KR20010099867A - Synthesis of histamine dihydrochloride - Google Patents

Abstract

The present invention relates to a method for preparing pharmaceutical grade histamine dihydrochloride using a two step non-enzymatic synthesis method. The present invention discloses a method for synthesizing histamine dihydrochloride by non-enzymatically decarboxylating histidine and then converting the decarboxylated product into the form of a dihydrochloride salt. In the present invention, the final product of histamine dihydrochloride containing 0.8% or less of L-histidine HCl monohydrate, 0.1% or less of impurities in each chromatography, and 2% or less of total impurities is acceptable for pharmaceutical use. watching.

Description

Synthesis method of histamine dihydrochloride {SYNTHESIS OF HISTAMINE DIHYDROCHLORIDE}

Histamine is a compound that possesses important biological activity mediated by pharmacological receptors. Histamine has long been treated as a molecule, mainly with negative biological effects. Recently, however, new uses of histamine as a potent pharmaceutical formulation have been discovered. For example, histamine activates NK cells with interferon a in the presence of monocytes. See US Pat. No. 5,728,378. In order to take full advantage of the therapeutic properties of histamine, it is necessary to obtain large amounts of pharmaceutical grade compounds.

Histamine is a naturally occurring substance as a result of the decay process and its derivative, histamine dihydrochloride, is currently commercially available as a standard for assays and as a component of certain allergy diagnostic kits. The source of histamine is often natural and contains many contaminants and is not suitable for pharmaceutical use. In addition, methods for synthesizing histamine dihydrochloride are also known in the art.

Histamine dihydrochloride can be readily synthesized by decarboxylation of histidine. Using this synthetic method, the histamine is decarboxylated to form the dihydrochloride salt of the molecule. For example, Hashimoto et al. Have discussed a method for preparing histamine using cyclohexanone as a catalyst for decarboxylation of histidine (Hashimoto, M., et al., Chemistry Letters, 893-896 (1986)). In Hashimoto et al., A reaction involving 1% v / v 2-cyclohexen-1-one in 10 parts of histidine and reflux (26 hours) cyclohexanol using 2-cyclohexen-1-one as a catalyst Has been reported for the separation of histamine dihydrochloride in 95% yield. The Hashimoto method also teaches using toluene to precipitate and collect the final histamine dihydrochloride product and foaming HCl gas with the resulting decarboxylation solution.

Attempts to reconstruct the Hashimoto method to produce pharmaceutically pure amounts of histamine have failed. An additional amount of catalyst is required to carry out the process, and there is a significant amount of impurities in the final product. Moreover, the impurities are difficult to remove. From these results, it can be seen that the Hashimoto method is not suitable as a method for producing a large amount of pharmaceutically acceptable histamine.

A method of using acetophenone as a catalyst for decarboxylation of histamine has also been reported. Thus, the inventor reconstructed the method described in Japanese Patent No. 05,255,204 (1983) to Akimasa et al., And used 10 parts of diethylene glycol and 0.26 equivalents of acetophenone as a solvent for the decarboxylation reaction. Although the method of Akimasa et al. Is more efficient at converting histidine to histamine, the method cannot produce a pharmaceutical grade product consistently. Similar to the final product using the Hashimoto method, impurities were observed in the final product formed using the Akimasa method during HPLC analysis.

Although the conditions using acetophenone and diethylene glycol appear promising, there are also related problems when performing. Since both histamine free base and dihydrochloride salts can be readily dissolved in water, using any extraction technique that separates the product separated from the diethylene glycol solvent, usually by water extraction, It is difficult. In addition, since the histamine dihydrochloride is also readily dissolved in diethylene glycol, it is not possible to use a direct separation method by filtration.

Reaction conditions such as Takano, including pentan-3-one, were also reconstructed [ Heterocycles , 6: 1167 (1977)]. The results obtained from the experiments showed that the acetophenone conditions were not improved at all.

There is a need for a constant supply of pharmaceutical grade histamine, especially in view of the pharmaceutical use for newly discovered histamine. The standard method used by those skilled in the art of purifying histamine in its pure form from natural sources is also unable to produce histamine that is high enough for pharmaceutical use. Moreover, synthetic methods performed in the art also cannot produce histamine of sufficiently high quality. Therefore, there is a need in the art for an improved method of preparing pharmaceutical grade histamine dihydrochloride.

The invention disclosed herein relates to a method for preparing pharmaceutical grade histamine dihydrochloride using a two step non-enzymatic synthesis method. One embodiment of the present invention comprises the steps of decarboxylating a L- histidine containing solution under conditions where no decarboxylase is present to produce a histamine containing solution; Producing histamine monohydrochloride from the histamine containing solution; And generating a histamine dihydrochloride containing solution from a histamine monohydrochloride containing solution.

1 illustrates a method of reaction taught in the art.

2 shows a method of the invention disclosed herein referred to in Examples 5 and 6.

Summary of the Invention

The invention disclosed herein relates to a method for preparing pharmaceutical grade histamine dihydrochloride using a two step non-enzymatic synthesis method. One embodiment of the present invention comprises the steps of decarboxylating a L- histidine containing solution under conditions where no decarboxylase is present to produce a histamine containing solution; Producing histamine monohydrochloride from the histamine containing solution; And generating a histamine dihydrochloride containing solution from a histamine monohydrochloride containing solution.

