PL2970244T3

Novel 1, 2, 4-oxadiazol compounds active against gram-positive pathogens

Abstract

This record has no abstract on file.

PL2970244T3, drawing sheet 1 — Sheet 1 of 11

Term

7.5 yearsto projected expiry

Projected expiry 17 March 2034, counted from filing; an application has no term until it is granted.

1 claim: 1 independent, 0 dependent

1
Patent claims Zastrzeżenia patentowe 1. Compounds of general formula (I):1. Związki o wzorze ogólnym (I):

437 paragraphs in 25 sections, as filed

Description

BACKGROUND ART [0001] The use and misuse of antibacterial agents has resulted in the development of bacterial resistance to all antibiotics in clinical use, regardless of the chemical class or molecular use of the drug. Infections caused by multi-resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and Streptococcus pneumoniae resistant to penicillin (PNSSP) have become a serious public health problem both in hospitals and among people around the world. The need for new antibiotics has led to the American Society of Infectious Diseases (IDSA). Infectious Disease Society of America) took on the challenge of developing ten new antibiotics by 2020.

[0002] Oxazolidinones are a class of antibacterial agents that exhibit activity against a variety of Gram-positive pathogens and are highly effective against bacteria resistant to many drugs. In particular, oxazolidinones are used to treat skin and respiratory infections caused by Staphylococcus aureus and streptococcal strains, and they are also active against Vancomycin-resistant Enterococcus faecium. Linezolid (Fig. 1), the first oxazolidinone antibiotic approved for clinical use, inhibits translation at the initial stage of protein synthesis in bacteria by binding to the 50S ribosomal subunit. However, since 2001, resistance to linezolid has started to appear in clinical isolates of Staphylococcus aureus and Enterococcus faecium, and the resistance index has increased with use, particularly among enterococcal and Staphylococcus epidermidis strains. [1-4] Furthermore, linezolid treatment is not without side effects such as reversible myelosuppression and monoamine oxidase inhibition (MAO).

[0003] There are many solutions to the problem of bacterial resistance. Effective strategies include combining existing antibacterial agents with other drugs, as well as developing improved diagnostic procedures that can lead to the rapid identification of the causative pathogen and allow the use of narrow spectrum antibacterial agents. Another strategy is to discover new classes of antibacterial agents that work through new mechanisms of action. However, the most common and still the most promising approach is to modify existing classes of antibacterial agents to provide new analogs with improved performance, although the activity and toxicity of new analogs are not easily predictable.

[0004] In this context, many researchers have tried to modify the structure of linezolid to improve antimicrobial activity without obtaining results that would lead to consent for the use of new molecules. To optimize the modification site, the linezolid structure can be formally divided into four parts according to the nomenclature used for the antibacterial oxazolidinone compounds [5]: i) Ring A, which consists of a central heterocyclic oxazolidinone ring; ii) ring B, which consists of an N-aryl moiety linked to an oxazolidinone nitrogen; iii) the C ring, which consists of a carbon-linked not necessarily aromatic heterocyclic functional group; iv) a side chain, consisting of any linked functional group

VP / 6877 / AG

With the C (5) atom of oxazolidinone or being in the isosteric position with respect to ring A of the general formula (Figure 1).

Side chain connected to C (5)

Ring Ring PierCOMPONENTSeñ

CB a

linezolid

Figure 1 [0005] Various types of modifications have been described in the literature; the most common is for the C ring, but only a few modifications have been noted on the A ring and in some cases good activity has been maintained. [6-7] [0006] The inventors have previously noted that the replacement of oxazolidinone (ring A) with an isosteric 1,2,4-oxadiazole heteroaromatic ring resulted in inactivity [8]. Therefore, these compounds were chosen as references for inactive compounds similar to linezolid in virtual screening.

[0007] It is an object of the present invention to find new molecules suitable as drugs that overcome the limitations and disadvantages of prior art molecules with regard to antibacterial activity, in particular against resistant strains, and are harmless.

SUMMARY OF THE INVENTION [0008] The present invention is based on the finding that substitution of a C ring in linezolid derivative molecules by a five membered heterocyclic ring, also substituted, containing 2 or 3 heteroatoms, is effective in obtaining new oxazolidinone antibiotics with modifiable activity through the presence of further modifications in the B ring and in the C (5) side chain of the oxazolidinone core.

[0009] Therefore, the present invention relates to new compounds of general formula (I) for use in the treatment of infections caused by Gram-positive bacteria

Formula (I) in the form of a racemic mixture or pure enantiomers or mixtures enriched in one of the S or R enantiomers, where:

R = F, Cl, Br, I, (C1-C3) alkyl (methyl, ethyl, n-propyl, iso-propyl), (C3-C6) cycloalkyl, phenyl, aryl, heteroaryl, NH2, OH, SH, NHR6, N (R6)2, OR6 from R6 = (C1-C3) alkyl, (C3-C6) cycloalkyl, aryl, heteroaryl, (C1-C4) acyl; R1-4 = independently H, F, Cl, Br, CH3, OH, OCH3;

PZI6877IAG 3 EP 2 970 244 Β1

R5 = -NH2; -AND; -N3; OH; -NCS, -NHC (X) CH3 with X = O or S; -NHC (X) CH2Z with X = O, S, Z = F, Cl; -NHC (X) CHZ2 with X = O, S, Z = F, Cl; -NHC (X) CZ3 with X = O, S, Z = F, Cl; NHC (X) NHR7 with X = O, S, R7 = H, (C1-C3) alkyl, (C3-C6) cycloalkyl, aryl, heteroaryl, (C1-C3) acyl.

A specific embodiment of the invention includes compounds of the general formula (I) wherein R is methyl, ethyl, n-propyl, isopropyl;

or compounds of the general formula (I) in which at least one of R1, R2, R3 or R4 is fluoro while the others are H;

or compounds of the general formula (I) in which R5 is selected from: - NHC (= O) CH3, -NHC (= S) CH3, -NHC (= O) CH2F, -NHC (= S) CH2F, -NHC (= O) CH2Cl, -NHC (= S) CH2Cl, -NHC (= S) NH2, NHC (= O) NH2, -NHC (= O) NHCH3, -NHC (= S) NHCH3, -NHC (= O) NHC2H5, -NHC (= S) NHC2H5, -NCS; 1,2,3-triazol-1-yl;

or compounds of the general formula (I) in which R is methyl and R5 is selected from: -NHC (= O) CH3, -NHC (= S) CH3, -NHC (= O) CH2F, -NHC (= S) CH2F, -NHC (= O) CH2Cl, -NHC (= S) CH2Cl, -NHC (= S) NH2, NHC (= O) NH2, -NHC (= O) NHCH3, - NHC (= S) NHCH3, -NHC (= O) NHC2H.5, -NHC (= S) NHC2H5, -NCS; 1,2,3triazol-1-yl;

or compounds of the general formula (I) in which R1 is F, R2, R3 and R4 are H and R is methyl and R5 is selected from: -NHC (= O) CH3, -NHC (= S) CH3, -NHC (= O) CH2F, -NHC (= S) CH2F, -NHC (= O) CH2Cl, -NHC (= S) CH2Cl, -NHC (= S) NH2, NHC (= O) NH2, -NHC (= O) NHCH3, -NHC (= S) NHCH3, -NHC (= O) NHC2H5, -NHC (= S) NHC2H5, -NCS; 1,2,3 triazol-1-yl.

