HK1189000A - Crystalline d-isoglutamyl-d-tryptophan and the mono ammonium salt of d-isoglutamyl- d-tryptophan - Google Patents

Abstract

AMMONIUM SALT OF D-ISOGLUTAMYL- D-TRYPTOPHAN A process for making pure crystalline D-isoglutamyl-D-tryptophan is provided which includes the step of deprotecting essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan or its diester to yield essentially pure D-isoglutamyl-D- tryptophan. A process is also provided for the preparation of pure mono ammonium salt of D-isoglutamyl-D-tryptophan from essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan. D-isoglutamyl-D-tryptophan, ammonium salt (1 :1) is a stable pharmaceutical solid.

Description

Crystalline D-isoglutamyl-D-tryptophan and the mono ammonium salt of D-isoglutamyl-D-tryptophan

Technical Field

The present invention relates to a novel stable crystalline form of D-isoglutamyl-D-tryptophan and a process for the isolation of its pure form (free of inorganic salts). The invention also relates to a novel stable ammonium salt of D-isoglutamyl-D-tryptophan and a process for its production in pure form by recrystallization and/or conventional silica gel column chromatography.

Background

D-isoglutamyl-D-tryptophan (also known as H-D-iGlu-Trp-OH or Thymodepressin) is a synthetic blood regulatory dipeptide developed for the treatment of autoimmune diseases, including psoriasis (Sapuntsova, S.G., et al (May 2002), Bulletin of Experimental Biology and Medicine,133(5), 488-cott 490). In russia (US 5,736,519), Thymodepressin was considered an effective treatment for psoriasis, where the drug was then sold as the disodium salt in liquid formulations for injection and intranasal administration. It is an immunosuppressant and selectively inhibits the proliferation of bone marrow cells and thereby induces immunosuppression.

The solid form of the known D-isoglutamyl-D-tryptophan disodium salt is an amorphous powder which is hygroscopic and difficult to handle. The structure of the disodium salt of Thymodepressin is described in Kashirin, D.M., et al (2000), Pharmaceutical Chemistry Journal,34(11), 619-. The monosodium salt of D-isoglutamyl-D-tryptophan was identified by the chemical Abstract service agency (CAS) registry system and listed in CASREGISTRY^SMIn folders, but there is no disclosure concerning its preparation and physical properties. Powdered or amorphous forms of a compound intended for pharmaceutical use (e.g., D-isoglutamyl-D-tryptophan) can cause manufacturing problems due to bulk density problems, hygroscopicity, and variable water content that cannot be corrected by vacuum drying. D-isoglutamyl-D-tryptophan is a dipeptide and is not recommended to be used under vacuumThe amorphous form is dried at elevated temperature (e.g., 80 to 100 ℃).

The only synthesis of H-D-iGlu-D-Trp-OH reported in the literature is disclosed in U.S. Pat. No. 5,736,519. The cyclic anhydride (1.2) is given by reacting 1, 3-Dicyclohexylcarbodiimide (DCC) with Boc-D-Glu-OH (1.1) according to the method reported in example 1 of U.S. Pat. No. 5,736,519, which is described herein as scheme 1. When Dicyclohexylurea (DCU) was removed by filtration, H-D-Trp-OH was reacted with the anhydride (1.2) to give a mixture of the dipeptides Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4). The yield of combined crude Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) was 70%. However, this mixture contained only not more than 35% of the desired intermediate Boc-D-iGlu-D-Trp-OH (1.3). The Boc protecting group was removed by stirring a solution of (1.3) and (1.4) in formic acid as solvent for one hour at 40 ℃. The ratio of (1.3) and (1.4) to formic acid was about 1g:8mL (weight/volume). The product was 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) were present in the same amount, purification required ion exchange chromatography using pyridine acetate buffer. The yield of the desired product H-D-iGlu-D-Trp-OH (1.5) was 35% of Boc-D-iGlu-D-Trp-OH (1.3). Thus, the total yield of H-D-iGlu-D-Trp-OH from Boc-D-Glu-OH (1.5) was 12.25%.

Scheme 1: synthesis of H-D-iGlu-D-Trp-OH as described in U.S. Pat. No. 5,736,519

The process described in US5,736,519 has several disadvantages:

1. DCC in step 1A may lead to other by-products, for example

Byproducts from DCC coupling peptides have been reported in Marder, O., and Albericio, F. (June 2003), Chemical Oggi (Chemistry Today), 6-32.

Deprotection of Boc-D-iGlu-D-Trp-OH (1.3) requires elevated temperatures and final purification of H-D-iGlu-D-Trp-OH requires the use of a very toxic solvent, pyridine. The elevated temperature in the deprotection of (1.3) may result in the N-tert-butylindole derivative (1.7) as an impurity: (1.7)M., et al, (1978), Hoppe-Seyler's Z.Physiol.chem.,359(12): 1643-51). In addition, the peptide may be cyclized to give glutarimide (1.8) (Pandit, U.K. (1989), Pure&Appl.Chem.,Vol.61,No.3,pp.423-426）。

3. This coupling reaction produced only a 1:1 mixture of Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4). In the coupling step 1B, the maximum yield of (1.3) cannot exceed 50%. At the end of the synthesis, a mixture of D-Glu-D-Trp-OH and D-iGlu-D-Trp-OH was formed. These peptides must be separated by ion exchange chromatography and reverse phase preparative high pressure liquid chromatography. The overall yield of H-D-iGlu-D-Trp-OH (1.5) is 12.25%, and preparative HPLC purification is very time consuming and inefficient. The retention times of two similar isomers H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6) have not been reported. Repeated multiple separation cycles to enrich the purity of the desired isomer (1.5) are very inefficient. This method is not suitable for large-scale production.

4. The opposite diastereomer L-isoglutamyl-L-tryptophan (also known as H-L-iGlu-L-Trp-OH or Bestim) is an immunopotentiator (see US5,774,452). Bestim has been used for the treatment of ulcers. It reduces the inflammatory effects in the gastric and duodenal mucosa and promotes the regression of clinical symptoms, and scarring of ulcers (tkachova, a., et al (2004), Eksp Klin Gastroenterol (6):29-33,163). The synthesis of H-L-iGlu-L-Trp-OH, the monosodium salt (1: 1) is described in scheme 2 (U.S. Pat. No. 5,744,452).

Scheme 2: L-iGlu-L-Trp-O-]Na⁺Synthesis of (2)

In the US5,744,452 scheme, step 1 produces dicyclohexylurea as a by-product and must be removed in filtration. In the second step, trifluoroacetic acid is said to have removed (2.2) of the gamma-O-benzyl ester of glutamic acid units. The benzyl ester in (2.3) was removed by transfer hydrogenation using ammonium formate in isopropanol, palladium catalyst, sodium bicarbonate at elevated temperature to give H-L-iGlu-L-Trp-OH monosodium salt (2.4). The solid phase synthesis of (2.4) is also reported in the same patent, but the tryptophan moiety must be protected as a carboxamide and subsequently deprotected. Other diastereomers L-isoglutamyl-D-tryptophan and D-isoglutamyl-L-tryptophan are also known compounds (US 5,916,878).

The synthesis of H-D-iGlu-L-Trp-OH and H-L-iGlu-D-Trp-OH is reported in scheme 3 and scheme 4, respectively (US 5,916,878).

Scheme 3: synthesis of H-D-isoglutamyl-D-tryptophan as described in US5,916,878

Scheme 4: synthesis of H-L-isoglutamyl-D-tryptophan as described in US5,916,878

The methods reported in schemes 2, 3, and 4 may have overcome the regiospecific synthesis of the γ amide products (2.2), (3.2), and (4.2), without the formation of the α amide product, but they involve the removal of the hydrogenation step in the benzyl esters of compounds (2.2), (3.2), and (4.2). This requires the use of large amounts of palladium catalyst. The second concern is the partial reduction of the indole ring on a production scale. A third concern is the formation of glutarimide, 2- (3-amino-2, 6-dioxo-piperidin-1-yl) -3- (1H-indol-3-yl) -propionic acid during hydrogenation. A fourth concern is cost. The CBz-Glu-OBzl derivatives such as (3.1) and (4.1) are almost twice as expensive as the corresponding Boc-Glu-OBzl in the production of fine chemicals. The methods in schemes 3 and 4 require HPLC purification of the final product. The overall yields were 33% and 35.9%, respectively. Scheme 2 requires the use of trifluoroacetic acid, which introduces other impurities into the reaction. Furthermore, the method in scheme 2 uses dicyclohexylcarbodiimide as a peptide coupling agent. The removal of trace impurities from this reagent is a serious problem in chemical production. Therefore, this technique is not suitable for industrial production, and cannot be used for large-scale production of H-D-isoglutamyl-D-tryptophan.

Disclosure of Invention

The present invention relates to a novel stable crystalline form of D-isoglutamyl-D-tryptophan and a process for isolating said compound in pure form (free of inorganic salts) by precipitation from water without the use of reverse phase preparative high pressure liquid chromatography. A process has been reported for the preparation of pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan and its diesters, free of N-tert-butoxycarbonyl-D-glutamyl-D-tryptophan, and the conversion of N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan and its diesters to pure crystalline D-isoglutamyl-D-tryptophan. The novel crystalline D-isoglutamyl-D-tryptophan of the present invention is readily purified. When compared to the prior art methods described above, the present invention provides a number of advantages:

first, D-isoglutamyl-D-tryptophan is prepared in crystalline form without preparative high pressure liquid chromatography.

Second, key intermediates, Boc-D-iGlu-D-Trp-OH or H-D-Glu- (γ -D-Trp-OMe) - α -OBzl HCl salts, were prepared in high yield and high purity.

Third, a process for converting Boc-D-iGlu-D-Trp-OH or its diester or H-D-Glu- (γ -D-Trp-OMe) - α -OBzl HCl salt to D-isoglutamyl-D-tryptophan in high yield and purity is provided.

Fourth, the pure crystalline form of D-isoglutamyl-D-tryptophan of the present invention is not known in the prior art. It can be used directly in liquid formulation for pH adjustment, thus eliminating the need for the use of the extremely hygroscopic and unstable disodium salt of D-isoglutamyl-D-tryptophan.

The invention also relates to a novel stable ammonium salt of D-isoglutamyl-D-tryptophan and to a process for producing the ammonium salt from N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan and isolating this compound in pure form by crystallization and/or conventional silica gel column chromatography.

The monoamine salt of D-isoglutamyl-D-tryptophan is a stable solid and is easy to dispense for formulation purposes. A morphometric profile is provided to identify the species of salt forms at different pH.

The novel process for the production of D-isoglutamyl-D-tryptophan and its D-isoglutamyl-D-tryptophan, ammonium salt (1: 1) circumvents the above-mentioned production problems and makes possible the recovery and disposal of thymodepressin and thymodepressin mono-ammonium salt in conventional chemical processing facilities.

It is an object of the present invention to provide a good process for the production of D-isoglutamyl-D-tryptophan, yielding a pharmaceutical material which is completely free of the other non-corresponding isomers discussed above and which provides the material with the ability to be stored in a stable form for a long period of time before it is formulated.

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

It is a further object of the present invention to provide a process for the preparation of pure D-isoglutamyl-D-tryptophan (H-D-iGlu-D-Trp-OH) from an acid addition salt of H-D-iGlu-D-Trp-OH which gives a product which is substantially or completely free of organic solvent residues and which does not require reverse phase high pressure liquid chromatography purification. Solid D-isoglutamyl-D-tryptophan was isolated from water.

It is a further object of the present invention to provide a process for the preparation of pure D-isoglutamyl-D-tryptophan (H-D-iGlu-D-Trp-OH) from a base addition salt of H-D-iGlu-D-Trp-OH which gives a product which is substantially or completely free of organic solvent residues and which does not require reverse phase high pressure liquid chromatography purification. The solid D-isoglutamyl-D-tryptophan is isolated from water.

It is a further object of the present invention to provide a process which produces D-isoglutamyl-D-tryptophan which is substantially free of inorganic salt contaminants.

It is yet another object of the present invention to produce crystalline D-isoglutamyl-D-tryptophan having an X-ray powder diffraction (XRPD) pattern as shown in figure 1.

It is a further object of the present invention to produce D-isoglutamyl-D-tryptophan monoammonium salt from an acid addition salt of D-isoglutamyl-D-tryptophan which produces a product which is substantially or completely free of organic solvent residues and which does not require reverse phase high pressure liquid chromatography purification. After treatment with ion exchange resin to remove inorganic salts, the solid D-isoglutamyl-D-tryptophan ammonium salt is isolated from isopropanol and ammonia.

It is a further object of the present invention to produce D-isoglutamyl-D-tryptophan monoammonium salt with an XRPD pattern as shown in figure 2.

It is a further object of the present invention to produce an amorphous D-isoglutamyl-D-tryptophan monoammonium salt characterized by a fourier infrared (FTIR) spectrum substantially as shown in figure 5.

It is a further object of the present invention to provide a process which produces a D-isoglutamyl-D-tryptophan monoammonium salt which is substantially free of inorganic salt contaminants.

It is a further object of the present invention to provide a process for the production of acid addition salts, especially hydrochloride salts, of H-D-iGlu-D-Trp-OH from pure Boc-D-iGlu-D-Trp-OH.

It is a further object of the present invention to provide a process for the production of the pure dipeptide Boc-D-iGlu-D-Trp-OH without chromatographic separation.

It is still another object of the present invention to provide a simple silica gel column chromatographic separation process for purifying D-isoglutamyl-D-tryptophan and its mono-ammonium salt.

It is a further object of the present invention to provide a morphometric profile to determine the pH range for the isolation of D-isoglutamyl-D-tryptophan and its single monovalent salt. The preferred pH range for the precipitation of D-isoglutamyl-D-tryptophan in water is from about 2.5 to about 3.0.

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

wherein R is¹Is selected from the group consisting of₁-C₄Alkyl and benzyl, and R²Is C₁-C₄Alkyl radical with the proviso that C₄The alkyl group is not a tertiary butyl group,

the metal hydroxide in water was used as well as an inert solvent in the presence of methanol to give Boc-D-iGlu-D-Trp-OH without other diastereomers. The metal hydroxide is selected from the group consisting of: lithium hydroxide, sodium hydroxide, and potassium hydroxide.

