Field of the Invention

The present invention relates to processes, process steps and intermediates useful in the preparation of monobactam antibiotic 1-(((Z)-(1-(2-aminothiazol-4-yl)-2-oxo-2-(((3S,4R)- 2-oxo-4-((2-oxooxazolidin-3-yl)methyl)-1-sulfoazetidin-3-yl) amino)ethylidene) amino)oxy)cyclopropanecarboxylic acid including any tautomeric species, salts, solvates or hydrates thereof. The present invention also relates to intermediates useful in such processes.

Background

Over the past several decades, the frequency of antimicrobial resistances and their association with serious infectious diseases have increased at alarming rates. The enhanced prevalence of resistances among nosocomial pathogens is particularly disconcerting. Of the over 2 million (hospital-acquired) infections occurring each year in the United States, 50 to 60% are caused by antibiotic-resistant strains of bacteria. The high rate of resistance to commonly used antibiotics increases the morbidity, mortality, and costs associated with nosocomial infections. In the United States, nosocomial infections are thought to contribute to or cause more than 77,000 deaths per year and cost approximately $5 to $10 billion annually.

Resistance of Gram-negative bacteria against antibiotics may be caused by extended- spectrum beta-lactamases (ESBLs), serine carbapenemases (KPCs) and metallo-beta- lactamases (for example NDM-1) in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis, high-level third-generation cephalosporin (AmpC) beta-lactamase resistance among Enterobacter species and Citrobacter freundii, and multidrug-resistance genes observed in Pseudomonas, Acinetobacter, and Stenotrophomonas. The problem of antibacterial resistance is aggravated by the existence of bacterial strains resistant to multiple antibiotics. For example, Klebsiella pneumonia harboring an NDM-1 metallo- beta-lactamase carries frequently additional serine-beta-lactamases on the same plasmid that carries the NDM-1 .

Thus, there is a need for new antibiotics that are effective against existing drug-resistant microbes, or are less susceptible to development of new bacterial resistance. A new class of monobactam antibiotics is described in WO2015/148379, the content of which is herein incorporated by reference. In example 22, the preparation of 1-(((Z)-(1-(2-aminothiazol- 4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxooxazolidin-3-yl)methy l)-1-sulfoazetidin-3- yl)amino)ethylidene)amino)oxy)cyclopropanecarboxylic acid is described. WO201 7/050218, the content of which is herein incorporated by reference, discloses this compound, crystalline forms and hydrates thereof and compositions comprising thereof, a pH modifier and sucrose. The compound was found to be primarily effective against Gram-negative bacteria, including strains that show resistance to other monobactams.

The compound may be prepared using the process disclosed in example 22 of WO201 5/148379. Further processes for preparing the compound are disclosed in

WO201 9/026004, the content of which is herein incorporated by reference.

A drawback from these processes is that they are complex and difficult in up-scaling. It would thus be beneficial to develop improved processes for the production of this compound. It was an object of the present invention to develop more cost-effective and efficient processes for manufacturing the compound that do not suffer from some or all of these disadvantages.

Summary of the invention

The present invention is directed to a new process for the synthesis of a compound of the Formula (I) as described herein in a shorter time, and in an improved, economic and simplified fashion. The new process is amenable for clinical supply and commercial manufacture. A first aspect of the present invention relates to a process for the preparation of (1-(((Z)- (1-(2-aminothiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxoox azolidin-3-yl)methyl)-1- sulfoazetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropane carboxylic acid) of the Formula (I): including any tautomeric species, salt, solvate or hydrate thereof, comprising the step of reacting compound of the Formula (II): including any tautomeric species, salt, solvate or hydrate thereof, particularly its acid addition salt, more particularly its methane sulfonate addition salt, wherein AG is an activating group, and O-AG together denotes a leaving group with a compound of the Formula (III) including any tautomeric species, salt, solvate or hydrate thereof, in a coupling reaction.

A further aspect of the present invention relates to novel compounds suitable as starting materials and/or intermediates in a process for the manufacture of the compound of the Formula (I) (in the following compound (I)).

Still a further aspect of the present invention relates to a method of crystallizing the compound (I) from a water/solvent mixture.

Still a further aspect of the present invention relates to the compound (I) obtainable by a process as described herein.

General definitions The compounds as shown in the formulae of the present application can possess one or more asymmetric centers. The preferred absolute configurations are as indicated herein specifically by the designations All other absolute configurations are also included.

Unless otherwise indicated the names of molecules and groups are intended to include all possible diastereomers, the two enantiomers of any diastereomer also being included. The term stereoisomer means one of the absolute configurations of a single organic molecule having at least one asymmetric carbon. Included within the definition of a stereoisomer are enantiomers and diastereomers. The term tautomeric species of a compound depicted in a formula of the present application relates to compound which differs from the depicted compound in that one or more H ⁺ ions are located at positions different from those indicated in the respective formula. For example, an NH ₂ group may be in a protonated form, i.e. as an NH ₃ ⁺ group, a CO ₂ H group may be in a deprotonated form, i.e. as a CO ₂ - group, and/or an SO ₃ H group may be in a deprotonated form, i.e. as an S0 ₃ - group. In certain embodiments, a compound includes a plurality of different tautomeric species, which may be in an equilibrium with each other depending on the pH. The term tautomeric species also includes zwitterionic species comprising both a protonated group and a deprotonated group.

A salt of a compound depicted in a formula of the present application comprises positively and/or negatively charged counter-ions. Salts include base salts comprising a positively charged ion, e.g. inorganic base salts, organic base salts, and basic amino acid salts. Inorganic bases that can form the inorganic base salts include ammonium, alkali metals (e.g. sodium, potassium, lithium) and alkaline earth metals (e.g., calcium, magnesium); organic bases that can form the organic base salts include amines such as cyclohexylamine, benzylamine, octylamine, ethanolamine, diethanolamine, diethylamine, triethylamine, morpholine, pyrrolidine, piperidine, N-ethylpiperidine, N-methylmorpholine; basic amino acids that can form the basic amino acid salts include lysine, arginine, ornithine and histidine. Salts further include acid salts comprising a negatively charged ion, e.g. inorganic acid salts, for example, from halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid, and organic acid salts for example, from carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, lactic acid, fumaric acid, succinic acid, citric acid, amino acids such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, benzoic acid, methane- or ethane- sulfonic acid, ethane-1 , 2-disulfonic acid, benzene sulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, N-cyclohexylsulfamicacid, N-methyl-, N-ethyl- or N- propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid. Preferred salts of the compound (I) are sodium and arginine salts. Suitable salts of the compound (I) are described in WO 2017/050218, the content of which is herein incorporated by reference.

The term solvate refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules, i.e. organic solvent molecules or water molecules. The term hydrate specifically refers to a complex where the solvent molecule is water.

A preferred hydrate of the compound (I) is the trihydrate wherein the compound (I) comprises three molecules H ₂ 0 per molecule. Suitable solvates and hydrates of the compound (I) are described in WO 2017/050218, the content of which is herein incorporated by reference.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise.

Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes” and “including” are interchangeable and not intended to be limiting.

Detailed description

The present invention relates to a process of manufacturing a compound (I) by a coupling reaction between a compound of the Formula (II) and a compound of the Formula (III) or a derivative, particularly an amino-protected derivative thereof.