[0010] In a preferred embodiment of the invention, all the compounds indicated above are in the form of the pure S enantiomer or in a mixture enriched with the S enantiomer

[0011] In a further embodiment of the invention, compounds are intended for use in the treatment of infections caused by Gram-positive bacteria, preferably resistant to many antibiotics (also called multi-resistant), for example in the treatment of infections caused by Staphylococcus spp., Enterococcus spp. , Streptococcus spp., In particular infection caused by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae. Especially if they are resistant to one or more antibiotics among methicillin, vancomycin, penicillin, macrolides, fluoroquinolones and linezolid.

[0012] The second object of the invention is pharmaceutical compositions containing the compounds of the invention as active ingredients and a pharmaceutically acceptable excipient for use in the treatment of infections with Gram-positive bacteria, including multi-resistant strains.

[0013] A third object of the invention are methods for preparing the compounds of the invention which include the steps shown in schemes 1, 2 and 3.

[0014] In one embodiment of the invention, the methods comprise one or more steps of separating the S and R enantiomers or enriching the racemic mixture with one of the enantiomers, preferably the S enantiomer.

[0015] A fourth object of the invention are methods of making pharmaceutical compositions comprising the step of mixing the active ingredients with a pharmacologically acceptable excipient.

[0016] Another object of the invention is the use of the compounds according to the invention in the manufacture of a medicament for the treatment of infections with multi-resistant Gram-positive strains.

[0017] Advantageous features presented by the present invention consist in the preparation of new antibiotic compounds with activity equivalent to or comparable to that of linezolid against

VP / 6877 / AG

Bacterial strains sensitive to linezolid but more effective than linezolid against bacterial strains resistant to linezolid and / or other antibiotics. In addition, some of these substances have levels of cytotoxicity comparable or lower than linezolid. Finally, replacing the morpholine ring of linezolid with an oxadiazole ring as described herein prevents ring opening and formation of inactive metabolites such as PNU-142586 and PNU-142300.

Description of the figures [0018]

Figure 1. Pattern of linezolid with structural elements and naming constituting it.

Figure 2. Results of cell viability tests on PK15 cells treated with Compound A4b (Compound 23 of Table 1) and linezolid. Significance limits: * = P <0.05, ** = P <0.01.

Figure 3. Results of cell viability tests on HaCaT cells treated with A4b (compound 23 of Table 1) and linezolid. Significance limits: * = P <0.05, ** = P <0.01.

Figure 4. Cell viability results on HepG2 cells treated with compound A4b (compound 23 of table 1) and linezolid. Significance limits: * = P <0.05, ** = P <0.01.

Figure 5. Cell viability results on HepG2 cells treated with compounds B4a and B4b (compounds 106 and 107 of Table 1) in the form of their respective enantiomers.

Figure 6: Results of the OXPHOS assay on HepG2 cells treated with compounds A4aS and A4bS (compounds 22 and 23 of Table 1) in the form of their respective S-enantiomers

Figure 7: Scheme 1 of the chemical synthesis of compounds 1-5 and A1.

Figure 8: Scheme 2 for the chemical synthesis of compounds A and B.

Figure 9: Scheme 3 of the chemical synthesis of compounds A and B of interest.

DETAILED DESCRIPTION OF THE INVENTION

compounds:

[0019] The chemical structure of the compounds of the present invention [formulas (I)] consists of an oxazolidinone ring (ring A), a phenyl ring (ring B), an oxadiazole ring (ring C) and a side chain connected to the carbon in the C5 position of oxazolidinone (chain side connected to C5).

<td>R hk Zo</td><td>R2 aboutand</td><td>l3 ABOUT AND. NO \ - (</td><td>J Y-R</td>

<td>ring C</td><td>ring B</td><td>ring A</td><td>Side chain connected to C5</td>

Ring C.

PZ / 6877 / AG 5 EP 2 970 244 Β1 [0020] Ring C is a 1,2,4-oxadiazole heterocyclic system connected via a C (5) atom to ring B. The R substituent on the C ring can be a substituent selected from: F, Cl, Br, I, methyl, ethyl, n -propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, aryl, heteroaryl, -NH2, NHCH3, NHC2H5, -N (CH3)2, N (CH3)(DOWN2H5), -NC (= O) CH3, -NC (= O) C2H5, -NH (cyclopropyl), NH (cyclobutyl), NH (cyclopentyl), NH (cyclohexyl), -OH, -OCH3, -OC2H5, -On-Propyl, O - / - Propyl, -SH, SCH3.

Ring B [0021] Groups R1, R2, R3, R4 are independently H, F, Cl, Br, CH3, OH, OCH3. Preferably, at least one of them is halogen, for example R1 is F, Cl or Br, or R1 and R2 are F, Cl or Br, or R1, R2 and R3 are F or Cl. In a specific embodiment, the halogen atom is F and the remaining R groups are hydrogen. In a preferred formula, R1 or R2 are F and R3 and R4 mean H.

Side chain C5 [0022] Substituent R5 in the side chain connected to C5 at the 5-position of the oxazolidinone ring is as defined in this claim 1. For example, R5 may be selected from the group consisting of the following radicals: I, - N3, -NHC (= O) CH3, -NHC (= S) CH3, -NHC (= O) CH2F, -NHC (= S) CH2F, -NHC (= O) CH2Cl, -NHC (= S) CH2Cl, -NHC (= O) CH2Br, -NHC (= S) CH2Br, - NHC (= O) CHF2, -NHC (= S) CHF2, -NHC (= O) CHCl2, -NHC (= S) CHCl2, -NHC (= O) CHBr2, -NHC (= S) CHBr2, -NHC (= O) CF3, -NHC (= S) CF3, -NHC (= O) CCl3, -NHC (= S) CCl3, -NHC (= O) CBr3, -NHC (= S) CBr3, -NHC (= S) NH2, -NHC (= O) NH2, -NHC (= O) NHCH3, -NHC (= S) NHCH3, -NHC (= O) NHC2H5, -NHC (= S) NHC2H5, -NHC (= O) NH-nC3H7, -NHC (= S) NH-nC3H7, -NHC (= O) NH-iC3H7, -NHC (= S) NH-iC3H7, NHC (= S) NH-cyclopropyl, -NHC (= O) NHcyclopropyl, NHC (= S) NH-cyclobutyl, -NHC (= O) NH-cyclobutyl, NHC (= S) NH-cyclopentyl, -NHC (= O) NHcyclopentyl, NHC (= S) NH-cyclohexyl, -NHC (= O) NH-cyclohexyl, NHC (= O) NHC (= O) CH3,

NHC (= S) NHC (= O) CH3, NHC (= O) NHC (= O) C2H5, NHC (= O) NH-heteroaryl, -NCS, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazol-1-yl, 1,2,4-triazol-1-yl.

[0023] It has been observed that compounds containing a thio group, as indicated above, appear to exhibit better solubility and greater capacity to cross biological membranes.

[0024] Given the asymmetric configuration of the carbon atom at the 5-position of the A ring, all of the above identified compounds are optically active. Thus, the present invention relates to: racemic mixtures of these compounds, mixtures enriched in one of the enantiomers and enriched in one of the isolated enantiomers. For the scope of the present invention, a racemic mixture is a mixture of the two R and S enantiomers in a ratio of 50%: 50%. By a mixture enriched in one of the enantiomers, is meant a mixture containing more than 50% of one (or S or R) enantiomer, e.g. 55%, 60%, 65%, 70%, 75% or more. An isolated enantiomer is understood to mean a pure enantiomer, i.e. 100% or a mixture heavily enriched in this enantiomer, e.g. 98%, 95%, 93%, 90%, 88%, 85%, 80%.

