RU2484849C2 - Improved method for preparing hydrazides - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to improved methods for preparing a hydrazide product of hydrazine and acid chlorides, particularly to a method for preparing 3-methyl-3-mercaptobutanoic acid hydrazide. The method involves the stages as follows: (a) preparing a stirred, substantially uniform suspension containing hydrazine and an inert solvent, and (b) continuously adding acid chloride to said suspension, wherein acid chloride has the structure:

, wherein P represents a thiol-protective group; each of R₁ and R₂ are specified in a group consisting of C_1-C₅ alkyl; L represents an alkylene linker with continuously adding of the acid chloride solution adjusted so that a reaction temperature maintained within the range of -65°C to -75°C. The invention also refers to a method for preparing a hydrazine suspension involving the stages as follows: (a) cooling the inert solvent to a temperature within the range of -68°C to -75°C; and (b) adding hydrazine drop-by-drop to the specified cold inert solvent; and to a method for preparing an immunoconjugate of calicheamicin member and a monoclonal antibody as a carrier involving preparing monoacylated hydrazide.

EFFECT: method eliminates or limits the formation of the undesired bis-hydrazine side products.

31 cl, 15 ex, 9 tbl

Description

FIELD OF TECHNOLOGY

[0001] The present invention relates to an improved synthesis method for preparing hydrazides from hydrazine and acid chlorides. The resulting hydrazide products contain a protected thiol group, which is used to cross-link calicheimycin with monoclonal antibodies.

BACKGROUND

[0002] MYLOTARG® (gemtuzumab ozogamicin), also called CMA-676 or simply CMA, consists of a monoclonal antibody to the CD33 antigen, which is coupled to calichimycin via an acid-hydrolyzing linker group. When a calichimycin derivative binds to a small groove of DNA, the derivative disrupts the development of DNA and ultimately causes the death of cancer cells. The commercial product was introduced on the market as the first chemotherapeutic agent with targeted antibody, called MYLOTARG®, and is currently approved for the treatment of acute myelogenous leukemia (AML) in elderly patients.

[0003] Agents in the family of highly active antibacterial and antitumor agents, collectively known as calichemicins or the LL-E33288 complex, are described in US Pat. Nos. 4,974,198, 4,939,244 and 5,079,233. The agents in this family can be used to produce therapeutically useful immunoconjugates with monoclonal antibodies as carriers. These antibodies may be an antibody to the CD33 antigen (e.g., hp67.6), an antibody to CD22 antigen (e.g., G544), an antibody to Lewis Y antigen (e.g., G193), an antibody to 5T4 antigen (e.g., H8), or CD20 antigen (e.g. rituximab). An agent belonging to the calicheimycin family is preferably N-acylated calichimycin, preferably N-acetyl-gamma-calichimycin. Agents belonging to the calichimycin family contain methyltrisulfide, which can interact with the corresponding thiols to form disulfides, while at the same time providing the introduction of a hydrazide functional group suitable for attaching the calicheimycin derivative to the carrier. Examples of such a reaction with calichimycin are given in US patent No. 5053394. The calichimycin hydrazide derivative binds to the monoclonal antibody through the formation of hydrazone. For example, a general method for attaching hydrazide derivative drugs to oxidized antibodies is described by T.J. McKern and others (T.J. McKearn, et al.) In US Pat. No. 4,671,958. US patent No. 5770701 relates to a method for producing forms with directed action based on bisulfide compounds of the LL-E33288 complex. The linker, which is 4- (4-acetyl-phenoxy) butanoic acid, undergoes a condensation reaction with a hydrazide derivative of calichimycin, preferably an N-acetyl-γ-dimethylhydrazide derivative of calichimycin to form an acylhydrazone, which is then subjected to N-hydroxysuccinimide to obtain OSu ester (N-succinimidyloxy) suitable for conjugation with a selected biomacromolecule. Calichimycins contain an endiin end group, which is activated by the reduction of the -S-S- bond, leading to double strand DNA breaks. Thus, mono-acylated hydrazines, in which the acyl group contains a mercapto group, are well suited for binding of calichimycins to monoclonal antibodies. 3-Methyl-3-mercaptobutanoic acid hydrazide, also called a DMH linker or CL-332258, is a preferred mercapto containing N-acylhydrazine to bind calichimycin with monoclonal antibodies and produce, for example, gemtuzumab ozogamicaminogin or gemtuzumab ozogamicamin ) The calichimycin derivative is then activated to conjugate with a humanized monoclonal antibody to give CMA-676. Currently, the DMH linker can be obtained using a 5-step process through the formation of p-methoxybenzylthio-ester acid hydrazide 5 as an intermediate (Equations I-V). In modern industrial processes in the United States, Michael addition of p-methoxybenzylthiol to 3,3-dimethylacrylic acid is facilitated by the addition of piperidine (Equation I).

[0004] The obtained thioester acid (1) reacts with oxalyl chloride in methylene chloride to form p-methoxybenzylthioester acid chloride (2) (Equation II)

The acid chloride (2) is slowly added to the hydrazine / methylene chloride mixture (in a volume ratio of about 28%) at low temperature (-70 ° C). The corresponding p-methoxybenzylthioether hydrazide (3) is formed in approximately 74% yield (Equation III).

[0005] However, the target p-methoxybenzylthioether hydrazide product (3) typically contains about 20% of the undesired by-product of the methoxybenzylthioether bis-hydrazide (see Equation VI below). Removal of the protective benzyl group in an acidic medium (Equation IV) followed by neutralization of the acid salt and purification (Equation V) gives 3-methyl-3-mercaptobutanoic acid hydrazide (5) in 45% yield.

[0006] An undesired by-product of methoxybenzylthioether bis-hydrazide (6) is formed by reacting a product of p-methoxybenzylthioether hydrazide with the starting material, which is p-methoxybenzylthioester acid chloride (Equation VI). The formation of this by-product leads to a decrease in the yield and quality of the target product.

[0007] When using the initial production method, methoxybenzylthioether bis-hydrazide (6) is formed in an amount of about 20%. Obviously, the presence of such or even more by-product according to Equation III seems undesirable. The present invention provides a method to overcome this problem and reduce the yield of an undesirable by-product.

DESCRIPTION OF FIGURES

The figure 1 presents the technological scheme for the production of p-methoxybenzylthioether acid (1).

The figure 2 presents the technological scheme for the production of p-methoxybenzylthio-ester acid chloride (2) and p-methoxy-benzylthio-ester hydrazide (3).

The figure 3 presents the technological scheme for obtaining a crosslinking group DMH (5).

SUMMARY OF THE INVENTION

[0008] According to some embodiments of the present invention, methods for synthesizing hydrazides (eg, 3-methyl-3-mercaptobutanoic acid hydrazide (4)) while reducing the content of a by-product of methoxybenzylthioether bis-hydrazide (eg, (6)) are provided, c about 20% to about 3-5%. In one embodiment, the methoxybenzylthioether ester chloride solution (2) is added to the stirred hydrazine / methylene chloride mixture, which is more diluted than in the original method (for example, the volume ratio is about 14% versus 24-32% in the original method). The preferred dilution volume ratio is about 14%. In order to obtain a hydrazine-containing mixture to which the acid chloride is added, in the embodiments of the present invention, it is possible to use, without limitation, other non-reactive (or inert) halogenated solvents that can be used both in place of methylene chloride and with it. Preferred examples of such other solvents include carbon tetrachloride, chloroform, ethylene dichloride and chlorobenzene. In some embodiments, the amount of methylene chloride (or the corresponding inert solvent) is doubled, which significantly reduces the amount of unwanted bis-hydrazide by-product. In some embodiments, the methoxybenzylthioester acid chloride solution is added to the hydrazine / methylene chloride mixture continuously at a constant rate, rather than in parts. In some embodiments, the rate of addition is adjusted to maintain a reaction temperature in the range of -68 to -75 ° C. In some embodiments, a stirring rate of between 300-400 rpm in a round bottom flask or 270 rpm in a Morton flask is used. Some methods according to the present invention provide for the production of p-methoxybenzylthioether hydrazide (3) with a concentration of 91.1% and a yield of 85%, as well as with a methoxybenzylthioether bis-hydrazide (6) content of about 4-7%.

[0009] In another embodiment of the invention, it was found that despite the improvements achieved by using a more dilute methylene chloride system, there was still a need to scrap frozen crystallized hydrazine from the bottom and walls of the reaction vessel. Previously, it was standard practice to cool a methylene chloride / hydrazine solution to about -70 ° C. This led to significant amounts of hydrazine, which crystallized and precipitated on the walls of the vessel. To ensure the participation in the reaction of the entire amount of hydrazine, it was necessary to scrape off the substance from the walls of the vessel in order to ensure the possibility of the transition of this substance into a stirred suspension. To prevent such a situation, an alternative technique has been developed according to some embodiments of the present invention. This alternative technique involves cooling methylene chloride to a temperature of from -68 to -75 ° C, preferably to -70 ° C, followed by slow dropwise addition of hydrazine to the cooled methylene chloride to form a uniform suspension. Using this new technique, the formation of a much more uniform suspension of hydrazine is achieved, which minimizes the formation of crystallized hydrazine on the inner side of the flask walls and reduces or eliminates the need to scrap hydrazine from the flask. At the same time, the required amount of hydrazine is involved in the reaction, which helps to reduce the formation of methoxybenzylthioether bis-hydrazide (6).

[0010] In some embodiments of the invention, a method is provided comprising continuously adding a methoxybenzylthioether ester chloride solution (2) to a relatively dilute (compared to the known method) and stirred chilled heterogeneous hydrazine / methylene chloride mixture (preferably with a hydrazine concentration of about 14%). A solution of methoxybenzylthioester acid chloride is added to the hydrazine / methylene chloride suspension continuously at an approximately constant speed, rather than in portions. The rate of addition is adjusted so that the reaction temperature is from -68 to -75 ° C. The preferred stirring speed is 300-400 rpm in a round bottom flask or 270 rpm in a Morton flask. Improved methods according to some embodiments of the invention reduce the amount of a by-product of methoxybenzylthioether bis-hydrazide (6) from a previously achieved level of 20% to about 3-5% or less. An improved method for the synthesis of intermediate 1 provides an increase in the efficiency of the entire synthesis method of gemtuzumab ozogamicin.

[0011] In some embodiments of the invention, a method for producing hydrazide from hydrazine and an acid chloride is provided, comprising: a) preparing a stirred, uniform suspension of hydrazine and an inert solvent; b) continuously adding an acid chloride to said mixture. In another aspect of this embodiment, the acid chloride is added substantially dropwise to the suspension.

