RU2483077C2 - Crystalline d-isoglutamyl-d-tryptophan and monoammonium salt of d-isogutamyl-d-tryptophan - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing pure crystalline D-isoglutamyl-D-trytophan which involves a step of removing protection from essentially pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan or diester thereof to yield essentially pure D-isoglutamyl-D- tryptophan. An amorphous ammonium alt of D-isoglutamyl-D- tryptophan (1:1) is also disclosed. Also disclosed is a method of producing a pure monoammonium salt of D-isoglutamyl-D-tryptophan from essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan. Disclosed is a compound H-D-Glu-(γ-D-Trp-OR²)-α-OR¹

and pharmaceutically acceptable acid addition salts thereof. Disclosed is a solid pharmaceutical composition and use thereof as an immunodepressant or anti-psoriasis agent.

EFFECT: improved method.

51 cl, 14 ex, 8 dwg, 1 tbl

Description

FIELD OF TECHNOLOGY

This invention relates to a new stable crystalline form of D-isoglutamyl-D-tryptophan and a method for its isolation in pure form, free from inorganic salts. The present invention also relates to a new stable ammonium salt of D-isoglutamyl-D-tryptophan and a method for preparing it in pure form by crystallization and / or conventional silica gel column chromatography.

BACKGROUND

D-Isoglutamyl-D-tryptophan (also known as HD-iGlu-Trp-OH or thymodepressin [Thymodepressin]) is a synthetic hemoregulatory dipeptide designed to treat autoimmune diseases, including psoriasis (Sapuntsova, SG, et al. (May 2002), Bulletin of Experimental Biology and Medicine, 133 (5), 488-490). Timodepressin is considered in Russia as an effective treatment for psoriasis (US patent 5736519), where the drug is currently on the market as disodium salt in a liquid preparation for injection and intranasal administration. It is an immunosuppressant and selectively inhibits the proliferation of bone marrow cells, thus causing inhibition of immunity.

The known solid form of the disodium salt of D-isoglutamyl-D-tryptophan is an amorphous powder that is hygroscopic and very difficult to handle from a technological point of view. The structure of thymodepressin disodium salt is described in Kashirin, DM, et al. (2000), Pharmaceutical Chemistry Journal, 34 (11), 619-622. The monosodium salt of D-isoglutamyl-D-tryptophan was identified by the Chemical Abstracts Service (CAS) and is listed in the CAS REGISTRY ^SM registries, but there are no publications regarding its preparation and physical properties. A powder or amorphous form of a compound such as D-isoglutamyl-D-tryptophan for pharmaceutical use can create manufacturing problems due to problems with bulk density, hygroscopicity and varying water content that cannot be controlled by vacuum drying. D-Isoglutamyl-D-tryptophan is a dipeptide, and drying an amorphous form at elevated temperatures, for example 80-100 ° C, under vacuum is not recommended.

The only synthesis route for HD-iGlu-D-Trp-OH known from the literature is disclosed in US Pat. No. 5,736519. In accordance with the method described in Example 1 of US Pat. No. 5736519, shown herein as Scheme 1, Boc-D-Glu- OH (1.1) reacts with 1,3-dicyclohexylcarbodiimide (DCC) to produce cyclic anhydride (1.2). After dicyclohexylurea (DCM) is removed by filtration, the anhydride (1.2) reacts with HD-Trp-OH to form a mixture of the dipeptide Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp- OH (1.4). The combined yield of crude Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) is 70%. However, the mixture contains only not more than 35% of the desired intermediate Boc-D-iGlu-D-Trp-OH (1.3). The Boc protecting group is removed by mixing solutions of (1.3) and (1.4) in formic acid as a solvent at 40 ° C. for 1 hour. The ratio of (1.3) and (1.4) to formic acid is approximately 1 g: 8 ml (w / v). The product is a mixture of H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6). Since peptides (1.5) and (1.6) are isolated in equal amounts, purification requires ion exchange chromatography using a pyridine acetate buffer. The yield of the target product H-D-iGlu-D-Trp-OH (1.5) is 35% of the amount of Boc-D-iGlu-D-Trp-OH (1.3). Thus, the total yield of H-D-iGlu-D-Trp-OH (1.5) of the amount of Boc-D-Glu-OH is 12.25%.

Scheme 1: Synthesis of H-D-iGlu-D-Trp-OH in accordance with US patent 5736519.

The method described in US patent 5736519, there are several disadvantages, as described below:

1. The DCC in step 1A may lead to the formation of other by-products, such as

By-products from peptide coupling with DCC were reported in Marder, O., and Albericio, F. (June 2003), Chemical Oggi (Chemistry Today), 6-32.

2. Deprotection of Boc-D-iGlu-D-Trp-OH (1.3) requires elevated temperature, and final purification of H-D-iGlu-D-Trp-OH requires an extremely toxic pyridine solvent. Elevated temperatures during deprotection of (1.3) can lead to the formation of the N-tert-butylindole derivative (1.7) as an impurity (Löw, M., et. Al. (1978), Hoppe-Seyler's Z. Physiol. Chem., 359 (12): 1643-51). In addition, the peptide can cyclize to form glutarimide (1.8) (Pandit, U.K. (1989), Pure & Appl. Chem., Vol. 61, No. 3, pp. 423-426).

3. During the reaction of the compound, only a mixture of Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) is formed in a 1: 1 ratio. The maximum yield (1.3) cannot exceed 50% at the stage of compound 1B. A mixture of D-Glu-D-Trp-OH and D-iGlu-D-Trp-OH is formed at the end of the synthesis. Peptides must be separated by ion exchange chromatography and preparative reverse phase high performance liquid chromatography (HPLC). The total yield of H-D-iGlu-D-Trp-OH (1.5) is 12.25%, and purification by preparative HPLC is a very long and inefficient process. Retention times for two such isomers, H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6), are not shown. Repeated isolation cycles to increase the purity of the target isomer (1.5) are very ineffective. This method cannot be adapted to large-scale production.

4. The opposite diastereomer of L-isoglutamyl-L-tryptophan (also known as H-L-iGlu-L-Trp-OH or Bestim) is an immunostimulant (see US Pat. No. 5,774,452). Bestim is used in the treatment of ulcers. It reduces inflammation in the stomach and duodenal mucosa and causes a regression of clinical symptoms and ulceration (Tkacheva, A., et al. (2004), Eksp Klin Gastroenterol. (6): 29-33, 163).

The synthesis of the monosodium salt of H-L-iGlu-L-Trp-OH (1: 1) is shown in Scheme 2 (US patent 5744452).

Scheme 2: Synthesis of [L-iGlu-L-Trp-O ^- ] Na ⁺ in accordance with US patent 5744452.

In the method of US Pat. No. 5,744,452 in step 1, dicyclohexylurea is formed as a by-product, which must be removed by filtration. It is stated that in the second stage trifluoroacetic acid removes γ-o-benzyl ether from the glutamic acid fragment (2.2). Benzyl ether (2.3) is removed by hydrogenation with ammonium formate as a hydrogen donor, a palladium catalyst, sodium bicarbonate in isopropanol at elevated temperature with the formation of the monosodium salt H-L-iGlu-L-Trp-OH (2.4). Solid-phase synthesis (2.4) is also described in this patent, but the tryptophan fragment must be protected as formamide, and later the protection must be removed. Other diastereomers, L-isoglutamyl-D-tryptophan and D-isoglutamyl-L-tryptophan, are also known compounds (US Pat. No. 5,916,878).

The synthesis of H-D-iGlu-L-Trp-OH, and H-L-iGlu-D-Trp-OH is shown in Scheme 3 and Scheme 4, respectively (US patent 5916878).

Scheme 3: Synthesis of H-D-isoglutamyl-L-tryptophan in accordance with US patent 5916878.

Scheme 4: Synthesis of H-L-isoglutamyl-D-tryptophan in accordance with US patent 5916878.

The methods shown in Schemes 2, 3 and 4 can provide the synthesis of a regiospecific gamma-amide product (2.2), (3.2) and (4.2) without the formation of an alpha-amide product, but they include a hydrogenation step during the removal of benzyl ether in the compounds ( 2.3), (3.2) and (4.2). This requires the use of a large amount of palladium catalyst. The second aspect concerns the partial reduction of the indole ring on a production scale. A third aspect relates to the formation of glutarimide, 2- (3-amino-2,6-dioxo-piperidin-1-yl) -3- (1H-indol-3-yl) propionic acid, during the hydrogenation. The fourth aspect concerns cost. The cost of the CBz-Glu-OBzl derivative, such as (3.1) and (4.1), is almost twice the price of the corresponding Boc-Glu-OBzl in production using fine chemical synthesis technologies. The methods in schemes 3 and 4 require purification of the final product by HPLC. The output value is 33% and 35.9%, respectively. Scheme 2 requires the use of trifluoroacetic acid, which introduces other impurities into the reaction. In addition, the method of Scheme 2 uses dicyclohexylcarbodiimide as a peptide condensing agent. Removing trace amounts of impurities from this reagent is a serious problem in chemical production. The technology, therefore, cannot be adapted to industrial production and also cannot be adapted for large-scale production of H-D-isoglutamyl-D-tryptophan.

SUMMARY OF THE INVENTION

The present invention relates to a new stable crystalline form of D-isoglutamyl-D-tryptophan and a method for isolating said compound in a pure form free of inorganic salts by precipitation from water without preparative reverse phase high performance liquid chromatography. The method is intended to produce pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan and its diester free of N-tert-butoxycarbonyl-D-glutamyl-D-tryptophan and to convert N-tert-butoxycarbonyl-D-isoglutamyl- D-tryptophan and its diester into pure crystalline D-isoglutamyl-D-tryptophan. The new crystalline D-isoglutamyl-D-tryptophan of the present invention is easy to purify. In comparison with the methods of the prior art described above, this invention offers a number of advantages, as described below.

First, D-isoglutamyl-D-tryptophan is obtained in crystalline form without the use of preparative high performance liquid chromatography.

Secondly, the key intermediate Boc-D-iGlu-D-Trp-OH or the salt H-D-Glu- (γ-D-Trp-OMe) -α-OBzl HCl is obtained in high yield and high purity.

Thirdly, a method is proposed for converting Boc-D-iGlu-D-Trp-OH and its diester or HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl salts into D-isoglutamyl-D-tryptophan with high yield and high purity.

Fourth, the pure crystalline form of D-isoglutamyl-D-tryptophan according to this invention is not known from the prior art. It can be used directly in the form of a pH-adjusted liquid preparation, which eliminates the need to use the extremely hygroscopic and unstable disodium salt of D-isoglutamyl-D-tryptophan.

The present invention also relates to a new stable ammonium salt of D-isoglutamyl-D-tryptophan and a method for its preparation from N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan, as well as the isolation of such a compound in pure form by crystallization and / or conventional column chromatography with silica gel.

The monoammonium salt of D-isoglutamyl-D-tryptophan is a stable solid, suitable for dosing in order to obtain formulations. A composition identification schedule is proposed for identifying salt forms at different pH values.

A new method for producing D-isoglutamyl-D-tryptophan and the ammonium salt of D-isoglutamyl-D-tryptophan (1: 1) avoids the above-described production problems and makes it possible to isolate and treat thymodepressin and the monoammonium salt of thymodepressin in traditional equipment for chemical processes.

The object of this invention is to propose a suitable method for the production of D-isoglutamyl-D-tryptophan, which would give a drug substance completely free of other diastereomers, as discussed above, and the ability of the material to maintain a stable shape for a long period of storage before being introduced into the drug.

Another object of the present invention is to provide D-isoglutamyl-D-tryptophan free of the alpha-amide isomer of D-glutamyl-D-tryptophan.

Another object of the present invention is to provide a method for producing pure D-isoglutamyl-D-tryptophan (HD-iGlu-D-Trp-OH) from the acid addition salt of HD-iGlu-D-Trp-OH, which gives the product substantially or completely free from residues of organic solvents and not requiring purification by reverse phase high performance liquid chromatography. Solid D-isoglutamyl-D-tryptophan is isolated from water.

Another object of the present invention is to propose a method for producing pure D-isoglutamyl-D-tryptophan (HD-iGlu-D-Trp-OH) from the base addition salt of HD-iGlu-D-Trp-OH, which gives the product, essentially or completely free from residues of organic solvents and not requiring purification by reverse phase high performance liquid chromatography. Solid D-isoglutamyl-D-tryptophan is isolated from water.

Another object of the present invention is to propose a method by which D-isoglutamyl-D-tryptophan is formed, essentially free of contaminants in the form of inorganic salts.

Another object of the present invention is the preparation of crystalline X-ray powder D-isoglutamyl-D-tryptophan (XRPD), as shown in FIG. one.

Another object of the present invention is to obtain the mono-ammonium salt of D-isoglutamyl-D-tryptophan from the acid addition salt of D-isoglutamyl-D-tryptophan, which gives a product substantially or completely free from residues of organic solvents and not requiring purification of a reversed-phase highly effective liquid chromatography. The solid ammonium salt of D-isoglutamyl-D-tryptophan is isolated from isopropanol and ammonia after treatment with an ion exchange resin to remove inorganic salts.

Another object of the invention is the production of the monoammonium salt of D-isoglutamyl-D-tryptophan with XRPD shown in FIG. 2.

Another object of the present invention is to provide the amorphous mono-ammonium salt of D-isoglutamyl-D-tryptophan, which is essentially characterized by the Fourier transform infrared spectrum (IR-FP) shown in FIG. 5.

Another object of the present invention is to propose a method for producing a monoammonium salt of D-isoglutamyl-D-tryptophan, essentially free of contaminants in the form of inorganic salts.

Another object of the present invention is to provide a method for producing an acid addition salt, H-D-iGlu-D-Trp-OH, in particular hydrochloride, from pure Boc-D-iGlu-D-Trp-OH.

Another object of this invention is to provide a method for producing pure Boc-D-iGlu-D-Trp-OH dipeptide without chromatographic isolation.

Another object of the present invention is to propose a simple method of chromatographic separation on a column of silica gel for the purification of D-isoglutamyl-D-tryptophan and its monoammonium salt.

Another object of the present invention is to propose a composition identification schedule for determining a pH range for the isolation of D-isoglutamyl-D-tryptophan and its monovalent monosalt. The dominant pH range for the precipitation of D-isoglutamyl-D-tryptophan from water is from about 2.5 to about 3.0.

The acid addition salt of D-isoglutamyl-D-tryptophan is obtained from the dipeptide Boc-D-iGlu-D-Trp-OH, resulting from the basic hydrolysis of a compound of formula I:

,

,

where R ¹ selected from the group consisting of C ₁ -C ₄ alkyl and benzyl, and R ² represents C ₁ -C ₄ alkyl, provided that C ₄ alkyl is not tert-butyl,

using metal hydroxide in water and an inert solvent in the presence of methanol to form Boc-D-iGlu-D-Trp-OH, free of other diastereomers. The metal hydroxide is selected from the group consisting of lithium hydroxide, sodium hydroxide and potassium hydroxide.

The compound of formula I, in turn, is obtained by peptide condensation of Boc-D-Glu (OH) -OR ¹ and D-Trp-OR ² , where R ¹ and R ² are as defined above, with peptide condensing reagents, such as HOBt and EDK. This method of synthesizing Boc-D-iGlu-D-Trp-OH provides significant advantages over the prior art described in US Pat. , and the alpha-peptide product Boc-D-Glu-D-Trp-OH cannot be formed during synthesis because Boc-D-Glu (OH) -OR ^{1 is used} .

Deprotection of the pure Boc-D-iGlu-D-Trp-OH dipeptide with an acid, such as hydrochloric acid, trifluoroacetic acid, gives an acid addition salt. The pH of the acid addition salt solution is from about 2.5 to about 3.0 to give thymodepressin as a solid.

Alternatively, the acid addition salt can be converted to an ammonium salt by treating an aqueous solution of the material with ion exchange chromatography with a sulfonic acid resin. When salt is removed by elution using water, the ion exchange resin is washed with a mixture of ammonia and isopropanol to obtain a crude ammonium salt, which is recrystallized from isopropanol and water to obtain a pure monoammonium salt.

A solution of the base addition salt of D-isoglutamyl-D-tryptophan is obtained by deprotecting with an acid, in particular deprotecting with HCl, from a compound of formula I, wherein each of R ¹ and R ^{2 is} independently selected from the group consisting of C ₁ -C ₄ alkyl and benzyl, to obtain the acid additive salt of the diester HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ , which is further treated with metal hydroxide in water and an inert solvent in the presence of methanol to form a basic -additive salt HD-iGlu-D-Trp-OH. The metal hydroxide is selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide. Extraction with a solvent not miscible with water removes the organic impurity in the organic phase; the aqueous fraction is separated and adjusted to a pH of from about 6 to about 7 using metal hydroxide. After evaporation of the solvent to reduce the amount of solvent to a ratio of solute to solvent of less than about 1: 8, where the solute is a peptide D-isoglutamyl-D-tryptophan in the form of a base addition salt, the pH of the resulting solution of the base addition salt is from about 2.5 to about 3.0 with an inorganic acid to cause precipitation of D-isoglutamyl-D-tryptophan.

Although it is not intended in any way to limit the context and practice of the present invention to the theory or method of practice of the invention and its possible explanations, it is believed that at a pH of from about 2.5 to about 3.0, the composition identification chart of FIG. 7 and FIG. Figure 8 shows that the main forms of thymodepressin are the free peptide (H-D-iGlu-D-Trp-OH) and the monovalent salt. Since the solubility of H-D-iGlu-D-Trp-OH in water that does not contain organic solvents is low (solubility in water <23 mg / ml), the compound precipitates from the solution in pure form. The XRPD of the material is shown in FIG. one.

In the production of the mono-ammonium salt, a solution of thymodepressin at a pH of from about 6.0 to about 8.0 is purified by ion exchange to remove the salt. An ammonia-based regenerating solution gives a pure mono-ammonium salt after crystallization from isopropanol and water. Although it can be assumed that the mono-ammonium salt is unstable and can again turn into a free dipeptide, in practice, the compound actually remains stable for more than two years. The inventor has invented a monoammonium salt of thymodepressin, which is a stable new chemical entity that can easily crystallize from isopropanol and water. The properties of the obtained crystalline material are shown in FIG. 2.