[0025] A specific embodiment of the invention means compounds consisting of the S enantiomer or compositions containing the S enantiomer in the form of an enriched mixture or pure

The enantiomer is PZ / 6877 / AG. A second specific form of embodiment of the invention includes compounds consisting of R / S racemic mixtures or compositions containing R / S racemic mixtures. A further embodiment of the particular embodiment, less preferred, is a mixture enriched with the R-enantiomer. [0026] Preferred compounds of general formula (I) are listed in Table 1 below.

Table 1

PZI6877IAG

EP 2 970 244 B1

PZI6877IAG

EP 2 970 244 B1

PZJ6877IAG

EP 2 970 244 B1

VP / 6877 / AG

EP 2 970 244 B1

<td>155 (B1a)</td><td>CH3</td><td>H</td><td>H</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td>156 (B1b)</td><td>CH3</td><td>F</td><td>H</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td> 157</td><td>CH3</td><td>F</td><td>F</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td> 158</td><td>CH3</td><td>cl</td><td>H</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td> 159</td><td>CH3</td><td>cl</td><td>cl</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td> 160</td><td>CH3</td><td>F</td><td>F</td><td>F</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td> 161</td><td>CH3</td><td>br</td><td>H</td><td>H</td><td>H</td><td>1,2,3-triazol-1-yl</td>

[0027] Each compound identified above is considered an S enantiomer, as well as a mixture enriched in S enantiomer or a racemic mixture. For compounds 127-133 and 148-161, it should be understood that the R enantiomer, both pure and in a mixture enriched in R enantiomer, is preferred.

Preparation of compounds of the invention [0028] The synthesis of compounds A and B of interest and appropriate intermediates are described below. Compounds of the invention were synthesized starting from the 1,2,4-oxadiazole ring structure, following the pathway using amidoxime (Scheme 1) as given in [9]. Thus, amidoxime 1 was reacted with the corresponding benzoyl chloride 2 to form 1,2,4-oxadiazole 3. Such 1,2,4-oxadiazoles in which the para position was activated to undergo an aromatic nucleophilic substitution reaction, [10-13] reacted with allylamine to give compounds 4. Reaction with di- (f-butyl) carbonate, followed by cyclization [14] of the resulting derivatives 5, leads to the preparation of oxazolidinones of interest A1 as suitable precursors for further side chain modifications.

Diagram 1

VP / 6877 / AG

[0029] Later lateralization of the side chain (Scheme 2) included the acetamidomethyl A3 moiety as well as the corresponding thioamide A4, thiourea B4 and azole derivatives A5-7, B1.

[0030] A2 azide precursors were obtained by reacting compounds A1 with an azide source. Their subsequent reduction led to obtaining the corresponding amine derivatives 6 [15]. Amine derivatives 6 were easily reacted with acetyl chloride or acetic anhydride to give compounds A3. A3 acetamidomethyl derivatives were reacted with sulfurizing reagents (i.e. Lawesson's reagent or P2S5) to give A4 thioamide derivatives (Scheme 2).

[0031] Azole derivatives A5-7, B1, were obtained by nucleophilic substitution starting from iodo derivatives A1, while (thio) urea B4 was obtained by reaction of amines 6 with iso (thio) cyanates (Scheme 2).

Scheme 2 [0032] The compounds thus obtained, synthesized as racemic mixtures, were separated into their corresponding (S or R) enantiomers by HPLC separation using a chiral stationary phase.

Pharmaceutical compositions

PZJ6877IAG [0033] Pharmaceutical compositions suitable for administering the compounds of the invention are compositions intended for oral, parenteral or topical use.

[0034] Oral compositions may be, for example, in the form of a tablet, coated tablet, hard capsule, soft capsule, syrup, solution, suspension, emulsion. Parenteral compositions may, for example, be in the form of an aqueous or oily solution or emulsion. Topical compositions may for example be in the form of an ointment, cream, gel, solution, O / W or W / O emulsion or suspension.

[0035] In a specific embodiment, the compositions are administered by inhalation.

[0036] In the preparation of pharmaceutical compositions, one or more compounds of the invention are mixed with various therapeutically acceptable excipients suitable for solid, liquid or paste compositions.

[0037] Suspensions / emulsions, regardless of their route of administration, may contain nanoparticles and / or liposomes as a vehicle or drug carrier.

[0038] Because some persistent lung infections often show a low response rate to typical therapy, partly because of the lack of drug selectivity, partly because of their low bioavailability, especially when administered systemically, particular attention has been paid to the present invention in the context of the present invention. endotracheal administration as an alternative to the non-invasive systemic delivery of the compounds described herein.

[0039] Therefore, a specific embodiment of the invention comprises intratracheal administration of a drug, preferably encapsulated with nanoparticles.

[0040] Indeed, nanocapsulation of drugs and their release in the lungs promotes higher accumulation and retention of the drug in the lungs. The main advantage of such a formulation and route of administration is that it is possible to treat locally located diseases such as those located in the lungs or bronchi, allowing high doses of the drug to act in the area and showing reduced systemic toxicity, and therefore reduced likelihood of systemic side effects;

[0041] Nanoparticle-based formulations provide the additional advantage of improving passage through biological membranes, such as the outer membrane of bacteria, which extends the spectrum of action of drugs also active against Gram-negative bacteria;

[0042] In particular, the inventors have developed a nebulizer for solid lipid nanoparticles (a nebulizer compatible with solid lipid nanoparticles (SLN) for the release of antimicrobials according to the invention. SLN can be used as a carrier for the release into the lung or bronchial antimicrobial drugs , improving stability, as well as retention time in vivo in the lungs, thereby allowing increased bioavailability.

[0043] SLN pharmacokinetic and biodistribution studies have shown that interstitial macrophages in lung tissue that are in closer contact with the circulation compared to alveolar macrophages significantly contribute to the capture of SLN. In addition, factors such as slowed blood flow, lower drug exposure in the kidneys, and markedly increased deposition in lung tissues have been observed to be important features of antimicrobial compounds that are also effective in the treatment of pneumonia caused by beta-lactam resistant bacteria (e.g., Staphylococcus aureus methicillin resistant (MRSA)).

Therapeutic applications

PZJ6877IAG

[0044] The claimed compounds are new antibiotics intended for use in the treatment of infections caused by bacteria, especially highly resistant Gram-positive bacteria. For example, inter alia, Staphylococcus spp., Enterococcus spp., Streptococcus spp., In particular in the treatment of infections caused by Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae The compounds of the invention have also been shown to be active against bacteria resistant to other antibiotics or resistant to the reference compound, linezolid. Preferably, the compounds of the invention are effective even against bacteria resistant to more than one antibiotic, against bacteria resistant to many, e.g. two or more antibiotics selected from methicillin, vancomycin, penicillin, macrolides, fluoroquinolones or linezolid.

[0045] In addition, the new compounds of the invention combine inhibitory or bactericidal activity against bacteria susceptible or (multi) resistant to known antibiotics with completely acceptable toxicity or even less than the toxicity of the reference compound, linezolid, thereby giving a completely beneficial clinical effect / therapeutic profile.