[0012] In some embodiments of the invention, a method for producing hydrazides from halides and hydrazines is provided. In this embodiment, the preparation is carried out by a chemical reaction between the electrophilic acylcarbonyl in the composition of the acid chloride and the nucleophilic nitrogen of hydrazine. Particular substituents attached to the acylcarbonyl suitable for the purposes of the present invention may be any moieties that do not interfere with hydrazide bond formation, including moieties containing protecting groups that eliminate interference with hydrazide bond formation. The use and removal of protecting groups is described in McOmie, Protecting Groups in Organic Chemistry, Plenum Press, NY, 1973, and Greene and Wuts, Protecting Groups in Organic Synthesis, 4nd. Ed., John Wiley & Sons, NY, 2006. For example, in some embodiments, the acid chloride contains a protected thiol. Examples of protected thiols include, but are not limited to, benzylthioesters.

[0013] In another aspect of the invention, acid chlorides have the structure:

,

,

where P is a thiol-protecting group, each of R ₁ and R _{2 is} selected from the group consisting of C ₁ -C ₅ alkyl, and L is an alkylene linker group. Examples of alkylene linker groups L include, but are not limited to, the linker group - (CH ₂ ) _n -, where n is an integer from 1 to 5. In some embodiments, R ₁ and R ₂ are the same and, for example, are identical C ₁ -C ₅ alkyls. Examples of C ₁ -C ₅ alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, including both linear and branched isomers of said alkyl. Examples of thiol protecting groups include, but are not limited to, a benzyl group in which the phenyl moiety may be substituted. Examples of possible substituents include, but are not limited to, an alkoxy group such as methoxy, ethoxy, etc. Thus, in one embodiment of the invention, the acid chloride has the following structure:

.

.

[0014] Another embodiment of the present invention is a method for producing hydrazide from hydrazine and an acid chloride, comprising, in a first step, preparing a stirred substantially uniform suspension comprising hydrazine and an inert solvent. In another aspect of this embodiment, the inert solvent is methylene chloride.

[0015] Another embodiment of the present invention is a method for producing a hydrazide from hydrazine and an acid chloride. In another aspect of this embodiment, the hydrazide product has the structure:

,

,

where P represents a thiol protecting group, and L, R ₁ and R ₂ may be as described above. In yet another aspect of this embodiment, P is a benzyl group optionally substituted on the phenolic ring. In another aspect of this embodiment, P is an n-methoxybenzyl group, R ₁ and R ₂ are selected from the group consisting of C ₁ -C ₅ alkyl, and L is an alkylene linker group. Examples of alkylene linker groups L include, but are not limited to the —CH ₂ - linker group. In one embodiment, each of R ₁ and R ₂ is independently methyl.

[0016] Another embodiment of the present invention are hydrazide products obtained in accordance with the methods of the present invention. In one embodiment, the implementation of the target hydrazide has the structure:

or is the corresponding salt. In yet another embodiment, the hydrazide is 3-methyl-3-mercaptobutanoic acid hydrazide.

[0017] In another aspect of the invention, the target hydrazide contains less than 5% bis-hydrazide by-product having the structure

,

,

where R and R ′ represent a possibly substituted alkyl, heteroalkyl or heteroalkaryl group.

[0018] In another aspect of this embodiment, each of the R and R ′ fragments in the bis-hydrazide by-product is

,

,

where P is a thiol-protecting group, each of R ₁ and R _{2 is} selected from the group consisting of C ₁ -C ₅ alkyl, and L is an alkylene linker group. Examples of alkylene linker groups L include, but are not limited to, —CH ₂ - groups. In another aspect of this embodiment, each of R ₁ and R ₂ is independently methyl. In another aspect of this embodiment, P is a benzyl group which may be substituted on the phenyl ring; examples include, but are not limited to, a p-methoxybenzyl group.

[0019] Another embodiment of the invention is a method for producing hydrazide from hydrazine and acid chloride, according to which the hydrazide product contains less than 5% bis-hydrazide by-product having the following structure:

[0020] Another embodiment of the present invention is a method for producing hydrazide from hydrazine and an acid chloride, comprising in the first step producing a stirred substantially uniform suspension containing hydrazine and an inert solvent, and in a second step, subsequent continuous addition of the acid chloride to said suspension. According to another aspect of this embodiment, the continuous addition of the acid chloride solution is controlled to maintain a reaction temperature in the range of about -68 ° C to about -75 ° C. In yet another aspect of this embodiment, the hydrazine suspension is substantially uniform.

[0021] In another embodiment, a hydrazide bond is prepared in accordance with a process comprising the steps of: a) cooling a reaction vessel containing a stirred inert solvent to a predetermined low temperature; b) continuously adding hydrazine to said reaction vessel to obtain a stirred substantially uniform suspension containing hydrazine and an inert solvent; c) continuously adding an acid chloride to said suspension to form a hydrazide bond. In another aspect of this embodiment, the inert solvent is methylene chloride.

[0022] In another embodiment, a hydrazine suspension is prepared by a process comprising the steps of: a) rapidly cooling an inert solvent to a temperature of from about -68 to about -75 ° C; and b) adding dropwise hydrazine dissolved in an inert solvent to said chilled inert solvent. In another aspect of this embodiment, the inert solvent is methylene chloride. In yet another aspect of this embodiment, the hydrazine suspension is mixed at a rate of from about 270 to about 400 rpm.

[0023] Another embodiment of the invention is a method for producing an immunoconjugate of an agent belonging to the calichimycin family with a monoclonal antibody as a carrier, said method comprising preparing mono-acylated hydrazine in which the acyl group contains an S-protected mercapto group according to the proposed method; removing said protective group and using the obtained hydrazide to obtain said immunoconjugate. Another embodiment of the invention is a method for producing gemtuzumab ozogamicin or inotuzumab ozogamicin, comprising the steps of producing a linker compound that is 3-methyl-3-mercaptobutanoic acid hydrazide in accordance with the method of the invention and using said linker to produce gemtuzumab ozogamicin or inotuzumab ozogamicin.

DETAILED DESCRIPTION OF THE INVENTION

[0024] p-Methoxybenzylthiol reacts Michael with 3,3-dimethylacrylic acid in piperidine. Amounts of reagents affect the yield of the reaction. In one embodiment, the amount of n-methoxybenzylthiol is in a small molar excess relative to the amount of 3,3-dimethylacrylic acid, for example, in the range of 0.354 kg (2.3 mol) and 0.362 kg (2.35 mol). If the amounts are less than the lower limit of this range, subsequent reactions may not proceed completely. If the amount is greater than the upper limit of this range, then an excess of reagents can complicate the process. The reaction mixture is heated so that its temperature does not exceed 98 ° C for at least 15 hours to prevent increased formation of impurities. Piperidine is removed by dilution with methylene chloride and washing with aqueous hydrochloric acid and then with water. Maintaining the temperature above 50 ° C, but below 90 ° C is necessary before and during the addition of HCl to prevent precipitation of the reaction product. The reaction mixture was further cooled and then extracted with methylene chloride, as indicated in the experimental part.

[0025] The amount of solvent used is proportional to the scale of the reaction for optimal results and purification. The resulting solution of the product in CH ₂ Cl _{2 was} dried with magnesium sulfate, purified, concentrated in vacuo, then diluted with heptane to precipitate the crude intermediate, which was filtered and washed with heptane. Purification is completed by re-dissolving the crude in methylene chloride and reprecipitation in heptane. The purified p-methoxybenzylthioether acid (1) is isolated by filtration, washed with heptane and dried under vacuum.

[0026] p-Methoxybenzylthioester acid (1) is converted to the corresponding acid chloride using oxalyl chloride and methylene chloride as solvent. Oxalyl chloride must be present in a molar excess with respect to n-methoxybenzylthioether acid for the reaction to proceed completely. The acid chloride product is isolated by concentration in vacuo to convert the methylene chloride and excess oxalyl chloride to an oily state. The resulting oil is diluted with methylene chloride and slowly added over a period of about 3-5 hours at a temperature ranging from 65 to 75 ° C to a diluted mixture of hydrazine and methylene chloride.

[0027] One aspect of the present invention is the formation of a uniform suspension containing hydrazine and an inert solvent, such as methylene chloride. In accordance with one embodiment of the invention, a uniform suspension is obtained by slowly adding dropwise liquid hydrazine to methylene chloride, pre-cooled to about -68 to -75 ° C, preferably -70 ° C, before hydrazine is added. On the contrary, cooling a pre-mixed solution of hydrazine and methylene chloride at the same temperature leads to a less preferred formation of crystalline hydrazine, which is collected on the walls of the reaction vessel. Not wishing to be bound by any particular theory, it is believed that the slow dropwise addition of hydrazine to pre-cooled methylene chloride and the control of the maximum concentration of hydrazine in methylene chloride results in the formation of smaller, more uniform hydrazine crystals that remain suspended in the stirred mixture and essentially do not freeze on the walls of the vessel. The formation of a substantially uniform suspension helps ensure that hydrazine is in contact with the stirred methylene chloride and that hydrazine is available for reaction with the added acid chloride solution. The formation of a uniform suspension eliminates the need for scraping off the substance from the inner surface of the reaction flask, which was required in the original method. Moreover, this formation of a substantially uniform suspension allows the required amount of hydrazine to participate in the reaction, which also leads to a decrease in the amount of methoxybenzylthioether bis-hydrazide formed (6).

[0028] The concentration of hydrazine in methylene chloride affects the amount of methoxybenzylthioether bis hydrazide (6), which is formed as a by-product. In known methods, the concentration of hydrazine / methylene chloride was from about 24 to 32% vol. Halving the ratio of hydrazine / methylene chloride (more dilute hydrazine) to about 12 to 16% vol., Preferably to about 14%, leads to a reduction in the amount of undesired methoxybenzylthioether bis-hydrazide formed (6) (see Table 1).