In this coupling reaction, the amino group of the compound (III) selectively reacts with the activated ester bearing the leaving group O-AG of compound (II) whereby the target compound (I) is obtained.

The term activating group refers to any group, which enhances the reactivity of a carboxylic acid group towards an amino group. In certain embodiments, the group O-AG is an O-N-succinimide group, which may be obtained by reaction with N-hydroxy succinimide. In further embodiments, the group O-AG is an O-N-benzotriazole group, which may be obtained by reaction with N-hydroxybenzotriazole.

The coupling reaction may be carried out in any suitable solvent where reaction partners and products are at least partially soluble. In certain embodiments, a aqueous/non- aqueous solvent mixture, particularly in a single phase aqueous/non-aqueous solvent mixture is used. In certain embodiments, the solvent is an alcohol/water mixture, e.g. a water/ethanol mixture, or a water/methanol mixture. The ratio of water/alcohol in the solvent mixture may be in the range of about 10:1 (v/v) to about 1 :20 (v/v), of about 2:1 (v/v) to about 1 :10 (v/v), or of about 1.5: 1 (v/v) to about 1 :3 (v/v). In certain embodiments, the reaction is performed in water/methanol, e.g. water/methanol 1 :1 (v/v).

The base to be used in the coupling reaction may be an organic or inorganic base, e.g. an amine base such as triethylamine. Typically, the base is added in a molar excess. The coupling reaction is typically performed at a temperature of about -5°C to about 5°C. After coupling, compound (I) may be isolated by crystallization from the reaction mixture, particularly by crystallization from an aqueous/non-aqueous solvent mixture as described above. In certain embodiments, the compound (I) is isolated from the coupling reaction mixture by pH-driven crystallization in high purity wherein crystallization is induced by acidification and optionally addition of compound (I) seed material. In certain embodiments, the coupling reaction and the crystallization comprise at least one of the following steps:

(i) performing the coupling reaction in a water/alcohol mixture, e.g. in the range of about 10:1 (v/v) to about 1 :20 (v/v), of about 2:1 (v/v) to about 0.5:1 (v/v), or of about 1 :1 (v/v), preferably in a water/methanol mixture;

(ii) acidifying the reaction mixture with HCI, particularly to a pH of about 0.8 to about 1.2, more particularly to about 1 ;

(iii) adding compound (I) seed material at a temperature of at least about 12°C, particularly at temperature of at least about 15°C; (iv) adding methanol to obtain a water/methanol mixture of at least about 1 :1.5 (v/v); particularly of at least about 1 :2 (v/v);

(v) cooling the mixture after step (iii) or - if present - step (iv) to a temperature of about 10°C or less, particularly between about 0°C and about 5°C; and (vi) optionally subjecting the reaction mixture to at least one warming-cooling cycle, e.g. 2 or 3 warming-cooling cycles, after step (iii), wherein a warming-cooling cycle comprises a warming step of increasing the temperature of the reaction mixture by at least 5°C, particularly by at least 10°C and a cooling step of reducing the temperature of the reaction mixture by at least 5°C, particularly by at least 10°C, and particularly performing a warming step and/or a cooling step for a time of at least about 30 min, of at least 1 h, or of at least about 2 h.

In certain embodiments, compound (I) is subjected to further purification steps, e.g. recrystallization and/or reslurrying, e.g. in a water/THF mixture.

In certain embodiments, compound (I) is obtained in the form of a trihydrate.

In certain embodiments, compound (I) is obtained in a purity of at least 95 area% by HPLC, of at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or more determined as area% by HPLC. In certain embodiments, compound (I) is obtained substantially free from impurities, i.e. having 1% or less impurities, particularly 0.5% or less impurities determined as area % by HPLC.

In certain embodiments, the impurities comprise the monomethyl ester of the compound (II), i.e. a compound of the Formula (IV), in an amount of up to about 1%, up to about 0.5% or up to about 0.3% determined as area % by HPLC.

In further embodiments, the compound of the Formula (III) is obtained from a compound of the Formula (Ilia) as starting material, an amino-protected derivative of the compound (III): including any tautomeric species, salt, solvate or hydrate thereof, wherein PG1 is a nitrogen-protecting group, by removal of PG1.

The nitrogen-protecting group PG1 is any group, which can be removed under deprotection conditions as known in the art. Suitable nitrogen-protecting groups are described e.g. in the relevant chapters of standard reference works such as J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973; T. W. Greene and P. G. M. Wuts, "Greene's Protective Groups in Organic Synthesis", Fourth Edition, Wiley, New York 2007; in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981 , and in "Methoden der organischen Chemie" (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15/1, Georg Thieme Verlag, Stuttgart 1974.

For example, the nitrogen-protecting group PG1 can be a group removable by acid hydrolysis, base hydrolysis, or reduction, e.g. hydrogenation. Groups PG1 that can be removed by hydrogenation are preferred, especially those that can be removed in the presence of hydrogen and Pd/C. The term hydrogenation is used to describe a chemical reaction, which refers to the action of reducing another compound in the presence of hydrogen. The source of hydrogen can be selected from gaseous hydrogen (H ₂ ), hydrogen donors (transfer hydrogenation, e.g. formic acid or salts thereof), hydride reagent (BH ₃ , B ₂ H ₆ or NaBH ₄ ) or the like. Preferably, the nitrogen-protecting group PG1 can be removed by hydrogenation in the presence of a catalyst, e.g. Pd, Pd/C or another catalyst, and a hydrogenation reagent, e.g. , e.g. formic acid or a salt thereof or hydrogen, in a suitable solvent. More preferably, the nitrogen-protecting group PG1 is benzyloxycarbonyl (Cbz) or tert-butyloxycarbonyl (Boc).

In certain embodiments, a salt of compound (Ilia), i.e. a compound of the Formula (I I lb) is used as a starting material for obtaining the compound (III): wherein PG1 is a nitrogen protecting group as described above and M is a cation. The cation M may be an organic cation, e.g. a quaternary ammonium ion, particularly N(Bu) ₄ ⁺ , or an inorganic cation, e.g. an alkaline metal cation, particularly K ⁺ or Na ⁺ .

In particular embodiments, a potassium salt of the Formula (II lc) is used: In certain embodiments, compound (III) or compound (Ilia), (I I lb) or (111 c) as described above are prepared from a compound of the Formula (V): including any tautomeric species, salt, solvate or hydrate thereof, wherein PG1 is a nitrogen protecting group as described above, particularly Cbz.

The preparation of compound (III), (Ilia), (I I lb) or ( 111 c) from compound (V) may comprise at least one of the steps of (i), (ii), (iii) and (iv) as outlined below.

Step (i) comprises reacting compound (V) to a compound of the Formula (VI): including any tautomeric species, salt, solvate or hydrate thereof, wherein PG1 is a nitrogen protecting group, particularly Cbz, and wherein OMs is a leaving group, e.g. a methane sulfonate (mesylate) group.

