PROCESS FOR THE PREPARATION OF A MONOSODIUM SALT OF H-D-GLU-(L-TRP-OH)-

OH AND A CRYSTALLINE FORM THEREOF

Technical Field

This invention relates to a monosodium salt of H-D-Glu(L-Trp-OH)-OH, its crystalline form and the process for its manufacturing.

Background

The name Golotimod (CAS#: 229305-39-9) refers to a dipeptide H-D-Glu(L-Trp-OH)-OH with the lUPAC name (R)-2-amino-5-(((S)-1-carboxy-2-(1 H-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 2005/1 12639), 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)-0-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)-0-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 h (Scheme A).

a. Pd/C, MeOH, H ₂

b. Prep. HPLC purification

c. Lyophilized

CBz -D-Glu(L-Trp-OBzl)-0-Bzl H-D-Glu(L-Trp-OH)-OH

Scheme A

In this synthesis: (a) 200 mg of Pd/C is used to convert 600 mg of N-benzyloxycarbonyl- gamm a-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 acetoniti'ile; (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, 11 1 , 291-295), H-D- Glu(L-Trp- OH)-OH is synthesized from D-glutamine and L-tryptophan through the

transpepticiation reaction using Escherichia coii gamma-glutamyltranspeptidase (GGT) as a catalyst. T he optimal condition reported involved the use of 50 mM D-glutamine and 50 mM L- tryptop an. The conversion did not go to completion. The reaction mixture was purified on a Dovvex 1 8 column, washed with 150 ml of water, and then 5 N CH ₃ COOH. The fractions which contained only gamma-D-glutamyl-L-tryptophan were saved and lyophilized to produce 126 mg of product.

Summary

The present invention is based, at least in part, on a crystal form of a monosodium salt of H-D-Glu(L- » ^r rp-OH)-OH.

The present invention is based, at least in part, on processes for manufacturing large quantities of a momosodium salt of H-D-Glu(L-Trp-OH)-OH, which processes do not require chromatographic purification and/or hydrogenation conditions. The processes of the present invention are illustrated in Table 1. Table 1

crystalline material. crystalline material. crystalline material.

The starting material for processes described herein is H-D-Glu(L-Trp-0-T)-0-G wherein T is methyl, ethyl, propyl, isopropyl, or benzyl and G is methyl, ethyl, propyl, or isopropyl. A preferred starting material is the compound H-D-Glu(L-Trp-0-T)-0-G wherein T is methyl and G is benzyl, namely H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl, which can be synthesized in two simple steps from Boc-D-Glu-OBzl and L-H-Trp-OCH ₃ .

In process A, a solution of H-D-Glu(L-Trp-0-T)-0-G hydrochloride salt wherein G and T are as defined above in isopropanol and water, is reacted with sodium hydroxide. Upon reaction completion, an insoluble particulate is filtered off. The filtrate is acidified to pH 6 with acetic acid and the suspension is stirred with additional isopropanol. The precipitated solid is collected by filtration and then dried to give the monosodium salt of H-D-Glu(L-Trp-OH)-OH. In process B, a solution of H-D-Glu(L-Trp-0-T)-0-G hydrochloride salt wherein G and T are as defined above in ethanol is first reacted with 1 equivalent of ammonium hydroxide to give H-D-Glu(L-Trp-0-T)-0-G which is then reacted with sodium hydroxide. The filtrate is acidified to pH 6 with acetic acid and the suspension is stirred with additional ethanol. The precipitated solid is filtered and then dried to give the monosodium salt of H-D-Glu(L-Trp-OH)-OH.

Process C is similar to process B except methanol is used instead of ethanol. Since the monosodium salt of H-D-Glu(L-Trp-OH)-OH is more soluble in methanol, the overall yield of the isolated product in process C is slightly lower than in process B.

In each of processes A, B and C, extraction of the filtrate (see Table 1) with an organic solvent such as ethyl acetate is not required to remove the other reaction product, benzyl alcohol. Process of the present invention may afford the monosodium salt of golotimod in pharmaceutical grade. Further, processes of the present invention are suitable for industrial scale production. This is due, at least in part, to a step in which the monosodium salt of golotimod can be made and isolated from the base hydrolysis of the diester of golotimod, thereby reducing the need to isolate golotimod followed by conversion to the monosodium salt with sodium hydroxide in a separate process step. Additionally, processes of the present invention reduce and/or eliminate the use of hydrogenation and the use of palladium on charcoal, avoid the use of acetonitrile in the last step of isolation, and avoid the use of the more expensive intermediate CBz-D-Glu(L-Trp-OBzl)-OBzl and CBz-D-Glu-OBzl as a starting material to produce CBz-D-Glu(L-Trp-OBzl)-OBzl. A pure crystalline form of the mono-sodium salt of golotimod is the product of processes of the present invention.

Often, particularly when describing processes herein, the compound H-D-Glu(L-Trp-0- T)-0-G.HCI wherein G is benzyl and T is methyl is used as an illustrative example of the starting material for the process. Other starting materials may be substituted in place of this specific compound in processes described herein.

In illustrative embodiments of the present invention, there is provided a crystal form of the mono sodium salt of H-D-Glu(L-Trp-OH)-OH of formula:

characterized by an X-ray powder diffraction pattern comprising at least three peak positions (degrees 2-theta + 0.2) selected from the group consisting of: 12.24, 15.45, 20.61 , 25.78, and 31.03.