One aspect of this embodiment includes pulverizing the histamine containing solution, for example, the histamine containing solution can be pulverized with methylene chloride solution. In another aspect of this embodiment, the histamine monohydrochloride containing solution is produced by adding an effective amount of hydrochloric acid in the isopropanol solution. For example, an effective amount of hydrochloric acid means the case where the molar equivalent of hydrochloric acid with respect to a histamine base is about 0.1-0.9. In another example, an effective amount of hydrochloric acid means when the hydrochloric acid is about 0.6 molar equivalents relative to the histamine base. Another aspect of this embodiment further comprises the step of separating the pharmaceutical grade histamine dihydrochlorai from the histamine dihydrochloride containing solution.

Another embodiment of the invention disclosed herein comprises decarboxylating an L-histidine containing solution under conditions where no decarboxylase is present to produce a histamine containing solution; Generating a histamine monohydrochloride containing solution from the histamine containing solution; Generating a histamine dihydrochloride containing solution from the histamine monohydrochloride containing solution; And separating the histamine dihydrochloride from the histamine dihydrochloride-containing solution.

In one aspect of this embodiment, the histamine dihydrochloride contains 0.8% or less of L-histidine HCl monohydrate, 0.1% or less of each chromatography impurity, and 2% or less of total impurities.

Detailed description of the invention

The invention disclosed herein relates to a method for preparing pharmaceutical grade histamine dihydrochloride using a two step non-enzymatic synthesis method. The invention disclosed herein describes a method for non-enzymatically decarboxylating histidine to synthesize histamine dihydrochloride and then converting the decarboxylated product to the form of the dihydrochloride salt. In the invention disclosed herein, the final product of histamine dihydrochloride contains 0.8% or less of L-histidine HCl monohydrate, 0.1% or less of each chromatographic impurity (as defined below), and 2% or less of total impurities. It is considered to be acceptable for pharmaceutical use.

Synthetic methods of histamine dihydrochloride known in the art do not yield a pure product sufficient to be used as a pharmaceutical compound. 1 illustrates a decarboxylation method taught in the art. The steps of the inventive method disclosed herein are shown in FIG.

Prior art methods have been used to produce histamine dihydrochloride from L-histidine starting materials in an attempt to produce pharmaceutical grade synthetic histamine dihydrochloride. The starting material was reacted with the prior art catalysts, α-tetraron and cyclohexane. After completion of the reaction, the sample was cooled and hydrochloric acid was bubbled into the solution to convert the histamine base into the form of the dihydrochloride salt. The precipitate formed was then filtered, washed and dried. The final product produced by the methods of the prior art was found to contain unacceptably much contaminants.

The purity of the crude material produced using the prior art methods was 92-94%, of which the main impurities were 3-5% and the remaining 5-8 impurities accounted for ≥0.1%. It is substantially effective to carry out further purification or recrystallization steps to substantially remove most of these contaminants. Nevertheless, two unidentified impurities may remain at levels above 0.1%. These impurities are released after the histamine dihydrochloride is produced and are called chromatographic impurities or chromatographic contaminants. Thus, the product produced by this method is not suitable for pharmaceutical use.

Based on the above results, a novel method of synthesizing histamine dihydrochloride with the desired purity has been devised. This novel method involves the decarboxylation of L-histidine (α-amino-4 (or 5) -imidazolpropionic acid (C ₆ H ₉ N ₃ O ₂ ) to produce histamine. The solution containing the muhistamine base was triturated with methylene chloride to precipitate the product, which was then washed by filtration The filtered product was then treated with hydrochloric acid in isopropanol to precipitate the crude histamine monohydrochloride salt. The product was isolated by filtration The crude salt can then be purified by recrystallization techniques or proceed to the final modification of the process of the invention Next, the monohydrochloride salt form is again treated with hydrochloric acid / isopropanol solution Resulting in the histamine dihydrochloride salt of the molecule followed by decolorization and The steps, i.e. steps called recrystallization, may be excessively repeated to obtain histamine dihydrochloride with pharmaceutical purity, all of which are performed under a nitrogen gas atmosphere. It was analyzed by a number of analytical methods including the analytical method.

The invention disclosed herein contemplates the use of a number of catalysts or radical initiators to promote the decarboxylation reaction. Suitable catalysts are those which effectively catalyze the decarboxylation of histamine when the precursor compound is in a neutral solvent and heated for a long time to produce a final product with acceptable purity. Electron-concentrated ketones that have the property of reducing large amounts of impurities present in the final product are preferred. For example, a suitable group of catalysts are benzoyl peroxide, 2,2'-azobisisobutyronitrile (AIBN), 2-cyclohexen-1-one, acetophenone, 4'-bromoacetophenone, benzophenone, p Nitroacetophenone, p-methylacetophenone, p-methoxyacetophenone, p-methylacetophenone / 1-methyl-4-piperidone and p-methylacetophenone / AcOH.

Decarboxylation reaction conditions promote decarboxylation of the starting material while minimizing the formation of undesirable contaminants. The reaction conditions include performing several steps in the presence of an inert gas, for example nitrogen. The reaction conditions further include, for example, performing a decarboxylation step in a temperature range of about 145 to 170 ° C. The reaction is preferably carried out at a temperature range of about 150 to 165 ° C, or about 160 to 165 ° C.

