The invention relates to specific 12-epi-mutilin compounds, a process for preparing these compounds and a use of said compounds in a process for preparing 12-epi-mutilin derivatives.

Background of the invention

(Pleuro)mutilins are compounds of e.g. formulae:

Pleuromutilin is a naturally occurring antibiotic, e.g. produced by the basidiomycetes Pleurotus mutilus and P. passeckerianus , see e.g. The Merck Index, 13th edition, item 7617. Pleuromutilin is manufactured by a fermentation process, extracted from the fermentation broth and finally isolated as crystalline solid compound. It is noted that in all of the marketed pleuromutilin derivatives the stereochemistry of the methyl and vinyl group at the C-12 position in the mutilin ring is the same as in the naturally occurring (pleuro)mutilin.

Pharmaceutical active compounds derived from Pleuromutilin (semi synthetic compounds) are inhibitors of ribosomal protein synthesis in bacteria. Representatives of semisynthetic pleuromutilins for human use are Retapamulin (approved as AltargoP ^® , AltabaxP ^® ), a topical agent approved for short term treatment of impetigo and infected small lacerations, abrasions or sutured wounds, and Lefamulin (approved as Xenleta ^® ) for the treatment of adults with community-acquired bacterial pneumonia (CABP). Tiamulin (Denagard ^® ) and Valnemulin (Econor ^® ) are two other semi-synthetic Pleuromutilin derivatives which have been used systemically in veterinary medicine as antibiotics for many years.

Approved semisynthetic compounds derived from Pleuromutilin have shown excellent activity against bacterial organisms which include inter alia Streptococcus pneumoniae , Haemophilus influenzae, Staphylococcus aureus (including MRS A), Moraxella catarrhalis , Legionella pneumophila , Chlamydophila pneumoniae and Mycoplasma pneumoniae.

Increasing rates of antibiotic resistance, in combination with the emergence of highly virulent pathogens, underpin the urgent need for novel antibacterial agents to combat serious bacterial infections caused by resistant organisms. Given their significant impact on morbidity and mortality, multi-drug resistant microbes are considered a substantial threat to public health by national academies and task forces including the Infectious Diseases Society of America, Centers for Disease Control and the World Health Organization.

In H. Berner et al., Monatshefte fur Chemie, 1986, 117, 1073-1080, for the first time a pleuromutilin derivative was described, in which the methyl group at position 12 of the mutilin ring has the opposite stereochemistry to that in the naturally occurring pleuromutilin. The second substituent in C-12 has also the inverted stereochemistry e.g. a vinyl group. However, the vinyl group can be transformed into a different substituent with impact on stereochemistry nomenclature (R and S). Therefore the C-12 methyl group qualifies best to define the stereochemistry at that position. Mutilins in which the methyl group at position 12 of the mutilin ring have the opposite stereochemistry to that in the naturally occurring (pleuro)mutilin are herein also designated as “12-epi-(pleuro)mutilin(s)”. WO 2015/110481 discloses semisynthetic 12-epi-(pleuro)mutilin compounds, such as 14-0- [((alkyl-, cycloalkyl-, heterocycloalkyl-, heteroaryl-, or aryl)-sulfanyl)-acetyl]-12-epi- mutilins, or 14-0-[((alkyl-, cycloalkyl-, heterocycloalkyl-, heteroaryl-, or aryl)-oxy)-acetyl]- 12-epi-mutilins of formula V which show interesting antimicrobial, e.g. antibacterial, activity.

Further 12-epi-(pleuro)mutilin(s) are e.g. described in:

• Dissertation of S. Schindler, “Funktionalisierung des tricyclischen Geriistes des Antibioticums Pleuromutilin”, University of Vienna, 2003, page 26 and 31.

• Murphy, S. K.; Zeng, M.; Herzon, S. B. “A modular and enantioselective synthesis of the pleuromutilin antibiotics.” Science 2017, 356, 956.

• Zeng, M.; Murphy S. K.; Herzon, S. B. “Development of a modular synthetic route to (+)-pleuromutilin, (+)-12-epi-mutilins, and related structures.” J. Am. Chem. Soc., 2017, 139 (45), pp 16377-16388.

• Herzon, S.; Murphy S. K.; Zeng, M. “New Pleuromutilin antibiotic compounds, composition and methods of use and synthesis” WO2018/144717.

For pharmaceutical use it is often essential to have an active compound comprising an asymmetric carbon atom in one of the diastereomerically pure forms, since one isomer may differ, e.g. in several aspects from another isomer, e.g. one isomer may be more active than the other isomer. Separation of isomers is often burdensome. Chromatography which, for example, may be useful for diastereomeric separation, can be difficult on a technical scale and often needs sophisticated and expensive means.

Especially, in view of the advantageous properties of 12-epi-(pleuro)mutilin(s) it is a critical task to provide enantiomerically pure intermediates with the 12-epi-stereochemistry to optimize the process for preparing the same.

Short description of the invention

The present invention provides the novel compound of formula I

wherein Ro is Cl or I, preferably Cl.

The inventors surprisingly found that the compound of formula I, wherein Ro is Cl, may be produced and separated as the diastereomerically pure compound, particularly on a technical scale. Surprisingly, the crystalline compound of formula I, wherein Ro is Cl, has been found to have unexpected chemical and diastereomeric purity, which is important for the production processes avoiding chromatographic purification and separation steps. The compound of formula I, wherein Ro is I, can also be used as key intermediate for the production of other 12-epi-(pleuro)mutilin(s). The compound of formula I, wherein Ro is Cl, is also designated as 12-epi-14-0-chloroacetyl-mutilin or chloroacetyl-12epi-mutilin or compound according to formula Ici

The compound of formula I, wherein Ro is I, is designated as 12-epi-14-0-iodooacetyl- mutilin or iodoacetyl-12epi-mutilin or compound according to formula Ii

A compound according to formula I, i.e. covering both Ro is Cl or I, is herein also referred to as a compound of or according to the present invention. The compound of formula Ici can be crystallized as a solid with only minor content of the diastereomer of formula IIci which compound is also referred to as 14-O-chloroacetyl-mutilin.

