HRP990317A2 - A process for the preparation of a thrombus imaging agent - Google Patents

Description

The field of technology

This invention generally relates to the efficient and practical commercial synthesis of new reagents for the preparation of radiopharmaceuticals useful as imaging agents in the diagnosis of cardiovascular disorders, infections, inflammation and cancer, as well as intermediates useful for the preparation of these reagents. These reagents consist of stable hydrazine-modified biologically active molecules that react with gamma-emitting radioisotopes to form radiopharmaceuticals that selectively accumulate at disease sites, enabling imaging of those sites by gamma scintigraphy.

State of the art

There is a need for non-invasive diagnostic procedures for various diseases such as thromboembolic disease, atherosclerosis, infection and cancer. Radiopharmaceuticals consisting of biologically active molecules labeled with a radionuclide that emits gamma radiation meet this need. Biologically active molecules serve to accumulate radionuclides at disease sites and thus enable these sites to be visualized by gamma scintigraphy. Molecules are generally proteins, antibodies, antibody fragments, peptides, polypeptides or peptidomimetics. The molecules react with a receptor or binding site expressed at the site of disease, or with a receptor or binding site on an endogenous blood component, such as platelets and leukocytes, that accumulates at those sites. This interaction leads to the selective accumulation of a certain percentage of the injected radiopharmaceutical, while the rest is removed via the kidneys or the hepatobiliary system. The accumulated radiopharmaceutical is then imaged externally, using gamma scintigraphy.

Numerous radiopharmaceuticals consisting of radionuclide-labeled proteins, antibodies or antibody fragments are in the development phase. A combination of factors complicates the development of these radiopharmaceuticals, and among them are obtaining and quality control, unfavorable rates of sequestration and removal, as well as the occurrence of antigenic or allergic responses to radiopharmaceuticals. These difficulties are mainly due to the macromolecular nature of proteins, antibodies and antibody fragments. Their high molecular weight makes direct chemical synthesis impractical, thus requiring synthesis by recombination or cloning techniques, which typically give low yields and require extensive isolation and purification protocols.

The use of peptides, polypeptides or peptidomimetics of lower molecular weight as biologically active molecules solves many of the mentioned problems. These compounds have a tendency to disappear more quickly from the bloodstream through active excretion, which leads to less background in the images. In addition, they are usually not immunogenic.

The use of hydrazine and hydrazide as chelators to modify proteins for labeling with radionuclides was described by Schwartz et al. in US Patent 5206370. The protein is modified by reaction with bifunctional aromatic hydrazines or hydrazides that have a protein-reactive substituent. For labeling with technetium-99m, the hydrazine-modified protein reacts with a reduced technetium compound, obtained by reacting pertechnetate with a reducing agent in the presence of a chelating dioxygen ligand. Technetium binds to protein bonds believed to be hydrazide or diazenide, with a coordination sphere filled with auxiliary dioxygen ligands.

US Patent 5750088 refers to the value of the compound of formula (I) as a novel reagent for the described imaging agents. Therefore, a feasible chemical synthesis that enables its production for the market is desirable. The present invention describes a process close to that for the preparation of (I) that reduces the time required for obtaining it by combining several key chemical transformations. The procedure described here suppresses the formation of undesirable side products, thereby maximizing the purity of the product. In addition, this invention describes a more environmentally friendly alternative to the use of purification reactions and protocols involving the use of carcinogenic and other hazardous solvents.

[image]

Presentation of the essence of the invention

This invention generally relates to procedures for the efficient preparation of (I), which have been achieved by the author's invention that the compound of formula (I), which is an important precursor of radiopharmaceuticals useful as imaging agents, as well as intermediates useful for the preparation of these reagents, can be obtained in high yield and marketable processes described here.

Detailed description of the invention

[1] In its first form, this invention describes a new process for the preparation of a compound of formula (I):

[image]

or its pharmaceutically acceptable salts, and this procedure consists of:

(11) cyclization of the compound of formula (XV):

[image]

or its salts, thereby obtaining the compound of formula (XVI):

[image]

(12) deprotection of the compound of formula (XVI), thereby obtaining the compound of formula (XVII):

[image]

or its salt;

(13) bringing the compound of formula (XVII) into contact with the compound of formula (IX):

[image]

in the presence of the first base. (13-i) bringing the reaction solution from step (13) into contact with the quenching agent, thereby obtaining the compound of formula (XVIII):

[image]

or an alternative salt thereof; and

(14) desalting of the compound of formula (XVIII), thereby obtaining the compound of formula (I).

[2] In the preferred form, the cyclization consists of contacting a compound of formula (XV) with a cyclization agent in a cyclization solvent, in the presence of a second base;

deprotection consists of contacting the compound of formula (XVI) with a strong acid at a suitable temperature; And

desalting consists of dissolving the compound of formula (XVIII) in an aqueous base solution, and then adjusting the pH of the solution, which results in the compound of formula (I).

[3] In a more preferred form, the cyclizing agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate;

the cyclization solvent is acetonitrile;

the second base is N,N-diisopropylethylamine;

strong acid is a mixture of trifluoroacetic acid and trifluoromethanesulfonic acid;

suitable temperature is from about -30°C to about 5°C;

the compound of formula (XVII) is in the form of a salt of di(trifluoromethanesulfonic acid);

the first base is N,N-diisopropylethylamine;

the extinguishing agent is ammonium acetate;

an aqueous base solution consists of sodium hydroxide in water; And

adjusting the pH consists of adding aqueous hydrochloric acid to the solution, until reaching a pH value in the range 4.1 to 4.4.

[4] In the next more recommended form, the compound of formula (XVI) is purified using a C-18 column, charging about 369 g of the reaction solution from step 11 per liter of bed volume.

[5] In the next more recommended form, the compound of formula (XVIII) is purified using a C-18 column, charging about 33 g of the reaction product from step 13i per liter of bed volume.

[6] In the following recommended form, the compound of formula (XV) is prepared by a process consisting of:

(9) coupling of the compound of formula (VI):

[image]

or its salts, with the compound of formula (XIII):

[image]

or its salt, thereby obtaining the compound of formula (XIV):

[image]

(10) deprotection of the compound of formula (XIV), thereby obtaining the compound of formula (XV) or its salt.

[7] In a further preferred embodiment, coupling consists of contacting a compound of formula (VI) with a compound of formula (XIII) in the presence of a coupling agent and a third base; And

deprotection consists of hydrogenating the compound of formula (XIV) at a suitable hydrogen pressure, in the presence of a hydrogenation catalyst, in a hydrogenation solvent.

[8] In an even more preferred form, the compound of formula (VI) is in the form of a salt of trifluoroacetic acid;

the coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

the third base is N,N-diisopropylethylamine;

suitable pressure is about 14.7 psi;

the hydrogenation catalyst is palladium on carbon; and the hydrogenation solvent is methanol.

[9] In the following recommended form, the compound of formula (IX) is prepared by a process consisting of:

(4) contacting the compound of formula (VII):

[image]

with hydrazine, resulting in the compound of formula (VIII):

[image]

(5) bringing the compound of formula (VIII) into contact with the compound of formula (VIIIa):

[image]

and N-hydroxysuccinimide in a polar, aprotic solvent, in the presence of another coupling agent, to give a compound of formula (IX).

[10] In the next more recommended form, the polar aprotic solvent is N,N-dimethylformamide, and the second coupling agent is N,N'-dicyclohexylcarbodiimide.

[11] In another form, this invention describes a new process for the preparation of a compound of formula (VI):

[image]

or its salts, and this procedure consists of:

(3) contacting the compound of formula (V):

[image]

or its salts, with a compound of formula (Va):

[image]

in the presence of a coupling agent and a base, resulting in a compound of formula (V-b):

[image]

(3-i) deprotection of the compound of formula (V-b), thereby obtaining the compound of formula (VI), or its salt.