In step (i), the OH group of compound (V) is converted to a leaving group OMs by reaction with an activating reagent. A leaving group is a group that enhances the reactivity of an OH group since it can be cleaved off in a bond-breaking step. Examples of leaving groups include, but are not limited to, sulfonates, nitrates or phosphates, carboxylates, phenoxides, and alkoxides. Preferred leaving groups are sulfonates including, without limitation, nonaflate (-O-SO ₂ C ₄ F ₉ ), triflate (-O-SO ₂ CF ₃ ), fluorosulfonate (-O-SO ₂ F), tosylate (-O-SO ₂ C ₆ H ₄ CH ₃ ), mesylate (-O-SO ₂ CH ₃ ) or besylate (-O-SO ₂ C ₆ H ₆ ). A preferred leaving group is mesylate.

The leaving group is introduced by reacting compound (V) with a suitable activation reagent, particularly a sulfonyl halide orsulfonyl anhydride reagent, e.g. methane sulfonyl chloride (MsCI) or methane sulfonyl anhydride (Ms ₂ 0) in the presence of an organic or inorganic base as herein described above, e.g. triethylamine or diisopropylethylamine. The reaction may be carried out in any suitable solvent where reaction partners and products are at least partially soluble, e.g. in an organic solvent, particularly in an aprotic polar solvent such as dichloromethane (DCM) or tetrahydrofuran (THF). Step (ii) comprises reacting compound (VI) to a compound of the Formula (VII): including any tautomeric species, salt, solvate or hydrate thereof, wherein PG1 is a nitrogen protecting group as herein described above, particularly Cbz, and wherein OMs is a leaving group as herein described above. Step (ii) is preferably carried out by sulfonating compound (VI) with a sulfonylation reagent, e.g. a halosulfonic acid, particularly chlorosulfonic acid, optionally in the presence of an organic or inorganic base as herein described above, e.g. pyridine bases such as 2,6-lutidine or 2-picoline.

Compound (VI) is preferably reacted in a solvent with a sulfonating reagent to obtain compound (VII). The temperature during the addition of compound (VI) is typically below 0°C, e.g. below -10°C, and then the reaction mixture is warmed up above 0°C, e.g. to about 20°C.The reaction may be carried out in any suitable solvent where reaction partners and products are at least partially soluble. Examples of suitable solvents are dichloromethane (DCM) and dimethylformamide (DMF). The sulfonating agent is preferably prepared in situ, by reacting a halosulfonic acid, preferably chlorosulfonic acid, with a base as herein described above, e.g. with an amine base, preferably a pyridine base, e.g. 2,6-lutidine.

Step (iii) comprises the intramolecular cyclization of compound (VII) to compound (Ilia) or the salt (I I lb) thereof by addition of a base, e.g. a hydroxide or (hydrogen) carbonate such as NaOH, Na ₂ CO ₃ , KHCO ₃ , K ₂ CO ₃ , Na ₂ HPO ₄ and adjusting the pH of the solution above pH 7, e.g. about pH.7.4. In the case that a potassium-containing base such as potassium hydroxide is used, potassium (K) salt ( 111 c) is obtained. In particular embodiments, the potassium salt (II lc) may be transformed into e.g. a corresponding ammonium salt, particularly the tetrabutylammonium (TBA) salt (II Id), preferably by addition of tetrabutylammonium hydrogen sulphate, before the respective product is employed in step (iv).

Step (iii) is preferably carried out by cyclizing compound (VII), particularly under slightly basic conditions, e.g. at a pH between about 7.2 and about 7.6, particularly about 7.4 in an aqueous solvent or solvent mixture.

In certain embodiments, the sulfonylation step (ii) and the cyclization step (iii) are performed in a single sequence without isolating compound (VII).

Step (iv) comprises reacting Compound (Ilia), e.g. in the form of a salt (I I lb), particularly as TBA salt (II Id) or potassium salt ( 111 c) to compound (III) as described above, including any tautomeric species, salt, solvate or hydrate thereof.

Step (iv) comprises removing the nitrogen-protecting group PG1 according to known methods, preferably by hydrogenation as described above.

In certain embodiments, the process of the invention comprises the successive application of steps (ii), (iii) and (iv) without isolation of the respective reaction products. Thus, the reaction mixture obtained after step (iv) can be used directly in the coupling step (v). In certain embodiments, the process of the invention comprises preparing compound (II) as described above from a compound of the Formula (VIII) as a starting material: including any tautomeric species, salt, solvate or hydrate thereof. In certain embodiments, an acid addition salt of compound (VIII), e.g. an addition salt with hydrochloric acid (HCI), is used as a starting material. This salt may be prepared by acidification with a suitable acid, e.g. HCI.

The preparation further comprises introduction of an active ester group by forming the group O-AG, particularly an O-succinimide group as described above. For this purpose, compound (VIII) or a salt thereof is reacted with an active ester reagent, particularly N- hydroxy succinimide, in the presence of a coupling reagent in a suitable solvent, e.g. a non-aqueous solvent, particularly a polar aprotic solvent such as DMF.

Suitable coupling reagents are known in the art, and may e.g. be selected from carbodiimides, chloroformates, carboxylic acid chlorides, and organophosphorus reagents. Preferably, the coupling reagent is N,N ^' -dicyclohexylcarbodiimide (DCC) optionally in combination with a silylation reagent such as chloro(trimethyl)silane.

In certain embodiments, the starting compound, e.g. the HCI addition salt of compound (VIII) is used in substantially water free state, e.g. having with a water content of about 0.5%, about 0.3% or about 0.1% by weight or less. For this purpose, residual water may be removed by azeotropic distillation e.g. with a suitable organic solvent such as ethyl acetate or dimethyl formamide (DMF). The reaction product, i.e. compound (II), may be obtained as solvate, e.g. DMF solvate having a content of about 9% to about 12% by weight of DMF.

In certain embodiments, the DMF solvate of compound (II) is subjected to a treatment wherein the DMF content is reduced, e.g. by re-slurrying the compound with a volatile organic solvent such as acetone or ethyl acetate.

In certain embodiments, compound (II) having a DMF content of about 1% by weight or less, e.g. a DMF content of about 0.05% to about 0.5% by weight, is obtained. This product is preferably used in the coupling reaction with compound (III) or a derivative thereof to obtain compound (I) as described above. A further aspect of the present invention relates to a process comprising preparing a compound of the Formula (VIII) as described above: including any tautomeric species, salt, solvate or hydrate thereof, e.g. an addition salt thereof with an acid, particularly an addition salt with hydrochloric acid (HCI), from a compound of the Formula (IX): wherein PG2 is a nitrogen-protecting group, e.g. a nitrogen-protecting group as described above, by deprotection, preferably with trifluoroacetic acid (TFA)/anisole/DCM. Preferably, PG2 is an acid-labile protecting group such as tert-butyloxycarbonyl (Boc).

In certain embodiments, an acid addition salt of compound (VIII), e.g. an acid addition salt with hydrochloric acid (HCI), is obtained.

A further aspect of the present invention relates to a process comprising preparing a compound of the Formula (VIII) as described above: including any tautomeric species, salt, solvate or hydrate thereof, e.g. an acid addition salt thereof, particularly an addition salt with hydrochloric acid (HCI), by reacting a compound of the Formula (X): including any tautomeric species, salt, solvate or hydrate thereof, with a compound of the Formula (XI): including any tautomeric species, salt, solvate or hydrate thereof, in a condensation reaction.

In certain embodiments, the reaction is carried out at ambient temperature (20°C to 25°C) in the presence of a base such as TEA and in a polar aprotic solvent such as dimethylacetamide (DMA).