TABLE 1 METHOD RESULTS AFTER IMPROVEMENTS No. p / p The concentration of hydrazide * (%) By-product bis-hydrazide product (%) The adjusted yield of hydrazide (%) 1.1 91.61 9.69 85,4 1.2 95.08 5.76 87.7 1.3 96.16 4.13 89.6 1.4 93.76 7.36 85.7 * determined by high pressure liquid chromatography

[0029] In some embodiments, the acid chloride solution is added to the hydrazine / methylene chloride suspension continuously at a constant rate rather than in batches. The amount of acid chloride added, as well as the rate of addition, affect the yield of the target methoxybenzylthioether hydrazide (3). If too little acid chloride is added, an excessive amount of a byproduct of methoxybenzylthioether bis-hydrazide may form (6). Also, if the time for adding the acid chloride is too short, amounting to less than 3 hours, an excessive amount of a by-product, which is methoxybenzylthioether bis-hydrazide, can form (6). The rate of addition is adjusted to maintain the reaction temperature in the range from -68 to -75 ° C. If the reaction temperature rises to higher values, an excess amount of methoxybenzylthioether bis hydrazide may also form (6). The mixing speed of the hydrazine suspension is preferably 300 to 400 rpm in a round bottom flask or 270 rpm in a Morton flask. Both aspects of the improved process, namely the use of a more dilute hydrazine / methylene chloride mixture and the formation of a uniform suspension, reduce the amount of the methoxybenzylthioether bis-hydrazide by-product (6) from about 20% to about 3 to 5%. Improved hydrazine production steps increase the overall efficiency of the synthesis of the linker, which is 3-methyl-3-mercaptobutanoic acid hydrazide, which also increases the overall production efficiency of MYLOTARG® (gemtuzumab ozogamicin).

[0030] After completion of the reaction, the reaction mixture was concentrated in vacuo and the precipitate was treated with a solution of sodium hydroxide in methanol (about 4-5%). The specified solution was concentrated in vacuo, diluted with methylene chloride, washed with water, dried over magnesium sulfate, purified and concentrated in vacuo to give a concentrate. It is necessary to use a sufficient amount of magnesium sulfate to completely dry the product, so that in the next stages there is no decomposition of the product or difficulties in its crystallization. The final concentrate is diluted with methylene chloride in an amount 1.33 times the mass of p-methoxybenzylthio-ester acid (1), and this solution is added to diethyl ether taken in an amount of 7.6 times the mass of p-methoxybenzylthio-ester acid (1). An aliphatic hydrocarbon solvent, such as heptane, hexane, octane or isohexane, preferably heptane, is added to the resulting suspension in an amount 1.83 times the mass of p-methoxybenzylthio-ester acid (1) to complete the precipitation. The n-methoxybenzylthioether hydrazide (6) was isolated by filtration, washed with heptane and dried under vacuum.

[0031] The p-methoxybenzylthioester hydrazide (3) is treated with trifluoromethanesulfonic acid in the presence of anisole using trifluoroacetic acid as a solvent. It is necessary to ensure that during the addition and subsequent course of the reaction, the reaction temperature does not exceed about 20 ° C to prevent the formation of undesirable impurities. Upon completion of the removal of the p-methoxybenzyl protecting group, the reaction mixture is quenched by pouring into methanol and filtered to remove solid by-products. The filtrates are concentrated in vacuo, dissolved in water, washed with methylene chloride and treated with an anion exchange resin to obtain 3-methyl-3-mercaptobutanoic acid hydrazide (5). The resin is removed by filtration, then an aqueous hydrochloric acid solution is added to the crude product solution to form the hydrochloride salt. The resulting amount of the product is concentrated in vacuo, dissolved in ethanol, purified by filtration and concentrated in vacuo. This concentrate was diluted with ethyl acetate and concentrated in vacuo. Then again, the precipitate was diluted with ethyl acetate and isolated by filtration. The wet cake is heated with ethyl acetate to about 48 to 55 ° C, cooled, filtered and dried under vacuum. The dried hydrochloride salt is converted to the free base by treatment with an anion exchange resin in water. The resin was removed by filtration, and the filtrates were concentrated in vacuo. The concentrate was dissolved in ethanol, concentrated in vacuo, suspended in diethyl ether and concentrated in vacuo. As a final purification, 3-methyl-3-mercaptobutanoic acid hydrazide (5) is dissolved in methylene chloride, purified by filtration and treated with silica, which is then removed by filtration. The purified product is isolated from the solution by concentration in vacuo. According to the preferred purification method shown in Example 15, 3-methyl-3-mercaptobutanoic acid hydrazide (5) is dissolved in 50 parts (v / w) methylene chloride at 20 ° C ± 3 ° C, stirred for 30 minutes and filtered. The resulting solution was treated with 0.7-1 parts (w / w relative to the weight of the crude linker compound) of silica gel, stirred for 30 minutes, filtered and concentrated to a dry residue in a rotary evaporator. The resulting solid is triturated with heptane. After isolation and drying under vacuum, 3-methyl-3-mercaptobutanoic acid hydrazide (5) is obtained as a free-flowing powder in approximately 76% yield.

[0032] One aspect of the present invention is a method for producing p-methoxybenzylthioether hydrazide with less than 5% content of an undesired by-product of methoxybenzylthioether bis-hydrazide (6). This improved process includes a modified process for coupling a thioester acid chloride with hydrazine to form p-methoxybenzylthioester hydrazide. The steps of this process are shown schematically in Equation I. An undesired by-product of methoxybenzylthioether bis-hydrazide (6) is formed by combining a product of n-methoxybenzylthio-ester hydrazide (3) with the starting material of n-methoxybenzylthio-acid chloride (n-methoxybenzylthioether 2). The formation of the undesired methoxybenzylthioether bis-hydrazide (6) leads to a decrease in the quality and yield of the product.

[0033] According to another aspect of the present invention, the proposed method as a whole may include wider application. A specific reaction sequence (Equation III) can be summarized as Equation VII.

[0034] When it is indicated in the present description that at one stage of the process, the substance is added continuously, this means that said addition is carried out continuously for some time, and not in portions and not at a time. Examples of continuous addition are dropwise addition of liquid or continuous liquid addition. In some embodiments, continuous addition is performed by controlling the rate of addition of a substance that reacts exothermically at a rate sufficient to maintain the reaction temperature within a given temperature range.

[0035] The term "suspension" in the present description refers to a combination of solid and liquid phases, which are thoroughly mixed together and, as a rule, cooled to a temperature at which both solid and liquid phases are present, while this mixture is exclusively a liquid at ambient temperature. Sometimes a suspension is called a liquid / solid mixture of the same substance, for example, an ice / water mixture in which the ice is relatively well crushed and mixed with liquid water. In the context of the present invention, a suspension may include a solid / liquid mixture formed by a combination of two substances, such as hydrazine and a solvent, for example, methylene chloride. It is believed that in a cooled suspension of hydrazine / methylene chloride, the liquid phase contains a mixture of methylene chloride and hydrazine, while the solid phase contains mainly hydrazine.

[0036] The term “alkyl” includes linear or branched alkyls of 1-10 carbon atoms, with lower alkyls containing 1 to 5 carbon atoms being preferred. For example, alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 2-methylbutyl, n-hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, decyl and the like. The term “alkylene” includes linear and branched diradicals of alkanes containing from 1 to 10 carbon atoms, such as methylene (—CH ₂ -), ethylene (—CH ₂ CH ₂ -), propylene, butylene and pentylene.

[0037] The term “heteroalkyl” refers to the above-described alkyl group in which one or more carbon atoms are replaced by a heteroatom (oxygen, sulfur, nitrogen or phosphorus) and which may contain additional heteroatoms. The term “heteroalkaryl” refers to a heteroalkyl moiety described above, but which is further substituted by an aryl moiety, wherein said aryl moiety may be substituted. Optionally substituted aryl includes phenyl and substituted phenyl. In substituted phenyl, one, two, or three possible substituents can replace hydrogen in the phenolic ring and are located in the ortho, meta, or para position with respect to the methylene group at the benzyl carbon atom (or at another point of attachment). In Example 1, the methoxy group is located in the para position to the methylene group. Non-limiting examples of possible substituents in aryl include C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ haloalkyl, C ₁ -C ₅ haloalkoxy, in which hydrogen atoms attached to carbon atoms in an alkyl or alkoxy group may be replaced by halogen atoms, such as —CF ₃ and —OCF ₃ .

The following non-limiting examples illustrate the present invention.

EXAMPLE 1

Initial Preparation of p-Methoxythioetheric Acid (1)

[0038] In accordance with Equation I, 400 g, 465 ml, 4, were placed in a 5-liter round-bottom flask equipped with a thermocouple, a mechanical stirrer, a reflux condenser with an inlet for supplying N ₂ in the upper part and a 250 ml dropping funnel with pressure compensator, 70 moles of piperidine. 3,3-Dimethylacrylic acid (215 g, 2.15 mol) was added portionwise to a 5 liter reaction flask with stirring. The mixture was vigorously stirred under N ₂ . During the addition, the reaction temperature was kept below 35-40 ° C (Note: strongly exothermic, gas evolution). p-Methoxybenzylthiol (386 g, 323 ml, 2.32 mol) was added via a dropping funnel with a pressure compensator for 15 minutes to a (5 liter) reaction flask. The mixture was heated to 82-88 ° C with stirring in an atmosphere of N ₂ . The reaction temperature was maintained in this range for 15 minutes. Note: exothermic. The clear yellow mixture was heated to 92-95 ° C and stirred in an atmosphere of N ₂ for at least 15 hours. A 1 ml sample was taken for HPLC. The reaction was considered complete when less than 3% 3,3-dimethylacrylic acid remained, as determined by peak area. The reaction mixture was cooled to 70-75 ° C, removing the heating mantle.

[0039] A 3M hydrochloric acid solution (1900 ml, 2090 g) was added via a liter dropping funnel with a pressure compensator to the stirred yellow solution, keeping the temperature below 90 ° C. The final temperature was 70-75 ° C. The mixture was cooled to 20-25 ° C by placing it in a water bath. Methylene chloride (1600 g, 1210 ml) was added to the heterogeneous mixture. The mixture was stirred for 5 minutes. The pH of the upper aqueous layer in the flask was checked and the required amount of a 3M HCl solution was added until the pH was below 2. The entire contents of the 5-liter reaction flask were transferred to a 4-liter separatory funnel and the two layers were allowed to separate for at least 10 minutes. The organic (lower) layer was transferred from the separatory funnel back to the 5-liter reaction flask. The prominent (upper) layer was transferred from a separatory funnel to a 4-liter Erlenmeyer flask. A 3M hydrochloric acid solution (1050 ml, 1154 g) was added to a 1000 ml dropping funnel with a pressure compensator to a solution of methylene chloride in a 5-liter flask over 10 minutes. The pH of the upper aqueous layer in the flask was checked and the required amount of 3M HCl was added until the pH of the aqueous layer was below 2. The entire contents of the 5 liter reaction flask were transferred to a 4 liter separatory funnel. The value of the total volume was recorded. The two layers were allowed to separate for at least 10 minutes. The organic (lower) layer was transferred from a separatory funnel to a clean 4-liter Erlenmeyer flask. The aqueous (upper) layer was transferred from a separatory funnel to a clean 4-liter Erlenmeyer flask. The aqueous layers were combined in a 5 liter reaction flask.