[12] In the following preferred form, the compound of formula (VI) is in the form of a salt of trifluoroacetic acid;

the coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

the base is N,N-diisopropylethylamine;

deprotection consists of contacting the compound of formula (V-b) with trifluoroacetic acid, at a temperature ranging from about -20°C to about 0°C; And

the compound of formula (V) is in the form of a hydrochloric acid salt.

[13] In the following recommended form, the compound of formula (V) is prepared by a process consisting of:

(2) contacting the compound of formula (IV):

[image]

with the compound of formula (IV):

[image]

or its salt; in the presence of a second coupling agent and a second base, resulting in a compound of formula (IV-b):

[image]

(2-i) deprotection of the compound of formula (IV-b), thereby obtaining the compound of formula (V), or its salt.

[14] In the next more recommended form, the compound of formula (IV-a) is in the form of a salt of p-toluenesulfonic acid;

another coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

the second base is N,N-diisopropylethylamine;

deprotection consists of contacting the compound of formula (V-b) with hydrochloric acid, at a temperature ranging from about -20°C to about 0°C; and the compound of formula (V) is in the form of a hydrochloric acid salt.

[15] In a third form, this invention describes a new process for the preparation of the compound of formula (XIII):

[image]

or its salts, and this procedure consists of:

(8) saponification of the compound of formula (XII):

[image]

or its salt, and bringing the resulting product into contact with the compound of formula (XIIa):

[image]

thus obtaining the compound of formula (XII-b):

[image]

or its salt; and

(8-i) bringing the compound of formula (XII-b) into contact with the compound of formula (III):

[image]

thereby obtaining a compound of formula (XIII) or a salt thereof.

[16] In the following recommended form, the compound of formula (XII) is in the form of a di(hydrochloric acid) salt; And

the compound of formula (XII-b) is in the form of a sodium salt.

[17] In the following recommended form, the compound of formula (XII) is prepared by a process consisting of:

(6) contacting the compound of formula (X):

[image]

with thionyl chloride, tetramethylene sulfone and sulfamide, at a temperature suitable for the formation of the compound of formula (XI):

[image]

(7) reduction of the compound of formula (XI), thereby obtaining the compound of formula (XII) or its salt.

[18] In a further preferred embodiment, a suitable temperature is in the range of about 100°C to about 125°C;

the compound of formula (XII) is a di(hydrochloric acid) salt; And

the reduction consists of hydrogenating the compound of formula (XI) at a hydrogen pressure of about 50 psi to about 70 psi, in the presence of platinum (IV) oxide as a hydrogenation catalyst.

[19] In the following recommended form, the compound of formula (III) is prepared by a process consisting of:

(1) contacting the compound of formula (II):

[image]

with a coupling agent to obtain the compound of formula (III).

[20] In the next more preferred form, the coupling agent is 1-(3-dimethylamino propyl)-3-ethylcarbodiimide, and the coupling takes place in acetonitrile, at a temperature ranging from about -10°C to about 10°C.

[21] In the following recommended form, the compound of formula (XII-b) is prepared by a process consisting of:

(8a) bringing into contact the compound of formula (XII-c):

[image]

or its salts, with a base, and bringing the resulting product into contact with the compound of formula (XIIa):

[image]

thereby obtaining the compound of formula (XII-b) or its salt.

DEFINITIONS

The following are the terms and abbreviations used, with their respective meanings. The abbreviation "Ts" or "tosyl", as used herein, denotes a 4-methylphenylsulfonyl protecting group, "Boc" as used herein denotes a butyloxycarbonyl protecting group, "DMF" as used herein denotes N,N-dimethylformamide, “THF” as used herein means tetrahydrofuran, “EtOAc” as used herein means ethyl acetate, “MTBE” as used herein means methyl t-butyl ether, “TFA” as used herein means trifluoroacetic acid, “ TFMSA” as used herein means trifluoromethanesulfonic acid, “Osu” as used herein means N-hydroxysuccinimido, “room temp.” as used herein means room temperature, “psi” as used herein means pounds per square inch, “HPLC” as used herein means high performance liquid chromatography, “DIEA” as used herein means N,N-diisopropylethylamine, “HBTU ” as used herein means O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, “DCC” as used herein means N,N'-dicyclohexylcarbodiimide and “EDC” in as used herein means 1-(3-dimethylamino propyl)-3-ethylcarbodiimide.

The reactions of the synthetic procedures of this patent are carried out in suitable solvents that will be readily selected by one skilled in the art of organic synthesis, a suitable solvent being any solvent that is essentially unreactive with the starting materials (reactants), intermediates and products, at temperatures at which reactions are carried out, i.e. at temperatures that can range from the freezing point of the solvent to the boiling point of the solvent. A single reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the individual reaction step, suitable solvents can be selected for the individual reaction step. These suitable solvents may include, by way of example and without limitation, chlorinated solvents, hydrocarbon solvents, ethereal solvents, polar protic solvents and polar aprotic solvents.

Suitable halogenated solvents include, without limitation, carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane, tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,2-trichloroethane. 1-dichloroethane, 2-chloropropane, hexafluorobenzene, 1,2,4-trichlorobenzene, o-dichlorobenzene, chlorobenzene, fluorobenzene, fluorotrichloromethane, chlorotrifluoromethane, bromotrifluoromethane, carbon tetrafluoride, dichlorofluoromethane, chlorodifluoromethane, trifluoromethane, 1,2-dichlorotetrafluoroethane, and hexafluoroethane.

Suitable hydrocarbon solvents include, without limitation, benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o- or p-xylene, octane, indane, nonane, chlorobenzene or naphthalene.

Suitable ether solvents include, but are not limited to, dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether , triethylene glycol dimethyl ether, anisole or t-butyl methyl ether.

Suitable polar protic solvents include, but are not limited to, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2- or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether , cyclohexanol, benzyl alcohol, phenol and glycerol.

Suitable polar aprotic solvents include, but are not limited to, dimethylformamide (DMF), dimethylacetamide (DMAC), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), 1,3 -dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP), formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate, isopropyl acetate, t-butyl acetate, sulfolane, N,N-dimethylpropionamide, nitromethane, nitrobenzene, hexamethylphosphoramide.

As used herein, "coupling solvent" refers to any solvent in which an amine can react with a carboxylic acid derivative to form an amide. Examples of such coupling solvents include, but are not limited to, ether, halogenated, hydrocarbon, and polar aprotic solvents.

As used herein, "strong acid" includes, without limitation, mineral acids such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, lithium bisulfate, potassium bisulfate, sodium bisulfate, and ammonium chloride; and organic acids such as formic acid, acetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, ethanoic acid, propionic acid, butyric acid, valeric acid and caproic acid.

As used herein, "suitable bases" include any of a variety of bases, the presence of which in the reaction facilitates the synthesis of the desired product. Suitable bases can be selected by those skilled in the art of organic synthesis. Suitable bases include, but are not limited to, inorganic bases, such as alkali metal, alkaline earth metal, thallium, and ammonium hydroxides, alkoxides, phosphates, and carbonates, as well as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate , thallium hydroxide, thallium carbonate, tetra-n-butylammonium carbonate and ammonium hydroxide. Suitable bases also include organic bases, including, without limitation, aromatic and aliphatic amines, such as pyridine; trialkyl amines such as triethylamine, N,N-diisopropylethylamine, N,N-diethylcyclohexylamine, N,N-dimethylcyclohexylamine, N,N,N'-triethylenediamine, N,N-dimethyloctylamine; 1,5-diazabicyclo [4.3.0] non-5-ene (DBN); 1,4-diazabicyclo [2.2.2] octane (DABCO); 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); tetramethylethylenediamine (TMEDA); substituted pyridines such as N,N-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine, 4-piperidinopyridine and morpholines such as N-methyl morpholine.

As used herein, "aqueous base" includes, without limitation, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium bicarbonate, sodium bicarbonate, and potassium bicarbonate. As used herein, “aqueous base solution” includes, without limitation, aqueous systems in which an aqueous base is dissolved, which may or may not include additional organic solvent ingredients. For example, an aqueous base solution includes, but is not limited to, sodium hydroxide in water.