In certain embodiments, an acid addition salt of compound (VIII), e.g. an addition salt with hydrochloric acid (HCI), is obtained.

The HCI addition salt of compound (VIII) may be obtained by crystallization in the presence of chloride ions, particularly under acidic conditions. Alternatively, compound (II) may be prepared from a compound of the Formula (XII), which is an unprotected derivative of Compound (IX): including any tautomeric species, salt, solvate or hydrate thereof, preferably by treatment with TFA/anisole/DCM. This reaction comprises introduction of an active ester group in the presence of a coupling agent, e.g. DCC or another suitable coupling agent as described above.

A further aspect of the present invention is a novel compound of the Formula (I lie) as described above:

wherein PG1 is a nitrogen protecting group, particularly Cbz.

A further aspect of the present invention is a novel compound of the Formula (II) as described above: including any tautomeric species, salt, solvate or hydrate thereof, particularly an addition salt thereof with methane sulfonic acid.

In certain embodiments, compound (II) is a DMF solvate, which may have a content of DMF of about 9 to about 12% by weight. In further embodiments, compound (II) has a DMF content of about 1% or less by weight, particularly a DMF content of about 0.05% to about 0.5% by weight.

A further aspect of the present invention is a novel compound of the Formula (VIII) as described above: including any tautomeric species, salt, solvate or hydrate thereof, e.g. an acid addition salt, particularly an addition salt thereof with hydrochloric acid (HCI), or an addition salt thereof with trifluoroacetic acid (TFA). The compounds of Formula (lllc), (II) and/or (VIII) may be used as a starting material and/or synthesis intermediate for the preparation of (1-(((Z)-(1-(2-aminothiazol-4-yl)-2- oxo-2 -(((3S,4R)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)-1 -sulfoazetidin-3- yl)amino)ethylidene)amino)oxy)cyclopropane carboxylic acid) of the Formula (I) as described above. A further aspect of the present invention relates to a process for the preparation of (1- (((Z)-(1-(2-aminothiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2 -oxooxazolidin-3-yl)methyl)- 1-sulfoazetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropane carboxylic acid) of Formula (I) as described above, comprising using a compound of Formula (lllc), (II) and/or (VIII) as a starting material and/or synthesis intermediate.

Still a further aspect of the present invention is a process of obtaining (1-(((Z)-(1-(2- aminothiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxooxazolid in-3-yl)methyl)-1- sulfoazetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropane carboxylic acid) of Formula (I) in a crystalline form: comprising the steps: (a) providing a solution of compound (I) in a solvent, which is a single-phase aqueous/non-aqueous solvent mixture; and

(b) crystallizing compound (I).

The solvent is a mixture of water and a water-miscible organic solvent, which forms a single phase. Particularly, the solvent is a water/alcohol mixture, e.g. a water/ethanol mixture, or a water/methanol mixture.

The ratio of water/non-aqueous solvent in the solvent mixture is typically in the range of about 10:1 (v/v) to about 1:20 (v/v), of about 2:1 (v/v) to about 1 :10 (v/v), or of about 1.5:1 (v/v) to about 1 :3 (v/v).

In certain embodiments, step (b) comprises a pH-driven crystallization, particularly an acidification, more particularly an acidification to a pH of about 2 or less, or about 1.5 or less. In certain embodiments, the crystallization is induced by addition of compound (I) seed material.

In certain embodiments, the crystallization comprises at least one of the following steps:

(i) providing a solution of compound (I) in a water/alcohol mixture, particularly a water/methanol mixture, more particularly a water/methanol mixture in the range of about 2:1 (v/v) to about 0.5:1 (v/v), even more particularly a water/methanol mixture of about 1 :1 (v/v);

(ii) acidifying the reaction mixture with HCI, particularly to a pH of about 0.8 to about 1.2, more particularly to about 1 ; (iii) adding compound (I) seed material at a temperature of at least about 12°C, particularly at temperature of at least about 15°C;

(iv) adding methanol to obtain a water/methanol mixture of at least about 1 :1.5 (v/v); particularly of at least about 1 :2 (v/v);

(v) cooling the mixture after step (iii) or - if present - step (iv) to a temperature of about 10°C or less, particularly between about 0°C and about 5°C; and

(vi) optionally subjecting the reaction mixture to at least one warming-cooling cycle, e.g. 2 or 3 warming-cooling cycles, after step (iii), wherein a warming-cooling cycle comprises a warming step of increasing the temperature of the reaction mixture by at least 5°C, particularly by at least 10°C and a cooling step of reducing the temperature of the reaction mixture by at least 5°C, particularly by at least 10°C, and particularly performing a warming step and/or a cooling step for a time of at least about 30 min, of at least 1 h, or of at least about 2 h.

In certain embodiments, compound (I) is subjected to further purification steps, e.g. recrystallization, e.g. in a water/THF mixture.

In certain embodiments, compound (I) is obtained in the form of a trihydrate.

In certain embodiments, compound (I) is obtained in a purity of at least 95%, of at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or more determined as area % by HPLC.

In certain embodiments, compound (I) is obtained substantially free from impurities, i.e. having 1% or less impurities, particularly 0.5% or less impurities determined as area % by HPLC. The processes of manufacturing compound (I) as described hereinabove do not only minimize the use of specialized facility for monobactam, but also reduces operations in handling and isolating labile monobactam API and intermediates, thereby maximizing isolation yield. Of particular advantage is the use of compound ( 111 c) directly in the de- protection step for removing the protecting group PG1 , e.g. Cbz, by hydrogenation, e.g. with Pd/C/H ₂ and transferring the resulting compound (III) process solution directly into the coupling step without isolation of compound (III), yielding high quality compound (I) after pH driven crystallization. The quality of isolated compound (I) was typically >99.5% and the only new impurity was identified as compound (IV), i.e. the monomethyl ester of compound (II), in an amount of about 0.2% to 0.3% determined as area % by HPLC.

A synthesis scheme of a particular embodiment of the novel process is as follows:

The synthesis scheme and modifications thereof will be described in detail in the following examples, which are merely illustrative and should not be considered as limiting the scope of the disclosure in any way. General Experimental Details

Synthesis

Generally, compounds according to the present disclosure can be synthesized by the route described in the Scheme as shown herein.

The skilled person will appreciate that the general synthetic routes detailed in the application show common reactions to transform the starting materials as required. When specific reactions are not provided the skilled person will know that such reactions are well known to those skilled in the art and appropriate conditions considered to be within the skilled person’s common general knowledge. The starting materials are either commercially available compounds or are known compounds and can be prepared from procedures described in the organic chemistry art. General Conditions

The following HPLC method can be used for the detection of Compound (I) and other compounds as described herein.