[0046] Without combining the invention with some specific scientific theories, the effectiveness of the compounds of the invention in the treatment of bacterial infections, especially those caused by bacteria also resistant to other antibiotics, appears to be based on mechanisms of action involving the modulation and / or inhibition of bacterial protein synthesis and / or activity. Theoretically, it seems that the effectiveness of the molecules of the invention is associated not only with the interaction with the proteins responsible for the emergence of immune mechanisms developed by bacteria, such as, for example, the PBP2a protein expressed by MRSA (Staphylococcus aureus resistant to metacillin) strains, but also with the interaction compounds of the invention with mechanisms for the synthesis of ribosomal proteins.

EXPERIMENTAL PART

Assessment of pharmacological activity

Microbiological tests (i) Bacterial strains [0047] Several Staphylococcus aureus isolates, well characterized for the phenotype of susceptibility to antibiotics, were used to determine the antibacterial activity of the compounds tested in vitro. In particular, reference strains S. aureus ATCC 29213 and S. aureus M923 (collection strain) were used as MSSA strains. Of the MRSA, the standard reference strain S was used to determine sensitivity. aureus MU50 (ATCC 700699) and two collection strains (433 and F511).

[0048] In particular, eleven coagulase negative staphylococci (CoNS) resistant to linezolid (ten S. epidermidis and one S. hominis) were tested. Eleven strains resistant to linezolid were isolated in several hospitals in 2010-2011 from positive blood cultures. A collection of forty sensitive to was used to compare the antimicrobial activity of various compounds

VP / 6877 / AG

EP 2 970 244 Β1 MRSA linezolid, isolated from cystic fibrosis patients showing different multidrug resistance profiles to different classes of antimicrobials (Tables 4 and 5).

(ii) Determination of minimum inhibitory concentrations (MIC) [0049] The in vitro antibacterial activity of new agents was tested by determining their minimum inhibitory concentrations (MIC) using a microdilution method in broth according to guidelines developed by the Institute of Clinical Standards and Laboratory (CLSI, Clinical and Laboratory Standards Institute). [16] Briefly, serial 2-fold dilutions of each compound were made using Mueller-Hinton broth with appropriate cation concentration (CAMHB) on 96-well microtiter plates. Dimethyl sulfoxide (DMSO) was used as the solvent for all synthesized compounds. An equal volume of bacterial inoculum was added to each well on a microtiter plate containing 0.05 ml serial dilutions of antibiotics (1x 106 cfu / ml). The microtiter plate was then incubated at 37 ° C for 18-24 hours, after which each well was analyzed for the presence of bacterial growth. The MIC was defined as the lowest concentration of antimicrobial capable of inhibiting bacterial growth, as indicated by the lack of turbidity of the culture medium. The in vitro antibacterial activity of new 1,2,4-oxadiazoles similar to linezolid was tested and compared with the reference oxazolidinone in clinical application: Linezolid (Sigma-Aldrich). Final DMSO concentrations were also included in all bioassays.

Minimum inhibitory concentration test [0050] Fourteen new compounds in the form of a racemic mixture (Group A), which are shown below, were analyzed for their antibacterial activity against Staphylococcus aureus strains with respect to standard reference strains and clinical strains both methicillin sensitive (MSSA) and methicillin-resistant (MRSA).

Α1-7> Λ

<td>A5a</td><td>H</td><td>pyrazol-1-yl</td>

<td>A5b</td><td>F</td><td>pyrazol-1-yl</td>

<td>A6a</td><td>H</td><td>imidazol-1 -i</td>

<td>A6b</td><td>F</td><td>imidazol-1 -i</td>

<td>A7a</td><td>H</td><td>1,2,4-triazol-1 -i</td>

<td>A7b</td><td>F</td><td>1,2,4-triazol-1 -i</td>

PZJ6877IAG

[0051] Antimicrobial activities, summarized in Table 2, were determined by the "gold standard" method of microdilution in broth, in accordance with the guidelines of the Institute of Clinical and Laboratory Standards (CLSI) (see experimental section). Minimum inhibitory concentration (MIC) values were expressed in pgIml and cell viability tests were performed to assess selective antibacterial toxicity for most active compounds. Linezolid was used as a reference antibiotic. Specifically, bacterial strains were tested: Staphylococcus aureus ATCC 29213, methicillin-sensitive S. aureus clinical strain (M923), S. aure us MU50 (methicillin resistant strain - MRSA) and two methicillin resistant clinical strains, 433 and F511. All strains tested were found to be sensitive to linezolid. Of these molecules, compounds A4a and A4b in racemic form proved to be the most active.

Table 2

MIC (pgIml)

<td>Relationship A</td><td> 29213</td><td>M923</td><td>MU50</td><td> 433</td><td>F511</td>

<td>linezolid</td><td> <0,4</td><td> 3,13</td><td> 0,8</td><td> 1,6</td><td> 3,13</td>

Compounds A3a, A3b, A4a, A4b, A1a, A1b correspond to compounds 15, 16, 22, 23, 148 and 149 of Table 1. [0052] Four of the fourteen test compounds (see Table 2) showed MIC values for both MSSA strains and MRSA, with comparable or higher potency than linezolid. In addition, sulfur-containing derivatives A4a and A4b showed better activity against strains MSSA and MRSA compared to linezolid, whereas compounds A3a and A3b proved to be less active

PZ / 6877 / AG than linezolid, with the exception of MRSA strain 433. The comparison with linezolid should take into account the fact that the tested compounds were used in the form of a racemic mixture, therefore it is assumed that the antibacterial effect of A3a, A3b, A4a and A4b is underestimated considering the pure, more active enantiomer.

[0053] From the other compounds (group B) shown below, the activity of both the racemic mixture and the S and R enantiomers was evaluated.

H · ·

<td>B1a</td><td>H</td><td>1,2,3-triazol-1-yl</td>

<td>B1b</td><td>F</td><td>1,2,3-triazol-1-yl</td>

[0054] Antimicrobial activities, summarized in Table 3, were determined by the "gold standard" method of microdilution in broth, in accordance with the guidelines of the Institute of Clinical and Laboratory Standards (CLSI) (see experimental section). The minimum inhibitory concentration (MIC) values are expressed in pg / ml. Linezolid was used as a reference antibiotic. In detail, the bacterial strains tested were: Staphylococcus aureus ATCC 29213, clinical strain S. methicillin-sensitive aureus (M923), strain S. aureus MU50 (methicillin resistant strain - MRSA) and two methicillin resistant clinical strains, 433 and F511. All strains tested were found to be sensitive to linezolid. Of the new molecules tested, the most active were compounds in racemic form B4a and B4b, followed by B1a and B1b with significant activity (Table 3).

Table 3

MIC (pg / ml)

<td>Relationship B</td><td>ATCC 29213</td><td>MSSA M923</td><td>MRSA MU50</td><td>MRSA 433</td><td>MRSA F511</td>

PZI6877IAG

EP 2 970 244 B1

MIC (pgIml)

<td>Relationship B</td><td> 29213</td><td>M923</td><td>MU50</td><td> 433</td><td>F511</td>

<td>linezolid</td><td> <0,4</td><td> 3,125</td><td> 0,8</td><td> 1,6</td><td> 3,125</td>

[0055] Among these compounds, compounds B4a and B4b (corresponding to compounds 106 and 107 in Table 1) showed antimicrobial activity very similar to that of linezolid relative to linezolid sensitive S. aureus strains.