As used herein, the term "coupling agent" refers to any agent, known in the art of organic synthesis or peptide chemistry, capable of promoting the formation of an amide bond from a carboxylic acid and an amine. Examples of these agents include, without limitation, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), carbonyldiimidazole (CDI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), O-(1H-benzotriazol-1-yl )-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (benzotriazolyloxy)tris (dimethylamino)phosphonium hexafluorophosphate (BOP), 2,3- dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

As used herein, “cyclizing agent” includes any agent that promotes the formation of an intramolecular amide bond between a carboxylic acid and an amine. Examples of these agents include, without limitation, those referred to herein as coupling agents.

As used herein, “suitable pressures” range from atmospheric to any pressure achievable in a laboratory or industrial facility.

As used herein, "hydrogenation catalysts" are those which promote the removal of a protecting group known in the art of organic synthesis to be hydrogen labile. These catalysts also include those known in the art to reduce a nitro group to an amino group and a cyano group to an aminomethyl group. These hydrogenation catalysts include, without limitation, palladium on carbon, palladium hydroxide on carbon, palladium on lead-poisoned calcium carbonate, platinum on carbon, and platinum (IV) oxide.

As used herein, "hydrogenation solvents" include solvents known in the art of organic synthesis to be useful for reactions occurring in a hydrogen atmosphere, such as hydrogenation. Examples of such solvents include, without limitation, polar protic solvents such as methanol, ethanol, propanol, isopropanol and the like, ethers, hydrocarbons, etc.

The compounds described here may have asymmetric centers. Unless otherwise indicated, all chiral, diastereomeric and racemic forms are included in this invention. It is apparent that the compounds of this invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Processes for the preparation of active forms from optically active starting materials are known in the art, some of which are resolution of racemic forms or synthesis. All chiral, diastereomeric, racemic forms, as well as all geometrically isomeric forms of the structure are included.

This invention includes all isotopes of atoms occurring in intermediate compounds or final compounds. Isotopes include those atoms that have the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium; carbon isotopes include 13C and 14C.

As used herein, "counterion" refers to a negative or positive ion that serves to pair in the salts of the described intermediates or products. Examples of counterions include, without limitation, ammonium, trifluoroacetate, chloride, and sodium.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the described compounds in which the intermediates or final compound have been altered by forming acid or basic salts of the intermediates or final compounds. Examples of pharmaceutically suitable salts include, without limitation, mineral or organic acid salts or basic residues such as amines; alkalis or organic salts of acidic residues, such as carboxylic acids; and similar.

It should be noted that the compounds of this invention contain different ionic centers. Therefore, depending on the pH of the solution, it is possible that more than one ion will be present in some of the compounds of this invention (ie, zwitterions). Accordingly, both external and internal salts are considered part of the present invention.

Pharmaceutically suitable salts of intermediates or final compounds include the usual non-toxic quaternary ammonium salts of non-toxic inorganic or organic acids. For example, these common non-toxic salts include those derived from inorganic acids such as hydrochloric, bromic, sulfate, sulfamic, phosphate, nitrite, and the like; as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic and similar.

Pharmaceutically acceptable salts are generally prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired inorganic or organic salt-forming acid or base, in a suitable solvent or in various combinations of solvents.

Pharmaceutically acceptable acid salts of the intermediates or final compounds are prepared by combining with an appropriate amount of a base, such as an alkali or alkaline earth metal hydroxide, e.g. sodium, potassium, lithium, calcium or magnesium, or an organic base such as an amine, e.g. dibenzylethylenediamine, trimethylamine, piperidine , pyrrolidine, benzylamine and the like, or quaternary ammonium hydroxide such as tetramethylammonium hydroxide and the like.

As set forth above, pharmaceutically acceptable salts of the compounds of the invention may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base, or acid, in water or an organic solvent, or in a mixture of the two; anhydrous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally recommended. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985, p. 1418, the contents of which are incorporated herein by reference.

The field of application of this invention is considered to be at least several grams, then kilograms, several kilograms, as well as industrial. Multigram order of magnitude, as used herein, refers to that extent where at least one starting substance is present in an amount of 10 grams or more, preferably at least 50 grams or more, even more preferably at least 100 grams or more. Multi-kilogram order of magnitude, as used here, refers to the extent where more than one kilogram of at least one starting substance is used. Industrial order of magnitude, in the sense in which it is used here, refers to that scale that is different from the laboratory, and is suitable for obtaining a product in a quantity sufficient either for clinical trials or for supplying consumers.

The processes of this invention, by way of example and without limitation, can be further traced to Schemes 1 through 9, which depict the general synthesis of a compound of formula (I). The compounds of formula (II), (VI), (VII) and (X) are either available on the market, or can be obtained by procedures, well known to those skilled in synthetic organic chemistry, on substances available on the market.

SYNTHESIS

[image]

Step 1: A compound of formula (III) can be prepared by contacting a compound of formula (II) with a first coupling agent. The first coupling agent is recommended 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The coupling reaction preferably takes place in a polar aprotic solvent (eg CH3CN), at a temperature ranging from about -10°C to about 10°C.

[image]

Step 2: By contacting the compound of formula (IV) with the compound of formula (IV-a) or its salt (e.g. p-toluenesulfonic acid salt) in the presence of another coupling agent and another base, the compound of formula (IV-b) is prepared . Preferably, the second coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and the second base is N,N-diisopropylethylamine. If desired, the in situ interface (IV-b) can be isolated. This interface can also be purged.

The compound of formula V or its salt (e.g. hydrochloric acid salt) can be obtained by deprotection (IV-b). Deprotection can be carried out by contacting the compound of formula (V-b) with a strong acid (eg hydrochloric acid) at a temperature ranging from about -20°C to about 0°C.

[image]

Step 3: By contacting a compound of formula (V) or its salt (e.g. HCl salt) with a compound of formula (Va in the presence of a coupling agent and a base, it is possible to prepare a compound of formula (V-b). The coupling agent is recommended O- (1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the base is preferably N,N-diisopropylethylamine.If desired, intermediate (V-b) can be isolated in situ and then purified .

Step 3-i: By deprotection of the compound of formula (V-b), the compound of formula (VI) or its salt (eg salt of trifluoroacetic acid) is obtained. Deprotection consists of bringing the compound of formula (V-b) into contact with a strong acid (eg trifluoroacetic acid), preferably at a temperature ranging from about -20°C to about 0°C.

[image]

Step 4: A compound of formula (VIII) can be formed by contacting a compound of formula (VII) with hydrazine.

Step 5: Contacting a compound of formula (VIII) with a compound of formula (VIIIa) and N-hydroxysuccinimide, in a polar aprotic solvent, in the presence of another coupling agent, gives a compound of formula (IX). A polar aprotic solvent is recommended DMF, and another coupling agent is recommended DCC.

[image]

Step 6: A compound of formula (XI) can be prepared by contacting a compound of formula (X) with thionyl chloride, tetramethylene sulfone and sulfamide. It is recommended to use at least one mole equivalent of each, ie, thionyl chloride, tetramethylene sulfone, and sulfamide. Even more recommended is 1.6 eq of thionyl chloride, 9.2 eq of sulfolane and 1.2 eq of sulfamide. A suitable temperature is in the range from about 100°C to about 125°C.

Step 7: Reduction of a compound of formula (XI) gives a compound of formula (XII) or a salt thereof (eg di-HCl salt). The reduction preferably involves hydrogenating the compound of formula (XI) under a hydrogen pressure of about 50 psi to about 70 psi, in the presence of a hydrogenation catalyst (eg, platinum (IV) oxide).

Step 8: Saponification of the compound of formula (XII) or its salt (e.g. di-HCl salt) and bringing the resulting compound into contact with the compound of formula (XII-a) yields the compound of formula (XII-b) or its salt (e.g. On salt). Saponification involves bringing the compound of formula (XII) or its salt into contact with an aqueous base solution (eg NaOH/H2O) and adjusting the appropriate pH of the aqueous base solution. The appropriate pH is recommended in the range of about 7.5 to about 8.5, more recommended 7.9-8.1, and the adjustment is recommended to be achieved by using a strong acid such as HCl. The compound of formula XII-b can then be converted to the compound of formula XIII via Step 8-i.