The compound of formula I is coupled in turn from the peptide to Boc-D-Glu (OH) -OR¹andD-Trp-OR²In which R is¹And R²Peptide coupling reagents, such as HOBt and EDC, are used, as defined above. This method of synthesizing Boc-D-iGlu-D-Trp-OH provides significant advantages over the prior art in U.S. Pat. No. 5,736,519 because the product does not contain the gamma peptide product Boc-D-iGlu-D-Trp-OH, and because Boc-D-Glu (OH) -OR is used¹Therefore, the alpha peptide product Boc-D-Glu-D-Trp-OH cannot be formed during synthesis.

Acid deprotection of the pure dipeptide Boc-D-iGlu-D-Trp-OH using, for example, hydrochloric acid, trifluoroacetic acid, provides an acid addition salt. The solution of the acid addition salt is adjusted to a pH of about 2.5 to 3.0 to obtain thymodepressin as a solid precipitate.

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

Preparation of a solution of a base addition salt of D-isoglutamyl-D-tryptophan by acid deprotection, in particular HCl deprotection, of a compound of formula I wherein R is¹And R²Each independently selected from the group consisting of: c₁-C₄Alkyl and benzyl to give the diester H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹Followed by treatment with a metal hydroxide in water and an inert solvent in the presence of methanol to give a base addition salt of H-D-iGlu-D-Trp-OH. The metal hydroxide is selected from the group consisting of: sodium hydroxide, lithium hydroxide, and potassium hydroxide. Extraction with a water immiscible solvent to remove organic impurities into the organic phase and separation of the aqueous phase and adjustment of the aqueous phase to a pH of about 6 to about 7 with a metal hydroxide. After evaporation of the solvent, reducing the amount of solvent to the expected ratio of solute to solvent at a ratio of less than about 1:8, where the solute is the peptide D-isoglutamyl-D-tryptophan in base addition salt form, the solution of this base addition salt is adjusted to a pH of about 2.5 to 3.0 with a mineral acid to effect precipitation of D-isoglutamyl-D-tryptophan.

While it is anticipated that the scope and operation of the present invention should not be limited by theory or by its manner of operation and its possible interpretation in any way, it is believed that the morphometric analysis of FIGS. 7 and 8 at a pH of about 2.5 to 3.0 shows that the main species of thymodepressin is the free peptide (H-D-iGlu-D-Trp-OH), rather than the monovalent salt. Since H-D-iGlu-D-Trp-OH is not very soluble in anhydrous organic solvents (solubility in water <23 mg/mL), the compound precipitates out in pure form. The XRPD pattern of this material is shown in figure 1.

In the production of the mono ammonium salt, a solution of thymodepressin is purified by ion exchange at a pH of about 6.0 to about 8.0 to remove the salt. After recrystallization from isopropanol and water, the ammonia-based regenerant solution yielded a pure mono-ammonium salt (mono ammonium salt). Although it may be speculated that the mono-ammonium salt is unstable and may in fact revert to the free dipeptide, in practice the compound is stable for more than two years. The applicant has invented the mono ammonium salt of thymodepressin, which is a stable novel chemical entity that can be easily recrystallized from isopropanol as well as water. The properties of the obtained crystalline material are shown in fig. 2.

The above process yields pure thymodepressin and mono ammonium salt without reverse phase HPLC on an industrial scale, but thymodepressin and mono ammonium salt can be purified by conventional silica gel column chromatography using the conditions described above. Thus, without removing any thymodepressin from the mother liquor from the recrystallization, the filtrate can be concentrated and further purified by silica gel column chromatography (if so desired).

Brief description of the drawings

The crystalline salts of the present invention are illustrated in the following examples.

Figure 1 is a characteristic XRPD pattern of crystalline D-isoglutamyl-D-tryptophan. The XRPD pattern may also be represented by: interplanar spacing d, bragg angle 2 θ, and relative intensity (expressed as a percentage relative to the strongest ray):

by normal pre-filling techniquesPowdered samples were prepared and run on a D8Discovery Diffractometer system operating with a Cu-. kappa.source at 45kV/45 mA. The system is equipped with a 2D-proportional area detector (GADDS). Experimental data was collected on two frames covering a range of 3 ° to 35 ° (2 θ) with 600s exposure per frame. The obtained 2D diffraction images are then integrated to obtain a standard, I and 2 theta, diffraction pattern. Data is processed by various Bruker AXS data processing software, including Eva^TM8.0 and Topas^TMv.2.1 (for feature suitability analysis and applications, when necessary)

Figure 2 is a characteristic XRPD pattern of the crystalline mono-ammonium salt of D-isoglutamyl-D-tryptophan. The XRPD pattern may also be represented by: interplanar spacing d, bragg angle 2 θ, and relative intensity (expressed as a percentage relative to the strongest ray):

the X-ray powder diffraction spectra of D-isoglutamyl-D-tryptophan and its ammonium salt are shown in fig. 1 and fig. 2 below. It is said that the 2 θ values of the X-ray powder diffraction patterns may vary slightly from one machine to another or from one sample to another, and so the values quoted are not to be understood as absolute.

Figure 3 is a characteristic XRPD pattern of the amorphous form of D-isoglutamyl-D-tryptophan.

FIG. 4 is a characteristic Infrared (IR) absorption spectrum of the crystalline mono-ammonium salt of D-isoglutamyl-D-tryptophan.

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

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

FIG. 7 is a graph of pKa using estimates and the software Hyperquad Simulation&Specification analysis of the formation of thymodepressin shows the formation analysis calculations for the dipeptide H-D-iGlu-D-Trp-OH and its salts using the predicted pKa of the acid as well as the amine group. LH₂Is the diacid form of the peptide H-D-iGlu-D-Trp-OH, and LH is the monocarboxylate of H-D-iGlu-D-Trp-OH. An example of this is the mono ammonium salt. L refers to the diacid salt form, and one such example is the disodium salt of the peptide H-D-iGlu-D-Trp-OH.

FIG. 8 is a graph of pKa's estimated experimentally using the software Hyperquad Simulation&Specification analysis of the formation of thymodepressin shows the analytical calculation of the formation of the dipeptide H-D-iGlu-D-Trp-OH and its salts using an acid and an amine group, an experimentally determined pKa. LH₂Is the diacid form of the peptide H-D-iGlu-D-Trp-OH, and LH is the monocarboxylate of H-D-iGlu-D-Trp-OH. An example of this is the mono ammonium salt. L refers to the diacid salt form, and one such example is the disodium salt of the peptide H-D-iGlu-D-Trp-OH.

Detailed description of the invention

The term "Boc-D-Glu (OH) -OR" as used herein¹"refers to the structure:

when R is¹When it is benzyl, it is the chemical 2-tert-butoxycarbonylamino-D-glutamic acid alpha-benzyl ester.

The term "D-Trp-OR" as used herein²"means the followingThe structure is as follows:

when R is²When methyl, the compound is D-tryptophan methyl ester.

As used herein, the term "Boc-D-Glu- (γ -D-Trp-OR)²)-α-OR¹"refers to the structure:

the term "H-D-Glu- (gamma-D-Trp-OR) as used herein²)-α-OR¹"refers to the structure:

when R is¹Is benzyl, R²Is methyl, the compound is

When R is¹Is methyl, R²Is benzyl, the compound is

The term "thymodepressin" as used herein refers to the dipeptide H-D-iGlu-D-Trp-OH, having the chemical structure:

it can also be written as H-D-Glu- (γ -D-Trp-OH) -OH.

An acid addition salt is a salt formed after the reaction of an amine of H-D-iGlu-D-Trp-OH with a variety of inorganic acids including hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or with a variety of 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, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid. It can also be formed from the acid deprotection of Boc-D-iGlu-D-Trp-OH derivatives.

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

The present invention is directed to a process for the production of H-D-iGlu-D-Trp-OH and its ammonium salts (free of inorganic salts) from the acid addition salt of H-D-iGlu-D-Trp-OH, preferably from the Boc-D-iGlu-D-Trp-OH peptide. From Boc-D-Glu (OH) -OR¹And D-Trp-OR²The preparation of Boc-D-iGlu-D-Trp-OH in which R is¹Is selected from the group consisting of: benzyl and C₁-C₄Alkyl and R²Is C₁-C₄Alkyl radical with the proviso that C₄The alkyl group is not a tert-butyl group.

The invention also relates to a method for producing H-D-iGlu-D-Trp-OH from a solution of a base addition salt of H-D-iGlu-D-Trp-OH, preferably from the dipeptide H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹The acid addition salt of (a) is prepared from a solution of the H-D-iGlu-D-Trp-OH base addition salt, wherein R is¹And R²Each independently selected from the group consisting of: benzyl and C₁-C₄An alkyl group.

Preferred embodiments

In several aspects of the invention (which are given in the summary of the invention), the order of the method steps and their relative preference are described as follows:

in one embodiment of the present invention, there is provided an aqueous phase process for the preparation of H-D-iGlu-D-Trp-OH (without inorganic salts) comprising:

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

(b) the pH is adjusted to a pH dominated by the diacid form with an alkali metal hydroxide solution or a mineral acid solution to cause precipitation of H-D-iGlu-D-Trp-OH.

(c) Recovering the precipitated H-D-iGlu-D-Trp-OH; and is

(d) Vacuum drying the product from step (c) to give H-D-iGlu-D-Trp-OH.

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

In another embodiment of the invention, there is provided crystalline H-D-iGlu-D-Trp-OH, which is D-isoglutamyl-D-tryptophan, characterized by the XRPD pattern as shown in figure 1.

In another embodiment of the present invention, there is provided a process for preparing a mono-ammonium salt of H-D-iGlu-D-Trp-OH free of inorganic salts, comprising the steps of:

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

(b) the pH is adjusted to a pH dominated by the monovalent salt with a metal hydroxide solution.

(c) Subjecting the solution from step (b) to an ion exchange resin and elution with water to exchange metal ions from the salts in the solution for hydrogen ions 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 regenerant solution operative to exchange ions therein with the H-D-iGlu-D-Trp-OH of interest contained in the ion exchange resin, thereby forming a regenerant eluate comprising the ammonium salt of H-D-iGlu-D-Trp-OH; and is

(e) Evaporating the solvent from the solution of step (d) to give the crude ammonium salt;

(f) dissolving the ammonium salt from step (e) in water and slowly adding isopropanol such that a precipitate of the mono-ammonium salt is formed; and is

(g) The product from step (f) was dried in vacuo to give H-D-iGlu-D-Trp-OH, a crystalline form of the ammonium salt (1: 1).

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

(h) subjecting the material from step (e) to silica gel chromatography with isopropanol and ammonia solution as eluent; and is

(i) The product from step (H) was freeze-dried to give the amorphous form of H-D-iGlu-D-Trp-OH, ammonium salt (1: 1).

In another embodiment of the present invention, there is provided a process for preparing the mono-ammonium salt of H-D-iGlu-D-Trp-OH from crystalline H-D-iGlu-D-Trp-OH containing no inorganic salts, the process comprising the steps of:

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

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

(c) evaporating the solvent to give an oil; adding isopropanol and stirring to cause precipitation of the mono ammonium salt;

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

(e) Vacuum drying the product from step (c) to give the H-D-iGlu-D-Trp-OH monoammonium salt.

In another embodiment of the invention, there is provided crystalline H-D-iGlu-D-Trp-OH, ammonium salt (1: 1), characterized by the XRPD pattern expressed in the figure legends.

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

In another embodiment of the present invention, there is provided amorphous H-D-iGlu-D-Trp-OH, ammonium salt (1: 1), characterized by FTIR (infrared) spectra as shown in FIG. 5.

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

(i) basic hydrolysis of compounds of formula I:

wherein R is¹Is selected from the group consisting of₁-C₄Alkyl and benzyl, and R²Is C₁-C₄Alkyl radical with the proviso that C₄The alkyl group is not tert-butyl, and metal hydroxide in water and an inert solvent in the presence of methanol are used to give Boc-D-iGlu-D-Trp-OH without other diastereomers.

(ii) (ii) deprotecting the Boc-D-iGlu-D-Trp-OH from step (i) in an inert organic solvent with hydrogen chloride; and the solvent was evaporated to give the hydrochloride salt of H-D-iGlu-D-Trp-OH.

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

(a) hydrogenation of the Compound of formula II

Wherein R is¹Is benzyl and R²Is selected from the group consisting of: a benzyl group and hydrogen,

using palladium on charcoal in methanol or ethanol;

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

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

In both of the above processes, the preparation of the acid addition salt of D-isoglutamyl-D-tryptophan from the compound of formula I is preferred over the acid addition salt of D-isoglutamyl-D-tryptophan from the compound of formula II due to the cost of chemical intermediates.

In another embodiment of the present invention, there is provided a process for preparing a solution of a base addition salt of H-D-iGlu-D-Trp-OH, wherein the process comprises:

(a) dipeptide N-Boc-D-Glu- (gamma-D-Trp-OR)²)-a-OR¹Deprotection of the acid of (1), wherein R¹And R2 are each independently selected from the group consisting of: c₁-C₄Alkyl and benzyl;

(b) alkaline 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 water-insoluble solvent and separating the aqueous layer;

(d) adjusting the pH of the aqueous phase from step (c) to from about 6 to about 7; and is

(e) Evaporating the solvent of the solution from step (D) to produce a solution comprising an estimated ratio of about one part solute to less than about 8 parts water, wherein the solute is a base addition salt of D-isoglutamyl-D-tryptophan.

In another embodiment of the present invention, there is provided a novel dipeptide derivative H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹Hydrochloride of the general formula (I), wherein R¹And R²Each independently selected from the group consisting of: benzyl and C₁-C₄An alkyl group.

Compound H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹Are not known in the prior art. These compounds can be used as intermediates in the preparation of the dipeptide D-isoglutamyl-D-tryptophan. Alternatively, H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹The hydrochloride may be used in pharmaceutical formulations wherein hydrolysis of the ester occurs in situ in preparation for formulation to give D-isoglutamyl-D-tryptophan.

In one embodiment of the invention, a morphometric profile is provided, shown in FIG. 8, for the isolation of H-D-iGlu-D-Trp-OH at a pH of about 2.5 to about 3.0.