[0056] In a completely unexpected way, the same compounds, separated into their enantiomers, were 8 to 32 times more effective than linezolid against linezolid resistant strains of Staphylococcus spp. The results are shown in Tables 4 and 5. In one case (A4bS), total reversal of linezolid resistance to susceptibility to this compound. Of these molecules, enantiomeric separations allowed attribution of the potency of the S enantiomer, while the R enantiomer appeared to be inactive (see Table 4).

[0057] Compounds B4a and B4b correspond to the racemic mixtures of these two compounds B4a and B4b, compounds B4bS and B4bR and B4aS and B4aR are respectively separated S and R enantiomers.

<td colspan="9">MIC range <0.06> 128 pgIml</td>

<td colspan="9">Test strains: 6 ATCC (4 MSSA, 2 MRSA) plus 45 MRSA all sensitive to LZD</td>

<td>Relationship</td><td>b4b</td><td>B4bS</td><td>B4bR</td><td>b4a</td><td>B4aS</td><td>B4aR</td><td>EF</td><td>DA</td>

<td>Range MIC</td><td> 0,5-16</td><td> 0,5-8</td><td> 64->128</td><td> 1-16</td><td> 0,5-8</td><td> 128- >128</td><td> 0,25-16</td><td> <0,06- >128</td>

<td colspan="9">Test strains: 12 MRSE all sensitive to LZD</td>

<td>Range MIC</td><td> 32->128</td><td> 8,16</td><td> >128</td><td> 32->128</td><td> 8-32</td><td> >128</td><td> 32-64</td><td> 0,12-1</td>

PZI6877IAG

EP 2 970 244 B1

Table 5

<td></td><td colspan="5">MIC range 0.06 - 128 g / ml</td>

<td>strains</td><td>A4aS</td><td>A4aR</td><td>A4bS</td><td>A4bR</td><td>EF</td>

<td>ATCC S. aureus 29213</td><td> 8</td><td> 128</td><td> 4</td><td> 64</td><td> 4</td>

<td>ATCC E. faecalis 29212</td><td> 4</td><td> > 128</td><td> 2</td><td> 32</td><td> 1</td>

<td>11 CoNS resistant to linezolid</td><td></td><td></td><td></td><td></td><td></td>

<td>Strain 1 S. epidermidis</td><td> 8</td><td> > 128</td><td> 8</td><td> 128</td><td> 64</td>

<td>S. epidermidis strain 2</td><td> 32</td><td> > 128</td><td> 8</td><td> > 128</td><td> 64</td>

<td>Strain 3 S. epidermidis</td><td> 4</td><td> > 128</td><td> 4</td><td> > 128</td><td> 64</td>

<td>Strain 4 S. epidermidis</td><td> 32</td><td> > 128</td><td> 4</td><td> 128</td><td> 64</td>

<td>Strain 5 S. epidermidis</td><td> 4</td><td> > 128</td><td> 2</td><td> 128</td><td> 64</td>

<td>S. epidermidis strain 6</td><td> 4</td><td> > 128</td><td> 4</td><td> 64</td><td> 32</td>

<td>Strain 7 S. epidermidis</td><td> 32</td><td> > 128</td><td> 8</td><td> > 128</td><td> 32</td>

<td>Strain 8 S. epidermidis</td><td> 32</td><td> > 128</td><td> 2</td><td> 128</td><td> 32</td>

<td>Strain 9 S. epidermidis</td><td> 1</td><td> > 128</td><td> 1</td><td> > 128</td><td> 32</td>

<td>Strain 10 S. epidermidis</td><td> 8</td><td> > 128</td><td> 4</td><td> 128</td><td> 32</td>

<td>Strain 11 S. hominis</td><td> 8</td><td> > 128</td><td> 4</td><td> 128</td><td> 32</td>

<td>MIC range</td><td> 1-32</td><td> >128</td><td> 1-8</td><td> 64->128</td><td> 32 - 64</td>

<td>45 MRSA sensitive to linezolid</td><td></td><td></td><td></td><td></td><td></td>

Cell viability (cytotoxicity study) [0058] To assess whether an activity demonstrated against bacterial cells may be associated with a selected toxicity or more generally a toxic effect, the inventors conducted a first level test in different types of eukaryotic cell lines to screen for new compounds for their overall cytotoxic activity.

Cell viability [0059] The effect of compound A4b (compound 23 of Table 1) and linezolid on cell viability was tested in vitro on cell lines PK15 (pig kidney epithelium), HaCaT (human keratinocytes) and HepG2 (human hepatocellular carcinoma). [17-19] HepG2 and HaCat cells were cultured in Dulbecco's modified Eagle's medium (DMEM), while PK15 in DMEMIM199 (1: 1). All media were supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units milliliter penicillin and 100 pglml streptomycin. Cells were maintained at 37 ° C in an atmosphere of 5% CO2. All cell culture reagents were from Euroclone (Pero, Italy).

[0060] Cell viability was measured by MTT test. [20] Briefly, MTT [3- (4,5-dimethythiazol-2-yl) -2,5-diphenyltetrazolium bromide] solution (5 mg / ml) was added to each well to a final concentration of 1.2 mM and cells incubated for 1 hour and 30 minutes at 37 ° C. After removing the MTT solution, the reaction was stopped by adding 90% ethanol. The suspended cells were centrifuged 10 minutes at 800 x g. Absorbance was measured using a Victor 3 multilabel spectrophotometer (Perkin Elmer, Turku, Finland) at 570 nm. Data are means ± SE of 3 separate experiments performed in triplicate.

Statistical analysis [0061] Statistically significant results were obtained by Student's t test compared to controls * = P <0.05, ** = P <0.001. Data are means ± SE of 3 separate experiments performed in triplicate.

[0062] All cell lines tested were treated with increasing concentrations (5-400 gg / ml) of A4a and linezolid as reference compound. Another control was DMSO used as a solvent.

[0063] The A4b molecule induced a moderate decrease in viability (less than 10%) in the PK15 cell line, with a statistically significant result at the concentrations 25 (P <0.01), 50 (P <0.05) and 200 g / ml ( P <0.05) (Figure 2). This relationship is comparable to that obtained for linezolid at the same concentrations.

[0064] The decrease in cell viability induced by the A4b molecule was slightly more pronounced in the HaCaT cell line, achieving levels statistically significant for mortality compared to values obtained for linezolid only at a concentration of 400 gg / ml (P <0.01; Figure 3) .

[0065] HepG2 cells showed a decrease in viability from a concentration of 50 gg / ml for compound A4b (Figure 4).

[0066] The in vitro effect of compounds B4a and B4b on cell viability relative to the human hepatoma cell line, HepG2 was then assessed in vitro and compared with linezolid-induced cytotoxicity (negative control).

[0067] Cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), L-glutamine to a final concentration of 2 mM, 100 units / ml penicillin and 100 gg / ml streptomycin. Cells were maintained at 37 ° C in an atmosphere of 5% CO2.

[0068] Cytotoxic treatment: cells plated on 40,000 cell / cm density plates2 and maintained in culture for two days, treated for 48 hours with increasing concentrations (25-100 g / ml) of both enantiomers of compounds B4a and B4b.