Step 8a: As an alternative to step 8, instead of saponifying the compound of formula (XII), the compound of formula (XII-c) or a salt thereof (e.g. di-HCl salt) may be contacted with a base (e.g. NaOH) and the resulting product is brought into contact with the compound of formula (XII-a), thereby obtaining the compound of formula (XII-b) or its salt (eg Na salt).

Step 8-i: By contacting the compound of formula (XII-b) with the compound of formula (III), the compound of formula (XIII) or its salt is obtained. Bringing it into contact is recommended to take place at a temperature in the range of about 0-5°C.

[image]

Step 9: Coupling a compound of formula (VI) or a salt thereof (eg TFA salt) with a compound of formula (XIII) or a salt thereof gives a compound of formula (XIV). Coupling in this step tends to involve bringing the two compounds into contact in the presence of a coupling agent and a third base. Preferably, the coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and the third base is N,N-diisopropylethylamine.

[image]

Step 10: Deprotection of a compound of formula (XIV) gives a compound of formula (XV) or a salt thereof. Deprotection in this step involves hydrogenating a compound of formula (XIV) at a suitable hydrogen pressure (eg, about 14.7 psi) in the presence of a suitable hydrogenation catalyst (eg, Pd/C) in a suitable hydrogenation solvent (eg, MeOH). Preferably, hydrogen is bubbled through the solution. However, pressures ranging from atmospheric to 60 psi can also be used.

[image]

Step 11: A compound of formula (XVI) can be prepared by cyclization of a compound of formula (XV). In this step, cyclization involves contacting a compound of formula (XV) with a cyclization agent (eg, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) in a solvent to cyclization (eg CH3CN), in the presence of another base (eg N,N-diisopropylethylamine). It is more recommended that the cyclization solvent be a mixture of CH3CN and THF.

Step 11 is recommended to be performed by reverse addition. In this context, the reverse addition is preferably carried out by adding a solution of a compound of formula (XV) (eg XV in CH3CN) to a solution of a cyclizing agent, a cyclizing solvent and another base. More preferably, the solution containing the compound of formula (XV) also contains a second base. It is even more recommended that the cyclizing agent be dissolved in CH3CN and THF. Preferably, the solution containing the cyclizing agent is heated to a temperature of 50-70°C before adding the solution containing the compound of formula (XV). It is more advisable to heat the solution containing the cyclizing agent to a temperature of 57-63°C before adding XV.

The compound of formula (XVI) can be purified by column chromatography, preferably a C-18 column (eg Shandon Hyperprep HS C-18 (12 μ) column (10x55.5 cm)). It is recommended that about 300-400 g, more recommended about 369 g, of the reaction mixture per liter of bed volume be loaded onto the column. Although a large amount of substance is applied to the column in this way, it is possible to obtain a purified substance by using an appropriate eluent and gradient. It is recommended to use 65% water/35% acetonitrile with a gradient bringing the mixture to 42% water/58% acetonitrile over approximately 120 minutes. Although the flow rate affects the rate of elution of the compound of formula (XVI), at 430 mL/min, elution can be expected when it contains a solvent of about 40-55% acetonitrile.

[image]

Step 12: Deprotection of the compound of formula (XVI) gives the compound of formula (XVII) or a salt thereof (eg di(trifluoromethanesulfonic acid) salt). This step is preferably carried out by contacting the compound of formula (XVI) with a strong acid, at a suitable temperature. Recommended a strong acid is a mixture of trifluoroacetic acid and trifluoromethanesulfonic acid. This acid mixture is preferably in a ratio of about 1 molar equivalent of TFA to about 4 molar equivalents of TFMSA. The recommended suitable temperature is from about -30 to 5°C.

Step 13: The compound of formula (XVIII), or its alternative salt, can be prepared by contacting the compound of formula (XVII) with the compound of formula (IX) in the presence of a first base, and then quenching (Step 13-i). Preferably, the first base is N,N-diisopropylethylamine. The compound of formula (IX) may be different from the sodium salt (eg potassium).

Step 13-i: Step 13 is completed by contacting the compound of formula (XVIII) with a quenching agent. Recommended extinguishing agent is ammonium acetate.

The compound of formula (XVIII) can be purified by column chromatography, preferably a C-18 column (eg Shandon Hyperprep HS C-18 (12 μ) column (10x55.5 cm)). It is recommended that about 30-40 g, more preferably about 33 g, of the reaction product per liter of bed volume is loaded onto the column. Although a large amount of substance is applied to the column in this way, it is possible to obtain a purified substance by using an appropriate eluent and gradient. Recommended gradient conditions are, initially: 10% acetonitrile/90% 50 mM aqueous sodium acetate; finally: 17% acetonitrile/83% 50 mM aqueous sodium acetate for approximately 35 min. This is followed by a second gradient to achieve 25% acetonitrile/75% 50 mM aqueous sodium acetate over approximately 260 minutes. Although the flow rate affects the rate of elution of the compound of formula (XVI), at 450 mL/min, elution can be expected when the solvent contains about 19 to 21% acetonitrile.

Step 14: Desalting of the compound of formula (XVIII) gives the compound of formula (I). In this context, desalting consists of dissolving the compound of formula (XVIII) in an aqueous base solution (eg NaOH/H 2 O) and then adjusting the pH of the solution, thereby obtaining the compound of formula (I). Adjusting the pH of the solution consists of adding a strong aqueous acid (eg HCl/H2O) to the solution until the desired pH value is reached, preferably in the range of about 4.1 to about 4.4. Under conditions of the desired pH, the compound of formula (I) precipitates in the zwitterionic form.

EXAMPLES

Other features of the invention will be apparent from the following descriptions of the forms set forth by way of example, which are given to illustrate the invention and are not intended to limit it.

EXAMPLE 1

Synthesis of 6-[[(1,1-dimethylethoxy)carbonyl]amino]hexanoic anhydride (III)

Boc-6-aminohexanoic acid (II; 1553 g) was dissolved in acetonitrile (13663 g) and then cooled to 0-4°C. Ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride (EDC; 786 g) was added and the solution was stirred for 3 h at 0-5°C. After rotary evaporation (bath temperature 20-30°C), the oil is redissolved in ethyl acetate (14934 g) and water (8392 g) and then partitioned. The organic phase is washed two more times with water (8392 g each), twice with sodium bicarbonate solution (each washing solution is prepared by adding 772 g of sodium bicarbonate to 8124 g of water) and once with salt solution (prepared by adding 2652 g of sodium chloride to 7386 g of water). The organic layer is dried over magnesium sulfate (235 g) for 15-30 min., filtered and then concentrated by circular evaporation (bath temperature 20-30°C). The residue was dissolved in cyclohexane as a thick solution (3525 g) for 60-90 minutes at 20-25°C. The solids are isolated by filtration or centrifugation, washed with cyclohexane (1 x 3525 g; 1 x 2585 g) and dried in a vacuum oven at 25-30°C, which gives 1222 g (82%) of compound (III).

