SEQUANAMYCIN MACROLIDES USEFUL IN THE TREATMENT OF

TUBERCULOSIS

The present invention relates to macrolides, and to the preparation and therapeutic use thereof. The compounds according to the present invention have substantial antimicrobial activity on mycobacteria, and are useful especially in the treatment of tuberculosis.

Due to the appearance of resistance, the development of novel antibacterial agents is necessary to make it possible to kill or to prevent the growth of mycobacteria, especially those which induce tuberculosis.

Tuberculosis is a disease which, at the present time, is still a worldwide health threat.

Globally, a third of the human population is infected with Mycobacterium tuberculosis.

Despite the fact that treatments exist and that the disease is curable, tuberculosis killed approximately 1 .8 million people in 2015, including 0.4 million of Mycobacterium tuberculosis and HIV co-infected patents, and its global incidence increases by 1 % per year, with an estimation in 2015 of 10.4 million annual new cases of declared disease.

Added to this are the difficulties of correct prescription and of adherence to the treatment protocols, and also the emergence of multi-resistant strains of M. tuberculosis. Drug-drug interactions also interfere with the optimum treatment of AIDS and tuberculosis in the case of co-infected patients.

The common treatment protocols for combating sensitive strains of M. tuberculosis are mainly based on a combination of three or, more frequently, of four molecules: isoniazide (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB). These drugs constitute the "first-line" treatment.

In recent decades, strains of M. tuberculosis have become resistant to each of these molecules. Strains that are resistant at least to isoniazide and to rifampicin are referred to as "multi-resistant" (MDR-TB). Recently, novel strains have appeared which are resistant to a larger number of molecules: those that are resistant to isoniazide, to rifampicin, to fluoroquinolones and to at least one injectable second-line drug are defined as being "ultra-resistant" (XDR-TB).

According to an estimation made by the WHO in 2016, there were 0.58 million cases of MDR-TB in 2015. Other evaluations report a relative incidence of about 1 1 % of multi- resistant strains among all new cases of tuberculosis.

Another therapeutic drawback in the treatment of tuberculosis is the interaction of rifampicin with treatments for combating HIV (human immunodeficiency virus), which represents an obstacle in the treatment of patients co-infected with tuberculosis and HIV. The current anti-HIV therapeutic recommendations favour, as a first-line treatment, an anti-retroviral triple therapy combining a protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) with two nucleoside reverse transcriptase inhibitors (NRTI). PI and NNRTI are metabolized by CYP3A4. Metabolic interactions between anti-retrovirals (ATRV) and certain combined drugs have been demonstrated. Thus, rifampicin, which is a powerful inducer of intestinal and hepatic CYP3A4, reduces the concentrations of ATRV. There is an urgent need to develop improved therapies for combating tuberculosis. Ideally, the novel anti-tuberculosis treatments should be capable of satisfying one or more of the following criteria:

• shorten the treatment time to improve the compliance to the treatment protocols and reduce the appearance of resistant bacteria,

· be well tolerated, acting via novel mechanisms of action and thus effective against multi-resistant and/or ultra-resistant strains,

• be active against tuberculosis,

• have a shortened latent tuberculosis (asymptomatic first infection) treatment time, so as to address the problem of the biological reservoir of M. tuberculosis.

FR 2 126 108 and Arnoux et al. (Journal of the American Chemical Society 102(10), 1980, 3605) describe sequanamycin (A), having the following formula:

(3S,4S,5R,7S,9S, 10S, 1 1 R, 12S, 13R)-12-[(4,5-dihydroxy-4,6-dimethyltetrahydro-2H-pyran- 2-yl)oxy]-7-hydroxy-2-{1 -[(5-hydroxy-3,4-dimethoxy-6-methyltetrahydro-2H-pyran-2- yl)oxy]propan-2-yl}-10-[(3-hydroxy-6-methyl-4-oxotetrahydro- 2H-pyran-2-yl)oxy]- 3,5,7,9,1 1 ,13-hexamethyl-6, 14-dioxooxacyclotetradecan-4-yl) 3-methylbutanoate.

(A) This compound is described therein as an antimicrobial agent and especially enables the treatment of tuberculosis. However, this compound may show instability, in particular in acidic or basic aqueous medium, and/or may also show metabolic instability, which makes it difficult to use as a drug.

WO 2014/044645 describes sequanamycin compounds of formula:

It still remains to find new compounds with potent anti-Mycobacterum tuberculosis activity and fulfilling the oral bioavailability requirements.

A subject of the present invention is in particular new macrolides, which have bacteriostatic and/or bactericidal action, on mycobacteria, especially against strains of sensitive Mycobacterium tuberculosis that are resistant to the first-line antibiotics, and the preparation and therapeutic uses thereof.

[COMPOUNDS]

According to a first object, the present invention thus concerns a compound of formula (I) :

z

(I)

in which:

- Y1 and Y2, identical or different, independently represents a hydrogen atom or a group -Ci.p-alkyl provided that at least one of Y1 and Y2 is not H ;

Z represents:

• a group -Ci. ₆ -alkyl, which is unsubstituted or substituted with one or more groups R ₄ ,

• a group -C ₃ - _/ -cycloalkyl,

- R ₄ independently represents a group chosen from:

• a hydroxyl group,

• a group -C _{1 4} -alkoxy

* indicates that the Carbon atom can exhibit any stereochemistry;

or a pharmaceutically acceptable salt thereof.

The compounds of general formula (I) may comprise one or more asymmetric carbons. They may therefore exist in the form of enantiomers or diastereoisomers. These enantiomers, diastereoisomers, and also mixtures thereof, including racemic mixtures, form part of the invention. The compounds of formula (I) may exist in the form of bases or acid-addition salts. Such addition salts form part of the invention. Pharmaceutically acceptable salts of the compounds of formula (I) do form part of the invention.

According to one embodiment, distinguished compounds are those of formula (I) wherein Y1 and Y2 are different, and where one of Y1 and Y2 represents a -Ci- ₆ -alkyl, then the other of Y1 and Y2 represents a hydrogen atom. According to another embodiment, distinguished compounds are those of formula (I) wherein Y1 and Y2 are different, and where one of Y1 and Y2 represents a methyl, then the other of Y1 and Y2 represents a hydrogen atom.

According to another embodiment, distinguished compounds are those of formula (I) wherein Y1 and Y2 independently represent an identical or different -C _{1 6} -alkyl.

According to one embodiment, distinguished compounds are those of formula (I) wherein Y1 and Y2, identical, represent a methyl. According to another embodiment, distinguished compounds are those of formula (I) wherein Y1 represents a hydrogen atom and Y2 represents a -Ci ₆ -alkyl.

According to another embodiment, distinguished compounds are those of formula (I) wherein Y2 represents a hydrogen atom and Y1 represents a -Ci- ₆ -aikyl.

According to another embodiment, distinguished compounds are those of formula (I) wherein Y1 represents a hydrogen atom and Y2 represents a methyl.

According to another embodiment, distinguished compounds are those of formula (I) wherein Y2 represents a hydrogen atom and Y1 represents a methyl.

According to another embodiment, distinguished compounds are those of formula (I) wherein Z represents a cyclobutyl, a methyl, a methoxyethyl, 2-methyl-butyl, 2,2-dimethyl- propyl or 2-hydroxy-2-methyl-propyl. According to another embodiment, distinguished compounds are those chosen from the compounds of formu -A):

z

(l-A)

wherein Y1 , Y2, Z are defined as in formula (I) as defined above.

According to one embodiment, distinguished compounds are those of formula (l-A) wherein Y1 and Y2 are different, and where one of Y1 and Y2 represents a -C :. ₆ -alkyl, then the other of Y1 and Y2 represents a hydrogen atom.

According to another embodiment, distinguished compounds are those of formula (l-A) wherein Y1 and Y2 are different, and where one of Y1 and Y2 represents a methyl, then the other of Y1 and Y2 represents a hydrogen atom.

According to another embodiment, distinguished compounds are those of formula (l-A) wherein Y1 and Y2 independently represent an identical or different -Ci ₆ -alkyl.

According to one embodiment, distinguished compounds are those of formula (l-A) wherein Y1 and Y2, identical, represent a methyl.

According to another embodiment, distinguished compounds are those of formula (l-A) wherein Y1 represents a hydrogen atom and Y2 represents a -d ₆ -alkyl. According to another embodiment, distinguished compounds are those of formula (I- A) wherein Y2 represents a hydrogen atom and Y1 represents a -Ci. ₆ -alkyl.

According to another embodiment, distinguished compounds are those of formula (I- A) wherein Y1 represents a hydrogen atom and Y2 represents a methyl.

According to another embodiment, distinguished compounds are those of formula (I- A) wherein Y2 represents a hydrogen atom and Y1 represents a methyl.

According to another embodiment, distinguished compounds are those of formula (l-A) wherein Z represents a cyclobutyl, a methyl, a methoxyethyl, 2-methyl-butyl, 2,2-dimethyl- propyl or 2-hydroxy-2-methyl-propyl.

According to a particular embodiment, the compound of formula (I) or (l-A) corresponds to one of the following compounds:

[(2R,3S,4R,5R,7S,9S, 10S, 1 1 R, 12S, 1 3R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-1 0-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9, 1 1 , 1 3-hexamethyl-4- (3-methylbutanoyloxy)-6, 14-dioxo-1 2-[[(2S,5R,7R)-2,4,5-trimethyl-1 ,4-oxazepan-7-yl]oxy]- oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S, 10S, 1 1 R, 12S, 1 3R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-1 0-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9, 1 1 , 1 3-hexamethyl-4- (3-methylbutanoyloxy)-6, 14-dioxo-1 2-[[(2S,5R,7R)-2,4,5-trimethyl-1 ,4-oxazepan-7-yl]oxy]- oxacyclotetradec-7-yl] (2S,6R)-2,6-dimethylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S, 10S, 1 1 R, 12S, 1 3R)-12-[[(2S,5R,7R)-4-cyclobutyl-2,5-dimethyl-1 ,4- oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyl- tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-1 0-[(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6- methyl-tetrahydropyran-2-yl]oxy-3,5,7,9, 1 1 , 13-hexamethyl-4-(3-methylbutanoyloxy)-6, 14- dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S, 10S, 1 1 R, 12S, 1 3R)-12-[[(2S,5S,7R)-4-cyclobutyl-2,5-dimethyl-1 ,4- oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyl- tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6- methyl-tetrahydropyran-2-yl]oxy-3,5,7,9, 1 1 , 13-hexamethyl-4-(3-methylbutanoyloxy)-6, 14- dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-12- [[(2S,5R,7R)-4-(2-hydroxy- 2-methyl-propyl)-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-3,5,7,9,1 1 ,13-hexamethyl-4-(3- methylbutanoyloxy)-6, 14-dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4- carboxylate