In illustrative embodiments of the present invention, there is provided a crystal form described herein, characterized by an X-ray powder diffraction pattern comprising at least one peak position (degrees 2-theta + 0.2) selected from the group consisting of 12.24, 15.45, and

20.61.

In illustrative embodiments of the present invention, there is provided a crystal form described herein, characterized by an IR absorption spectrum comprising peaks, in cm ^"1 , at 745, 810, 940, 1008, 1103, 1234, 1341 , 1402, 1457, and 3423.

In illustrative embodiments of the present invention, there is provided a crystal form described herein, characterized by a differential scanning calorimetry thermogram having an endotherm in the range of from about 175°C to about 225°C with a peak onset at about 209.0 ± 2.5 °C and a peak apex at about 218.9 ± 2.5 °C.

In illustrative embodiments of the pres form of a

mono sodium salt of H-D-Glu(L-Trp-OH)-OH: cterized by a solid state C NMR spectrum comprising peaks, in ppm, at 24.98, 30.69, 31.67, 33.73, 109.43, 110.79, 118.07, 123.66, 126.25, 134.41, 175.76, 176.88, 180.85, and 181.98.

In illustrative embodiments of the present invention, there is provided a crystal form described herein, characterized by an IR absorption spectrum having peaks, in cm , at 745,

810, 940, 1008, 1103, 1234, 1341, 1402, 1457, and 3423.

In illustrative embodiments of the present invention, there is provided a crystal form described herein, characterized by a differential scanning calorimetry thermogram having an endotherm in the range of from about 175°C to about 225°C with a peak onset at about 209.0 ± 2.5 °C and a peak apex at about 218.9 ± 2.5 °C.

In illustrative embodiments of the present invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a crystal form as described herein.

In illustrative embodiments of the present invention, there is provided a process for the preparation of pharmaceutical grade of the mono sodium salt of H-D-Glu(L-Trp-OH)-OH:

, the process comprising: (a) forming an aqueous layer by reacting sodium hydroxide with either (i) a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in isopropanol and water, or (ii) a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in ethanol and one equivalent of ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI, or (iii) a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in methanol and one equivalent of ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI; (b) filtering the aqueous layer and collecting a filtrate; (c) acidifying the filtrate to pH 6 with acetic acid, thereby forming a mixture; (d) adding isopropanol, ethanol or methanol to the mixture and stirring thereby forming a solid suspension; (e) filtering the solid suspension and washing a solid collected from the filtering of the solid suspension with isopropanol, methanol or ethanol; and (f) drying the solid under vacuum at 40 to 45°C, thereby forming the monosodium salt of H-D-Glu(L-Trp-OH)-OH.

In illustrative embodiments of the present invention, there is provided a process described herein wherein the forming the aqueous layer comprises reacting sodium hydroxide with a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in isopropanol and water; the adding comprises adding isopropanol; and the washing comprises washing with isopropanol.

In illustrative embodiments of the present invention, there is provided a process described herein wherein the forming the aqueous layer comprises reacting sodium hydroxide with a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in ethanol and one equivalent of ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI; the adding comprises adding ethanol; and the washing comprises washing with ethanol.

In illustrative embodiments of the present invention, there is provided a process described herein wherein the forming the aqueous layer comprises reacting sodium hydroxide with a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in methanol and one equivalent of ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI; the adding comprises adding methanol; and the washing comprises washing with methanol.

In illustrative embodiments of the present invention, there is provided a process for the preparation of the mono sodium salt of H-D-Glu(L-Trp-OH)-OH: the process comprising: (a) milling H-D-Glu(L-Trp-OH)-OH, thereby forming a fine powder; (b) adding, in an ice bath, 1 equivalent of sodium hydroxide solution to a suspension of 1 equivalent of the fine powder in isopropanol, thereby forming a reaction mixture; (c) adding isopropanol slowly to the reaction mixture and stirring thereby forming a thick suspension; (d) filtering off the mono sodium salt of H-D-Glu(L-Trp-OH)-OH from the think suspension and washing the mono sodium salt of H-D-Glu(L-Trp-OH)-OH with isopropanol; (e) drying the mono sodium salt of H-D-Glu(L-Trp-OH)-OH at 40°C-45°C under vacuum.

In illustrative embodiments of the present invention, there is provided a process described herein wherein the monosodium salt of H-D-Glu(L-Trp-OH)-OH is characterized by an X-ray powder diffraction pattern comprising at least three peak positions (degrees 2-theta + 0.2) selected from the group consisting of: 12.24, 15.45, 20.61 , 25.78, and 31.03.

In illustrative embodiments of the present invention, there is provided a process described herein wherein the monosodium salt of H-D-Glu(L-Trp-OH)-OH is characterized by a solid state ³ C NMR spectrum comprising peaks, in ppm, at 24.98, 30.69, 31.67, 33.73, 109.43, 110.79, 118.07, 123.66, 126.25, 134.41 , 175.76, 176.88, 180.85, and 181.98.

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,

FIG. 1 shows the powder X-ray diffraction pattern of Apo8 6Na Form A, the

monosodium salt of H-D-Glu(L-Trp-OH)-OH.

FIG. 2 shows the solid state ³ C NMR spectrum for Apo8 6Na Form A, the monosodium salt of H-D-Glu(L-Trp-OH)-OH.