Preparation method

As illustrated in scheme 5 below, the present invention provides a reliable methodology for the high-yield synthesis of pure N- (tert-butoxycarbonyl) -D-isoglutamyl-D-tryptophan which is not present in the prior art (e.g., US5,736,519).

Scheme 5

In the method of the present invention, Boc-D-Glu (OH) -OR¹In an inert solvent (wherein in the inert solvent, R¹Is benzyl) reacts with N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), hydroxybenzotriazole (HOBt) and Diisopropylethylamine (DIPEA). The preferred temperature is from about 5 ℃ to about-5 ℃, and the preferred solvent is dichloromethane. After mixing for about 5 minutes to about 30 minutes, preferably about 15 minutes, D-Trp-OR is added dropwise²(wherein R is²Methyl) with Diisopropylethylamine (DIPEA). The resulting solution is stirred at ice-cold temperature, preferably from about-5 ℃ to about 5 ℃, for 1 hour and then at room temperature for about 12 hours to about 20 hours, preferably about 16 hours. The product Boc-D-iGlu- (D-Trp-OR) was purified by conventional means²)-α-OR¹Separation is carried out. The compound can be easily crystallized from ethyl acetate and hexane. Two synthetic impurities present in trace amounts are compounds (a) and (B), which can be removed by recrystallization. Both compounds are believed to be derived from the reagent HOBt.

Diester Boc-D-Glu- (gamma-D-Trp-OR) in ethanol²)-α-OR¹Mixing with sodium hydroxide solution. The preferred amount of sodium hydroxide is from about 2.5 to about 5 equivalents per equivalent of diester. Preferably, a molar ratio of about 2 moles to about 3.5 moles of NaOH per mole of the diester compound is used. The preferred solvent ratio is about 2mL of ethanol per mL of water, and the preferred NaOH to water ratio is about 1g to 20 mL. This separation procedure involves extraction of the reaction mixture with ethyl acetate, thus removing any organic impurities at this stage of the synthesis. When the acidification is performed, the aqueous portion is extracted with an organic solvent (e.g., ethyl acetate). The diester N- (tert-butoxycarbonyl) -D-isoglutamyl-D-tryptophan (Boc-D-iGlu-D-Trp-OH) was isolated as a solid by conventional means. The isolated yield of the combination of the two steps was 89% yield. This is an advantage over the prior art procedure (US 5,736,519). The novel method of the present invention is further shown in the following examples.

Through the detailed studies and monitoring of the hydrolysis step by the applicant, it was shown that methanol first reacts with the compound Boc-D-iGlu- (D-Trp-OR)²)-OR¹(wherein R is¹Is benzyl and R²Is methyl) to give Boc-D-iGlu- (D-Trp-OMe) -OMe, and then the compound is hydrolyzed to the diacid. The applicants have determined that methanol is a necessary condition for rapid hydrolysis of the alpha benzyl ester. In view of the large volume of ethyl acetate required to extract the product, applicants have invented a biphasic procedure for Boc-D-iGlu- (D-Trp-OR)²)-OR¹Efficient hydrolysis of (3). Boc-D-iGlu- (D-Trp-OR) in p-tert-butyl methyl ether (MTBE)²)-OR¹With a metal hydroxide solution, such as lithium hydroxide (lithum hydroxide) or sodium hydroxide solution. Metal hydroxide ratio Boc-D-iGlu- (D-Trp-OR)²)-OR¹Is between about 2.0 and 2.5 and 1.Methanol is added and the mixture is stirred vigorously for about 1 to about 6 hours, preferably from about 1.5 hours to about 2.5 hours. The mixture is separated from the organic phase by conventional means. This procedure excludes the use of large amounts of ethyl acetate for extraction and is shown in the examples below.

In the conventional method, the compound Boc-D-iGlu-D-Trp-OH was deprotected with an organic acid to give the dipeptide H-D-iGlu-D-Trp-OH, which required thorough purification. The prior art procedures (U.S. Pat. No. 5,736,519) suffer from a number of disadvantages, these procedures deprotecting a mixture of Boc-D-iGlu-D-Trp-OH and Boc-D-Glu-D-Trp-OH at 40 ℃ using formic acid to give a mixture of H-D-iGlu-D-Trp-OH and H-D-Glu-D-Trp-OH. Ion exchange chromatography and reverse phase HPLC were used to isolate the product. The recovery is low and the procedure is not suitable for large scale production. Deprotection of the N-tert-butoxycarbonyl group with trifluoroacetic acid or formic acid yields a tert-butyl carbonium ion which can react with the indole nitrogen to form the tert-butyl product: (M., et al, (1978), Hoppe-Seyler's Z.Physiol.chem.,359(12): 1643-51). Glutarimide formation (1.8) (Pandit, U.K. (1989), Pure&Appl.chem., vol.61, No.3, pp.423-426) is another concern.

The applicants have determined that the acid addition salts of the present invention, particularly the crude hydrochloride salt, can be readily prepared using HCl in an inert solvent such as ethyl acetate at low temperatures, preferably from about 0 ℃ to about room temperature. Evaporation of the solvent yields thymodepressin hydrochloride, which can be used to prepare thymodepressin.

In predicting the pH required for precipitation of thymodepressin its diacid form H-D-iGlu-D-Trp-OH, applicants theorized calculations on the morphometric plot and concluded that thymodepressin exists in the diacid form at a pH of about 2.5 to about 3. This inference led us to find a method for isolating thymodepressin without chromatography.

Fig. 7 illustrates such a calculation. In FIG. 7, LH₂Is a dicarboxylic acid form of the peptide H-D-iGlu-D-Trp-OH (i.e., thymodepressin), and LH is a monocarboxylate form of the peptide H-D-iGlu-D-Trp-OH; one such example is the mono-ammonium salt, L is the dicarboxylate form of the peptide H-D-iGlu-D-Trp-OH; one such example is the disodium salt, and LH₃Is an acid addition salt of thymodepressin. The X-axis provides the pH of the solution. The Y-axis provides% formation relative to L (the default term for the software) and reports the molar fraction of the species present at a particular pH. At a pH of about 2.5 to about 3.0, the majority (80%) of the dipeptide is present as a dicarboxylic acid and can precipitate out of solution if it is not soluble in water. Our studies show that the dicarboxylic acid form prepared by this method has an aqueous solubility of about 23 mg/mL. At a pH of about 7.0, 100% of the species is in the monocarboxylic acid form. If the counterion is sodium, then the species is thymodepressin monosodium.

In practice, when a solution of the hydrochloride salt of thymodepressin is dissolved in water and the pH is adjusted to about 3.0 with stirring, a solid slowly appears and is filtered from the mixture. The LC purity of the material is over 97% and falls within pharmaceutical grade purity as an active pharmaceutical ingredient. We determined that this method is superior to the prior art in producing and isolating pure thymodepressin (H-D-iGlu-D-Trp-OH). Ion exchange and reverse phase preparative column chromatography are not required because the only by-product is sodium chloride, which is soluble in water. This is a straightforward procedure for isolation and purification of thymodepressin. It is predicted using theoretical morphometric profiles and the method is superior to prior art procedures which require lengthy purification.

The pKa of the acid and amine groups of H-D-iGlu-D-Trp-OH are determined experimentally. FIG. 8 shows a morphological analysis of the dipeptide using pKAs determined experimentally. In FIG. 8, LH₂Is thymodepressin, LH is a monocarboxylate, L is a dicarboxylate, and LH₃Is an acid addition salt of thymodepressin. X-axis provides pH of solution. The Y-axis provides% formation relative to L (the default term for the software) and reports the molar fraction of the species present at a particular pH. A concentration of 0.5M was used to reflect the equivalent of 1gm thymodepressin in 6ml of water during separation. This figure shows that at pH 2.7, approximately 75% thymodepressin (LH)₂) In the form of dicarboxylic acids. For this reason, thymodepressin precipitates at pH 2.7 and can be filtered. The mother liquor can be concentrated to obtain a second batch of thymodepressin. This figure confirms the theoretical prediction that the diacid form of H-D-iGlu-D-Trp-OH predominates at a pH of about 2.5 to about 3.0, which is the pH used to isolate the diacid from water as a precipitate. Since only about 80% of the material will precipitate in pure form, the mother liquor should be reduced in volume and subjected to a second round of precipitation at a pH of 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 yields the trifluoroacetate salt. The inert solvent is dichloromethane, and often a 1:1 mixture of trifluoroacetic acid and dichloromethane is used. Solvent evaporation yielded an oil that was vacuum dried to remove residual solvent. The oil is dispersed in water. When the pH was adjusted to about 3.0, a white solid appeared after stirring continued for about 12 to 16 hours.

In the preparation of the acid addition salts, it is preferred to use HCl in an inert solvent to produce the hydrochloride salt. Alternatively, trifluoroacetate can be produced using the above process. The use of this hydrochloride salt as an acid addition salt is preferred because deprotection of Boc-D-iGlu-D-Trp-OH is more efficient using HCl in an inert solvent (e.g., 3M HCl in ethyl acetate). With trifluoroacetic acid, the reaction time is significantly longer. In addition, the trifluoroacetate salt of D-iGlu-D-Trp-OH contains several synthetic impurities that transfer to D-iGlu-D-Trp-OH when precipitated in water at a pH of about 2.5 to about 3.0. These impurities must be removed by thorough recrystallization.

Using this methodology, the starting material Boc-D-iGlu-D-Trp-OH was prepared as described earlier. The acid addition salt should be dried under vacuum to ensure that it is free of organic solvents and volatile impurities. During precipitation of thymodepressin in water, a solution of acid addition in water is prepared. The ratio of acid addition salt to water is in the range of about 1:5 to about 1: 10. But preferably the ratio of acid addition salts is in the range of about 1:6 to about 1: 8. A metal hydroxide solution, typically sodium hydroxide solution, is used to precipitate the product, but potassium hydroxide as well as other metal hydroxide solutions may be used.

Crude H-D-iGlu-D-Trp-OH can also be prepared by hydrogenation of the compound of formula II.

Wherein R is¹Is benzyl and R²Is selected from the group consisting of benzyl and hydrogen, using palladium on activated carbon in methanol or ethanol. After filtration of the catalyst, the filtrate was evaporated to an oil, which was further purified by silica gel chromatography using isopropanol and water as eluent. The H-D-iGlu-D-Trp-OH obtained can be converted into the H-D-iGlu-D-Trp-OH hydrochloride salt with hydrochloric acid in water.

By dipeptide Boc-D-Glu- (gamma-D-Trp-OR²)-α-OR¹Deprotection of the acid of (a) to prepare a solution of a base addition salt of D-isoglutamyl-D-tryptophan, wherein R is¹And R²Each independently selected from the group consisting of: benzyl and C₁-C₄An alkyl group. For example, Boc-D-Glu- (gamma-D-Trp-OR) in an inert solvent (e.g., dichloromethane)²)-α-OR¹Deprotection of HCl to give H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹The HCl salt of (1). For the combination, wherein R¹Is benzyl and R²Is methyl and the product is HCl. H-D-Glu- (gamma-D-Trp-OR)²)-α-OR¹Precipitating from dichloromethane and canCan be removed by filtration. Treating the acid addition salt with a metal hydroxide in an inert solution (e.g. methanol) for single phase homogeneous hydrolysis; or tert-butyl methyl ether for biphasic hydrolysis to give the base addition salt of H-D-iGlu-D-Trp-OH in solution. When the reaction mixture is extracted with a water-insoluble solvent (e.g., ethyl acetate or t-butyl methyl ether), the aqueous phase is neutralized to a pH of about 6 to about 7, and the solution is evaporated, reducing the volume to the desired ratio of less than about 1 part solute to 8 parts water. This solute, predicted by morphological calculations as shown in FIG. 8, is a base addition salt of H-D-iGlu-D-Trp-OH (in the monocarboxylate form). If sodium hydroxide is used as the metal hydroxide, the solute will be the mono-sodium form of H-D-iGlu-D-Trp-OH in water. Adjusting this solution to a pH of about 2.5 to about 3.0 results in precipitation of solid thymodepressin, H-D-iGlu-D-Trp-OH.

The mono-ammonium salt of thymodepressin can be directly prepared from Boc-D-iGlu-D-Trp-OH dipeptide. The crude acid addition salt (e.g. hydrochloride salt) prepared as described above is treated with an ion exchange resin to remove inorganic salts. Thus, a solution of crude thymodepressin hydrochloride was dissolved in water and the pH was adjusted to about 6 to about 8. The solution was treated with an ion exchange resin. The preferred resin is a sulfonic acid-based resin. An example of this is15. The inorganic salts are removed by washing with water until the pH is about 5.7 to about 7. Ammonia is used as a regenerant to recover the ammonium salt of thymodepressin from the resin. Concentrated ammonia and isopropanol are preferably used as regenerants. The preferred ratio is concentrated ammonia to isopropanol in a ratio of about 1 to about 3 to 4, with the final wash being performed using concentrated ammonia in a ratio of 1 part concentrated ammonia to 1 part water to 2 parts isopropanol. Ammonia wash (ammonia wash) was evaporated under reduced pressure to an oil which was crystallized from isopropanol and water to give the mono ammonium salt (m) as a white solidono ammonium salt). The preferred ratio of isopropanol to water for recrystallization is in the range of about 5:1 to about 10: 1. Column chromatography is not required.

No other methods for the purification of thymodepressin other than reverse phase preparative liquid chromatography have been reported in the prior art. This process is very time consuming and expensive and is not suitable for large scale production. The applicants have determined that crude thymodepressin can also be purified to pharmaceutical grade purity by flash silica gel chromatography using isopropanol and water as eluent. The preferred mobile phase is isopropanol to water in the range of about 10:1 to about 5: 1. The product is isolated by conventional means.

In a similar manner, the monoammonium salt can also be purified by flash silica chromatography using isopropanol and concentrated ammonia as eluent. The preferred mobile phase is isopropanol to ammonia in the range of about 10:1 to about 5: 1. The product is isolated by conventional means.

The D-isoglutamyl-D-tryptophan monoammonium salt obtained from crystallization using isopropanol and water is crystalline. In another aspect, the amorphous material is obtained when a solution of the D-isoglutamyl-D-tryptophan monoammonium salt is freeze-dried.