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-12- [[(2S,7R)-4-isopentyl-2,5- dimethyl- 1 ,4-oxazepan-7-yl]oxy]-3,5,7,9, 1 1 , 13-hexamethyl-4-(3-methylbutanoyloxy)-6, 14- dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-12- [[(2S,7R)-4-(2- methoxyethyl)-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-3, 5,7,9,1 1 ,13-hexamethyl-4-(3- methylbutanoyloxy)-6, 14-dioxo-oxacyclotetradec-7-yl] (2S,6R)-2,6-dimethylmorpholine-4- carboxylate

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-12-[[(2S,7R)-4-cyclobutyl-2,5-dimethyl-1 ,4- oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyl- tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6- methyl-tetrahydropyran-2-yl]oxy-3,5,7,9, 1 1 , 13-hexamethyl-4-(3-methylbutanoyloxy)-6, 14- dioxo-oxacyclotetradec-7-yl] (2S,6R)-2,6-dimethylmorpholine-4-carboxylate

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9,1 1 ,13-hexamethyl-4- (3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5R,7R)-2,4,5-trime thyl-1 ,4-oxazepan-7-yl]oxy]- oxacyclotetradec-7-yl] (2R)-2-methylmorpholine-4-carboxylate [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9,1 1 ,13-hexamethyl-4- (3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5R,7R)-2,4,5-trime thyl-1 ,4-oxazepan-7-yl]oxy]- oxacyclotetradec-7-yl] (2S,6S)-2,6-dimethylmorpholine-4-carboxylate or a pharmaceutically acceptable salt thereof.

According to a more particular embodiment, the compound of the invention is:

[(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9,1 1 ,13-hexamethyl-4- (3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5R,7R)-2,4,5-trime thyl-1 ,4-oxazepan-7-yl]oxy]- oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4-carboxylate or a pharmaceutically acceptable salt thereof. As used herein, certain terms have the following definitions:

- an alkyl group: a saturated, linear or branched hydrocarbon-based aliphatic group comprising, unless otherwise mentioned, from 1 to 6 and preferably from 1 to 4 carbon atoms. Examples that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, etc. groups;

- a cycloalkyl group: a cyclic alkyl group comprising, unless otherwise mentioned, from 3 to 6 carbon atoms. Examples that may be mentioned include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. groups;

- an alkoxy group: a radical -O-alkyl in which the alkyl group is as defined previously, including -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-tert-butyl, - O-pentyl, -O-isopentyl, -O-hexyl.

[PROCESS OF PREPARATION] According to a further object, the present invention also concerns the process of preparation of the compound of formula (I). In the steps described below, the usual organic chemistry reactions may be followed, especially those described in "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" by Richard C. Larock, published by John Wiley & Sons Inc.

In the process described below, the protecting groups are preferably chosen from those described in "Protective Groups in Organic Chemistry, J.F.W. McOmie, Plenum Press, 1973 or in "Greene's Protective Groups in Organic Synthesis", by Theodora W. Greene, published by John Wiley & Sons Inc., 2006. The processes for the protection and deprotection of the protecting groups are typically those described in these books.

The described processes make it possible to prepare compounds according to the invention in which:

In the text herein below, the term "protecting group PG" means a group that can, firstly, protect a reactive function such as a hydroxyl or an amine during the synthesis and, secondly, regenerate the intact reactive function at the end of the synthesis. Examples of protecting groups and also protection and deprotection methods are given in particular in Protective Groups in Organic Synthesis, supra.

In the text hereinbelow, the term "leaving group LG" means a group that can be readily cleaved from a molecule by breaking a heterolytic bond, with loss of an electron pair. This group may thus be readily replaced with another group, for example during a substitution reaction. Such leaving groups are, for example, halogens or an activated hydroxyl group such as a methanesulfonate, benzenesulfonate, p-toluenesulfonate, triflate, acetate, etc. Examples of leaving groups and also references for their preparation are given in Advanced Organic Chemistry, M.B. Smith and J. March, 6th Edition, Wiley Interscience, 2007, pp. 496-501 .

The order of the steps depends on the nature of the substituents to be introduced and on the need or otherwise to introduce protecting groups for the hydroxyl functions present. Thus, steps of protection/deprotection of the various hydroxyl groups present on the sequanamycin compounds may be added before or after each of the steps mentioned above (or between two steps). Determining the order of the steps and the need or otherwise to protect the hydroxyl functions are routine operations for a person skilled in the art, who knows, in the light of his general knowledge, whether or not a hydroxyl group is liable to react during the steps below and whether or not it needs to be protected. According to the invention, the preparation of the compounds may be achieved by either of the two following routes: route A called the "oxime-mycarose route", and

route B called the "oxime oxazepan route".

Both routes may start from sequanamycin (A). · According to route A.

The compound of formula (I) may be obtained by an oxidative cleavage and reductive amination from a corresponding 7-carbamate, oxime compound of formula (VI):

(VI)

by reacting with a compound of formula (C):

Z-NH2

(C)

in which Y1 , Y2 and Z are defined as in formula (I).

According to a further object, the present invention thus concerns the process of preparation of a compound of formula (I) comprising reacting the corresponding compound of formula (VI)

(VI) with a compound of formula (C):

Z-NH ₂

(C)

in which Y1 , Y2 and Z are defined as in formula (I).

This reaction may be conducted by reacting an oxidative reagent such as sodium periodate (Nal0 ₄ ) followed by reacting a reducing reagent such as Sodium cyanoborohydride (NaBH ₃ CN), or NaBH(OAc) ₃ .

The reaction may be conducted in two successive steps, in a solvent such as tetrahydrofuran (THF) or methanol (MeOH).

Typically, in an illustrative embodiment to compound (VI) in THF is added a solution of Nal0 ₄ in H ₂ 0 and the reaction mixture is filtered. Then the amine (C) in acetic acid (AcOH) (or the hydrochloride salt of the amine) and NaBH ₃ CN are added to the filtrate. The compound of formula (VI) may be obtained by deprotection of the corresponding compound of formula (VII):

(VII)

where Y1 and Y2 are defined as in formula (I).

The reaction may be conducted in a basic medium, such as potassium carbonate (K ₂ C0 ₃ ) in MeOH/H ₂ 0. The compound (VII) may be obtained from the corresponding 7 imidazole carbamate compound of formula (VIII):

by reacting a corresponding morpholine compound of formula (B):

where Y1 and Y2 are defined as in formula (I).

The reaction may be conducted in the presence of N-hydrosuccinimide (NHS), in an organic solvent such as acetonitrile.

The compound of formula (VIII) may be obtained from the corresponding imidazole carbamate-compound of formula (IX):

(IX) by hydrolysis.

The reaction may be conducted in an acidic medium, e.g. by adding an aqueous solution of hydrochloric acid (HCI). The reaction may be conducted in an organic solvent such as THF, THF- cyclohexane, or acetonitrile. The compound (IX) may be obtained from the corresponding oxime compound of formula

(V):

(V)

by reacting Ν,Ν'-carbonyldiimidazole (GDI) The reaction may be conducted in an organic solvent such as toluene, or cyclohexane, or cyclohexane-THF or cyclohexane- methyl- THF.

The compound (V) may be obtained from sequanamycin (A) by an oximation reaction. Said oximation reaction may be conducted by reacting the compound of formula (D):

MeONH ₂ HCI

(D)

This reaction may be typically carried out in the presence of a base, generally an organic base, such as triethylamine (Et ₃ N) in an organic solvent such as methanol.

Sequanamycin (A) may be obtained by application of the process disclosed in the examples.

• According to route B:

The compound of formula (I) may be obtained from the corresponding 7 imidazole carbamate compound of formula (II):

(II) by reacting the corresponding morpholine compound of formula (B):

HN O

Y ₂

(B)

where Y1 , Y2 and Z are defined as in formula (I).

According to a further object, the present invention also concerns the process of preparation of a compound of formula (I) comprising reacting a compound of formula (II)

(ll) with a compound of formula (B):

HN O

(B)

In which Z, Y1 and Y2 are defined as in formula (I), or a salt thereof. Typically, in an illustrative embodiment, the reaction may be conducted by:

- adding a solution of compound (B) in an organic solvent such as acetonitrile, optionally in the presence of a base such as an organic base, typically triethylamine if a salt of compound (B) is used;

to

- a solution of compound (II) and N-hydroxysuccinimide in an organic solvent such as acetonitrile.

The compound of formula (II) may be obtained by hydrolysis of the corresponding imidazole carbamate compound of formula (III):

(III)

in which Z is defined as in formula (I).

Said hydrolysis may be conducted by adding an aqueous solution of an acid (such as HCI) to a solution of compound (II) in an organic solvent such as THF, THF-Cyclohexane. The compound of formula (III) may be obtained from the corresponding 7-membered oxazepan compound of formula (IV):

(IV)

In which Z is defined as in formula (I) by reacting with N.N'-carbonyldiimidazole (GDI). The reaction may be conducted in an organic solvent such as toluene, or cyclohexane, or cyclohexane-THF or cyclohexane- methyl-THF.

The compound of formula (IV) may be obtained from the corresponding oxime compound of formula (V):

(V)

by reacting a compound of formula (C):

Z-NH2

(C)

where Z is defined as in formula (I), in an oxidative cleavage and double reductive amination reaction.

This reaction may be conducted by reacting an oxidative reagent such as NalCv followed by reacting a reducing reagent such as NaBH ₃ CN, or NaBH(OAc) ₃ .

The reaction may be conducted in two successive steps, in a solvent such as THF or

MeOH. Typically, in an illustrative embodiment, to compound (V) in THF is added a solution of Nal0 ₄ in H ₂ 0 and the reaction mixture is filtered. Then the amine (C) in AcOH (or a hydrochloride salt of the amine (C)) and NaBH ₃ CN are added to the filtrate. The oxime compound (V) may be obtained from sequanamycin (A) by an oximation reaction, as disclosed above

The processes of preparation of the invention may further comprise the step of isolating and/or purifying the obtained compound of formula (I).

According to a further object, the present invention also concerns novel intermediates involved in the above synthetic processes.

Accordingly, the invention also concerns the following compounds (VI) and (VII):

(VI) (VII)

wherein Y1 , Y2 are defined as in formula (I).

EXAMPLES

MATERIALS AND METHODS The following Examples describe the preparation of certain compounds in accordance with the invention. These examples are not limiting and merely illustrate the present invention.