The above methods produce pure thymodepressin and a monoammonium salt without reverse phase HPLC on an industrial scale, but thymodepressin and a monoammonium salt can be purified by conventional silica gel column chromatography using the above conditions. Thus, instead of discarding a portion of thymodepressin in the crystallization mother liquor, the filtrate can be evaporated and optionally further purified by silica gel column chromatography.

BRIEF DESCRIPTION OF THE FIGURES

The crystalline salts of this invention are described in the Examples below.

FIG. 1 is an XRPD graph of crystalline D-isoglutamyl-D-tryptophan. XRPD parameters can also be represented in terms of interplanar spacings d, Bragg angle 2θ and relative intensity (expressed as a percentage of the most intense line), as shown below.

Angle [° 2θ] The value of d [Å] Relative intensity [%] 6.67 13,239 3 11.09 7,975 4.4 11.77 7,515 1,2 13.29 6,655 four 14.26 6,205 11.3 15,58 5,685 33.3 16.81 5,269 28.9 17.27 5.13 30,4 18.35 4,832 12,2 18.87 4.7 95.8 20.05 4,424 63.6 20.9 4,247 33,2 22.03 4,032 17.1 22.88 3,884 one hundred 23.74 3,744 97.9 24.54 3,625 41.9 25.44 3,499 20.3 25.69 3,465 12.1 26.31 3,384 16,4 27 3.3 27.4 27.75 3,212 24.9 28.18 3,164 19.3 28.79 3,099 6.8 29.13 3,063 6.2 29.91 2,985 79.2 31.04 2,879 8.6 31.49 2,839 33.7 32.54 2,749 4.4 33.29 2,689 9.3 33.97 2,637 10.5 34,99 2,562 17.3 35.54 2,524 21.8 36.14 2,483 5.1 36.74 2,444 5.9 37.35 2,406 7.7 38.31 2,348 25.6 39.01 2,307 20.3

The powder samples were obtained by the usual front packing technique and analyzed on a D8 Discovery diffractometer system with a Cu-kα source operating in the 45 kV / 45 mA mode. The system is equipped with a 2D proportional area detector (GADDS). The experimental data were obtained on two frames with a contact of 600 sec for each, which covered the interval 3 ^o -35 ^o (2θ). The obtained 2D diffraction images were further integrated to obtain a standard, I vs. 2θ, of the diffraction pattern. Data was processed using a variety of Bruker AXS data processing software, including: Eva ™ 8.0 and Topas ™, version 2.1 (for analysis of profile approximation and, if necessary, scaling).

FIG. 2 represents the XRPD of the crystalline monoammonium salt of D-isoglutamyl-D-tryptophan. XRPD parameters can also be represented in terms of interplanar spacings d, Bragg angle 2θ and relative intensity (expressed as a percentage of the most intense line), as follows:

Angle [° 2θ] The value of d [Å] Relative intensity [%] 9.29 9,517 4.1 12.19 7,258 4,5 13.93 6,354 76,2 15.17 5,837 27.4 16.49 5,371 9.8 17.18 5,157 3 18.56 4,778 31.6 18.88 4,696 10.5 20.02 4,431 one hundred 22.28 3,986 3 23.31 3,814 4.6 23.66 3,757 9.8 24.03 3,7 52.9 24.37 3,649 26.3 25.07 3,549 11,4 25.61 3,475 5,6 25.96 3.43 5 27.62 3,227 29.7 28.12 3.17 55.7 28.49 3,131 12,2 29.52 3,023 23.1 30.27 2,951 3,7 30.64 2,915 7.9 31.31 2,854 11.8 31.7 2,821 29.6 32.16 2,781 19,2 32.81 2,728 16 33.78 2,652 7.4 34.14 2,625 5.1 35.76 2,509 16 36.94 2,431 11.6 37.58 2,391 25.6 38.03 2,364 12.5 39.22 2,295 1.9

X-ray powder spectra for D-isoglutamyl-D-tryptophan and its ammonium salt are shown in FIG. 1 and FIG. 2 below. It should be understood that the values of 2 theta for the graph of the powder x-ray may vary somewhat from one device to another or from one sample to another, that is, the values given should not be considered absolute.

FIG. 3 represents the XRPD of an amorphous form of D-isoglutamyl-D-tryptophan.

FIG. 4 represents a characteristic infrared (IR) absorption spectrum of the crystalline monoammonium salt of D-isoglutamyl-D-tryptophan.

FIG. 5 shows a characteristic infrared (IR) absorption spectrum of the amorphous monoammonium salt of D-isoglutamyl-D-tryptophan.

FIG. 6 represents a characteristic infrared (IR) absorption spectrum of crystalline D-isoglutamyl-D-tryptophan.

FIG. 7 illustrates the calculation of the identification of the composition of the HD-iGlu-D-Trp-OH dipeptide and its salt using pKa values for acid and amine groups. LH ₂ is a dibasic acid form of the HD-iGlu-D-Trp-OH peptide; LH is a monocarboxylic acid salt of HD-iGlu-D-Trp-OH. An example of LH is a monoammonium salt. L denotes the form of a dibasic salt, and its example is the disodium salt of the HD-iGlu-D-Trp-OH peptide.

FIG. 8 illustrates the calculation of the identification of the composition of the HD-iGlu-D-Trp-OH dipeptide and its salt using experimentally determined pKa values for acid and amine groups. LH ₂ is a dibasic acid form of the HD-iGlu-D-Trp-OH peptide; LH is a monocarboxylic acid salt of HD-iGlu-D-Trp-OH. An example of LH is a monoammonium salt. L denotes the form of a dibasic salt, and its example is the disodium salt of the HD-iGlu-D-Trp-OH peptide.

DETAILED DESCRIPTION OF THE INVENTION

In this description, the term “Boc-D-Glu (OH) -OR ¹ ” refers to the structure:

if R ¹ is benzyl, then from a chemical point of view, the compound is 2-tert-butoxycarbonylamino-D-glutamic acid alpha-benzyl ester.

In this description, the term “D-Trp-OR ² ” refers to the structure:

if R ² is methyl, the compound is D-tryptophan methyl ester.

In this description, the term “Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ " refers to the structure:

In this description, the term “HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ ” refers to the structure:

if R ¹ is benzyl, R ² is methyl, then the compound is

If R ¹ is methyl, R ² is methyl, the compound is

As used herein, the term “thymodepressin” refers to the chemical structure of an H-D-iGlu-D-Trp-OH dipeptide:

.

.

It may also be designated as H-D-Glu- (γ-D-Trp-OH) -OH.

An acid addition salt is a salt formed by the reaction of an amine HD-iGlu-D-Trp-OH with inorganic acids, including hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid and the like, or organic acids, including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, trifluoroacetic acid, benzoic acid one salicylic acid, benzenesulfonic acid and toluenesulfonic acid. It can also be formed by deprotection of the Boc-D-iGlu-D-Trp-OH derivative with an acid.

The base addition salt is a salt formed by the reaction of an H-D-iGlu-D-Trp-OH carboxylic acid with inorganic bases, including sodium hydroxide, lithium hydroxide, potassium hydroxide, and the like.

The present invention is directed to a method for producing HD-iGlu-D-Trp-OH and its inorganic salt free of inorganic salts from the acid addition salts of HD-iGlu-D-Trp-OH, which are preferably derived from the Boc-D-iGlu dipeptide -D-Trp-OH. Boc-D-iGlu-D-Trp-OH is obtained from Boc-D-Glu (OH) -OR ¹ and D-Trp-OR ² , where R ^{1 is} selected from the group consisting of benzyl and C ₁ -C ₄ alkyl, and R ² is C ₁ -C ₄ alkyl, provided that C ₄ alkyl is not tert-butyl.

The present invention is also directed to a method for producing HD-iGlu-D-Trp-OH from a solution of the base addition salt of HD-iGlu-D-Trp-OH, which is preferably obtained from the acid addition salt of the dipeptide HD-Glu- (γ-D- Trp-OR ² ) -α-OR ¹ where each of R ¹ and R ^{2 is} independently selected from the group consisting of benzyl and C ₁ -C ₄ alkyl.

PREFERRED OPTIONS

Within several aspects of the present invention, which is set forth in the summary of the invention, the sequence of process steps and their relative preference are described below.

In an embodiment of the present invention, there is provided a process for the preparation of an inorganic salt-free H-D-iGlu-D-Trp-OH in the aqueous phase, which comprises:

(a) preparing a solution of the acid addition salt of H-D-iGlu-D-Trp-OH in an aqueous medium substantially free of organic solvent; or obtaining a solution of the base addition salt of H-D-iGlu-D-Trp-OH in an aqueous medium substantially free of organic solvent;

(b) adjusting the pH to a pH corresponding to the predominant form for the dibasic form using an alkali metal hydroxide or inorganic acid solution to precipitate H-D-iGlu-D-Trp-OH;

(c) recovering precipitated H-D-iGlu-D-Trp-OH; and

(d) vacuum drying the product obtained in step (c) to form H-D-iGlu-D-Trp-OH.

In another embodiment of the present invention, there is provided a crystalline form of H-D-iGlu-D-Trp-OH, which is D-isoglutamyl-D-tryptophan, which is characterized by the XRPD shown in the description of the figures.

In another embodiment of the present invention, there is provided crystalline H-D-iGlu-D-Trp-OH, which is D-isoglutamyl-D-tryptophan, which is characterized by the XRPD illustrated in FIG. one.

In another embodiment of the present invention, a method for producing a monoammonium salt of H-D-iGlu-D-Trp-OH free of inorganic salts is provided, wherein the method comprises the following steps:

(a) preparing a solution of the acid addition salt of H-D-iGlu-D-Trp-OH in an aqueous medium substantially free of organic solvent;

(b) adjusting the pH to a pH corresponding to the predominant form for the monovalent salt form using a metal hydroxide solution;

(c) treating the solution of step (b) with an ion exchange resin and eluting with water to exchange the metal ion from the salt in the solution to a hydrogen ion until the pH is from about 5.7 to about 7.0;

(d) contacting the ion exchange resin with an ammonia-based regenerating solution that exchanges ions in solution for the target HD-iGlu-D-Trp-OH contained in the ion exchange resin, thereby forming an eluate of the regenerating solution containing the HD ammonium salt -iGlu-D-Trp-OH;

(e) evaporating the solvent from the solution from step (d) to obtain a crude ammonium salt;

(f) dissolving the ammonium salt from step (e) in water and slowly adding isopropanol such that a monoammonium salt precipitates; and

(g) vacuum drying the product from step (f) to form a crystalline form of the ammonium salt H-D-iGlu-D-Trp-OH (1: 1).

Alternatively, instead of steps (f) and (g), the process comprises the following steps:

(h) processing the material from step (d) by silica gel chromatography using isopropanol and an ammonia solution as an eluent; and

(i) freeze drying the product from step (h) to form an amorphous form of the ammonium salt H-D-iGlu-D-Trp-OH (1: 1).

In another embodiment of the present invention, there is provided a method for producing the mono-ammonium salt of H-D-iGlu-D-Trp-OH from crystalline H-D-iGlu-D-Trp-OH free of inorganic salts, wherein the method comprises the following steps:

(a) adding H-D-iGlu-D-Trp-OH to less than one equivalent of an ammonium hydroxide solution;

(b) adjusting the pH to 6-7 with ammonium hydroxide;

(c) evaporating the solvent to form an oil; adding isopropanol with stirring to precipitate the monoammonium salt;

(d) recovering precipitated ammonium salt H-D-iGlu-D-Trp-OH and

(e) vacuum drying the product obtained in step (c) to obtain the monoammonium salt of H-D-iGlu-D-Trp-OH.

In another embodiment, the present invention provides a crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1: 1), which is characterized by XRPD presented in the description of the figures.

In another embodiment of the present invention, there is provided a crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1: 1), which is characterized by the XRPD shown in FIG. 2.

In another embodiment of the present invention, there is provided an amorphous ammonium salt of H-D-iGlu-D-Trp-OH (1: 1), which is characterized by the IR-FP (IR) spectrum shown in FIG. 5.

In another embodiment of the present invention, there is provided a method for preparing an acid addition salt of D-isoglutamyl-D-tryptophan, wherein the salt is H-D-iGlu-D-Trp-OH hydrochloride and wherein the method comprises:

(i) basic hydrolysis of a compound of formula I:

,

,

where R ¹ selected from the group consisting of C ₁ -C ₄ alkyl and benzyl, and R ² represents C ₁ -C ₄ alkyl, provided that C ₄ alkyl is not tert-butyl,

using metal hydroxide in water and an inert solvent in the presence of methanol to form Boc-D-iGlu-D-Trp-OH free of other diastereomers;

(ii) deprotecting with Boc-D-iGlu-D-Trp-OH from step (i) in an inert organic solvent; and evaporating the solvent to form H-D-iGlu-D-Trp-OH hydrochloride.

In another embodiment of the present invention, a method for producing an acid addition H-D-iGlu-D-Trp-OH hydrochloride solution is provided, wherein the method comprises:

(a) hydrogenation of a compound of formula II:

,

,

where R ¹ represents benzyl, and R ² selected from the group consisting of benzyl and hydrogen,

using palladium on charcoal in methanol or ethanol;

(b) purification of the crude H-D-iGlu-D-Trp-OH from step (a) silica gel chromatography using isopropanol and water as an eluent; and

(c) treating the material from step (b) with hydrochloric acid in water to form a solution of H-D-iGlu-D-Trp-OH hydrochloride in water.

In the above two methods, the preparation of the acid addition salt of D-isoglutamyl-D-tryptophan from a compound of formula I is preferable to the preparation of a compound of formula II from the point of view of the cost of the intermediate chemical compounds.

In another embodiment of the present invention, a method for producing a solution of the base addition salt of H-D-iGlu-D-Trp-OH, where the method includes:

(a) deprotecting the N-Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ dipeptide, where each of R ¹ and R ^{2 is} independently selected from the group consisting of C ₁ -C ₄ alkyl and benzyl;

(b) basic hydrolysis of the product from step (a) with a metal hydroxide in water and an inert solvent in the presence of methanol, wherein the metal hydroxide is selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide;

(c) extracting the material from step (b) with a solvent not miscible with water and separating the aqueous fraction;

(d) adjusting the pH of the aqueous fraction from step (c) to values in the range of from about 6 to about 7; and

(e) evaporating the solvent from the solution from step (d) to form a solution containing a ratio of about one part of the dissolved substance to less than about 8 parts of water, where the dissolved substance is a basic addition salt of D-isoglutamyl-D-tryptophan.

In another embodiment, the present invention provides a new diester derivative HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ hydrochloride, where each of R ¹ and R ^{2 is} independently selected from the group consisting of benzyl and C ₁ - C ₄ alkyl.

Compounds HD-Glu- (γ-D-Trp-OR ² ) -α-OR ^{1 are} not known in the prior art. These compounds can be used as intermediates for the preparation of the D-isoglutamyl-D-tryptophan dipeptide. Alternatively, HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ hydrochloride can be used in a pharmaceutical preparation where ester hydrolysis occurs in situ to produce D-isoglutamyl-D-tryptophan in the manufacture of the preparation.

In an embodiment of the present invention, there is provided a composition identification graph illustrated in FIG. 8 to isolate H-D-iGlu-D-Trp-OH at a pH of from about 2.5 to about 3.0.

METHOD FOR PRODUCING

The present invention provides, as depicted in Scheme 5 below, a reliable method for the synthesis of pure N- (tert-butoxycarbonyl) -D-isoglutamyl-D-tryptophan in high yield, and this reliable method is not available in the prior art (e.g., US Pat. No. 5,736519 )

Scheme 5

In the method of this invention, a solution of Boc-D-Glu (OH) -OR ¹ , where R ¹ is benzyl, in an inert solvent, reacts with N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), hydroxybenzotriazole (HOBt ) and diisopropylethylamine (DIPEA). The preferred temperature is from about 5 to about -5 ° C, and dichloromethane is the preferred solvent. After stirring for about 5 to about 30 minutes, preferably about 15 minutes, a solution of D-Trp-OR ² HCl salt, where R ² is methyl, with diisopropylethylamine (DIPEA) is added dropwise. The resulting solution was stirred at ice temperature, preferably from about -5 to about 5 ° C for about 1 hour, and then at room temperature from about 12 to about 20 hours, preferably about 16 hours. The product Boc-D-iGlu- (D-Trp-OR ² ) -α-OR ^{1 is} isolated by conventional means. The compound can be easily crystallized from ethyl acetate and hexane. Two synthetic impurities are present in trace amounts - compounds (A) and (B), which can be removed by recrystallization. Both compounds are believed to be formed from the HOBt reagent.

The Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ diester in alcohol is mixed with sodium hydroxide solution. A preferred amount of sodium hydroxide is from about 2.5 to about 5 equivalents per equivalent of diester. More preferably, a molar ratio of from about 2 to about 3.5 mol of NaOH per mole of diester compound is used. A preferred solvent ratio is about 2 ml of alcohol per 1 ml of water, and a preferred ratio of NaOH and water is about 1 g per 20 ml. Such an isolation technique involves extraction of the reaction mixture with ethyl acetate, and thus, any organic impurity is removed at this stage of the synthesis. After acidification, the aqueous fraction is extracted with an organic solvent, such as ethyl acetate. The dibasic N- (tert-butoxycarbonyl) -D-isoglutamyl-D-tryptophan (Boc-D-iGlu-D-Trp-OH) is isolated by conventional means as a solid. The isolated yield for the two combined stages is 89%. This figure is higher than the methodology of the prior art (US patent 5736519). The new method of this invention is further illustrated in the examples below.