Extensive studies were performed to confirm that no racemization of chiral centers was present in column purification and throughout the reaction sequence, and detailed information is shown in the examples below.

According to the present invention, a method is provided for the synthesis of Boc-D-iGlu-D-Trp-OH (without the alpha amide isomer). A method is provided for converting Boc-D-iGlu-D-Trp-OH to an acid addition salt, particularly the hydrochloride salt, of thymodepressin. Morphogram prediction provides a method for precipitating thymodepressin in pure form at a pH of about 3 in water. In addition, a method is provided for purifying thymodepressin having a purity of less than 97% by flash column chromatography using isopropanol and water as an eluent. Another aspect of the present invention relates to a conventional process for preparing the mono ammonium salt from the hydrochloride salt of thymodepressin. The inorganic salts are removed by ion exchange resins and the mono-ammonium salt is recovered by using an ammonia-based regenerant solution. The mono-ammonium salt can be obtained by crystallization in pure form. Also provided is a method for purifying low purity monoammonium salt by flash silica gel column chromatography using isopropanol and water as eluent.

Furthermore, the method for the synthesis of thymodepressin disclosed in the prior art (US 5,736,519) produces crude thymodepressin which must be purified by ion exchange chromatography as well as reverse phase preparative liquid chromatography. The isolation 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 a very serious production problem. The purification sequences listed in the prior art are unsuitable for large-scale production.

Application and administration

The disodium salt of Thymodepressin has been used in the treatment of psoriasis. Thus, the crystalline thymodepressin and thymodepressin monoammonium salt of the present invention may be formulated into pharmaceutical compositions for administration to a subject in a therapeutically effective amount and in a biologically suitable form suitable for in vivo administration, i.e., in the form of a peptide, wherein the therapeutic effect outweighs any toxic effect.

According to the morphological analysis chart as shown in FIG. 8, the predominant species at neutral pH is the monocarboxylate form of thymodepressin, if the counterion is sodium, i.e., the monosodium salt of the dipeptide D-isoglutamyl-D-tryptophan. The disodium salt of D-isoglutamyl-D-tryptophan is very hygroscopic and difficult to handle for dispensing. The crystalline thymodepressin of the present invention has the XRPD pattern as detailed in fig. 1 and has a water solubility of about 20mg per ml in water. It is an ideal candidate to replace the disodium salt in the preparation of different formulations. Although the solution of D-isoglutamyl-D-tryptophan has a pH of about 3 in solution, the pH can be adjusted to about 7 to about 7.4 with sodium hydroxide, sodium carbonate, or sodium bicarbonate. The monoammonium salts of the present invention exist in both crystalline and amorphous forms. Both of these mono-ammonium salt forms are very soluble in water. It is therefore also an excellent candidate for formulation.

As described herein, the novel crystalline thymodepressin and/or its mono-ammonium salt may be administered in any of the acceptable administration forms for systemically active therapeutic agents. These methods include oral, parenteral and other systemic, aerosol or topical forms.

Depending on the intended form of administration, the compositions employed may be in solid, semi-solid, or liquid dosage forms, e.g., tablets, suppositories, pills, capsules, powders, liquids, aerosols, suspensions, and the like, preferably in unit dosage form, suitable for single administration of precise dosages. These compositions will comprise at least one conventional pharmaceutical carrier or excipient and crystalline thymodepressin or its pharmaceutically acceptable mono-ammonium salt and, in addition, may comprise other pharmaceutical agents (medicinal agents), pharmaceutical agents (pharmaceutical agents), carriers, adjuvants and the like.

For solid compositions, conventional non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. The active compounds as defined above may be formulated as suppositories using, for example, polyalkylene glycols (e.g., propylene glycol) as the carrier. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispensing, etc., the active substance as defined above, and the optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, ethanol, etc.), thereby forming a solution or suspension. If desired, the pharmaceutical compositions to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine acetateSodium, triethanolamine oleate, and the like. The actual methods of making such dosage forms are known, or will be apparent, to those skilled in the art; see, for example, Remington, The Science and Practice of Pharmacy, David B& Wilkins,Philadelphia,PA,21^stEdition,2006 in any event, the composition or formulation to be administered will contain a large amount of one or more compounds in an amount effective to alleviate the symptoms of the subject being treated.

Generally, parenteral administration is characterized by injection, whether subcutaneous, intramuscular, or intravenous. Injectables can be prepared in conventional forms, whether as liquid solutions or suspensions, solid forms suitable for liquid solutions or suspensions prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethane and the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.

Either oral or intranasal (bronchial) administration is preferred for thymodepressin or its mono-ammonium salt, depending on the nature of the discomfort being treated.

For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the addition of any of the commonly employed excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, 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 comprise from about 1% to about 95% active ingredient, preferably from about 25% to about 70%.

Oral as well as intranasal administration to the lung can also be effected by aerosol delivery forms. For aerosol administration, the active ingredient is preferably provided in finely divided form together with a surfactant and a propellant. Typical active ingredient percentages are from about 0.01% to about 20%, preferably from about 0.04% to about 1.0% by weight.

The surfactant must of course be non-toxic and preferably soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, eleostearic acid (olestauric acid) and oleic acid with fatty polyols or its cyclic anhydrides, such as ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, hexitol anhydrides derived from sorbitol (sorbitol esters, toThe name of (d) and polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides, may also be employed. Preferred surfactants are oleates or sorbitans, e.g. toC (sorbitan sesquioleate),80 (sorbitan monooleate) and85 (sorbitan trioleate). The surfactant may be comprised of from about 0.1% to about 20%, preferably from about 0.25% to about 5%, by weight of the composition.

The remainder of the composition is typically a propellant. Liquefied propellants are typically gases at ambient conditions and are compressed under pressure. Among the suitable liquid propellants are the lower alkanes, which contain up to 5Carbon, such as butane and propane; and preferably fluorinated or fluorochloroalkanes, e.g. inThose sold under the name. Mixtures of the above may also be employed.

In the production of aerosols, a container fitted with a suitable valve is filled with a suitable propellant containing a finely divided active ingredient and a surfactant. The components are thus maintained at elevated pressure until released by the action of the valve.

For topical administration, these compositions comprise an effective amount of such compounds in admixture with at least one pharmaceutically acceptable, non-toxic carrier. A suitable range of compositions may be from about 0.1% to about 10% active ingredient, with the balance being the carrier, preferably from about 1% to about 2% active ingredient. The concentration of the active ingredient in the pharmaceutical composition suitable for topical administration will vary depending upon the particular activity of the compound employed, in combination with the condition to be treated and the subject. Suitable carriers or pharmaceutical vehicles for topical administration of these compounds include creams, ointments, lotions, emulsions, solutions, and the like.

For example, for topical application of the compounds of the present invention, suitable ointments include from about 15 to about 45 percent of saturated fatty alcohols having 16 to 24 carbon atoms, such as cetyl alcohol, stearyl alcohol, behenyl alcohol, and the like, and from about 45 to about 85 percent by weight of an ethylene glycol solvent, such as propylene glycol, polyethylene glycol, dipropylene glycol, and mixtures thereof. The ointment may contain from about 0 to about 15 weight percent of a plasticizer, such as polyethylene glycol, 1,2, 6-hexanetriol, sorbitol, glycerin, and the like; from about 0 to about 15 weight percent of a coupling agent, for example saturated fatty acids having from 16 to 24 carbon atoms, such as stearic acid, palmitic acid, behenic acid, fatty acid amides, such as oleamide, palmitamide, stearamide, behenamide, and esters of fatty acids having from 16 to 24 carbon atoms, such as sorbitol monostearate, polyethylene glycol monostearate, polypropylene glycol or monoesters of corresponding other fatty acids, such as oleic acid and palmitic acid; and from about 0 to about 20 percent by weight of a penetrant such as dimethyl sulfoxide or dimethylacetamide.

The therapeutically active amount of crystalline thymodepressin or its ammonium salt may vary depending on various factors, such as the disease state, age, sex, and weight of the individual. The dosage regimen may be varied to provide the optimum therapeutic response. Generally, the daily regimen should be in the range of from about 1 to about 200mg of peptide.

The following are examples of representative formulations and in no way limit the scope of formulations for preparing different pharmaceutical compositions.

The above ingredients are mixed thoroughly and pressed into a single scored tablet.

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

The above ingredients are intimately mixed and pressed into a single scored tablet.

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

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

An injectable formulation buffered to a pH of about 7 is prepared having the following ingredients:

an injectable formulation buffered to a pH of about 7 is prepared having the following components:

an oral suspension is prepared having the following composition:

topical formulations

All the above ingredients (except water) were combined and heated to about 45 ℃ with stirring. A sufficient amount of water is then added at about 45℃, with vigorous stirring to emulsify the ingredients, and the water is then added to the appropriate amount of 100 g.

The present invention is explained in detail below with reference to examples, but the present invention is not limited thereto in any way.

Example 1

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

Procedure 1A:

pure reference sample N-t-Boc-D-Glu- (γ -D-Trp-OMe) - α -OBzl was prepared using silica gel chromatography.

EDC (5.11 g, 26.6 mmol), HOBt (3.60 g, 26.6 mmol) and DIPEA (4.60 mL, 26.6 mmol) were added sequentially to CH₂Cl₂(70 mL) of a stirred ice-cooled solution of Boc-D-Glu-OBzl (6.00 g, 17.8 mmol). Then, dropwise adding into CH₂Cl₂(50 mL) of H-D-Trp-OMe. HCl (6.77 g, 26.6 mmol) and DIPEA (4.60 mL, 26.6 mmol). The resulting mixture was stirred at an ice-cold temperature (-3 ℃ to 0 ℃) for 1 hour, then it was heated to room temperature and stirred for 16 hours. The reaction mixture was evaporated to dryness. The residue was washed with EtOAc and NaHCO₃Are separated from each other. The organic portion was collected, washed with 10% citric acid, then brine. Organic layer in Na₂SO₄Dried, filtered and concentrated to a viscous oil. The residue was purified by column chromatography on silica gel using hexane and EtOAc (8/2, 7/3 and 3/7 ratios, v/v) as eluents to give the title product as a white solid (9.40 g, 98%).¹H NMR（DMSO-d₆）δ 10.86（s,1H）、8.31（d,J=7.4Hz,1H）、7.49（d,J=7.7Hz,1H）、7.31-7.35（m,7H）、7.14（d,J=2.0Hz,1H）、7.06（t,J=7.9Hz,1H）、6.98（t,J=6.8Hz,1H）、5.12（q,J=5.9Hz,2H）、4.5（q,J=6.5Hz,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）；¹³C NMR（DMSO-d₆） 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（CH₂）、53.3（CH）、53.16（CH）、51.7（CH₃）、31.3（CH₂）、28.2（CH₃）、27.1（CH₂）、26.4（CH₂）；MS（m/z）538[M+1]⁺(ii) a To C₂₉H₃₅N₃O₇.0.5H₂And O, carrying out analysis calculation: c, 63.72; h, 6.64; n, 7.69; the following are found: c, 63.79; h, 6.06; and N, 7.65.

Procedure 1B:

pure sample N-t-Boc-D-Glu- (gamma-D-Trp-OMe) -alpha-OBzl was prepared by recrystallization.

Will be in CH₂Cl₂A suspension of Boc-D-Glu-OBzl (60.26 g, 178.6 mmol) in (335 mL) was cooled to about-1 deg.C and stirred for 15min. Next, DIPEA (46.70 mL, 268.0 mmol), HOBt (36.20 g, 268.0 mmol), and EDC (51.38 g, 268.0 mmol) were added in this order. Then, dropwise adding into CH₂Cl₂(187 mL) of H-D-Trp-OMe.HCl (68.25 g, 268.0 mmol) and DIPEA (4.60 mL, 268.0 mmol). In cold conditionThe resulting mixture was stirred at temperature (-1 ℃ to-5 ℃) for 2 hours, then it was warmed to room temperature and stirred under a blanket of nitrogen overnight.

The reaction mixture was evaporated to dryness. The residue was washed with EtOAc (200 mL), Na₂CO₃Saturated solution of (3) (100 mL) and H₂O (150 mL) was separated. The aqueous layer was re-extracted with EtOAc (200 mL). Collecting the organic fraction with H₂O (100 mL), 10% citric acid (2 × 200 mL), and brine (60 mL) were washed. Organic layer in Na₂SO₄Dried, filtered and evaporated to dryness. The residue was dissolved in EtOAc (141 mL) followed by the addition of hexane (106 mL). The resulting suspension was stirred for 6h and filtered. The solid was washed thoroughly with hexane (100 mL) and then dried in an oven under vacuum at 40 ℃ overnight. An off-white solid was obtained (81.67 g, 85%).¹H NMR data were consistent with the structure (see example 1, procedure 1A).

Procedure 1C:

without chromatographic purification, a pure sample of N-t-Boc-D-Glu- (γ -D-Trp-OMe) - α -OBzl was prepared and the synthetic impurities were determined.

Boc-D-Glu-OBzl (48.0 g, 142.2 mmol) was dissolved in 270mL of dichloromethane and then it was cooled to 0 to 5 ℃ 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 min. EDC (38 g, 199.1 mmol) and a premixed H-D-Trp-OMe solution (prepared from H-D-Trp-OMe.HCl (39.7 g, 156.4 mmol) in 150mL of dichloromethane and DIPEA (27.0 mL, 156.4 mmol) stirred at room temperature for 20 min) were added to the solution in succession. The reaction was continued at0 ℃ for 2 hours, and then at room temperature overnight. The reaction was poured into 250mL of distilled water and extracted. With 250mL each of 10% citric acid, 2X 5% NaHCO₃The organic layer was washed with the solution and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to yield a pale yellow foamy solid.

The solid was dissolved in about 250mL of ethyl acetate and evaporated to dryness. The reaction was carried out twice to form a waxy solid. To the solid material was added 100mL of ethyl acetate, and allowed to stir at room temperature. The mixture was stirred at moderate to rapid speed until a slurry-like suspension was formed-this process took about 45min (long stirring could cause the solution to solidify into a gelatin-like material). Then, 75mL of hexane was added, and the mixture was stirred for another 10 min. At this point, a further 20mL of ethyl acetate was added and the slurry was immediately filtered to give a loose grey solid. The solid was immediately washed 3 times with 30mL of hexane, which helped the solid to remove pink color. The filtrate was collected and allowed to stand for 40 minutes. The particulate solid precipitated from the filtrate. The mixture was filtered and the solid was washed 3 times with 10mL of hexane.