FIG. 3 shows the FTIR (KBr) of Apo816Na Form A, the monosodium salt of H-D-Glu(L- Trp-OH)-OH in KBr disk. FIG. 4 shows the differential scanning calorimetry thermogram of the monosodium salt of H-D-Glu(L-Trp-OH)-OH (Apo816Na From A).

FIG. 5 shows the Thermogravimetric Analysis thermogram of the monosodium salt of H- D-Glu(L-Trp-OH)-OH (Apo816Na From A).

FIG. 6 shows the speciation plot of H-D-Glu(L-Trp-OH)-OH. L is H-D-Glu(L-Trp-0 0\ The counter ion in this case is Na ⁺ .

FIG. 7 shows the overlays of the powder X-ray diffraction patterns of the materials from Examples 2A, 2B and 3.

Detail Description of Invention

As used herein, Apo816 refers to the dipeptide H-D-Glu(L-Trp-OH)-OH with the chemical structure

As used herein, Apo816Na refers to the monosodium salt of H-D-Glu(L-Trp-OH)-OH with the chemical structure

As used herein, H-D-Glu(L-Trp-OMe)-OBzl or H-D-Glu(L-Trp-OCH ₃ )-OCH ₂ Ph refers to the chemical structure:

In one embodiment, there is provided a method for the preparation of pharmaceutical grade monosodium salt of H-D-Glu(L-Trp-OH)-OH from a diester H-D-Glu(L-Trp-0-G)-0-T or its hydrochloride salt and sodium hydroxide. The product monosodium salt of H-D-Glu(L-Trp-OH)- OH is isolated in a crystalline form. The crystalline form may be characterized by an X-ray powder diffraction pattern substantially similar to the X-ray powder diffraction pattern set out in Figure 1. This crystalline form of the sodium salt may be termed Apo816Na Form A.

Apo816Na Form A may be characterized by at least one of the following characteristics:

(a) an X-ray powder diffraction pattern comprising at least three peaks (degrees 2-theta + 0.2), when measured using CuK alpha radiation, selected from the group consisting of 12.24, 15.45, 20.61 , 25.78, and 31.03.

(b) a solid state ¹³ C NMR spectrum comprising peaks at 24.98, 30.69, 3 .67, 33.73, 109.43, 110.79, 118.07, 123.66, 126.25, 134.41 , 175.76, 176.88, 180.85, 181.98 ppm and the NMR peaks are referenced with respect to tetramethylsilane (delta( ¹³ C) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm. Alternatively, or

13

additionally, the solid state C NMR spectrum may be substantially similar to that spectrum set out in Figure 2.

(c) an IR spectrum comprising absorption maxima at approximately 745, 810, 940, 1008, 1103, 1234, 1341 , 1402, 1457, 3423 cm ^"1 . Alternatively, or additionally, the IR spectrum may be substantially similar to that spectrum set out in Figure 3.

(d) a differential scanning calorimetry thermogram having an endotherm in a range of from about 175°C to about 225°C with a peak onset at about 209.0 ± 2.5 °C and peak apex at about 218.9 ± 2.5 °C. Alternatively or additionally, the thermogram may be substantially similar to that thermogram set out in Figure 4.

In another aspect of the invention, there is provided a method, process A, for the manufacturing of Apo816Na Form A, monosodium salt of H-D-Glu(L-Trp-OH)-OH without chromatographic purification which comprises:

(1) Reacting a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in isopropanol and water with sodium hydroxide solution;

(2) Filtering the aqueous layer from step (1 ) and collecting the filtrate;

(3) Acidifying the filtrate from step (2) to pH 6 with acetic acid;

(4) Adding isopropanol and stirring of the resulting mixture to give a solid suspension;

(5) Filtering the solid from step (4) and washing the solid with isopropanol

(6) Drying the material from step (5) under vacuum at 40 to 45°C to give the monosodium salt of H-D-Glu(L-Trp-OH)-OH; In another aspect of the invention, there is provided a method, process B, for the manufacturing of Apo816Na Form A, monosodium salt of H-D-Glu(L-Trp-OH)-OH without chromatographic purification which comprises:

(1 ) Reacting a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in ethanol and one equivalent ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI;

(2) Adding sodium hydroxide solution to the solution in step (1 );

(3) Filtering the aqueous layer from step (2) and collecting the filtrate;

(4) Acidifying the filtrate from step (3) to pH 6 with acetic acid;

(5) Adding ethanol and stirring of the resulting mixture to give a solid suspension;

(6) Filtering the solid from step (5) and washing the solid with ethanol;

(7) Drying the material from step (6) under vacuum at 40 to 45°C to give the monosodium salt of H-D-Glu(L-Trp-OH)-OH;

In another aspect of the invention, there is provided a method, process C, for the manufacturing of Apo816Na Form A, monosodium salt of H-D-Glu(L-Trp-OH)-OH without chromatographic purification which comprises:

(1 ) Reaction a solution of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI in methanol and one equivalent ammonium hydroxide per equivalent of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI;

(2) Adding sodium hydroxide solution to the solution in step (1 );

(3) Filtering the aqueous layer from step (2) and collecting the filtrate;

(4) Acidifying the filtrate from step (3) to pH 6 with acetic acid;

(5) Adding methanol and stirring of the resulting mixture to give a solid suspension;

(6) Filtering the solid from step (5) and washing the solid with methanol;

(7) Drying the material from step (6) under vacuum at 40 to 45°C to give the monosodium salt of H-D-Glu(L-Trp-OH)-OH.