Detailed investigation and monitoring of the hydrolysis step by the applicant showed that the compound Boc-D-iGlu- (D-Trp-OR ² ) -OR ¹ , where R ¹ is benzyl and R ² is methyl, first reacts with methanol to form Boc- D-iGlu- (D-Trp-OMe) -OMe, after which the compound is hydrolyzed to a dibasic acid. Applicant has determined that methanol is necessary for the rapid hydrolysis of alpha-benzyl ether. In view of the large volume of ethyl acetate required for the extraction of the product, the applicant invented a two-phase procedure for the efficient hydrolysis of Boc-D-iGlu- (D-Trp-OR ² ) -OR ¹ . Stir the mixture of Boc-D-iGlu- (D-Trp-OR ² ) -OR ¹ in tert-butyl methyl ether (MTBE) and a solution of a metal hydroxide such as a solution of lithium hydroxide or sodium hydroxide. The preferred ratio of metal hydroxide to Boc-D-iGlu- (D-Trp-OR ² ) -OR ¹ is in the range of from about 2.0-2.5 to 1. Methanol is added and the mixture is vigorously stirred for a period of from about 1 to about 6 hours, preferably from about 1.5 to about 2.5 hours. The organic phase is isolated from the mixture by conventional means. This technique eliminates the need to use a large amount of ethyl acetate for extraction and is illustrated in the examples below.

In the traditional method, the Boc-D-iGlu-D-Trp-OH compound is deprotected with organic acids to form the H-D-iGlu-D-Trp-OH dipeptide, which requires serious purification. There are numerous drawbacks of prior art techniques (US Pat. No. 5,736519) that use formic acid at 40 ° C to deprotect a mixture of Boc-D-iGlu-D-Trp-OH and Boc-D-Glu-D-Trp- OH to form a mixture of HD-iGlu-D-Trp-OH and HD-Glu-D-Trp-OH. Ion exchange chromatography and reverse phase HPLC are used to isolate the product. The yield is low, and the technique cannot be adapted to large-scale production. Deprotection of the N-tert-butoxycarbonyl group with trifluoroacetic acid or formic acid forms a tert-butyl carbonium ion, which can react with indole nitrogen to form an N-tert-butyl product (Löw, M., et. Al. (1978), Hoppe-Seyler's Z. Physiol. Chem., 359 (12): 1643-51). The formation of glutarimide (1,8) (Pandit, U.K. (1989), Pure & Appl. Chem., Vol. 61, No. 3, pp. 423-426) is another problem.

It has been determined by the Applicant that the acid addition salt of the present invention, in particular crude hydrochloride, can be readily prepared with HCl in an inert solvent such as ethyl acetate at low temperature, preferably from about 0 ° C to ambient temperature. Evaporation of the solvent gives thymodepressin hydrochloride, which is used to prepare thymodepressin.

In order to predict the required pH for precipitation of thymodepressin in the form of HD-iGlu-D-Trp-OH dibasic acid, the applicant carried out a theoretical calculation using the composition identification schedule and concluded that thymodepressin exists in dibasic form at pH values from approximately 2.5 to about 3.0. This conclusion allowed us to invent a method for the isolation of thymodepressin without chromatography.

FIG. 7 illustrates such a calculation. In FIG. 7 LH ₂ is a dicarboxylic acid form of the HD-iGlu-D-Trp-OH peptide (i.e. thymodepressin), LH is a monocarboxylic acid salt form of the HD-iGlu-D-Trp-OH peptide; one such example is a mono-ammonium salt, L is a dicarboxylic acid salt form of the HD-iGlu-D-Trp-OH peptide; one such example is a disodium salt, and LH ₃ is an acid addition salt of thymodepressin. The X axis shows the pH of the solution. The Y axis shows% formation relative to L (typical software terminology) and gives the molar fraction of the form present at a particular pH. At a pH of from about 2.5 to about 3.0, most (80%) of the dipeptide exists as a form of a dicarboxylic acid, and can be precipitated from solution if it is insoluble in water. Our study shows that the form of dicarboxylic acid obtained in this way reveals a solubility in water of approximately 23 mg / ml. At a pH of approximately 7.0, 100% of the forms are represented by the form of monocarboxylic acid. If the counterion is sodium, then the form is thymodepressin monosodium.

In practice, if a solution of thymodepressin hydrochloride is dissolved in water and the pH is adjusted to approximately 3.0 with stirring, a solid slowly forms, which is filtered from the mixture. The purity of the material according to LC exceeds 97% and is within the category of pharmaceutical purity as an active pharmaceutical ingredient. The authors determined that this method surpasses the prior art in terms of production and isolation of pure thymodepressin (H-D-iGlu-D-Trp-OH). There is no need for ion-exchange and reverse-phase chromatography of the drug on a column, since the only by-product is water-soluble sodium chloride. The described technique is a direct technique for the isolation and purification of thymodepressin. It provides a theoretical schedule for identifying the composition, and the method is superior to the methods of the prior art, which require cumbersome cleaning.

The pKa values of the acid and amine groups of HD-iGlu-D-Trp-OH are determined experimentally. A graph of the composition of the dipeptide constructed using experimentally determined pKa values is shown in FIG. 8. In FIG. 8 LH ₂ is thymodepressin, LH is a salt of monocarboxylic acid, L is a salt of dicarboxylic acid, and LH ₃ is an acid addition salt of thymodepressin. The X axis shows the pH of the solution. The y axis represents the% of formation with respect to L (typical software terminology), and the molar fraction of the forms present at a particular pH is recorded. A concentration of 0.5 M is used to display the equivalence of 1 g of thymodepressin in 6 ml of water for isolation. This figure shows that approximately 75% of thymodepressin (LH ₂ ) exists in the form of a dicarboxylic acid at a pH of 2.7. For this reason, thymodepressin precipitates at pH 2.7 and can be filtered. The mother liquor can be evaporated to obtain a second portion of thymodepressin. The graph confirms the theoretical prediction - the form of the dibasic acid HD-iGlu-D-Trp-OH prevails at a pH of from about 2.5 to about 3.0; the indicated range is a pH value for isolating a form of dibasic acid from water as a precipitate. Since only about 80% of the material will precipitate in pure form, the volume of the mother liquor should be reduced and subjected to a second round of precipitation at a pH of from about 2.5 to about 3.0, preferably at a pH of about 2.7.

Deprotection of Boc-H-D-iGlu-D-Trp-OH with trifluoroacetic acid in an inert solvent gives the trifluoroacetic acid salt. The inert solvent is dichloromethane, and a mixture of trifluoroacetic acid and dichloromethane (1: 1) is usually used. Evaporation of the solvent gives an oil which is dried under vacuum to remove residual solvent. The oil is dispersed in water. Upon adjusting the pH to about 3.0, a white solid precipitates after stirring for a period of from about 12 to about 16 hours.

In the preparation of the acid addition salt, it is preferable to use HCl in an inert solvent to form the hydrochloride. Alternatively, a trifluoroacetic acid salt may be formed using the method described above. The use of a hydrochloric acid salt as an acid addition salt is preferred since deprotection of Boc-D-iGlu-D-Trp-OH is more effective when using HCl in an inert solvent, for example 3 M HCl in ethyl acetate. The reaction time is significantly longer when using trifluoroacetic acid. In addition, the trifluoroacetic acid salt of D-iGlu-D-Trp-OH contains several synthetic impurities that are converted to D-iGlu-D-Trp-OH when precipitated at a pH of from about 2.5 to about 3.0 in water. Impurities must be removed by repeated recrystallization.

Boc-D-iGlu-D-Trp-OH starting material was prepared using the previously described methodology. The acid addition salt must be dried under vacuum to ensure the absence of organic solvents and volatile impurities. In the case of precipitation of thymodepressin from water, a solution of an acid addition salt in water is obtained. The ratio of acid addition salt to water is in the range of from about 1: 5 to about 1:10. Even more preferably, the ratio of the acid addition salt is in the range of from about 1: 6 to about 1: 8. A metal hydroxide solution, usually a sodium hydroxide solution, is used to precipitate the product, but potassium hydroxide and other metal hydroxide solutions can be used.

Crude H-D-iGlu-D-Trp-OH can also be obtained by hydrogenation of a compound of formula II:

,

,

where R ¹ represents benzyl, and R ² selected from the group consisting of benzyl and hydrogen, using palladium on carbon in methanol or ethanol. After filtering the catalyst, the filtrate was evaporated to an oil, which was further purified by silica gel chromatography using isopropanol and water as an eluent. The resulting HD-iGlu-D-Trp-OH can be converted to the salt of HD-iGlu-D-Trp-OH hydrochloride in water using hydrochloric acid.

A solution of the base addition salt of D-isoglutamyl-D-tryptophan is obtained by deprotecting the acid with the dipeptide Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ , where each of R ¹ and R ² independently selected from the group consisting of benzyl and C ₁ -C ₄ alkyl. For example, deprotection with HCl with Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ in an inert solvent such as dichloromethane gives the HD-Glu- (γ-D-Trp- salt OR ² ) -α-OR ¹ HCl. In the case of a combination where R ¹ is benzyl and R ² is methyl, the product HCl.HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ precipitates from dichloromethane and can be removed by filtration. Treatment of the acid addition salt with a metal hydroxide in an inert solvent such as methanol for the purpose of single-phase homogeneous hydrolysis or tert-butyl methyl ether for the purpose of two-phase hydrolysis gives the basic addition salt HD-iGlu-D-Trp-OH in solution. When the reaction mixture is extracted with a solvent not miscible with water, such as ethyl acetate or tert-butyl methyl ether, the aqueous fraction is neutralized to a pH of from about 6 to about 7 and the solution is evaporated to reduce the volume to a ratio of less than about 1 part of the solute to 8 parts water. The dissolved substance, as predicted by the calculation of composition identification, as shown in FIG. 8, is a base addition salt (in the form of a monocarboxylate) of HD-iGlu-D-Trp-OH. If sodium hydroxide is used as metal hydroxide, the solute will be the monosodium form of HD-iGlu-D-Trp-OH in water. Correction of the pH of this solution to values from about 2.5 to about 3.0 will result in precipitation of solid thymodepressin, HD-iGlu-D-Trp-OH.

The monoammonium salt of thymodepressin can be obtained directly from the dipeptide Boc-D-iGlu-D-Trp-OH. A crude acid addition salt, such as a hydrochloride salt obtained in the manner described above, is treated with an ion exchange resin to remove the inorganic salt. Thus, the crude thymodepressin hydrochloride solution was dissolved in water and the pH adjusted to from about 6 to about 8. The solution was treated with an ion exchange resin. Advantageously, the resin is a sulfonic acid resin. An example is AMBERLYST® 15. An inorganic salt is removed by washing with water to a pH of from about 5.7 to about 7. Ammonia is used as a regenerating agent to extract the ammonium salt of thymodepressin from the resin. Mostly, concentrated ammonia and isopropanol are used as a regenerating agent. The preferred ratio is a concentrated solution of ammonia and isopropanol in a ratio of from about 1 to about 3-4, with a final wash concentrated solution of ammonia and isopropanol in a ratio of 1 part concentrated ammonia solution / 1 part water / 2 part isopropanol. The washing ammonia solution was evaporated under reduced pressure to an oil which crystallized using isopropanol and water to give the monoammonium salt as a white solid. The preferred ratio of isopropanol to water for recrystallization is in the range of from about 5: 1 to about 10: 1. Column chromatography is not required.

In the prior art, there is no method for purifying thymodepressin other than reverse phase liquid chromatography of a preparation. This method is extremely time-consuming and expensive and cannot be adapted to large-scale production. The Applicant has determined that crude thymodepressin can also be purified to pharmaceutical grade by flash chromatography on silica gel using isopropanol and water as an eluent. A preferred mobile phase is an isopropanol / water mixture (from about 10: 1 to about 5: 1). The product is isolated by traditional means.

In a similar way, the mono-ammonium salt can also be purified by flash chromatography on silica gel using isopropanol and concentrated ammonia as an eluent. A preferred mobile phase is an isopropanol / ammonia solution (from about 10: 1 to about 5: 1). The product is isolated by traditional means.

The mono-ammonium salt of D-isoglutamyl-D-tryptophan, obtained by crystallization using isopropanol and water, is a crystalline substance. On the other hand, if the solution of the mono-ammonium salt of D-isoglutamyl-D-tryptophan is freeze-dried, an amorphous material is obtained.

Extensive research was conducted to confirm that there was no racemization of chiral centers during purification on the column and reaction sequence; Details are shown in the examples below.

In accordance with this invention, a method for the synthesis of Boc-D-iGlu-D-Trp-OH, free of the alpha isomer of the amide. A method is proposed for converting Boc-D-iGlu-D-Trp-OH to an acid addition salt of thymodepressin, in particular hydrochloride. The identification schedule provides a method for precipitating thymodepressin in pure form at a pH of about 3 in water. In addition, a method for purifying thymodepressin with a purity of less than 97% flash column chromatography using isopropanol and water as an eluent is provided. Another aspect of the present invention includes a conventional method for producing a monoammonium salt from thymodepressin hydrochloride. The inorganic salt is removed by ion exchange resin, and the mono-ammonium salt is mined using an ammonia-based regenerating solution. Monoammonium salt can be obtained by crystallization in pure form. A method for purifying lower purity monoammonium salt by flash chromatography on a silica gel column using isopropanol and water as an eluent is also provided.

Additionally, the thymodepressin synthesis method disclosed in the prior art (US Pat. No. 5,736519) provides crude thymodepressin, which should be purified by ion exchange chromatography and reverse phase liquid chromatography of the preparation. The separation of the alpha-amide product H-D-Glu-D-Trp-OH from the gamma-amide product thymodepressin D-i-D-Glu-D-Trp-OH remains the most serious production problem. The purification sequence given in the prior art is unsuitable for large-scale production.

Application and Introduction

Thymodepressin disodium salt was used to treat psoriasis. Thus, the crystalline thymodepressin and the mono-ammonium salt of thymodepressin according to this invention can be incorporated into pharmaceutical compositions for administration to a subject in a therapeutically active amount and in a biocompatible form suitable for in vivo administration, i.e., in the form of peptides for administration, in which the therapeutic effect is superior any toxic effects.

According to the composition identification schedule, as shown in FIG. 8, the predominant forms at neutral pH are the monocarboxylate form of thymodepressin, i.e., the monosodium salt of the dipeptide D-isoglutamyl-D-tryptophan, if the counterion is sodium. The disodium salt of D-isoglutamyl-D-tryptophan is extremely hygroscopic and very complex in terms of processing and preparation of dosage forms. The crystalline thymodepressin of the present invention has XRPD, as shown in detail in FIG. 1, and its solubility in water is approximately 20 mg / ml. The compound is an ideal candidate for disodium salt replacement in the manufacture of a variety of preparations. Although the pH of the D-isoglutamyl-D-tryptophan solution is about 3, a pH correction can be carried out using a solution of sodium hydroxide, sodium carbonate or sodium bicarbonate to a pH of from about 7 to about 7.4. The mono-ammonium salt of this invention exists in both crystalline and amorphous forms. Both forms of monoammonium salt have extremely high solubility in water. Thus, they are also suitable candidates for formulations.

The introduction of a new crystalline thymodepressin and / or its monoammonium salt, as described herein, can be carried out by any of the generally accepted methods of administering therapeutic drugs with systemic activity. These methods include dosage forms for oral, parenteral and other types of systemic or local application or use in the form of an aerosol.

Depending on the intended route of administration, the compositions for use may exist in the form of solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, aerosols, suspensions, and the like, preferably in dosage forms, suitable for single administration of exact doses. The compositions will contain at least one traditional pharmaceutical carrier or excipient and crystalline thymodepressin or a pharmaceutically acceptable mono-ammonium salt thereof, and may further comprise other drugs, pharmaceuticals, carriers, adjuvants and the like.

For solid compositions, conventional non-toxic solid carriers can be used which include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharinate, talc, cellulose, glucose, sucrose, magnesium carbonate and the like, in the pharmaceutical category. The active compound, as defined above, can be introduced into suppositories using, for example, polyalkylene glycols, for example, propylene glycol, as a carrier. Liquid compositions suitable for pharmaceutical administration may, for example, be prepared by dissolution, dispersion, and the like. an active compound as defined above and optional pharmaceutical adjuvants in a carrier such as water, saline, aqueous dextrose, glycerin, ethanol and the like, to form a solution or suspension in this way. Optionally, the pharmaceutical composition for administration may also contain minor amounts of non-toxic adjuvants, such as moisturizing or emulsifying agents, buffers and the like, for example sodium acetate, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate and the like. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, David B. Troy (Ed.), Lipincott Williams & Wilkins, Philadelphia, RA, 21st Edition, 2006. In any case, the composition or formulation for administration will contain the amount of active (s) ) compound (s) in an amount effective to alleviate the symptoms of the subject to be treated.

Parenteral administration is generally represented by injection, subcutaneous, intramuscular or intravenous. Injectable agents can be made in traditional forms, such as liquid solutions or suspensions, solid forms suitable for the manufacture of a solution or suspension in a liquid prior to injection, or as emulsions. Suitable excipients, for example, are water, saline, glucose, glycerin, ethanol, and the like. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic adjuvants, such as moisturizing or emulsifying agents, buffering agents and the like, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and the like.

For thymodepressin or its monoammonium salt, oral or nasal (intrabronchial) administration is preferred depending on the nature of the disorder to be treated.

For oral administration, a pharmaceutically acceptable non-toxic composition is prepared by combining any of the commonly used excipients of the pharmaceutical category, such as mannitol, lactose, starch, magnesium stearate, sodium saccharinate, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations, and the like. Such compositions may contain from about 1% to about 95% of the active ingredient, preferably from about 25% to about 70%.

Oral and nasal administration to the lungs can also be carried out using aerosol dosage forms. For aerosol administration, the active ingredient is preferably supplied in finely divided form together with a surfactant and a propellant. A typical percentage of active ingredients is from about 0.01 to about 20 wt.%, Preferably from about 0.04% to about 1.0%.

Surfactants should generally be non-toxic and preferably soluble in the propellant. A typical example of such agents is esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olestearic and oleic acids with polyhydric aliphatic alcohol or its cyclic anhydride, such such as ethylene glycol, glycerin, erythritol, arabitol, mannitol, sorbitol, hexitol anhydride derived from sorbitol (sorbitan esters available on the market under the name SPAN®), and polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides, may be used. Preferred surface active agents are oleates or sorbitan, for example, those available on the market under the names ARLACEL®C (sorbitan sesquioleate), SPAN®80 (sorbitan monooleate) and SPAN®85 (sorbitan trioleate). The surfactant content may be from about 0.1% to about 20% by weight of the composition, preferably from about 0.25% to about 5%.