Many solvents are contemplated for use in the invention disclosed herein. Solvents that advance any step of the reaction can act on the reaction time required to catalyze the decarboxylation of histidine. Solvents that may be used in the invention disclosed herein include cyclohexanol, n-methylpyrrolidinone (NMP), di (ethylene glycol), di (ethylene glycol) methyl ether, 2-methyloxyethyl ether, 1-butanol, Methoxyethanol, cyclohexanol / NMP (3: 1 ratio), dimethylformamide and tetramethylenesulphone.

Another variable of the reactions disclosed herein is the method of treating the reaction mixture with hydrogen chloride to produce histamine in salt form. The impurity profile of the final product was found to be affected by the molar equivalents of acid added during the precipitation of the monohydrochloride crude salt. A hydrogen chloride solution having a known concentration in isopropanol can be prepared and the reaction mixture can be treated to control the degree of generation of impurities.

A molar equivalent of hydrogen chloride (HCl) in a range of isopropanol (ISA) can be used to practice the methods of the invention disclosed herein. A molar equivalent range of about 0.01 to 2 may be applied to produce the salt form of histamine. Alternatively, a molar equivalent range of about 0.05 to 1.4 can also be applied. In another example, a molar equivalent range of about 0.1-0.9 can be applied. In another example, about 0.5 molar equivalents may be applied. The ratio selected for carrying out the invention disclosed herein should ultimately produce an end product in which the level of impurities is acceptable so that the end product can be used as a pharmaceutical composition.

The concentration of the acidic solution used to produce the salt form is not critical. For example, the HCl concentration in ISA is about 6-9 N. However, the number of moles of acid introduced is important for separating the final product in a pharmaceutically acceptable grade. If an excessive amount of acid is added, impurities that are very difficult to remove when the histamine dihydrochloride salt is subsequently produced can precipitate with the monohydrochloride salt. The relationship between salt formation methods and pollutant production methods is not known in the art.

During the addition of HCl in isopropanol a number of co-solvents are used to precipitate the monohydrochloride salt form of the molecule (salt precipitate). Co-solvents that can be used in the invention disclosed herein include methylene chloride, cyclohexanol, toluene and tertiary butyl methyl ether (TBME).

Of particular interest is the final purity of the final product. Also contemplated are steps of a further method of purifying the final product. For example, recrystallization is an repeated crystallization process to purify the material. Many solvents for use in such purification methods are contemplated. The solvents include methyl chloride, 2-propanol, methanol, ethanol (ETOH), methanol / acetone, water, methanol / ethyl acetate, water / acetone, methanol / ethanol, water / methanol, methanol / hexane, water / methanol / acetone, Methanol / methylene chloride, 2-propanol / ethanol, methanol / 2-propanol, acetone / 2-propanol, acetone / ethanol. From a toxicological point of view, non-toxic solvents such as ETOH are preferred.

It was observed that many of the solutions obtained in the synthetic route were colored. Activated carbon may be added before or at the same time as the recrystallization process to remove the color.

The invention disclosed herein contemplates an inductive chemical reaction to aid in the purification of histamine dihydrochloride. Accordingly, it should also be taken into account that the chemical derivatives of the various impurities are produced in the histamine dihydrochloride production method of the present invention which promotes the removal of the impurities. The production of such derivatives involves the addition of tertiary butoxycarbonyl groups to the target molecule. Other modification groups such as benzyloxycarbonyl group (CBZ) are also contemplated.

The following examples refer to methods for decarboxylation of histamine and separation of histamine products. Also mentioned are methods for purifying crude histamine dihydrochloride product using multiple recrystallization steps. Mention is also made of charcoal bleaching methods.

The efficiency of the various steps of this method and the purity of the final product can be analyzed by the methods described below. One or more monitoring steps may be used to analyze the efficiency of the decarboxylation step. In addition, many assays well known in the art can be used to analyze the purity of the final product. An example of such a monitoring step is the process well known in the art, ie, thin layer chromatography (TLC) on a number of reaction products. For example, the reactions can be monitored using TLC (mobile phase = CH ₃ CN: H ₂ O: NH ₄ OH; 7.5: 2.0: 0.5; and ninhydrin spray). The monitoring step may be performed at any point after the decarboxylation step.

Specific embodiments of the invention are described in detail below. The following examples are for illustrative purposes only and should not be construed as limiting the claims of the invention. There are a number of alternative techniques and methods well known to those skilled in the art in which the invention of the invention is successfully performed in a similar form.

Preparation of Histamine Dihydrochloride

The following example describes a method for synthesizing histamine dihydrochloride from the precursor compound L-histidine. Existing histamine synthesis methods have the limitation that while histamine dihydrochloride can be obtained, an impure end product is produced. The following examples describe a number of improvements in the synthesis of histamine dihydrochloride and teach how to prepare a pharmaceutically acceptable grade of histamine dihydrochloride.

Example 1

Preparation of 500 g of crude histamine dihydrochloride

Hereinafter, a method of synthesizing 500 g of histamine dihydrochloride sample will be described.

A 12 liter four-necked round bottom flask equipped with a thermometer, mechanical stirrer, condenser and nitrogen foamer was charged with 7.5 l of cyclohexanol (solvent), 750 g of L-histidine (substrate) and 113 ml of acetophenone (catalyst). The suspension was continued to stir under nitrogen atmosphere while the reaction proceeded.