[0069] Cell viability was assessed using the PrestoBlue® Cell Viability Reagent test, a solution containing resazurin that penetrates the cells and is reduced by the cells when they are alive and metabolically active. Briefly, PrestoBlue® solution is applied directly to the cell medium in culture according to the instructions of the manufacturer who supplied the product. Cells are incubated for 1 hour at 37 ° C, during which time PrestoBlue® solution metabolized by living cells changes color from blue to red. Absorbance is measured using a Victor3 multifunctional spectrophotometer (Perkin Elmer, Turku, Finland) at 570 nm. The results obtained and presented in the graph correspond to the mean ± SE of independent experiments carried out in triplicate.

PZ / 6877 / AG 20 EP 2 970 244 Β1 [0070] The HepG2 cell line was exposed to increasing concentrations (25-100 gg / ml) of both enantiomers of compounds B4a and B4b. Linezolid was used as a reference molecule only to a final concentration of 100 micrograms / ml. In addition, as an additional control, cells are also treated with 0.9% DMSO, used as a solvent for test substances.

[0071] Both enantiomers of B4b induced a moderate decrease in viability (<12%) in the HepG2 cell line at all concentrations tested (Figure 5).

[0072] The S enantiomer of B4a has a slight, concentration independent cytotoxic effect in HepG2 cells (visible only at a concentration of 25 microgramIml), whereas the R enantiomer does not determine a noticeable reduction in cell viability. As expected, 20% of HepG2 cells die after treatment with 100 micrograms / ml linezolid.

Oxidative phosphorylation study (OXPHOS) [0073] This study (Nadaciva S. et al., 2010) is used to monitor the level of mitochondrial protein synthesis for some key proteins in the oxidative phosphorylation process of eukaryotic cells, comparing it to the level of nuclear-encoded mitochondrial protein synthesis. GOUT. This study analyzes the effect of A4bS on proteins encoded by mitochondrial DNA (mtDNA).

[0074] The results shown in figure 6 confirm that linezolid (100 g / ml) negatively affects the synthesis of mitochondrial proteins. In fact, proteins of complex I, III (core 2 subunit) and IV (synthesized by mtDNA) are significantly reduced after treatment with linezolid. In parallel, you can compare the A4bS molecule (10-100 pg / ml), which, like linezolid, causes a reduction in protein synthesis of complex I and IV relative to control (untreated cells). It should be noted, however, that the decrease in protein synthesis induced by the A4bS compound has a lower value than that induced by linezolid, this effect highlights the reduction of the side effect that is associated with reversible bone marrow suppression. The results shown in Figure 6 were obtained as follows: levels of proteins encoded by mitochondrial DNA (mtDNA), which are synthesized on the mitochondrial ribosome (complex IV, complex I) and proteins encoded by nuclear DNA, which are synthesized on the ribosome in the cytosol (complex with the V subunit of complex II) and imported into mitochondria with the WB MitoProfile® Total OXPHOS human antibody after treatment of HepG2 cells (human hepatocellular carcinoma cells) with A4bS. Data represent the mean ± SEM of three separate experiments performed in triplicate. Statistical significance was obtained using the Student test in comparison with compounds.

[0075] * = p <0.05; ** = p <0.01.

Chemical synthesis [0076] Melting points were determined with a Reichart-Thermovar apparatus and remain uncorrected. IR spectra (in Nujol) were determined using a Shimadzu FTIR-8300 instrument; H NMR spectra were recorded on a Bruker 300 Avance spectrometer using TMS as an internal standard. Flash chromatography was performed using silica gel (0.040-0.063 mm) and mixtures of ethyl acetate and petroleum ether (boiling fraction in the range of 40-60 ° C) in various proportions. In all cases

VP / 6877 / AG

The purity of the compounds was above 95%, which was checked in both NMR and HPLC analyzes. Separation of racemates was carried out by HPLC with a chiral stationary phase (Daicel, Chiralpak-IA), using hexane-iPrOH (70:30) as the mobile phase and a flow rate of 1 ml / min. In each case, ee> 99% was obtained. [0077] The most interesting compounds:

A1a (compound 148 from table 1), A1b (compound 149 from table 1), A3a (compound 15 from table 1), A3b (compound 16 from table 1), A4a (compound 22 from table 1), A4b (compound 23 from table 1) Table 1), B1a (compound 155 from table 1), B1b (compound 156 from table 1), B4a (compound 106 from table 1), B4b (compound 107 from table 1); listed in table 2 (group A) and 3 (group B) and the corresponding intermediates 1-6, were obtained according to the general methodologies shown in schemes 1 and 2, in accordance with the specifications indicated below and in scheme 3.

[0078] General procedure for the preparation of compounds 3a, b

A solution of hydroxylamine hydrochloride (1.00 g, 14.4 mmol) and NaOH (0.57 g, 14.4 mmol) in water (5 mL) was added to 15 mL of CH3CN (in about 15 minutes). The reaction mixture was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure and the residue was treated

PZI6877IAG 22 EP 2 970 244 Β1 ethanol; the resulting suspension was filtered and the solvent removed under reduced pressure to give 1.659 g of acetamidoxime 1 (77%). Then, to a solution of 1 (1.00 g; 13.5 mmol) in acetone (35 ml) also containing K2WHAT3 (2.05 g, 14.8 mmol) either 4-fluorobenzoyl chloride (2a) or 2,4-difluorobenzoyl chloride (2b) (14.8 mmol) was added. The mixture was stirred at room temperature for about 90 minutes, after which the solvent was removed under reduced pressure. The residue was treated with water and the solid precipitate was filtered off. The resulting O-acylamidoxime was heated without further purification at about 130 ° C for 90 minutes in a sealed tube. The resulting residue was chromatographed to provide the corresponding 1,2,4-oxadiazoles 3a and 3b.

[0079] 3-Methyl-5- (4'-fluorophenyl) -1,2,4-oxadiazole (3a): Yield (72%); mp. 80,0-81,0 ° C;11 H NMR (300 MHz; CDCl 3) 2.45 (s, 3H, Me); 7.16-7.23 (m, 2H, Ar); 8.08-8.14 (m, 2H, Ar). Element analysis Found (calculated) for C9H7FN2O (%): C, 60.65 (60.67); H 3.90 (3.96); N 15.70 (15.72).

[0080] 3-methyl-5- (2 ', 4'-difluorophenyl) -1,2,4-oxadiazole (3b): Yield (72%); mp. 57,0-60,0 ° C; 1 H-NMR (300 MHz; CDCl3) 2.46 (s, 3H, Me); 6.95-7.07 (m, 2H, Ar); 8.04-8.14 (m, 1H, Ar). Element analysis Found (calculated) for C9H6F2N2O (%): C, 55.15 (55.11); H 3.10 (3.08); N, 14.25 (14.28).

Preparation of N-allyl-4- (3'-methyl-1,2,4-oxadiazol-5'-yl) -aniline (4a) [0081] Compound 3a (0.61 g; 3.43 mmol) was heated with allylamine (3.0 ml; 2.28 g; 40.0 mmol) and K2WHAT3 (2.00 g 14.5 mmol), at about 60 ° C for 8 days. The reaction mixture was treated with water and extracted with EtOAc. The organic layers were collected, dried over anhydrous Na2SO4, filtered and the solvent removed. The residue was chromatographed to give compound 3a: Yield (54%); mp. 63,9-65,5 ° C; IR (Nujol) 3335 (NH), 1607 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 2.31 (s, 3H, Me); 3.76-3.79 (m, 2H, CH2);

5.12 (dd, 1H, J1 = 10.5 Hz, J2 = 1.8 Hz, -CH = CH2); 5.22 (dd, 1H, J1 = 17.1Hz, J2 = 1.8 Hz, -CH = CH2); 5,825,93 (m, 1H, -CH = CH2); 6.68 (d, 2H, J = 9.0 Hz, Ar); 6.87 (t, 1H, J = 5.7 Hz, NH, ex. From D2ABOUT); 7.76 (d, 2H, J = 9.0 Hz, Ar). Element analysis Found (calculated) for C12H13N3O (%): C 66.95 (66.96); H, 6.10 (6.09); N 19.45 (19.52).