The rest of the composition is usually a propellant. Liquefied propellants are usually gaseous at ambient conditions and condense under pressure. Suitable liquefied propellants include lower alkanes containing up to five carbon atoms, such as butane and propane, and preferably fluorinated or fluorinated alkanes, such as those available on the market under the name FREON®. Mixtures of the above components may also be used.

In aerosol production, a container equipped with a suitable valve is filled with an appropriate propellant containing a finely divided active ingredient and a surfactant. Thus, the ingredients are held at elevated pressure prior to release when the valve is activated.

For topical use, such compositions contain an effective amount of a compound of this class in a mixture with at least one pharmaceutically acceptable non-toxic carrier. A suitable range of the composition is from about 0.1% to about 10% of the active ingredient, and the rest are carriers, preferably from about 1% to about 2% of the active ingredient. The concentration of the active ingredient in pharmaceutical compositions suitable for topical application will vary depending on the particular activity of the compound, which is used depending on the condition present and the subject to be treated. Suitable carriers or solvents for the topical administration of these compounds include creams, ointments, lotions, emulsions, solutions, and the like.

For example, a suitable topical ointment for the compounds of this invention contains from about 15 to about 45% saturated fatty alcohol containing 16-24 carbon atoms, such as cetyl alcohol, stearyl alcohol, behenyl alcohol and the like, and from about 45 up to about 85 wt.% glycol solvent, such as propylene glycol, polyethylene glycol, dipropylene glycol and mixtures of these components. The ointment may also contain from about 0 to about 15 wt.% A plasticizer, such as polyethylene glycol, 1,2,6-hexanetriol, sorbitol, glycerin, and the like; from about 0 to about 15 wt.%, a binder, such as saturated fatty acids containing from 16 to 24 carbon atoms, for example stearic acid, palmitic acid, behenic acid, fatty acid amides, for example oleamide, palmitamide, stearamide, behenamide, and fatty acid esters containing 16-24 carbon atoms, such as sorbitol monostearate, polyethylene glycol monostearate, polypropylene glycol or the corresponding monoester of other fatty acids such as oleic acid and palmitic acid; and from about 0 to about 20 wt.% penetration enhancing agents such as dimethyl sulfoxide or dimethylacetamide.

The therapeutically active amount of crystalline thymodepressin or its ammonium salt may vary according to factors such as the pathological condition, age, gender and body weight of the individual. The treatment regimen can be changed in such a way as to provide an optimal therapeutic response. In general, the daily administration schedule should include a dose in the range of from about 1 to about 200 mg of the peptide.

The following are examples of representative formulations that in no way limit the context of the preparation of a variety of pharmaceutical compositions:

Ingredients Amount per tablet (mg) Active ingredient 25 Spray Dried Lactose twenty Corn starch 153 Magnesium stearate 2

The above ingredients are thoroughly mixed and pressed into tablets with the same risk.

Ingredients Amount per tablet (mg) Active ingredient one hundred Spray Dried Lactose 148 Magnesium stearate 2

The above ingredients are mixed and introduced into a hard gelatin capsule.

Ingredients Amount per tablet (mg) Active ingredient 200 Lactose 145 Corn starch fifty Magnesium stearate 5

The above ingredients are thoroughly mixed and compressed into single-risk tablets.

Ingredients Amount per tablet (mg) Active ingredient 108 Lactose fifteen Corn starch 25 Magnesium stearate 2

The above ingredients are mixed and introduced into a hard gelatin capsule.

Ingredients Amount per tablet (mg) Active ingredient 150 Lactose 92

The above ingredients are mixed and introduced into a hard gelatin capsule.

An injection preparation buffered to a pH of approximately 7 is prepared with the following composition:

Ingredients Active ingredient 0.2 g KH ₂ PO ₄
KOH (1 N. solution) 2 ml
required amount up to pH 7 Water (distilled, sterile) required amount up to 20 ml

An injection preparation buffered to a pH of approximately 7 is prepared with the following composition:

Ingredients Active ingredient 0.01 g Water (distilled, sterile) required amount up to 1 ml NaOH (0.2 N) required amount up to pH 7

A suspension for oral administration is prepared with the following composition:

Ingredients Active ingredient 0.1 g Fumaric acid 0.5 g Methylparaben 2.0 g Granulated sugar 0.1 g Sorbitol (70% solution) 25.5 g VEEGUM® K (Vanderbilt Co.) 12.85 g Flavoring 1.0 g Dyes 0.035 ml Distilled water required amount up to 100 ml

Topical preparation:

Ingredients Grams Active connection 0.2-2 SPAN® 60 2 TWEEN® 60 2 Mineral oil 5 Petroleum jelly 10 Methylparaben 0.15 Propylparaben 0.05 BHA (bottled
hydroxyanisole) 0.01 Distilled water required amount up to 100 ml

All of the above ingredients, except water, are combined and heated to approximately 45 ° C with stirring. Then add a sufficient amount of water with a temperature of approximately 45 ° C with vigorous stirring to emulsify the ingredients and then add the required amount of water to 100 g.

Below the invention is explained in detail with reference to Examples, but the invention is not limited to them in any way.

Example 1

Preparation of N-α-tert-butoxycarbonyl-γ-D-glutamyl (α-benzyl ether) -D-tryptophan methyl ester or (2R) -tert-butoxycarbonylamino (4R) - [2- (1H-indol-3-yl ) -1-methoxycarbonyl-ethylcarbamoyl] butyric acid benzyl ester or N-tert-Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl.

Method 1A:

Obtaining a clean reference sample of N-tert-Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl with purification by chromatography on silica gel.

While stirring, to an ice-cooled solution of Boc-D-Glu-OBzl (6.00 g, 17.8 mmol) in CH ₂ Cl ₂ (70 ml), EDC (5.11 g, 26.6 mmol), HOBt (3.60 g, 26.6 mmol) and DIPEA (4.60 ml, 26.6 mmol). Then, a solution of HD-Trp-OMe.HCl (6.77 g, 26.6 mmol) and DIPEA (4.60 ml, 26.6 mmol) in CH ₂ Cl ₂ (50 ml) was added dropwise. The resulting mixture was stirred at ice temperature (from -3 ° C to 0 ° C) for 1 hour, after which it was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was evaporated to dryness. The residue was partitioned between EtOAc and saturated NaHCO ₃ solution. The organic fraction was collected, washed with 10% citric acid solution and then with saturated saline. The organic fraction was dried over sodium sulfate, filtered and evaporated to give a thick oil. The residue was purified by silica gel column chromatography using a hexane / EtOAc solvent mixture gradient (ratio 8: 2, 7: 3 and 3: 7, v / v) as an eluent to obtain the title product (9.40 g, 98 %) as a white solid. ¹ H NMR (DMSO-d ₆ ) δ ppm 10.86 (s, 1H), 8.31 (d, J = 7.4 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.31-7.35 (m , 7H), 7.14 (d, J = 2.0 Hz, 1H), 7.06 (t, J = 7.9 Hz, 1H), 6.98 (t, J = 6.8 Hz, 1H ), 5.12 (q, J = 5.9 Hz, 2H), 4.5 (q, J = 6.5 Hz, 1H), 3.96-4.03 (m, 1H), 3.55 (s, 3H), 2.98-3.16 (m, 2H), 2.18-2.24 (m, 2H), 1.86-1.95 (m, 1H), 1.71-1 80 (m, 1H); 1.37 (s, 9H); ¹³ C NMR (DMSO-d ₆ ) δ ppm: 172.4 (C), 172.3 (C), 171.4 (C), 155.6 (C), 136.1 (C), 136.0 (C), 128.4 (CH), 127.9 (CH), 127.7 (CH), 127.1 (C), 123.6 (CH), 120.9 (CH), 118 4 (CH), 117.9 (CH), 111.4 (CH), 109.5 (C), 78.2 (C), 65.8 (CH ₂ ), 53.3 (CH), 53 16 (CH), 51.7 (CH ₃ ), 31.3 (CH ₂ ), 28.2 (CH ₃ ), 27.1 (CH ₂ ), 26.4 (CH ₂ ); MS (mass to charge ratio) 538 [M + 1] ⁺ ; analytically calculated for C ₂₉ H ₃₅ N ₃ O ₇ .0.5H ₂ O: C, 63.72; H, 6.64; N, 7.69; Found: C, 63.79; H, 6.06; N, 7.65.

Method 1B:

Obtaining a pure sample of N-tert-Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl by recrystallization.

A suspension of Boc-D-Glu-OBzl (60.26 g, 178.6 mmol) in CH ₂ Cl ₂ (335 ml) was cooled to about -1 ^° C and stirred for 15 minutes. Then, DIPEA (46.70 ml, 268.0 mmol), HOBt (36.20 g, 268.0 mmol), EDC (51.38 g, 268.0 mmol) are successively added. Then, a solution of HD-Trp-OMe.HCl (68.25 g, 268.0 mmol) and DIPEA (46.70 ml, 268.0 mmol) in CH ₂ Cl ₂ (187 ml) was added dropwise. The resulting mixture was stirred at ice temperature (from -1 ° C to -5 ° C) for 2 hours, after which it was allowed to warm to room temperature and was stirred overnight in a nitrogen atmosphere.

The reaction mixture was evaporated to dryness. The residue was partitioned between EtOAc (200 ml), saturated Na ₂ CO ₃ solution (100 ml) and H ₂ O (150 ml). The aqueous fraction was again extracted with ethyl acetate (200 ml). The organic fractions were collected, washed with H ₂ O (100 ml), 10% citric acid solution (2 × 200 ml) and brine (60 ml). The organic fraction was dried over sodium sulfate, filtered and evaporated to dryness. The residue was dissolved in ethyl acetate (141 ml), then hexane (106 ml) was added. The resulting suspension was stirred for 6 hours and filtered. The solid is washed thoroughly with hexane (100 ml), then dried under vacuum in an oven at 40 ° C, keeping at this temperature overnight. A solid is obtained that is substantially white (81.67 g, 85%). ¹ H NMR data confirm the structure (see Example 1, Procedure 1A).

Method 1C:

Obtaining a pure sample of N-tert-Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl without chromatographic purification and determination of synthetic impurities.

Boc-D-Glu-OBzl (48.0 g, 142.2 mmol) was dissolved in 270 ml of dichloromethane, and then cooled to 0-5 ° C using an ice bath. HOBt (23.8 g, 156.4 mmol) was added followed by DIPEA (27.0 ml, 156.4 mmol) and stirred for 10 minutes. EDC (38 g, 199.1 mmol) and pre-mixed solution of HD-Trp-OMe (prepared from HD-Trp-OMe.HCl [39.7 g, 156.4 mmol] and DIPEA [27.0 ml, 156, 4 mmol] in 150 ml of dichloromethane is stirred at room temperature for 20 minutes) successively added to the solution. The reaction mixture was stirred at 0 ° C, keeping at this temperature for 2 hours and then overnight at room temperature. The reaction mixture was poured into 250 ml of distilled water and extracted. The organic layer was washed with 250 ml of a 10% citric acid solution, 2 × 5% NaHCO ₃ solution and brine. The organic layer was dried over sodium sulfate and evaporated in vacuo to give a pale yellow foamy solid.

The solid is dissolved in approximately 250 ml of ethyl acetate and evaporated to dryness. The operation is carried out twice with the formation of a waxy solid. 100 ml of ethyl acetate was added to the solid and stirred at room temperature. The mixture is stirred at moderate or high speed until a suspension is formed with the consistency of a liquid batter - this process takes approximately 45 minutes (mixing for a long period of time can lead to the hardening of the solution in the form of a gelatinous substance). Then 75 ml of hexane was added and the mixture was stirred for another 10 minutes. At this point, another 20 ml of ethyl acetate is added and the suspension is filtered immediately to give a pale pink fluffy solid. The solid is immediately washed three times with 30 ml of hexane, which helps to remove the pinkish color. The filtrate is collected and allowed to stand still for 40 minutes. A granular solid precipitates from the filtrate. The mixture is filtered and the solid washed three times with 10 ml of hexane.

The filtrate was collected and evaporated to a solid. The solid is dissolved in 20 ml of ethyl acetate and stirred until a slurry forms with the consistency of a batter. Then 40 ml of hexane are added and the mixture is stirred for 5 minutes. The mixture was filtered and the collected solid was washed with hexane. The combined solids were dried overnight in an oven (35 ° C) under vacuum to constant weight. Thus, 59.0 g (77.2%) of the title compound are obtained. Mp: 83.1-87.5 ° C. ¹ H NMR data are identical to those described in Example 1, Procedure 1A. Purity by HPLC (% peak area): 97.2%; retention time: 7.56 min; HPLC conditions: Waters Symmetry C 18 column, 3.9 × 150 mm, 5 μm; mobile phase: 0.035% HClO ₄ , pH 2 / CH ₃ CN, gradient (min-% CH ₃ CN) 0-35, 10-90, 12-90; flow rate: 1 ml / min; λ: 230, 260, 280 nm.

Analysis of impurities in the mother liquor.

TLC analysis of the mother liquor (ethyl acetate / hexane 50:50 mixture) is shown in the table below. Both spots A and B fluoresce in UV light, but give a negative result of ninhydrin tests. Product stains and starting line give positive ninhydrin test results. Samples A and B were isolated by silica gel column chromatography, and their structure was clarified by ¹ H NMR and MS / MS. Structure A and B are shown below.

The value of Rf in a mixture of ethyl acetate / hexane 50:50:

Spot Rf value BUT 0.80 B 0.60 Product 0.39

Spots A and B correspond to impurities associated with HOBt. Impurities can be removed by recrystallization. Any trace impurities can be removed in the next hydrolysis step.

Example 2

Preparation of N-α-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan or (2R) -tert-butoxycarbonylamino (4R) - [1-carboxy-2- (1H-indol-3-yl) ethylcarbamoyl] oil acids or N-tert-α-Boc-D-iGlu-D-Trp-OH.

Method 2A:

Single phase hydrolysis with NaOH.

While stirring to a solution of N-tert-Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl (3.7 g, 6.9 mmol) from Example 1, Procedure 1A, in methanol (40 ml ) add a solution of NaOH (1.0 g, 25 mmol) in H ₂ O (20 ml). The resulting solution was stirred at room temperature overnight. The reaction mixture was poured into 1N. NaOH solution (100 ml) and the aqueous mixture washed with ethyl acetate (2 × 100 ml). The aqueous fraction is acidified with 3 N. HCl solution, then extracted with ethyl acetate (2 × 50 ml). The organic fractions are combined, dried over sodium sulfate and evaporated to dryness under reduced pressure. A white solid is obtained (2.7 g, 91%). Mp: 148-158 ° C; ¹ H NMR (DMSO-d ₆ ) δ ppm: 12.47 (broad, 2H), 10.82 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7 53 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.06 (t, J = 7.5 Hz, 2H), 6.98 (t, J = 7.4 Hz, 1H), 4.46 (q, J = 5.3 Hz, 1H), 3.88-3 83 (m, 1H), 3.17-2.97 (dd, J = 5.2 and 8.4 Hz, 2H), 2.23-2.10 (m, 2H), 1.90-1 82 (m, 1H); 1.75-1.68 (m, 1H); 1.38 (s, 9H); ¹³ C NMR (DMSO-d ₆ ) δ ppm: 173.9 (C), 173.4 (C), 171.5 (C), 155.6 (C), 136.1 (C), 127.2 (C), 123.5 (CH), 120.9 (CH), 118.4 (CH), 118.2 (CH), 111.4 (CH), 109.9 (C), 78 0 (C), 53.1 (CH), 52.9 (CH), 31.7 (CH ₂ ), 28.2 (CH ₃ ), 27.2 (CH ₂ ), 26.7 (CH ₂ ); IR-FT (KBr) ν: 3415, 3338, 2986, 1719, 1686, 1654, 1534, 1424, 1366, 1252, 1169, 1069, 744, 634, 429 cm ^-1 ; MS (mass to charge ratio) 434 [M + 1] ⁺ .

Method 2B:

Single phase hydrolysis using LiOH.

While stirring, to a solution of Boc-D-Glu- (γ-D-Trp-OCH ₃ ) -α-OBzl (46.06 g, 85.68 mmol) in methanol cooled in ice (0 ° C to 5 ° C) (200 ml) add a solution of LiOH (10.78 g, 257.0 mmol) in H ₂ O (136 ml). The resulting solution was stirred and kept at a temperature of 0 ° C to 10 ° C, keeping at this temperature for 3 hours. The reaction mixture was poured into a saturated solution of Na ₂ CO ₃ (100 ml) and H ₂ O (150 ml), the aqueous mixture was washed with ethyl acetate (2 × 150 ml). The aqueous fraction is acidified to pH 2-3 with 3 N. HCl solution, and then extracted with ethyl acetate (2 × 200 ml). The organic fractions are combined, dried over sodium sulfate and evaporated to dryness under reduced pressure. A white solid is obtained (36.65 g, 98.7%). ¹ H NMR and MS / MS data confirm the structure (see Example 2, Method 2A).

Method 2C:

Obtaining Boc-D-iGlu-D-Trp-OH without chromatographic purification using a two-phase hydrolysis process.

Lithium hydroxide (4.1 g, 97.7 mmol) is dissolved in 35 ml of distilled water. Then add 65 ml of methyl tert-butyl ether (MTBE), followed by the addition of the dipeptide Boc-D-Glu- (γ-D-Trp-OCH ₃ ) -α-OBzl (25 g, 46.5 mmol), obtained as described in Example 1. Immediately a very thick suspension forms, and 15 ml of methanol and 15 MTBE are added with vigorous stirring. An additional 2 ml of methanol was added and the solid was slowly dissolved for approximately 5 minutes. Immediately after dissolving the material, the solution becomes yellow / green, while the upper organic fraction becomes pale green and the aqueous fraction yellow. The reaction mixture was vigorously stirred at room temperature for 80 minutes; at this point in time, no starting material remains in the organic fraction and the aqueous fraction contains the product (TLC control: EtOAC / hexane [1: 1], v / v). The solution was poured into a dividing watering can and 2 fractions were separated. The organic fraction was washed with 15 ml of water. The organic fraction becomes pink when washed with water. The combined aqueous fraction was washed twice with 30 ml of ethyl acetate. The aqueous fraction was acidified to approximately pH 2 by dropwise adding 16.6 ml of 6N hydrochloric acid at room temperature. The aqueous fraction was extracted twice with 50 ml of ethyl acetate. The maximum amount of methanol is added during the second extraction in order to improve the solubility of the product in the organic fraction. The combined organic fractions were dried over sodium sulfate and evaporated in vacuo from a yellow liquid to give a white solid. The solid was dried overnight in an oven (28 ° C) under vacuum to constant weight.