The suspension was then heated to reflux and maintained at a temperature (150-165 ° C.) for at least 40 hours. In order to perform an in-process assay, a small amount of samples was collected and the degree of decarboxylation of histidine was measured. The suspension was then cooled to below 80 ° C. and filled with 1875 mL of toluene. Further the mixture was cooled to room temperature. The mixture was filtered through a Buchner funnel into a fresh 12 L four neck round bottom flask.

The new flask containing the filtrate was equipped with a thermometer, mechanical stirrer, hydrogen chloride trap and vacuum trap, and then hydrogen chloride gas was added. After stirring, the solution was cooled to 10 ° C or lower. The batch temperature was kept below 20 ° C. and charged with at least 441 g (2.5 equivalents) of hydrogen chloride gas. After complete addition of the hydrogen chloride gas, the resulting pale yellow suspension was stirred at room temperature for 1 hour.

The suspension was then filtered again through a Buchner funnel. Thereafter, the filter cake was washed with a mixture of 375 ml of cyclohexanol and 375 ml of toluene, then washed twice with 750 ml of toluene and twice with 750 ml of hexane. The cake was dried by suction on the filter for at least 30 minutes. The filter cake contained a significant amount of cyclohexanol that was removed through the grinding process.

A 12 liter four necked round bottom flask equipped with a mechanical stirrer and nitrogen foamer was charged with the wet filter cake. 7.5 liters of ethanol (ETOH) were charged. The suspension was stirred at rt for 4 h. The suspension was then filtered with a Buchner funnel and the filter cake was washed with 400 ml of hexane. The filter cake was then dried overnight in a vacuum oven at 60-65 ° C. The product of this method (purity measured using high performance liquid chromatography (HPLC) was 94.4% a / a) produced 504 g of crude histamine dihydrochloride (yield 56.6%), which was recrystallized to give a final product. The purity of the product was improved.

A 12 liter four necked round bottom flask equipped with a thermometer, mechanical stirrer, condenser, addition funnel and nitrogen foamer was charged with 503 g of crude histamine dihydrochloride product synthesized as described above. In addition, 4.5 liters of ETOH and 200 ml of water were added to the reaction flask to dissolve the filter cake. The suspension was stirred under a nitrogen atmosphere.

The suspension was then heated to reflux. The suspension was kept under reflux and charged to the suspension by dropwise addition of water until most of the solid was dissolved. The solution was then cooled to 75 ° C. or below. The solution was charged with a mixture of 50 g of NUCHAR SA (West Baco, NY, NY) and 50 g of CELITE (J.T. Baker, Hayward, CA). The suspension was then heated to reflux and maintained at this temperature for 30 minutes. The suspension was cooled to 65-75 ° C. and again filtered through a CELITE layer into a clean, dried 12 L four neck round bottom flask. The filter cake was washed with a mixture of 450 ml ETOH and 50 ml water.

The filtered solution was slowly cooled to room temperature with stirring overnight. The solution was then further cooled to 0-5 ° C. for 2 hours. At 0-5 ° C. the suspension was filtered with a Buchner funnel. The filter cake was washed three times with 200 ml of ETOH cooled to 0-5 占 폚. The filter cake was then dried in a vacuum oven at 60-65 ° C.

After recrystallization, 299 g (59.4% yield) of the final product with HPLC purity of 99.1% a / a were obtained. The HPLC method is discussed in Example 7 below. Recrystallization was performed several more times to increase the purity of the final product. However, two unknown impurities (RRt, 1.3, 1.5) were still present above the threshold level of 0.1% even after recrystallization.

Typically, the purity of the first impurity (RRt 1.3) was 0.2 to 0.4% a / a, and the purity of the second impurity (RRt 1.5) was 0.5 to 0.6% a / a. As a result of two recrystallizations of the above-described samples, the respective impurity levels were reduced to 0.1 to 0.2% and 0.4 to 0.5%, respectively. When the samples were re-analyzed after several days, the impurities appeared to be grown, suggesting that they were related to the stability to the final product. The instability of the product can explain why the above-mentioned wet filter cake had an HPLC purity of 99.9% a / a but only 99.1% after drying the batch. Post-treatment or charcoal treatment with dichloromethane could not remove the impurities.

Example 2

Catalyst for decarboxylation of L-histidine

As can be seen from the above results, many modified synthesis methods could be performed. These modifications were performed to reduce the unknown impurities to acceptable levels. One modified review of the nature of the catalyst used in the decarboxylation reaction was performed. A number of other catalysts, including acetophenones, have been considered, allowing us to grasp the role of these catalysts in the formation of chromatographic impurities. Table 1 shows the catalysts used in this study. 0.3 equivalents of catalyst were used.