H-D-Glu(L-Trp-OH)-OH is available from Chem-lmpex International as well as Bachem

AG.

In another aspect of the present invention, there is provided a method, process D, for the preparation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH from H-D-Glu(L-Trp-OH)-OH without chromatography which comprises:

(a) Adding one equivalent of H-D-Glu(L-Trp-OH)-OH to about one equivalent of sodium hydroxide solution; (b) Adding isopropanol with stirring to precipitate the mono sodium salt of H- D-Glu(L-Trp-OH)-OH;

(c) Filtering the insoluble solid and drying the material under vacuum at 40 to

45°C.

In process D, a large volume of isopropanol (5 to 20 volume of isopropanol to the volume of water in the aqueous solution is required. In process D, the Apo816Na Form A can be obtained in pharmaceutical grade without chromatographic purifications.

In another aspect of the present invention, there is provided a method, process E, for the preparation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH from H-D-Glu(L-Trp-OH)-OH without chromatography which comprises:

(a) Milling of H-D-Glu(L-Trp-OH)-OH to a fine powder;

(b) Adding 1 equivalent of sodium hydroxide solution to a suspension of 1 equivalent of the fine powder of H-D-Glu(L-Trp-OH)-OH from step (a) in isopropanol an ice-bath;

(c) Adding isopropanol slowly to the reaction mixture in step (b) with stirring to maintain a thick suspension;

(d) Filtering off the mono sodium salt of H-D-Glu(L-Trp-OH)-OH and washing the solid with isopropanol;

(e) Drying the solid under at 40-45°C under vacuum.

Process E avoids the use of large amount of isopropanol and water to prepare the Apo816Na Form A. In process E, the Apo816Na Form A can be obtained in pharmaceutical grade without chromatographic purifications. Process E is a convenient process for the preparation of Apo816Na Form A from H-D-Glu(L-Trp-OH)-OH (Apo816).

Depending on the pH of the solution, H-D-Glu(L-Trp-OH)-OH can exist in different protonated forms, namely H ₃ L, H ₂ L, HL, L. The definition of these species is shown in Scheme 1 below:

H ₂ L Drawn in zwitterion format HL Drawn in zwitterion format

Scheme 1

The speciation distribution of H ₃ L, H ₂ L, 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 (Protonic Software, UK). The compound H-D-Glu(L-Trp-OH)-OH has three pKas (2.1, 3.45, & 9.33). The speciation plot analysis is shown in Figure 6. Thus, the monosodium salt HL (monosodium salt of H-D-Glu(L-Trp-OH)-OH) is the predominant species when the pH is between 5.0 and 8.5, preferably between 5.5 and 7.2, most preferably between 5.9 and 6.7. These computation results are shown in Table 2 below. Table 2: Speciation composition of H-D-Glu(L-Trp-OH)-OH and its sodium salt.

In one embodiment, solvent media are devised to allow the formation of the desired crystalline form as a solid in the reaction mixture, at the optimal pH for the formation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH, as per computed data shown in Table 2. The total concentration of H-D-Glu(L-Trp-OH)-OH of 2 x 10 ^"3 M, regardless of its protonated or unprotonated species: H-D-Glu(L-Trp-OH)-OH.HCI (H ₃ L), H-D-Glu(L-Trp-OH)-OH (H ₂ L), monosodium salt (HL), disodium salt (L), and the log beta values of 9.33, 12.78, 4.88 are used for the HL, H ₂ L and H ₃ L respectively in this computation with the software HySS 2009.

In another embodiment, the pure monosodium salt of H-D-Glu(L-Trp-OH)-OH is formed as a solid between pH 5.0 and 8.5, preferably between 5.5 and 7.2 and most preferably between 5.9 and 6.7, and slowly precipitated out of the reaction media. The solvent media for such precipitation is a mixture of alcohol and water. Examples of those alcohols are methanol, isopropanol and ethanol.

In a particular embodiment, the speciation data as shown in Table 2 supports that the monosodium salt of H-D-Glu(L-Trp-OH)-OH is the main species formed in over 99% in the mixture at pH 5.5 and 7.2, and most preferably between 5.9 and 6.7. An original selection of the solvent media such as a mixture of alcohol and water allows the pure monosodium salt of H-D- Glu(L-Trp-OH)-OH to be precipitated as a crystalline material, as characterized by the powder X-ray diffraction pattern substantially in accordance with FIG. 1.