Thus, 18.6 g (yield 92%) of the title compound are obtained. Mp: 179.0-184.6 ° C; ¹ H NMR data were identical to those described in Example 2A; purity by HPLC (% peak area): 98.3%; retention time: 5.33 min; HPLC conditions: Waters Symmetry C 18 column, 3.9 × 150 mm, 5 μm; mobile phase: 0.035% HClO ₄ , pH 2 / CH ₃ CN, gradient (T, min-% CH ₃ CN) 0-20, 10-90, 12-90; flow rate: 1 ml / min; λ: 230, 260, 280 nm.

Control of the above reaction can be carried out using HPLC. In Method 2C above, the presence of benzyl alcohol resulting from the hydrolysis of a benzyl ether fragment can be observed by TLC (EtOAC / hexane [1: 1, v / v] as eluent). Benzyl alcohol is formed by hydrolysis of a benzyl ether fragment. The impurity corresponding to the first spot is the same as in Example 1, Method 1C. HPLC control and LC / MS analysis show that Boc-D-Glu- (γ-D-Trp-OCH ₃ ) -α-OBzl first reacts with base and methanol to form Boc-D-Glu- (γ- D-Trp-OCH ₃ ) -α-OCH ₃ , which is then rapidly hydrolyzed to produce the dibasic acid, Boc-D-Glu- (γ-D-Trp-OH) -OH or Boc-D-iGlu-D-Trp- OH.

Example 3:

Obtaining D-isoglutamyl-D-tryptophan.

Method 3A:

Obtaining D-isoglutamyl-D-tryptophan and its purification by recrystallization.

Boc-D-iGlu-D-Trp-OH (20.0 g, 46.14 mmol, from Example 2) was placed in a 1 L 3-neck round-bottom flask equipped with a magnetic stirrer. Ethyl acetate (300 ml) was added and the resulting suspension was cooled to -10 ° C in an ice bath. HCl gas was passed through a cold suspension. The temperature range from -4 ° C to -10 ° C is maintained during the reaction and the progress of the reaction is monitored by HPLC. At a certain point, the heterogeneous reaction mixture turns into a clear, homogeneous solution of a light pink color. After the conversion of the starting material, the reaction mixture again turns into a suspension. Volatile materials are then removed in vacuo to give a light pink solid. The solid is dissolved in 60 ml of demineralized water and the resulting solution is washed with dichloromethane (2 × 25 ml). Then, the pH of the aqueous solution was adjusted to approximately 3.0 by the addition of NaOH (10 M, approximately 3.6 ml) under cooling. The resulting solution was filtered to remove any residual solid particles. The filtrate is collected and vigorously stirred. The solid is separated by filtration. The filtrate is left for later use. Then, the solid was again placed in a round bottom flask and 30 ml of demineralized water was added. The mixture was vigorously stirred and the solid was separated by filtration. The filtrate is left for later use. The solid is then washed with ice cold cold demineralized water (4 × 15 ml). The third wash water solution does not contain chlorides, which is confirmed by the negative test result using AgNO ₃ (4% solution). The solid is dried in air and then placed in a vacuum oven at 36 ° C, keeping at this temperature overnight to obtain 8.5 g (purity by HPLC,% peak area: 98.3%).

The filtrates from the above steps are combined and the same recrystallization procedure is carried out to obtain another 3.2 g of product (purity by HPLC,% peak area: 98.7%). The combined yield for 2 servings is 11.7 g (75%).

Additional processing of the final filtrate gives a third portion (1.0 g, purity by HPLC,% peak area: 82.0%).

¹ H NMR (D ₂ O-NaOD, pH 7.0) δ ppm: 7.64 (d, J = 7.9 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.19-7.16 (m, 2H), 7.10 (t, J = 7.4 Hz, 1H), 4.52-4.48 (m, 1H), 3.48 (t , J = 6.1 Hz, 1H), 3.34-3.29 (m, 1H), 3.08-3.02 (m, 1H), 2.30-2.17 (m, 2H), 1.92-1.75 (m, 2H). The HRPD spectrum for the resulting material is shown in FIG. 1. HPLC method: XTerra MS C18 column; 5 μm, 4.6 × 250 mm; mobile phase: A = aqueous fraction: 4 mM Tris, 2 mM EDTA, pH 7.4; B = organic fraction: CH ₃ CN; gradient program: B%: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate = 1 ml / min; injection volume = 5 μl; λ: 222, 254, 282, 450 nm; product retention time = 6.4 min.

Method 3B:

Ethyl acetate (250 ml), pre-cooled to 0 ° C, was saturated with gaseous HCl for 25 minutes. Boc-D-iGlu-D-Trp-OH (15.0 g, 34.6 mmol) was added to form a suspension. The solution was stirred for 90 minutes at ice bath temperature. The solvent was evaporated in vacuo to give a white solid. The solid is dissolved in 35 ml of distilled water with the formation of a thick solution of light brown color. The aqueous fraction is washed with 30 ml of dichloromethane, after which it is transferred into a 100 ml beaker. Using a pH electrode to control acidity, the pH was adjusted from 1.28 to 2.96 using 3.2 ml of 10 N. NaOH solution. The solution is stirred for 1 hour at room temperature, a white precipitate forms slowly. The solid is suction filtered and washed thoroughly with water. The crude solid is suspended in 20 ml of distilled water, stirring for 2 hours at room temperature. The mixture was filtered, the solid was collected and dried to constant weight in an oven in vacuo overnight (40 ° C). Thus, 8.6 g (yield 74.5%) of the title compound are obtained. Purity by HPLC (% peak area): 98.8%. Retention time: 4.21 min; HPLC conditions: Waters Symmetry C 18 column, 3.9 × 150 mm, 5 μm; mobile phase: 0.035% HClO ₄ , pH 2 / CH ₃ CN, gradient (T, min-% CH ₃ CN) 0-10, 10-90, 12-90; flow rate: 1 ml / min; λ: 230, 260, 280 nm; ¹ H NMR data are consistent with structure.

Example 4:

Synthesis of monoammonium salt of D-isoglutamyl-D-tryptophan (1: 1) and its isolation by purification by column chromatography.

Method 4A:

HCl gas was passed with stirring through a solution of Boc-D-Glu - (- D-Trp-OH (2.5 g, 5.8 mmol) in ethyl acetate (60 ml) cooled in ice (0-5 ° C) for 2 ., 5 hours, the reaction mixture was then evaporated to dryness Purification of the residue by column chromatography on silica using a gradient solvent mixture of isopropanol and ammonium hydroxide (28-30% NH ₄ OH) (ratio 8:. 2 and 7: 3, v /. vol.) as the eluent gives the title product (1.8 g, 84.7%) as a white solid after evaporation of the solvent, mp: 124-128 ° C; ¹ H NMR (DM СО-d ₆ ) δ ppm: 10.98 (s, 1H), 8.25 (d, J = 5.9 Hz, 1H), 7.53 (7.8 Hz, 1H), 7, 30 (d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.92 (t, J = 7.2 Hz, 1H), 4.28 (m, 1H), 3.22-3.31 (m, 2H), 2.90-2.96 (m, 1H), 2.23-2.25 (m, 2H), 1.97-1.98 (m, 1H), 1.84-1.86 (m, 1H); ¹³ C NMR (DMSO-d ₆ ) δ ppm: 175.5 (C) , 171.6 (C), 171.4 (C), 136.0 (C), 127.6 (C), 123.5 (CH), 120.5 (CH), 118.3 (CH), 117.9 (CH), 111.5 (C), 111.3 (CH), 55.3 (CH), 53.7 (CH), 32.5 (CH ₂ ), 27.8 (CH ₂ ) 27.4 (CH ₂ ); ¹⁴ N NMR (D ₂ O) δ ppm: 20.4 (s); IR-FP (KBr) ν: 3406, 3055, 1581, 1456, 1399, 1341, 1096, 1009, 744, 535, 426 cm ^-1 ; MS (mass to charge ratio) 334 [dibasic acid + 1] ⁺ ; analytically calculated for C ₁₆ H ₂₂ N ₄ O ₅ .H ₂ O: C, 52.17; H, 6.57; N, 15.21; Found: C, 51.95; H, 6.84; N, 14.85. The substance is a mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1). The resulting material is amorphous, as confirmed by XRPD data.

Method 4B:

HCl gas was condensed in cold ethyl acetate (133.4 g) at a temperature of -2 ^° C (temperature of an external ice bath) for 16 minutes. The weight gain of the solution is 21 g. Boc-D-iGlu-D-Trp-OH (3.8 g, 8.73 mmol) is dissolved in 50 ml of the above solution. The temperature of the resulting mixture was maintained in the range of 0-5 ° C for 55 minutes. The reaction mixture was monitored by TLC, and then evaporated under reduced pressure (rotary evaporator temperature: 51-52 ° C) until dry. Purification by flash chromatography on silica gel using a gradient of a mixture of solvents of isopropanol and a solution of ammonium hydroxide (28-30%) (ratio 8: 2 and 7: 3, vol./about.) As an eluent gives the product (2.0 g, 62 %) in the form of a solid almost white. Data ¹ H NMR similar to the data given in Example 4, Method 4A. The resulting material is amorphous, as confirmed by XRPD data.

Example 5

Synthesis of monoammonium salt of D-isoglutamyl-D-tryptophan (1: 1).

Method 5A:

Synthesis of the mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1) by removal of inorganic salts using an AMBERLYST®15 resin, followed by purification by column chromatography.

HCl gas was passed under stirring through an ice-cooled (0-5 ° C) suspension of the resulting Boc-D-iGlu-D-Trp-OH (10.82 g, 24.96 mmol) in ethyl acetate (200 ml) for 2 hours. The reaction mixture was further evaporated to dryness. The residue was dissolved in H ₂ O (30 ml) and neutralized to pH 6-7 with 6 N. NaOH solution. The resulting solution was loaded onto a chromatographic column filled with AMBERLYST®15 resin, followed by elution with water to a pH of 5-5.5, then with 100% isopropanol (pH 7) and finally with a mixture of 25% NH ₄ OH / IPA (pH 10).

The fractions that contain the product are combined and evaporated to dryness under reduced pressure. Further purification of the residue by silica gel column chromatography using a gradient of a mixture of isopropanol solvent and concentrated ammonium hydroxide solution (ratio 17: 3, 4: 1 and 7: 5, v / v) as an eluent gives the title product (6.68 g, 72.7%) as a foamy solid, light yellow in color. ¹ H NMR and MS / MS data confirm the structure (see Method 4A). The water content according to the Karl Fischer test is 3.7%.

Method 5B:

Synthesis of the mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1) by removal of inorganic salts using Amberlyst15 resin, followed by purification by recrystallization.

Under stirring, a suspension of Boc-D-iGlu-D-Trp-OH (10.75 g, 24.80 mmol) in ethyl acetate (200 ml) was passed through ice-cold (0-5 ° C) and gaseous HCl was passed. The reaction mixture was kept in an ice bath (0-5 ° C) for 2 hours. TLC analysis (30% ammonia in isopropanol) demonstrates the complete conversion of the starting material. The reaction mixture is evaporated to dryness in vacuo, the residue is dissolved in H ₂ O (30 ml) and neutralized to pH 6-7 with 10 N. NaOH. The resulting homogeneous solution was loaded onto a chromatographic column filled with AMBERLYST®15 resin, followed by elution with water (2450 ml) to pH 4-5.5, isopropanol (1000 ml) and a mixture of 25% NH ₄ OH / isopropanol. The fractions that contain the product are combined and evaporated to dryness. A colorless foamy solid was obtained, to which isopropanol (150 ml) and H ₂ O (30 ml) were added. The resulting suspension was stirred at room temperature overnight. The solid was suction filtered, washed thoroughly with isopropanol (2 × 60 ml), then EtOAc (2 × 60 ml) and finally dried in a vacuum in an oven at 42 ° C, keeping at this temperature overnight. A substantially white solid is obtained (6.60 g, 72.2%). Data ¹ H NMR and MS / MS correspond to the structure (see Example 5). The XRPD data of the obtained crystalline material are shown in FIG. 2. The water content according to the Karl Fischer method is 5.9%.

Example 6

Synthesis of the mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1) from H-D-iGlu-D-Trp-OH.

Method 6A:

Obtaining a monoammonium salt of D-isoglutamyl-D-tryptophan (1: 1) using CBz-D-Glu- (γ-D-Trp-OH) -γ-OBzl as an intermediate.

EDC (562 mg, 2.93 mmol) was added to a solution of ZD-Glu-OBz (990 mg, 2.67 mmol) and N-hydroxysuccinimide (337 mg, 2.93 mmol) in DMF (50 ml) in an ice bath; the resulting clear solution was stirred overnight at room temperature. HD-Trp-OH (640 mg, 3.13 mmol) and Et ₃ N (1 ml) were added at room temperature. After 20 minutes, the material is mixed with water and extracted with ethyl acetate. The combined EtOAc fractions were washed with 10% citric acid solution and then with saturated brine, dried over sodium sulfate, filtered, evaporated to dryness, dried in vacuo to give 1.49 g of CBz-D-iGlu- (γ-OBzl) -D- Trp-OH. The resulting material was hydrogenated using 33% w / w. 10% Pd / C at atmospheric pressure. After 4 hours, the catalyst was filtered off with CELITE® and the filtrate was evaporated to give an oil. The crude product was purified by flash column chromatography using an isopropanol / NH ₄ OH mixture (80:20 to 70:30 v / v) as eluent to give the title compound (813 mg). Data MS / MS and ¹ H NMR are similar to the compound obtained using the method shown in Example 4 above.

Method 6B:

Obtaining a mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1) from H-D-iGlu-D-Trp-OH (see Example 3).

H-D-iGlu-D-Trp-OH (1 g from Example 3) was mixed with ammonium hydroxide (0.55 M, 6 ml). The mixture is stirred and a pH value of approximately 4.5 is measured. A solution of ammonium hydroxide (0.55 M) is added dropwise to a solution pH of 7.0-7.5. Volatile materials are removed in vacuo and the residual oil is mixed with isopropanol. A white precipitate forms. After 2 hours, the solid (ammonium salt) was separated by vacuum filtration. The solid is dried to constant weight (1 g) under high vacuum for 12 hours to obtain the ammonium salt of D-isoglutamyl-D-tryptophan (1: 1). The water content according to the Karl Fischer method is 4.6%.

Example 7

Purification of H-D-iGlu-D-Trp-OH silica gel column chromatography using a mixture of isopropanol and water.

A. Preparation of Cbz-D-Glu- (γ-D-Trp-OBzl) -α-OBzl.

To an ice-cooled solution of 2.67 g of Cbz-D-Glu-OBzl in 50 ml of DMF was added 0.91 g of N-hydroxysuccinimide (1.1 eq.) And 1.52 g of EDC (1.1 eq.) And the resulting the solution was stirred in an ice bath for 1 hour, and then at room temperature overnight. To the resulting reaction mixture, 2.50 g of HD-Trp-OBzl.HCl (1.05 equiv.) And 3 ml of Et ₃ N were added at room temperature. The reaction is completed after 1 hour, which is confirmed by HPLC data.

The reaction mixture was quenched with demineralized water in an ice bath, and then extracted several times with ethyl acetate. The combined ethyl acetate extracts were washed with 10% citric acid solution and then with saturated brine, dried over sodium sulfate, filtered and evaporated to dryness. The residue was applied to silica gel in methanol and the mixture was evaporated in vacuo. The residue was applied to the top of a wet silica gel column and the desired product, Cbz-D-Glu- (γ-D-Trp-OBzl) -α-OBzl, was eluted using a gradient of a solvent mixture (ethyl acetate / hexane, 80:20 to 100 : 0). The desired fractions were combined and evaporated in vacuo to give 4.64 g (99.6% yield) of the title compound. Purity by HPLC (% peak area): 93%; HPLC conditions: column: Symmetry C18, 3.9 × 150 mm, 5 μm; mobile phase: 0.035% HClO ₄ (pH 2.5) CH ₃ CN = gradient (min-CH ₃ CN%: 0-10, 10-100, 12-100, 14-50); flow rate: 1 ml / min; λ: 280 nm; retention time: 9.7 minutes

B. Purification of D-isoglutamyl-D-tryptophan by column chromatography using a mixture of isopropanol and water.

To a suspension of 4.0 g of Cbz-D-Glu- (γ-D-Trp-OBzl) -α-Obzl, prepared as described in Example 7A above, in 150 ml of a mixture of MeOH / H ₂ O (95: 5, v / v) ./vol.) add 1.5 g of Pd / C (37.5% wt./wt.). The mixture was hydrogenated at a hydrogen pressure of 30 lbs / ^in2. The reaction is completed after 75 min, which is confirmed by HPLC data. The catalyst was filtered on a CELITE® bed and the filtrate was evaporated under reduced pressure and a temperature of 45 ° C. Next, the residue was purified by flash chromatography on silica gel using an isopropanol / H ₂ O mixture (ratio 80:20 v / v). The purest fractions (according to HPLC) are combined and evaporated in vacuo. Thus, the title compound (1.1 g, 52%) was obtained in the form of a pale yellow powder. Purity by HPLC (% peak area): 99%. MS / MS and ¹ H NMR data correspond to the target structure.

Less pure fractions (by HPLC) were combined, evaporated in vacuo to give approximately 1.0 g (48% yield) of the title compound. Purity by HPLC (% peak area): 96.7%. MS / MS and ¹ H NMR data correspond to the target structure.