Principle RP UHPLC method with ion pairing and UV detection

Reagents Grade/Source

Acetonitrile: gradient grade, e.g. Merck LiChrosolv No. 100030

Methanol: gradient grade, e.g. Merck LiChrosolv No. 106007

Water deionized, for HPLC

Tetrabutylammonium HPLC grade, e.g. Sigma-Aldrich No. 86853 hydrogen sulfate (TBAHS)

Trifluoroacetic acid (TFA) HPLC grade, e.g. Sigma-Aldrich No. 302031

1 M Hydrochloric acid reagent grade, e.g. Fluka No. 35328 1 M Sodium hydroxide reagent grade, e.g. Fluka No. 35256

Diluent Mobile Phase A

Materials

Glassware Amber colored volumetric glass flasks and UHPLC vials

Equipment

Apparatus UHPLC system with gradient elution and UV detector, e.g. Agilent 1290 with UV detector or equivalent

Column Waters Acquity HSS T3

Length: 100 mm, internal diameter: 2.1 mm

Particle size: 1.8 pm

Chromatographic conditions

Separation mode Gradient

Mobile Phase A Water + 0.05% TFA + 5 mM TBAHS

Mobile Phase B Methanol + 0.05% TFA + 5mM TBAHS

Flow rate 0.4 mL/min Detection UV 260 nm

Column Temperature 40° C Autosampler Temperature 5°C Injection volume 2.0 pL of the test and reference solutions, equivalent to a column loading of approximately 2.4 pg of Compound (I) drug substance (free acid).

Retention time (RT) Compound (I) about 10.3 min

Calculation Percent of related substance (%RS): where

PA _T Peak area of individual peaks in the test solution

Zi UV response factor

(ratio of the detector signal of a given amount of drug substance as salt free form divided by the detector signal of the same amount of related substance i as salt free form)

For unknown compounds Z = 1.0 Examples

Example 1: Preparation of Compound (VI)

Compound (V) Compound (VI) In a 50 L glass reactor equipped with a mechanical stirrer and a thermometer, 20 L DCM (dichloromethane) were charged and temperature was adjusted at 0~5°C. 1.0 Kg Compound (V) was charged, followed by 2.0 L DCM.

365.5 ml TEA (triethylamine) were charged. At the end of the addition, 161.9 ml methane sulfonyl chloride (MsCI) were charged over 15 minutes while keeping temperature at

0~5°C. The suspension was stirred for 30 minutes. This procedure was repeated two times. The resulting solution was stirred at 0~5°C until the reaction was complete.

1.5 L DIW (deionized water) were charged into reactor. A subsequent distillation was carried out under reduced pressure to reduce the solution volume to about 15 L.

Temperature was kept below 40°C throughout the concentration phase. During this operation, a solid precipitate was obtained.

8.0 L MIBK (methyl isobutyl ketone) were charged into reactor, followed by 6.5 L DIW and then distillation was continued by reducing the volume to about 15.0 L. Temperature was kept below 54°C throughout the concentration phase.

The suspension was cooled to 0~5°C over 1 hour and the slurry was stirred over at least 2 h. The crystal slurry was filtered through a Büchner funnel equipped with filter paper under vacuum and then washed twice with 4.0 L DIW and once with 4.0 L MIBK. The wet cake was dried under vacuum at 60°C. The yield was 85.6%~87.1%.

Example 2: Preparation of Compound (Ilia)

Compound (VI) Compound (VII)

2.1 Preparation of Compound (VII) tetrabutylammonium (TBA) salt In a 250 ml_ four-necked round-bottom flask (R1) with a magnetic stirrer, a thermometer, a nitrogen inlet; 70.0 ml_ dichloromethane (DCM) and 20.8 ml_ 2,6-lutidine were charged and the mixture was cooled to -22~-20°C.

5.2 ml_ chlorosulfonic acid were added under stirring and the reaction mixture temperature was controlled not to exceed -10~0°C.

The mixture was cooled to -11~-10°C and held at this temperature for 30 min, under stirring. 10.0 g Compound (VI) were charged under stirring over 15 min. The temperature was adjusted at 13~20°C and the mixture was held at this temperature over 4 hours under stirring. Then, the reaction mixture was cooled to 0~5°C

In a 1 L four-necked jacketed reactor (R2) equipped with a chiller, a mechanical stirrer, a thermometer, a pH meter and an addition funnel, a quenching mixture was prepared by combining 53.3 ml_ water and 15.5 ml_ DCM and by adjusting the temperature at 0~5°C.

The reaction mixture was then slowly poured from (R1) into (R2) under stirring, keeping temperature between 0~5°C. Then, the reaction mixture was warmed to room temperature. Next, the reaction mixture was poured into a mixture of water (142.0 ml_), DCM (182.0 ml_) and tetrabutylammonium hydrogen sulfate (NBu ₄ HSO ₄ , 8.99 g) at 5 ^° C. After addition, the mixture was stirred for 30 min~1 h and the aqueous phase was 2x extracted with 50 ml_ DCM. The combined organic phase was 2x washed with 50 ml_ water, concentrated under vacuum to dryness and then purified by column chromatography (DCM/methanol =75/1-15/1) to obtain 12.6 g compound (VII) TBA salt as pale yellow solid with a purity of 90.7% determined by HPLC.

2.2 Preparation of Compound (VII) potassium (K) salt

In a 10 L four-necked round-bottom flask (R1) with a mechanical stirrer, a thermometer, a nitrogen inlet; 3.51 L DCM and 1.03 L 2,6-lutidine were charged and the mixture was cooled to -22~20°C. 248.6 ml_ chlorosulfonic acid were added under stirring over 50 min, the reaction mixture temperature was controlled not to exceed -10~0°C. Then, the mixture was cooled to -11 ~-10°C and held at this temperature for 30 min understirring.

500.0 g Compound (VI) were charged into reactor R1 under stirring over 15 min. The temperature was adjusted at 13~20°C and the mixture was held at this temperature over 4 hours under stirring. Then the reaction mixture was cooled to 0~5°C.

In a 20 L four-necked jacketed reactor (R2) equipped with a chiller, a mechanical stirrer, a thermometer, a pH meter and an addition funnel, a quenching mixture was prepared by combining 2.0 L water and 808 ml_ DCM and by adjusting the temperature at 0~5°C.

The reaction mixture was then slowly poured from R1 into R2 under stirring, keeping temperature between 0~5°C. The quenched reaction mixture was stirred in R2 for 10 min at 0~5°C. 2.63 L of a 5.0 M KOH aqueous solution were charged into R2 over 30 min under stirring at 0~5 °C. After addition, the pH of the mixture was 7.2 at 0~5 °C.

The mixture was heated to 37~38°C. 105 ml_ of a 5.0 M KOH aqueous solution were charged into R2 over 5 min under stirring at 37~38°C. After addition, the pH of the mixture was 7.8 at 37~38°C. Next, the mixture was stirred for 5 min at 37~38°C and then settled for 5 min.

The organic phase (lower layer) was transferred back into R1. The aqueous phase was left in R2 and washed with 906 ml_ DCM. The combined organic phases were extracted by charging 300.0 ml_ water into R1 . The mixture was stirred for 5 min at 37-38 °C and transferred into a separation funnel. The organic phase (lower layer) was removed and the rich aqueous phase (upper layer) combined with the rich aqueous phase in R2 was used as starting solution for the following step. 2.3 Cyclization and Preparation of Potassium (K) salt (lllc)

Compound (VII) Compound (lllc)

The combined aqueous phases from the sulfonation step of Example 2.2 were stirred and heated to 40°C. The mixture was titrated under stirring at 40°C to pH 7.4 over 20 h, with overall 1160.0 ml_ of a 0.5 M KOH aqueous solution. The product, i.e. potassium salt (lllc) crystallized out as a white solid.