In the Preparations and in the Examples, the following abbreviations are used:

EtOAc: ethyl acetate

TLC: thin-layer chromatography

DCM: dichloromethane

DMF: N,N-dimethylformamide

TEA: triethylamine Nal0 ₄ : sodium metaperiodate,

K ₂ C0 ₃ : potassium carbonate

MeOH: methanol

MgS0 ₄ : magnesium sulfate

NaBH ₃ CN: sodium cyanoborohydride

NaCI: sodium chloride

NaHC0 ₃ : sodium bicarbonate

Na ₂ S0 _4: sodium sulfate

NH ₄ CI: ammonium chloride

NH ₄ Ac: ammonium acetate

THF: tetrahydrofuran

RT: room temperature

The progress of the synthetic reactions is monitored by TLC. The plates are made of glass and are coated with Merck 60 F ₂₅₄ silica gel. After elution, the plates are observed under ultraviolet light at 254 nm and then revealed by spraying with a 5M sulfuric acid/water solution followed by heating.

The microwave reactions were performed using a Biotage Initiator 8 EXP microwave machine.

The products were purified, when necessary, on a Biotage SP-1 chromatograph or a Spot

2 chromatograph from Merck. The columns used are Merck 15-40 μηι silica columns (2.5 g to 400 g).

ANALYSES

Mass Spectrometry (MS):

Method a:

The spectra were acquired on a Waters UPLC-SQD machine;

Ionization: electrospray in positive and/or negative mode (ES+/-);

Chromatographic conditions:

Column: Acquity BEH C18 - 1 .7 μηι - 2.1 x 50 mm,

Solvents: A: H ₂ 0 (0.1 % formic acid) B: CH ₃ CN (0.1 % formic acid),

Column temperature: 50°C,

Flow rate: 0.8 ml/min, • Gradient (2.5 min): from 5% to 100% B over 1 .8 min; 2.40 min: 100% B; 2.45 min: 100% B; from 100% to 5% B over 0.05 min.

Method b:

· The spectra were acquired on a Waters ZQ machine;

• Ionization: electrospray in positive and/or negative mode (ES+/-);

• Chromatographic conditions:

• Column: XBridge C18- 2.5 pm - 3 x 50 mm,

• Solvents: A: H ₂ 0 (0.1 % formic acid) B: CH ₃ CN (0.1 % formic acid),

· Column temperature: 70°C,

• Flow rate: 0.9 ml/min,

• Gradient (7 min): from 5% to 100% B over 5.3 min; 5.5 min: 100% B; 6.3 min:

5% B. Method c:

• The spectra were acquired on a Waters ZQ machine;

• Ionization: electrospray in positive and/or negative mode (ES+/-);

• Chromatographic conditions:

• Xselect C18 column 3.5 μηι - 3 x 50 mm,

· Solvents: A: H20 (0.1 % formic acid) B: CH3CN (0.1 % formic acid),

• Column temperature: 60°C,

• Flow rate: 1 ml/min,

• Gradient (7 min): from 10% to 100% B over 4.5 min; 4.85 min: 100% B; 6.5 min: 10% B.

Method d:

• The spectra were acquired on a Waters UPLC-SQD machine;

• Ionization: electrospray in positive and/or negative mode (ES+/-);

• Chromatographic conditions:

· Column: Acquity CSH C18 - 1 .7 μηι - 2.1 x 50 mm,

• Solvents: A: H20 (0.1 % formic acid) B: CH3CN (0.1 % formic acid),

• Column temperature: 50°C,

• Flow rate: 0.9 ml/min,

• Gradient (2.5 min): from 5% to 100% B over 1 .8 min; 2.4 min: 100% B; 2.45 min: 5% B. Method e:

• The spectra were acquired on an Shimadzu 2020-2010 machine

• Ionization: electrospray in positive and/or negative mode (ES+/-);

· Chromatographic conditions:

• Xtimate C18 column 2.1 X 30 mm, 3 μιτι,

• Solvents: A: H20 (4L) + TFA (1 .5 ml_) B: CH3CN (4L) + TFA (0.75 ml_),

• Column temperature: 50°C,

• Flow rate: 1 .2 ml/min,

· Wavelength :UV 220nm

• Gradient (2 min): from 10% to 80% B over 0.9 min: holding at 80% for 0.6 minutes

Method f :

• The spectra were acquired on an Shimadzu 2020-2010 machine

· Ionization: electrospray in positive and/or negative mode (ES+/-);

• Chromatographic conditions:

• Xtimate C18 column 2.1 X 30 mm, 3 μηι,

• Solvents: A: H20 (4L) + TFA (1 .5 ml_) B: CH3CN (4L) + TFA (0.75 ml_),

• Column temperature: 50°C,

· Flow rate: 0.8 ml/min,

• Wavelength :UV 220nm

• Gradient (7 min): from 10% to 80% B over 6 min; holding at 80% for 0.5 minutes

Method g:

· The spectra were acquired on an Agilent 1200-6120 machine

• Ionization: electrospray in positive and/or negative mode (ES+/-);

• Chromatographic conditions:

• Waters Xbridge C18 30 ^* 2.0mm,3.5um

• Solvents: A) 0.04%TFA in Water; B) 0.02%TFA in ACN

· Column temperature: 50°C,

• Flow rate: 1 .0 ml/min,

• Wavelength :UV 220nm

• Gradient (7 min): 0%B increase to 95% B within 5.8 min; hold at 95% B for 1 .1 min; then back to 0%B at 6.91 min and hold for 0.09 min. Method h:

• The spectra were acquired on an Agilent 1200-6140 machine

• Ionization: electrospray in positive and/or negative mode (ES+/-)

• Chromatographic conditions:

• Waters Xbridge C18 30 ^* 2.0mm, 3. Sum

• Solvents: A) 0.1 %FA in Water; B) 0.1 %FA in ACN

• Column temperature: 50°C,

• Flow rate: 1 .0 ml/min,

• Wavelength :UV 220nm & ELSD

• Gradient (7 min): 0%B increase to 95% B within 5.8 min; hold at 95% B for 1 .1 min; then back to 0%B at 6.91 min and hold for 0.09 min

1 H Nuclear magnetic resonance (NMR)

The 1 H NMR spectra were recorded on a Bruker Avance and/or Varian G spectrometer (300 MHz, 400 MHz, 500 MHz or 600 MHz) in deuterated DMSO. The chemical shifts are expressed in units δ (ppm) using tetramethylsilane (TMS) as internal reference. For the interpretation of the spectra, the following abbreviations were used: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, dd = doubled doublet, ddd = doublet of doubled doublets, m = multiplet, ax. = axial, equat. = equatorial.

PREPARATION

All the following compounds were synthesized according to the protocols described below.

Preparation of sequanamycin:

The strain described in FR 2 126 108 deposited at the Northern Regional Research Laboratory (NRRL) under the number NRRL 3892 may be used to prepare sequanamycin.

The fermentation and purification process described in FR 2 126 108 may be followed.

The fermentation and purification processes may also be conducted using Allokutzneria albata to isolate sequanamycin (A). The strain known as Allokutzneria albata deposited on 20 December 2010 at the Deutsche Sammlung Von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Inhoffenstr. 7b 38124 Braunschweig, Germany) by the group Sanofi- Aventis (Sanofi Aventis Deutschland GmbH, Industriepark Hochst H831 , 65926 Frankfurt am Main) under the identification reference ST108942 and the order number DSM24416 may also be used to prepare sequanamycin.

This may be performed by application or adaptation of the protocol below, which is given as a non-limiting illustration.

Thus, the fermentation process described below was performed for 500 liters, but may be adapted to smaller or larger proportions.

The free culture medium (known as "5294 medium") used is typically as follows:

The pH of the medium before sterilization is 7.2.

The main culture medium (known as "5254-Seq01 medium") used is typically as follows:

The fermentation process is typically as follows:

1 vial of the Working Cell Bank (WCB)

I

Step 1 : Preculture 1

500 μΙ_ of WCB were placed in a 300 mL conical flask comprising twice 100 mL of 5294 medium. The mixture was stirred for 96 hours at 28°C.

I

Step 2: Preculture 2

25 mL of the culture medium from step 1 were placed in 4 times 500 mL of 5294 medium in a 2-liter conical flask, and the mixture was then stirred for 72 hours at 28°C I

Step 3: Preculture 3

1 .5 L of the culture medium from step 2 were placed in 30 liters of 5294 medium in a 42 L bioreactor, and the mixture was then stirred and aerated for 24 hours at 28°C without monitoring the pH

I

Step 4 (main culture):

30 kg of culture medium from step 3 were placed in 500 L of 5254-Seq1 medium in an 800 L bioreactor, and the mixture was then stirred and aerated for 96±5 hours at 28°C without monitoring the pH

I

Harvesting

The fermentation process described above was performed for 500 liters, but may be adapted for smaller or larger proportions. It was, for example, used on a scale of 7000 liters as follows, using the same culture media:

Preculture 1 = 250 ml_, inoculum: one vial of WCB

Preculture 2 = 5 L in the flasks (2 x 2.5 L), inoculum of 0.5% starting with Preculture 1 Preculture 3 = 400 L of medium in a 600 L bioreactor, seeding proportion of 1 .25% starting with Preculture 2

Main culture = 7000 L of medium in a 10 000 L bioreactor, seeding proportion of 5.7% starting with Preculture 3.

Fermentation process is followed by the purification process below (performed on the 500 liters fermentation broth described above).

After having terminated the fermentation, the fermentation broth was separated as a culture supernatant and mycelium using a cylindrical seeding sorter. The separation gave about 440L of culture supernatant.

In separate batches, 100-120L of culture supernatant comprising sequanamycin (A) were loaded onto a column filled with adsorption resin (glass column filled with styrene- divinylbenzene copolymer, inside diameter of 200, a length of about 180 mm, flow rate of 250 ml/minute). The resin was then washed with 30 percent 2-propanol.

Sequanamycin (A) were isolated by eluting the column with the following elution gradient: 30-70% B over 45 minutes, 70% B over 10 minutes, 100% B over 25 minutes; with A = H ₂ 0, B = 2-propanol, modifier: 1 vol% NH ₄ Ac 50g/L adjusted to pH 7). The fractions comprising sequanamycin (A) were combined and the 2-propanol was evaporated off. The pH of the solution obtained was adjusted to above 7.5 and the solution was then extracted twice with ethyl acetate. The organic phases were combined and the solvents were evaporated off. The oil obtained (about 10 grams per 100 L of culture supernatant) was purified on silica gel (40 mm χ 260 mm column), eluting with a column with gradient from n-heptane to 30/70 n-heptane/ethyl acetate over 45 minutes, and then 30/70 n-heptane/ethyl acetate maintained for about 40 minutes (at a flow rate of 100 ml/minute). Monitoring of the purification may be performed by thin-layer chromatography, eluting with ethyl acetate and revealing the sequanamycins (in the form of blue spots) with a reagent such as vanillin.