This unexpected purity and crystallizability of the compound of formula Ici was not observed for other intermediates used for the production of 12-epi-(pleuro)mutilin(s), such as 12-epi- pleuromutilin or 12-epi-pleuromutilin tosylate. In WO 2015/110481 a compound of formula III (pleuromutilin tosylate) was epimerized to obtain a mixture of a compound of formula III and its respective diastereomer compound of formula IV (12-epi-pleuromutilin tosylate) wherein it was necessary for the compound of formula IV to be separated from the compound of formula III by chromatography.

In another aspect, the invention relates to a process for preparing a compound of formula I, the process comprising the steps of isolating a compound of formula I in solid form, in particular a compound of formula Ici in crystalline form. The invention also relates to a compound of formula I made (or obtainable) by said process comprising the steps of isolating a compound of formula I in solid form, in particular a compound of formula Ici in crystalline form.

Moreover, the present invention refers to the use of a compound of formula I in a process of preparing 12-epi-mutilin derivatives, as well as a process for preparing 12-epi-mutilin derivatives, the process comprising providing a compound of formula I, wherein R ₀ is Cl, for the preparation of such derivatives. In particular, the 12-epi-mutilin derivatives are compounds of formula V wherein R ₁ is an organic group comprising 1 to 16 carbon atoms, optionally comprising one or more heteroatoms selected from N, O, S, halogen, in particular N;

X is sulfur or oxygen, in particular sulfur; and

R ₂ is an organic group comprising 1 to 22 carbon atoms, optionally comprising one or more heteroatoms selected from N, O, S, and halogen.

The invention also relates to a compound of formula V made (or obtainable) by said process for preparing 12-epi-mutilin derivatives, the process comprising providing a compound of formula Ici.

Detailed description of the invention

In the following, the mutilin numbering system described by H. Berner (Berner, H.; Schulz, G.; Schneider H. Tetrahedron 1980, 36, 1807-1811) is used.

Stereochemistry of (pleuro)mutilin(s) may be characterized by its proton nuclear magnetic resonance ( ¹ H-NMR) spectrum. The spectrum of compound of formula I is comprising inter alia peaks at 3.45 ppm ± 0.05 ppm obtained in CDCh using a 400 MHz NMR spectrometer. This signal corresponds to the CH in position 11 of the mutilin ring (H-l 1) and is a multiplet. The signal for the H-l 1 allows to differenciate between the naturally occurring (pleuro)mutilin configuration in compound of formulae II or III (3.35 ppm ± 0.05 ppm in CDCh at 400 MHz) and the 12-epi-(pleuro)mutilin(s) in compound of formulae I (both compound of formula Ici and compound of formula Ii) or IV (3.45 ppm ± 0.05 ppm in CDCh at 400 MHz). If both compounds are investigated in a mixture these peaks are quantitatively separated. In consequence, the diastereomeric ratio may be determined using NMR.

Alternatively, the diastereomeric ratio can be determined after separation of the compounds using HPLC analytics, for example using a reversed phase column and UV detection, such as described in the Example section below.

In one embodiment of the present invention, the compound of formula Ici is provided in a mixture with the compound of formula IIci, wherein the diastereomeric ratio between the compound of formula Ici and the compound of formula IIci (epi:non-epi) is 80:20 or higher, preferably 90:10 or higher and more preferably 95:5 or higher. The diastereomeric ratio can be determined by a method according to present art, in particular using NMR or HPLC analytics as described above. Accordingly, in a preferred embodiment, the compound of formula I is provided with a purity of > 80%, or > 90%, most preferably > 95%.

In a preferred embodiment, the compound of formula Ici is provided in a crystalline form. The crystalline form may be in the form of colorless to off-white crystals and the crystal nature of the compound is determined by standard means such as visual assessment and/or X-ray powder diffraction (XRPD). Different approaches for preparing a compound of formula Ici are described herein, e.g., as shown in Reaction Schemes 1-3 and Examples 1-3.

Reaction Scheme 1

Reaction Scheme 2 The approaches for the preparation of a compound of formula Ici of the present invention may comprise the steps of: a) providing a pleuromutilin wherein the hydroxy group of the side chain in position 14 is activated e.g., activated as a tosylate, e.g., a compound of formula III; b) epimerizing position 12 of the mutilin ring in a mutilin yielded from the previous step to obtain the inverse stereochemistry of said position; c) substituting the activated hydroxy group of the side chain in position 14 from e.g., tosylate to chloride; and d) isolating a compound of formula Ici of the present invention from the reaction mixture.

In one embodiment, the steps are performed in the sequence a) to d) (Reaction Scheme 1). In another embodiment of the invention step c) is performed prior to step b), thus involving the steps in the sequence a), c), b) and d) (Reaction Scheme 2).

Intermediates (or starting materials) in the preparation of a compound of the present invention are known or may be prepared according, e.g., analogously, to a method as conventional or as specified herein.

Preparing an activated pleuromutilin as performed in step a), e.g., pleuromutilin tosylate of formula III, is disclosed (e.g. in DE2036027).

Substituting the hydroxy group of the side chain in position 14 from, e.g. tosylate, to another activating group, e.g., chloride as in step c), has been described (e.g., see DE2036027).

Suitable conditions for epimerizing position 12 of the mutilin ring, i.e. as in step b), are disclosed in e.g. Monatshefte fiir Chemie, 1986, 117, 1073-1080 or in WO 2015/110481. This step can be carried out by reacting (pleuro)mutilin or a mutilin- or pleuromutilin derivative with e.g., ethyl zinc iodide or diethyl zinc in an organic solvent, as described in example 1A and B step 1 and example 2 step 2 of the present invention.

A feature in these approaches is a step of isolating a compound of formula I as solid, preferably a compound of formula Ici in crystalline form. Example 1 A and B, Step 3, Example 2, Step 3 and Example 3, last step, disclose different conditions to obtain the desired solid form of a compound of formula Ici. This step yields the unexpected and surprising diastereomerically pure or enriched compound and allows its convenient separation, e.g., in solid form, such as in crystalline form, from the (diastereomeric) reaction mixture. Thus it is the essential feature of the process.

Accordingly, the process according to one aspect of the invention comprises the step of isolating a compound of formula I in solid form, preferably the compound of formula Ici in crystalline form.

Preferably, the step of isolating the compound of formula Ici includes at least one step of crystallizing and isolating the compound of formula Ici in crystalline form.