Study on Decarboxylation Catalyst catalyst Reaction time (h) HPLC Purity (% a / a) First impurity Second impurity Acetophenone (Control) 21 4.5 4.0 4'-bromoacetophenone 21 7.0 3.4 Benzophenone 21 14.5 2.9 p-nitroacetophenone 17 decomposition decomposition p-methylacetophenone 16 1.65 0.71 p-methyloxyacetophenone 16 1.9 2.9 p-methylacetophenone / 1-methyl-4-piperidone 7.5 3.0 2.4 p-methylacetophenone / AcOH 7.5 9.6 3.5

As can be seen from the results in Table 1, the impurity levels found in the final product were more reduced in p-methylacetophenone than in acetophenone. In contrast, the use of p-methylacetophenone with base (1-methyl-4-piperidone) did not increase the level of impurity generation, while the addition of acid (acetic acid) significantly increased the level of impurities in the final product. It can be seen that. In addition, p-methoxyacetophenone provided an advantage over acetophenone in the generation of contaminants but did not provide a significant advantage over p-methylacetophenone when separating monohydrochloride salts. The data suggest that catalysts with electron deficient ketones increase the impurity production found in the final product, while electron-enriched ketones reduce the impurity production. Based on these results, acetophenone is replaced by p-methylacetophenone when the catalyst is used in the decarboxylation reaction of the invention disclosed herein.

Example 3

Method of preparation of the form of histamine salt

Regarding the production of histamine dihydrochloride that is tolerably pure, other variables considered include the molar equivalents of acid added during precipitation of the crude salt. One of the surprising findings is the correlation between the reduction of the amount of contaminants present in the final product and the amount of acid used to produce the salt form of the molecule. In the process according to the prior art, 2.5 molar equivalents of hydrogen chloride (HCl) gas were introduced into a solution containing decarboxylated histidine (muhistamine base) to produce crude dihydrochloride salt. This example introduces an acid dissolved in isopropanol (ISA) to observe the effect of adding various molar equivalents of hydrochloric acid to the histamine base solution.

Various concentrations of HCl were dissolved in ISA and tested for their effect on impurity production. This synthesis was carried out in the same manner as described above, except that 0.3 equivalent of p-methylacetophenone and toluene were used as auxiliary solvents to add HCl. Impurity presence was measured using the HPLC method of Example 7 below. The concentration of the acid used at this time is shown in Table 2 below.

Equivalence of HCl and Their Effect on Impurity Generation Molar equivalents of HCl / IPA HPLC Purity (% a / a) First impurity Second impurity Condensation products 2 (control) 2.5 2.35 22.0 1.4 2.0 2.1 6.5 0.9 0.55 1.15 2.1 0.5 0.06 0.83 0.45

The results shown in Table 2 show how dramatically the amount of acid charged in the solution containing the histamine base alters the two impurity levels present in the product. It is thought that the decrease is observed because the impurity is inferior in base characteristics than in the case of the histamine base. As a result, it is believed that the histamine base is first protonated and then the impurities are protonated.

The use of 0.5 equivalents of HCl provides the best results in limiting the level of impurities found in the product. Under these conditions, the crude product isolated was a monohydride salt measured by titration to chlorine content. Thus, in order to synthesize the dihydrochloride form of histamine with an acceptable degree of purity, an intermediate purification step was employed, including producing the monohydride salt as intended. However, using this method, it is necessary to add additional equivalents of HCl to the later synthesis step in order to produce the dihydrochloride form of the molecule.

Further experiments were conducted to investigate the effect of small changes in acid concentration on product purity and yield. The results of the experiment are shown in Table 3. The results were obtained from the resulting product from 100 ml of reaction mixture with 0.3 equivalents of p-methylacetophenone and the co-solvent, CH ₂ Cl ₂ . The selection of the CH ₂ Cl ₂ is described in Example 4.

Small changes in acid equivalents and their effects on product formation Crude salt Final product HCl (equivalent) yield(%) HPLC Purity (% a / a) yield(%) HPLC Purity (% a / a) First impurity Second impurity First impurity Second impurity 0.57 43.3 0.05 0.14 57.7 0.03 0.07 0.67 50.6 0.06 0.17 61 0.05 0.10 0.76 53.5 0.09 0.20 59 0.05 0.1

The equivalent range was narrowed to investigate the effect of relatively small changes in acid content on the impurity profile of the crude salt. The data shown in Table 3 supports the previous observation that decreasing the amount of charged acid not only reduces the amount of impurities present in the final product, but also reduces the yield. Future experiments used 0.6 equivalents of HCl relative to the starting material. As the amount of starting material used increased, the amount of product also increased, and as measured by the assay, the required amount of acid was 0.85 molar equivalents of HCl per mole of free base. The calculated HCl amount was about 0.6 molar equivalents relative to the starting material L-histidine.

Example 4

Auxiliary solvent used during salt formation

The following variables investigated to improve the synthesis of histamine dihydrochloride relate to the co-solvent used in the acid addition step of the process. As in the previous case, toluene was used as an auxiliary solvent. In order to consider the possible effects of the cosolvent on the purity of the final product, various cosolvents were used in the precipitation step. As described above, the purity of the resulting samples was analyzed using the HPLC method described in Example 7. The results are shown in Table 4.

Effect of Methylene Chloride and Other Cosolvents on Final Product Purity Auxiliary solvent yield(%) HPLC Purity (% a / a) First impurity Second impurity Toluene (Control) 49.5 0.13 0.31 CH ₂ Cl ₂ 45.9 0.10 0.15 TBME 51.9 0.33 0.51 none 43.5 0.12 0.18

The reaction conditions for the results shown in Table 4 were 0.3 equivalent p-methylacetophenone, 0.6 equivalent HCl / IPA and 5 parts co-solvent for precipitation. As can be seen from the results in Table 4, methylene chloride provided superior results for impurity production compared to other co-solvents.