Depending on the concentration of sequanamycin (A) in the individual 100-liter batches, about 2.5 to 3.5 g of sequanamycin (A) in a purity of 68-75% (determined by NMR) were obtained per batch.

If a higher purity is required, the sequanamycin (A) may be repurified by reverse-phase chromatography on a WatersAtlantis machine with a 50x 100 mm, 5 μ column. An elution gradient of H ₂ 0 (A) and acetonitrile (B) and 1 vol% NH ₄ Ac 50 g/L adjusted to pH 7 was used (40-60% B over 30 minutes, flow rate of 140 ml/minute). The chromatography was monitored by the light scattering electrical signal. The fractions comprising sequanamycin (A) were combined and freeze-dried after evaporating off the acetonitrile. The yield of sequanamycin (A) after this final purification step was 57% with an 85% pure compound according to the NMR analyses.

Sequanamycin (A) serves as starting materials for synthesizing the compounds of formula (I) according to the invention.

Preparation of the intermediates for the examples described below:

Preparation 1 : Intermediate B (Compound of formula (V))

(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-12-{[(2R,4R,5S,6S)-4,5-dihydroxy-4,6- dimethyltetrahydro-2H-pyran-2-yl]oxy}-7-hydroxy-2-(1 -{[(2R,3R,4R,5R,6R)-5-hydroxy-3,4- dimethoxy-6-methyltetrahydro-2H-pyran-2-yl]oxy}propan-2-yl)- 10-{[(2S,3R,4E,6R)-3- hydroxy-4-(methoxyimino)-6-methyltetrahydro-2H-pyran-2-yl]ox y}-3,5,7,9,1 1 ,13- hexamethyl-6, 14-dioxooxacyclotetradecan-4-yl 3-methylbutanoate.

12 g of sequanamycin (A) are placed in 175 ml of MeOH with stirring, and 5.3 ml of TEA, and 3 g of methylhydroxylamine hydrochloride are then added, in this order. The stirring is continued at RT for 20 hours and the MeOH is then evaporated off under vacuum. The crude reaction product is taken up in 150 ml of DCM and washed with 100 ml of water and then with 100 ml of saturated aqueous NaCI solution. The aqueous phases are extracted with 150 ml of DCM. The organic phases are combined, dried over MgS0 ₄ , filtered and concentrated under vacuum. 12.7 g of the product obtained are suspended in 70 ml of a petroleum ether (40-60°C)/isopropanol mixture (2/1 ). The mixture is heated to 70°C, the insoluble matter is filtered off while hot and the product is then left to precipitate out at RT over 20 hours. It is filtered off by suction and rinsed with 20 ml of a petroleum ether (40- 60°C)/isopropanol mixture (2/1 ). The precipitate is dried under vacuum at 35°C to give 10.62 g of expected product. MS method b:

Retention time Tr (min) = 4.87; [M+Na]+: m/z 1014; [M-H+HC0 ₂ H]-: m/z 1036.

1 H NMR spectrum (500 MHz, in ppm, DMSO-d ₆ ): 0.81 (d, J=6.8 Hz, 3 H); 0.93 to 1 .01

(m, 15 H); 1 .07 (d, J=7.0 Hz, 3 H); 1 .09 to 1 .13 (m, 9 H); 1 .17 (d, J=6.0 Hz, 3 H); 1 .18 (d,

J=6.0 Hz, 3 H); 1 .24 (s, 3H); 1 .44 (dd, J=10.8 and 14.4 Hz, 1 H); 1 .68 to 1 .76 (m, 2 H); 1 .81 (d, J=14.4 Hz, 1 H): 1 .88 (dd, J=1 1 .5 and 15.9 Hz, 1 H); 1 .96 to 2.06 (m, 3 H); 2.07 to

2.20 (m, 4 H); 2.73 (quint, J=7.0 Hz, 1 H); 2.81 (t, J=9.0 Hz, 1 H); 2.89 to 2.97 (m, 2 H);

3.03 (ddd. J=2.5 and 7.3 and 9.5 Hz. 1 H); 3.18 (q, J=6.8 Hz, 1 H); 3.34 to 3.36 (m, 2 H);

3.37 (s, 3 H); 3.45 (s, 3 H); 3.52 (dq, J=6.2 and 9.4 Hz, 1 H); 3.60 (s, 1 H); 3.62 to 3.65

(m, 1 H); 3.66 (t, J=2.5 Hz, 1 H); 3.71 to 3.77 (m, 1 H); 3.78 (m, 1 H); 3.80 (s. 3 H); 3.81 to 3.84 (m, 1 H); 3.87 (m, 1 H); 4.39 to 4.46 (m, 3 H); 4.50 (s, 1 H); 4.72 (d, J = 8.3 Hz, 1 H);

4.78 (d, J=8.3 Hz, 1 H); 4.84 (d, J=7.3 Hz, 1 H); 4.87 (d, J=3.8 Hz, 1 H); 5.19 (d, J=4.4 Hz,

1 H). Preparation 2: Intermediate C (Compound of formula (IV))

Compound 1 -a: (2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-7-hydroxy-2-(1 -

{[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyltetrah ydro-2H-pyran-2- yl]oxy}propan-2-yl)-10-{[(2S,3R,4E, 6R)-3-hydroxy-4-(methoxyimino)-6-methyltetrahydro- 2H-pyran-2-yl]oxy}-3,5,7,9,1 1 ,13-hexamethyl-6,14-dioxo-12-{[(2S,5R,7R)-2,4,5-trimethyl- 1 ,4-oxazepan-7-yl]oxy}oxacyclotetradecan-4-yl 3-methylbutanoate. Intermediate C

Compound 1 -b: (2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-7-hydroxy-2-(1 -

{[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyltetrah ydro-2H-pyran-2- yl]oxy}propan-2-yl)-10-{[(2S,3R,4E, 6R)-3-hydroxy-4-(methoxyimino)-6-methyltetrahydro- 2H-pyran-2-yl]oxy}-3,5,7,9,1 1 ,13-hexamethyl-6,14-dioxo-12-{[(2S,5S,7R)-2,4,5-trimethyl- 1 ,4-oxazepan-7-yl]oxy}oxacyclotetradecan-4-yl 3-methylbutanoate.

3 g of the Intermediate B of Preparation 1 are placed in 68 ml of MeOH. The reaction medium is cooled in an ice bath to a temperature of +4°C, followed by dropwise addition of a solution of 3.23 g of sodium periodate in 68 ml of water. The mixture is stirred for

6 hours at room temperature.

The medium is saturated with NaCI and filtered, and the filtrate is extracted with DCM (3x200 ml). The organic phases are combined, washed with saturated aqueous NaCI solution, dried over MgS0 ₄ , filtered and finally concentrated under reduced pressure. The oily residue obtained is dissolved, under argon, in 680 ml of MeOH. The pH is adjusted to

7 by addition of acetic acid, followed by addition of 2 M methylamine dissolved in 12.1 ml of THF. The pH is maintained at 7 with acetic acid. After stirring for 30 minutes at room temperature, 0.95 g of NaBH ₃ CN is added in a single portion, and the mixture is stirred for a further 16 hours at room temperature. The reaction medium is filtered and rinsed with MeOH. The filtrate is concentrated under reduced pressure and then taken up in 600 ml of DCM. The resulting mixture is washed with saturated aqueous NaCI solution (3 χ 60 ml). The organic phase is dried over MgS0 ₄ , filtered and then evaporated to dryness under vacuum. 3.5 g of product are purified by chromatography on a Merck cartridge (150 g of 15-40 μηι silica) with a 100/0 to 90/10 DCM/MeOH elution gradient. 530 mg of diastereoisomer 1-a, 380 mg of diastereoisomer 1-b and 661 mg of a mixture of the two isomers are obtained.

Compound 1 -a: Intermediate C

MS method a:

Retention time Tr (min) = 1 .26; [M+H]+: m/z 989; [M-H+HC0 ₂ H]-: m/z 1033 (base peak). 1 H N R spectrum (500 MHz, in ppm, DMSO-d ₆ ): 0.79 (d. J=6.8 Hz, 3 H); 0.89 to 1 .01 (m, 15 H);1 .03 (d, J=6.8 Hz, 3 H); 1 .05 to 1 .10 (m, 9 H); 1 .1 1 (d, J=6.1 Hz, 3 H); 1 .13 (d, J=6.1 Hz, 3 H); 1 .24 (s, 3H); 1 .48 (dd, J=1 1 .4 and 14.7 Hz, 1 H); 1 .70 to 2.08 (m, 8 H); 2.10 to 2.22 (m, 3 H); 2.18 (broad s, 3 H); 2.36 (m, 1 H); 2.57 (m, 1 H); 2.70 (d, J=13.6 Hz, 1 H); 2.75 (m, 1 H); 2.83 (dd, J=2.9 and 16.6 Hz, 1 H);2.92 (dd, J=2.7 and 8.0 Hz, 1 H); 3.03 (m, 1 H); 3.12 (q, J=6.8 Hz, 1 H); 3.30 (partially masked m, 1 H); 3.38 (s, 3 H); 3.45 (s. 3 H); 3.52 (m, 1 H); 3.58 to 3.72 (m, 4 H); 3.80 (s, 3 H); 3.89 (m, 2 H); 4.26 (m, 1 H); 4.31 (s, 1 H); 4.45 (d. J=8.0 Hz, 1 H); 4.65 (broad d, J=9.8 Hz, 1 H); 4.70 (d, J=4.6 Hz, 1 H); 4.74 (d, J=9.6 Hz, 1 H); 4.86 (d, J=7.1 Hz, 1 H); 4.93 (dd, J=3.1 and 9.5 Hz, 1 H); 5.33 (d, J=4.6 Hz. 1 H).

Compound 1 -b:

MS method a:

Retention time Tr (min) = 1 .26; [M-H+HC0 ₂ H] ^~ : m/z 1033 (base peak).