The HPLC conditions are the same as described in section 7A above; the retention time of H-D-iGlu-D-Trp-OH is 4.0 minutes

Example 8

A. Preparation of H-D-Pyr-D-Trp-OH (5-oxo-D-prolyl-D-tryptophan) with Z-D-Glu- (γ-OEt) -OH.

The title compound is a possible synthetic impurity of H-D-iGlu-D-Trp-OH. It is synthesized independently and used as a reference sample for HPLC analysis of the products described in Examples 3-7 above.

To an ice-cooled solution of ZD-Glu- (γ-OEt) -OH (1 g, 3.23 mmol) in DMF (60 ml) was added N-hydroxysuccinimide (409 mg, 3.56 mmol), EDCI (682 mg, 3.56 mmol) and the resulting solution was stirred in an ice bath for 1 hour, after which it was stirred at room temperature overnight. To the resulting reaction mixture was added HD-Trp-OH (792 mg, 3.88 mmol) and Et ₃ N (1 ml) at room temperature. Water is added after 1.5 hours at the temperature of the ice bath. The mixture was extracted several times with ethyl acetate. The combined ethyl acetate extracts were washed with 10% citric acid solution, followed by brine, dried over sodium sulfate, filtered, evaporated to dryness and dried in vacuo to give 410 mg of crude product A. The aqueous fraction was evaporated in vacuo in a 55 ° bath. C. The residue was dissolved in CH ₂ Cl ₂ , and the organic fraction was washed with 10% citric acid solution (1 × 20 ml) and saturated saline, dried over sodium sulfate, filtered, evaporated to a practically dry state at a temperature of 50 ° C to obtain a crude product fraction B. Both fractions of crude product A and B were combined and hydrogenated over 10% Pd / C (40% w / w Pd / C) at atmospheric pressure using a hydrogen cylinder at room temperature for 2.75 hours. The reaction mixture was filtered through CELITE®. The filtrate containing the product HD-iGlu (γ-OEt) -D-Trp-OH was analyzed by HPLC (the same method as described in 7A above). The retention time for the product peak is 4.54 minutes. The filtrate was evaporated to dryness under reduced pressure in a bath at a temperature of approximately 45 ° C. HPLC analysis showed a partial conversion of the product with R _{t of} 4.54 min to another product with a peak of R _{t of} 4.45 min. The crude product is purified by flash column chromatography using isopropanol and concentrated NH ₄ OH solution (elution with a gradient of 90:10 to 80:20 v / v). An HPLC analysis of the resulting material (670 mg) showed a peak with an R _t value of 4.45 minutes. Clarification of the structure by ¹ H NMR spectroscopy showed that the product after treatment was a cyclic compound shown below:

¹ H NMR (CD ₃ OD) δ ppm: 7.60 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.10 ( s, 1H), 7.07 (t, J = 7.8 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 4.68-4.65 (m, 1H), 4.05-4.02 (m, 1H), 3.44 (dd, J = 14.6 Hz, J = 4.6 Hz, 1H), 3.23-3.18 (m, 1H), 2 , 34-2.24 (m, 1H), 2.13-2.05 (m, 1H), 2.00-1.92 (m, 1H) and 1.76-1.68 (m, 1H) . MS (mass to charge ratio): 316 [M + 1] ⁺ , 188 (100%).

¹³ C NMR (CD ₃ OD) δ ppm: 181.6, 177.5, 174.2, 138.0, 129.3, 124.5, 122.4, 119.8, 119.5, 112.3, 111.7, 58.3, 56.3, 30.2, 28.8 and 26.5.

HPLC method: Symmetry C18 column, 5 μm, 3.9 × 150 mm, WAT 046980; mobile phase - 0.035% HClO ₄ / CH ₃ CN, gradient; method: min-CH ₃ CN%: 0-10%, 10-100%, 12-100%, 14-50%; flow rate: 1.0 ml / min; λ detector: 254 nm.

B. Independent synthesis of 5-oxo-D-prolyl-D-tryptophan from (R) (+) - 2-pyrrolidine-2-5-carboxylic acid (H-D-Pyr-D-Trp-OH).

(R) (+) - 2-Pyrrolidine-2,5-carboxylic acid (2.09 g, 0.016 mol) is added to 250 ml of dichloromethane. Diisopropylethylamine (3.12 ml, 0.018 mol) was added and the solution became clear. Hydroxybenzotriazole hydrate (HOBt.H ₂ O, 2.48 g, 0.016 mol) was added at 0 ° C. EDCI (4.64 g, 0.025 mol) was added. After 30 minutes, HD-Trp-OBzl.HCl (4.76 g, 0.016 mol) was added, followed by diisopropylethylamine (3.12 ml, 0.018 mol). The solution remains clear at a time point of 5 minutes and is stirred for 16 hours. The solvent was evaporated to dryness and the material was partitioned between ethyl acetate and 10% hydrochloric acid. The ethyl acetate fraction was washed with 10% sodium bicarbonate solution, followed by saturated saline. The organic fraction was dried over sodium sulfate and evaporated to give a foamy solid. The resulting solid is benzyl 5-oxo-D-prolyl-D-tryptophanate. Without purification, a sample of the crude benzyl 5-oxo-D-prolyl-D-triptofanata (1.55 g) was dissolved in methanol (30 mL) and hydrogenated over 10% Pd / C under a hydrogen pressure of 45 lb / in ² for 2 hour . The catalyst was filtered through celite and the filtrate was evaporated to give a red foam. The resulting material was purified by column chromatography (elution gradient: 10% methanol in dichloromethane and then 50% isopropanol in dichloromethane) to give a solid (419 mg). The purity of the material can be checked by TLC (30% aqueous ammonia / isopropanol solution). The resulting material is 5-oxo-D-prolyl-D-tryptophan with the same NMR data as for the material from section A.

Example 9

Synthesis of mono-ammonium salt of L-isoglutamyl-L-tryptophan (1: 1).

A: Synthesis of Boc-L-Glu- (γ-L-Trp-O-tert-Bu) -α-O-tert-Bu.

A solution of Boc-L-Glu-O-tert-Bu (1.50 g, 4.9 mmol) in CH ₂ Cl ₂ (50 ml) was cooled to -3 ° C and stirred for 15 minutes. Next, 1-hydroxybenzotriazole (HOBt, 1.00 g, 7.4 mmol), N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC, 1.42 g, 7.4 mmol) and diisopropylethylamine (DIPEA) are successively added. 1.30 ml, 7.4 mmol). After this, a solution of HL-Trp-O-tert-Bu.HCl (2.20 g, 7.4 mmol) and DIPEA (1.30 ml, 7.4 mmol) in CH ₂ Cl ₂ (20 ml) was added dropwise. . The resulting mixture was stirred at ice temperature (from -3 ° C to 0 ° C) for 1 hour and then allowed to warm to room temperature and stirred overnight in a nitrogen atmosphere.

The reaction mixture was evaporated to dryness. The residue was partitioned between EtOAc (40 ml) and saturated NaHCO ₃ solution (100 ml). The organic fraction was separated, washed with 10% citric acid solution and then with saturated brine (30 ml). The organic fraction was dried over sodium sulfate, filtered and evaporated to give a thick oil. Purification of the residue by silica gel column chromatography using a gradient of hexane / EtOAc solvent mixture (ratio 85:15, 80:20 and 60:40, v / v) as an eluent gave the title product (2.55 g, 95%) ) as a white solid. ¹ H NMR (DMSO-d ₆ ) δ ppm: 10.84 (s, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 7, 7 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.12-7.15 (m, 2H), 7.06 (t, J = 7.6 Hz, 1H) 6.98 (t, J = 6.8 Hz, 1H), 4.41 (q, J = 6.7 Hz, 1H), 3.73-3.80 (m, 1H), 2.94- 3.12 (m, 2H), 2.13-2.21 (m, 2H), 1.60-1.85 (m, 2H), 1.28-1.38 (m, 27H); ¹³ C NMR (DMSO-d ₆ ) δ ppm: 171.5 (C), 171.4 (C), 171.1 (C), 155.5 (C), 136.1 (C), 127.2 (C), 123.5 (CH), 120.9 (CH), 118.3 (CH), 118.1 (CH), 111.3 (CH), 109.7, 80.2, 78.0, 53.9 (CH), 53.6 (CH), 31.5 (CH ₂ ), 28.2 (CH ₃ ), 27.9 (CH ₃ ), 27.6 (CH ₃ ), 27.5 (CH ₃ ), 27.2 (CH ₂ ), 26.6 (CH ₂ ); MS (mass to charge ratio) 546 [M + 1] ⁺ ; analytically calculated for C ₂₉ H ₄₃ N ₃ O ₇ .0.5H ₂ O: C, 62.80; H, 8.00; N, 7.58; Found: C, 62.69; H, 8.56; N, 7.57.

B: Synthesis of the mono-ammonium salt of L-isoglutamyl-L-tryptophan (1: 1).

HCl gas was passed under stirring through an ice-cooled (0-5 ° C) solution of Boc-L-Glu - (- L-Trp-O-tert-Bu) -γ-O-tert-Bu, prepared as described above, (2.38 g, 4.4 mmol) in CH ₂ Cl ₂ (40 ml) for 5 hours. The reaction mixture becomes cloudy during the passage of gaseous HCl. The reaction temperature is maintained below 30 ° C by cooling on ice. The reaction mixture was further evaporated to give a white solid (mass of crude material = 2.80 g).

Purification of the crude sample (482 mg) using reverse phase high performance flash chromatography (HPFC ™, Biotage) using a C18HS M + 40 column and a solvent mixture gradient of 15 mM NH ₄ OAc / CH ₃ CN solution as eluent gave the title compound after evaporation of the solvent and freeze drying of the material (150 mg). ¹ H NMR (DMSO-d ₆ ) δ ppm: 7.57 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.11 -7.14 (m, 2H), 7.05 (t, J = 7.2 Hz, 1H), 4.44-4.48 (m, 1H), 3.42 (t, J = 5.7 Hz, 1H), 3.25 (dd, J = 14.7, 4.7 Hz, 1H), 2.97-3.03 (m, 1H), 2.14-2.19 (m, 2H) 1.72-1.85 (m, 2H); IR-FT (KBr) ν: 3057, 1581, 1400, 745 cm ^-1 ; MS (mass to charge ratio) 334 [dibasic acid + 1] ⁺ ; analytically calculated for C ₁₆ H ₂₂ N ₄ O ₅ .H ₂ O: C, 52.17; H, 6.57; N, 15.21; Found: C, 51.92; H, 6.80; N, 14.94. The substance is a mono-ammonium salt of L-isoglutamyl-L-tryptophan (1: 1).

Example 10

Synthesis of monoammonium salt of H-D-isoglutamyl-L-tryptophan (1: 1).

A. Synthesis of Boc-D-Glu- (γ-L-Trp-O-tert-Bu) -α-O-tert-Bu.

Apply the methodology described in Example 1A. With stirring, an ice-cold solution of Boc-D-Glu-O-tert-Bu (4.00 g, 13.2 mmol) in CH ₂ Cl ₂ (75 ml) was subsequently added with EDC (3.80 g, 19.8 mmol), HOBt (2.68 g, 19.8 mmol) and DIPEA (3.50 ml, 19.8 mmol). The resulting mixture was stirred at ice temperature for 20 minutes. Next, a solution of HL-Trp-O-tert-Bu.HCl (5.88 g, 19.8 mmol) and DIPEA (3.50 ml, 19.8 mmol) in CH ₂ Cl ₂ (50 ml) was added dropwise to within 10 minutes The resulting mixture was stirred at ice temperature for 1 hour, after which it was allowed to warm to room temperature and stirred overnight.

The reaction mixture was evaporated to dryness. The thick residual oil was transferred into ethyl acetate (50 ml) and the organic fraction was washed successively with saturated NaHCO ₃ solution (100 ml), 10% citric acid solution (100 ml), saturated saline (100 ml) and water (100 ml). The organic fraction was dried over sodium sulfate, filtered and evaporated in vacuo. Purification of the residue by silica gel column chromatography using a gradient of hexane / EtOAc solvent mixture (8: 2, 7: 3 and 6: 4 v / v) as an eluent gave the title product (5.88 g, 82%) in the form of a solid substance of almost white color. ¹ H NMR (DMSO-d ₆ ) δ ppm: 10.85 (s, 1H), 8.18 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7, 7 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.14-7.05 (m, 3H), 6.99 (t, J = 7.1 Hz, 1H) 4.40 (q, J = 7.5 Hz, 1H), 3.81-3.75 (m, 1H), 3.12-3.07 (m, 1H), 3.01-2.95 (m, 1H), 2.18-2.15 (m, 2H), 1.86-1.83 (m, 1H), 1.73-1.65 (m, 1H), 1.39-1 29 (m, 27H); MS (mass to charge ratio) 568 [M + Na] ⁺ ; 546 [M + 1] ⁺ ; analytically calculated for C ₂₉ H ₄₃ N ₃ O ₇ .0.75H ₂ O: C, 62.29; H, 8.02; N, 7.51. Found: C, 62.43; H, 7.95; N, 7.08.

B. Synthesis of the monoammonium salt of H-D-Glu - (- L-Trp-OH (1: 1).

Apply the methodology described in Example 1B. HCl gas was passed under stirring through an ice-cooled (approximately -5 ° C) solution of Boc-D-Glu- (γ-L-Trp-O-tert-Bu) -α-O-tert-Bu, prepared as described above , (1.59 g, 2.91 mmol) in ethyl acetate (100 ml) for 45 minutes. The solution of colorless becomes a dull yellow. The mixture was stirred at ice temperature for 1 hour, after which it was allowed to warm to room temperature and stirred for another 2 hours. According to HPLC, the reaction is complete (column: Waters C18, 3.9 × 150 mm, WAT046980, mobile phase: gradient of a solvent mixture of 0.035% HClO ₄ [pH 2-2.5] / acetonitrile, flow rate: 1 ml / min, 8 : 210-270 nm).

The reaction mixture was evaporated under reduced pressure to obtain a solid. The solid is dissolved in acetone and the volatiles are removed under reduced pressure. The last procedure is repeated twice more. Purification of the residue by column chromatography using a gradient of a mixture of solvents of isopropanol and a solution of ammonium hydroxide (28-30% NH ₄ OH) (ratio 85:15 and 70:30, v / v) as an eluent gives the title product (0, 42 g, 39%) as a foam almost white. Mp: 120-130 ° C; ¹ H NMR (D ₂ O) δ ppm: 7.67 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.18- 7.22 (m, 2H), 7.13 (t, J = 7.1 Hz, 1H), 4.53 (q, J = 3.8 Hz, 1H), 3.45 (t, J = 5 , 8 Hz, 1H), 3.33 (dd, J = 14.7 and 4.75 Hz, 1H), 3.07 (dd, J = 14.7 and 8.8 Hz, 1H), 2.19 -2.31 (m, 2H); 1.78-1.98 (m, 2H); ¹³ C NMR (D ₂ O) δ ppm: 181.4 (C), 176.6 (C), 176.5 (C), 138.8 (C), 129.9 (C), 126 9 (CH), 124.5 (CH), 121.9 (CH), 121.4 (CH), 114.5 (CH), 113.2 (C), 58.6 (CH), 56, 7 (CH), 34.2 (CH ₂ ), 30.3 (CH ₂ ), 28.9 (CH ₂ ); MS (mass to charge ratio) 334 [dibasic acid + 1] ⁺ .

Example 11

Synthesis of mono-ammonium salt of L-isoglutamyl-D-tryptophan (1: 1).

A. Synthesis of Boc-L-Glu - ((- D-Trp-OMe) -α-O-tert-Bu.

Apply the methodology described in Example 1A. With stirring, an ice-cold solution of Boc-L-Glu-O-tert-Bu (3.45 g, 11.4 mmol) in CH ₂ Cl ₂ (120 ml) was subsequently added with EDC (3.31 g, 17.3 mmol), HOBt (2.36 g, 17.5 mmol) and DIPEA (3.0 ml, 17.1 mmol). The resulting mixture was stirred at ice temperature for another 25 minutes. Next, a solution of HD-Trp-OMe.HCl (2.20 g, 7.40 mmol) and DIPEA (3.0 ml, 17.1 mmol) in CH ₂ Cl ₂ (40 ml) was added. The resulting mixture was stirred at ice temperature for 1 hour, after which it was allowed to warm to room temperature and stirred overnight.

After the usual workup as described in Example 1A, purification of the residue by column chromatography using a gradient of hexane / EtOAc solvent mixture (8: 2 and 6: 4, v / v) as an eluent gave the title product (4.25 g, 74%) as a white foam. ¹ H NMR (CDCl ₃ ) δ ppm: 8.64 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 8.0 Hz , 1H), 7.15 (t, J = 7.2 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 6.61 (d , J = 7.2 Hz, 1H), 5.30 (d, J = 7.8 Hz, 1H), 4.92 (q, J = 6.8 Hz, 1H), 4.16-4.17 (m, 1H), 3.67 (s, 3H), 3.28-3.34 (m, 2H), 2.16-2.27 (m, 2H), 2.05-2.14 (m , 1H); 1.79-1.89 (m, 1H); 1.42-1.43 (m, 18H); ¹³ C NMR (CDCl ₃ ) δ ppm: 172.6 (C), 172.1 (C), 171.6 (C), 171.3 (C), 155.9 (C), 136, 3 (C), 127.7 (C), 123.2 (CH), 122.2 (CH), 119.6 (CH), 118.6 (CH), 111.5 (CH), 109.9 (C), 82.3 (C), 79.9 (C), 53.7 (CH), 53.3 (CH), 52.4 (CH), 32.6 (CH ₂ ), 28.9 (CH ₂ ), 28.4 (CH ₃ ), 28.1 (CH ₃ ), 27.7 (CH ₂ ); MS (mass to charge ratio) 504 [M + 1] ⁺ ; analytically calculated for C ₂₆ H ₃₇ N ₃ O ₇ .0.25H ₂ O: C, 61.46; H, 7.44; N, 8.27; Found: C, 61.36; H, 7.50; N, 7.84.