The processes according to reactions schemes 1 and 2, i.e., as exemplified in Example 1 and 2, are preferred embodiments. Accordingly, there is no need for a step of separating two diastereomeric compounds by chromatography. The diastereomeric separation is preferably performed at the level of the substituted mutilin derivative. The substituted mutilin derivative of the compound of formula Ici was shown to solidify (e.g., crystallize) with a clear preference for the desired 12-epi-stereochemistry (chiral enrichment).

Accordingly, in a preferred embodiment a step of crystallizing the compound of formula Ici is performed starting from a reaction mixture comprising a compound of formula Ici and a compound of formula IIci (14-O-chloroacetyl -mutilin)

Steps b) and c) (epimerizing and substituting) can be performed in reverse order in accordance to reaction schemes 1 or 2, respectively. The process according to reaction scheme 1 results in higher yields for compound I. The process according to the reaction scheme 2 has the benefit of involving fewer intermediate products.

Accordingly, in one preferred embodiment the reaction mixture comprising a compound of formula Ici and a compound of formula IIci is obtained by the steps of: providing a compound of formula III (pleuromutilin tosylate);

epimerizing a compound of formula III to obtain a mixture of a compound of formula III and a compound of formula IV (12-epi-pleuromutilin tosylate) substituting the tosylate group of the mixture of the compound of formula III and the compound of formula IV with a chloride group using an inorganic chloride to obtain a mixture comprising a compound of formula Ici and a compound of formula IIci.

In another preferred embodiment, the reaction mixture comprising a compound of formula Ici and a compound of formula IIci is obtained by the steps of:

- providing a compound of formula III (pleuromutilin tosylate) substituting the tosylate group of a compound of formula III with a chloride group using an inorganic chloride to obtain a compound of formula IIci; and epimerizing a compound of formula IIci to obtain a mixture of a compound of formula Ici and a compound of formula IIci.

In some embodiments, the step of crystallizing may include seeding with a compound of formula Ici in crystalline form. As shown in comparison between example 1A and example IB, the seeding may allow for obtaining a higher yield of the desired product and/or a better diastereomeric enrichment of the compound of formula Ici. Pure seeding crystals may be obtained in the process according to Reaction Scheme 3 as shown in Example 3, wherein the substitution is performed starting from the purified 12-epi- pleuromutilintosylat. In this embodiment, the process according to the invention comprises the steps of:

- providing a compound of formula III epimerizing a compound of formula III to obtain a mixture of a compound of formula III and a compound of formula IV separating the compound of formula IV from the compound of formula III, preferably by use of chromatography; substituting the tosylate group of the compound of formula III with a chloride group using an inorganic chloride to obtain a compound of formula Ici; and crystallizing and isolating the compound of formula Ici.

The step of crystallizing refers to a process, wherein 14-0-chloroacetyl-12-epi-mutilin is obtained via crystallization from a reaction mixture in crystalline form. In certain embodiments, the crystalline 14-0-chloroacetyl-12-epi-mutilin is obtained as a mixture of 14- O-chloroacetyl-mutilin and 14-0-chloroacetyl-12-epi-mutilin. The desired 14-0- chloroacetyl-12-epi-mutilin can be further enriched via re-crystallization(s). In the process according to the present invention, (re-)crystallization can be repeated until the desired degree of purification is reached.

In a preferred embodiment, the step of crystallizing is repeated more than once. In this embodiment, the process includes at least one step of re-crystallization. The (re-)crystallization(s) is/are carried out in a solvent, a solvent mixture, or a solvent/antisolvent combination, wherein use of a single organic solvent is preferred over a solvent mixture, more preferably a single organic solvent. During crystallization, the crude starting material, i.e., a mixture of 14-O-chloroacetyl-mutilin and 14-0-chloroacetyl-12-epi-mutilin, may be dissolved, partially dissolved or suspended in the solvent, the solvent mixture, or the solvent/anti-solvent combination. Accordingly, the reaction mixture from which the 14-0- chloroacetyl-12-epi-mutilin is obtained in crystalline form is a solution or suspension. In a preferred embodiment, the reaction mixture for (re-)crystallizations is a suspension.

In certain embodiments, the crystalline form obtained by crystallization may be a solvate, i.e., the crystal form incorporates solvent molecules. For example, the compound of formula I can be obtained as a hydrate, e.g., when the crystal is obtained from a reaction mixture comprising water. Accordingly, in certain embodiments, the compound of formula I may be in a crystalline form, which is a solvate. In another embodiment, the crystalline form obtained by crystallization may be free of solvents, e.g., anhydrous, and accordingly, the compound of formula I may be free of solvents.

According to the present invention, a preferred organic solvent for any step of (re)- crystallizing is an aromatic apolar solvent, e.g. an organic solvent having at least one aromatic ring system such as toluene or xylene, preferably toluene, or an alcoholic solvent, e.g., an organic solvent having at least one hydroxyl functionality, such as a linear or branched, primary, secondary or tertiary Cl -C6 alcohol. In a preferred embodiment, the alcoholic solvent is selected from the group consisting of methanol, ethanol, isopropanol, and butanol.

In a preferred embodiment, at least one step of crystallizing is carried out in an organic solvent being an aromatic apolar solvent, preferably toluene, or an alcoholic solvent, preferably selected from the group consisting of methanol, ethanol, isopropanol, and butanol.

The following table summarizes the results for re-crystallizing 20 g of an input mixture comprising compounds of formula Ici and IIci in a diastereomeric ratio of 63:37, wherein the diastereomeric ratio was determined via NMR (400 MHz, CDC1 ₃ ), using different (re-)- crystallization solvents.

Diastereomeric ratio determined via NMR (400 MHz, CDC1 ₃ )

Thus, the process provides the 14-0-chloroacetyl-12-epi-mutilin (compound of formula Ici) by means of crystallization and one or more re-crystallization steps with a purity of the compound of formula Ici of > 80%, or > 90%, or > 95%.

Preparation of 14-0-iodooacetyl-12-epi-mutilin, a compound of formula Ii, is disclosed in Example 4. In step 1 of example 4, the compound of formula I, wherein Ro is I, is isolated as amorphous solid.

The compound of formula Ii can be obtained by converting the 14-0-chloroacetyl-12-epi- mutilin, preferably with the advantageous purity and diastereomeric ratio as described above, in a substitution reaction (Finkelstein reaction). The reaction involves iodide salt, preferably an inorganic iodide salt such as Nal, and can be performed in an aprotic, polar organic solvent, such as acetone, using moderate reaction conditions.