[0040] Methylene chloride (305 ml, 400 g) was added to the aqueous solution obtained in the previous step. The mixture was stirred for at least 5 minutes. The entire contents of the 5-liter reaction flask were transferred to a 6-liter separatory funnel, and the value of the total volume was recorded. The mixture was allowed to settle to separate the two layers for at least 5 minutes. The organic (lower) layer was transferred from a separatory funnel to a 4 liter Erlenmeyer flask. The methylene chloride solution was washed with 1000 ml of water. The mixture was thoroughly mixed for 1-2 minutes and left to settle for at least 10 minutes. The aqueous layer was separated. The pH value of the aqueous layer in the flask was measured. The water layers were combined and discarded. Anhydrous magnesium sulfate (110 g) was added to the methylene chloride solution and stirred for at least 15 minutes. On a 5-liter flask, 800, 900 and 1000 ml level marks were applied. Using vacuum filtration, the yellow mixture was filtered through a 15 cm Buchner funnel with filter paper (Whatman # 1) into a 5 liter reaction flask. The flask and filter cake were washed with 300 ml, 400 g of methylene chloride. The methylene chloride solution was concentrated as described below: a 5 liter round bottom flask was equipped with a mechanical stirrer and a Kleisen distillation head equipped with a thermocouple. The distillation nozzle was connected to a 30 cm simple condenser refrigerator, which was connected to a lead connected to a 1-liter inlet flask cooled in an ice bath. Alonge was connected to a trap cooled to 0 ° С. A cooled trap was connected to a vacuum pump.

[0041] Methylene chloride from a solution in a 5-liter flask was distilled off at a temperature of 15-35 ° C under vacuum until a volume of liquid in the vessel of 900 ml was reached. The distillate was discarded. The temperature of the contents of the 5-liter flask was set between 15-20 ° C. Heptane (2442 ml, 1670 g) was added via a dropping funnel to the stirred concentrated solution for at least 10 minutes. A precipitate formed after the addition of approximately 1000 ml (684 g) of heptane. The heterogeneous mixture was cooled with stirring to a temperature of 0-5 ° C for at least 20 minutes and kept at that temperature for at least 30 minutes. The contents of the 5 liter reaction flask were filtered through a 30 cm Buchner funnel with Whatman # 1 filter paper. The filtrate was collected in a 4 liter flask for vacuum filtration. The 5-liter reaction flask was washed with two 310 ml, 212 g, heptane portions and the solution was added to the filter cake. The precipitate was dried by vacuum filtration until the filtrate collection had substantially stopped, and for at least 25 minutes. The height of the filter cake was approximately 20 mm. The mass of sediment was approximately 601 g. The filtrate was discarded. The precipitate was placed in a 5-liter round-bottom flask equipped with a thermocouple, a mechanical stirrer, a supply of N ₂ and a 1-liter dropping funnel with a pressure compensator. Methylene chloride (750 ml, 990 g) was placed in a 5-liter reaction flask, and stirring was carried out until the solids were completely dissolved (about 10 minutes). Heptane (1060 ml, 725 g) was placed in a 5-liter reaction flask. The heterogeneous solution was cooled to 0-5 ° C. using an ice bath for at least 15 minutes, then stirred for at least 30 minutes. Got a thick heterogeneous solution. The contents of the 5 liter reaction flask were filtered through a 30 cm Buchner funnel with Whatman # 1 filter paper. The filtrate was collected in a 4 liter flask for vacuum filtration. The 5-liter reaction flask was washed with two 310 ml, 212 g, heptane portions and the solution was added to the filter cake. The precipitate was dried by vacuum filtration until the filtrate was substantially stopped (minimum 20 minutes). The height of the filter cake was approximately 20 mm. The mass of sediment was approximately 632 g. The filtrate was discarded. A wet cake was placed in a desiccator. The desiccator with p-methoxybenzylthioester acid was covered with clean filter paper. The product was dried in a vacuum oven at 38-40 ° C and 28-30 inches (711.2-762.0 mm Hg) for 20 hours.

EXAMPLE 2

Initial Preparation of n-Methoxybenzylthioether Acid Chloride (2)

[0042] In accordance with Equation II, p-methoxybenzylthio-ester acid (400 g, 1.57 mol) was placed in a 5-liter round bottom flask equipped with a thermocouple, a mechanical stirrer, a reflux condenser with an opening for supplying N ₂ in the upper part and 0.5 liter drip funnel with pressure compensator. Methylene chloride (1600 g, 1212 ml) was placed in a 5 liter reaction flask. The clear solution was heated to 20-25 ° C. Methylene chloride (300 g) and oxalyl chloride (110 g, 78 ml) were placed in a 0.5-liter dropping funnel. 350 ml of a solution of oxalyl chloride / methylene chloride was added using a dropping funnel, maintaining the reaction temperature of 20-30 ° C. The clear yellow solution was stirred at 20-25 ° C for at least 30 minutes until the bubbling ceased. The addition of oxalyl chloride was repeated. 350 ml of a solution of oxalyl chloride / methylene chloride was added via a dropping funnel to the reaction flask, maintaining the reaction temperature of 20-30 ° C (addition time about 45 minutes). The reaction mixture was heated to 32-38 ° C. The stirred solution was kept in this temperature range for at least 1 hour. A 1 ml sample was taken for HPLC. The reaction was considered complete when less than 3% of the starting material, methoxybenzylthioether acid, remained. The reaction mixture was cooled to 23-28 ° C for at least 5 minutes. The solution was transferred to a calibrated 3 liter round bottom flask. The reaction flask was washed with 100 ml, 132 g, methylene chloride, the solution was placed in a 3-liter flask. The reaction solution was concentrated under vacuum in a rotary evaporator with a set temperature of 33-36 ° C at a pressure of 25-28 inches (635.0-711.2 mm Hg) to remove volatiles. The final weight was 1367 g, and the net weight of p-methoxybenzylthio-ester acid chloride was 500.3 g. The distillate was discarded.

EXAMPLE 3

Initial preparation of p-methoxybenzylthioether hydrazide (3)

[0043] In accordance with Equation III, a 5-liter round-bottom flask of Morton was equipped with a thermocouple, a mechanical stirrer, a reflux condenser with an opening for supplying N ₂ in the upper part and a 0.5-liter dropping funnel with a pressure compensator. P-methoxybenzylthioether ester was dissolved in 500 ml, 660 g, methylene chloride. The solution was transferred to a 2 liter Erlenmeyer flask. 500 ml of methylene chloride was added to obtain a solution with a total volume of 1300 ml.

[0044] 2400 g, 1818 ml, methylene chloride and 256 g, 245 ml, 7.8 mol of 98% anhydrous hydrazine were placed in a 5-liter round-bottom flask of Morton. The speed of the mechanical stirrer was set at 255-270 rpm. The turbid suspension was cooled to a temperature of from -69 to -72 ° C using a dry ice / acetone mixture.

[0045] The acid chloride solution was added dropwise to a 5 liter flask using a 0.5 liter dropping funnel with a pressure compensator, maintaining the reaction temperature from -68 to -72 ° C. It was important to maintain the rate of addition of the p-methoxybenzylthio-ester acid chloride solution to the stirred hydrazine / methylene chloride suspension at a level that maintained the reaction temperature below -67 ° C. The addition was completed after about 3 hours. The stirred reaction mixture was kept at a temperature of from -68 to -72 ° C for at least 30 minutes. A 1 ml sample was taken for HPLC. The solution was warmed to room temperature (20-30 ° C), removing the ice bath.

[0046] The reaction mixture was transferred to a calibrated 3 L round bottom flask. The reaction solution was concentrated under vacuum in a rotary evaporator at a temperature of 32-36 ° C and a pressure of 25-28 inches (635.0-711.2 mm Hg). All volatiles have been removed. The final mass was 1490.7 g, the net mass of the crude solid p-methoxybenzylthioether solid hydrazide was 630 g. The distillate was discarded.

[0047] Methanol (1250 g, 1580 ml) was added to the crude solid p-methoxybenzylthioether ether hydrazide, and the heterogeneous mixture was stirred in a 5 liter round bottom flask at 33-36 ° C for at least 5 minutes until a clear solution was formed. The solution of the crude p-methoxybenzylthioether / methanol hydrazide solution was transferred to a 5 liter reaction flask.

[0048] 1312 g of a 4% solution of sodium hydroxide in methanol was added to the indicated 5-liter reaction flask at 28-34 ° C. over 8 minutes. The clear mixture was stirred at 33-36 ° C for 20 minutes. A loose precipitate formed.

[0049] The contents of the 5 liter reaction flask were filtered through a 30 cm Buchner funnel with filter paper (Whatman # 1). A (5 liter) reaction flask was washed with 200 ml, 158 g, methanol. The filtrate was transferred to a calibrated 3 liter round bottom flask. The reaction solution was concentrated under vacuum in a rotary evaporator at a temperature of 36-40 ° C and a pressure of 25-28 inches (635.0-711.2 mm Hg). All volatiles have been removed. The final mass was 1484 g, the net mass of the untreated solid p-methoxybenzylthioether hydrazide was 622.7 g. The distillate was discarded. The solid was dissolved in 700 g, 530 ml of methylene chloride. The heterogeneous mixture was stirred in a rotary evaporator (without vacuum) at 33-36 ° C for at least 10 minutes until a clear solution was formed.

[0050] The solution was concentrated under vacuum in a rotary evaporator at a temperature of 36-40 ° C and a pressure of 25-28 inches (635.0-711.2 mm Hg). All volatiles have been removed. The final weight was 1494.2 g, the net weight of the untreated solid solid hydrazide of n-methoxybenzylthioether was 632.5 g. The distillate was discarded. The solid was dissolved in 2100 ml, 2772 g, methylene chloride. The heterogeneous mixture was stirred at 20-25 ° C for at least 5 minutes until a clear solution was formed. To the methylene chloride solution was added 110 g of anhydrous magnesium chloride, the mixture was stirred for 1 hour. The yellow mixture was suction filtered through a 15 cm Buchner funnel with filter paper (Whatman # 1) into a 5 liter reaction flask. The flask and filter cake were washed with 500 ml, 660 g of methylene chloride. The filtrate was transferred to a calibrated 3 liter round bottom flask. The solution was concentrated under vacuum in a rotary evaporator at a temperature of 32-35 ° C and a pressure of 20-25 inches (508.0-635.0 mm Hg). All volatiles have been removed. The final mass was 1090 g, the net mass of the untreated solid p-methoxybenzylthioether hydrazide was 643 g. The distillate was discarded.