EXAMPLE 2

Synthesis of N-[N5-[imino[[(4-methylphenyl)sulfonyl]amino]methyl]-N2-methyl-L-ornityl]-glycine, phenylmethyl ester monohydrochloride (V)

The reaction vessel is filled successively with ethyl acetate (2805 g), (IV; 850 g). (IV-a in the form of the tosylate salt; 765 g), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 850 g), and more ethyl acetate (1131 g) . N,N-diisopropylethyl amine (DIEA; 757 g) was added dropwise, maintaining the temperature at 20-30°C. After addition, the mixture is stirred until analytical HPLC confirms the disappearance of (IV). A solution of citric acid (327 g) in water (2665 g) is added, followed by an additional amount of ethyl acetate (748 g). After mixing, the layers are separated and the organic layer is washed two more times with citric acid solution, then twice with sodium bicarbonate solution (each washing solution is prepared by adding 122 g of sodium bicarbonate to 1264 g of water), and twice with salt solution (each is prepared by adding 408 g of sodium chloride to 1139 g of water). The organic phase is dried over magnesium sulfate for 30-60 minutes and then filtered. The solids are washed with ethyl acetate (1326 g) and the filtrate (containing in situ intermediate (IV-b)) is transferred to a reaction vessel (washing with 128 g of ethyl acetate). After cooling to -15 to -21°C, gaseous hydrogen chloride (approximately 1308 g) is added to the mixture, with the temperature not exceeding -10°C. The solution is stirred for 15 minutes and then analyzed by analytical HPLC to determine the disappearance of intermediate (IV-b). If necessary, the reaction vessel is charged with additional hydrogen chloride gas (approximately 131 g). After completion, methyl t-butyl ether (MTBE; 4956 g) was added while maintaining a temperature of -10 to -15°C. After the addition is complete, the thick solution is heated to -6 to 0°C. The solids are isolated by filtration or centrifugation and then washed with additional MTBE (1 x 2508 g and 1 x 5908 g). The solids are redissolved in MTBE (6800 g) as a thick solution for 12-15 h at 20-25°C and then filtered or centrifuged once more, washing with MTBE (2465 g). Drying in a vacuum oven (30°C) gives 1082 g (107%) of compound (V) in the form of the hydrochloride salt.

EXAMPLE 3

Synthesis of N-[N5-[imino[[4-methylphenyl)sulfonyl]amino]methyl]-N2-methyl-N2-D-valyl-L-ornityl]-glycine, phenylmethyl ester of trifluoroacetate (VI)

The reaction vessel was sequentially charged with (V) as the hydrochloride salt (1077 g), (V-a) (641 g), HBTU (1118 g) and ethyl acetate (8190 g). After the dissolution is complete, DIEA is added at such a rate that the temperature is maintained at 20-30°C. The mixture is then stirred until the end of the reaction according to analytical HPLC (typically 18-20 h). A solution of citric acid (645 g) in water (5251 g) is added, followed by an additional amount of ethyl acetate (205 g). After mixing, the layers are separated and the organic phase is washed once more with citric acid solution, then twice with sodium bicarbonate solution (each is prepared by adding 530 g of sodium bicarbonate to 5525 g of water), and twice with salt solution (each is prepared by adding 931 g of sodium chloride in 2595 g of water). The organic phase is dried over magnesium sulfate (676 g) for 30-60 minutes and then filtered. The solids are washed with ethyl acetate (1280 g) and the filtrate is partially concentrated by rotary evaporation (bath temperature 25-35°C) to a target weight of 2590 g. If necessary, more ethyl acetate can be added to reach the target weight. This solution contains an in situ intermediate (V-b).

The second reaction vessel is filled with trifluoroacetic acid (2436 g) and cooled to -15°C. A portion of the above solution (V-b) (1295 g) is further diluted with ethyl acetate (160 g) and then added to the reaction vessel at such a rate that the temperature is maintained at -15 to -10°C. Upon completion of the addition, the additional funnel was washed with ethyl acetate (60 g) and the solution was stirred at -7 to -3°C, until confirmation of completion by analytical HPLC (typically 22-62 h). A special reaction vessel is charged with MTBE (9322 g) and cooled to 0-5°C. The above reaction mixture is added to the container with MTBE over 50-70 minutes. After washing with additional MTBE (266 g), the mixture is stirred for 30-60 minutes at 0-5°C, after which the solids are collected by filtration or centrifugation. The solids are washed once with MTBE (5412 g) and once with a mixture of MTBE in cyclohexane (1623 g each), dissolved to form a thick solution in a mixture of MTBE and cyclohexane (5520 g each) for 2-4 h, at 20-25° C. The solids are isolated once again, by filtration or centrifugation, and then washed once with a mixture of MTBE and cyclohexane (5520 g each) and another smaller amount of MTBE and cyclohexane (2165 g each). Drying in a vacuum oven (30°C) yields 680 g (92%) of (VI) in the form of its TFA salt.

EXAMPLE 4

Synthesis of 6-hydrazino-3-pyridinecarboxylic acid (VIII)

A solution of (VII) (340 g) in hydrazine monohydrate (1532 g) is heated to 98-102°C for 4 h. After cooling to 45-55°C, the solution is concentrated by rotary evaporation (bath temperature 60°C) and the residue is redissolved in water (1618 g). With cooling (10-20°C), the pH is adjusted to 5.4-5.6 by adding concentrated hydrochloric acid (approximately 1160 g) and the precipitate is collected by filtration or centrifugation. The solids are washed with water (194 g), ethanol (788 g) and MTBE (486 g) and dried in a vacuum oven (35-40°C), which gives 301 g (91%) of compound (VIII).

EXAMPLE 5

Synthesis of 2-[[[5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid, monosodium salt (IX)

Compound (VIIIa) (400 g) was stirred in DMF (11172 g) for 10-20 minutes at 20-25°C, and then (VIII) (294 g) was added. The suspension is stirred for 4-5 h at 20-25°C, then N-hydroxysuccinimide (HOSu; 221 g) is added. After 15-20 min. stirring at 20-25°C, N,N'-dicyclohexylcarbodiimide (DCC; 813 g) is added and stirring is continued for another 14-24 h at 20-25°C. After filtering through a pad of Hyflo Super-Cel or Celite (147 g) and washing with DMF (1682 g), the filtrate is concentrated by rotary evaporation (35-45°C). The residue was dissolved as a thick solution in DMF (559 g) over 10-30 min. at 20-25°C and MTBE (9937 g) was added. The mixture is kept under reflux (external temperature 60-70°C) for 30-40 minutes, cooled to 45-52°C and then filtered. The solid was washed with MTBE (1411 g) and then redissolved as a thick solution in methanol (6997 g) at 70-75°C (external) for 50-70 minutes. After hot filtration, the solid is washed with methanol (3058 g) and MTBE (882 g) and then dried in a vacuum oven at 87-93°C until the vapor impurity at 3.2 ppm becomes invisible by 1H NMR spectroscopy, giving 639 g (76%) of compound (IX).

EXAMPLE 6

Synthesis of 3-cyano-5-nitrobenzoic acid, methyl ester (XI)

The reaction vessel is charged with (X) (904 g), sulfonamide (463 g), sulfolane (4416 g) and thionyl chloride (1194 g), and heated to 115-122°C for 14-22 h. The mixture is cooled to 50-60°C and an additional aliquot of thionyl chloride (177 g) is added, followed by reheating to 115-122°C for 5-8 h.

After cooling to 40-45°C, the reaction mixture is processed in two parts as follows: a part of the reaction solution (2874 g) is added to a flask containing water (16260 g) while maintaining a temperature of 15-25°C. After the addition is complete, the solution is stirred for 2-3 h at 15-25°C and the solid part is filtered off or centrifuged, washing the solid part with an additional amount of water (2710 g). This gives a total of 771 g of raw product.

The crude product (770 g) was dissolved in ethyl acetate (19423 g) by heating to reflux (external temperature 85-95°C) and cooling to 55-65°C. The solution is filtered through Hyflo Super-Cel or Celite (77 g), which is then washed with ethyl acetate (511 g). The filtrate is concentrated by rotary evaporation (bath temperature 60°C) and the obtained residue is prepared as a thick solution in ethanol (475 g) and concentrated again. The ethanol slurry preparation/concentration procedure was repeated once more before suspending the solids in ethanol (19109 g) and heating at 74-78°C for 12-18 h. The hot solution is filtered through Hyflo Super-Cel or Celite (110 g) which is then washed with ethanol (511 g). The filtrate is partially concentrated (target weight 10230 g; more ethanol can be added if necessary), refluxed (external temperature 85-95°C) for 30 minutes, and then cooled to 0-5°C. After 2-3 h, the solids are isolated by filtration or centrifugation, and washed with ethanol (730 g), yielding 591 g (71%) of compound (XI).