The reaction conditions do not affect the stereochemistry at the mutilin ring and thus allow to maintain the desirable diastereomeric ratio. Hence, the present invention discloses a compound of formula I _I in a diastereomeric mixture with a compound of formula II _I (14-0- iodooacetyl-mutilin), wherein the diastereomeric ratio between the compound of formula Ii and the compound of formula II _I is 80:20 or higher, preferably 90: 10 or higher and more preferably 95:5 or higher Accordingly, in a preferred embodiment, the process for preparing the compound of formula Ii comprises the steps of: providing a compound of formula Ici reacting a compound of formula Ici with a iodide salt, preferably in an aprotic, polar organic solvent, and isolating the compound of formula Ii as solid.

Compounds of the present invention are suitable as key intermediates for the production of pharmaceutically active compounds as disclosed in WO 2015/110481, e.g., for the treatment and prevention of diseases which are mediated by microbes, e.g., by bacteria.

Preferably, the invention relates to a use of a compound of formula I in a process for preparing 12-epi-mutilin derivatives (intermediates and/or final products) and a process for preparing 12-epi-mutilin derivatives.

The term “12-epi-mutilin derivatives” as used herein is understood as including any compound that is a semisynthetic derivative other than the compound of formula I, which can be formali stically derived from a compound of the following formula.

This definition of 12-epi-mutilin derivatives is in analogy to the common understanding of the term “(pleuro)mutilin derivative” (denoting compounds of the (pleuro)mutilin class being compounds derived from (pleuro)mutilin), with the difference that in the 12-epi-mutilin derivatives the methyl group at position 12 of the mutilin ring has the opposite stereochemistry to that in the naturally occurring (pleuro)mutilin as shown in the background section of the present application.

Preferably, the 12-epi-mutilin derivatives are compounds of formula V wherein

Ri is an organic group comprising 1 to 16 carbon atoms, optionally comprising one or more heteroatoms selected from N, O, S, halogen, in particular N;

X is sulfur or oxygen, preferably sulfur; and

R ₂ is an organic group comprising 1 to 22 carbon atoms, optionally comprising one or more heteroatoms selected from N, O, S, and halogen.

The term “organic group” denotes a moiety as known in organic chemistry and includes a linear, branched or cyclic as well as saturated, unsaturated or aromatic hydrocarbon (an alkyl-, cycloalkyl-, or aryl-group) and as indicated by the optional presence of the heteroatoms also an heterocycloalkyl- or heteroaryl-group. The group may be further substituted with heteroatoms or functional groups such as acyl, carboxy, hydroxyl, alkoxy, amino, carbamoyl, amido, and guanidine.

In one embodiment, R ₂ is an alkyl-, cycloalkyl-, heterocycloalkyl-, heteroaryl-, or aryl- group, optionally further substituted.

In one embodiment, Ri is either (C _1-16 )alkyl or (C _2-16 )alkenyl, substituted by (C _1-13 )- heterocyclyl, including aliphatic heterocyclyl and aromatic heterocyclyl comprising 1 to 4 heteroatoms selected from N, O, S, with the proviso that at least one heteroatom is a nitrogen atom, or

R ₁ is a group of formula wherein Y-N(R ₃ R ₄ ) is:

- (C _1-16 )alkyl-N(R ₃ R ₄ ); in particular (C _1-12 )alkyl-N(R ₃ R ₄ );

- (C _1-16 )alkyl-(C _6-14 )aryl-N(R ₃ R ₄ );

- (C _1-16 )alkyl-(C _6-14 )aryl-(C _1-16 )alkyl-N(R3R4) in particular (C _1-4 )alkyl-phenyl-(C _1-6 )alkyl-N(R ₃ R ₄ );

- (C _1-16 )alkyl-(C _1-13 )heterocyclyl -N(R ₃ R ₄ );

- (C _1-16 )alkyl-(C _1-13 )heterocyclyl-(C _1-16 )alkyl-N(R ₃ R ₄ );

- carbonyl -N(R ₃ R ₄ );

- (C _1-4 )alkyl-carbonyl-N(R ₃ R ₄ );

- (C _2-16 )alkenyl-N(R ₃ R ₄ );

- (C _2-16 )alkenyl-(C _6-14 )aryl-N(R ₃ R ₄ ), in particular (C _2-4 )alkenyl-phenyl-N(R3R4), in particular ethenyl -phenyl -N(R ₃ R ₄ );

- (C _2-16 )alkenyl-(C ₆ - ₁₄ )aryl-(C _1-16 )alkyl-N(R ₃ R ₄ ); in particular (C _2-16 )alkenyl-phenyl-(C _1-16 )alkyl-N(R3R4);

- (C _2-16 )alkenyl-(C _1-13 )heterocyclyl-N(R3R4); and/or

- (C ₂ - ₁₆ )alkenyl-(C _1-13 )heterocyclyl-(C _1-16 )alkyl-N(R ₃ R ₄ ) _.

For example, the 12-epi-mutilin derivative is a compound according to the group comprising Examples 1 to 160 as disclosed in the example section of WO 2015/110481, said compounds being incorporated herein by reference. The 12-epi-mutilin derivatives as disclosed in WO 2015/110481 have interesting activity against Gram negative and Gram positive bacteria, in particular improved activity against Gram negative bacteria, in particular activity against Escherichia coli.

In the process for preparing the 12-epi-mutilin derivatives, the compound of formula I, wherein Ro is Cl, is provided and subsequently processed according to one of the reaction path ways summarized in Reaction Scheme 4, wherein PG refers to a protection group as known in the present art. Reaction Scheme 4

Accordingly, in one aspect, the present invention relates to a process for preparing the 12- epi-mutilin derivatives, the process comprising providing a compound of formula I wherein R ₀ is Cl, subsequently at least two steps of chemical processing, wherein a moiety R ₁ and a moiety R ₂ -X are introduced independently to each other, and isolating a 12-epi-mutilin derivative, wherein the 12-epi-mutilin derivative is a compound of formula V.

In a preferred embodiment of the use or the process for preparing the 12-epi-mutilin derivatives according to the invention, the compound of formula I, wherein R ₀ is Cl, is provided in crystalline form.