Preparation of N-allyl-3-fluoro-4- (3'-methyl-1,2,4-oxadiazol-5'-yl) -aniline (4b) [0082] To compound 3b (0.86g 4.38 mmol) allylamine (1.64 ml; 1.25 g; 22.0 mmol) was added in DMF (2.0 mL). The reaction mixture was stirred for 2 days, then the solution was treated with water and extracted with EtOAc. The organic layers were collected, dried over anhydrous Na2SO4, filtered and the solvent removed. The residue was chromatographed to give compound 4b: Yield (49%); 57,9-59,9 ° C; IR (Nujol) 3335 (NH), 1626 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 2.34 (s, 3H, Me); 3.77-3.81 (m, 2H, CH2);

5.13 (dd, 1H, J1 = 13.2 Hz, J2 = 1.2 Hz, -CH = CH2); 5.23 (dd, 1H, J1 = 17.4 Hz, J2 = 1.2 Hz, -CH = CH2); 5,815,93 (m, 1H, -CH = CH2); 6.46 (dd, 1H, J1 = 14.4 Hz, J2 = 1.8 Hz, Ar); 6.56 (dd, 1H, J1 = 8.7 Hz, J2 = 1.8 Hz, Ar); 7.17-7.21 (bs, 1H, NH, ex. From D.2ABOUT); 7.72-7.77 (m, 1H, Ar). Element analysis Found (calculated) for C12H12FN3O (%): C, 61.80 (61.79); H, 5.10 (5.19); N 18.15 (18.02).

General procedure for the preparation of compounds 5a, b

PZI6877IAG 23 EP 2 970 244 Β1 [0083] Compound 4a or 4b (2.15 mmol) was dissolved in CH3CN (25 ml); di- (t-butyl) bicarbonate (0.51 g; 2.36 mmol) and 4-dimethylaminopyridine (0.29 g; 2.36 mmol) were added and the mixture was stirred for 2 days or 2.5 hours, respectively. The solvent was removed under reduced pressure and the obtained residue was chromatographed to give the corresponding compounds 5a and 5b.

[0084] Tert-butyl N-allyl- (4- (3'-methyl-1,2,4-oxadiazol-5'-yl) -phenyl) -carbamate (5a): oil; efficiency (73%); IR (Nujol) 1711 (NCO2), 1614 (C = N) cm-1; 1 H-NMR (300 MHz; CDCl 3) 1.27 (s, 9H, t-Bu); 2.25 (s, 3H, Me); 4.10 (d, 2H, J = 5.1 Hz, CH2); 4.95-4.97 (m, 1H, -CH = CH2); 4.99-5.01 (m, 1H, -CH = CH2); 5,675,78 (m, 1H, -CH = CH2); 7.23 (d, 2H, J = 9.0 Hz, Ar); 7.84 (d, 2H, J = 9.0 Hz, Ar). Element analysis Found (calculated) for C17H21N3ABOUT3 (%): C, 64.70 (64.74); H 6.80 (6.71); N 13.35 (13.32).

[0085] Tert-butyl N-allyl- (3-fluoro-4- (3'-methyl-1,2,4-oxadiazol-5'-yl) -phenyl) -carbamate (5b): oil; efficiency (72%); IR (Nujol) 1713 (NCO2), 1615 (C = N) cm-1; 1 H-NMR (300 MHz; CDCl 3) 1.53 (s, 9H, tBu); 2.53 (s, 3H, Me); 4.36 (d, 2H, J = 5.1 Hz, CH2); 5.21-5.28 (m, 2H, -CH = CH2); 5.91-6.02 (m, 1H, -CH = CH2); 7.28-7.36 (m, 2H, Ar); 8.02-8.08 (m, 1H, Ar). Element analysis Found (calculated) for C17H20FN3ABOUT3 (%): C, 61.25 (61.25); H, 6.10 (6.05); N 12.65 (12.61).

General procedure for the preparation of compounds A1a, b [0086] For a solution of 1.70 mmol of any compound 5a or 5b in CH2cl2 (10 ml) sublimated I was added2 (1.29 g; 5.10 mmol). The solution was stirred for 24 hours and then treated with a Na solution2SO3; the organic layer was dried over anhydrous Na2SO4, filtered and the solvent removed. The residue was chromatographed to give the corresponding compounds A1a and A1b.

3- (4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (iodomethyl) oxazolidin-2-one (A1a): Yield (89%) ; mp. 145,0-147,0 ° C; IR (Nujol) 1763 (NCO2), 1618 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d 6) 2.47 (s, 3H, Me); 3.62-3.73 (m, 2H, CH 2 -I); 3.80 (dd, 1H, J1 = 9.3 Hz, J2 = 6.0 Hz, C4-H); 4.34 (dd, 1H, J1 = 9.3 Hz, J2 = 9.0 Hz, C4-H); 4.81-4.90 (m, 1H, C5-H); 7.88 (d, 2H, J = 9.0 Hz, Ar); 8.17 (d, 2H, J = 9.0 Hz, Ar). Element analysis Found (calculated) for C13H12IN3O3 (%): C 40.55 (40.54); H 3.15 (3.14); N 10.85 (10.91). 3- (3'-fluoro-4 '- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) -5- (iodomethyl) oxazolidin-2-one (A1 b) : Efficiency (76%); 148,0-149,0 ° C; IR (Nujol) 1743 (NCO2), 1637 (C = N) cm-1; 1 H-NMR (300 MHz; DMSOd 6) 2.48 (s, 3H, Me); 3.61-3.72 (m, 2H, CH 2 -I); 3.81 (dd, 1H, J1 = 9.6 Hz, J2 = 6.0 Hz, C.4-H); 4.33 (dd, 1H, J1 = 9.6 Hz, J2 = 9.0 Hz, C.4-H); 4.83-4.93 (m, 1H, C5-H); 7.68 (dd, 1H, J1 = 8.7 Hz, J2 = 2.1 Hz, Ar); 7.80 (dd, 1H, J1 = 13.8 Hz, J2 = 2.1 Hz, Ar); 8.16 (dd, 1H, J1 = 8.7 Hz, J2 = 8.5 Hz, Ar). Element analysis Found (calculated) for C13H11FIN3O3 (%): C 38.75 (38.73); H 2.55 (2.75); N, 10.35 (10.42).

General procedure for the preparation of compounds A2a, b [0089] To a solution of 0.75 mmol of A1a or A1b in DMF (6 ml) was added NaN3 (0.39 g 6.00 mmol). The solution was stirred for 24 hours, then the reaction was treated with water and extracted with EtOAc; organic layers were dried over anhydrous Na2SO4, filtered and the solvent removed. The residue was chromatographed to give the corresponding compounds A2a and A2b.