Example 5

Preparation of Crude Histamine Monohydrochloride

The method described below teaches how to prepare histamine monohydrides. A 2-liter three necked round bottom flask (reactor) was equipped with a thermometer, a mechanical stirrer, a condenser and a nitrogen purification system and charged with 1 liter of cyclohexanol, 100 g of L-histidine and 25.9 ml of p-methylacetophenone. The melting point of cyclohexanol was 22-22 ° C. and heating is required to produce a liquid that can be transferred to the reactor. The suspension turned white, with a temperature of 20-25 ° C. and a volume of 1050 ml. The suspension was stirred throughout the reaction in the presence of a nitrogen atmosphere.

The suspension was heated to reflux by heating (160-165 ° C.) and kept at reflux for 30 hours. A small amount of sample was taken to determine what percentage of the starting material was decarboxylated. The suspension contained L-histidine of ≦ 1% a / a. In the case of incomplete reaction, the sample was taken again after further heating for 3 to 5 hours while refluxing the suspension. The production of a clear and homogeneous solution suggests that the starting material has been consumed and the decarboxylation reaction is complete.

Once the reaction was complete, the suspension was cooled to about 20-25 ° C. The reactor was then charged with 300 ml of methylene chloride. The mixture was further cooled to room temperature. The mixture was then filtered using a Buchner funnel into another two liter three neck round bottom flask. The first reactor was then washed twice with 100 ml of methylene chloride used to clean the filter. The filtration step removed any residual L-histidine.

The second reactor containing the filtrate was equipped with a thermometer, a mechanical stirrer, an addition funnel and a nitrogen purification system. After performing a washing step and a restablishment step of nitrogen atmosphere in the reactor, the filtrate was heated to 30-35 ° C. A small amount of solution was taken and analyzed for the content of histamine base. The results obtained from this assay were used to determine the amount of acid needed to produce the monohydrochloride salt. The amount of acid required was 0.85 equivalents of HCl per mole of histamine base.

While stirring vigorously, 50.5 ml of a 7.65 M HCl isopropanol (HCl / ISA) solution was added dropwise at a rate such that the temperature of the solution did not exceed 40 ° C. In the step of the method with exothermic properties, HCl / ISA solution was added over time. The resulting light beige suspension was cooled to 20-25 ° C. over 1 hour and stirred for a minimum of 2 hours. 7.65 M HCl in isopropanol solution was prepared by blowing 27.9 g of HCl gas into 100 mL of isopropanol cooled to 5-10 ° C.

The cooled suspension was filtered through a Buchner funnel under a stream of nitrogen and the filter cake was washed three times with 100 ml of a 1: 1 solution of methylene chloride / cyclohexanol. The filter cake was then washed three times with 100 ml of methylene chloride. Since the monohydrochloride salt is easily soluble in water, laboratory humidity can affect the yield of the product. Therefore, the filter cake was operated under a nitrogen nitrogen stream to minimize exposure of the filter cake to moisture.

The wet filter cake was then charged to a 1 L three-necked round bottom flask equipped with thermometer, mechanical stirrer and nitrogen purification system for methylene grinding. The solid was suspended in 500 ml of methylene chloride and stirred under nitrogen atmosphere for 1 hour. As indicated in the subsequent steps described below, methylene grinding aided in the removal of residual cyclohexanol, which resulted in more efficient drying of the product.

Under a stream of nitrogen, the suspension was filtered and the solid material washed twice with 75 ml of methylene chloride. The filter cake was dried in a vacuum oven at 55-60 ° C. for 16 hours.

Table 5 below shows the results of the method described in this example. The method was carried out three times, after which the products obtained each time were compared.

Crude product yield of histamine monohydrochloride Experiment 1 Experiment 2 ^‡ Experiment 3 ^‡ Weight of anhydrous solids ^† 50.28 52.29 50.28 Crude product yield (%) 52.9 55.0 52.9

†: Solid weight corrected for solvent content and corresponding yield%

‡: In experiment 2 and 3, filter cake dried for 8 hours instead of 16 hours

Example 6

Preparation of Histamine Dihydrochloride by Decarboxylation of L-Histidine

Example 6 shows a method for synthesizing histamine dihydrochloride from the monohydrochloride precursor produced by the method of Example 5.

A 1 L three-necked round bottom flask (reactor) equipped with a mechanical stir bar, addition funnel, condenser, nitrogen purge system and thermometer was left in the heating mantle. The reactor was charged with 40 g of histamine monohydride, 32 ml of H ₂ O (distilled water), and 280 ml of a 1 × ETOH solution consisting of 99.5% ETOH and 0.5% toluene. The histamine monohydrochloride salt was very hygroscopic and therefore maintained under nitrogen atmosphere throughout the reaction.

The next step of the method involves adding HCl / ISA solution to convert the histamine monohydrochloride salt into the dihydrochloride form. To the reactor was added 41.5 mL of 6.85 M HCl / ISA solution (1.05 equiv). As mentioned above, since an acid solution was added, it generated heat, and acid was added over 15 minutes. In the early stages of the acid addition process, a clear solution was produced, but soon after about 75% of the acid was introduced, it soon turned into a pale off-white suspension.