The filtrate was collected and concentrated to a solid. The solid was dissolved in 20mL of ethyl acetate and stirred until a slurry was formed. Then, 40mL of hexane was added, and the mixture was stirred for 5min. The mixture was filtered and the collected solid was washed with hexane. The combined solids were dried in an oven (35 ℃) under vacuum overnight to constant weight. Then, 59.0g (77.2%) of the title compound was obtained. And Mp: 83.1 to 87.5 ℃;¹the H NMR data were the same as those described in example 1, procedure 1A; HPLC purity (peak area percentage): 97.2 percent; retention time: 7.56 min; HPLC conditions: column Waters Symmetry C18, 3.9x150mm, 5 μm; a flow box: 0.035% HClO₄、pH2/CH₃CN gradient (min-% CH)₃CN) 0-35, 10-90 and 12-90; flow rate: 1 mL/min; λ: 230. 260 nm and 280 nm.

Analysis of impurities in mother liquor

The results of the mother liquor analysis by TLC (50/50 EtOAc/hexanes) are shown in the following table. Spots A and B are both UV active, but give a negative ninhydrin test. Both the product and the baseline spot gave a positive ninhydrin test. Separating samples A and B by column chromatography on silica gel, and passing their structures¹H NMR and MS/MS are elucidated. The structures of A and B are shown below.

Rf value in 50/50 EtOAc/hexanes:

spots A and B are both HOBt related impurities. These impurities can be removed by recrystallization. Any trace impurities can be removed in a subsequent 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 ] -butyric acid or N-t- α -Boc-D-iGlu-D-Trp-OH.

Procedure 2A:

single phase hydrolysis with NaOH.

A solution of NaOH (1.0 g, 25 mmol) in water (20 mL) was added to a stirred solution of N-t-Boc-D-Glu- (γ -D-Trp-OMe) - α -OBzl (3.7 g, 6.9 mmol) from example 1, procedure 1A in MeOH (40 mL). The resulting solution was stirred at room temperature overnight. The reaction mixture was poured into a 1N solution of NaOH (100 mL) and the aqueous mixture was washed with EtOAc (2X 100 mL). The aqueous layer was acidified with 3N HCl solution, followed by extraction with EtOAc (2X 50 mL). Combining the organic moieties in Na₂SO₄Dried and evaporated to dryness under reduced pressure. An off-white solid was obtained (2.7 g, 91%). M.p.148 to 158 ℃;¹H NMR（DMSO-d₆）δppm：12.47（br,2H）、10.82（s,1H) 8.21 (d, J =7.8Hz, 1H), 7.53 (d, J =7.8Hz, 1H), 7.34 (d, J =8.1Hz, 1H), 7.13 (d, J =2.0Hz, 1H), 7.06 (t, J =7.5Hz, 2H), 6.98 (t, J =7.4Hz, 1H), 4.46 (q, J =5.3Hz, 1H), 3.88-3.83 (m, 1H), 3.17-2.97 (dd, J =5.2 and 8.4Hz, 2H), 2.23-2.10 (m, 2H), 1.90-1.82 (m, 1H), 1.75-1.68 (m, 1H), 1.38 (s, 9H);¹³C NMR（DMSO-d₆）δppm：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（CH₂）、28.2（CH₃）、27.2（CH₂）、26.7（CH₂）；FT-IR（KBr）v：3415、3338、2986、1719、1686、1654、1534、1424、1366、1252、1169、1069、744、634、429cm^-1；MS（m/z）434[M+1]⁺。

procedure 2B:

single phase hydrolysis using LiOH

LiOH (10.78 g, 257.0 mmol) in water (136 mL) was added to Boc-D-Glu- (. gamma. -D-Trp-OCH) in MeOH (200 mL)₃) -a stirred ice-cooled (0 ℃ to 5 ℃) solution of a-OBzl (46.06 g, 85.68 mmol). The resulting solution was stirred and maintained at between 0 ℃ and 10 ℃ for 3 h. The reaction mixture was poured over Na₂CO₃(100 mL) and H₂O (150 mL) in a saturated solution, the water mixture was washed with EtOAc (2X 150 mL). The aqueous layer was acidified to pH =2 to 3 with 3N HCl solution, followed by extraction with EtOAc (2 × 200 mL). The organic fractions were combined over Na₂SO₄Dried and evaporated to dryness under reduced pressure. A white solid was obtained (36.65 g, 98.7%).¹H NMR and MS/MS data were in agreement with the structure (see example 2, procedure 2A).

Procedure 2C:

Boc-D-iGlu-D-Trp-OH was prepared using a biphasic hydrolysis method without chromatographic purification.

Lithium hydroxide (4.1 g, 97.7 mmol) was dissolved in 35mL of distilled water. Next, 65mL of methyl tert-butyl ether (MTBE) was added, followed by the dipeptide Boc-D-Glu- (γ -D-Trp-OCH obtained as described in example 1₃) - α -OBzl (25 g, 46.5 mmol). A very viscous suspension formed immediately, and 15mL of methanol and 15mL of MTBE were added with vigorous stirring. An additional 2mL of methanol was added and the solid was slowly dissolved for about 5min. Once all the material was dissolved, the solution was yellow/green, with the upper organic phase being grayish green and the aqueous phase being yellow. The reaction was stirred vigorously at room temperature for 80min, at which time no starting material remained in the organic phase and the aqueous phase contained product (TLC monitor: 1/1 EtOAC/hexane, v/v). The solution was poured into a separatory funnel and the 2 phases were separated. The organic phase was washed with 15mL of water. The organic phase turned pink when washed with water. The combined aqueous phases were washed twice with 30mL of ethyl acetate. The aqueous phase was acidified to about pH2 by dropwise addition of 16.6mL of 6N hydrochloric acid at room temperature. The aqueous phase was extracted twice with 50mL of ethyl acetate. A minimum amount of methanol was added during the second extraction to aid in the dissolution of the product in the organic layer. The combined organics were dried over sodium sulfate and concentrated in vacuo from a yellow liquid to yield a white solid. The solid was dried in an oven (28 ℃) under vacuum overnight to constant weight.

Then, 18.6g (92% yield) of the title compound was obtained. And Mp: 179.0 to 184.6 ℃;¹h NMR data were identical to those illustrated in example 2A; HPLC purity (peak area percentage): 98.3 percent; retention time: 5.33 min; HPLC conditions: column Waters Symmetry C18, 3.9x150mm, 5 μm; mobile phase: 0.035% HClO₄、pH2/CH₃CN, gradient (T min-% CH)₃CN) 0 to 20, 10 to 90, 12 to 90; flow rate: 1 mL/min; λ: 230. 260 nm and 280 nm.

The reaction was monitored by HPLC. In the above procedure 2C, benzene evolution was observed on TLC (1/1 EtOAC/hexane, v/v as eluent)Presence of benzyl alcohol resulting from partial hydrolysis of the methyl ester. Benzyl alcohols are derived from the hydrolysis of benzyl ester moieties. The first spot impurity was the same as in example 1, procedure 1C. Monitoring by HPLC and demonstration of Boc-D-Glu- (γ -D-Trp-OCH by LC/MS analysis₃) - α -OBzl is first reacted with a base and methanol to give Boc-D-Glu- (γ -D-Trp-OCH)₃)-α-OCH₃It is then rapidly hydrolyzed to give the diacid Boc-D-Glu- (γ -D-Trp-OH) -OOrBoc-D-iGlu-D-Trp-OH.

Example 3:

preparation of D-isoglutamyl-D-tryptophan

Procedure 3A:

preparation of D-isoglutamyl-D-tryptophan and purification thereof by recrystallization

Boc-D-iGlu-D-Trp-OH (20.0 g, 46.14mmol, from example 2) was placed in a 1L-3 neck round bottom flask equipped with a mechanical stirrer. Ethyl acetate (300 mL) was added and the resulting suspension was cooled to-10 ℃ in an ice salt bath. The HCL gas is bubbled (bubble) into the cold suspension. The temperature was maintained in the range of-4 ℃ to-10 ℃ during the reaction, and the progress of the reaction was monitored by HPLC. The heterogeneous reaction mixture became a clear, pale pink homogeneous solution at some point. And after consumption of the starting material, the reaction mixture becomes a suspension again. The volatile material was then removed in vacuo to give a pale pink solid. The solid was dissolved in 60mL of deionized water and the resulting solution was washed with dichloromethane (2 × 25 mL). The pH of the aqueous solution was then brought to about 3.0 by the addition of NaOH (10M, about 3.6 mL) under cooling. The resulting solution was filtered to remove any residual solid particles. The filtrate was collected and stirred vigorously as the solids separated. The solid was collected by filtration. The filtrate was set aside for subsequent use. The solid was then placed back in the round bottom flask and 30mL of deionized water was added. The mixture was stirred vigorously and the solid was collected by filtration. The filtrate is set aside and then used. The solid was then washed with ice cold deionized water (4 x15 mL). As by negative AgNO₃The third water wash solution was chloride free as determined by assay (using a 4% solution). The solid was air-dried and then placed in a vacuum oven overnight at 36 ℃ to give 8.5g (HPLC purity (peak area percentage): 98.3%). The filtrates from the above steps were combined and subjected to the same recrystallization procedure to give an additional 3.2g of product (HPLC purity (peak area percentage): 98.7%). The combined yield of these 2 batches was 11.7g (75%).

The final filtrate was further processed to yield a third batch (1.0 g, HPLC purity (peak area percentage: 82.0%).

¹H NMR（D₂O-NaOD,pH7.0） ppm: 7.64 (d, J =7.9Hz, 1H), 7.43 (d, J =8.1Hz, 1H), 7.19-7.16 (m, 2H), 7.10 (t, J =7.4Hz, 1H), 4.52-4.48 (m, 1H), 3.48 (t, J =6.1Hz, 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 XRPD pattern of this material is shown in figure 1. HPLC method: column: XTerra MS C18; 5 μm, 4.6x250 mm; mobile phase: a = aqueous phase: 4mM Tris, 2mM EDTA, pH7.4; b = organic phase: CH (CH)₃CN; gradient program: b%: 0min.5%, 15min.55%, 30min.55%, 32min.5%, 40 min.5%. Flow rate =1 mL/min; injection volume =5 μ Ι _; λ: 222. 254, 282, 450 nm; retention time of product =6.4 min.

Procedure 3B:

ethyl acetate (250 mL), precooled to 0 ℃ and saturated with HCl gas for 25 min. Boc-D-iGlu-D-Trp-OH (15.0 g, 34.6 mmol) was added and a suspension was formed. The solution was stirred at ice bath temperature for 90 min. The solvent was evaporated under vacuum to form a white solid. The solid was dissolved in 35mL of distilled water to form a viscous light brown solution. The aqueous layer was washed twice with 30mL of dichloromethane, followed byThe aqueous layer was transferred to a 100mL beaker. The acidity was monitored using a pH electrode, which was adjusted from 1.28 to 2.96 using 3.2mL of 10N NaOH. The solution was stirred at room temperature for 1 hour and a white precipitate slowly formed. The solid was collected by suction filtration and washed thoroughly with water. The crude solid was suspended in 20mL of distilled water and allowed to stir at room temperature for 2 hours. The mixture was filtered, the solid collected and dried in an oven overnight (40 ℃) under vacuum to constant weight. Next, 8.6g (74.5% yield) of the title compound was obtained. HPLC purity (peak area percentage): 98.8 percent; retention time: 4.21 min; HPLC conditions: column Waters Symmetry C18, 3.9x150mm, 5 μm; mobile phase: 0.035% HClO₄、pH2/CH₃CN, gradient (T min-% CH)₃CN) 0-10, 10-90 and 12-90; flow rate: 1 mL/min; λ: 230. 260 nm and 280 nm;¹h NMR data were in agreement with the structure.

Example 4:

D-isoglutamyl-D-tryptophan monoamine salt (1: 1) was synthesized and isolated by column chromatography purification.

Procedure 4A:

HCl gas was bubbled into a stirred ice-cooled (0 ℃ to 5 ℃) solution of Boc-D-Glu- (-D-Trp-OH (2.5 g, 5.8 mmol) in EtOAc (60 mL) for 2.5 h, then the reaction mixture was evaporated to dryness using isopropanol and ammonium hydroxide (28 to 30% NH)₄OH) (8/2 and 7/3 ratio, v/v) as eluent, the residue was purified by column chromatography on silica gel to yield the title product (1.8 g, 84.7%) as a white solid after evaporation of the solvent. M.p.124 to 128 ℃;¹H NMR（DMSO-d₆）δppm：10.98（s,1H）、8.25（d,J=5.9Hz,1H）、7.53（7.8Hz,1H）、7.30（d,J=8.0Hz,1H）、7.17（s,1H）、7.00（t,J=7.7Hz,1H）、6.92（t,J=7.2Hz,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）；¹³C NMR（DMSO-d₆）δppm：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（CH₂）、27.8（CH₂）、27.4（CH₂）；¹⁴N NMR（D₂O） ppm：20.4（s）；FT-IR（KBr）v：3406、3055、1581、1456、1399、1341、1096、1009、744、535、426cm^-1；MS（m/z）334[Diacid+1]⁺(ii) a To C₁₆H₂₂N₄O₅.H₂Analytical calculation of O: c, 52.17; h, 6.57; n, 15.21; the following are found: c, 51.95; h, 6.84; n, 14.85. This material is the mono-ammonium salt of D-isoglutamyl-D-tryptophan (1: 1). This material was determined to be amorphous by XRPD.