1 H NMR spectrum (500 MHz, in ppm, DMSO-d ₆ ): 0.82 (d, J=6.8 Hz, 3 H); 0.91 to 1 .32 (m, 36 H); 1 .44 to 1 .51 (m. 1 H); 1 .80 to 1 .87 (m, 1 H); 1 .96 to 2.37 (m, 10 H); 2.76 to 2.80 (m, 2 H); 2.77 (s, 3 H); 2.85 (dd, J=3.1 and 16.8 Hz, 1 H); 2.93 (dd, J=2.7 and 8.0 Hz, 1 H); 3.00 to 3.07 (m, 2 H); 3.12 to 3.18 (m, 1 H); 3.28 (d, J=13.7 Hz, 1 H); 3.31 to 3.36 (m, 1 H); 3.40 (s, 3 H); 3.48 (s, 3 H); 3.51 to 3.58 (m, 2 H); 3.64 to 3.70 (m, 2H); 3.82 (s, 3 H); 3.86 to 3.90 (m, 1 H); 3.94 to 4.02 (m, 2 H); 4.31 to 4.37 (m, 1 H); 4.47 (d, J=7.9 Hz, 1 H);4.66 to 4.77 (m, 3 H); 5.12 (dd, J=5.8 and 8.9 Hz, 1 H). Preparation 3: Intermediate D (Compound of formula (III))

1 .35 g of Intermediate C and 1 .1 1 g of Ν,Ν'-carbonyldiimidazole are placed in 8 ml of cyclohexane. The mixture is heated at 100°C for 35 minutes by microwave. The heterogeneous medium is taken up in 60 ml of DCM and washed with 40 ml of water and then with 40 ml of saturated NaCI solution. The aqueous phases are re-extracted with 60 ml of DCM. The organic phases are combined, dried over MgS0 ₄ , filtered and then evaporated to dryness under vacuum. 1 .7 g of the expected compound is obtained.

MS method c:

Retention time Tr (min) = 3.67; [M+H] ⁺ : 1271 Preparation 4: Intermediate E (Compound of formula (II))

1 .7 g of the intermediate D prepared in preparation 3 are placed in 17 ml of THF. 6.84 ml of 1 M HCI are added. The mixture is stirred for 3 hours at room temperature. 50 ml of DCM are added and the resulting mixture is washed with saturated NaHC0 ₃ solution (20 ml) and then with saturated NaCI solution (20 ml). The aqueous phases are re-extracted with 50 ml of DCM. The organic phases are combined, dried over MgS0 ₄ , filtered and then evaporated to dryness under vacuum. 1 .48 g of the expected product are recovered.

MS method c:

Retention time Tr (min) = 3.41 ; [M+H] ⁺ : 1083 Preparation 5: Intermediate F (Compound of formula (IX))

1 ml of toluene, 0.2 g of the intermediate B obtained in Preparation 1 and 196 mg of Ν,Ν'- carbonyldiimidazole are placed under argon. The reaction medium is heated for 3 hours at 80°C and then concentrated under vacuum. DCM is added and the resulting mixture is then washed with saturated aqueous NaCI solution. The organic phase is dried over MgS0 ₄ , filtered and then evaporated to dryness under vacuum. The residue is purified by chromatography (10 g of 15-40 μηι silica) with a 98/2 to 95/5 DCM/MeOH elution gradient. 1 12 mg of the expected compound is obtained.

MS method a :

Retention time Tr (min) = 1 .63; [M+H] ⁺ : 1300

Preparation 6: Intermediat

3 ml of THF, 240 mg of the intermediate F prepared in preparation 5 and 369 μΙ of 1 M HCI are stirred together under argon. The pale yellow homogeneous medium is stirred overnight at room temperature. A further 369 μΙ of 1 M HCI are added and stirring is continued for 24 hours. The reaction medium is neutralized with saturated aqueous sodium bicarbonate solution. The resulting mixture is extracted with EtOAc. The organic phase is dried over MgS0 ₄ , filtered and then evaporated to dryness under vacuum. 181 mg of the residue obtained are purified by chromatography (10 g of 15-40 μηι silica) with a 50/50 to 70/30 EtOAc/heptane elution gradient. 87 mg of the expected compound is obtained. MS method b: Retention time Tr (min) = 1 .56; [M-H+HC0 ₂ H] ^~ : m/z 1 156 (base peak).

Example 1 A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10

[(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahyd ropyran-2-yl]oxy- 3,5,7,9,1 1 ,13-hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5 R,7R)-2,4,5- trimethyl-1 ,4-oxazepan-7-yl]oxy]-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4- carboxylate

To a solution of intermediate E (0,4g, 369 μηιοΙ) and N-hydroxysuccinimide (0,13 g, 618,29 μηιοΙ) in CH ₃ CN (8 ml) was added dropwise a solution of (2S)-2-methylmorpholine hydrochloride (0,107 g, 738 μπιοΙ) in CH ₃ CN (4 ml) and TEA (155 μΙ, 1 .1 1 mmol) at 70°C. The resulting mixture was stirred at 70°C for 20h. After cooling to room temperature, the reaction was diluted with EtOAc (10ml), washed with H ₂ 0 (10 ml), brine (10 ml), dried over MgS0 ₄ , filtered and the filtrate was concentrated under reduced pressure to afford 0.48 g of crude product as pale oil. The residue was purified by silica gel column (20 g SiOH 15- 40 μηι, EtOAc-NEt ₃ 98/2) to afford 0.36 g of the expected product as a white solid.

NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.8 Hz, 3 H); 0.91 (m, 6 H); 0.96 (d, J=6.6 Hz, 6 H); 0.99 to 1 .14 (m, 21 H); 1 .65 to 2.06 (m, 12 H); 2.08 to 2.23 (m, 6 H); 2.39 (m, 2 H); 2.58 (m, 2 H); 2.65 to 2.95 (m, 7 H); 3.03 (m, 2 H); 3.28 to 3.40 (m, 4 H); 3.46 (s, 3 H); 3.52 (m, 1 H); 3.59 to 3.83 (m, 8 H); 3.90 (s large, 1 H); 4.19 (m, 1 H); 4.45 (d, J=7.9 Hz, 1 H); 4.63 (m, 3 H); 4.86 (d, J=7.1 Hz, 2 H); 5.17 to 5.39 (m, 1 H) MS method d:

Retention time

Tr (min): 1 .02 ; [M+H]+ 1 1 16 ; [M-H+HCOOH]-: m/z 1 160 (base peak)

Example 2A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10- [(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahydrop yran-2-yl]oxy- 3,5,7,9,1 1 ,13-hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5 R,7R)-2,4,5- trimethyl-1 ,4-oxazepan-7-yl]oxy]-oxacyclotetradec-7-yl] (2S,6R)-2,6-dimethylmorpholine- 4-carboxylate

Step 2A.1

To a mixture of Intermediate G (23.00 g, 20.68 mmol, 1 .00 eq) and 1 -hydroxypyrrolidine- 2,5-dione (5.43 g, 47.15 mmol, 2.28 eq) in CH ₃ CN (150 mL) was added dropwise a solution of (2S,6R)-2,6-dimethylmorpholine (3.67 g, 31 .85 mmol, 1 .54 eq) in CH ₃ CN (50 mL) at 70 °C, then the resulting mixture was stirred at 80 °C for 12 hour. LCMS showed the starting material was consumed and one new peak with desired product was detected. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to remove most of CH ₃ CN. The residue was diluted with EtOAc (500 mL), then washed with H ₂ 0 (200 mL x 3), brine (200 mLx3), dried over anhydrous Na ₂ S0 ₄ , filtered and the filtrate was concentrated under reduced pressure to give desired product (22.00 g, crude) as yellow solid.

MS method e:

Retention time Tr (min): 1 .67 ; m/z 1 181 .5 [M+Na] ⁺

Step 2A.2

A mixture of compound step 2A.1 (22.00 g, 18.98 mmol) and K ₂ C0 ₃ (7.87 g, 56.94 mmol) in MeOH (220.00 mL) and H ₂ 0 (22.00 mL) was stirred at 25 °C for 2 hour. The reaction mixture was diluted with EtOAc (200 mL), then washed with brine (50 mL x 3), dried over anhydrous Na ₂ S0 ₄ , filtered. The filtrate was concentrated under reduced pressure to give 21 .00 g of the desired crude compound as white solid.

MS method e:

Retention time Tr (min): 1 .32 ; m/z 1 155.5 [M+Na] ⁺

Step 2A.3 (Example 2A)

To a mixture of compound step 2A.2 (10.00 g, 8.82 mmol) in THF (100 mL) was added a solution of Nal0 ₄ (9.44 g, 44.10 mmol, 2.45 mL) in H ₂ 0 (60.00 mL) at 0 °C over 10 min. The resulting mixture was stirred at 25 °C for 2 hr. Then the reaction mixture was filtered and the cake was washed with THF (50 mL). Then to the above combined filtrate was added methylamine (2 M, 22.05 mL) and AcOH (6.09 g, 101 .43 mmol, 5.80 mL). The resulting mixture was stirred at 25 °C for 1 hr, then to the above reaction mixture was added NaBH ₃ CN (1 .39 g, 22.05 mmol) at 0 °C and stirred at 25 °C for 2 hr. The combined mixture was diluted with EtOAc (200 mL), washed with NaHC0 ₃ solution (100 mL), brine (100 ml_ x 3), dried over anhydrous Na ₂ S0 ₄ , filtered. The filtrate was concentrated under reduced pressure to afford a crude product as a yellow solid. The residue was purified by column chromatography (SiOH, ethyl acetate/MeOH = 1 /0 to 50/1 ) To give 4g of the desired compound Example 2A as a white solid.

NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.8 Hz, 3 H); 0.91 (m, 6 H); 0.96 (d, J=6.6 Hz, 6 H); 0.99 to 1 .14 (m, 27 H); 1 .69 to 2.07 (m, 12 H); 2.15 (m, 6 H); 2.32 to 2.45 (m, 3 H); 2.57 (m, 1 H); 2.70 (m, 2 H); 2.86 (m, 1 H); 2.92 (dd, J=2.8 et 8.0 Hz, 1 H); 3.02 (m, 2 H); 3.28 (m masked, 1 H); 3.38 (s, 3 H); 3.44 (m, 5 H); 3.52 (m, 1 H); 3.63 (dd, J=4.4 et 9.8 Hz, 1 H); 3.67 (t, J=2.5 Hz, 1 H); 3.68 to 3.94 (m, 9 H); 4.21 (m, 1 H); 4.45 (d, J=7.9 Hz, 1 H); 4.61 (m, 3 H); 4.86 (d, J=7.3 Hz, 1 H); 4.88 (dd, J=3.3 et 9,3 Hz. 1 H); 5,30 (m, 1 H).

MS method d: Retention time Tr (min): 1 .05 ; [M+H]+ 1 130 ; [M-H+HCOOH]-: m/z 1 1 16 (base peak).