[0051] The obtained solid was dissolved in 400 ml, 528 g of methylene chloride. The heterogeneous mixture was stirred at 35-40 ° C for at least 5 minutes until a clear yellow solution formed. 4260 ml, 3040 g of diethyl ether was placed in a 12-liter round bottom flask equipped with a thermocouple, a mechanical stirrer, inlet No. ₂ and a dropping funnel with pressure compensator. The ether in a 12-liter flask was cooled to a temperature of 0 to -10 ° C using saturated saline and ice. A yellow solution of n-methoxybenzylthioether hydrazide in methylene chloride (prepared above) was added via a dropping funnel to a rapidly stirred (300-400 rpm) diethyl ether, maintaining the temperature from 0 to -10 ° С. 1070 ml, 732 g, heptane were added. The heterogeneous mixture was stirred at a temperature of from 0 to -5 ° C for 20 minutes. The contents of the (12-liter) reaction flask were filtered through a 30 cm Buchner funnel with filter paper (Whatman # 1). The filtrate was collected in a 4 liter flask for vacuum filtration. A 5 liter flask was washed with 1070 ml, 732 g, heptane, the solution was added to the precipitate. The filtrate was discarded. The filter cake was dried by vacuum filtration for at least 50 minutes, until essentially stopping the filtrate collection. The height of the sediment on the filter was 15 cm. The mass of the sediment was 429 g. The wet sediment was transferred to a desiccator. The desiccator with n-methoxybenzylthioester acid was covered with clean filter paper. The product was dried in a vacuum oven at 38-40 ° C and 28-30 inches (711.2-762.0 mm Hg) for at least 18 hours.

[0052] The results of a series of typical reactions used in Examples 1-3 are combined in Table 2.

TABLE 2 TYPICAL REACTIONS BEFORE USING IMPROVED METHODS Series No. The concentration of hydrazide (%) Bis-hydrazide by-product (%) The adjusted yield of hydrazide (%) 2.1 78.5 22.1 67.8 2.2 78,4 19.3 74.8

EXAMPLE 4

Modified preparation of n-methoxybenzylthioether hydrazide (3)

[0053] In order to reduce the content of a by-product of methoxybenzylthioether bis-hydrazide (6), reaction parameters were studied that affect the formation of a by-product in the isolated product of n-methoxybenzylthioether hydrazide. The procedure of Example 3 was repeated. At -78 ° C, the hydrazine / CH ₂ Cl ₂ solution is a non-mixed frozen mixture. When the viscous mass was placed in the reaction flask, the stirrer blade rotated in the air. A solution of thioester chloride in CH ₂ Cl _{2 was} added dropwise to a frozen hydrazine / CH ₂ Cl ₂ mixture (28% v / v), keeping the temperature at about -72 ° C. HPLC at the end of the addition (temperature was -72 ° C) showed that the reaction was not significant. This contradicted the expected rapid reaction. The indicated mismatch could occur due to the lack of necessary mixing of the reaction mixture. For the most part, the unreacted reaction mixture was allowed to warm. When the temperature reached about -50 ° C, a mixed heterogeneous mixture was formed, followed by rapid heating, which led to an instant increase in temperature to -28 ° C, while the color of the reaction mixture changed from yellow to white with a yellowish tint. Based on the above, it was postulated that inefficient mixing can lead to a localized reaction that promotes the formation of bis-hydrazide. Heating to room temperature and carrying out the reaction as in Example 3 led to the formation of bis-hydrazide as the main product (82%, HPLC), which is much higher than the standard level of undesirable product of 20%. It was concluded that there was no efficient mixing of the solution of added thioester acid chloride (2) in CH ₂ Cl ₂ with a frozen mass of hydrazine.

EXAMPLE 5

The effect of temperature on the preparation of p-methoxybenzylthioether hydrazide (3)

[0054] In Example 5, the reaction from Example 4 was repeated, but was carried out at 0 ° C, and not at about -72 ° C. A solution of thioester acid chloride in CH ₂ Cl _{2 was} added dropwise to a stirred homogeneous solution of hydrazine / CH ₂ Cl ₂ (28% v / v). In this case, 39% (% of area according to HPLC) of bis-hydrazide was formed. These conditions allow us to conclude that lower temperatures and mixing are factors that influence the formation of an undesirable by-product. The results of examples 3.1 and 3.2 are presented in Table 3.

TABLE 3 PRODUCTION OF P-METHOXIBENZYLTHYOESTER HYDRAZIDE AS A TEMPERATURE FUNCTION No. of Example Hydrazine (equivalent) Hydrazine / CH ₂ Cl ₂ (V / V) (%) Temperature (° C) Bis-hydrazide (%) Comments 3.1 5 28 ~ -72 82 Poor mixing 3.2 5 28 ~ 0 39 Mix suspension but. The acid chloride was added to a mixture of hydrazine / CH ₂ Cl ₂ at a rate of 0.25 ml / min

EXAMPLE 6

The effect of hydrazine concentration on the preparation of p-methoxybenzylthioether hydrazide (3)

[0055] The effect of using hydrazine at a lower concentration at low temperatures was then investigated. The results are presented below in Table 4. A stirred heterogeneous mixture of hydrazine / CH ₂ Cl ₂ at a temperature of from -65 to -72 ° C was obtained by diluting hydrazine in CH ₂ Cl ₂ to concentrations of 5% and 19% against 28% (vol./about. ) Experiments 4.1 and 4.2 were carried out at hydrazine / CH ₂ Cl ₂ concentrations of 19% and 5%, respectively, with the formation of a stirred heterogeneous mixture, subjected to interaction with thioester acid chloride to form the target product containing 3% and 5% bis-hydrazide by-product, respectively. When repeating the same reaction using less hydrazine (in Experiment 4.3, Table 4 used 5 against 10 mol equivalents), only 3% bis-hydrazide was formed. The standard amount of hydrazine is 5 mol equivalents relative to thioester acid. Doubling the amount of hydrazine to 10 mol equivalents (Table 4: Experiments 4.1 and 4.2) did not significantly affect the level of bis-hydrazide in the final product.

[0056] Adding the acid chloride to the diluted heterogeneous hydrazine / CH ₂ Cl ₂ mixture (5% and 19%) at a higher rate (Experiments 4.4 and 4.5 in Table 4, in which the rate was 1, rather than 0.25 ml / min) led to the formation of bis β-hydrazide in the amount of 3% and 9%, respectively. A sharp decrease in the amount of bis-hydrazide (from 82% to 3%, Experiment 3.1 in Table 3 and Experiment 4.4 in Table 4) can be attributed to one or more of the following factors: temperature, concentration, amount of hydrazine, rate of addition, and mixing. The formation in Experiment 4.5 in Table 4 of 9% bis-hydrazide may be due to initial ineffective mixing.

TABLE 4 PREPARATION OF P-METHOXIBENZYLTHYOESTER HYDRAZIDE AT VARIOUS DILUTION OF HYDRAZINE IN CH ₂ С ₁₂ No. Exp. (Per 5 g) Hydrazine (equiv.) The rate of addition (ml / min) Hydrazine / CH ₂ Cl ₂ (vol.%) Bis-hydrazide (%) Comments 4.1 10 0.25 19 four Mix suspension 4.2 10 0.25 5 5 Mix suspension 4.3 5 0.25 5 3 Mix suspension 4.4 5 1,0 5 3 Mix suspension 4.5 5 1,0 19 9 Started with non-mixed suspension During the addition, the reaction temperature was maintained in the range from -68 to -73 ° C.

EXAMPLE 7

The effect of temperature on the preparation of p-methoxybenzylthioether hydrazide (3)

[0057] Table 5 examined the effect of temperature on the content of bis-hydrazide in the final product. In experiments in which thioester acid chloride was added to a hydrazine / CH ₂ Cl ₂ mixture (19 volume%) at -20 ° C and -72 ° C (Table 5: Experiments 5.1 and 5.2), bis-hydrazide was formed in an amount of 28% and 4%, respectively. Similar results were observed in Experiments 5.3 and 5.4 (Table 5). These results indicate that a lower reaction temperature

(~ -70 ° C) is necessary to obtain a lower (3-5%) content of bis-hydrazide.

TABLE 5 INFLUENCE OF TEMPERATURE ON THE CONTENT OF THE FORMED BIS HYDRASIDE Experiments ^a (calculation for 5 g) Hydrazine / CH ₂ Cl ₂ (V / V) (%) Temperature (° C) Bis-hydrazide (%) Comments 5.1 19 -twenty 28 Mix suspension 5.2 19 -72 four Mix suspension 5.3 5 -38 16 Mix suspension 5.4 5 -72 3 Mix suspension but. The above experiments were performed using 5 equivalents of hydrazine.

EXAMPLE 8

The effect of hydrazine concentration on the preparation of p-methoxybenzylthioether hydrazide (3)

[0058] A study of the experiments in Table 4 showed that at a temperature of addition of about −70 ° C. and a concentration of hydrazine in CH ₂ Cl ₂ of both 19% and 5%, comparable results were obtained. This observation was further verified in Table 6. In Experiments 6.1, 6.2 and 6.4 in Table 6, in which thioester acid chloride was added to a heterogeneous mixture of hydrazine / CH ₂ Cl ₂ with a concentration of 19%, 14% and 10%, bis was obtained β-hydrazide in the amount of 6%, 13% and 4%, respectively. The volume of the reaction mixture, the size of the flask and the stirring speed were kept constant. Results at 19% concentration were comparable with results at 10% concentration. The experiment with a 14% concentration of the hydrazine / CH ₂ Cl ₂ mixture was repeated with a sample weight of 30 g (Experiment 6.3 in Table 6), while the obtained target product contained only 3% bis-hydrazide. The highest bis-hydrazide content in Experiment 6.2 was due to the rapid addition of acid chloride, which caused a jump in the reaction temperature to -57 ° C before it was quickly returned to the required value.

TABLE 6 INFLUENCE OF HYDRAZINE CONCENTRATION IN CH ₂ Cl ₂ ON THE CONTENT OF THE FORMED BIS-HYDRAZIDE Experiment No. ^a (hitch) Hydrazine / CH ₂ Cl ₂ (V / V) (%) Temperature (° C) Bis-hydrazide (%) Comments (fluid volume / round bottom flask size) 6.1 (5 g) 19 -68 - -70 6 20 ml / 100 ml 6.2 (4.2 g) fourteen -57 - -65 13 Fast addition of 20 ml / 100 ml 6.3 (30 g) fourteen -68 - -71 3 140 ml / 500 ml 6.4 (2.9 g) 10 -61 - -68 four 20 ml / 100 ml but. The above experiments were performed using 5 equivalents of hydrazine.