Procedure 4B:

the HCl gas was compressed into cold ethyl acetate at-2 ℃ (external ice bath temperature) for 16 minutes. The weight gain of the solution was 21 g. Boc-D-iGlu-D-Trp-OH (3.8 g, 8.73 mmol) was dissolved in 50mL of the above solution. The resulting mixture was maintained between 0 and 5 ℃ for 55 minutes. The reaction was monitored by TLC and then evaporated to dryness under reduced pressure (rotary evaporator temperature: 51 to 52 ℃). Purification by flash chromatography on silica gel using a solvent gradient of a mixture of isopropanol and ammonium hydroxide (28 to 30%) (8/2 and 7/3 ratios, v/v) as eluents gave the product as an off-white solid (2.0 g, 62%).¹H NMR data were similar to those reported in example 4, procedure 4A. This material was determined to be amorphous by XRPD.

Example 5

D-isoglutamyl-D-tryptophan, mono ammonium salt (1: 1) were synthesized.

Procedure 5A:

use of15Resin was purified by column chromatography to remove inorganic salts and to synthesize D-isoglutamyl-D-tryptophan, mono ammonium salt (1: 1).

HCl gas was bubbled into a stirred ice-cooled (0 ℃ to 5 ℃) suspension of the obtained Boc-D-iGlu-D-Trp-OH in EtOAc (200 mL) for 2 hours. The reaction mixture was then evaporated to dryness. The residue was dissolved in water (30 mL) and neutralized with 6N NaOH to pH =6 to 7. The resulting solution was loaded and filled15 column chromatography of resin, followed by H₂O eluted until pH =5 to 5.5, followed by 100% isopropanol (pH = 7) and finally 25% NH₄OH/IPA（pH=10）。

The fractions containing the product were combined and evaporated to dryness under reduced pressure. The residue was further purified by column chromatography on silica gel using a solvent gradient of isopropanol and a mixture of concentrated ammonium hydroxide (17/3, 4/1 and 7/5 ratios, v/v) as eluent to yield the title product as a light yellow foamy solid (6.68 g, 72.7%).¹H NMR and MS/MS data were in agreement with the structure (see procedure 4A). The water content was 3.7% as determined by the Karl-Fisher test.

Procedure 5B:

D-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1) was synthesized using Amberlyst15Resin to remove inorganic salts followed by purification by recrystallization.

HCl gas was bubbled into a stirred ice-cooled (0 ℃ to 5 ℃) suspension of Boc-D-iGlu-D-Trp-OH (10.75 g, 24.80 mmol) in EtOAc (200 mL). The reaction mixture was kept in an ice bath (0 ℃ to 5 ℃) for 2 hours. TLC analysis (in iso-phase)30% ammonia in propanol) showed complete conversion of the starting material. The reaction mixture was evaporated to dryness under vacuum, the residue was dissolved in water (30 mL) and neutralized with 10N NaOH to pH =6 to 7. The obtained homogeneous solution is loaded and filled15 column of resin, followed by H₂O (2450 mL) eluted until pH =4 to 5.5 with isopropanol (1000 mL) and 25% NH₄OH/isopropanol elution. The fractions containing the product were combined and concentrated to dryness. A colorless foamy solid was obtained by mixing isopropanol (150 mL) with H₂O (30 mL) was added. The resulting solution was stirred at room temperature overnight. The solid was collected by suction filtration, washed thoroughly with isopropanol (2 x60 mL), followed by EtOAc (2 x60 mL), and finally dried in a 42 ℃ oven under vacuum overnight. An off-white solid was obtained (6.60 g, 72.2%).¹H NMR and MS/MS data were in agreement with the structure (see example 5). The XRPD of this crystalline material is shown in fig. 2. The water content was determined to be 5.9% by the Karl-Fisher test.

Example 6

D-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1) was synthesized from H-D-iGlu-D-Trp-OH.

Procedure 6A:

D-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1) was prepared using CBz-D-Glu- (γ -D-Trp-OH) - γ -OBzl as an intermediate.

EDC (562 mg, 2.93 mmol) was added to Z-D-Glu-OBz (990 mg, 2.67 mmol) and N-hydroxysuccinimide (337 mg, 2.93 mmol) in DMF (50 mL) in an ice-water bath and the resulting clear solution was stirred at room temperature overnight. H-D-Trp-OH (640 mg, 3.13 mmol) and Et were added at room temperature₃N (1 mL). After 20 minutes, the material was mixed with water and extracted with ethyl acetate. The combined EtOAc was washed with 10% citric acid and then brine, over Na₂SO₄The mixture was dried, filtered, evaporated to dryness and dried under vacuum to give 1.49g of CBz-D-iGlu- (gamma-OBzl) -D-Trp-OH. This material was hydrogenated with 33% (w/w) of 10% Pd/C at atmospheric pressure. After 4 hours, the catalyst was filteredAnd the filtrate was evaporated to give an oil. Using isopropanol/NH₄The crude product was purified by flash column chromatography using OH (80/20 to 70/30, v/v) as eluent to give the title compound (813 mg). MS/MS and¹h NMR data was similar to the compound obtained using the method shown in example 4 above.

Procedure 6B:

D-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1) was synthesized from H-D-iGlu-D-Trp-OH (see example 3).

H-D-iGlu-D-Trp-OH (1 g from example 3) was combined with ammonium hydroxide (0.55M, 6 mL). The mixture was stirred and the pH was measured to be about 4.5. Ammonium hydroxide (0.55M) was added dropwise until the pH of the solution reached between 7.0 and 7.5. Volatiles were removed under vacuum and the residual oil was mixed with isopropanol. A white precipitate appeared. After 2 hours, the solid ammonium salt was collected by suction filtration. The solid was dried under high vacuum for 12 hours to constant weight (1 g) to give D-isoglutamyl-D-tryptophan, ammonium salt (1: 1). The water content was determined to be 4.6% by the Karl-Fisher test.

Example 7

The H-D-iGlu-D-Trp-OH was purified by column chromatography on silica gel using a mixture of isopropanol and water.

A. Preparation of Cbz-D-Glu- (gamma-D-Trp-OBzl) -alpha-OBzl

0.91g of N-hydroxysuccinimide (1.1 eq.) and 1.52g of EDC (1.1 eq.) are added to 2.67g Cbz-D-Gl in 50mL DMFu-OBzl in ice-cooled solution, and in the ice water bath stirring the solution for 1 hours, and then at room temperature overnight. 2.50g H-D-Trp-OBzl. HCl (1.05 eq.) and 3mL Et₃N was added to this reaction mixture at room temperature. The reaction was complete after 1 hour as monitored by HPLC.

The reaction mixture was quenched with deionized water in an ice water bath and then extracted several times with EtOAc. The combined EtOAc extracts were washed with 10% citric acid followed by brine, over Na₂SO₄Dried, filtered and evaporated to dryness. The residue was applied to silica gel in MeOH, and the mixture was concentrated in vacuo. The latter was applied to the top of a wet-packed silica gel column and the desired product Cbz-D-Glu- (γ -D-Trp-OBzl) - α -OBzl was eluted using a solvent gradient mixture (EtOAc/Hexanes, from 80/20 to 100/0). The desired fractions were combined and concentrated in vacuo to yield 4.64g (99.6% yield) of the title compound. HPLC purity (peak area percentage): 93 percent; HPLC conditions: column Symmetry C18, 3.9x150mm, 5 μm; mobile phase: 0.035% HClO₄（pH=2.5）/CH₃CN = gradient (min-CH)₃CN%: 0 to 10, 10 to 100, 12 to 100, 14 to 50); flow rate: 1 mL/min; λ: 280 nm; retention time: 9.7 min. B. D-isoglutamyl-D-tryptophan was purified by column chromatography using a mixture of isopropanol and water.

1.5g of Pd/C (37.5% w/w) was added to 150mL95/5MeOH/H₂Suspension of 4.0g Cbz-D-Glu- (γ -D-Trp-OBzl) - α -OBzl in O (v/v) as described in example 7A above. The mixture was hydrogenated at 30psi hydrogen pressure. The reaction was complete after 75min as monitored by HPLC. Catalyst in one layerThe bed was filtered off and the filtrate was concentrated at 45 ℃ under reduced pressure. Then, isopropyl alcohol/H was used₂O (80/20 ratio, v/v), the residue was purified by flash chromatography on silica gel. The purest fraction is taken (by HPLC)) Combined and concentrated in vacuo. Thus, the title compound (1.1 g, 52%) was obtained as a pale yellow powder. HPLC purity (peak area percentage): 99 percent. MS/MS and¹h NMR corresponds to the desired structure.

The less pure fractions were combined (by HPLC) and concentrated in vacuo to yield another approximately 1.0g (48% yield) of the title compound. HPLC purity (peak area percentage): 96.7 percent. MS/MS and¹h NMR corresponds to the desired structure.

HPLC conditions were the same as in section 7A above; the retention time of H-D-iGlu-D-Trp-OH was 4.0 min.

Example 8

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

The title compound is a possible synthetic impurity of H-D-iGlu-D-Trp-OH. It can be synthesized separately and used as a reference in HPLC analysis of the products described in examples 3 to 7 above.

N-hydroxysuccinimide (409 mg, 3.56 mmol), EDCI (682 mg, 3.56 mmol) were added to an ice-cooled solution of Z-D-Glu- (γ -OEt) -OH (1 g,3.23 mmol) in DMF (60 mL), and the resulting solution was stirred in an ice-water bath for 1 hour, then at room temperature overnight. H-D-Trp-OH (792 mg, 3.88 mmol) and Et were added at room temperature₃N (1 mL) was added to this reaction mixture. After 1.5 hours water was added at ice bath temperature. The mixture was extracted several times with EtOAc. The combined EtOAc extracts were washed with 10% citric acid followed by brine, over Na₂SO₄Dried, filtered, concentrated to dryness, and dried under vacuum to yield 410mg of crude product fraction a. The aqueous layer was concentrated in vacuo at a bath temperature of 55 ℃. Dissolving the residue in CH₂Cl₂And the organic portion was washed with 10% citric acid (1 × 20 mL) and brine over Na₂SO₄Drying, filtering, at 50Concentrate to almost dryness at deg.C to give crude fraction B. Crude product fractions A and B were combined and hydrogenated over 10% Pd/C (40% w/w Pd/C) at atmospheric pressure using a hydrogen filled balloon at room temperature for 2.75 hours. The reaction mixture is filteredThe filtrate contained the product H-D-iGlu (γ -OEt) -D-Trp-OH and was analyzed by HPLC (same method as one described in 7A above). The retention time of the product peak was 4.54 min. The filtrate was concentrated to dryness under reduced pressure at a bath temperature of about 45 ℃. HPLC analysis showed R_tPartial conversion of the product at 4.54min to have R_tAnother peak at 4.45 min. Using isopropanol and concentrated NH₄The crude product was purified by flash column chromatography on OH (elution gradient: 90/10 to 80/20, v/v). HPLC analysis of this material (670 mg) showed R_tThe peak was 4.45 min. By passing¹The structural elucidation of H NMR spectroscopy indicated that the product was the cyclized compound shown below after the work-up procedure:

¹H NMR（CD₃OD) δ ppm: 7.60 (d, J =7.8Hz, 1H), 7.31 (d, J =8.0Hz, 1H), 7.10 (s, 1H), 7.07 (t, J =7.8Hz, 1H), 7.00 (t, J =7.4Hz, 1H), 4.68-4.65 (m, 1H), 4.05-4.02 (m, 1H), 3.44 (dd, J =14.6 Hz, J =4.6Hz, 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 (m/z): 316[ M +1 ]]⁺，188（100%）。

¹³C NMR（CD₃OD) 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: Column-Symmetry C18, 5 μm, 3.9x150mm, WAT 046980; mobile phase-0.035% HClO₄/CH₃CN, gradient; the method comprises the following steps: min-CH₃CN%: 0-10%, 10-100%, 12-100%, 14-50%; flow rate: 1.0 mL/min; detecting lambda: 254 nm.

B. 5-oxo-D-prolyl-D-tryptophan was independently synthesized 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) was added to 250ml of dichloromethane. Diisopropylethylamine (3.12 ml, 0.018 mol) was added and the solution was clear at this point. Hydroxy benzotriazole hydrate (HOBt. H) was added at0 deg.C₂O, 2.48g, 0.016 mol). EDCI (4.64 g, 0.025 mol) was added. After 30min, H-D-Trp-OBzl. HCl (4.76 g, 0.016 mol) was added followed by diisopropylethylamine (3.12 ml, 0.018 mol). The solution cleared within 5min and was stirred all the way to the left for 16 hours. The solvent was evaporated to dryness and the material was partitioned between ethyl acetate and 10% hydrochloric acid. The ethyl acetate layer was washed with 10% sodium bicarbonate solution and then with brine. The organic layer was dried over sodium sulfate and evaporated to give a foamy solid. This solid is benzyl 5-oxo-D-prolyl-D-tryptophan. Without purification, a sample of crude benzyl 5-oxo-D-prolyl-D-tryptophan (1.55 g) was dissolved in methanol (30 ml) and hydrogenated over 10% Pd/C under 45psi of hydrogen for 2 hours. The catalyst was filtered through Celite and the filtrate was evaporated to give a red foam. This material was purified by column chromatography (elution gradient: 10% methanol: dichloromethane followed by 50% isopropanol in dichloromethane (dichoromethane)) to give a solid (419 mg). The purity of the material can be monitored by TLC (30% ammonia: isopropanol). This material is 5-oxo-D-prolyl-D-tryptophan, which has the same NMR data as the material in part a.

Example 9

L-isoglutamyl-L-tryptophan, mono ammonium salt (1: 1) were synthesized.

A: synthesis of Boc-L-Glu- (gamma-L-Trp-O-t-Bu) -alpha-O-t-Bu

Boc-L-Glu-O-t-Bu (1.50 g, 4.9 mmol) in CH₂Cl₂The solution (50 mL) was cooled to-3 ℃ and stirred for 15min. Subsequently, 1-hydroxybenzotriazole (HOBt, 1.00g, 7.4 mmol), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC, 1.42g, 7.4 mmol) and diisopropylethylamine (DIPEA, 1.30mL, 7.4 mmol) were added in this order. Then, H-L-Trp-O-t-Bu. HCl (2.20 g, 7.4 mmol) and DIPEA (1.30 mL, 7.4 mmol) in CH were added dropwise₂Cl₂Solution of (1) (20 mL). The resulting mixture was stirred at ice-cold temperature (-3 ℃ to 0 ℃) for 1 hour, then it was warmed to room temperature and stirred under a blanket of nitrogen overnight.