Example 3A & 3A' : [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-12-[[(2S,5R,7R)-4- cyclobutyl-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy- 3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3- hydroxy-4-methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3,5 ,7,9,1 1 ,13-hexamethyl-4- (3-methylbutanoyloxy)-6, 14-dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4- carboxylate & [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-12-[[(2S,5S,7R)-4-cyclobutyl-2,5- dimethyl-1 ,4-oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6- methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3-hydroxy-4- methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3, 5,7,9,1 1 ,13-hexamethyl-4-(3- methylbutanoyloxy)-6, 14-dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4- carboxylate

Example 3A Example 3 A' Step 3A.1

To a mixture of Intermediate G (5.00 g, 4.50 mmol, 1 .00 eq) and 1 -hydroxypyrrolidine- 2,5-dione (1 .18 g, 10.26 mmol, 2.28 eq) in CH ₃ CN (40 mL) was added dropwise a solution of (2S)-2-methylmorpholine (910.35 mg, 9.00 mmol, 2.00 eq) in CH ₃ CN (10 mL) at 70 °C, then the resulting mixture was stirred at 80 °C for 12 hour. LCMS showed the starting material was consumed and one new peak with desired product was detected. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to remove most of CH ₃ CN. The residue was diluted with EtOAc (100 mL), then washed with H ₂ 0 (50 mL x 3), brine (50 mLx3), dried over anhydrous Na ₂ S0 ₄ , filtered and the filtrate was concentrated under reduced pressure to give desired compound (5.00 g, crude) as a yellow solid. MS method e:

Retention time Tr (min): 1 .29; m/z 1 167.9 [M+Na] ⁺

Step 3A.2

A mixture of compound step 3A.1 (7.50 g, 6.55 mmol, 1 .00 eq) and K ₂ C0 ₃ (2.72 g, 19.65 mmol, 3.00 eq) in MeOH (75.00 mL) and H ₂ 0 (7.50 mL) was stirred at 25 °C for 2 hour. LCMS showed the starting material was consumed and one new peak with desired product was detected. The mixture was concentrated under reduced pressure to remove most of MeOH, and the reaction mixture was diluted with EtOAc (200 ml_), then washed with brine (100 ml_ x 3), dried over anhydrous Na ₂ S0 ₄ , filtered. The filtrate was concentrated under reduced pressure to give desired compound (7.00 g, crude) as yellow solid.

MS method e:

Retention time Tr (min): 1 .23 ;m/z 1 141 .8 [M+Na] ⁺ Step 3A.3 (Example 3A & 3A')

To a mixture of compound of compound step 3A.2 (250 mg, 223,34 μηιοΙ) in THF (2.5 ml) and water (1 .25 ml) was added Nal0 ₄ (241 ,27 mg, 1 ,12 mmol). The resulting mixture was stirred at room temperature for 3h. The reaction was diluted with THF (7.5 ml) and the Cyclobutylamine hydrochloride (37,16 mg, 335,02 μηιοΙ) was added. The resulting mixture was stirred for 45 min then to the above reaction was added NaBH ₃ CN (44,32 mg, 670,03 μηιοΙ) and stirred at room temperature for 16h. The reaction was diluted with EtOAc (20 ml), washed with NaHC0 ₃ solution (15 ml), dried with anhydrous MgS0 ₄ , filtered. The filtrate was concentrated under reduced pressure to afford 0.268 g of crude product. The residue was purified by silica gel column (20 g SiOH 15-40 μηι, EtOAC-TEA 98.5/1 .5) to afford 0,180 g of mixture 3A and 3A'. The mixture was purified by silica gel column (10 g of 15-40 μηι silica) eluted with the following elution solvent: DCM-MeOH- NH ₄ OH 96/4/0.2) to afford 65 mg of the desired compound example 3A and 17 mg of 3A'.

Example 3A

NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.8 Hz, 3 H); 0.93 (m, 15 H); 1 .04 (m, 12 H); 1 .1 1 (d, J=6.1 Hz, 6 H); 1 .15 (d, J=6.1 Hz, 3 H); 1 .52 (m, 2 H); 1 .66 to 2.07 (m, 16 H); 2.14 (m, 3 H); 2.23 (dd, J=7.5 et 14.6 Hz, 1 H); 2.42 (m, 0.5 H); 2.58 (m, 0.5 H); 2.71 (m, 1 H); 2.76 to 2.95 (m, 5 H); 3.01 (m, 2 H); 3.26 to 3.42 (m, 7 H); 3.46 (s, 3 H); 3.52 (m, 1 H); 3.59 to 3.99 (m, 13 H); 4.45 (d, J=8.0 Hz, 1 H); 4.58 (m, 2 H); 4.66 (d, J=9.5 Hz, 1 H); 4.88 (m, 2 H); 5.15 (m, 1 H).

MS method d:

Retention time Tr (min): 1 .1 ; [M+H]+ 1 156 ; [M-H+HCOOH]-: m/z 1200 (base peak). Example 3A'

NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.9 Hz, 3 H); 0.93 (m, 15 H); 1 .00 to 1 .16 (m, 21 H); 1 .47 to 2.07 (m, 18 H); 2.14 (m, 3 H); 2.28 (dd, J=9.2 et 13.5 Hz, 1 H); 2.42 (m, 1 H); 2.71 (m, 1 H); 2.85 (m, 4 H); 3.05 (m, 3 H); 3.25 to 3.42 (m, 7 H); 3.45 (s, 3 H); 3.52 (m, 1 H); 3.57 to 3.99 (m, 12 H); 4.15 (m, 1 H); 4.44 (d, J=8.0 Hz, 1 H); 4.57 (m, 2 H); 4.65 (m, 1 H); 4.86 (d, J=7.1 Hz, 1 H); 4.94 (t, J=6.9 Hz, 1 H); 5.41 (m, 1 H)

MS method d:

Retention time Tr (min): 1 .1 ; [M+H]+ 1 156 ; [M-H+HCOOH]-: m/z 1200 (base peak).

Example 4A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10- [(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahydrop yran-2-yl]oxy-12- [[(2S,5R,7R)-4-(2-hydroxy-2-methyl-propyl)-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-

3,5,7,9,1 1 ,13-hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-oxacyclote tradec-7-yl] (2S)- 2-methylmorpholine-4-carboxylate

Procedure: see example 3A & 3A'

Purification: The mixture (277 mg) was purified by silica gel column (20 g SiOH 15-40 μηι, DCM-MeOH-NH ₄ OH 96/4/0.2) to afford 70 mg of the desired compound Example 4A. NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.8 Hz, 3 H); 0.94 (m, 15 H); 1 .05 (m, 21 H); 1 .1 1 (d, J=6.1 Hz, 3 H); 1 .16 (d, J=5.8 Hz, 3 H); 1 .72 (m, 6 H); 1 .81 to 2.06 (m, 6 H); 2.13 (m, 3 H); 2.25 (m, 1 H); 2.44 (m, 3 H); 2.75 (m, 2 H); 2.90 (m, 3 H); 3.03 (m, 2 H); 3.19 (m, 1 H); 3.25 to 3.45 (m, 6 H); 3.46 (s, 3 H); 3.52 (m, 1 H); 3.59 to 3.85 (m, 1 1 H); 3.91 (m, 2 H); 4.33 (s large, 1 H); 4.45 (d, J=8.0 Hz, 1 H); 4.57 (m, 2 H); 4.65 (d, J=9.5 Hz, 1 H); 4.86 (m, 2 H); 5.14 (m, 1 H).

MS method d:

Retention time Tr (min): 1 .06 ; [M+H]+ 1 174; [M-H+HCOOH]-: m/z 1218 (base peak).

Example 5A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-

[(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahyd ropyran-2-yl]oxy-12-

[[(2S,7R)-4-isopentyl-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-3,5,7,9,1 1 ,13-hexamethyl-4-(3- methylbutanoyloxy)-6, 14-dioxo-oxacyclotetradec-7-yl] (2S)-2-methylmorpholine-4- carbox late

Procedure: see example 3A & 3A'

Purification: The mixture (277 mg) was purified by silica gel column (20 g SiOH 15-40 μηι, DCM-MeOH-NH ₄ OH 96/4/0.2) to afford 98 mg of the desired compound example 5A (mixture of dias 80/20 R/S).

NMR 1 H (500MHz, δ in ppm , DMSO-d6): 0.78 (d, J=6.7 Hz, 3 H); 0.85 (t, J=6.5 Hz, 6 H); 0.92 (m, 6 H); 0.96 (d, J=6.6 Hz, 6 H); 1 .00 to 1 .16 (m, 24 H); 1 .24 (m, 2 H); 1 .53 (tt, J=6.5 et 13.2 Hz, 1 H); 1 .70 to 2.05 (m, 12 H); 2.13 (m, 3 H); 2.34 to 2.58 (m maskeds, 5 H); 2.72 (m, 1 H); 2.78 to 2.95 (m, 4 H); 3.03 (dt, J=2.9 et 6.5 Hz, 2 H); 3.31 to 3.42 (m, 6 H); 3.46 (s, 3 H); 3.53 (m, 1 H); 3.58 to 3.97 (m, 12 H); 4.16 (m, 1 H); 4.45 (d, J=8.0 Hz, 1 H); 4.54 to 4.71 (m, 3 H); 4.85 (m, 2 H); 5.14 (m, 1 H).

MS method d:

Retention time Tr (min): 1 .15 ; [M+H]+ 1 172 ; [M-H+HCOOH]-: m/z 1216 (base peak). Example 6A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10- [(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahydrop yran-2-yl]oxy-12- [[(2S,7R)-4-(2-methoxyethyl)-2,5-dimethyl-1 ,4-oxazepan-7-yl]oxy]-3,5,7,9,1 1 ,13- hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-oxacyclotetrad ec-7-yl] (2S,6R)-2,6- dimethylmorpholine-4-carboxylate

To a solution of compound 2A.2. (300.00 mg, 264.70 μπιοΙ) in THF (8.00 mL) was added a solution of Nal0 ₄ (283.08 mg, 1 .32 mmol) in water 3 mL at 0 °C. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered, and the cake was washed with THF 2 mL. Then to the above combined filtrate was added AcOH (182.79 mg, 3.04 mmol) and 2-methoxyethanamine (99.41 mg, 1 .36 mmol). The resulting mixture was stirred at 25 °C for 1 hour. NaBH ₃ CN (41 .58 mg, 661 .75 μηιοΙ was added, the resulting mixture was stirred at 25 °C for another 1 hour. The reaction mixture was quenched by addition saturated aqueous NaHC0 ₃ 10 mL at 15°C, and then diluted with water 10 mL and extracted with EtOAc (10 mL X 2). The combined organic layers were washed with brine 10 mL, dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOH, Petroleum ether/Ethyl acetate=10:1 to 1 :4). 106.00 mg of the desired compound was obtained as a white solid.