EXAMPLE 9

Effect of stirring speed on the preparation of p-methoxybenzylthioether hydrazide (3)

[0059]

A study was conducted of the effect of mixing (table 7). Experiments 7.2 and 7.1 showed that accelerated mixing (400 rpm./min. Against 200 rpm.) Leads to a decrease in the amount of bis-hydrazide (22% against 40%). A higher than usual bis-hydrazide content in both experiments could be caused by inefficient mixing, given the initial content (36 ml) of the hydrazine / CH ₂ Cl ₂ mixture in a 50 ml flask compared to 20 ml of liquid in a 100 ml flask, as shown in Table 8. This means that in addition to the mixing speed, the shape of the reaction vessel and the liquid content must be taken into account.

TABLE 7 INFLUENCE OF MIXING ON THE CONTENT OF THE FORMED BIS HYDRAZIDE Experiment (5 g sample) Hydrazine / CH ₂ Cl ₂ (V / V) (%) Temperature (° C) Bis-hydrazide (%) The rate of addition (ml / min) Mixing Speed (rpm) 7.1 19 -60 40 one 200 7.2 19 -65 22 one 400 The final ratio of the volume of liquid to the size of the reaction vessel was 72%

EXAMPLE 10

The effect of zooming in on the preparation of p-methoxybenzylthioether hydrazide (3)

[0060] The preparation of p-methoxybenzylthioether hydrazide yielded comparable results using a hydrazine / CH ₂ Cl ₂ mixture at both 19% and 5% concentrations. Although a hydrazine / CH ₂ Cl ₂ mixture with a concentration of 19% is usually a stirred mixture at -70 ° C, there is a risk that it will turn into a frozen mixture that is difficult to mix. If the process is carried out on an industrial scale (400 g sample) with a hydrazine / CH ₂ Cl ₂ mixture with a concentration of 5%, it may be necessary to use a larger reaction vessel (20-liter Morton flask). However, if the process is carried out using a hydrazine / CH ₂ Cl ₂ mixture with a concentration of 14%, the reaction can be carried out in a glass reactor: a 5-liter round-bottom Morton flask. When the process was carried out using a hydrazine / CH ₂ Cl ₂ mixture with a concentration of 14% and a weighed portion of 20 g (Experiment 8.1 in Table 8), the isolated product, which was p-methoxybenzylthioether hydrazide, contained 4.4% (% of area, HPLC) of the by-product a methoxybenzylthioether bis-hydrazide product. The application of these conditions (a hydrazine / CH ₂ Cl ₂ mixture with a concentration of 14%) on a larger scale (Experiment 8.2 in Table 8) led to the production of p-methoxybenzylthioether hydrazide contaminated with 4.2% (% of the area, HPLC) of a bis by-product methoxybenzylthioether β-hydrazide.

TABLE 8 PRODUCTION OF P-METHOXIBENZYLTHYOESTER HYDRAZIDE BY CONCENTRATION OF HYDRAZINE / CH ₂ Cl ₂ MIX, EQUAL 14% Experiment No. (hitch) Flask Size (ml) Initial fluid volume / reactor size (%) HPLC peak area of bis-hydrazide (%) 8.1 ^a (20 g) 250 39 ^c 4.4 8.2 ^b (400 g) 5000 (Morton) 39 ^c 4.4 a. the concentration of the hydrazine / CH ₂ Cl ₂ mixture is 14% (v / v) b. the concentration of the mixture hydrazine / CH ₂ Cl ₂ - 14% (vol./vol.) c. The initial ratio of the volume of the reaction mixture to the size of the reactor = 39%, the final ratio of the volume of the reaction mixture to the size of the reactor = 64%

[0061] In the original method (Table 9, Experiment 9.1), a product of p-methoxybenzylthio-ester hydrazide was obtained with a concentration of 78.4%, said product containing 19.3% of a by-product of bis-hydrazide. In the modified method (Table 9, Experiment 9.2), continuous addition of a methoxybenzylthioether ester chloride solution to a more dilute and stirred heterogeneous mixture (with a concentration of 14%) hydrazine / methylene chloride was used. Received a product representing hydrazide p-methoxybenzylthioether, with a concentration of 91.1%, containing 4.7% by-product bis-hydrazide product.

TABLE 9 COMPARISON OF THE MODIFIED AND ORIGINAL METHOD FOR PRODUCING P-METHOXIBENZYLTHIOETHER HYDRAZIDE Method (hitch) Mixing Speed (rpm) Flask Size (ml) P-methoxybenzylthioether hydrazide (% concentration) Bis-hydrazide (%) 9.1 ^a (400 g) There is no data 5000 ^c 78,4 19.3 9.2 ^b (400 g) 270 5000 ^d 91.1 4.7 a. The concentration of the hydrazine / CH ₂ Cl ₂ mixture is 28% (v / v) b. The concentration of the hydrazine / CH ₂ Cl ₂ mixture is 14% (v / v) c. The initial ratio of volume to reactor size = 21%. Final ratio of liquid volume to reactor size = 47% d. The initial ratio of volume to reactor size = 39%. Final ratio of liquid volume to reactor size = 64%

[0062] The following conditions provide a low content of the resulting bis-hydrazide by-product and are recommended for large-scale production of p-methoxybenzylthioether hydrazide. These reaction conditions are considered as aspects of certain embodiments of the present invention:

1. The rate of addition of methoxybenzylthioacid chloride solution to a hydrazine / CH ₂ Cl ₂ mixture was adjusted to maintain a temperature in the range of -68 to -75 ° C.

2. Effective mixing (30-40% ratio of the initial volume of liquid to the size of the reactor) with a high mixing speed (300-400 rpm). Mixing at 260-270 rpm in a 5-liter Morton flask.

3. A stirred homogeneous mixture of hydrazine / CH ₂ Cl ₂ (concentration from 5 to 19%, vol./about.). In one example, the ratio of hydrazine / CH ₂ Cl ₂ was 14%, vol./about.

4. 30-40% ratio of the initial volume of liquid to the size of the reactor.

[0063] The following Examples 11-15 illustrate a preferred embodiment of the invention.

EXAMPLE 11

Modified Preparation of Intermediate Compound of p-Methoxybenzylthioether Acid (1)

[0064] An assembly containing a 5 liter reaction flask equipped with a refrigerator, supply No. ₂ , a stirrer and a temperature sensor / controller was assembled. Piperidine (0.402 g) was placed in a vessel in an atmosphere of N ₂ . 3,3-Dimethylacrylic acid (0.215 g) was added portionwise with stirring, then p-methoxybenzylthiol (0.358 g) was added. The reaction mixture was gradually heated to 82-88 ° C for at least 15 minutes and the indicated reaction temperature was maintained until signs of an exothermic reaction appeared. The temperature should not exceed 95 ° C. When the exothermic reaction was completed, heating was continued at 92-98 ° C and the indicated temperature was maintained for at least 15 hours.

[0065] A 3 liter 3M aqueous HCl solution was prepared. The mantle heater was removed and the reaction mixture was cooled to 70-75 ° C. 1.9 L of HCl solution was slowly added. Cooling was continued in a water bath until the temperature of the contents of the flask was 20-30 ° C. 1.64 kg of CH ₂ Cl _{2 was} added and the contents of the flask were mixed for at least 5 minutes. The pH was checked and maintained at <2 with a HCl solution. The reaction mixture was transferred to a separatory funnel, the phases were separated, and the lower organic layer was transferred back to the reaction flask. The upper aqueous layer was transferred to a separate flask. The remaining HCl solution was added to the organic phase and stirred for at least 5 minutes. The pH was checked and maintained at <2 using a freshly prepared HCl solution.

[0066] The contents of the reaction flask were placed back into the separatory funnel and the layers were allowed to separate for at least 5 minutes. The lower organic phase was poured into a clean Erlenmeyer flask, the aqueous layer was poured into a reaction flask. The aqueous phase from the previous extraction was added to the reaction flask together with CH ₂ Cl ₂ (0.400 kg). The reaction mixture was stirred for at least 5 minutes, then the contents of the reaction flask were transferred to a separatory funnel and the layers were allowed to separate for at least 5 minutes. The resulting lower organic phase was combined with the previously obtained organic phase and placed in a reaction flask. The combined aqueous phases were discarded. Water (1.00 kg) was added to the reaction flask and stirred for at least 5 minutes. The mixture was placed in a separatory funnel and the layers were allowed to separate for at least 5 minutes. The lower organic phase containing the product was transferred to an Erlenmeyer flask. The aqueous phase was discarded. The organic product solution was dried over anhydrous MgSO ₄ , then filtered under vacuum into a 5 liter 4-neck flask.

[0067] The Erlenmeyer flask and filter cake were washed with CH ₂ Cl ₂ (0.350 kg) in a 5 liter 4-necked flask. CH ₂ Cl _{2 was} distilled to a volume of 900 ± 50 ml. The temperature of the concentrate was maintained in the range of 15-20 ° C, then the concentrate was precipitated by the addition of heptane (1.67 kg). The mixture was cooled to 5 ° C and stirred for at least 30 minutes, then it was filtered under vacuum, the precipitate was washed with heptane (2 × 0.272 kg). A product sample was taken for drying loss analysis (LOD), and the filtrate was checked for solute content. If the filtrate contained> 0.110 kg, then it was concentrated and treated with heptane to precipitate a second portion of the product, as previously indicated. The wet product was weighed and the dry product weight was calculated using LOD data. The dry weight of the product was equivalent to constant A (kg). The wet product was returned to a 5 liter flask and dissolved in CH ₂ Cl ₂ (minimum 1.75 A ^× kg, maximum 2.20 ^× A kg). Heptane (4.56 ^× A kg) was slowly added, causing precipitation. The suspension was cooled to 0-5 ° C, then kept for at least 30 minutes, after which it was filtered under vacuum, and the filter cake was washed with heptane (2 × 0.272 kg). Filtration was continued until the filtrate was substantially isolated. The wet cake was transferred to calibrated dishes, the mass was recorded, then the filtrates were placed in the appropriate waste containers. The precipitate was dried in a vacuum oven at a temperature of no more than 38 ° C until then, when the loss during drying did not reach <1.0%. The sample was given for analysis.