EXAMPLE 7

Synthesis of 3-amino-5-(aminomethyl)benzoic acid, methyl ester of dihydrochloride (XII)

The hydrogenation vessel is charged with (XI) (587 g), 1-propanol (25629 g) and concentrated hydrochloric acid (701 g). A thick solution of platinum (IV) oxide (29 g) in 1-propanol (949 g) was added and the mixture was hydrogenated at approximately 60 psi and 55-60°C. After analytical HPLC indicates completion of the reaction (an additional 6 g aliquot of platinum(IV) oxide may be required), water (5695 g) is added and the mixture is cooled to 25-35°C for 30-60 minutes. Filtration through a Halar filter or Celite gives a filtrate that is concentrated by circular evaporation (bath temperature 45°C). The residue is prepared as a thick solution in methanol (1898 g) for approximately 10 minutes and concentrated again. The process of preparing a thick solution/concentrating with methanol is repeated once more before adding a mixture of acetonitrile (5430 g) and methanol (664 g) and heating to reflux (external temperature 75-85°C) for 2-3 h. After cooling to 20-25°C, the mixture is stirred for an additional 2-3 h and the solids are isolated by filtration or centrifugation. The product is washed with a mixture of acetonitrile (1481 g) in methanol (150 g) and then dried in a vacuum oven (45°C), which gives 616 g (86%) of (XII) dihydrochloride salt.

EXAMPLE 8

Synthesis of (S)-3-[[6-[[(1,1-dimethylethoxy)carbonyl]amino]-1-oxohexyl]amino]-5-[[[4-(1,1-dimethylethoxy)-1,4 -dioxo-2-[[(phenylmethoxy)carbonyl]amino]butyl]amino]methyl] benzoic acid (XIII)

Compound (XII) in the form of dihydrochloride salt (305 g) is saponified by adding a solution of sodium hydroxide pellets (204 g) in water (1649 g) at 30°C. After the addition is complete, the mixture is stirred for 60-80 minutes, and then a solution of concentrated hydrochloric acid (284 g) in water (767 g) is added at 18-22°C until pH 7.9-8.1 is reached.

The reaction vessel is then charged with (XII-a) (493 g) and acetonitrile (4144 g). The pH is periodically adjusted to 7.9-8.1 by adding sodium hydroxide solution (prepared by dissolving 120 g of sodium hydroxide pellets in 1084 g of water). After 40-45 minutes, the reaction is monitored by analytical HPLC and, if necessary, more aliquots (XIIa) (24 g) are added until the reaction is complete. The mixture is partially concentrated (target weight 4305 g) by rotary evaporation (bath temperature 25-35°C) and sufficient water is added to give a total volume of approximately 8914 mL. Ethyl acetate (9251 g) was added and the pH was adjusted to 3.4-3.6 by adding a solution of citric acid (474 g) in water (1133 g). The aqueous layer is removed, and the organic phase is washed three times with water (1245 g each time) and twice with brine (each washing solution is prepared by adding 588 g of sodium chloride to 1641 g of water). The organic solution is then dried over magnesium sulfate (169 g) for 15-30 minutes, and filtered (washing with 530 g of ethyl acetate). The filtrate was concentrated by rotary evaporation (bath temperature 25-35°C) to give (XII-b), redissolved in THF (458 g) and concentrated again (bath temperature 25-35°C). The THF addition/concentration procedure is repeated one more time, when the residue is re-dissolved in a larger amount of THF (2590 g) and cooled to 0-3°C.

A solution of (III) (501 g) in THF (1527 g) was added over 20-60 minutes, maintaining the temperature at 0-5°C. After the addition is complete, the solution is cooled to 20-25°C for 50-70 minutes and then stirred for 16-20 hours. By circular evaporation (bath temperature 30-40°C) an oil is obtained, which is redissolved in ethyl acetate at 30-40°C (1482 g) and concentrated again. The obtained oil is re-dissolved in a larger volume of ethyl acetate (8301 g) and then washed twice with sodium bicarbonate (once a solution was prepared from 623 g of sodium bicarbonate in 6493 g of water, and the second time from 311 g of sodium bicarbonate in 3246 g of water), once with a solution of hydrochloric acid (prepared by dissolving 129 g of concentrated hydrochloric acid in 6394 g of water), and once with a salt solution (prepared by adding 1017 g of sodium chloride to 2837 g of water). The organic phase is dried over magnesium sulfate (163 g) for 15-30 minutes, filtered, the solid part is washed with ethyl acetate (667 g), and the filtrate is partially concentrated (target weight 4471 g) by circular evaporation (bath temperature 30-40°C ). The solution is cooled to 20-25°C for 16-20 h, after which the solid part is filtered off or centrifuged and washed with ethyl acetate (697 g). The solid part is suspended in a mixture of ethyl acetate (504 g) and n-heptane (7412 g), and heated to 40-46°C for 40-60 minutes. After cooling to 20-25°C, the solid part is isolated by filtration and washed with additional n-heptane. After drying in a vacuum oven (25°C), 530 g (65%) of (XIII) are obtained.

EXAMPLE 9

Synthesis of N-[N2-[N-[3-[[6-[[(1,1-dimethylethoxy)carbonyl]amino]-1-oxohexyl]amino]-5-[[[4-(1,1-dimethylethoxy )-1,4-dioxo-2-[[(phenylmethoxy)carbonyl]amino]butyl]amino]methyl]-benzoyl]-D-valyl]-N5-[imino[[(4-methylphenyl)sulfonyl]amino]methyl ]-N2-methyl-L-ornityl]-glycine, phenylmethyl ester (XIV)

The reaction vessel is charged with acetonitrile (3191 g), HBTU (325 g), (XIII) (510 g), (VI) (602 g) and additional acetonitrile (287 g). After 5-10 minutes of stirring, DIEA (443 g) was added while maintaining the temperature at 20-30°C. After the addition is complete, the reaction is stirred at 20-25°C for 6-20 h and monitored by analytical HPLC. If necessary, an additional aliquot of VI (30 g) can be added. Ethyl acetate (6516 g) is added, followed by a solution of citric acid (530 g of citric acid in 4774 g of water). After mixing, the aqueous phase is removed, and the organic phase is washed two more times with citric acid solution, twice with sodium bicarbonate solution (each washing solution is prepared by adding 473 g of sodium bicarbonate to 4935 g of water), and twice with salt solution (each the washing solution is prepared by adding 1613 g of sodium chloride to 4507 g of water). The organic phase is dried over sodium sulfate (1112 g), the solid part is washed with ethyl acetate (1627 g), and the filtrate is concentrated by rotary evaporation (bath temperature 35-45°C), which gives (XIV) in the form of an oily foam ( 1250 g; 134%) which is used without extensive drying in the next reaction.

EXAMPLE 10

Synthesis of N-[N2-[N-[3-[[[2-amino-4-(1,1-dimethylethoxy)-1,4-dioxobutyl]amino]methyl]-5-[[6-[[(1 ,1-dimethylethoxy)-carbonyl]amino]-1-oxohexyl]amino]benzoyl]-D-valyl]-N5-[imino[[(4-methylphenyl)sulfonyl]amino]methyl]-N2-methyl-L-ornityl ]glycine (XV)

Compound (XIV) (2385 g) was dissolved in methanol (18889 g), palladium on carbon (239 g) was added and hydrogen gas bubbled through the mixture. Upon completion of the reaction (analytical HPLC), the mixture was filtered through Hyflo Super-Cel or Celite (239 g) and the solid was washed with methanol (1322 g). The filtrate was concentrated to a viscous oil by rotary evaporation (bath temperature 25-45°C) and further dried on a high vacuum line at 20-25°C for 18-72 h to give 1751 g (118%) of (XV).