The reaction mixture was evaporated to dryness. The residue was partitioned between EtOAc (40 mL) and NaHCO₃(100 mL) of saturated solution. The organic portion was collected, washed with 10% citric acid, then brine (30 mL). Organic layer in Na₂SO₄Dried, filtered and concentrated to a viscous oil. The residue was purified by column chromatography on silica gel using a solvent gradient of a mixture of hexane and EtOAc (85/15, 80/3 and 20/40ratio, v/v) as eluent to give the title product as a white solid (2.55 g, 95%).¹H NMR（DMSO-d₆）δppm：10.84（s,1H）、8.18（d,J=7.5Hz,1H）、7.52（d,J=7.7Hz,1H）、7.35（d,J=8.0Hz,1H）、7.12-7.15（m,2H）、7.06（t,J=7.6Hz,1H）、6.98（t,J=6.8Hz,1H）、4.41（q,J=6.7Hz,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）；¹³C NMR（DMSO-d₆）δppm：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（CH₂）、28.2（CH₃）、27.9（CH₃）、27.6（CH₃）、27.5（CH₃）、27.2（CH₂）、26.6（CH₂）；MS（m/z）546[M+1]⁺(ii) a To C₂₉H₄₃N₃O₇.0.5H₂And O, carrying out analysis calculation: c, 62.80; h, 8.00; n, 7.58; the following are found: c, 62.69; h, 8.56; and N, 7.57.

B: synthesis of L-isoglutamyl-L-tryptophan, monoammonium salt (1: 1)

Bubbling HCl gas into the CH gas obtained as described above₂Cl₂A stirred ice-cooled (0 ℃ to 5 ℃) solution of Boc-L-Glu- ((-L-Trp-O-t-Bu) - γ -O-t-Bu (2.38 g, 4.4 mmol) in (40 mL) for 5 hours.

Reverse phase High Performance Flash Chromatography (HPFC) Using C18HS M +40 column^TM) (Biotage) and 15mM NH as eluent₄OAc and CH₃CN, crude sample (482 mg), evaporation of the solvent and lyophilization of the material (150mg) gave the title compound.¹H NMR（DMSO-d₆）δppm：7.57（d,J=7.8Hz,1H）、7.39（d,J=8.1Hz,1H）、7.11-7.14（m,2H）、7.05（t,J=7.2Hz,1H）、4.44-4.48（m,1H）、3.42（t,J=5.7Hz,1H）、3.25（dd,J=14.7,4.7Hz,1H）、2.97-3.03（m,1H）、2.14-2.19（m,2H）、1.72-1.85（m,2H）；FT-IR（KBr）v：3057、1581、1400、745cm^-1；MS（m/z）334[diacid+1]⁺(ii) a To C₁₆H₂₂N₄O₅.H₂And O, performing calculation analysis: c, 52.17; h, 657; n, 15.21; the following are found: c, 51.92; h, 6.80; n, 14.94. The substance is the mono-ammonium salt of L-isoglutamyl-L-tryptophan (1: 1).

Example 10

Synthesis of H-D-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1).

A. Synthesis of Boc-D-Glu- (gamma-L-Trp-O-t-Bu) -alpha-O-t-Bu

The procedure described in example 1A was used. EDC (3.80 g, 19.8 mmol), HOBt (2.68 g, 19.8 mmol) and DIPEA (3.50 mL, 19.8 mmol) were added sequentially to stirred Boc-D-Glu-O-t-Bu (4.00 g, 13.2 mmol) in CH₂Cl₂(75 mL) in ice-cooled solution. The resulting mixture was stirred at an ice-cold temperature for 20 minutes. Next, H-L-Trp-O-t-Bu. HCl (5.88 g, 19.8 mmol) and in CH were added dropwise over a period of 10 minutes₂Cl₂DIPEA (3.50 mL, 19.8 mmol) in (50 mL). The resulting mixture was stirred at an ice-cold temperature for 1 hour, and then it was warmed to room temperature and stirred overnight.

The reaction mixture was evaporated to dryness. The residual viscous oil was taken up in EtOAc (50 mL) and saturated NaHCO₃The organic layer was washed sequentially with solution (100 mL), 10% citric acid solution (100 mL), brine (100 mL), and water (100 mL). Organic layer in Na₂SO₄Dried, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel using a solvent gradient of a mixture of hexane and EtOAc (8/2, 7/3 and 6/4 ratios, v/v) as eluent to give the title product as an off-white solid (5.88 g, 82%).¹H NMR（DMSO-d₆）δppm：10.85（s,1H）、8.18（d,J=7.4Hz,1H）、7.50（d,J=7.7Hz,1H）、7.33（d,J=8.0Hz,1H）、7.14-7.05（m,3H）、6.99（t,J=7.1Hz,1H）、4.40（q,J=7.5Hz,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（m/z）568[M+Na]⁺；546[M+1]⁺(ii) a To C₂₉H₄₃N₃O₇.0.75H₂And O, performing calculation analysis: c, 62.29; h, 8.02; n, 7.51. The following are found: c, 62.43; h, 7.95; and N, 7.08. .

B. Synthesizing H-D-Glu- (-L-Trp-OH and monoammonium salt (1: 1).

The procedure described in example 1B was used. HCl gas was bubbled into a stirred, cold (ca. -5 ℃ C.) solution of Boc-D-Glu- (γ -L-Trp-O-t-Bu) - α -O-t-Bu (1.59 g, 2.91 mmol) obtained as described above in EtOAc (100 mL) for 45 minutes. The solution changed from colorless to gray yellow (cloudy yellow). The resulting mixture was stirred at an ice-cold temperature for 1 hour, then it was heated to room temperature, and stirred for another 2 hours. The reaction was complete as monitored by HPLC (column: Waters C18, 3.9X150mm, WAT046980, mobile phase: 0.035% HClO₄(pH =2 to 2.5) and acetonitrile, flow rate: 1mL/min, 8:210 to 8:270 nm).

The reaction mixture was concentrated to a solid under reduced pressure. The solid was dissolved in acetone and the volatiles were removed under reduced pressure. The latter procedure was repeated twice more. Isopropanol and ammonium hydroxide (28 to 30% NH) were used as eluents₄OH) (85/15 and 70/30 ratio, v/v) the residue was purified by column chromatography to yield the title product as an off-white foam (0.42 g, 39%). Mp120 to 130 ℃;¹H NMR（D₂O）δppm：7.67（d,J=7.9Hz,1H）、7.46（d,J=8.2Hz,1H）、7.18-7.22（m,2H）、7.13（t,J=7.1Hz,1H）、4.53（q,J=3.8Hz,1H）、3.45（t,J=5.8Hz,1H）、3.33（dd,J=14.7and4.75Hz,1H）、3.07（dd,J=14.7and8.8Hz,1H）、2.19-2.31（m,2H）、1.78-1.98（m,2H）；.¹³C NMR（D₂O）δppm：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（CH₂）、30.3（CH₂）、28.9（CH₂）；MS（m/z）334[Diacid+1]⁺。

example 11

L-isoglutamyl-D-tryptophan, the mono ammonium salt (1: 1) was synthesized.

A. Synthesis of Boc-L-Glu- ((-D-Trp-OMe) -alpha-O-t-Bu

The procedure described in example 1A was used. EDC (3.31 g, 17.3 mmol), HOBt (2.36 g, 17.5 mmol) and DIPEA (3.0 mL, 17.1 mmol) were added sequentially to Boc-L-Glu-O-t-Bu (3.45 g, 11.4 mmol) in stirred, cold CH₂Cl₂Solution (120 mL). The resulting mixture was stirred at ice-cold temperature for another 25 min. H-D-Trp-OMe.HCl (2.20 g, 7.40 mmol) and DIPEA (3.0 mL, 17.1 mmol) in CH were then added₂Cl₂(40 mL) of the solution. The resulting mixture was stirred at an ice-cold temperature for 1 hour, and then it was heated to room temperature and stirred overnight.

Following the general procedure as described in example 1A, purification of the residue by column chromatography using a solvent gradient of a mixture of hexane and EtOAc (8/2 and 6/4 ratio, v/v) as eluent yielded the title product as a white foam (4.25 g, 74%).¹H NMR（CDCl₃） ppm：8.64（s,1H）、7.53（d,J=7.7Hz,1H）、 7.33（d,J=8.0Hz,1H）、7.15（t,J=7.2Hz,1H）、7.08（t,J=7.5Hz,1H）、6.98（s,1H）、6.61（d,J=7.2Hz,1H）、5.30（d,J=7.8Hz,1H）、4.92（q,J=6.8Hz,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）；¹³C NMR（CDCl₃）δppm：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（CH₂）、28.9（CH₂）、28.4（CH₃）、28.1（CH₃）、27.7（CH₂）；MS（m/z）504[M+1]⁺(ii) a To C₂₆H₃₇N₃O₇.0.25H₂And O, carrying out analysis calculation: c, 61.46; h, 7.44; n, 8.27; the following are found: c, 61.36; h, 7.50; and N, 7.84. .

B. Synthesizing Boc-L-iGlu-D-Trp-OH.

A solution of NaOH (654 mg, 16.4 mmol) in water (20 mL) was added to a stirred solution of Boc-L-Glu- ((-D-Trp-OMe) - α -O-t-Bu (3.94 g, 7.82 mmol) in MeOH (50 mL.) the resulting solution was stirred overnight at room temperature 1N NaOH solution (150 mL) was added to the reaction mixture and the aqueous material was washed with EtOAc (3X 100 mL.) the aqueous layer was acidified to a pH of about 2 with 3N HCl solution followed by extraction with EtOAc (3X 100 mL.) the organic moieties were combined, Na₂SO₄Dried and concentrated under reduced pressure. Using CH as eluent₂Cl₂And MeOH (85/15, 70/3 ratio, v/v), by column chromatography on silica gel to give the title product as a pink foam (0.55 g, 97%).¹H NMR（MeOD-D₄） ppm：7.58（d,J=7.7Hz,1H）、7.31（d,J=8.0Hz,1H）、7.05-7.09（m,2H）、6.99（t,J=7.3Hz,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）； ¹³C NMR（MeOD-d₄） 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（CH2）、28.9（CH₃）、28.7（CH₂）； MS（m/z）490[M+1]⁺。

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

Bubbling HCl gas into CH₂Cl₂(20 mL) of a stirred, cold (approx. 0 ℃) solution of Boc-L-iGlu-D-Trp-OH (500 mg, 1.0 mmol) as described above in a solvent mixture with EtOAc (10 mL). The reaction mixture was stirred at ice-cold temperature for 1 hour. The reaction was complete as monitored by HPLC (column: Waters C18, 3.9X150mm, WAT046980, mobile phase: 0.035% HClO₄(pH =2.0 to 2.5) solvent gradient of a mixture with acetonitrile, flow rate: 1mL/min, 8:210 to 270 nm).

The reaction mixture was evaporated to dryness to a dark purple foam. Isopropanol and ammonium hydroxide (28 to 30% NH) were used as eluents₄OH) (85/15 and 70/30 ratio, v/v) by column chromatography to yield the title product as an orange viscous oil (333 mg, 88%).¹H NMR（D₂O）δppm：7.64（d,J=7.9Hz,1H）、7.44（d,J=8.0Hz,1H）、7.16-7.19（m,2H）、7.10（t,J=7.5Hz,1H）、4.52（q,J=3.7Hz,1H）、3.44（t,J=6.3Hz,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）；¹³C NMR（D₂O）δppm：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（CH₂）、30.2（CH₂）、28.9（CH₂）；MS（m/z）356[Diacid+Na]⁺、334[Diacid+1]⁺To C₁₆H₂₂N₄O₅.2.35H₂And O, carrying out analysis calculation: c, 48.94; h, 6.85; n, 14.27; the following are 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 four diastereoisomeric diacids were analyzed on a chiral column HPLC. The D, D-and L, D-diastereomers are from the above examples, whereas the D, L-isomer is from Bachem and the L, L-isomer is from Sigma. Analysis using a chiral HPLC column (table 1) showed that the obtained D-isoglutamyl-D-tryptophan did not contain the other diastereomers named (D, L), (L, L) and (L, D).

Table 1: HPLC analysis of H-iGlu-Trp-OH

The method A comprises the following steps:

column:TAG5μM，4.6x250mm

mobile phase: 20mM ammonium acetate (pH = 4.1)/MeOH (80/20)

Flow rate: 0.8mL/min

Detecting lambda: 222. 254, 282, 450nm

Column temperature: 45 deg.C

The method B comprises the following steps:

column: symmetry C18, part No.: WAT046980

Mobile phase: HClO₄（pH=2）/CH₃CN（85/15）

Flow rate: 1.0mL/min

Detecting lambda: 210 to 280nm

In method A, a sample is analyzed using a chiral column. The retention times for all four diastereomers are clearly different. In method B, the samples were analyzed using a normal reverse phase column, and there was no substantial difference in the retention time of the samples. The mono-ammonium salt of D-isoglutamyl-D-tryptophan disclosed in the present invention is stable after 2 years of storage. HPLC analysis by method B showed a purity of 99.8% at 254 nm.