In another aspect of the invention, conditions are designed to optimize the isolated yield and purity of the monosodium salt of H-D-Glu(L-Trp-OH)-OH by precipitation and the salts such as sodium chloride or sodium acetate are removed by further washing with alcohols.

peptide coupling

Boc-D-Glu-OBzl + H-L-Trp-OMe * Boc-D-Glu(L-Trp-OCH ₃ )-OBzl

HCl/EtOAc H-D-Glu(L-Trp-OCH ₃ )-OBzl.HCl

Process A, or B

or C (Table 1)

Scheme 2

The starting material for the production of the monosodium salt of H-D-Glu(L-Trp-OH)- OH is Boc-D-Glu(L-Trp-0-T)-0-G. For illustration purpose, the compound Boc-D-Glu(L-Trp-0- T)-0-G wherein G is benzyl and T is methyl is chosen as a starting material (Scheme 2). Both reagents Boc-D-Glu-OBzl and H-L-Trp-OMe. HCI are available in kg amounts. Boc-D-Glu-OBzl and H-L-Trp-OMe can be coupled with EDCI or DCC or EDCI/HOBt to give the dipeptide Boc-D- Glu(L-Trp-0-CH ₃ )-OBzl which can be isolated in more than 97% purity (HPLC, area under curve) without chromatographic purification. Treatment of Boc-D-Glu(L-Trp-OCH ₃ )-OBzl with HCI in ethyl acetate affords H-D-Glu(L-Trp-0-CH ₃ )-OBzl.HCI, which is isolated by filtration of the reaction mixture. As a result, H-D-Glu(L-Trp-0-CH ₃ )-OBzl.HCI can be produced in >0.5 kg amounts in two simple steps from commercially available materials.

Any of process A or B or C may be used to convert H-D-Glu(L-Trp-0-CH ₃ )-OBzl.HCI to the monosodium salt of H-D-Glu(L-Trp-OH)-OH.

Hydrochloric acid is often used to adjust the pH of a basic solution to 6 or 7 after the sodium hydroxide hydrolysis of H-D-Glu(L-Trp-0-CH ₃ )-0-Bzl.HCI. This results in the formation of sodium chloride as an inorganic by-product which co-precipitates with the Apo816Na from the alcohol and water based reaction mixture. Since both sodium chloride and Apo816Na are very soluble in water, the sodium chloride cannot be removed from the solid product mixture by pulping in water without significant loss of Apo816Na in the aqueous washings. Acetic acid may be used to neutralize the sodium hydroxide reaction mixture in any of processes A or B or C. Sodium acetate is formed as a by-product of this neutralization using acetic acid. Since sodium acetate is very soluble in alcohol (5.3 g of NaOAc.3H ₂ 0 dissolves in 100 mL ethanol), the co-precipitated sodium acetate can be easily removed by washing the crude Apo816Na with ethanol or methanol.

In all three processes A, B and C, procedures may be designed to improve the product yield and to facilitate the removal of sodium chloride and/or sodium acetate by-products in the reaction mixture.

In general, H-D-Glu(L-Trp-OCH ₃ )-OBzl.HCI is mixed with alcohol solvent such as isopropanol or ethanol or methanol and sodium hydroxide solution to hydrolyze the diester H-D- Glu(L-Trp-OCH ₃ )-OBzl.HCI.

Conditions in all three processes A, B and C may be designed in such a manner to wash out the sodium chloride or sodium acetate with alcohol solvents. The effluents from each washing are collected separately and are tested with silver nitrate solution to ensure that inorganic sodium chloride is no longer present in the final Apo816Na product.

All three processes A, B and C afford Apo816Na in more than 99% HPLC purity.

Process B provides the highest recovery of the Apo816Na (-60% yield).

Apo816Na is a distinct crystalline material (Apo816Na Form A) characterized by the PXRD pattern, solid state ³ C NMR, FTIR and DSC as shown in the Examples and as set out in the Figures.

H-D-Glu(L-Trp-OH)-OH may also react with one equivalent of sodium hydroxide in water to give Apo816Na, the monosodium salt of H-D-Glu(L-Trp-OH)-OH. Since the sodium salt is soluble in water, solvent evaporation of water is required to give crude Apo816Na. The purity of the crystalline form Apo816 Form A cannot be assured by using this method. Processes D and E may be adapted for the production of Apo816 Form A from H-D-Glu(L-Trp-OH)-OH without chromatographic purifications.

In process D, a solution of H-D-Glu(L-Trp-OH)-OH in one equivalent of sodium hydroxide is reacted to give Apo816Na in solution. The monosodium salt of H-D-Glu(L-Trp- OH)-OH may be isolated by using large amount of isopropanol as an antisolvent (1 mL of aqueous solution of Apo816Na to 20 mL of isopropanol) to precipitate the Apo816Na. This material has similar FTIR and PRXD pattern to the material Apo816 (Apo816Na Form A).

Apo816Na may be prepared from a suspension of milled fine particle of H-D-Glu(L-Trp- OH)-OH in isopropanol and sodium hydroxide solution to give pharmaceutical grade Apo816Na (Apo816Na Form A). This avoids the use of large amount of isopropanol and water, as required in process D.

Processes of the present invention may afford a simple method for the production of Apo816Na and eliminate the use of chromatographic purification and hydrogenation conditions. Furthermore, the use of pure H-D-Glu(L-Trp-OH)-OH as a starting material is not required. Processes of the present invention may also eliminate concerns regarding removing inorganic chloride by-products. Apo816Na Form A is water soluble and can be manufactured in large amounts with a short production cycle time using processes of the present invention.

Compounds described herein, such as Apo816Na may be used to prepare

pharmaceutical compositions. Pharmaceutical compositions of the present invention contain Apo816Na Form A or mixtures of Apo816Na Form A and/or other crystalline or amorphous forms thereof, optionally in mixtures with one or more other active ingredient(s).

