monochromated Μο-Κα radiation and were measured using a combination of Φ scans and ω scans with κ offsets, to fill the Ewald sphere. The data were processed using the Denzo-SMN package (Otwinowski, Z. and Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A edited by C. W. Carter and R. M. Sweet pp. 307-326. London: Academic press). Absorption corrections were carried out using SORTAV (Blessing, R. H. (1995). Acta Cryst. A51 , 33-38). The structure was solved and refined using SHELXTL V6.1 (Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122) for full-matrix least-squares refinement that was based on F2.

Apo816DL- Form A: Crystal data, data collection and structure refinement parameters are summarized in Table 4. Selected bond lengths ( Å) and bond angles (°) are displayed in Table 5. The single crystal X-ray structure of Apo816DL - Form A of Example 5 is displayed in FIG 4 and 5.

Apo816DL- Form B: Crystal data, data collection and structure refinement parameters are summarized in Table 6. Selected bond lengths ( Å) and bond angles (°) are displayed in Table 7. The single crystal X-ray structure of Apo816DL - Form B of Example 6 is displayed in FIG 10 and 11.

Example 9

Crystalline form stability study of H-D-Glu(L-Trp-OH)-OH - Form A (Apo816DL - Form A)

A. A suspension of H-D-Glu(L-Trp-OH)-OH - Form A (1 g) in de-ionized water (8 ml_) was stirred at room temperature for 16 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material (863 mg) with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form A.

B. A suspension of H-D-Glu(L-Trp-OH)-OH - Form A (500 mg) in dichloromethane (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and air-dried for 2.5 days to give a material with identical XRPD to the starting material H-D-Glu(L-Trp- OH)-OH - Form A.

C. A suspension of H-D-Glu(L-Trp-OH)-OH - Form A (500 mg) in tetrahydrofuran (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and air-dried for 2.5 days to give a material with identical XRPD to the starting material H-D-Glu(L-Trp- OH)-OH - Form A.

D. A suspension of H-D-Glu(L-Trp-OH)-OH - Form A (500 mg) in a 1 : 1 mixture of

methanol and water (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and air-dried for 2.5 days to give a material with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form A.

E. A suspension of H-D-Glu(L-Trp-OH)-OH - Form A (201.78 mg) in de-ionized water (3 mL) was tumbled at room temperature for 20 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material (167 mg) with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form A.

Example 10

Crystalline form stability study of H-D-Glu(L-Trp-OH)-OH - Form B (Apo816DL - Form B)

A. A suspension of H-D-Glu(L-Trp-OH)-OH - Form B (1 g) in de-ionized water (8 mL) was stirred at room temperature for 16 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material (863 mg) with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form B.

B. A suspension of H-D-Glu(L-Trp-OH)-OH - Form B (500 mg) in dichloromethane (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form B.

C. A suspension of H-D-Glu(L-Trp-OH)-OH - Form B (500 mg) in tetrahydrofuran (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form B.

D. A suspension of H-D-Glu(L-Trp-OH)-OH - Form B (500 mg) in a 1 : 1 mixture of

methanol and water (5 mL) was stirred at room temperature for 16 h. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form B.

E. A suspension of H-D-Glu(L-Trp-OH)-OH - Form B (204.4 mg) in de-ionized water (3

mL) was tumbled at room temperature for 20 hr. The solid was suction filtered and dried under high vacuum at 40°C for 16 h to give a material (185 mg) with identical XRPD to the starting material H-D-Glu(L-Trp-OH)-OH - Form B.

Example 11

Intrinsic Dissolution Rate of Form A and Form B

The method listed in USP <1087> with our setup is as per Fig 1 & Fig 2 is used.

The intrinsic dissolution apparatus from Aligent Technologies (Ref:

http.7/www.chem.agilent.com/Library/datasheets/Public/5990- 7402EN_lntrinsic%20Apparatus.pdf) was installed onto a Distek 2100C or a Vankel VK 7000 dissolution bath for the test. About 200mg of the test article Form A or Form B was used for each test respectively, and all samples were compressed under 1.5 ton of force for 1 minute. 1 mL of media was withdrawn at predetermined time points. All the samples were analyzed by Agilent 1100 series HPLC system against a known concentration standard. The results were all calculated based on the Apo816 H-D-Glu(L-Trp-OH)-OH. The IDR for Apo816DL Form A and Form B are 2.834 mg/cm2/min (r2 = 0.99) and 1.806 mg/cm2/min (r2 = 0.99) in 0.1 N HCl dissolution medium respectively.

Example 12

Stability of Apo816DL Form A & Form B vs. Humidity at Room Temperature and Examined by FTIR Technology

A. Stability of Apo816DL Form A at Relative Humidity at 11%, 57% and 86%

300 mg of Apo816DL-Form A was gently ground to a fine powder. The powder was divided evenly to four parts. The first part of the sample was placed in a 3-mL amber vial with the cap hand-tight closed (regular storage). Each of the other three parts was placed in a 30-mL amber bottle. The latter uncapped bottles were placed in three relative humidity chambers at room temperature: RH = 1 1 %, 57%, and 86%, respectively.

The IR spectrum of the sample in regular storage was recorded on Day 1. The IR spectra of all four samples were recorded on Day 3, Day 7, and Day 13. Prior to FTIR recording, the sample bottles were removed from the RH chambers and capped tightly by hand. After FTIR recording, the samples were returned back uncapped to the corresponding RH chambers. The FTIR instrument used is The Perkin Elmer Spectrum Two equipped with the Attenuated Total Reflectance (ATR) unit as the sampling technique. The data were analyzed by Spectrum Two software (Application Version: 10.03.09.0139). Employing this FTIR technique, the FTIR spectrum of the chemical substance is recorded directly. The preparation of a KBr pellet is not required for each test article chemical substance.

Based on the FTIR analysis results of the various spectra (Figure 15) and the correlation value of > 0.99, the polymorph Form A remains unchanged at Day 3, 7 and 13 with the RH set at 11 %, 57% and 86% respectively. Form A is stable under the above storage conditions.

B. Stability of Apo816DL Form B at Relative Humidity at 11%, 57% and 86%

Proceeding in the same manner, the stability of Apo816DL Form B was monitored at RH at 11%, 57% and 86% at Day 3, 7 and 13. Based on FTIR analysis results of the various spectra (Figure 16) and the correlation value of > 0.99, the polymorph Form B

remains unchanged at Day 3, 7 and 13 with the RH set at 1 1 %, 57% and 86% respectively. Form B is stable under the above storage condition.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in

accordance with the common general knowledge of those skilled in this art. Such

modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. Furthermore, numeric ranges are provided so that the range of values is recited in addition to the individual values within the recited range being specifically recited in the absence of the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. Furthermore, material appearing in the background section of the specification is not an admission that such material is prior art to the invention. Any priority document(s) are incorporated herein by reference as if each individual priority document were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.