Example 13

A. Preparation of H-D-Glu- (gamma-D-Trp-OMe) -alpha-OBzlHCl salt { (2R) -amino- (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl-ethylcarbamoyl ] -butyric acid benzyl ester hydrochloride }

Boc-D-Glu- (γ -D-Trp-OMe) - α -OBzl (20 g, 0.037 mol) and 100mL of methylene chloride were placed in a 250-mL3N round bottom flask equipped with a magnetic stir bar, which gave a clear solution when stirred. The solution was cooled to-10 ℃ in an ice-NaCl cooling bath. HCL gas was bubbled into the cold solution. The temperature during the reaction is in the range of-4 ℃ to-10 ℃. The reaction was completed in about 1 hour. A white solid appeared from the solution. The solid product was collected by filtration. The solid was washed with dichloromethane (40 mL x 2), air dried, then dried in a vacuum oven at 42 ℃ to give 16.4g (94%, HPLC purity 98.2%).¹H NMR（DMSO-d₆） ppm: 10.93 (s, 1H), 8.61 (b, 3H), 8.50 (d, J =7.4Hz, 1H), 7.47 (d, J =7.4Hz, 1H), 7.40-7.32 (m, 6H), 7.17 (s, 1H), 7.06 (t, J =7.4Hz, 1H), 6.97 (t, J =7.4Hz, 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 C185 mu m4.6x250 mm; mobile phase: a = aqueous phase: 4mM Tris, 2mM EDTA, ph7.4, B = organic phase: CH (CH)₃And (C) CN. Gradient program: b%: 0min.5%, 15min.55%, 30min.55%, 32min.5%, 40 min.5%. Flow rate: 1 ml/min; injection volume =5 μ Ι _; wavelength: 222. 254, 282 and 450 nm. R of the starting Material_t=25.1 min; r of the product_t=17.2min。

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

Boc-D-Glu- (γ -D-Trp-OMe) - α -OMe (2.8 g, 6.06 mmol) and methanol (30 mL) were placed in a 250-mL3N round bottom flask equipped with a magnetic stir bar to give a clear solution when stirred. The solution was cooled to-12 ℃ by an ice-NaCl cooling bath. HCL gas was bubbled into the cold solution. The temperature during the reaction is in the range of-12 ℃ to +9 ℃. The reaction was completed in about 30 minutes. About half of the reaction mixture was concentrated to dryness to give 1.3g of solid (HPLC purity 96.8%).¹H NMR（DMSO-d₆） ppm: 10.90 (s, 1H), 8.48-8.46 (m, 4H), 7.48 (d, J =7.8Hz, 1H), 7.34 (d, J =8.0Hz, 1H), 7.16 (s, 1H), 7.07 (t, J =7.4Hz, 1H), 6.98 (t, J =7.4Hz, 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 MSC185 μm 4.6X250mm; mobile phase: a = aqueous phase: 4mM Tris, 2mM EDTA, pH7.4; b = organic phase: CH (CH)₃And (C) CN. Gradient program: b%: 0min.5%, 15min.55%, 30min.55%, 32min.5%, 40 min.5%. Flow rate: 1 ml/min. Injection volume =5 μ Ι _; wavelength: 222. 254, 282, 450 nm; r of the starting Material_t=18.7min, R of product_t=13.0min。

C. Preparation of H-D-Glu- (gamma-D-Trp-OMe) -alpha-OBzlHCl salt { (2R) -amino- (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl-ethylcarbamoyl ] -butyric acid benzyl ester hydrochloride }

A solution of HCl in ethyl acetate was prepared with bubbling HCl gas by adding 50mL of concentrated HCl dropwise to concentrated H₂SO₄Thus, cold (0 ℃ C. to 4 ℃ C.) ethyl acetate (100 mL) was produced. This cold acid solution was placed in a dropping funnel and added to a mild suspension of Boc-D-Glu (D-Trp-OMe) -OBzl (36.45 g, 67.80 mmol) in 150mL ethyl acetate. A viscous suspension was obtained within 5min. After all the HCl solution was added, HCl gas was generated as described above and bubbled directly into the resulting suspension. The internal temperature was kept between 5 ℃ and 10 ℃ with ice cooling. The stirred suspension was maintained at 5 ℃ to 15 ℃ for 2.5 hours, then it was heated to room temperature. The progress of the reaction was monitored by TLC (hexane/EtOAc, 1/1, v/v as eluent). The solid was collected by suction filtration, washed with EtOAc (2 × 100 mL), and then dried in a vacuum oven overnight. A pale pink solid was obtained (27.49 g, 86% yield). Analytical data were similar to those reported in example 13A above, except that some residual EtOAc was monitored by 1H NMR. Residual EtOAc can be removed by air drying for 24 to 48 hours before drying in a vacuum oven.

D. Preparation of H-D-Glu- (gamma-D-Trp-OMe) -alpha-OBzlHCl salt { (2R) -amino- (4R) - [2- (1H-indol-3-yl) -1-methoxycarbonyl-ethylcarbamoyl ] -butyric acid benzyl ester hydrochloride }

The 3L round bottom flask was filled with Boc-D-Glu-OBzl (200.0 g, 0.593 mol) and EtOAc (1.6L). Diisopropylethylamine (206.5 mL, 1.186 mol) was added to the resulting white suspension. The internal temperature was about 21 ℃ and a viscous suspension was observed. The suspension gradually becomes thinner and dissolves within 15 minutes to give a clear solution. N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (136.4 g EDC, 0.711 mol) was then added as the suspension formed. HCl.H-D-TrpOMe (166.1 g, 0.652 mol) was added portionwise over 30 minutes. An exotherm was observed and the internal temperature reached 28 ℃. After 2 hours, the internal temperatureThe temperature was reduced to 25 ℃. The resulting suspension was stirred vigorously at room temperature overnight. The reaction mixture was diluted with EtOAc (200 mL), followed by washing sequentially with 1N HCl (600 mL) and 5% HCl solution (2X 256 mL). By TLC and¹h NMR concentrated and analyzed a sample of the crude mixture. The water content was 2.3% by Karl-Fisher test.

The contents of the flask were cooled in an ice bath and the internal temperature was about 0 ℃. HCl gas was bubbled into the cold solution for 1 hour. An exotherm was observed within 10 minutes after bubbling HCl gas, and the internal temperature reached 19 ℃. After bubbling for 20 minutes, the internal temperature was 22 ℃. The resulting suspension was stirred for a further 1 hour. The solid was collected by suction filtration, washed with EtOAc (2 x640 mL), then dried under vacuum to constant weight (243.8 g). The analytical data were similar to those reported in example 13A above. By passing¹H NMR also observed the presence of EtOAc (about 10% by proton incorporation). Trapped EtOAc can be removed by air drying the solid for 24 to 48 hours before oven drying.

Example 14

A solution of a base addition salt of D-isoglutamyl-D-tryptophan is prepared and converted to Thymodedepressin D-isoglutamyl-D-tryptophan.

Procedure 14A:

the starting material, H-D-Glu- (γ -D-Trp-OMe) - α -OBzl HCl salt (4.0 g, 8.4 mmol), was placed in a 250mL3N round bottom flask equipped with a magnetic stir bar. Methanol (20 mL) was added to give a clear solution. The solution was cooled to-10 ℃ with an ice-NaCl salt bath. NaOH solution (3N, 8.4mL,25.2 mmol) was added. The reaction was monitored using HPLC. After 2 hours, HPLC analysis of the reaction mixture indicated that the reaction was not complete. NaOH solution (3N, 1.4mL,4.2 mmol) was added. At this point, a total of 29.4mmol of NaOH were added. After a further 4 hours, HPLC analysis of the reaction mixture indicated a product in the reaction mixture of greater than 95.2%. The reaction was stopped. Using an ice-water bath in cold conditionsNext, the reaction mixture was acidified to pH6.5 by the addition of hydrochloric acid (6N, to 1.3mL,7.8 mmol). The resulting solution was concentrated to remove most of the methanol to a volume of 15 mL. The solution was washed with ethyl acetate (15 mL x 2). The solution was filtered and the filtrate was collected. The filtrate was further acidified to pH3 by addition of hydrochloric acid (6N to 1.3mL,7.8 mmol). At this time, about 15.6mmol of hydrochloric acid was used in total. When stirred at room temperature, a solid formed. The mixture was stirred overnight. The solid was collected by filtration. The solid was air-dried to give 2.4g of crude product. The solid was then placed back into the round bottom flask. Deionized water (15 mL) was added and the mixture was stirred for 2 hours. The solid was collected by filtration. The solid was again air dried and then placed back into the round bottom sintered plate again. Deionized water (15 mL) was added and the mixture was stirred for 1 hour. The solid was collected by filtration and then washed with ice-cold deionized water (6 mL x 3). The solid was confirmed to be chloride free by silver nitrate assay. The solid was air dried and then placed in a vacuum oven at 42 ℃ for 19 hours to give 1.3g (46%, HPLC purity 98.8%). The filtrate and the water wash solution were combined and concentrated for isolation of the second crop of product.¹H NMR（D₂O-NaOD,pH7.0） ppm：7.59（d,J=7.6Hz,1H）、7.38（d,J=7.6Hz,1H）、7.15-7.12（m,2H）、7.05（t,J=7.2Hz,1H）、4.47-4.44（m,1H）、3.40（t,J=6.1Hz,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（m/z）334.3[M+1]⁺. HPLC method: column: XTerra MS C185. mu. m4.6X250mm. Mobile phase: a = aqueous phase: 4mM Tris, 2mM EDTA, pH7.4; b = organic phase: CH (CH)₃And (C) CN. Gradient program: b%: 0min.5%, 15min.55%, 30min.55%, 32min.5%, 40 min.5%. Flow rate: 1 ml/min. Injection volume =5 μ L. Wavelength: 222. 254, 282 and 450 nm. R of the product_t=6.5min。

Program 14B:

lithium hydroxide monohydrate (0.374 g,8.9 mmol) was dissolved in 3.5mL of deionized water. The solution was placed in a 100mL1N round bottom flask equipped with a magnetic stir bar. 6.5mL of methyl tert-butyl ether was added to the solution. The starting material H-D-Glu- (γ -D-Trp-OMe) - α -OBzl HCl salt (2.0 g, 4.2 mmol) was added at room temperature to form a suspension. Methanol (2 mL) was added and most of the solid was dissolved. The reaction was monitored using HPLC. The starting material was still present in the reaction mixture after stirring overnight at room temperature. Lithium hydroxide monohydrate (0.190 g, 4.5 mmol) was dissolved in 2mL of deionized water and added to the reaction mixture after addition of 2mL of methanol. At this point, a total of 13.4mmol of LiOH was added. After 4 hours, HPLC analysis of the reaction mixture indicated that the reaction was not complete. Lithium hydroxide monohydrate (0.100 g, 2.4 mmol) was dissolved in 1mL of deionized water and added to the reaction mixture. At this point, a total of 15.8mmol of LiOH was added. After a further 2.5 hours, HPLC analysis of the reaction mixture indicated a product in the reaction mixture of greater than 97.5%. The reaction was stopped. The solution was poured into a separatory funnel and the 2 phases were separated. The aqueous phase was washed with ethyl acetate (15 mL x 2). The aqueous phase was acidified to pH6 by the addition of hydrochloric acid (6N, ca. 650. mu.L, 3.9 mmol) using an ice-water bath under cooling conditions. The aqueous phase was concentrated to 5mL and filtered and the filtrate collected. The filtrate was further acidified to pH3 by addition of hydrochloric acid (6N, ca. 700. mu.L, 4.2 mmol). At this time, about 8.1mmol of hydrochloric acid was used in total. When stirred at room temperature, a solid formed. The solid was collected by filtration. The solid was air dried and then placed back into the round bottom flask. Deionized water (6 mL) was added and the mixture was stirred for 15 minutes. The solid was collected by filtration and then washed with ice-cold deionized water (6 mL x 6). The solid was confirmed to be chloride free by silver nitrate assay. The solid was air dried and then placed in a vacuum oven at 42 ℃ for 12 hours to give 0.44g (31%, HPLC purity 98.5%). The filtrate and the water wash solution were combined and concentrated for further isolation of the second crop of product.¹H NMR（D₂O-NaOD,pH6.0） ppm：7.59（d,J=7.7Hz,1H）、7.38（d,J=7.7Hz,1H）、7.15-7.12（m,2H）、7.05（t,J=7.2Hz,1H）、4.47-4.44（m,1H）、3.41（t,J=6.0Hz,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 C185. mu. m4.6X250mm. Mobile phase: a = aqueous phase: 4mM Tris, 2mM EDTA, pH 7.4: b = organic phase: CH (CH)₃CN。

Gradient program: b%: 0min.5%, 15min.55%, 30min.55%, 32min.5%, 40 min.5%. 1 ml/min. Injection volume =5 μ L. Wavelength: 222. 254, 282 and 450 nm. R of the product_t=6.5min。

Thymodepressin prepared according to the invention has a solubility in water of from about 20mg to 23mg per ml of water. The water wash in procedures a & B serves to remove inorganic salts (e.g. sodium chloride or lithium chloride). In large scale preparations, the volume of the aqueous wash (aqueous washing) can be controlled by calculating the amount of inorganic salt present and using solubility to determine the amount of water required to wash the product.

As many changes can be made in the preferred embodiments of the invention without departing from the scope thereof, it is intended that all matter contained herein be interpreted as illustrative and not in a limiting sense.

Claims (17)

1.H-D-Glu-(γ-D-Trp-OR²)-α-OR¹Of (a) a compound

And pharmaceutically acceptable acid addition salts thereof, wherein R¹And R²Each independently selected from benzyl and C₁-C₄An alkyl group.

2. The compound of claim 1, wherein said C is₁-C₄The alkyl group is selected from methyl, ethyl, isopropyl and tert-butyl.

3. The compound of claim 1 or 2, wherein R¹And R²The same is true.

4. A compound according to any one of claims 1 to 3, wherein R is¹And R²Is methyl.

5. A compound according to any one of claims 1 to 3, wherein R is¹And R²Is ethyl.

6. A compound according to any one of claims 1 to 3, wherein R is¹And R²Is isopropyl.

7. A compound according to any one of claims 1 to 3, wherein R is¹And R²Is benzyl.

8. A compound according to any one of claims 1 to 3, wherein R is¹And R²Is a tert-butyl group.

9. A compound according to any one of claims 1 to 3, wherein R is¹Is ethyl, and R²Is methyl.

10. The compound of claim 1 or 2, wherein R¹Is ethyl, and R²Is isopropyl.

11. The compound of claim 1 or 2, wherein R¹Is tert-butyl, and R²Is isopropyl.

12. The compound of claim 1 or 2, wherein R¹Is benzyl, and R²Is ethyl.

13. A compound according to claim 1 or 2, wherein if R is¹Is benzyl or methyl, then R²Is ethyl, isopropyl or tert-butyl.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and at least one pharmaceutically acceptable excipient.

15. Use of a compound according to any one of claims 1 to 13 in the manufacture of an anti-psoriasis agent.

16. A process for preparing a pharmaceutical composition comprising a compound of any one of claims 1 to 13 and at least one pharmaceutically acceptable excipient comprising mixing the compound with the at least one pharmaceutically acceptable excipient.

17. Use of a compound according to any one of claims 1 to 13 in the preparation of an immunosuppressant.