After completion of the addition of the acid, the resulting cloudy off-white suspension was heated to reflux in an oil bath (78-80 ° C.). The solid material in the suspension was gradually dissolved to form an amber solution. Once the solid material was completely dissolved, the reactor was removed from the oil bath. The reactor was then charged with NUCHAR SA charcoal (2 g) and CELITE (2 g). The suspension was heated to reflux for 25 minutes. Since the product precipitates at about 60 ° C., it is important to maintain the temperature.

The hot, black suspension was filtered through a CELITE layer into a fresh 1 L three-necked round bottom flask equipped with a mechanical stirrer and thermometer. The CELITE layer was used as a barrier to prevent char flow through the filtration unit. The new reactor was preheated in an oil bath and also charged to the reactor in the oil bath.

The first reactor containing the reaction mixture was washed twice with 40 mL of ETOH 1X solution at a temperature between 60 and 65 ° C. The solution was filtered and then added to the filtrate prepared as above. The addition of the wash volume resulted in some precipitate in the filtrate. The total volume solution was then stirred at 60-65 ° C. for 30 minutes.

The suspension (histamine dihydrochloride) was then slowly cooled to 25 ° C. over 1 hour, stirred at 20-25 ° C. for 2 hours and again cooled to 0-5 ° C. for at least 2 hours. The suspension was then filtered under a stream of nitrogen and the filter cake washed three times with cold ETOH 1 ×, 40 mL. The filter cake was then weighed and dried in a vacuum oven at 55-60 ° C. for 16 hours. The results of three different experiments for converting histamine monohydrochloride to the dihydrochloride salt form are shown in Table 6.

Yield of Histamine Dihydrochloride Experiment 4 Experiment 5 Experiment 6 Wet Cake Weight (g) 48.8 45.6 46.4 Dry solid weight (g) 34.9 33.3 33.6 Yield ^- (%) 70 66.7 67.4

^-: Yield (%) value Is that are based on the histamine monohydrochloride corrected for solvent content.

Example 7

HPLC method for analyzing, confirming and measuring the purity of histamine dihydrochloride

This example discusses the use of HPLC to quantify and identify histamine dihydrochloride and to quantify degradation agents in the relevant and final products. The method takes a gradient and uses a complete HPLC system with UV detection capability. In chromatographic purity measurement, a system including a computerized data acquisition system is used. Other apparatus used include Waters Symmetry C-18, 5 μm, 4.6 × 350 mm columns; Analytical balances having a minimum measurement limit of 0.01 mg or 0.01 g; Glass containers in volume units; And column heaters. Reagents and criteria used herein include, but are not limited to, USP histamine dihydrochloride reference standards or equivalents; Methanol for HPLC; Acetonitrile for HPLC; HPLC or equivalent 1-heptane sulfonic acid, sodium salt from Fisher Scientific (Pittsburgh, Pennsylvania, USA); Sodium phosphate, monobasic, monohydrate of ACS reagent grade; D-, L-histidine monohydrochloride, monohydrate (Sigma, St. Louis, MO); 1N sodium hydroxide solution; 1N hydrochloric acid solution; Purified water; And benzyl alcohol, ACS reagent grades or equivalents.

Two mobile phase buffers were prepared. The pH of mobile phase A (MPA) containing 0.02 M sodium phosphate monobasic and 0.005 M heptanesulfonic acid was adjusted to 3.0. Mobile phase B (MPB) contained acetonitrile (ACN) / methanol (MeOH) at 20/15 (v / v).

Standards and samples were prepared for analysis and chromatographic purity measurements. Analytical standards include preparing a histamine dihydrochloride standard solution at three concentrations, 0.88 mg / ml, 0.80 mg / ml and 0.72 mg / ml. D, L-histidine monohydrochloride, monohydrate standard was prepared at 0.008 mg / ml. Similarly, analytical samples were prepared in duplicate to contain 0.8 mg / ml of synthesized histamine dihydrochloride, while a LOQ solution was prepared at 0.0006 mg / ml of histamine dihydrochloride. The sensitivity of the method to histamine was determined to be 0.07% for the limit of quantitation and 0.03% for the limit of detection. The results of the photodiode sequence peaks explain the specificity for histamine.

After preparing a number of standards and samples, the HPLC system was equilibrated. Once equilibrated, the flow rate from the discharge line was examined at the initial condition setting (ie, 10% MPB at 1.5 mL / min). The flow rate at this time was 1.5 ml / min ± 0.15 ml / min. Water blanks were injected after equilibrating the system to the conditions of the column prior to performing this assay.

Once this preparation was completed, 0.7 ± 0.1 mg / ml of histamine dihydrochloride as a sea solution was injected. The resolution "R" between the peak of the 1 mg / ml benzyl alcohol solution and the histamine peak was measured. In addition, the method was repeated five times to find the standard deviation.

In the assay, standard curves were drawn. Four standard tests were performed on six sample injections with the following parameters: That is, the tailing factor was not> 2.0; The resolution was> 1.5 and the relative standard deviation of the histamine peak was not> 2.0%; The correlation coefficient of the standard curve was greater than 0.995.

To correct the chromatographic purity, the aqueous solution was injected once. The resolution "R" between the benzyl alcohol and the histamine peak was measured to determine the tailing factor of the histamine peak. The resolution and tailing factors are shown in the specification and the LOQ samples were injected continuously.