The reaction in the first row of Reaction Scheme 4 is exemplarily shown in Example 4. The reaction may involve the conversion of 14-O-chloroacetyl- 12-epi-mutilin to the intermediate product 14-O-iodoacetyl- 12-epi-mutilin and further to 12-epi-mutilin tosylate. The compound of formula I, wherein Ro is I, i.e. compound of formula I _I , is a convenient intermediate product, as it can be isolated as solid and shows the desired reactivity towards tosylation. Thus, 14-O-iodoacetyl- 12-epi-mutilin allows to obtain a compound of formula IV in high yield and purity.

Accordingly, one embodiment of the use or the process for preparing the 12-epi-mutilin derivatives according to the invention comprises reacting a compound of formula I, wherein Ro is Cl, with a iodide salt, preferably in an aprotic, polar organic solvent, and isolating a compound of formula I, wherein Ro is I.

In a preferred embodiment, the use or the process for preparing the 12-epi-mutilin derivatives according to the invention comprises converting a compound of formula I, wherein Ro is I, into a compound of formula IV Example 5 of the present invention exemplifies an alternative embodiment of the use or the process for preparing the 12-epi-mutilin derivatives according to the invention. The reaction scheme 4 shows the steps of this embodiment starting with the left part of the reaction scheme e.g. direct substitution of the C1 with PG-R ₂ -X group (without requirement of tosylation).

Example 5 also includes the final step towards a compound of the generic formula V. The reaction yields 12-epi-12-Desvinyl-14-0-[(Piperidin-4-ylsulfanyl)-acetyl]-12 -[(E)-2-(3- methyl-pyrazin-2-yl)-ethenyl]-mutilin, a compound of formula VI

Examples

Herein, including the examples and the reaction scheme the following abbreviations are used:

¹ H-NMR proton nuclear magnetic resonance spectroscopy

°c degrees Celsius

DMSO dimethylsulfoxide eq equivalents h hour(s)

HPLC High Performance Liquid Chromatography

IPA isopropyl alcohol, isopropanol

M molarity

MS mass spectrometry m/z mass/charge ratio vol volume(s), mL/kg starting material The trivial name “mutilin” refers to the IUPAC systematic name (IS, 2R, 3S, 4S, 6R, 7R, 8R, 14R)-3,6-di hydroxy-2, 4, 7, 14-tetram ethyl -4-vinyl-tricyclo[5, 4, 3, 0 ^{1 8} ]tetradecan-9-one as shown below (mutilin).

In the following examples, again the pleuromutilin derivatives are numbered in analogy to the mutilin numbering system described by H. Berner (Berner, H.; Schulz, G.; Schneider H. Tetrahedron 1980, 36, 1807-1811) as follows:

Mutilin

In the compounds of the present invention, e.g., in the products of examples 1 to 3, the stereochemistry - at position 12 - is inverted to 12-epi-mutilin(s) as follows:

12-epi-Mutilin

The NMR method to analyze the epi / non-epi ratio uses the integral of the CH of position 11 in the mutilin ring (H-11) from its proton nuclear magnetic resonance ( ¹ H-NMR) spectrum in CDC1 ₃ using a 400 MHz NMR spectrometer.

The HPLC method to analyze the epi / non-epi ratio comprises aNucleosil 100-5 C18 4.0 x 125 mm 5μm HPLC column, detection at 200 or 254 nm, an eluent A (0.92 g sulfamic acid in 1 L of HPLC-water) and eluent B (0.92 sulfamic acid in 700 mL of acetonitrile and 300 mL of HPLC-water) at a flow rate of 1.01 mL/min. No correction for a potential difference in relative response factor was applied.

Characterization of compounds

The compounds pleuromutilin tosylate and 12-epi-pleuromutilin tosylate as well as the chloroacetyl-(epi)-mutilins as used in Examples 1 to 3 were characterized by NMR (mutilin numbering system used) and MS.

14-O-Chloroacetyl- 12-epi-mutilin. compound of formula Ici

¹ H-NMR (400 MHz, CDC1 ₃ , d, ppm, characteristic signals, mutilin numbering system): 5.79- 5.70 (m, 1 H, H-19), 5.65 (d, 1H, H-14, J=8.4 Hz), 5.25 - 5.17 (m, 2H, H-20), AB (2H, H- 22, VA=3.98, _VB =3.97, J=15 HZ), 3.46-3.44 (m, 1H, H-11), 1.47 (s, 3H, CH ₃ -15), 1.25 (s, 3H, CH ₃ -18), 0.97 (d, 3H, CH ₃ -17, J=7.1 Hz), 0.73 (d, 3H, CH ₃ -16, J=7.0 Hz).

MS m/e: 419 [M + Na ⁺ ], 815 [2M + Na ⁺ ],

14-O-Chloroacetyl -mutilin. compound of formula IIci

¹ H-NMR (400 MHz, CDC1 ₃ , δ, ppm, characteristic signals, mutilin numbering system): 6.48 (dd, 1 H, H-19, J _E =17.4 HZ, J _Z =11.0 H _z ), 5.79 (d, 1H, H-14, J=8.5 Hz), 5.40 - 5.18 (m, 2H, H-20), AB (2H, H-22, v _A =3.99, _VB =3.97, J=14 HZ), 3.39-3.34 (m, 1H, H-l l), 1.48 (s, 3H, CH ₃ -15), 1.18 (s, 3H, CH ₃ -18), 0.89 (d, 3H, CH ₃ -17, J=7.1 Hz), 0.73 (d, 3H, CH ₃ -16, J=7.0 Hz).

MS m/e: 419 [M + Na ⁺ ], 815 [2M + Na ⁺ ],

14-O-Iodoacetyl-l 2-epi-mutilin. compound of formula L

¹ H-NMR (400 MHz, CDC13, δ, ppm, characteristic signals, mutilin numbering system): 5.80-5.68 (m, 1 H, H-19), 5.60 (d, 1H, H-14, J=8.4 Hz), 5.26-5.16 (m, 2H, H-20), 3.66-3.55 (m, 2H, H-22), 3.44 (d, 1H, H-l l, J=6.4Hz), 1.45 (s, 3H, CH ₃ -15), 1.20 (s, 3H, CH ₃ -18),

0.95 (d, 3H, CH ₃ -17, J=6.8 Hz), 0.73 (d, 3H, CH ₃ -16, J=6.8 Hz).