Pharmaceutical compositions of the present invention may include, for example, excipients such as diluent, binder, disintegrant, glidant, and lubricant may be added to produce a solid dose formulation such as a tablet. In liquid pharmaceutical compositions of the present invention, Apo816 Form A and any other solid excipients are dissolved or suspended in a liquid carrier such as water, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin. In some cases, emulsifying agents, viscosity enhancing agent, sweetening agents may be added to prepare the liquid formulation. The selection of excipients and the amounts to use may be readily determined by the experience of the formulation scientist.

Typical finished dosage forms include, but are not limited to, solid dosage forms like tablets, powders, capsules, suppositories, sachets, troches and lozenges as well as liquid syrups, suspensions and elixirs.

A pharmaceutical composition of Apo816Na Form A contains Apo816Na Form A and at least one pharmaceutical excipient.

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 (Process A)

Preparation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH (Apo816Na) from D-Glu(L-

H-D-Glu(L-Trp-0-Me)-0-Bzl. HCI Apo816Na

To an ice-water bath cooled suspension of H-D-Glu(L-Trp-OMe)-OBzl HCI salt (6.0 g, 12.66 mmol) in isopropanol (12 mL) and de-ionized (Dl) water (3.56 mL) was added a 6N NaOH solution (8.44 mL) dropwise. The mixture was stirred for 2 h. The reaction mixture was filtered and the filtrate was collected. The reaction vessel was rinsed with a mixture of IPA and water (6 mL), and filtered. The filtrates were combined, and then acidified to pH about 6 with the dropwise addition of a 6N acetic acid solution. The mixture was diluted with isopropanol (12 mL), and the resulting suspension was stirred at RT for overnight. The solid was collected by suction filtration, and washed with isopropanol (2 x 10 mL). The solid was dried under vacuum at about 40 °C for 4h to afford 3.05 g of a solid product. A small sample of the solid was dissolved in water and it gave a very slight positive silver nitrate test.

A suspension of 1.0 g of the solid in 10 mL of Dl water and 20 mL of isopropanol was stirred at RT for overnight. The solid was collected, washed with EtOH (8 mL), air-dried for overnight, then further dried under vacuum at 40°C for 4h to give 830 mg of Apo816Na. A small sample of this material in water gave a negative silver nitrate test. This material displayed similar solution H N R pattern (D ₂ 0, 400MHz) as that described in Example 2, Procedure A below. The PXRD pattern and the FT-IR spectrum in KBr are very similar to those described in Example 2, Procedure A below,

Example 2 (Process B)

Preparation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH (Apo816Na) from D-Glu(L- Trp-0-Me)-0-Bzl. HCI in EtOH

H-D-Glu(L-Trp-0- e)-0-Bzl. HCI Apo816Na

To a suspension of H-D-Glu(L-Trp-0-Me)-0-Bzl. HCI salt (30.0 g, 63.3 mmol) in EtOH (60 mL) was added a solution of cone. (28-30%) NH ₄ OH (4.16 mL) in water (24.2 mL). Then, a 6N NaOH solution (31.65 mL, 189.9 mmol) was added dropwise while the internal temperature was maintained at below 7°C using an ice-water bath. The reaction was completed after 95 min. After clarification filtration, the filtrate was acidified to pH 6 with dropwise addition of a 6N acetic acid under cooling in an ice-water bath. EtOH (60mL) was added to the resulting suspension, and the mixture was then stirred at RT for overnight. The solid was collected by suction filtration, and washed with EtOH (2 x 40mL). The solid was dried under vacuum at 40 °C for 4h to afford 13.6 g (60.6% yield) of Apo816Na. A small sample of the solid product was dissolved in water and it was shown to be chloride free by the silver nitrate test. HPLC purity (peak percent area) = 99.2%; ¹ H NMR (D ₂ 0, 400MHz): δ (ppm) 7.69 (d, J = 8.1 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1H), 7.19 - 7.27 (m, 2H), 7.11 - 7.19 (m, 1H), 4.55 (dd, J = 8.1 , 5.1 Hz, 1 H), 3.46 (t, J = 6.1 Hz, 1H), 3.37 (dd, J = 15.2, 5.1 Hz, 1 H), 3.09 (dd, J = 14.7, 8.6 Hz, 1H), 2.19 - 2.35 (m, 2H), 1.89 - 2.01 (m, 1H), 1.77 - 1.89 (m, 1H); MS-ESI (m/z): 355.8 [M + 1f , 334.2 [M-Na + 2] 205.2, 188.1(100%); Anal. Calcd. for C ₁₆ H _{18 3} a05 : C, 54.08; H, 5.1 1 ; N, 11.83; adjusted for C ₁₆ H ₁₃ N ₃ NaO ₅ .0.55 H ₂ 0: C, 52.62; H, 5.27; N, 1.50; Found: C, 52.66; H, 5.28; N, 11.48. The water content of this material was 2.9% as determined by the Karl-Fischer test. The PXRD pattern is displayed in FIG.1 , the solid state ³ C NMR spectrum in FIG. 2, the FT-IR spectrum in KBr in FIG.3, the differential scanning calorimetry (DSC) pattern in FIG. 4, and the