MS method f :

Retention time Tr (min): 3.83 ; m/z: 1 174.7 [M+H] ⁺

1 H NMR (400MHz, DMSO-d6) 5.23 (br d, J=12.1 Hz, 1 H), 4.91 - 4.81 (m, 2H), 4.68 - 4. (m, 3H), 4.44 (d, J=7.9 Hz, 1 H), 4.21 (br s, 1 H), 3.89 (br t, J=5.0 Hz, 1 H), 3.79 (s, 51 3.74 - 3.66 (m, 3H), 3.63 (br d, J=5.7 Hz, 1 H), 3.52 (br dd, J=6.4, 9.3 Hz, 1 H), 3.45 (s, 4H), 3.37 (s, 3H), 3.31 - 3.27 (m, 2H), 3.21 (s, 3H), 3.06 - 2.99 (m, 2H), 2.95 - 2.83 (m, 4H), 2.74 - 2.56 (m, 4H), 2.45 - 2.32 (m, 2H), 2.19 - 2.08 (m, 3H), 2.06 - 1 .94 (m, 3H), 1 .90 - 1 .69 (m, 9H), 1 .17 - 1 .1 1 (m, 6H), 1 .10 - 0.97 (m, 24H), 0.96 (d, J=6.6 Hz, 6H), 0.91 (br d, J=4.0 Hz, 6H), 0.77 (br d, J=6.8 Hz, 3H)

Example 7A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-12-[[(2S,7R)-4-cyclobutyl-2,5- dimethyl-1 ,4-oxazepan-7-yl]oxy]-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6- methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10-[(2S,3R,4E,6R)-3-hydroxy-4- methoxyimino-6-methyl-tetrahydropyran-2-yl]oxy-3, 5,7,9,1 1 ,13-hexamethyl-4-(3- methylbutanoyloxy)-6,14-dioxo-oxacyclotetradec-7-yl] (2S,6R)-2,6-dimethylmorpholine-4- carboxylate

To a solution of compound 2A.2: (300.00 mg, 264.70 μηιοΙ in THF (8.00 mL) was added a solution of Nal0 ₄ (283.08 mg, 1 .32 mmol) in water 3 mL at 0 °C. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was filtered, and the cake was washed with THF 2 mL. Then, to the above combined filtrate was added AcOH (182.79 mg, 3.04 mmol) and cyclobutanamine (94.13 mg, 1 .32 mmol). The resulting mixture was stirred at 25 °C for 1 hour. NaBH ₃ CN (41 .58 mg, 661 .75 μηιοΙ) was added. The resulting mixture was stirred at 25 °C for another 1 hour. The reaction mixture was quenched by addition sat. aq. NaHC0 ₃ 10 mL at 15°C, diluted with water 10 mL and extracted with EtOAc (10 mL X 2). The combined organic layers were washed with brine 10 mL, dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOH, Petroleum ether/Ethyl acetate=10:1 to 1 :2). 101 .00 mg of the desired compound was obtained as a white solid.

MS method g: Retention time Tr (min): 4.07; m/z: 1 170.8 [M+H] ⁺ 1 H NMR (400MHz, DMSO-d6) 5.50 - 5.10 (m, 1 H), 4.88 (br d, J=7.1 Hz, 2H), 4.69 - 4.61 (m, 1 H), 4.57 (br d, J=8.6 Hz, 2H), 4.44 (d, J=7.9 Hz, 1 H), 4.01 - 3.87 (m, 1 H), 3.86 - 3.73 (m, 6H), 3.70 - 3.57 (m, 4H), 3.52 (br dd, J=6.4, 9.5 Hz, 1 H), 3.45 (s, 5H), 3.37 (s, 3H), 3.29 - 3.24 (m, 1 H), 3.06 - 3.00 (m, 2H), 2.96 - 2.78 (m, 4H), 2.68 (br d, J=10.1 Hz, 2H), 2.41 - 2.25 (m, 2H), 2.21 - 2.08 (m, 4H), 2.04 - 1 .85 (m, 8H), 1 .73 (s, 6H), 1 .56 - 1 .44 (m, 2H), 1 .18 - 1 .10 (m, 9H), 1 .04 (br d, J=3.7 Hz, 18H), 0.97 - 0.88 (m, 15H), 0.77 (br d, J=6.6 Hz, 3H)

Example 8A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10- [(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahydrop yran-2-yl]oxy- 3,5,7,9,1 1 ,13-hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5 R,7R)-2,4,5- trimethyl-1 ,4-oxazepan-7-yl]oxy]-oxacyclotetradec-7-yl] (2R)-2-methylmorpholine-4- carboxylate

Step 8A.1

To a mixture of Intermediate G (3.00 g, 2.70 mmol and 1 -hydroxypyrrolidine-2,5-dione (708.49 mg, 6.16 mmol) in CH ₃ CN (20 mL) was added dropwise a solution of (2R)-2- methylmorpholine hydrochloride (572.18 mg, 4.16 mmol) in CH ₃ CN (10 mL) and TEA (683.03 mg, 6.75 mmol, 935.66 μΙ_, 2.50 eq) at 70 °C, then the resulting mixture was stirred at 80 °C for 12 hour. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to remove most of CH ₃ CN. The residue was diluted with EtOAc (150 mL), then washed with H ₂ 0 (100 mL), brine (100 mLx3), dried over anhydrous Na ₂ S0 ₄ , filtered and the filtrate was concentrated under reduced pressure to give 3.00 g of the desired crude product as a yellow solid.

MS method e:

Retention time Tr (min): 1 .45 ;m/z 1 167.8 [M+Na] ⁺

Step 8A.2

A mixture of Step 8A.1 (3.00 g, 2.62 mmol) and K ₂ C0 ₃ (1 .09 g, 7.86 mmol) in MeOH (30.00 mL) and H ₂ 0 (3.00 mL) was stirred at 25 °C for 2 hour. The reaction mixture was diluted with EtOAc (100 mL), then washed with brine (50 mL x 3), dried over anhydrous Na ₂ S0 ₄ , filtered. The filtrate was concentrated under reduced pressure to give 2.9 g of the desired crude product as yellow solid.

MS method e:

Retention time Tr (min): 1 .39 m/z 1 141 .6 [M+Na] ⁺

Step 8A.3 (Example 8A)

To a mixture of Step 8A.2 (2.90 g, 2.59 mmol) in THF (35 mL) was added a solution of Nal0 ₄ (2.77 g, 12.95 mmol) in H ₂ 0 (19.00 mL) at 0 °C over 10 min. The resulting mixture was stirred at 25 °C for 1 hr. Then the reaction mixture was filtered and the cake was washed with THF (20 mL). Then to the above combined filtrate was added methanamine (2 M, 6.48 mL) and AcOH (1 .79 g, 29.79 mmol). The resulting mixture was stirred at 25 °C for 1 hr. Then to the above reaction mixture was added NaBH ₃ CN (407.02 mg, 6.48 mmol) at 0 °C and stirred at 25 °C for 1 hr. The combined mixture was diluted with EtOAc (100 mL), washed with NaHC0 ₃ solution (50 mL), brine (50 mL x 3), dried over anhydrous Na ₂ S0 ₄ , filtered. The filtrate was concentrated under reduced pressure to afford a crude product as a yellow residue. The residue was purified by column chromatography (SiOH, ethyl acetate/MeOH = 1 /0 to 50/1 ). 900 mg of the desired compound was obtained as a white solid.

MS method h:

Retention time Tr (min): 3.14 ;m/z 1 1 16.6 [M+H] ⁺ 1 H NMR (400MHz, DMSO-d6) 5.07 (br d, J=5.5 Hz, 1 H), 4.85 (d, J=7.3 Hz, 1 H), 4.79 (br s, 1 H), 4.59 (br dd, J=9.8, 14.4 Hz, 2H), 4.42 (d, J=7.9 Hz, 1 H), 4.34 (br d, J=7.9 Hz, 1 H), 4.07 (br s, 1 H), 3.83 (br s, 4H), 3.73 - 3.56 (m, 5H), 3.54 - 3.45 (m, 2H), 3.43 (s, 3H), 3.35 (s, 3H), 3.26 (br d, J=4.6 Hz, 2H), 3.05 - 2.95 (m, 3H), 2.90 (br d, J=8.4 Hz, 1 H), 2.82 - 2.62 (m, 4H), 2.45 - 2.38 (m, 1 H), 2.32 (br d, J=15.7 Hz, 1 H), 2.21 (br s, 3H), 2.15 - 2.09 (m, 3H), 2.00 - 1 .88 (m, 3H), 1 .80 - 1 .71 (m, 5H), 1 .17 (br d, J=5.7 Hz, 3H), 1 .1 1 - 0.98 (m, 27H), 0.93 (dd, J=2.6, 6.6 Hz, 9H), 0.89 (br d, J=6.8 Hz, 3H), 0.75 (br d, J=6.4 Hz, 3H).

Example 9A: [(2R,3S,4R,5R,7S,9S,10S,1 1 R,12S,13R)-2-[(1 S)-2-[(2R,3R,4R,5R,6R)-5- hydroxy-3,4-dimethoxy-6-methyl-tetrahydropyran-2-yl]oxy-1 -methyl-ethyl]-10- [(2S,3R,4E,6R)-3-hydroxy-4-methoxyimino-6-methyl-tetrahydrop yran-2-yl]oxy- 3,5,7,9,1 1 ,13-hexamethyl-4-(3-methylbutanoyloxy)-6,14-dioxo-12-[[(2S,5 R,7R)-2,4,5- trimethyl-1 ,4-oxazepan-7-yl]oxy]-oxacyclotetradec-7-yl] (2S,6S)-2,6-dimethylmorpholine-4- carbox late

To a solution of Intermediate G (3.00 g, 2.70 mmol) and Et ₃ N (73.00 mg, 721 .42 μπιοΙ) in CH ₃ CN (20.00 mL) was added 1 -hydroxypyrrolidine-2,5-dione (708.49 mg, 6.16 mmol). Then a solution of (2S,6S)-2,6-dimethylmorpholine (478.88 mg, 4.16 mmol) in CH ₃ CN (5.00 mL) was added dropwise into the above solution at 70°C under N ₂ , then the mixture was stirred at 80 °C for 12 hour. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to remove most of CH ₃ CN. The residue was diluted with EtOAc (50 mL), then washed with H ₂ 0 (20 mL x 3), brine (20 mL x 3), dried over anhydrous Na ₂ S0 ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was used into next step without purification. 2.90 g of the desired compound was obtained as a yellow solid. MS method e:

Retention time Tr (min): 1 .46 ; m/z 1 182.6[M+Na] ⁺

Step 9A.2

To a solution of Step 9A.1 (2.90 g, 2.50 mmol) in MeOH (30.00 mL) and H ₂ 0 (3.00 mL) was added K ₂ C0 ₃ (1 .04 g, 7.50 mmol). Then the mixture was stirred at 20 °C for 1 .5 hour. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to remove most of MeOH. The residue was diluted with EtOAc (50 mL), then washed with H ₂ 0 (20 mL x 3), brine (20 mL x 3), dried over anhydrous Na ₂ S0 ₄ , filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was used into next step without purification. 2.90 g of the desired crude compound was obtained as a yellow solid.