MS m/z: 511 [M + Na ⁺ ], 999 [2M + Na ⁺ ], 533 [M + HCOO ^' ].

Pleuromutilin tosylate. compound of formula III

¹ H-NMR (400 MHz, CDC1 ₃ , δ, ppm, characteristic signals, mutilin numbering system): 7.81 (d, 2H, arom., J=8.3 Hz), 7.35 (d, 2H, arom., J=8.0 Hz), 6.46-6.35 (m, 1 H, H-19), 5.76 (d, 1H, H-14, J=8.5 Hz), 5.35 - 5.15 (m, 2H, H-20), 4.47 (m, 2H, H-22), 3.36-3.32 (m, 1H, H- 11), 2.45 (s, 3H, -CH ₃ ), 1.40 (s, 3H, CH ₃ -15), 1.15 (s, 3H, CH ₃ -18), 0.87 (d, 3H, CH ₃ -17, J=7.0 Hz), 0.62 (d, 3H, CH ₃ -16, J=7.0 Hz).

MS m/e: 550 [M ⁺ + NH ₄ ],

12-epi-Pleuromutilin tosylate. compound of formula IV

¹ H-NMR (400 MHz, CDC1 ₃ , δ, ppm, characteristic signals, mutilin numbering system): 7.81 (d, 2H, arom., J=8.2 Hz), 7.35 (d, 1H, arom., J=8.3 Hz), 5.71 (dd, 1 H, H-19, J _E =17.4 Hz, Jz=10.8 Hz), 5.64 (d, 1H, H-14, J=8.4 Hz), 5.25 - 5.17 (m, 2H, H-20), 4.47 (bs, 2H, H-22), 3.43-3.41 (m, 1H, H-ll), 2.45 (s, 3H, -CH ₃ ), 1.40 (s, 3H, CH ₃ -15), 1.18 (s, 3H, CH ₃ -18),

0.95 (d, 3H, CH ₃ -17, J=7.0 HZ), 0.62 (d, 3H, CH ₃ -16, J=7.0 Hz).

MS m/e: 550 [M ⁺ + NH ₄ ],

Example 1A

14-0-Chloroacetyl-12-epi-mutilin (Reaction Scheme 1, w/o seeding )

Step 1: Pleuromutilin tosylate and 12-epi-Pleuromutilin tosylate

Pleuromutilin tosylate (1.016 kg, 1.91 mol) was suspended in toluene (5 vol) and heated to 80-85 °C. Diethylzinc (1 mol/L in heptane, 0.80 L) was charged, the reaction was stirred at 85 °C for 2 h, then cooled to 35°C and stirred at this temperature overnight. Water (28.8 mL) was added and the organic phase was washed with 1 M citric acid solution (2 x 1 vol) followed by 0.5 M NaHCO ₃ solution (1 vol) and concentrated under vacuum to dryness. A mixture of pleuromutilin tosylate and 12-epi-pleuromutilin tosylate (diastereomeric ratio epi:non-epi of 68:32 ratio by NMR), quantitative yield containing residual solvent, was isolated in the form of a dark yellow to orange oil.

Step 2: 14-O-Chloroacetyl-mutilin and 14-O-Chloroacetyl-12-epi-mutilin Pleuromutilin tosylate / 12-epi-pleuromutilin tosylate mixture (3.93 mol) containing residual toluene (approximately 0.25 vol) was dissolved in acetone (6 L). Lithium chloride (0.2 kg) was charged, the reaction mixture was heated to reflux and held overnight, then cooled to room temperature. After evaporation of solvents the residue was distributed between ethyl acetate and water and the phases were separated. The organic phase was washed with water and saturated sodium chloride solution, dried and evaporated to dryness. A mixture of 14-O- chloroacetyl-mutilin and 14-O-chloroacetyl-12-epi -mutilin (1.489 kg, diastereomeric ratio epi:non-epi of 67:33 ratio by NMR) was yielded in the form of colorless to light brown crystals. Step 3: 14-O-Chloroacetyl-12-epi-mutilin

14-O-chloroacetyl-mutilin / 14-O-chloroacetyl-12-epi-mutilin mixture (1.42 kg, 67:33 epi:non-epi ratio by NMR) was dissolved in IPA (2 vol) and heated to 75 °C. The resulting solution was slowly cooled to 5 °C and stirred at 5 °C overnight. The suspension was filtered and the precipitate was isolated and dried. The precipitate was suspended in IPA (2 vol) and heated to 75 °C, then cooled to 5 °C over 2 hours and stirred at 5 °C overnight. The resulting suspension was filtered and the precipitate was isolated and dried. 14-0-chloroacetyl-12-epi- mutilin (0.419 kg, diastereomeric ratio epi:non-epi of 93:7 by NMR, 29.5 % yield and 41.7 % of theory,) was isolated in the form of colorless crystals.

Alternative Step 3: 14-O-Chloroacetyl-12-epi-mutilin

14-O-chloroacetyl-mutilin / 14-O-chloroacetyl-12-epi-mutilin mixture (2.00 kg, 67:33 epi:non-epi ratio by NMR) was suspended in denatured ethanol (2 vol) and heated to reflux. The resulting turbid solution was slowly cooled to 5 °C over 6 h and stirred at 5 °C overnight. The suspension was filtered and the precipitate was isolated and dried. The precipitate was suspended in denatured ethanol (2 vol) and heated to reflux, then cooled to 5 °C over 6 hours and stirred at 5 °C overnight. The resulting suspension was filtered and the precipitate was isolated and dried. 14-O-chloroacetyl-12-epi-mutilin (1.127 kg, diastereomeric ratio epi:non-epi of 94:6 by NMR, 56.4 % yield and 79.3 % of theory) was isolated in the form of colorless to light brown crystals.