EXAMPLE 11

Synthesis of N-[3-(aminomethyl)-5-[[(6-[[(1,1-dimethylethoxy)carbonyl]amino]-1-oxohexyl)amino]benzoyl]-D-valyl-N5-[imino[[ 4-methylphenyl)sulfonyl]amino]methyl]-N2-methyl-L-ornitylglycyl-L-aspartic acid, cyclic (41→1)-peptide (XVI)

The flask is charged with acetonitrile (1744 g), DIEA (498 g), (XV) (1744 g) and additional acetonitrile (302 g). The second reaction vessel is charged with HBTU (741 g) and acetonitrile (4773 g), and stirred at 15-25°C until everything becomes a solution. THF (1006 g) is added and the mixture is heated to 57-63°C, after which the aforementioned solution (XV) is added over 1.5-2.5 h. At the end of the addition, the addition funnel is washed with DIEA solution (60 g) and acetonitrile (273 g), the reaction mixture is stirred for 0.5-5 h and monitored by analytical HPLC. Upon completion of the reaction, the solution is cooled to 15-25°C (it can be stored for up to 3 weeks at -5°C). Parts are removed for purification by preparative HPLC.

Acetonitrile (13962 g) was mixed with water (33150 g). This mixture is added to the heated (external temperature 35°C) part of the above-mentioned reaction mixture (1625 g) with stirring for 15-30 minutes. After 30-90 minutes of stirring at 22-28°C, the solution is filtered and the filtrate is purified by preparative HPLC as follows:

A Shandon Hyperprep HS C-18 (12 μ) column (10 x 55.5 cm) is equilibrated with 65% water/35% acetonitrile. The upper solution is pumped onto the column. Upon completion of loading, the column was washed at 430 mL/min under isocratic conditions (65% water/35% acetonitrile) for 21 min. Followed by a linear gradient (up to 42% water/58% acetonitrile) for 120 min. The main peak typically elutes when the solution contains 40-55% acetonitrile. The major peak is fractionated and the fractions analyzed by analytical HPLC. Fractions >95% pure are extracted into ethyl acetate (0.48 g ethyl acetate per gram prep HPLC eluent) and the aqueous phase is back-extracted with ethyl acetate (0.24 g ethyl acetate per gram prep HPLC eluent; sodium chloride may be added to accelerated phase separation).

The mixed organic phases from all series of preparative HPLC are concentrated in one flask by circular evaporation (bath temperature 35-50°C). The product is further dried in a vacuum oven at 35-45°C, yielding 759 g (44%) (XVI).

EXAMPLE 12

Synthesis of N-[3-(aminomethyl)-5-[(6-amino-1-oxohexyl)amino]benzoyl]-D-valyl-N2-methyl-L-arginylglycyl-L-aspartic acid, cyclic (41→1) -peptide, bis (trifluoromethanesulfonate) (XVII)

The reaction vessel is filled with trifluoroacetic acid (1258 g) and cooled to 12-18°C. Add (XVI) (340 g) in portions over 10-30 min. and the mixture is stirred at 10-20°C until all solid particles are dissolved. After cooling to -15 to -20°C, trifluoromethanesulfonic acid (2883 g) was added at such a rate that the temperature was maintained between -10 and -20°C. After the addition was complete, anisole (274 g) was added, again keeping the temperature in the same range. The mixture is stirred at -5 to -15°C until the completion of the reaction is determined according to analytical HPLC, and then cooled to -30 to -25°C. The reaction was quenched by adding diisopropyl ether (9860 g) at such a rate that the temperature was maintained at -25°C. After the addition is complete, the mixture is brought to a temperature of -25 to -15°C. The solid is isolated by filtration or centrifugation, washed with cold (-15 to -5°C) diisopropyl ether (2465 g) and then dissolved in diisopropyl ether (4930 g) for 5-15 minutes to give a thick solution, at 0-20 °C. The solid part is isolated by filtration or centrifugation and washed with diisopropyl ether (1849 g). The sequence of thick solution/filtration/washing is repeated once more, and the resulting solid is dried in a vacuum oven (25°C), which gives 381 g (113%) of (XVII) in the form of bis(TFMSA) salt.

EXAMPLE 13

Synthesis of N-[3-(aminomethyl)-5-[[1-oxo-6-[[[6-[[(2-sulfonyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]amino]benzoyl ]-D-valyl-N2-methyl-L-arginylglycyl-L-aspartic acid, cyclic (41→1)-peptide, monoammonium salt (XVIII)

The reaction vessel was charged with (XVII) as the bis(TFMSA) salt (798 g), (IX) (414 g) and DMF (13694 g). The mixture is stirred at 15-30°C until all solid particles are dissolved. DIEA (363 g) is then added over 5-20 minutes, maintaining the temperature at 15-30°C, and stirring is continued at 15-30°C for 16-26 h. After completion of the reaction (analytical HPLC), ammonium acetate (160 g) was added to quench unreacted (IX) and the mixture was concentrated by rotary evaporation (bath temperature 35-45°C). The resulting oil is mixed with ethyl acetate (7980 g) for 20-45 min., and then transferred to the reaction vessel. An additional amount of ethyl acetate (15960 g) was added and the suspension was stirred at 20-25°C for 15-30 minutes. The crude (XVIII) (741 g; 92%) is isolated by filtration or centrifugation, and washed with ethyl acetate (10785 g) before drying in a vacuum oven (40-50°C) for 18-72 h.

The crude (XVIII) is purified in portions by preparative HPLC. The sample solution was prepared by dissolving crude (XVIII) (147 g) in a solution of 0.1 M aqueous ammonium acetate (4910 g) and acetonitrile (3838 g). After dilution with additional 0.1 M ammonium acetate (40250 g), the solution is loaded onto a Shandon Hyperprep HS C-18 (12 μ) column (10 x 55.5 cm), pre-equilibrated with 90% 50 mM ammonium acetate/10% acetonitrile. After completion of filling, the substance is washed under conditions of a linear gradient at 450 mL/min. Gradient conditions were, initially: 10% acetonitrile/90% 50 mM aqueous sodium acetate; at the end: 17% acetonitrile/83% 50 mM aqueous sodium acetate for 35 min. A second gradient is then used to achieve: 25% acetonitrile/75% 50 mM aqueous sodium acetate over 260 min. The main peak typically elutes when the solvent is 19-21% acetonitrile. The main peak is fractionated, and the resulting fractions can be stored in a refrigerator (<15°C) for up to two weeks.

Fractions from all series with purity >95% (analytical HPLC) are mixed for circular evaporation (bath temperature 40-50°C). The resulting solid is redissolved, at 40-50°C, in acetonitrile (1564 g) and water (2000 g), the solution is filtered and the filtrate is evaporated again at 40-50°C. The solid part is redissolved in acetonitrile/water once more and evaporated. Finally, acetonitrile (1546 g) was added and the suspension was evaporated. Drying in a vacuum oven (50-60°C) yields 529 g (65%) of purified (XVIII) in the form of an ammonium salt.

EXAMPLE 14

Synthesis of N-[3-(aminomethyl)-5-[[1-oxo-6-[[[6-[[(2-sulfonyl)methylene]hydrazino]-3-pyridinyl]carbonyl]amino]hexyl]amino]benzoyl ]-D-valyl-N2-methyl-L-arginylglycyl-L-aspartic acid, cyclic (41→1)-peptide (I)

The reaction vessel is filled with acetonitrile (1153 g) and a solution of sodium hydroxide pellets (8.4 g) dissolved in water (12105 g). Add (XVIII) in the form of an ammonium salt (525 g), in portions, over 15-45 min., at 15-30°C. The pH is adjusted to 6.1-6.5 by adding sodium hydroxide solution (prepared by dissolving 16 g of sodium hydroxide pellets in water, 315 g, 1.3M) and stirring until the solid part dissolves. Concentrated hydrochloric acid (301 g) is dissolved in water (902 g) and added to the reaction solution in portions at 15-30°C, bringing the pH to 4.1-4.4. After 10-20 minutes of mixing, if necessary, the pH is adjusted again and the solid substance is isolated by vacuum or centrifugal filtration. The reactor is washed with water (5402 g), and the solid is washed with an additional amount of water (5439 g). The product is dried in a vacuum oven at 50-60°C until a water content of <5% is obtained to give (I) (294 g; 57% yield).