Thermogravimetric Analysis (TGA) spectrum in FIG. 5,

Similarly as described in Example 2A above, the experiment was also performed on a 6g scale of the starting material (H-D-Glu(L-Trp-O-Me)-O-Bzl. HCI salt) to afford Apo816Na (2.94 g, 65.4% yield). HPLC purity (peak percent area) = 99.1 %. This material displayed similar solution ¹ H NMR pattern (D ₂ 0, 400MHz) and MS spectra as those described in Example 2A above. Anal. Calcd. for C ₆ H ₁₈ N ₃ Na0 ₅ : C, 54.08; H, 5.1 1 ; N, 11.83; adjusted for C ₁₆ H ₁₈ N ₃ Na0 ₅ .0.6H ₂ O: C, 52.49; H, 5.29; N, 1 1.48; Found: C, 52.46; H, 5.31 ; N, .41. The water content of this material was 3.0% as determined by the Karl-Fischer test. The PXRD pattern, the solid state ³ C NMR spectrum, the FT-IR spectrum in KBr, the differential scanning calorimetry (DSC) pattern, and the Thermogravimetric Analysis (TGA) thermogram of this Apo816Na are very similar to those described in Example 2, Procedure A above,

H-D-Glu(L-Trp-0-Me)-0-Bzl. HCI Apo816Na

To a suspension of H-D-Glu-(L-Trp-OMe)-OBzl.HCI (30 g, 63.3 mmol) in EtOH (60 mL) was added a mixture solution of 6N NaOH solution (36.9 mL, 221.6 mmol) and 23.1 mL of de- ionized water dropwise while the internal temperature was maintained at below 8°C using an ice-water bath. The reaction was completed after 1 h based on HPLC analysis. After clarification filtration, the filtrate was acidified to pH 6 with the dropwise addition of a 6N acetic acid under cooling in an ice-water bath. To the resulting suspension was added EtOH (60mL), and the mixture was then stirred at RT for overnight. The solid was collected by suction filtration, and washed with EtOH (1 x40mL, 1 x30mL). The solid was air dried for overnight, and then dried under vacuum at 40-41 °C for 4h to afford 17.32 g (yield = 77%) of Apo816Na. A small sample of the product was dissolved in water and it was shown to be chloride free by the silver nitrate test. This material displayed similar solution ¹ H NMR pattern (D ₂ 0, 400MHz) and MS spectra as those described in Example 2, Procedure A above. Anal. Calcd. for

C ₁₆ H ₁₈ N3Na05: C, 54.08; H, 5.11 ; N, 1 1.83; Found: C, 52.37; H, 5.32; N, 11.48; adjusted for Ci ₆ H _{8 3} Na050.65H ₂ O: C, 52.36; H, 5.30; N, 1 1.45. The water content of this material was 3.3% as determined by the Karl-Fischer test. The PXRD pattern and the FT-IR spectrum in KBr are very similar to those described in Example 2, Procedure A above, Example 3 (Process C)

Preparation of the monosodium salt of H-D-Glu(L-Trp-OH)-OH (Apo816Na) from D-Glu(L- -0-Me)-0-Bzl. HCI in MeOH

H-D-Glu(L-Trp-0-Me)-0-Bzl. HCI Apo816Na

To a suspension of H-D-Glu(L-Trp-OMe)-OBzl. HCI salt (6.0 g, 12.66 mmol) in MeOH (12 mL) was added a solution of cone. (28-30%) NH ₄ OH (0.83 ml_) in water (4.8 ml_). Then, a 3N NaOH solution (6.33 ml_, 38 mmol) was added dropwise with cooling using an ice-water bath, and the resulting mixture was then stirred for 2h. After clarification filtration, the filtrate was acidified to pH 6 with the dropwise addition of a 6N acetic acid under cooling in an ice-water bath. To the resulting suspension was added MeOH (12mL), and the mixture was stirred at RT for overnight. The solid was collected by suction filtration, and washed with MeOH (2 x 10mL). A small sample of the product was dissolved in water and it was shown to be chloride free by the silver nitrate test. The solid was dried under vacuum at 40°C for 4.5h to afford 2.16 g (yield = 48%) of Apo816Na. HPLC purity (peak percent area) = 99.1 %. The solution Ή NMR spectrum in D ₂ 0 (400MHz) and MS spectra of this material were similar to those as described in Example 2, Procedure A above. Anal. Calcd. for Ci ₆ H _{18 3} a05 : C, 54.08; H, 5.1 1 ; N, 1 1.83; Found: C, 52.51 ; H, 5.40; N, 11.39; adjusted for C ₁₆ H _{18 3} Na0 ₅ .0.6H ₂ O: C, 52.49; H, 5.29; N, 1 1.48; The water content of this material was 3.1 % as determined by the Karl-Fischer test. The PXRD pattern, the solid state ¹³ C NMR spectrum, the FT-IR spectrum in KBr, the differential scanning calorimetry (DSC) pattern, and the Thermogravimetric Analysis (TGA) thermogram of this Apo816Na are very similar to those described in Example 2, Procedure A above.

Example 4: Preparation of the monosodium salt of D-Glu(L-Trp-OH)-OH (Apo816Na) from D-Glu(L-Trp-OH)-OH (Apo816DL).