MS method e:

Retention time Tr (min): 1 .37 m/z 1 155.6[M+Na] ⁺ Step 9.3 (Example 9A)

To a solution of Step 9.A2 (2.90 g, 2.56 mmol) in THF (30.00 mL) was added dropwise a solution of Nal0 ₄ (2.74 g, 12.79 mmol) in H ₂ 0 (18.00 mL) at 0 °C, then the mixture was stirred at 35 °C for 2 h. Then the mixture was filtered, the filter cake was washed with THF (20.00 mL). The resulting filtrate was added AcOH (1 .77 g, 29.43 mmol) and then MeNH ₂ (2 M in MeOH, 6.40 mL) was added dropwise at 25 °C. The resulting mixture was stirred at 25 °C for 1 h. Then NaBH ₃ CN (401 .98 mg, 6.40 mmol) was added into the above solution, the resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was quenched by addition saturated aqueous NaHC0 ₃ 10 mL at 15 °C, and then diluted with water 10 mL and extracted with EtOAc 50 mL (25 mL X 2). The combined organic layers were washed with brine 10 mL, dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiOH, MeOH/Ethyl acetate 0:1 to 1 :100). 1 .15 g of the desired product was obtained as white solid.

MS method h:

Retention time Tr (min): 3.39 m/z 1 130.6 [M+H] ⁺

1 H NMR (400MHz, DMSO-d6) 5.34 (br s, 1 H), 4.98 - 4.83 (m, 2H), 4.64 - 4.51 (m, 3H), 4.42 (d, J=7.9 Hz, 1 H), 4.22 (br s, 1 H), 3.92 - 3.80 (m, 4H), 3.77 (d, J=2.2 Hz, 4H), 3.69 - 3.58 (m, 3H), 3.54 - 3.45 (m, 2H), 3.43 (s, 3H), 3.35 (s, 3H), 3.25 (br s, 1 H), 3.09 - 2.92 (m, 4H), 2.90 (br d, J=7.7 Hz, 1 H), 2.82 (br d, J=17.6 Hz, 2H), 2.71 - 2.63 (m, 2H), 2.30 (br s, 1 H), 2.16 - 2.04 (m, 4H), 2.03 - 1 .91 (m, 4H), 1 .90 - 1 .66 (m, 8H), 1 .23 - 0.95 (m, 30H), 0.95 - 0.87 (m, 12H), 0.75 (br d, J=6.6 Hz, 3H)

[USES]

The compounds corresponding to the general formula (I) that are the subject of the invention underwent microbiological trials which showed their value as therapeutically active substances. Specifically, they have bacteriostatic and/or bactericidal action on mycobacteria, especially against strains of Mycobacterium, in particular strains of Mycobacterium which are sensitive and resistant to the first-line antibiotics.

More precisely, the compounds corresponding to the general formula (I) which are the subject of the invention are used for the prevention and/or treatment of bacterial infections caused by mycobacteria. Measurement of permeability (Caco-2):

The in vitro transport assays for the evaluation of the permeability characteristics is described hereafter. Permeability of a compound across the epithelium cells along the gastrointestinal tract is an important limiting factor for the oral absorption of the compound. An in vitro model utilizing Caco-2/TC7 cells is employed to assess the permeability characteristics of new chemical entities (NCEs).

• Permeability under non Standard Conditions. When discrepancies are observed for a chemical series between in vitro permeability and in vivo bioavailability, the non-standard permeability study is performed under conditions for which BSA or pH gradients, NCE concentration, inclusion of additive (e.g. transporters inhibitors, EGTA...) can differ in order to highlight factors that alter the compound transport or to mimic more predictive experimental conditions.

Here permeability studies are performed in the apical to basolateral (A→B) direction, without pH gradient (pH 7.4 in apical and basal compartment), with a BSA gradient (0.5% in apical and 5% in basal compartment). The NCE concentration is 20 μΜ. The addition of a large spectrum inhibitor of efflux pump (2 μΜ Elacridar) allows determining if the NCE is susceptible to be a substrate of efflux pump.

EXPERIMENTAL PROTOCOL: PERMEABILITY ASSAY CONDITIONS

1 Buffer and media

Asymmetrical conditions used for the screen with Apical medium (0.5% BSA at pH 7.4) / Basal medium (5% BSA at pH 7.4).

Test NCE concentration 20 μΜ.

2 Compound preparation and Incubation

conditions

• DMSO final concentration

1 % (v/v)

• Assay time is 2h, at 37°C, under optimized agitation,

without C02.

CALCULATIONS

[Z>o] x S * f

R2: Receiver quantity of compound after 2 hours (corresponding peak area or

calculated concentration)

[Do]: Donor concentration of test solution (corresponding peak area or calculated concentration)

S: insert area: (0.08 cm ² for BD HTS 96-well insert system)

t: time (2 hours ie 7200 seconds) Measurement of the inhibitory activity of the compounds according to the invention towards Mycobacterium tuberculosis The in vitro test used makes it possible to identify molecules having antimicrobial activity on the strain of Mycobacterium tuberculosis H ₃₇ R _V . This is a bacterium of biohazard category 3.

Materials and methods

The test used is Alamar blue (MABA). This is a colorimetric test which makes it possible to determine the MIC (minimum inhibitory concentration) of antibacterial agents. Alamar blue is a redox indicator which changes from blue to pink in the case of bacterial growth. Resazurin (blue and non-fluorescent) is reduced to resorufin (pink and fluorescent) by live bacteria. The plate is thus read visually or by fluorescence measurement. The fluorescence intensity is proportional to the number of live bacteria.

Thus, the more the fluorimetric MIC value tends towards zero, the less the amount of product necessary to inhibit the total growth of the bacteria. The experiments performed demonstrate that the compounds according to the present invention have activity on inhibiting the growth of M. tuberculosis. The MIC values are typically between 0.1 and 10 μΜ, or even between 0.1 and 1 μΜ. The compounds presented as examples in the present patent application generally have MIC values of less than 1 μΜ.

Table 1 : Table of results

Examples Mtb_MABA_MICjiM Caco2 nm/s

Example 1A 0.527 20.5

^"°'" W . \

Example 4A 0.7 32.6

Example 5A 0.863 24.1

Example 6A 0.688 69.8

As matter of comparison, example 78 of WO 2014/044645 was tested in the above tests. The obtained results are summarized in Table 2 below:

The results showed that example 78 with an unsubstitued morpholine was less potent in terms of inhibitory activity with a less favorable Caco2 parameter.

The compounds according to the invention, i.e. the compounds corresponding to formula (I), furthermore show good microbiological properties and are particularly suitable for use for the preparation of medicaments, in particular antibiotics with a narrow spectrum for the treatment and/or prevention of tuberculosis.

In particular, these antibiotics have antimicrobial action against M. tuberculosis for the treatment and/or prevention of tuberculosis.

Thus, according to another of its aspects, a subject of the invention is medicaments that comprise a compound of formula (I), or an addition salt with a pharmaceutically acceptable acid or base of the compound of formula (I).

These medicaments find their use in therapy, especially in the treatment and/or prevention of tuberculosis.

According to another of its aspects, the present invention relates to pharmaceutical compositions comprising, as active principle, a composition according to the invention. These pharmaceutical compositions contain an effective dose of at least one compound according to the invention, or a pharmaceutically acceptable salt of said compound, and also at least one pharmaceutically acceptable excipient.

Said excipients are chosen, according to the pharmaceutical form and the desired mode of administration, from the usual excipients known to those skilled in the art.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal or rectal administration, the active principle of formula (I) above or the salt thereof may be administered in unit administration form, as a mixture with standard pharmaceutical excipients, to man and animals for the prophylaxis or treatment of the above disorders or diseases.

The appropriate unit forms of administration include oral-route forms such as tablets, soft or hard gel capsules, powders, granules and oral solutions or suspensions, sublingual, buccal, intratracheal, intraocular, intranasal and inhalation administration forms, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants. For topical application, the compounds according to the invention may be used in creams, gels, ointments or lotions.

By way of example, a unit administration form of a compound according to the invention in tablet form may comprise the following components:

Compound according to the invention 50.0 mg

Mannitol 223.75 mg

Croscaramellose sodium 6.0 mg

Corn starch 15.0 mg

Hydroxypropylmethylcellulose 2.25 mg

Magnesium stearate 3.0 mg

There may be particular cases in which higher or lower dosages are appropriate: such dosages are not outside the scope of the invention. According to the usual practice, the dosage that is appropriate to each patient is determined by the doctor according to the mode of administration and the weight and response of said patient.

According to another of its aspects, the present invention also relates to the compounds of formula (I), or a pharmaceutically acceptable salt thereof, for use for the treatment and/or prevention of bacterial infections caused by mycobacteria. Thus, one of the aspects of the invention concerns the compounds of formula (I), or a pharmaceutically acceptable salt thereof, for use for the treatment and/or prevention of infectious diseases such as to tuberculosis, pulmonary mycobacterial infection, cutaneous mycobacterial infection, atypical mycobacterial infection and mycobacteriosis.

The term "tuberculosis" includes infections caused by bacilli of the tuberculosis complex (M. tuberculosis, M. bovis and M. africanum), which are all pathogens to man. Pulmonary tuberculosis is far and away the most common and the most widespread: this is tuberculosis of the lungs, of the larynx, of the trachea and of the bronchi, tuberculosis of the intrathoracic lymphatic ganglions, respiratory tuberculosis of the pleura, primary respiratory tuberculosis and any other respiratory tuberculosis. Although less common, ganglionic tuberculosis and extrapulmonary tuberculosis, tuberculosis of the nervous system such as tuberculous meningitis, tuberculous leptomeningitis, cerebral tuberculoma and any other tuberculosis of the nervous system, or alternatively bone or articular tuberculosis, tuberculosis of the genitourinary system, lymphadenopathic peripheral tuberculosis, intestinal or peritoneal tuberculosis and/or tuberculosis of the mesenteric glands, tuberculosis of the skin and of the subcutaneous tissues, tuberculosis of the eyes, of the ears or of the adrenal glands, and also disseminated tuberculosis, exist.

According to another of its aspects, the present invention also relates to a method for treating the pathologies indicated above, which comprises the administration, to a patient in need thereof, of an effective dose of a compound of formula (I).

The compounds of the invention are suitable for combined administration with one or more other active agent useful for treating and/or preventing bacterial infections caused by mycobacteria, especially caused by bacilli of the tuberculosis complex (M. tuberculosis, M. bovis and/or M. africanum).