Although the present invention has been described with respect to specific embodiments, the details of those embodiments should not be construed as limitations. Numerous equivalents, changes and modifications are possible without departing from the essence and scope of this invention, and it is understood that such, equivalent forms are also part of the invention.

Claims (21)

1. Procedure for the preparation of the compound of formula (I): [image] or its pharmaceutically acceptable salts, characterized in that it consists of: (11) cyclization of the compound of formula (XV): [image] or its salts, thereby obtaining the compound of formula (XVI): [image] (12) deprotection of the compound of formula (XVI), thereby obtaining the compound of formula (XVII): [image] or its salt; (13) bringing the compound of formula (XVII) into contact with the compound of formula (IX): [image] in the presence of first base. (13-i) bringing the reaction solution from step (13) into contact with the quenching agent, thereby obtaining the compound of formula (XVIII): [image] or an alternative salt thereof; and (14) desalting of the compound of formula (XVIII), thereby obtaining the compound of formula (I). 2. The procedure according to Claim 1, characterized by the fact that: - cyclization consists of bringing the compound of formula (XV) into contact with a cyclization agent in a cyclization solvent, in the presence of another base; - deprotection consists of contacting the compound of formula (XVI) with a strong acid at a suitable temperature; And - desalting consists of dissolving the compound of formula (XVIII) in an aqueous solution of the base, and then adjusting the pH of the solution, thereby obtaining the compound of formula (I). 3. Procedure according to Request 2, characterized by the fact that: - the agent for cyclization is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; - the solvent for cyclization is acetonitrile; - the second base is N,N-diisopropylethylamine; - strong acid is a mixture of trifluoroacetic acid and trifluoromethanesulfonic acid; - suitable temperature is from about -30°C to about 5°C; - the compound of formula (XVII) is in the form of a salt of di(trifluoromethanesulfonic acid); - the first base is N,N-diisopropylethylamine; - extinguishing agent is ammonium acetate; - the aqueous solution of the base consists of sodium hydroxide in water; And - adjusting the pH consists of adding aqueous hydrochloric acid to the solution, until reaching a pH value in the range of 4.1 to 4.4. 4. The process according to Claim 2, characterized in that the compound of formula (XVI) is purified using a C-18 column, charging about 369 g of the reaction solution from step 11 per liter of bed volume. 5. The process according to Claim 2, characterized in that the compound of formula (XVIII) is purified using a C-18 column, charging about 33 g of the reaction product from step 13i per liter of bed volume. 6. The process according to Claim 1, characterized in that the compound of formula (XV) is prepared by a process consisting of: (9) coupling of the compound of formula (VI): [image] or its salts, with the compound of formula (XIII): [image] or its salt, thereby obtaining the compound of formula (XIV): [image] (10) deprotection of the compound of formula (XIV), thereby obtaining the compound of formula (XV) or its salt. 7. The procedure according to Request 6, characterized by the fact that: - coupling consists of bringing the compound of formula (VI) into contact with the compound of formula (XIII) in the presence of a coupling agent and a third base; And - deprotection consists of hydrogenation of the compound of formula (XIV) at a suitable hydrogen pressure, in the presence of a hydrogenation catalyst, in a hydrogenation solvent. 8. The procedure according to Request 7, characterized by the fact that: - the compound of formula (VI) is in the form of a salt of trifluoroacetic acid; - the coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; - the third base is N,N-diisopropylethylamine; - suitable pressure is about 14.7 psi; - the catalyst for hydrogenation is palladium on carbon; And - the hydrogenation solvent is methanol. 9. The process according to Claim 1, characterized in that the compound of formula (IX) is prepared by a process consisting of: (4) contacting the compound of formula (VII): [image] with hydrazine, resulting in the compound of formula (VIII): [image] (5) bringing the compound of formula (VIII) into contact with the compound of formula (VIIIa): [image] and N-hydroxysuccinimide in a polar, aprotic solvent, in the presence of another coupling agent, to give a compound of formula (IX). 10. The method according to Claim 9, characterized in that the polar aprotic solvent is N,N-dimethylformamide, and the second coupling agent is N,N'-dicyclohexylcarbodiimide. 11. Procedure for the preparation of the compound of formula (VI): [image] or its salts, indicated by the fact that it consists of: (3) contacting the compound of formula (V): [image] or its salts, with a compound of formula (Va): [image] in the presence of a coupling agent and a base, resulting in a compound of formula (V-b): [image] and (3-i) deprotection of the compound of formula (V-b), thereby obtaining the compound of formula (VI), or its salt. 12. Procedure according to Claim 11, characterized in that: - the compound of formula (VI) is in the form of a salt of trifluoroacetic acid; - the coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; - the base is N,N-diisopropylethylamine; - deprotection consists of bringing the compound of formula (V-b) into contact with trifluoroacetic acid, at a temperature ranging from about -20°C to about 0°C; And - the compound of formula (V) is in the form of a salt of hydrochloric acid. 13. Process according to Claim 11, characterized in that the compound of formula (V) is prepared by a process consisting of: (2) contacting the compound of formula (IV): [image] with the compound of formula (IV): [image] or its salt; in the presence of a second coupling agent and a second base, resulting in a compound of formula (IV-b): [image] (2-i) deprotection of the compound of formula (IV-b), thereby obtaining the compound of formula (V), or its salt. 14. Procedure according to Claim 13, characterized in that: - the compound of formula (IV-a) is in the form of a salt of p-toluenesulfonic acid; - another coupling agent is O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; - the second base is N,N-diisopropylethylamine; - deprotection consists of bringing the compound of formula (V-b) into contact with hydrochloric acid, at a temperature ranging from about -20°C to about 0°C; And the compound of formula (V) is in the form of a hydrochloric acid salt. 15. Procedure for the preparation of the compound of formula (XIII): [image] or its salts, indicated by the fact that it consists of: (8) saponification of the compound of formula (XII): [image] or its salt, and bringing the resulting product into contact with the compound of formula (XIIa): [image] thus obtaining the compound of formula (XII-b): [image] or its salt; and (8-i) bringing the compound of formula (XII-b) into contact with the compound of formula (III): [image] thereby obtaining a compound of formula (XIII) or a salt thereof. 16. Procedure according to Claim 15, characterized in that: - the compound of formula (XII) is in the form of a di(hydrochloric acid) salt; And - the compound of formula (XII-b) is in the form of sodium salt. 17. Process according to Claim 15, characterized in that the compound of formula (XII) is prepared by a process consisting of: (6) contacting the compound of formula (X): [image] with thionyl chloride, tetramethylene sulfone and sulfamide, at a temperature suitable for the formation of the compound of formula (XI): [image] (7) reduction of the compound of formula (XI), thereby obtaining the compound of formula (XII) or its salt. 18. Procedure according to Claim 17, characterized in that: - suitable temperature is in the range from about 100°C to about 125°C; - the compound of formula (XII) is a salt of di(hydrochloric acid); And - reduction consists of hydrogenation of the compound of formula (XI) at a hydrogen pressure of about 50 psi to about 70 psi, in the presence of platinum (IV) oxide as a catalyst for hydrogenation. 19. Process according to Claim 15, characterized in that the compound of formula (III) is prepared by a process consisting of: (1) contacting the compound of formula (II): [image] with a coupling agent to obtain the compound of formula (III). 20. The method according to Claim 19, characterized in that the coupling agent is 1-(3-dimethylamino propyl)-3-ethylcarbodiimide, and the coupling takes place in acetonitrile, at a temperature ranging from about -10°C to about 10°C . 21. Process according to Claim 15, characterized in that the compound of formula (XII-b) is prepared by a process consisting of: (8a) bringing into contact the compound of formula (XII-c): [image] or its salts, with a base, and bringing the resulting product into contact with the compound of formula (XIIa): [image] thereby obtaining the compound of formula (XII-b) or its salt.