B. Synthesis of Boc-L-iGlu-D-Trp-OH.

While stirring, a solution of NaOH (654 mg) was added to a solution of Boc-L-Glu - ((- D-Trp-OMe) -α-O-tert-Bu (3.94 g, 7.82 mmol) in methanol (50 ml) , 16.4 mmol) in H ₂ O (20 ml). The resulting solution was stirred at room temperature overnight. 1N NaOH solution (150 ml) was added to the reaction mixture and the aqueous material was washed with ethyl acetate (3 × 100 ml). The aqueous fraction was acidified with 3 N HCl to a pH of about 2, and then extracted with ethyl acetate (3 × 100 ml). The organic fractions were combined, dried over sodium sulfate and evaporated under reduced pressure. silica gel chromatography using a gradient of a mixture of solvents CH ₂ Cl ₂ and MeOH (ratio 85:15, 70:30, v / v) as an eluent gives the title product (0.55 g, 97%) as a foam pink. ¹ H NMR (MeOD-d ₄ ) δ ppm: 7.58 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.05-7.09 (m, 2H), 6.99 (t, J = 7.3 Hz, 1H), 4.61-4.67 (m, 1H), 4.13 (br, 1H) , 3.30-3.38 (m, 2H), 3.12-3.18 (m, 1H), 2.21-2.27 (m, 2H), 1.98-2.06 (m, 1H), 1.81-1.88 (m, 1H), 1.42 (s, 9H); ¹³ C NMR (MeOD-d ₄ ) δ ppm: 158.2 (C), 138.1 (C), 129.1 (C), 124.5 (CH), 122.4 (CH), 119.9 (CH), 119.5 (CH), 112.3 (CH), 111.5 (C), 80.6 (C), 33.5 (CH ₂ ), 28.9 (CH ₃ ) , 28.7 (CH ₂ ); MS (mass to charge ratio) 490 [M + 1] ⁺ .

C. Synthesis of monoammonium salt H-L-iGlu-D-Trp-OH (1: 1).

HCl gas was passed under stirring through an ice-cold (approximately 0 ° C) solution of Boc-L-iGlu-D-Trp-OH as described above (500 mg, 1.0 mmol) in a solvent mixture of CH ₂ Cl ₂ (20 ml) and EtOAc (10 ml) for 30 minutes. The reaction mixture was stirred at ice temperature for 1 hour. According to HPLC, the reaction is complete (column: Waters C18, 3.9 × 150 mm, WAT046980, mobile phase: gradient of solvent mixture 0.035% HClO ₄ [pH 2.0-2.5] / acetonitrile, flow rate: 1 ml / min 8: 210-270 nm).

The reaction mixture was evaporated to dryness to give a dark purple foam. Purification of the residue by column chromatography using a gradient of a mixture of solvents isopropanol / ammonium hydroxide solution (28-30% NH ₄ OH) (ratio 85:15 and 70:30, vol./about.) As an eluent gives the title product (333 mg , 88%) as a thick orange oil. ¹ H NMR (D ₂ O) δ ppm: 7.64 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.16- 7.19 (m, 2H), 7.10 (t, J = 7.5 Hz, 1H), 4.52 (q, J = 3.7 Hz, 1H), 3.44 (t, J = 6 , 3 Hz, 1H), 3.29-3.34 (m, 1H), 3.03-3.09 (m, 1H), 2.17-2.30 (m, 2H), 1.76- 1.96 (m, 2H); ¹³ C NMR (D ₂ O) δ ppm: 181.2 (C), 176.6 (C), 176.5 (C), 138.8 (C), 129.9 (C), 126 9 (CH), 124.5 (CH), 121.9 (CH), 121.4 (CH), 114.5 (CH), 113.1 (C), 58.5 (CH), 56, 7 (CH), 51.6 (CH), 34.2 (CH ₂ ), 30.2 (CH ₂ ), 28.9 (CH ₂ ); MS (mass to charge ratio) 356 [dibasic acid + Na] ⁺ , 334 [dibasic acid + 1] ⁺ , analytically calculated for C ₁₆ H ₂₂ N ₄ O ₅ .2.35H ₂ O: C, 48.94; H, 6.85; N, 14.27; Found: C, 48.94; H, 6.64; N, 14.28.

Example 12

HPLC analysis of D-isoglutamyl-D-tryptophan, L-isoglutamyl-L-tryptophan, L-isoglutamyl-D-tryptophan and D-isoglutamyl-L-tryptophan.

The dibasic acid of the four diastereomers was analyzed on a chiral HPLC column. D, D and L, D diastereomers are taken from the examples above, while the D, L isomer is obtained from Bachem and the L, L isomer is obtained from Sigma. Analysis using a chiral HPLC column (Table 1) showed that the resulting D-isoglutamyl-D-tryptophan is free of other diastereomers, namely (D, L), (L, L) and (L, D) isomers.

Table 1
HPLC Analysis of H-iGlu-Trp-OH H-iGlu-Trp-OH Retention Time (HPLC, Method A, min) Retention Time (HPLC, Method B, min) (D, D) diastereomer 19.32 3.92 (D, L) diastereomer 9.46 3.92 (L, D) diastereomer 13,99 3.87 (L, L) diastereomer 6.70 3.91

Method A: Speaker: CHIROBIOTIC® TAG, 5 μm, 4.6 × 250 mm Mobile phase: 20 mm solution of ammonium acetate (pH 4.1) / MeOH (80:20) Flow rate: 0.8 ml / min Detector, λ: 222, 254, 282, 450 nm Column Temperature: 45 ° C Method B: Speaker: Symmetry C18 Part No. WAT 046980 Mobile phase: HClO ₄ (pH 2) / CH ₃ CN (85/15) Flow rate: 1.0 ml / min Detector, λ: 210-280 nm

In method A, samples were analyzed on a chiral column. The retention times for all four diastereomers are obviously different. In method B, samples were analyzed on a conventional reverse phase column; virtually no difference in retention time for the samples. The monoammonium salt of D-isoglutamyl-D-tryptophan disclosed in this invention is stable after 2 years of storage. HPLC analysis (method B) showed that purity at a wavelength of 254 nm is 99.8%.

Example 13

A. Preparation of the HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl {(2R) -amino (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl- salt ethylcarbamoyl] -butyric acid benzyl ester hydrochloride}.

In a 250 ml 3-neck round-bottom flask equipped with a magnetic stirrer, Boc-D-Glu- (γ-D-Trp-OMe) -α-OBzl (20 g, 0.037 mol) and 100 ml of dichloromethane are placed to form a clear solution at stirring. The solution was cooled in a cooling bath containing an ice / NaCl mixture to -10 ° C. HCl gas was passed through a cold solution. During the reaction, the temperature is in the range from -4 ° C to -10 ° C. The reaction ends in about 1 hour. A white solid precipitates from solution. The solid product is separated by filtration. The solid was washed with dichloromethane (2 × 40 ml), first dried in air and then dried in a vacuum oven at 42 ° C overnight to obtain 16.4 g (94%, purity by HPLC 98.2%). ¹ H NMR (DMSO-d ₆ ) δ ppm: 10.93 (s, 1H), 8.61 (broad, 3H), 8.50 (d, J = 7.4 Hz, 1H), 7 47 (d, J = 7.4 Hz, 1H), 7.40-7.32 (m, 6H), 7.17 (s, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.26-5.14 (m, 2H), 4.51-4.46 (m, 1H), 4.05-3 95 (m, 1H), 3.56 (s, 3H), 3.16-3.11 (m, 1H), 3.06-3.01 (m, 1H), 2.40-2.26 (m, 2H), 2.00-1.98 (m, 2H). HPLC method: column: XTerra MS C18 5 μm 4.6 × 250 mm; mobile phase: A = aqueous phase: 4 mM Tris, 2 mM EDTA, pH 7.4, B = organic phase: CH ₃ CN. Gradient program: B%: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml / min; injection volume = 5 μl; wavelength: 222, 254, 282, 450 nm. R _{t of} starting material = 25.1 min; R _{t of} product = 17.2 min.

B. Preparation of (2R) -amino (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl-ethylcarbamoyl] -butyric acid methyl ester hydrochloride; H-D-Glu- (γ-D-Trp-OMe) -α-OMe HCl salt.

Boc-D-Glu- (γ-D-Trp-OMe) -α-OMe (2.8 g, 6.06 mmol) and methanol (30 ml) are placed in a 250-ml 3-neck round bottom flask equipped with a magnetic stirrer. ) to obtain a clear solution with stirring. The solution was cooled in a cooling bath containing an ice / NaCl mixture to -12 ° C. HCl gas was passed through a cold solution. During the reaction, the temperature is in the range from -12 ° C to + 9 ° C. The reaction ends in approximately 30 minutes. About half of the reaction mixture was evaporated to dryness to give 1.3 g of a solid (purity according to HPLC 96.8%). ¹ H NMR (DMSO-d ₆ ) δ ppm: 10.90 (s, 1H), 8.48-8.46 (m, 4H), 7.48 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 7.07 (t, J = 7.4 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 4.52-4.47 (m, 1H), 4.05-3.99 (m, 1H), 3.69 (s, 3H), 3.58 (s , 3H), 3.17-3.12 (m, 1H), 3.07-3.01 (m, 1H), 2.37-2.23 (m, 2H), 1.98-1.91 (m, 2H). HPLC method: column: XTerra MS C18 5 μm 4.6 × 250 mm; mobile phase: A = aqueous phase: 4 mM Tris, 2 mM EDTA, pH 7.4; B = organic phase: CH ₃ CN. Gradient program: B%: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml / min. Injection volume = 5 μl; wavelength: 222, 254, 282, 450 nm; R _{t of} starting material = 18.7 min; R _{t of} product = 13.0 min.

C. Preparation of the HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl {(2R) -amino (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl- salt ethylcarbamoyl] -butyric acid benzyl ester hydrochloride}.

A solution of HCl in ethyl acetate was obtained by passing gaseous HCl formed by adding dropwise 50 ml of conc. HCl to conc. H ₂ SO ₄ , through ice-cold (0-4 ° C) ethyl acetate (100 ml). The resulting cold acid solution was placed in a dropping funnel and added to a thin suspension of Boc-D-Glu (D-Trp-OMe) -OBzl (36.45 g, 67.80 mmol) in 150 ml of ethyl acetate. A thick suspension forms within 5 minutes. Then the entire HCl solution is added, gaseous HCl is formed as described above and passed directly through the resulting suspension. The internal temperature is maintained in the range of 5-10 ° C, cooling on ice. With stirring, the temperature of the suspension is maintained at 5-15 ° C, keeping at this temperature for 2.5 hours, after which they allow to warm to room temperature. The progress of the reaction is monitored by TLC (hexane / EtOAc [1: 1 v / v] as eluent). The solid was collected by vacuum filtration, washed with ethyl acetate (2 × 100 ml), and then dried in a vacuum oven overnight. A pale pinkish solid is obtained (yield 27.49 g, 86%). Analytical data similar to those shown in Example 13A above, with the exception of some residual EtOAc detected from ¹ H NMR. Residual EtOAc can be removed by drying in air for 24-48 hours before drying in a vacuum oven.

D. Preparation of the HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl {(2R) -amino (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl- salt ethylcarbamoyl] -butyric acid benzyl ester hydrochloride}

Boc-D-Glu-OBzl (200.0 g, 0.593 mol) and EtOAc (1.6 L) were placed in a 3-necked round bottom flask. Diisopropylethylamine (206.5 ml, 1.186 mol) was added to the resulting white suspension. The internal temperature is approximately 21 ° C, and a thick suspension is observed. The suspension is gradually diluted and within 15 minutes turns into a clear solution. Next, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (136.4 g EDC, 0.711 mol) was added as a suspension formed. HCl.HD-TrpOMe (166.1 g, 0.652 mol) was added portionwise over 30 minutes. Heat is observed and the internal temperature reaches 28 ° C. After 2 hours, the internal temperature drops to 25 ° C. The resulting suspension was vigorously stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate (200 ml), and then washed sequentially with 1 N. HCl (600 ml) and 5% HCl solution (2 × 256 ml). A sample of the crude mixture was evaporated and analyzed by TLC and ¹ H NMR. The water content according to the Karl Fischer method is 2.3%.

The contents of the flask are cooled in an ice bath and the internal temperature is approximately 0 ° C. HCl gas was passed through an ice-cooled solution for 1 hour. Heat is observed within 10 minutes after passing gaseous HCl and the internal temperature reaches approximately 19 ° C. After 20 minutes of transmission, the internal temperature is 22 ° C. The resulting suspension was stirred for another hour. The solid was suction filtered and washed with ethyl acetate (2 × 640 ml), then dried under vacuum to constant weight (243.8 g). The analytical data are similar to those in Example 13A above. The presence of ethyl acetate (approximately 10% according to proton integration) is confirmed by ¹ H NMR. Trapped EtOAc can be removed by drying the solid in air for 24-48 hours before drying in an oven.

Example 14

Preparation of a solution of the base addition salt of D-isoglutamyl-D-tryptophan, and its conversion to thymodepressin (D-isoglutamyl-D-tryptophan).

Method 14A:

Starting material, HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl salt (4.0 g, 8.4 mmol) was placed in a 250 ml 3-neck round-bottom flask equipped with a magnetic stirrer. Methanol (20 ml) was added to give a clear solution. The solution was cooled in a bath containing ice and NaCl salt to -10 ° C. NaOH solution (3N, 8.4 ml, 25.2 mmol) was added. HPLC is used to monitor the progress of the reaction. After 2 hours, analysis of the reaction mixture by HPLC shows that the reaction is not yet complete. NaOH solution (3N, 1.4 ml, 4.2 mmol) was added. At this point, a total of 29.4 mmol NaOH is added. After another 4 hours, analysis of the reaction mixture by HPLC showed that the content of the product in the reaction mixture was more than 95.2%. The reaction is stopped. The reaction mixture was acidified while cooling with a bath containing ice-water to pH 6.5 by the addition of hydrochloric acid (6N, ~ 1.3 ml, 7.8 mmol). The resulting solution was evaporated to a volume of 15 ml to remove most of the methanol. The solution was washed with ethyl acetate (15 ml × 2). The solution was filtered and the filtrate was collected. The filtrate was further acidified to pH 3 by the addition of hydrochloric acid (6 N, ~ 1.3 ml, 7.8 mmol). At this point, a total amount of ~ 15.6 mmol of hydrochloric acid is used. A solid is formed during stirring at room temperature. The mixture was stirred overnight. The solid is separated by filtration. The solid was dried in air to give 2.4 g of a crude product. Next, the solid is again placed in a round bottom flask. Demineralized water (15 ml) was added and the mixture was stirred for 2 hours. The solid is separated by filtration. The solid is air dried again and then placed in a round bottom flask. Demineralized water (15 ml) was added and the mixture was stirred for 1 hour. The solid was separated by filtration and washed with ice-cold demineralized water (6 ml × 3). A silver nitrate test showed that the solid was chloride free. The solid is dried in air and then placed in a vacuum oven at 42 ° C, keeping at this temperature for 19 hours, to obtain 1.3 g (46%, purity according to HPLC 98.8%). The filtrates and aqueous washing solutions were combined and evaporated to isolate a second portion of the product. ¹ H NMR (D ₂ O-NaOD, pH 7.0) δ ppm: 7.59 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.15-7.12 (m, 2H), 7.05 (t, J = 7.2 Hz, 1H), 4.47-4.44 (m, 1H), 3.40 (t , J = 6.1 Hz, 1H), 3.30-3.25 (m, 1H), 3.03-2.97 (m, 1H), 2.3-2.1 (m, 2H), 1.84-1.69 (m, 2H). MS (mass to charge ratio) 334.3 [M + 1] ⁺ . HPLC method: column: XTerra MS C18 5 μm 4.6 × 250 mm. Mobile phase: A = aqueous phase: 4 mM Tris, 2 mM EDTA, pH 7.4; B = organic phase: CH ₃ CN. Gradient program: B%: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml / min. Injection volume = 5 μl. Wavelength: 222, 254, 282, 450 nm. R _{t of} product = 6.5 minutes

Method 14B:

Lithium hydroxide monohydrate (0.374 g, 8.9 mmol) is dissolved in 3.5 ml of demineralized water. The solution was placed in a 100 ml single-necked round-bottom flask equipped with a magnetic stirrer. 6.5 ml of methyl tert-butyl ether is added to the solution. At room temperature, the starting material, salt HD-Glu- (γ-D-Trp-OMe) -α-OBzl HCl, (2.0 g, 4.2 mmol) was added to form a suspension. Methanol (2 ml) was added, most of the solid was dissolved. HPLC is used to monitor the progress of the reaction. A certain amount of starting material remains in the reaction mixture after stirring at room temperature overnight. Lithium hydroxide monohydrate (0.190 g, 4.5 mmol) is dissolved in 2 ml of demineralized water and added to the reaction mixture, followed by 2 ml of methanol. At this point, a total amount of 13.4 mmol of LiOH is added. After 4 hours, analysis of the reaction mixture by HPLC shows that the reaction has not yet been completed. Lithium hydroxide monohydrate (0.100 g, 2.4 mmol) is dissolved in 1 ml of demineralized water and added to the reaction mixture. At this point, a total amount of 15.8 mmol of LiOH is added. After another 2.5 hours, analysis of the reaction mixture by HPLC shows that the content of the product in the reaction mixture exceeds 97.5%. The reaction is stopped. The reaction mixture was poured into a separatory funnel and 2 fractions were separated. The aqueous fraction was washed with ethyl acetate (15 ml × 2). The aqueous fraction was acidified by cooling with a bath containing ice water to pH 6 by the addition of hydrochloric acid (6 N, ~ 650 μl, 3.9 mmol). The aqueous fraction was evaporated to 5 ml and filtered, collecting the filtrate. The filtrate was further acidified to pH 3 by the addition of hydrochloric acid (6 N, ~ 700 μl, 4.2 mmol). At this point, a total amount of ~ 8.1 mmol of hydrochloric acid is used. A solid is formed during stirring at room temperature. The solid is separated by filtration. The solid is dried in air, after which it is again placed in a round bottom flask. Demineralized water (6 ml) was added and the mixture was stirred for 15 minutes. The solid was separated by filtration and washed with ice cold cold demineralized water (6 ml × 6). It is brought up that the solid does not contain chlorides, using the test with silver nitrate. The solid is dried in air and then placed in a vacuum oven at 42 ° C, keeping at this temperature for 12 hours, to obtain 0.44 g (31%, purity according to HPLC 98.5%). The filtrates and aqueous washing solutions are combined and evaporated to further isolate a second portion of the product. ¹ H NMR (D ₂ O-NaOD pH 6.0) δ ppm: 7.59 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H ), 7.15-7.12 (m, 2H), 7.05 (t, J = 7.2 Hz, 1H), 4.47-4.44 (m, 1H), 3.41 (t, J = 6.0 Hz, 1H), 3.29-3.25 (m, 1H), 3.03-2.97 (m, 1H), 2.3-2.1 (m, 2H), 1 83-1.58 (m, 2H).