Procedure A: To a finely ground suspension of Apo816DL (8.0g) in isopropanol (16ml_) was added dropwise a solution of 6N NaOH (4ml_) with cooling at ice water bath temperature. A further 56ml_ of isopropanol was added, and the resulting suspension was stirred for 1.5h. The solid was collected via filtration, washed with isopropanol, and then air-dried for overnight. The solid was further dried under vacuum at 40°C for 4.5h to afford 8.69g of Apo816Na. The solution ¹ H NMR pattern (D ₂ 0, 400MHz) and MS spectra of this material were similar to those as described in Example 2, Procedure A above. Anal. Calcd. for 0 ₆ Η ₁₈ Ν ₃ 3θ ₅ : C, 54.08; H, 5.11 ; N, 1 1.83; Found: C, 51.98; H, 5.17; N, 11.41 ; adjusted for C ₁₆ H ₁₈ N ₃ NaO ₅ .0.75H ₂ O: C, 52.10; H, 5.33; N, 11.39. The water content of this material was 3.3% as determined by the Karl-Fischer test. The PXRD pattern and the FT-IR spectrum in KBr are very similar to those described in Example 2, Procedure A above.

Procedure B: Apo816DL (2.0 g, 6.0 mmol) was added portionwise to a solution of 1 N NaOH (6 ml_, 6 mmol) cooled in an ice-water bath. After clarification filtration, the filtrate was added dropwise to 50mL of isopropanoi, and the mixture was then stirred for overnight. The solid was collected via suction filtration, and washed with isopropanoi (15 mt_x2). The solid was air dried for overnight and then under vacuum at 40°C for 4 h to give Apo816Na (1.9 g). A small sample of the product was dissolved in water and it was shown to be chloride free by the silver nitrate test. ¹ H The solution ¹ H NMR pattern (D ₂ 0, 400MHz) and MS spectra of this material were similar to those as described in Example 2, Procedure A above. The water content of this material was 3.7% as determined by the Karl-Fischer test. The PXRD pattern and the FT-IR spectrum in KBr are very similar to those described in Example 2, Procedure A above.

Example 5: Analytical Methods

A. Powder X-Ray Diffraction (PXRD) Analysis

The PXRD analysis of Apo816Na was performed on a PANalytical X'Pert Pro Multipurpose X-ray Diffraction (MPD) system with X'Pert Data Collector Ver. 2.2d using CuK alpha 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 10.3 s per step. The sample was loaded into Zero-background sample holder after grinding. As an illustrative example, the PXRD pattern of the material from Example 2, Procedure A is shown in FIG. 1. The respective peak information such as two-theta (2Θ) angles, d-values and relative intensities are reported in Table 3.

Table 3: Two-theta (2Θ) angles, d-values and relative intensities of Apo816Na of Example 2,

Procedure A

The PXRD pattern of compound from Example 2, Procedure A is shown in FIG. 1 Overlays of the PXRD patterns of the materials from Example 2 Procedure A, Example 2

Procedure B, and Example 3 are shown in FIG. 7. The three batches of material displayed similar PXRD patterns.

B. Solid State ¹³ C NMR

The solid-state ¹³ C 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, 1H 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 (δ(130) = 0.0 ppm) by setting the high-frequency resonance of adamantane to 38.56 ppm.

adamantane

In solid state studies, ¹³ C chemical shifts are referenced to an external adamantane sample, with the chemical shift of the methylene ¹³ C NMR line taken to be 29.51 and 38.56 ppm relative to tetramethylsilane (TMS).

As an illustrative example, the solid state ^, C NMR spectrum of Apo816Na of Example 2, Procedure A is shown in FIG. 2.

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 an illustrative example, the FT-IR spectrum in KBr disc for Apo816Na from Example 2, Procedure A is presented in FIG.3.

D. Differential Scanning Calorimetric (DSC)

DSC analysis was performed on a TA 2920 DSC unit with Universal Thermal Solutions V2.5H Software. The sample was weighed directly into an aluminum TZero holder with the lid placed on top and crimped. After an initial equilibration at 25°C the sample was heated to 250°C at a rate of 10°C/min. All activities were carried out under a N ₂ purge (50 mL/min). As an illustrative example, the DSC thermogram for Apo816Na from Example 2, Procedure A displaying the thermal events observed for the sample with onset and peak max. temperatures are presented in FIG. 4. E. Thermogravimetric (TGA) Analysis

TGA analysis was performed on a TA Instruments Q500 Q Series TGA unit. The sample was heated from ambient temperature to 300°C at a rate of 10°C/min using a dynamic high-resolution mode. All activities were carried out under a nitrogen purge (balance purge at 10 mlJmin, sample purge at 60 mL/min). The TGA thermogram for Apo816Na from Example 2, Procedure A displaying the thermal events and percent weight losses is shown in FIG. 5.

Example 6: Stability Studies

The monosodium salt of H-D-Glu(L-Trp-OH)-OH, prepared in accordance with an aspect of the present invention, was evaluated by PXRD Analysis at the time of preparation. The material was then stored in an amber bottle at ambient temperature. After 20 months of storage, the crystalline form was again evaluated by PXRD Analysis. The PXRD pattern after 20 months of storage remained unchanged from that at time of preparation (Table 4). Thus, the crystalline form of the monosodium salt of H-D-Glu(L-Trp-OH)-OH, prepared in accordance with an aspect of the present invention, is stable to storage.

Table 4: PXRD Analysis of two different batches of the crystalline form of the monosodium salt of H-D-Glu(L-Trp-OH)-OH (Apo816DL-Na) at time of preparation and after 20 months of storage

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.