The mixture was cooled to 0~5°C over 90 min and held at this temperature for 60 min understirring. The crystal slurry was filtered under reduced pressure using a B chner funnel.

After washing with 500 ml_ water at 0~5°C and then with 500 ml_ DCM, the wet cake of potassium salt (lllc) was grinded and directly used in the subsequent conversion to the tetrabutylammonium salt (I I Id).

2.4 Conversion to TBA salt (IIId)

I53 ml_ water, 3532 ml_ DCM and 279.0 g tetrabutylammonium hydrogen sulfate (NBU4HSO4 or TBAHSO4) were charged into reactor R2 and the temperature was adjusted at 0~5°C.

Wet potassium salt (lllc) obtained in Example 2.3 was added and the mixture was stirred for 10 min, until complete dissolution occurred. The stirring was stopped and two phases separated after 5 min. The rich organic phase (lower layer) was transferred into a 10 L four-necked jacketed reactor (R3), equipped with a mechanical stirrer, and a thermometer. The aqueous phase in R2 was washed with 506 ml_ DCM under stirring for 5 min. The organic phase was decanted for 5 min at 0~5°C and transferred into R3. The combined organic phases were concentrated under vacuum at 20~25°C until the mixture volume was 1500.0 mL.

2000.0 mL methyl tert-butyl ether (MTBE) were added over 15 min into R3 under stirring. The mixture was vigorously stirred, so that the two phases were well mixed at 20~25°C for 30 min. At this stage, crystallization occurred.

1000.0 mL MTBE were added over 15 min under stirring. The mixture was cooled to 0~5°C and stirred for 2 h. The crystal slurry was filtered under reduced pressure through a B ichner funnel. After washing R3 and the filter cake with 500.0 mL MTBE, the cake was dried under nitrogen flow at 20~25 ^° C to a constant weight. 437.63 g of TBA salt (llld) were obtained as white crystals (purity: 100%).

Example 3: Preparation of Compound (III)

TBA salt (llld) Compound (III)

3.1. Preparation of Compound (III) by transfer hydrogenation

The TBA salt (I I Id) was used as starting material. The Cbz protecting group was removed by transfer hydrogenation with formic acid/ammonium formate in the presence of a Pd/C catalyst.

In a 5 L four-necked flask (R1) equipped with a chiller, a mechanical stirrer and a thermocouple, 363.5 ml DIW (deionized water) and 509.0 ml EtOH were charged adjusting the temperature at 20~25°C. 71.54 g HCOONH ₄ were charged, followed by 53.26 g HCOOH and 43.05 g TEA. Temperature of the solution inside (R1) was adjusted at 0~3°C. 21.81 g Pd/C on activated charcoal (10% Pd basis, 50% wet) were added and the solution ware stirred for 1 min.

In a 2 L four-necked flask (R2) equipped with a thermometer and a mechanical stirrer, an ethanol/aqueous solution was prepared (218.0 ml DIW and 509.0 ml EtOH). 363.48 g Compound (I I Id) were added and the mixture was stirred until complete dissolution occurred.

The R2 solution was added over 30 minutes to R1 , maintaining the temperature of R1 at 0~3°C, followed by 218.0 ml ethanol (EtOH), to rinse off R2 walls and the line. The resulting reaction mixture was finally stirred at 0~3°C until reaction completion. The suspension was filtered through celite, equipping a B ichner funnel. The flask and the funnel were then washed with a solution of 218.0 ml DIW and 363.5 ml EtOH.

To the rich solution and wash, 40.08 g methane sulfonic acid (MsOH) were added to a pH of 5 and the solution was stirred for 5-10 min. Next, 156.63 g MsOH were added to a pH of 2. The crystallization occurred during addition. The suspension was cooled to -15~- 20°C and stirred for at least 1 hour.

The product slurry was filtered through a Buchner funnel (F2) equipped with filter paper and washed twice: first with a solution of 218.0 ml DIW and 363.5 ml_ EtOH and then with 727.0 ml_ EtOH. The wet cake was dried under nitrogen flow at 20~25 ^° C to a constant weight. 116.32 g of Compound (III) were obtained as white crystals (purity: 96.4%; yield: 73.8%)

3.2. Preparation of Compound (III) with molecular hydrogen

The TBA salt (llld) and the potassium salt (II lc) were used as starting materials. The Cbz protecting group was removed by hydrogenation with molecular hydrogen in an autoclave optionally under pressure using MeOH/H ₂ O (1 :1) as solvent and a Pd/C catalyst.

3.2.1 Preparation of Compound (III) from TBA salt (Mid)

TBA salt (llld) Compound (III) After 19 h, starting material was consumed completely. The solution pH increased from 2.41 to 5.65. The suspension was filtered to remove Pd/C. After addition of 0.5 g MsOH to pH 2.34, the solution started to become cloudy. Then the solution was cooled to -15~- 20 ^° C. After 2 h, the suspension was filtered and the filter cake was dried under nitrogen flow. The product yield was 75.5%.

3.2.2 Preparation of Compound (III) from potassium salt (lllc)

K salt (lllc) Compound (III)

After 3.5 h, the starting material was consumed completely. The solution pH was 5.35, which was almost same with the pH before reaction (5.30). Then the suspension was filtered to remove Pd/C. After adding 1.1 g MsOH to pH 2.03, the solution started to become cloudy. Next, the solution was cooled to -15~-20 ^° C. After 2 h, the solution was filtered and the isolated cake was dried under nitrogen flow.

The product yield was 61.4%. Example 4: Preparation of Compound (II)

The diacid Compound (VIII) or its HCI addition salt was used as the starting material for the preparation of Compound (II). Compound (VIII) may be obtained from Compound (IX) or from Compound (X) and Compound (XI).

Compound (X)

4.1. Preparation of Compound (VIII) as TFA salt

In a 3 L four-necked flask equipped with a mechanical stirrer and a thermometer, anisole (150 ml_) and DCM (1.5 L) were charged at 18~24°C. 300.0 g Compound (IX) were charged at 18~24 ^° C.The suspension solution was cooled to 0~5°C, and 600 ml_ trifluoroacetic acid (TFA) were added slowly, maintaining the temperature at 0~5°C. After addition, the temperature was increased to 20~25 ^° C and the solution was stirred overnight.

The product was filtered and washed with DCM (300 ml_). Next, the product was dried under nitrogen flow at 20~25°C to a constant weight to give Compound (VIII) as TFA salt in a purity of 97.8%. 4.2. Preparation of Compound (VIII) as HCI salt

In a 3 L four-necked flask equipped with a mechanical stirrer and a thermometer, water (1.0 L) and. 200.12 g Compound (VIII) were charged at 20~25 ^° C. Saturated. Na ₂ C0 ₃ solution (385 ml_) was added slowly to adjust pH >7.5, while maintaining the temperature 20~25°C.

Undissolved material was filtered off and the filtrate was stirred at 20~25°C. 6 N HCI (160 ml_) was added slowly to adjust a pH <2.0, while maintaining the temperature 20~25°C.

The solution stirred overnight at 0~5°C. The precipitated product was filtered and dried under nitrogen flush at 25°C to a constant weight to give Compound (VIII) HCI salt. The product purity was greater than 98.5%; KF was about 6%; Cl- content was about 6%. The yield was 83.3% to 84.1% respectively from Compound (IX).