Example IB

14-0-Chloroacetyl-12-epi-mutilin (Reaction Scheme 1 with seeding)

Step 1: Pleuromutilin tosylate and 12-epi-Pleuromutilin tosylate

Pleuromutilin tosylate (1.209 kg, 2.27 mol) was suspended in toluene (4 vol) and heated to 80-85 °C. Diethylzinc (1 mol/L in heptane, 0.80 L) was charged, the reaction mixture was stirred at 85°C for 2 h, then cooled to 35°C and stirred at this temperature overnight. Water (28.8 mL) was added and the organic phase was washed with 1 M citric acid solution (2 x 0.8 vol) followed by saturated NaCl solution (0.8 vol) and concentrated under vacuum to dryness. A mixture of pleuromutilin tosylate and 12-epi-pleuromutilin tosylate (diastereomeric ratio epi:non-epi of 68:32 ratio by NMR), containing residual solvent, was isolated in the form of a dark yellow to orange oil.

Step 2: 14-O-Chloroacetyl-mutilin and 14-O-Chloroacetyl-12-epi-mutilin

Pleuromutilin tosylate / 12-epi-pleuromutilin tosylate mixture (4.52 mol) containing residual toluene (approximately 0.25 vol) was dissolved in acetone (2.5 vol). Lithium chloride (0.23 kg) was charged, the reaction mixture heated to reflux and held for 2.5 h, then cooled to room temperature. After evaporation of solvents the residue was distributed between ethyl acetate and water and the phases were separated. The organic phase was washed with water and saturated sodium chloride solution, dried and evaporated to dryness. A mixture of 14-0- chloroacetyl-mutilin and 14-0-chloroacetyl-12-epi-mutilin (1.680 kg, diastereomeric ratio as in step 1) was yielded in the form of colorless to off-white solid.

Step 3: 14-O-Chloroacetyl-12-epi-mutilin

14-O-chloroacetyl-mutilin / 14-O-chloroacetyl-12-epi-mutilin mixture (1.68 kg, epi:non-epi of 68:32 ratio by NMR) was dissolved in IPA (2 vol) and heated to 75 °C. The resulting solution was slowly cooled to 5 °C (1.5 g of pure 14-0-chloroacetyl-12-epi-mutilin seed crystals were added at 52 °C) and stirred at 5 °C overnight. The suspension was filtered and the precipitate was isolated and dried. The precipitate was suspended in IPA (2.5 vol) and heated to 75 °C, then cooled to 5 °C over 2 hours and stirred at 5 °C overnight. The resulting suspension was filtered and the precipitate was isolated and dried. 14-O-chloroacetyl-12-epi- mutilin (0.718 kg, diastereomeric ratio epi:non-epi of 94:6 by NMR, 42.7 % yield and 61.0 % of theory,) was isolated in the form of colorless crystals.

Two additional maturation cycles in 2 Vol IPA at 75 °C further increased the diastereomeric ratio epi:non-epi of to >97 :<3.

Example 2

14-0-Chloroacetyl-12-epi-mutilin (Reaction Scheme 2)

Step 1: 14-O-Chloroacetyl-mutilin

Pleuromutilin tosylate (50 g) was dissolved in acetone (10 vol). Lithium chloride (4.77 g) was charged, the reaction mixture was heated to reflux overnight. After evaporation of solvents the residue was distributed between ethyl acetate and water and the phases were separated. The organic phase was washed with water and saturated sodium chloride solution, dried and evaporated to dryness. 14-O-chloroacetyl-mutilin (37.2 g) was isolated in the form of colorless to off-white crystals.

Step 2: 14-O-Chloroacetyl-mutilin and 14-O-Chloroacetyl-12-epi-mutilin 14-O-Chloroacetyl-mutilin (37.2 g) was suspended in toluene (50 mL) and heated to 80-85 °C. Diethylzinc (1 mol/L in hexane, 50 mL) was charged, the reaction mixture was stirred at 80 °C for 4 h, then cooled to room temperature. Water (0.9 mL) was added and the organic phase was washed with 1 M citric acid solution (2 x) followed by saturated NaCl solution and concentrated under vacuum to dryness. A mixture of 14-O-chloroacetyl-mutilin and 14- O-chloroacetyl-12-epi-mutilin (diastereomeric ratio epi:non-epi of 60:40 by NMR), quantitative yield containing residual solvent, was isolated.

Step 3: 14-O-Chloroacetyl-12-epi-mutilin

14-O-chloroacetyl-mutilin / 14-0-chloroacetyl-12-epi-mutilin mixture (20.1 g) of Step 2 was suspended in IPA (2 vol) and heated to reflux. The resulting solution was slowly cooled to room temperature (20 mg of pure 14-O-chloroacetyl-12-epi-mutilin seed crystals were added at 40 °C) and stirred at 2-8 °C overnight. The suspension was filtered and the precipitate was isolated and dried. The precipitate was suspended in IPA (2 vol) and heated to reflux, then slowly cooled to room temperature overnight and stirred at 5 °C for 2 h. The resulting suspension was filtered and the precipitate was isolated and dried. 14-O-chloroacetyl-12-epi- mutilin (3.30 g, diastereomeric ratio epi:non-epi of 92:8 by HPLC at 200 nm, 16.4 % yield and 29.4 % of theory,) was isolated in the form of colorless crystals.

Example 3

14-0-Chloroacetyl-12-epi-mutilin (Reaction Scheme 3)

12-epi-Pleuromutilin tosylate (10 g, see e.g. WO 2015/110481 Example 1 Step 2, obtained via chromatography) was dissolved in acetone. Lithium chloride (3.2 g) was charged, the reaction mixture was heated to reflux for 3 h. After evaporation of solvents the residue was distributed between ethyl acetate and water and the phases were separated. The organic phase was washed with water and saturated sodium chloride solution, dried and evaporated to dryness. 14-0-chloroacetyl-12-epi-mutilin (7.4 g) was isolated in the form of colorless crystals.

Example 4

12-epi-Pleuromutilin tosylate via 14-0-Iodoacetyl-12-epi-mutilin (Reaction Scheme 4)

Step 1: 14-O-Iodoacetyl-12-epi-mutilim compound of formula I _I

To 14-O-chloroacetyl-12-epi-mutilin (10 g) was added acetone and sodium iodide (4.53 g, 1.2 eq.) and stirred overnight at room temperature. The resulting reaction mixture was concentrated under reduced pressure, taken up in dichloromethane and washed with half- saturated NaCl and 5% NaCl solution. The organic phase was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness under reduced pressure to obtain the title compound (11.87 g) in the form of a pale yellow to yellow solid. Step 2: 12-epi-Pleuromutilin tosylate. compound of formula IV

To 14-O-iodoacetyl-12-epi-mutilin (11.87 g) was added dry acetonitrile and silver tosylate (8.14 g, 1.2 eq.) and heated to reflux for 3.5 hours. The resulting reaction mixture was cooled to room temperature, filtered and concentrated under reduced pressure. The concentrate was taken up in dichloromethane and washed with half-saturated NaCl solution and the aqueous phases was washed with dichloromethane (3x). Then all organic phases were combined, washed with water, dried over anhydrous Na ₂ SO ₄ and evaporated to dryness under reduced pressure to obtain the title compound (12.73 g) in the form of a colorless solid.