Relative standard deviations for three histamine peak responses were measured. In general, the tailing factor was not> 2.0, the resolution was greater than 1.5, and the relative standard deviation of the histamine peak was not greater than 10.0%.

When these parameters were met, the final histamine dihydrochloride sample was tested. Operating parameters for HPLC are listed in Table 7. Gradient variables are listed in Table 8.

Working variables Flow rate 1.5 ml / min Injection volume 20 μl detection 212 nm column Water Symmetry Column C-18, 5 μm, 4.6 mm x 250 mm Column temperature 50 ℃ Assay concentration 0.8 mg / ml histamine dihydrochloride Working time About 30 minutes Mobile phase A Buffer Mobile phase B ACN / MeOH 20/15 (v / v)

Gradient variables Minutes % Mobile phase B Flow rate (ml / min) 0 10 1.5 20 30 1.5 21 10 1.5 30 10 1.5

Retention time: histidine = about 3 minutes

Histamine = about 12 minutes

Using the analytical system provided in the method, it is necessary to measure the purity of the histamine dihydrochloride sample produced in the above-described examples.

Example 8

HPLC analysis of histamine dihydrochloride product

The histamine dihydrochloride product obtained in Example 6 was subjected to the HPLC assay described in Example 7 to determine the purity of the sample and to determine whether the final product conformed to the standards of purity defined by the methods of the invention disclosed herein. Confirmed. For use as a pharmaceutical preparation, the histamine dihydrochloride should contain minimal chromatographic impurities. Each impurity present at levels above 0.1% a / a generally possesses toxicological properties. Three histamine dihydrochloride quotas were generated using the methods of Examples 5 and 6. Their purity is described in Table 9.

HPLC analysis results Experiment Impurity Description History / detection amount Experiment 1 L-histidine HCl monohydrate <0.8% w / w; not detected Each chromatographic impurity First impurity Second impurity <0.1% w / w <0.05% w / w 0.13% w / w Total Chromatography Impurities <2.0% w / w; 0.2% w / w Experiment 2 L-histidine HCl monohydrate <0.8% w / w; not detected Each chromatographic impurity First impurity Second impurity <0.1% w / w <0.05% w / w 0.10% w / w Total Chromatography Impurities <2.0% w / w; 0.2% w / w Experiment 3 L-histidine HCl monohydrate <0.8% w / w; not detected Each chromatographic impurity First impurity Second impurity <0.1% w / w <0.05% w / w 0.06% w / w Total Chromatography Impurities <2.0% w / w; 0.1% w / w

As can be seen from the results shown in Table 9, it can be seen that the final histamine dihydrochloride product establishes acceptable criteria for the invention disclosed herein. First, the level of the first impurity was below the limit of quantification in the assay. Second, the second impurity will be defined throughout the toxicological test as slightly above the 0.1% threshold. The impurity breakdown level was <0.2% w / w. These results indicate that the synthetic methods of the invention disclosed herein provide a means to synthesize pharmaceutically acceptable forms of histamine dihydrochloride.

conclusion

The invention disclosed herein describes a novel, non-enzymatic method for preparing pharmaceutical grade histamine dihydrochloride. One important advantage with the methods described herein is that histamine dihydrochloride is obtained at a higher level of purity than is acceptable.

Finally, the foregoing embodiments are not intended to limit the scope of the invention, which is set forth in the following claims. In particular, many equivalent ranges and alternative ranges will be recognized by those skilled in the art, in light of the foregoing disclosure, which are deemed to be within the scope of the disclosed invention.

Claims (9)

Decarboxylating the L-histidine containing solution under conditions free of decarboxylase to produce a histamine containing solution; Generating a histamine monohydrochloride containing solution from the histamine containing solution; And Generating a histamine dihydrochloride containing solution from a histamine monohydrochloride containing solution Synthesis method of histamine dihydrochloride comprising a. The method for synthesizing histamine dihydrochloride according to claim 1, further comprising pulverizing the histamine containing solution. The method for synthesizing histamine dihydrochloride according to claim 2, wherein the histamine containing solution is triturated with a methylene chloride solution. The method for synthesizing histamine dihydrochloride according to claim 1, wherein the solution containing the histamine monohydrochloride is produced by adding an effective amount of hydrochloric acid in the isopropanol solution. The method of claim 4, wherein the effective amount of hydrochloric acid is about 0.1-0.9 molar equivalents of hydrochloric acid relative to the histamine base. 5. The method of claim 4, wherein the effective amount of hydrochloric acid is about 0.6 molar equivalents of hydrochloric acid relative to histamine free base. The method of claim 1, further comprising separating pharmaceutical grade histamine dihydrochloride from the histamine dihydrochloride containing solution. Decarboxylating the L-histidine containing solution under conditions free of decarboxylase to produce a histamine containing solution; Generating a histamine monohydrochloride containing solution from the histamine containing solution; Generating a histamine dihydrochloride containing solution from a histamine monohydrochloride containing solution; And Separating histamine dihydrochloride from histamine dihydrochloride containing solution Method of synthesizing histamine dihydrochloride of the pharmaceutical grade comprising a. 9. The histamine dihydrochloride of claim 8, wherein the histamine dihydrochloride contains 0.8% or less of L-histidine HCl monohydrate, 0.1% or less of each chromatography impurity and 2% or less of total impurities. Synthetic method.