EXAMPLE 12A

Obtaining p-methoxybenzylthioester acid chloride (2)

[0068] An assembly containing a 5 liter reaction flask equipped with a refrigerator, a water scrubber, a temperature sensor, a 1 liter dropping funnel, a No. ₂ inlet and a stirrer was assembled. CH ₂ Cl ₂ (1.6 kg) was placed in a flask under N ₂ , then p-methoxybenzylthio-ester acid (0.400 kg) was added with stirring. A solution of oxalyl chloride (0.220 kg) and CH ₂ Cl ₂ (0.600 kg) was prepared in a dropping funnel. About half of the oxalyl chloride solution was added while maintaining the temperature in the range of 20-30 ° C (exothermic!). During at least 30 minutes of stirring, the evolution of CO ₂ / CO was observed, then the remaining solution of oxalyl chloride was added, the temperature was maintained in the range of 20-30 ° C. The reaction mixture was stirred until gas evolution was complete (approximately 30 minutes), then the mixture was heated to 33-38 ° C. This temperature was maintained for approximately 60 minutes until gas evolution was complete. A portion of the reaction mixture was taken, and the amount of acid remaining was determined by HPLC. The reaction was considered complete when no more than 5% of the starting materials remained. If the reaction was not completed, stirring was continued at 33-38 ° C for one hour, then a sample was taken again and analysis was performed. The mantle heater was removed and the reaction mixture was cooled to 20-30 ° C. The mixture was placed in a 3 liter one-neck flask, then washed with CH ₂ Cl ₂ . The mixture was concentrated on a rotary evaporator until most of the volatiles were removed.

EXAMPLE 12B

Obtaining an intermediate compound, which is a p-methoxybenzylthioether hydrazide (3)

[0069] An assembly was assembled containing a 5-liter 4-neck Morton reaction flask equipped with a refrigerator, N ₂ supply, thermocouple, stirrer and 2-liter dropping funnel. CH ₂ Cl ₂ (1.6 kg) was placed in a flask in an atmosphere of N ₂ and cooled to a temperature of from -75 to -65 ° C. Anhydrous hydrazine (0.252 kg) was added to obtain a uniform suspension of ice hydrazine, with no hydrazine crystals forming on the walls of the flask. A solution of thioester acid chloride was placed in a dropping funnel and, if necessary, diluted with CH ₂ Cl ₂ to obtain a solution of 1.3 l.

[0070] The acid chloride solution was added dropwise at a constant rate for at least 3 hours, the temperature was maintained in the range of -65 to -75 ° C (preferably from -70 to -75 ° C). After complete addition, the reaction mixture was stirred at a temperature of from -65 to 75 ° C for at least 30 minutes. The mixture was heated to 20-25 ° C. A sample of the reaction mixture was taken for HPLC, and the reaction was considered complete when no more than 5% of the acid chloride remained. If at this point the reaction did not end, stirring was continued at 20-25 ° C for at least 1 hour, then the sample was taken again. A mixture of a 3 liter flask was concentrated in a rotary evaporator. In accordance with the requirements, the mixture in the flask was washed with CH ₂ Cl ₂ . The concentrate was diluted with methanol (1.25 kg) and transferred to a 5-liter reaction flask equipped with a stirrer, thermocouple, inlet No. ₂ and, if necessary, washed with methanol.

[0071] In an atmosphere of N ₂ , a solution of NaOH (0.0640 kg) in methanol (1.25 kg) was added and mixed for at least 20 minutes. The mixture was purified by vacuum filtration and washed with methanol as required. The filtrate was transferred to a calibrated 3-liter flask (washed with methanol as required) and concentrated in a rotary evaporator to remove volatile compounds, then continued to concentrate in vacuo for at least 30 minutes. After cessation of the application of vacuum, CH ₂ Cl ₂ (0.704 kg) was added. The rotation was continued until the concentrate was dissolved, then vacuum was continued and the mixture was concentrated until a solid precipitate formed. The mass of the precipitate was measured and the sediment was transferred to a separatory funnel using CH ₂ Cl ₂ (2.84 kg). The mixture was stirred until a solution formed.

[0072] The CH ₂ Cl ₂ solution was washed with two portions of water (1.00 kg each). Anhydrous MgSO ₄ (0.300-0.420 kg) was added to the CH ₂ Cl ₂ solution, stirring was carried out for approximately 15 minutes until a clear solution was formed. The mixture was filtered under vacuum and washed with CH ₂ Cl ₂ . The filtrate was transferred to a calibrated 3-liter flask and concentrated in a rotary evaporator until a solid precipitate formed. The mass of the precipitate was measured and dissolved in CH ₂ Cl ₂ (at least 0.532 kg). The solution was transferred to a 1-liter dropping funnel attached to a 12-liter four-necked reaction flask equipped with a stirrer, thermocouple and N ₂ inlet. Diethyl ether (3.04 kg) was placed in a flask in an atmosphere of N ₂ . The ether was cooled to a temperature of from 0 to -10 ° C.

[0074] The solution of p-methoxybenzylthioether hydrazide was added to the ether solvent while stirring said solvent at high speed, maintaining the temperature from -10 to 0 ° C. The added solution was transferred to the flask using CH ₂ Cl ₂ (0.0660 kg). Heptane (0.732 kg) was placed in a dropping funnel and slowly added to the liquid suspension, again maintaining the same temperature range. The resulting suspension was stirred at the same temperature for at least 60 minutes. The mixture was filtered under vacuum through paper. The precipitate was washed with heptane (2 portions of 0.366 kg each) and dried by vacuum filtration to form a filtered precipitate. The filtered precipitate was placed in a calibrated container and weighed. The precipitate was dried in a vacuum oven (at a temperature not exceeding 38 ° C) until then, when the loss during drying did not become lower than 2.0%. The mass of dry p-methoxybenzylthioether dry hydrazide was measured and samples were given for analysis.

EXAMPLE 13

Obtaining a thiol-unprotected intermediate compound (4)

[0075] The Dowex SRB OH anion exchange resin was prepared by adding 2.4 kg of resin to a large Büchner funnel and washing with water (4 portions of 2.4 kg) and then washing with methanol (4 portions of 1.92 kg). The resin was filled with water in a beaker so that the water covered it, and kept it for at least 1 hour, then the water was filtered. The resin was transferred to a container of suitable size. Assembled a unit containing a 5-liter flask equipped with a stirrer, thermocouple, supply No. ₂ and a 250 ml dropping funnel. Trifluoroacetic acid (2.8 kg) was placed in a flask in an atmosphere of N ₂ and cooled to 5-10 ° C. Thioether hydrazide (0.380 kg) was added in portions (exothermic!), The temperature was maintained in the range of 5-15 ° C. Then the solution was cooled to 0-5 ° C.

[0076] Trifluoromethanesulfonic acid (0.243 kg) was placed in a dropping funnel and added to the reaction mixture, maintaining the temperature at 0-10 ° C. After complete addition, anisole (0.0152 kg) was added. The reaction mixture was stirred at 10-15 ° C. for at least 2 hours until the color of the reaction mixture turned dark red and no longer changed. A sample was taken from the mixture, which was analyzed by TLC, and the reaction was considered complete if the reaction mixture contained no more than 4% of the starting materials. An assembly was assembled containing a 12-liter reaction flask equipped with a stirrer, inlet No. ₂ and a 2-liter dropping funnel. Methanol (3.01 kg) was added to the flask. The vessel was cooled to 0-5 ° C in a nitrogen atmosphere. The reaction mixture was transferred to a dropping funnel and then added to the cooled methanol at a low speed, maintaining the reaction temperature at 0-5 ° C. A white precipitate formed. The reaction flask was washed with additional methanol (0.0790 kg), the solution was placed in a dropping funnel. The white suspension was stirred for about 15 minutes at 0-5 ° C. The mixture was filtered under vacuum through paper, the precipitate was washed with methanol (two servings of 0.600 kg each). After essentially stopping the separation of the filtrate, it was placed in a calibrated 3-liter flask (washed with methanol as required) and concentrated in a rotary evaporator until a semi-solid precipitate formed.

[0077] The precipitate was redissolved in methanol (0.600 kg) and concentrated again as before. The precipitate was redissolved in CH ₂ Cl ₂ (0.600 kg) and concentrated again as before, and the mass of the obtained precipitate was measured. The precipitate was dissolved in water (1.52 kg), and the solution was transferred to a 6-liter separatory funnel. The precipitate was washed with CH ₂ Cl ₂ (three portions of 0.927 kg). The organic phases were transferred to appropriate waste containers. The aqueous phase containing the product was transferred to a beaker and the pH was adjusted to 6.5-7.5 pH units by adding a swollen resin. If necessary, the pH was adjusted with trifluoroacetic acid. After reaching the target pH range, the suspension was stirred for about 30 minutes, the pH was checked again and, if necessary, adjusted to the desired value. The mixture was filtered under vacuum, the resin was washed with water (2 servings of 0.600 kg). Then a 37% HCl solution (0.160 kg) was added. We measured the pH and made sure that the value was <1.5. If necessary, an additional amount of HCl was added. The aqueous solution was transferred to a calibrated 3-liter flask (washed with water as required) and concentrated in a rotary evaporator until a solid precipitate formed. The mass of sediment was measured. The precipitate was dissolved in absolute ethanol (1.20 kg) and concentrated again. The precipitate was redissolved in absolute ethanol (1.65-2.85 kg) and heated to 50-65 ° C. The warm solution was filtered under vacuum. The filtrate was transferred to a calibrated 3-liter flask, the ethanol solution was concentrated in a rotary evaporator until the distillation was substantially stopped. Ethyl acetate (8.21 kg) was added in portions and concentrated as before. A fourth portion of ethyl acetate (2.74 kg) was added and cooled to 20-25 ° C. The reaction mixture was stirred for about 15 minutes. The mixture was filtered under vacuum and washed with ethyl acetate (2 portions of 0.135 kg each). Vacuum filtration was continued until essentially stopping the separation of the filtrate. The precipitate was dried under vacuum for about 60 minutes. The filtrate was discarded.

[0078] The wet sediment mass was measured and the mass value (in kg) was used in the following calculations as constant B. The product was transferred to a 12-liter reaction flask equipped with a refrigerator, stirrer, inlet No. ₂ and a thermocouple. Ethyl acetate (45.1 × B kg) was added under nitrogen, the suspension was heated to 48-53 ° C with stirring. Heating was stopped when reaching 50 ° C. The mantle heater was removed, the suspension was cooled to 20-25 ° C. The mixture was filtered under vacuum and washed with ethyl acetate (2 portions of 0.270 kg). Vacuum filtration was continued until essentially stopping the separation of the filtrate. The precipitate was dried in a vacuum oven at a temperature not exceeding 38 ° C for at least 12 hours. The mass of sediment was measured and this mass (in kg) was used in further calculations.