4.3 Reduction of the water content of Compound (VIII) HCI salt

The water content of Compound (VIII) HCI salt was reduced to improve the subsequent selective protection of the carboxylic acid group. Azeotropic distillation of water by repeated addition of ethyl acetate under vacuum at 45 ^° C resulted in a reduction of the water content from 0.9% to 0.09% (measured by Karl Fischer (KF) titration).

4.4 Preparation of Compound (II) from Compound (VIII) HCI salt

In a 1 L four-necked flask equipped with a mechanical stirrer and a thermometer, DMF (400 ml_) and 50.00 g Compound (VIII) HCI salt were charged at 20~25°C.

EA (250 ml_) was added and the solution was concentrated under vacuum at 50°C to 400 ml_. This procedure was repeated five times. The solution was transferred into a 1 L four-necked flask. 27.64 g (1.38 equiv.) trimethylsilylchloride (TMSCI) were charged for 5 min and stirred at 20~25°C for 20 min. The solution was transferred to a single flask, rinsed with DMF (25.0 ml_) and concentrated under vacuum at 50 ^° C to 250 ml_. Next, the solution was transferred to four neck bottle rinsed with DMF (25.0 ml_) and cooled to -5~0°C. 22.92 g (1.08 equiv.) HOSu (N-hydroxy succinimide) were charged for 3 minutes at -5~0°C. Thereafter, a DCC (dicyclohexyl carbodiimide) solution (43.76 g DCC (1.15 equiv.) dissolved in 184 ml_ EA) was charged for 30 minutes at -5~0°C.

The reaction solution was stirred for 2 h at 0~5°C. Then, 13.97 g (0.75 equiv.) TEA were charged for 5 min. After stirring for 20 minutes and filtration, the filter cake was rinsed with EA/DMF (4:1 , 100 ml_) and EA (50 ml_). The reaction solution was concentrated to -200 ml_ and 16.6 ml_ MsOH (1.39 equiv.) were added at 20~25°C for 30 min.

Compound (II) was obtained from the solution after addition of EA (745 ml_) and cooling to 0~5°C, stirring for 90 min, filtering and drying under nitrogen flush to a constant weight to give 70.4 g Compound (II) as a white crystalline solid. Purity was 95.2%; DMF residual content was 9.2% (by weight); the yield was 70.7%.

4.5. Removal of DMF from Compound (II)

Compound (II) as prepared in Example 4.4 was a DMF solvate having a residual DMF content of about 10%.

In a 100 ml_ four-necked flask equipped with a mechanical stirrer and a thermometer, EA (20.0 ml_) or acetone (20.0 ml_) were charged at 20~25°C. 2.0 g Compound (II) were charged at 20~25°C and the suspension was stirred at 20~25°C for 16 h.

The product was filtered and dried under nitrogen flush at 20~25°C to a constant weight. After slurrying in acetone for 1 d, residual DMF was 2591 ppm, after 3 d, residual DMF was 1713 ppm. The recovery yield was 90.2%. After slurry in EA for 1 d, residual DMF was 8.2%, after 3 d, residual DMF was 4775 ppm. The recovery yield was 92.8%. Example 5: Alternative preparation of Compound (II)

In an alternative procedure, Compound (II) was prepared based on the procedure below from monoacid ester Compound (XII).

5.1 Preparation of Compound (XIII)

Compound (XII) Compound (XIII) In a 1 L four-necked flask equipped with a mechanical stirrer and a thermometer, DMF (323 ml_), monoacid ester (XII) (75.0 g, 171.44 mmol) and N-hydroxy succinimide (HO- Su) (20.70 g, 180.01 mmol) were charged at 18~23°C. A solution of DCC (38.92 g, 188.58 mmol) in DMF (50 ml_) was added slowly. The mixture is stirred at 20~25°C over 3 hours. The mixture was cooled to 0~5°C and stirred for 2 h. Dicyclohexylurea (DCU) was removed by suction filtration, and the cake was washed with ice-cold DMF.

The filtrate was cooled to 0°C, and MeOH (450 ml_) was added, keeping the temperature below 6°C. Then water (450 ml_) was slowly added, keeping the temperature below 6°C. After completion of the water addition the mixture was cooled to 0~5°C and stirred for 1 h.

The precipitated product was filtered, washed with ice-cold MeOH/H ₂ 0 (1 :1 , 3 ^x 50 ml_) and dried by nitrogen flow at 25°C to a constant weight. The yield was 92.8% to 94.7%. 5.2. Preparation of Compound (II)

In a 2 L four-necked flask equipped with a mechanical stirrer and a thermometer, anisole (52.1 ml_) and DCM (521 ml_) were charged at 20~25°C. The solution was cooled to 0°C, and MsOH (52.1 ml_) were added slowly, maintaining the temperature 0~5°C. Di-ester (XIII) (86.77 g, 162.33 mmol) was added portion wise, keeping the temperature at 0~5°C.

The mixture was stirred at 0~10°C for 3 h. Acetone (1039 ml_) was added at a rate such that the temperature did not exceed 5°C. Crystallization of the product started during the addition of acetone. After stirring for 1.5 h at 0~5°C, the product was filtered and washed with acetone. Then the product was dried under nitrogen flow at 25°C to a constant weight to give Compound (II). The yield was between 60% and 70% with a purity of about 98%.

Example 6: Preparation of Compound (I) from Compound (II) and Compound (III)

Compound (III) Compound (I) Trihydrate

Compound (I) was prepared from Compound (II) and Compound (III) by a coupling reaction. The resulting product, compound (I) in the form of the trihydrate, was crystallized from the reaction mixture.

6.1 Coupling reaction using Compound (II) from Example 5 as starting material

In a 100 ml_ four-necked flask equipped with a magnetic stirrer and a thermometer, a mixture (1 :1) of water and methanol (30.0 ml_) was charged at 20~25°C. 3.00 g Compound (III) (11.31 mmol, 1.00 equiv.) were charged at 20~25°C and the solution was cooled to -5~0°C. TEA (1.14 g, 11 .27 mmol, 0.99 equiv.) was added to adjust the pH to 8.0-8.5.

4.98 g Compound (II) (13.52 mmol, 1.20 equiv., from Example 5) was added portion-wise, and the pH was maintained in the range of 8.0-8.5 with addition of TEA (5.11 g, 50.50 mmol, 4.47 equiv.) at 0~5°C. The reaction solution was stirred for 3 h at 0~5°C.

The pH of the mixture was adjusted to 1.5 with concentrated HCI (4.08 g) for 30 min at 0~5°C, no solid was observed. Then 11 mg Compound (I) as seed material was added. After 3 minutes, a large amount of solid precipitated. Then additional MeOH (15.0 ml_) and additional concentrated HCI (6.51 g) to adjust pH to 0.48 were added. The solution was cooled to 0~5°C and stirred at 0~5°C for 1 h, then filtered. The product was dried under nitrogen flush at 25°C to a constant weight to give Compound (I). The yield was 71.0% with a purity of 84.2%.

6.2. Crystallization

In order to improve the product purity, crystallization was performed under the following conditions.

4.0 g Compound (III) were used as starting material to prepare Compound (I) in a 1 :1 (v/v) water/methanol mixture.