Example 5

12-epi-12-Desvinyl-14-0-[(Piperidin-4-ylsulfanyl)-acetyl] -12-[(E)-2-(3-methyl-pyrazin- 2-yl)-ethenyl]-mutilin dihydrochloride (Compound of formula V, in particular of formula VI, according to Reaction Scheme 4)

Step 1 : 12-epi-14-O-[(T-tert-Butoxycarbonyl-piperidine-4-ylsulfanv1) -acetv]-mutilin

To 14-O-chloroacetyl-12-epi-mutilin (3 g) was added methanol (22.5 mL), tetrahydrofuran (4.5 mL), tert-butyl 4-acetylsulfanylpiperidine-l-carboxylate (1.96 g) and potassium carbonate solution (5M in water, 3.02 mL) and stirred overnight at room temperature. The resulting reaction mixture was concentrated to dryness, taken up in ethyl acetate (50 mL) and washed twice with half-saturated NaCl solution (50 mL). The combined aqueous phases were extracted with ethyl acetate (50 mL), then the combined organic phases were dried over anhydrous Na ₂ SO ₄ and evaporated to dryness under reduced pressure. The evaporation residue was subjected to chromatography over silica gel using cyclohexane / EtOAc 3 : 2 to obtain the title compound (3.67 g) in the form of a pale orange to orange solid.

¹ H-NMR (400 MHz, CDC13, d, ppm, characteristic signals, mutilin numbering system): 5.79-5.68 (m, 1 H, H-19), 5.62 (d, 1H, H-14, J=8.0 Hz), 5.25-5.15 (m, 2H, H-20), 4.00-3.90 (m, 2H, H-22), 3.43 (d, 1H, H-11, J=6.4Hz), 1.50-1.40 (m, 12H, Boc, CH ₃ -15), 1.22 (s, 3H, CH ₃ -18), 0.95 (d, 3H, CH ₃ -17, J=7.2 Hz), 0.72 (d, 3H, CH ₃ -16, J=6.8 Hz).

MS m/z: 600 [M + Na ⁺ ], 612 [M + Cl-], 622 [M + HCOO-].

Step 2: 12-epi-12-desvinyl-14-O-[(T-tert-Butoxycarbonyl-piperidin-4- ylsulfanv]-acetv]-12-

[2-(3-methyl-pyrazin-2-v]-ethenv]-mutilin

2-Bromo-3 -methyl -pyrazine (1.20 g, 4 eq.) and bis-(benzonitrile)-palladium(II)-chlorid (266 mg, 0.4 eq.) were suspended in ethylene glycol (80 mL). Then example 5 step 1 product (1 g, 1 eq.) and N-methyl-morpholine (1.52 mL, 8 eq.) were added and the resulting mixture was stirred at 100°C for 48 hours. The reaction mixture was diluted with ethyl acetate (500 mL), extracted with HCl/NaCl solution (500 mL, 0.1 M aqueous HC1 + 500 mL 5% aqueous NaCl solution, 1:1) and with 5% aqueous NaCl solution (500 mL). The combined aqueous phases were washed with ethyl acetate (250 mL). All organic phases were combined, washed with saturated aqueous NaCl solution (250 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The evaporation residue was subjected to chromatography over silica gel using EtOAc as eluent to obtain the title compound (153 mg) as a pale yellow to yellow solid.

¹ H-NMR (400 MHz, CDC13, δ, ppm, characteristic signals, mutilin numbering system): 8.44-8.28 (m, 2H, aromat.), 6.97 and 6.66 (2d, 2 H, H-19, H-20, J=15.6 Hz), 5.64 (d, 1H, H- 14, J=8.0 Hz), 4.04-3.83 (m, 2H, H-22), 3.70 (d, 1H, H-l l, J=6.0Hz), 2.64 (s, 3H, CH ₃ - aromat.), 1.53-1.35 (m, 15H, BOC, CH ₃ -15, CH ₃ -18), 0.97 (d, 3H, CH ₃ -17, J=7.2 Hz), 0.74 (d, 3H, CH ₃ -16, J=7.2 Hz).

MS m/z: 670 [M + H ⁺ ], 714 [M + HCOO ^' ].

Step 3: 12-epi-12-desvinyl-14-O-[(Piperidin-4-ylsulfanyll-acetyll-12 -[2-(3-methyl-pyrazin-

2-v)-ethenv] -mutilin dihydrochloride

Example 5 step 2 product (153 mg) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (2 mL) was added. The reaction mixture was stirred for 2 hours at room temperature and evaporated to dryness. The resulting residual was dissolved in little dichloromethane and diethylether and hydrogen chlorid (2M in diethylether, 2 mL) was added, stirred for 30 minutes at room temperature and filtered. The precipitate was dissolved in water and lyophilized to obtain the title compound (113 mg) as pale yellow to yellow solid.

¹ H-NMR (400 MHz, DMSO-d6, δ, ppm, characteristic signals, mutilin numbering system): 9.10-8.77 (m, 2H, NH), 8.37-8.21 (m, 2H, aromat.), 7.08 and 6.57 (2d, 2H, H-19, H-20, J = 15.6 Hz), 5.52 (d, 1H, H-14, J = 8.0 Hz), 3.66 (d, 1H, H-11, J = 6.0 Hz), 2.50 (s, 3H, CH ₃ - aromat.), 1.35 (s, 3H, CH ₃ -15), 1.18 (s, 3H, CH ₃ -18), 0.80 (d, 3H, CH ₃ -17, J = 6.8Hz), 0.63 (d, 3H, CH ₃ -16, J = 6.4 Hz).

MS m/z: 570 [M + H ⁺ ], 604 [M + C1-], 614 [M + HCOO-].