EXAMPLE 14

Obtaining a free base of hydrazide (5)

[0079] The resulting crude hydrochloride product was mixed with water (20.0 × C kg) in a 12 liter 4-necked flask and mixed until a solution formed. The pH of the solution was adjusted using a resin in the range of 6.5-7.5 pH units. The mixture was stirred for about 15 minutes, then the pH was checked again and, if necessary, adjusted to a value of 6.5-7.5 pH units. The mixture was filtered under vacuum and washed with water (3.00 × C kg), then with absolute ethanol (2 portions of 5.30 × C kg). Vacuum filtration was continued until the filtrate was substantially isolated. The filtrate was transferred to a calibrated 3-liter flask and, if necessary, washed with absolute ethanol. The mixture was concentrated on a rotary evaporator until essentially the distillation was complete. The resulting precipitate was redissolved in absolute ethanol (1.58 × C kg) and concentrated as before. The resulting precipitate was redissolved in anhydrous diethyl ether (2.57 × C kg) and concentrated as before. As soon as the distillation ceased, drying was continued in a high vacuum and evaporation was carried out for at least 2 hours. The net weight of the sediment was taken as constant D. Using CH ₂ Cl ₂ (3.98 × D kg), the concentrate was transferred to a 12-liter flask equipped with a stirrer, temperature sensor and nitrogen supply. The mixture was stirred at 15-30 ° C for at least 30 minutes. The mixture was filtered, collecting the filtrates in another 12-liter flask. The first flask was washed with CH ₂ Cl ₂ (3.98 × D kg), the solution was placed in the second flask, passing through a filter. The second flask containing the mixture was equipped with a stirrer, temperature sensor and nitrogen supply. Silica gel (0.700 × D kg) was added with stirring, stirring was continued for approximately 30 minutes. The mixture was filtered under vacuum, the silica gel was washed with CH ₂ Cl ₂ (2 portions of 3.98 × D kg), collecting the obtained filtrates in a 10-liter vacuum filter flask. The mixture was concentrated on a rotary evaporator into a 1 liter flask at a temperature of about 30 ° C. In accordance with the requirements, a 10-liter flask was washed with CH ₂ Cl ₂ , the solution was placed in an evaporator. The distillation was carried out until its essentially complete cessation. The rotary evaporator was switched to the deep vacuum mode and evaporation was continued for 3 hours at 35-40 ° C.

[0080] The oily product was crystallized by adjusting the temperature of the rotary evaporator bath in the range of 0-5 ° C and rotating the flask at maximum speed. Evaporation continued for approximately 30 minutes after the product solidified. A sample of the product (base) was taken and examined for residual methylene chloride, the product was dried until an acceptable test result for the content of residual solvent. The final mass was recorded, the product was placed in yellow glass bottles with caps containing inert material inserts, and samples were taken for analysis.

EXIT

[0081] The final yield of the 5-stage process was at least 33% of the theoretical value (0.105 kg), while the difference between the highest and lowest yields was not more than 15%. The limiting value for the previously known method was 33-43% of theoretical value; however, it is expected that a decrease in the formation of a by-product caused by an increase in the amount of methylene chloride during the hydrazide formation reaction will increase the yield. The actual outputs of the test experiments will be used to determine the range of output values for industrial lots.

EXAMPLE 15

Final purification of 3-methyl-3-mercaptobutanoic acid hydrazide, Cl 332258 (DMH linker)

[0082] Methylene chloride (1000 ml, 1325 g) was placed in a 2 liter 4-necked flask equipped with a mechanical stirrer, N ₂ inlet, reflux condenser and temperature control device. A DMH linker (20 g) was placed in a reaction flask. The suspension was stirred in an atmosphere of N ₂ , at 20 ± 3 ° C for at least 30 minutes. The resulting turbid DMH-linker solution was filtered through a 350 ml Buchner funnel with a medium-porous filter made of porous glass. The filtrate was collected in a clean 2-liter, 4-necked round bottom flask equipped with a mechanical stirrer, N ₂ inlet, reflux condenser and temperature control device. The reaction flask was washed with 20 ml, 26.5 g of methylene chloride, and the solution was poured into a clean reaction flask. Silica gel (20 g) was placed in a solution in a reaction flask, maintaining a temperature of 15-25 ° C. The suspension was stirred in an atmosphere of N ₂ at 20 ± 3 ° C for at least 30 minutes. The heterogeneous mixture was filtered through (350 ml) a Buchner funnel with a medium-pore filter made of porous glass. The filtrate was collected in a clean 2-liter single-necked round-bottom flask. The reaction flask was washed with methylene chloride (50 ml, 66.3 g), the solution was poured into a filter cake, and the filtrate was collected in a one-necked flask. The filtrate was concentrated to a dry residue using a rotary evaporator (bath temperature 35 ± 5 ° C) and a water-jet pump (15-30 mm Hg), and then a deep vacuum (7-10 mm Hg). The resulting white solid was cooled to 0-5 ° C and dried in high vacuum at 7 mm Hg. within 2 hours. n-Heptane (100 ml, 68.4 g) was added to the solid and stirred at room temperature for at least 10 minutes until a homogeneous suspension formed.

[0083] The product was isolated by vacuum filtration through filter paper (# 1 Whatman) on a 15 cm Buchner funnel. A 2-liter flask was washed with a residual n-heptane solution, the solution was transferred to a filter cake, then the flask was washed with n-heptane (2 50 ml portions, 34.2 g). The precipitate was dried under vacuum at room temperature for at least 5 minutes. The precipitate was transferred to a yellow glass bottle placed in a vacuum desiccator. The wet cake was dried from 14.34 g to constant weight under vacuum (<10 mmHg) at 20-25 ° C for 3 hours. Yield: 14.21 g, 75.9%, theoretical yield: 18.7 g.

Test Method Expected Value Result Concentration HPLC, L18284-154 95% 102.8% Total number of impurities HPLC, L18284-148 5% 1.34% The largest single impurity HPLC, L18284-148 5% 1.03% Melting temperature USP TBD 51.5-52 ° C

Claims (31)

1. A method of obtaining a hydrazide product from hydrazine and acid chloride, comprising the steps of:
(a) obtaining a stirred substantially uniform suspension containing hydrazine and an inert solvent; and
(b) continuously adding an acid chloride to said suspension, wherein the acid chloride has the structure:

where P represents a thiol protecting group;
each of R ₁ and R _{2 is} selected from the group consisting of C ₁ -C ₅ alkyl;
L is an alkylene linker,
and the continuous addition of the acid chloride solution is controlled so that the reaction temperature is maintained in the range from -65 ° C to -75 ° C. 2. The method according to claim 1, characterized in that said acid chloride is added dropwise to the suspension. 3. The method according to claim 1, characterized in that said acid chloride contains a benzylthioether group. 4. The method according to claim 1, characterized in that L is —CH ₂ -. 5. The method according to claim 1, characterized in that each of R ₁ and R ₂ independently represents methyl. 6. The method according to claim 1, characterized in that P represents a benzyl group, possibly substituted on the phenyl ring. 7. The method according to claim 6, characterized in that P represents an n-methoxybenzyl group. 8. The method according to claims 1 or 2, characterized in that said acid chloride has the structure:

9. The method according to any one of claims 1 to 7, characterized in that said inert solvent is methylene chloride. 10. The method according to any one of claims 1 to 7, characterized in that said hydrazide compound has the structure:

where P represents a thiol protecting group;
each of R ₁ and R _{2 is} selected from the group consisting of C ₁ -C ₅ alkyl;
L is an alkylene linker. 11. The method according to claim 10, characterized in that L represents -CH ₂ -. 12. The method according to claim 10, characterized in that each of R ₁ and R ₂ independently represents methyl. 13. The method according to claim 10, characterized in that P represents a benzyl group, possibly substituted on the phenyl ring. 14. The method according to item 13, wherein P represents an n-methoxybenzyl group. 15. The method according to claim 10, characterized in that the target hydrazide is

or a salt of said compound. 16. The method according to any one of claims 1 to 7 or 11-14, characterized in that said hydrazide compound contains less than 5% bis-hydrazide by-product having the structure:

where R and R 'are

17. The method according to clause 16, wherein L is —CH ₂ -. 18. The method according to 17, characterized in that each of R ₁ and R ₂ is independently methyl. 19. The method according to clause 16, wherein P represents a benzyl group, possibly substituted on the phenyl ring. 20. The method according to claim 19, characterized in that P represents an n-methoxybenzyl group. 21. The method according to claim 20, characterized in that the bis-hydrazide by-product has the structure:

22. The method according to any one of claims 1 to 7, 11-15 or 17-21, characterized in that the continuous addition of the acid chloride solution is controlled to maintain the reaction temperature in the range from about -68 to about -75 ° C. 23. A method of obtaining a compound containing a hydrazide linker group, comprising the steps of:
(a) cooling the reaction vessel containing a stirred inert solvent to a temperature in the range of from -65 ° C to -75 ° C;
(b) continuously adding hydrazine to said reaction vessel, resulting in a stirred substantially uniform suspension containing hydrazine and said inert solvent;
(c) continuously adding an acid chloride to said suspension, leading to the formation of a hydrazide linker group, wherein the acid chloride has the structure:

where P represents a thiol protecting group;
each of R ₁ and R _{2 is} selected from the group consisting of C ₁ -C ₅ alkyl;
L is an alkylene linker. 24. The method according to item 23, wherein the specified inert solvent is methylene chloride. 25. A method of obtaining a suspension of hydrazine, comprising the steps of:
(a) cooling an inert solvent to a temperature in the range of -68 ° C to -75 ° C; and
(b) adding hydrazine dropwise to said cold inert solvent. 26. The method according A.25, characterized in that the inert solvent is methylene chloride. 27. The method according A.25 or 26, characterized in that the said suspension of hydrazine is stirred at a speed of from 270 to 400 rpm 28. A method of producing a 3-methyl-3-mercaptobutanoic acid hydrazide, comprising the step of producing a hydrazide according to the method of claim 14. 29. A method of producing an immunoconjugate of a member of the family of calichimycins with a monoclonal antibody as a carrier, comprising producing a mono-acylated hydrazide, wherein the acyl group contains an S-protected mercapto group, according to the method of claim 1, removing said protective group from an S-protected mercapto group and using the resulting hydrazide for obtaining the specified immunoconjugate. 30. The method according to clause 29, wherein said hydrazide obtained is 3-methyl-3-mercaptobutanoic acid hydrazide. 31. The method according to clause 29 or 30, wherein the 3-methyl-3-mercaptobutanoic acid hydrazide is used as a linker to produce gemtuzumab ozogamicin or inotuzumab ozogamicin.

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