3.0 g concentrated HCI was added to adjust pH 0.94 (target 1.0) at temperature 15 ^° C. 5.2 mg Compound (I) was added as seed and stirred at 15 ^° C for 10 min. Then methanol was added to obtain a 1 :2 (v/v) water/methanol ratio and the mixture was cooled to 10°C for 30 min and stirred at 10°C for 30 min. The mixture was cooled to 0 ^° C for 1 h and the product started to precipitate. Then the temperature was warmed to 15 ^° C for 4 h, cooled to 0 ^° C for 4 h, then again warmed to 15 ^° C for 4 h and cooled to 0 ^° C for 4 h. The mixture was stirred at 0 ^° C for 4 h and filtered. The filter and the wet cake were stored at -15~- 20 ^° C.

The product purity in the wet cake was 99.4 area-% by HPLC.

The X-ray powder diffractogram (XRPD) of the product Compound (I) Trihydrate was identical with a standard sample of Compound (I) T rihydrate, but the degree of crystallinity was slightly lower than the standard (Fig. 1).

6.3. Characterization and removal of impurities

The major impurities in the product Compound (I) were N-hydroxy succinimide (HOSu) and Compound (IV), i.e. the monomethyl ester of Compound (II), having the following structure:

Chemical Formula: C ₁₀ H ₁₁ N ₃ O ₅ S Exact Mass: 285.04

Compound (IV)

In order to reduce the amounts of impurities, the wet cake of Example 6.2 was re-slurried in THF/water at 9:1 (v/v). In a 100 mL four-necked flask equipped with a mechanical stirrer and a thermometer, water (2.92 mL, taking wet cake into account) and THF (26.3 mL) were charged at 20~25°C. 5.60 g compound (I) (wet cake, assay 52.09%, corrected weight 2.92 g) were charged at 20~25°C.

The suspension solution was stirred at 20~25°C for 2 h and then cooled to -8~-12°C for 40 min and stirred for 2 h. The product was filtered and dried under nitrogen flush (RH

30%~40%) at room temperature (15~20°C) to a constant weight.

The purity increased to 99.6%. The amount of HOSu decreased from 0.14% to 0.07%, the amount of the monomethyl ester (IV) decreased from 0.52% to 0.22%.

6.4 Coupling reaction using Compound (II) from Example 4 as starting material The preparation of Compound (I) was performed with Compound (III) and Compound (II) from Example 4.5 (re-slurry with EA) as starting materials.

In a 250 mL four-necked flask equipped with a mechanical stirrer and a thermometer, a mixture (1 :1) of water and methanol (100.0 mL) and 10.00 g Compound (III) (37.70 mmol, 1.00 equiv.) were charged at 20~25°C and the solution was cooled to -5~0°C.TEA (3.82 g, 37.75 mmol, 1 .00 equiv.) was added to adjust the pH to 8.42.

16.67 g Compound (II) (45.24 mmol, 1.20 equiv.) was added portion-wise, and the pH was maintained in the range of 8.0-8.5 by addition of TEA (13.82 g, 136.57 mmol, 3.62 equiv.) at 0~5°C. The reaction solution was stirred for 2~4h at 0~5°C and checked for completeness by HPLC.

The reaction solution was warmed to 14~16°C and the pH was adjusted to 1.0 with concentrated HCI (14.47 g) at 14~16°C. No solid was observed. 20.1 mg Compound (I) (0.2% wt) as seed were added and the solution was cooled to 10°C for 30 min. The temperature was kept at 10°C for 30 min. Additional 50 ml_ methanol were added since the reaction solution was difficult to stir.

The mixture was cooled to 0 ^° C for 1 h. Then the temperature was warmed to 15 ^° C for 4 h, cooled to 0 ^° C for 4 h, then again warmed to 15 ^° C for 4 h and cooled to 0 ^° C for 4 h. The mixture was stirred at -10 ^° C for 4 h, filtered and rinsed with water (10 ml_) to give 27.68 g wet cake. The filter and the wet cake were stored at -15~-20 ^° C. The product purity in the wet cake was 99.4% area % by HPLC.

In a 500 mL four-necked flask equipped with a mechanical stirrer and a thermometer, water (total 29.2 mL, taking wet cake into account) and THF (262.6 mL) were charged at 20~25°C. 27.62 g crude Compound (I) (wet cake, assay 52.84%, corrected weight 14.59 g) were charged at 20~25°C and the suspension was stirred at 20~25°C for 1 h. The solution was cooled to -8~-12°C for 40 min and stirred at -8~-12°C for 2 h. The product Compound (I) Trihydrate was filtered and dried under nitrogen flush (RH 30%~40%) at room temperature to a constant weight. The yield was 68.30% with 99.6% product purity. The XRPD was identical with a standard sample of Compound (I) Trihydrate (Fig. 2).

Preparation of Compound (I) Trihydrate from Compound (III) and Compound (II) from Example 4.5 (acetone re-slurry) gave the product with a yield of 70.16% and a purity of 99.47%. The XRPD was identical with a standard sample of Compound (I) Trihydrate (Fig. 3).

Example 7: Preparation of Compound (I) from TBA salt (IIId)

An alternative process for preparing the target compound Compound (I) starts from compound (Ilia), e.g. in the form of a tetrabutylammonium salt (llld).

The crude product yield was 48.23% from 24.35 g Compound (Ilia).

TBA salt (llld) Compound (III) 10.0 g TBA salt (llld) in (50 ml_ MeOH and 50 ml_ water) was used as starting material to prepare Compound (I) in the form of the trihydrate. After 23 h, the starting material was consumed completely, the purity was 98.4% and IPC assay yield was 95.7%. Then the reaction solution was used for a coupling reaction without purification. After 2.5 h, the reaction solution was used for work-up. The product purity was 99.0% and assay was 87.1%. The product assay yield was 51.6%, which was comparable with two separate steps procedure (~52%).

The product was re-slurried with water/methanol (1 :1 , 7.0 volumes) to remove impurities. The purity increased from 99.00% to 99.48%. HOSu decreased from 0.22% to 0.08%. Compound (II) monomethyl ester (IV) decreased from 0.51% to 0.20%.

Alternatively, after re-slurrying the product in THF/H2O (9/1), the purity was 99.7%. Example 8: Preparation of Compound (I) from Compound (Ilia) Potassium salt (lllc)

An alternative process for preparing the target compound (I) in the form of the trihydrate starts from the potassium salt (lllc) of Compound (Ilia).

Compound (I) Trihydrate Compound (VI) was used as staring material to prepare ( 111 c) , which was obtained as white solid.

3.00 g Compound ( 111 c) was used to prepare Compound (III) by hydrogenation in the presence of Pd/C as catalyst and molecular hydrogen in 30 ml_ of a 1 :1 (v/v) water/methanol mixture. After 4 h, Compound (I lie) was consumed completely.

Then additional water (15 ml_) and 1.4 equiv. Compound (II) (re-slurry by EA) were added for the coupling reaction. After 3 h, the remaining amount of Compound (III) was only 0.1%.

The reaction solution was filtered and crystallized. 4.95 g crude Compound (I) was obtained. After re-slurrying with THF/H ₂ 0 (9/1 , 20 vol) at -10°C, the recovery yield was 90.64%. The purity was 99.09%. Total yield was 54.05% from ( 111 c) .