HPLC method: column: XTerra MS C18 5 μm 4.6 × 250 mm. Mobile phase: A = aqueous phase: 4 mM Tris, 2 mM EDTA, pH 7.4; B = organic phase: CH ₃ CN. Gradient program: B%: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate 1 ml / min. Injection volume = 5 μl. Wavelength: 222, 254, 282, 450 nm. R _{t of} product = 6.5 minutes

The timodepressin obtained in this invention has a solubility in water of from about 20 to about 23 mg / ml. Water rinsing in Procedures A and B serves the purpose of removing inorganic salts such as sodium chloride or lithium chloride. Upon receipt of large quantities, the volume of water for washing can be controlled by calculating the amount of inorganic salt present and using solubility to determine the amount of water required for washing the product.

Since numerous changes to the preferred embodiments of the invention can be made without departing from the context of the invention, it is contemplated that all material contained herein should be interpreted as illustrating the invention and not limiting it.

Claims (51)

1. Crystalline D-isoglutamyl-D-tryptophan, characterized by peaks in a powder x-ray having the following 2θ values: 18.87, 20.05, 23.74 and 29.91. 2. Crystalline D-isoglutamyl-D-tryptophan, characterized by a powder x-ray shown in figure 1. 3. Crystalline D-isoglutamyl-D-tryptophan, characterized by the following parameters of the powder X-ray diffraction pattern presented in the values of interplanar spacings d, Bragg angle 2θ and relative intensity (expressed as a percentage relative to the most intense line):
Angle, ° 2θ Value d, A Relative intensity [%] 6.67 13,239 3 11.09 7,975 4.4 11.77 7,515 1,2 13.29 6,655 four 14.26 6,205 11.3 15,58 5,685 33.3 16.81 5,269 28.9 17.27 5.13 30,4 18.35 4,832 12,2 18.87 4.7 95.8 20.05 4,424 63.6 20.9 4,247 33,2 22.03 4,032 17.1 22.88 3,884 one hundred 23.74 3,744 97.9 24.54 3,625 41.9 25.44 3,499 20.3 25.69 3,465 12.1 26.31 3,384 16,4 27 3.3 27.4 27.75 3,212 24.9 28.18 3,164 19.3 28.79 3,099 6.8 29.13 3,063 6.2 29.91 2,985 79.2 31.04 2,879 8.6 31.49 2,839 33.7 32.54 2,749 4.4 33.29 2,689 9.3 33.97 2,637 10.5 34,99 2,562 17.3 35.54 2,524 21.8 36.14 2,483 5.1 36.74 2,444 5.9 37.35 2,406 7.7 38.31 2,348 25.6 39.01 2,307 20.3 4. A method of obtaining D-isoglutamyl-D-tryptophan, free of inorganic salts, comprising the following stages:
(a) preparing a solution of the acid addition salt of HD-iGlu-D-Trp-OH in an aqueous medium substantially free of an organic solvent; or obtaining a solution of a base addition salt of HD-iGlu-D-Trp-OH in an aqueous medium substantially free of an organic solvent;
(b) adjusting the pH to values in the range of from about 2.0 to about 3.2 with an alkali metal hydroxide or inorganic acid solution to cause precipitation of HD-iGlu-D-Trp-OH;
(c) recovering precipitated HD-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in step (c) to obtain HD-iGlu-D-Trp-OH. 5. The method according to claim 4, in which the acid addition salt is HD-iGlu-D-Trp-OH hydrochloride obtained by a method comprising the following steps:
(i) basic hydrolysis of a compound of formula I:

where R ¹ selected from the group consisting of C ₁ -C ₄ alkyl and benzyl, and R ² represents C ₁ -C ₄ alkyl, provided that C ₄ alkyl is not tert-butyl,
using metal hydroxide in water and an inert solvent in the presence of methanol to give Boc-D-iGlu-D-Trp-OH free of other diastereomers; and
(ii) deprotecting with Boc-D-iGlu-D-Trp-OH from step (i) in an inert organic solvent; and evaporating the solvent to obtain HD-iGlu-D-Trp-OH hydrochloride. 6. The method according to claim 4, characterized in that the acid addition salt is HD-iGlu-D-Trp-OH hydrochloride, and the acid addition salt solution in step (a) is prepared by a process comprising:
(i) hydrogenation of a compound of formula II

where R ¹ represents benzyl, and R ² selected from the group consisting of benzyl and hydrogen, using palladium on carbon in methanol or ethanol;
(ii) purification of the crude H-D-iGlu-D-Trp-OH from step (i) by silica gel chromatography using isopropanol and water as an eluent; and (iii) treating the material from step (ii) with hydrochloric acid in water to obtain a solution of the HD-iGlu-D-Trp-OH hydrochloride salt in water. 7. The method according to claim 4, in which the solution of the base addition salt of HD-iGlu-D-Trp-OH in stage (a) is obtained by a method including:
(i) deprotecting the Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ dipeptide with an acid addition salt of the HD-Glu- (γ-D-Trp-OR diester) ² ) -α-OR ¹ where each of R ¹ and R ^{2 is} independently selected from the group consisting of C ₁ -C ₄ alkyl and benzyl;
(ii) basic hydrolysis of the product from step (i) using a metal hydroxide in water and an inert solvent in the presence of methanol to give the base addition salt HD-iGlu-D-Trp-OH, where R ¹ and R ² are as defined above, and where the metal hydroxide is selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide;
(iii) extracting the material from step (ii) a solvent which is not miscible with water, and separating the aqueous layer;
(iv) adjusting the pH of the aqueous phase from step (iii) to a pH of from about 6 to about 7; and
(v) evaporating the solvent from the solution from step (iv) to form a solution containing about one part of the solute into less than about 8 parts of water, where the solute is a basic addition salt of D-isoglutamyl-D-tryptophan. 8. The method of obtaining free of inorganic salts HD-iGlu-D-Trp-OH according to claim 4, which includes:
(a) preparing a solution of the acid addition salt of HD-iGlu-D-Trp-OH in an aqueous medium substantially free of an organic solvent;
(b) adjusting the pH to about 2.0 to about 3.2 with an alkali metal hydroxide solution to cause precipitation of HD-iGlu-D-Trp-OH;
(c) recovering precipitated HD-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in step (c) to obtain HD-iGlu-D-Trp-OH. 9. The method of obtaining free of inorganic salts HD-iGlu-D-Trp-OH according to claim 4, which includes:
(a) preparing a solution of the base addition salt of HD-iGlu-D-Trp-OH in an aqueous medium substantially free of an organic solvent;
(b) adjusting the pH to values from about 2.0 to about 3.2 with an inorganic acid solution to cause precipitation of HD-iGlu-D-Trp-OH;
(c) recovering precipitated HD-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in step (c) to obtain HD-iGlu-D-Trp-OH. 10. The method of claim 8, wherein the acid addition salt of step (a) is H-D-iGlu-D-Trp-OH hydrochloride. 11. The method according to claim 8 or 9, in which the pH in stage (b) is from about 2.5 to about 3.0. 12. The method according to claim 5, in which R ¹ represents benzyl, and R ² represents methyl. 13. The method according to claim 5, in which the inert solvent in step (i) is selected from the group consisting of methanol and methyl tert-butyl ether; and the metal hydroxide is selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide. 14. The method according to claim 9, in which in the compound of the formula Boc-D-Glu- (γ-D-Trp-OR ² ) -α-OR ¹ in stage (a) R ¹ selected from the group consisting of methyl and benzyl and R ^{2 is} selected from the group consisting of methyl and ethyl. 15. The method according to claim 9, in which the inert solvent in step (b) is selected from the group consisting of methanol and methyl tert-butyl ether. 16. The crystalline ammonium salt H-D-iGlu-D-Trp-OH (1: 1), characterized by peaks in a powder x-ray having the following 2θ values: 13.93, 20.02, 24.03 and 28.12. 17. The crystalline ammonium salt H-D-iGlu-D-Trp-OH (1: 1), characterized by a powder x-ray, which is shown in Fig.2. 18. Crystalline ammonium salt HD-iGlu-D-Trp-OH (1: 1), characterized by the following parameters of the powder X-ray diffraction pattern, presented in the values of interplanar spacings d, Bragg angle 2θ and relative intensity (expressed as a percentage relative to the most intense line) :
Angle, ° 2θ Value d, A Relative intensity [%] 9.29 9,517 4.1 12.19 7,258 4,5 13.93 6,354 76,2 15.17 5,837 27.4 16.49 5,371 9.8 17.18 5,157 3 18.56 4,778 31.6 18.88 4,696 10.5 20.02 4,431 one hundred 22.28 3,986 3 23.31 3,814 4.6 23.66 3,757 9.8 24.03 3,7 52.9 24.37 3,649 26.3 25.07 3,549 11,4 25.61 3,475 5,6 25.96 3.43 5 27.62 3,227 29.7 28.12 3.17 55.7 28.49 3,131 12,2 29.52 3,023 23.1 30.27 2,951 3,7 30.64 2,915 7.9 31.31 2,854 11.8 31.7 2,821 29.6 32.16 2,781 19.2 32.81 2,728 16 33.78 2,652 7.4 34.14 2,625 5.1 35.76 2,509 16 36.94 2,431 11.6 37.58 2,391 25.6 38.03 2,364 12.5 39.22 2,295 1.9 19. Amorphous ammonium salt of D-isoglutamyl-D-tryptophan (1: 1). 20. Amorphous ammonium salt of D-isoglutamyl-D-tryptophan (1: 1), characterized by a powder x-ray, which is shown in Fig.3. 21. A method of obtaining a monoammonium salt of HD-iGlu-D-Trp-OH, free of inorganic salts, comprising the following steps:
(a) preparing a solution of the acid addition salt of HD-iGlu-D-Trp-OH in an aqueous medium substantially free of an organic solvent;
(b) adjusting the pH to values from about 3.9 to about 8.6 with a metal hydroxide solution;
(c) treating the solution of step (b) with an ion exchange resin and eluting with water to exchange the metal ion from the salt in the solution to a hydrogen ion until the pH of the eluate is from about 5.7 to about 7.0;
(d) contacting the ion-exchange resin with an ammonia-based regenerating solution used to exchange ions therein for the target HD-iGlu-D-Trp-OH contained in the ion-exchange resin so that an eluate of the regenerating solution containing the HD- ammonium salt is formed iGlu-D-Trp-OH; and
(e) evaporating the solvent from the solution from step (d) to obtain a crude ammonium salt;
and further comprising the following steps:
(f) dissolving the ammonium salt of step (e) in water and slowly adding isopropanol so that a monoammonium salt precipitates; and
(g) vacuum drying the product from step (f) to obtain a crystalline form of the ammonium salt HD-iGlu-D-Trp-OH (1: 1); or
(h) treating the material from step (e) with silica gel chromatography using a solution of isopropanol and ammonia as an eluent, and removing isopropanol by evaporation of the solvent or dissolving the product from step (g) in water to obtain a solution of the ammonium salt HD-iGlu-D- Trp-OH (1: 1) in water; and
(i) freeze drying the product from step (h) to obtain an amorphous form of the ammonium salt HD-iGlu-D-Trp-OH (1: 1). 22. The method according to item 21, in which at least one metal hydroxide solution selected from a solution of sodium hydroxide, a solution of lithium hydroxide and a solution of potassium hydroxide is used. 23. The method according to item 21 or 22, in which the acid addition salt is HD-iGlu-D-Trp-OH hydrochloride obtained by a method comprising the following steps:
(i) basic hydrolysis of a compound of formula I:

where R ¹ selected from the group consisting of C ₁ -C ₄ alkyl and benzyl, and R ² represents C ₁ -C ₄ alkyl, provided that C ₄ alkyl is not tert-butyl,
using metal hydroxide in water and an inert solvent in the presence of methanol to give Boc-D-iGlu-D-Trp-OH free of other diastereomers; and
(ii) deprotecting with Boc-D-iGlu-D-Trp-OH from step (i) in an inert organic solvent; and evaporating the solvent to obtain HD-iGlu-D-Trp-OH hydrochloride. 24. The method according to item 21 or 22, in which the acid addition salt is HD-iGlu-D-Trp-OH hydrochloride, and the acid addition salt solution in step (a) is prepared by a process comprising:
(i) hydrogenation of a compound of formula II

where R ¹ represents benzyl, and R ² selected from the group consisting of benzyl and hydrogen, using palladium on carbon in methanol or ethanol;
(ii) purification of the crude HD-iGlu-D-Trp-OH from step (i) by silica gel chromatography using isopropanol and water as an eluent; and
(iii) treating the material from step (ii) with hydrochloric acid in water to obtain a solution of the HD-iGlu-D-Trp-OH hydrochloride salt in water. 25. The method according to item 23, in which R ¹ represents benzyl, and R ² represents methyl. 26. The method according to item 23, in which the inert solvent in stage (i) is selected from the group consisting of methanol and methyl tert-butyl ether; and the metal hydroxide is selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide. 27. The method according to item 21 or 22, in which the acid addition salt in step (a) is HD-iGlu-D-Trp-OH hydrochloride, the pH value corresponding to the predominant form of the monovalent salt in step (b) is from from about 5.7 to about 7.0, and the ion exchange resin in step (c) is AMBERLYST®15. 28. A method of obtaining a mono-ammonium salt of HD-iGlu-D-Trp-OH from crystalline HD-iGlu-D-Trp-OH, free of inorganic salts, comprising the following steps:
(a) adding crystalline HD-iGlu-D-Trp-OH to less than about one equivalent of an ammonium hydroxide solution;
(b) adjusting the pH to values in the range of from about 6 to about 7 with ammonium hydroxide;
(c) evaporating the solvent to obtain an oil; adding isopropanol with stirring to precipitate the monoammonium salt;
(d) recovering precipitated ammonium salt HD-iGlu-D-Trp-OH; and
(e) vacuum drying the product obtained in step (c) to obtain a monoammonium salt of HD-iGlu-D-Trp-OH. 29. The method of separation of crystalline HD-iGlu-D-Trp-OH from an aqueous solution of HD-iGlu-D-Trp-OH using a graphical composition to determine the percentage of HD-iGlu-D-Trp-OH against pH, where the percentage forms of HD-iGlu-D-Trp-OH in solution is more than about 50% in solution with a pH of from about 2.0 to about 3.2, which leads to the precipitation of crystalline HD-iGlu-D-Trp-OH. 30. Compound HD-Glu- (γ-D-Trp-OR ² ) -α-OR ¹

and its pharmaceutically acceptable acid addition salts, wherein each of R ¹ and R ^{2 is} independently selected from the group consisting of benzyl and C ₁ -C ₄ alkyl. 31. The compound of claim 30, wherein the C ₁ -C ₄ alkyl is selected from the group consisting of methyl, ethyl, isopropyl, and tert-butyl. 32. The compound according to item 30 or 31, in which R ¹ has the same meaning as R ² . 33. The compound of claim 32, wherein R ¹ and R ² are methyl. 34. The compound of claim 32, wherein R ¹ and R ² are ethyl. 35. The compound of claim 32, wherein R ¹ and R ² are isopropyl. 36. The compound of claim 32, wherein R ¹ and R ² are benzyl. 37. The compound of claim 32, wherein R ¹ and R ² are tert-butyl. 38. The compound of claim 30 or 31, wherein R ¹ is ethyl and R ² is methyl. 39. The compound of claim 30 or 31, wherein R ¹ is ethyl and R ² is isopropyl. 40. The compound of claim 30 or 31, wherein R ¹ is tert-butyl and R ² is isopropyl. 41. The compound of claim 30 or 31, wherein R ¹ is benzyl and R ² is ethyl. 42. The compound of claim 30 or 31, wherein R ¹ is benzyl or methyl and R ² is ethyl, isopropyl or tert-butyl. 43. The compound of claim 30 or 31, wherein R ¹ is benzyl and R ² is methyl. 44. A pharmaceutical composition that is an immunosuppressant or anti-psoriasis agent, comprising a therapeutically effective amount of a compound according to any one of claims 30 to 43, at least one pharmaceutically acceptable excipient. 45. The use of a therapeutically effective amount of a compound according to any one of claims 30 to 43 as an immunosuppressant or anti-psoriasis agent. 46. A pharmaceutical composition that is an immunosuppressant or anti-psoriasis agent, comprising a therapeutically effective amount of an amorphous monoammonium salt according to claim 19 or 20 and at least one pharmaceutically acceptable excipient. 47. A solid pharmaceutical composition that is an immunosuppressant or anti-psoriasis agent, comprising a therapeutically effective amount of a crystalline salt according to any one of claims 16-18 and at least one pharmaceutically acceptable excipient. 48. The use of an amorphous monoammonium salt according to claim 19 or 20 as an immunosuppressant or anti-psoriasis agent. 49. The use of a crystalline salt according to any one of claims 16-18 as an immunosuppressant or anti-psoriasis agent. 50. A solid pharmaceutical composition that is an immunosuppressant or anti-psoriasis agent, comprising a therapeutically effective amount of a crystalline salt of a compound according to any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient. 51. The use of the crystalline compound according to any one of claims 1 to 3 as an immunosuppressant or anti-psoriasis agent.

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