3- (4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (azidomethyl) oxazolidin-2-one (A2a):

Yield (94%); mp. 133,9-135,0 ° C; IR (Nujol) 2095 (N3), 1765 (NCO2), 1727 (NCO2), 1618 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 2.46 (s, 3H, Me); 3.75-3.88 (m, 2H, CH2-N3); 3.92 (dd, 1H, J1 = 9.3 Hz, J2

PZJ6877IAG 24 EP 2 970 244 Β1 = 6.0 Hz, C.4-H); 4.28 (t, 1H, J = 9.3 Hz, C.4-H); 4.96-5.03 (m, 1H, C.5-H); 7.86 (d, 2H, J = 9.0 Hz, Ar); 8.16 (d, 2H, J = 9.0 Hz, Ar). Element analysis Found (calculated) for C13H12N6ABOUT3 (%): C, 52.05 (52.00); H 4.10 (4.03); N 27.85 (27.99).

3- (3'-fluoro-4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (azidomethyl) oxazolidin-2-one (A2b): Yield (99%); mp. 126,2-127,7 ° C; IR (Nujol) 2107 (N3), 1758 (NCO2), 1743 (NCO2), 1630 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 2.41 (s, 3H, Me); 3.69-3.82 (m, 2H, CH2-N3); 3.86 (dd, 1H, J1 = 9.3 Hz, J2 = 6.0 Hz, C.4-H); 4.21 (t, 1H, J = 9.3 Hz, C.4-H); 4.91-4.99 (m, 1H, C5-H); 7.60 (dd, 1H, J1 = 9.0 Hz, J2 = 1.8 Hz, Ar); 7.72 (dd, 1H, J1 = 13.5 Hz, J2 = 1.8 Hz, Ar); 8.08-8.14 (m, 1H, Ar). Element analysis Found (calculated) for C13H11FN6O3 (%): C, 49.10 (49.06); H 3.50 (3.48); N, 26.45 (26.41).

General procedure for the preparation of compounds 6a, b [0092] To a solution of 0.45 mmol of compound A2a or A2b in THF (15 ml) was added PPh3 (0.16 g 0.60 mmol). The solution was stirred for about 90 minutes, then 100 L of distilled water was added and the resulting mixture was heated to reflux for 4 hours. THF was removed under reduced pressure, the obtained residue was neutralized with hydrochloric acid and extracted with EtOAc. A solution of NaOH (pH ~ 9) was added to the aqueous phase, which was extracted with EtOAc; organic layers were dried over anhydrous Na2SO4, filtered and the solvent removed to give the corresponding compounds 6a and 6b.

3- (4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (aminomethyl) oxazolidin-2-one (6a): Yield (66%) ; mp. 139,3-141,3 ° C; IR (Nujol) 3390 (NH), 3361 (NH), 1748 (NCO2), 1616 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-ds) 2.22 (bs, 2H, NH2, dim. from D2ABOUT); 2.39 (s, 3H, Me); 2.77-2.91 (m, 2H, CH2NH2); 3.94 (dd, 1H, J1 = 9.0 Hz, J2 = 6.3 Hz, C.4-H); 4.13 (t, 1H, J = 9.0 Hz, C.4-H); 4.61-4.70 (m, 1H, C.5-H); 7.80 (d, 2H, J = 9.0 Hz, Ar); 8.09 (d, 2H, J = 9.0 Hz, Ar). Element analysis Found (calculated) for C13H14N4ABOUT3 (%): C, 56.90 (56.93); H 5.15 (5.14); N 20.45 (20.43).

3- (3'-fluoro-4 '- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) -5- (aminomethyl) oxazolidin-2-one (6b): Yield (88%); mp. 137,0-140,0 ° C; IR (Nujol) 3372 (NH), 1743 (NCO2), 1630 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-ds) 2.21 (bs, 2H, NH2, dim. from D2ABOUT); 2.41 (s, 3H, Me); 2.77-2.91 (m, 2H, CH2NH2); 3.93 (dd, 1H, J1 = 9.3 Hz, J2 = 6.3 Hz, C.4-H); 4.13 (t, 1H, J = 9.0 Hz, C.4-H); 4.63-4.71 (m, 1H, C5-H); 7.60 (dd, 1H, J1 = 9.0 Hz, J2 = 2.1 Hz, Ar); 7.73 (dd, 1H, J1 = 10.8 Hz, J2 = 2.1 Hz, Ar); 8.08-8.14 (m, 1H, Ar). Element analysis Found (calculated) for C13H13FN4ABOUT3 (%): C 53.40 (53.42); H 4.45 (4.48); N 19.25 (19.17).

[0095] General procedure for the preparation of compounds A3a, b.

[0096] For a solution of any of the compounds A3a or A3b (0.28 mmol) in CH2cl2 (3 mL) also containing pyridine (1 mL; 0.97 g; 12.3 mmol), acetyl chloride (40 µL; 44 mg; 0.56 mmol) was added. The solution was stirred for 30 minutes after which the solvent was removed and the residue was treated with 1M HCl (20 mL) and extracted with EtOAc; the organic layers were dried over anhydrous Na2SO4, filtered and the solvent removed. The residue was chromatographed to give the corresponding compounds A3a and A3b.

3- (4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (N-acetylaminomethyl) oxazolidin-2-one (A3a): Yield (58 %); mp. 214,0-216,0 ° C; IR (Nujol) 3257 (NH), 1751 (NCO2), 1646 (amide), 1616 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 1.89 (s, 3H, COMe); 2.46 (s, 3H, Me); 3.50 (t, 2H, J = 5.7 Hz, CH2NHCOMe); 3.88 (dd, 1H, J1 = 9.0 Hz, J2 = 6.6 Hz, C.4-H); 4.25 (t, 1H, J = 9.0 Hz, C.4-H); 4.79-4.87 (m, 1H, C5-H); 7.84 (d, 2H, J = 8.7 Hz, Ar); 8.16 (d, 2H, J = 8.7 Hz, Ar); 8.32 (t, 1H, J = 5.7 Hz, NH, ex. From D2ABOUT);

PZI6877IAG 25 EP 2 970 244 Β113C-NMR (75 MHz; DMSO-d6) 11.4, 22.6, 41.5, 47.2, 72.0, 118.1 (overlapping signals), 128.9, 142.6, 154.1, 167.7, 170.2, 174.5. Element analysis Found (calculated) for C15H16N4ABOUT4 (%): C, 56.95 (56.96); H 5.05 (5.10); N 17.85 (17.71).

3- (3'-fluoro-4 '- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) -5- (N-acetylaminomethyl) oxazolidin-2-one (A3b): Yield (62%); mp. 184,0-186,0 ° C; IR (Nujol) 3343 (NH), 1751 (NCO2), 1666 (amide), 1628 (C = N) cm-1; 1 H-NMR (300 MHz; DMSO-d 6) 1.89 (s, 3H, COMe); 2.48 (s, 3H, Me); 3.50 (t, 2H, J = 5.4 Hz, CH2-NHCOMe); 3.88 (dd, 1H, J1 = 9.3 Hz, J2 = 6.3 Hz, C4-H); 4.25 (t, 1H, J = 9.0 Hz, C4-H); 4.81-4.88 (m, 1H, C5-H); 7.64 (dd, 1H, J1 = 9.0 Hz, J2 = 1.8 Hz, Ar); 7.77 (dd, 1H, J1 = 13.8 Hz, J2 = 1.8 Hz, Ar); 8.15-8.21 (m, 1H, Ar), 8.31 (m, 1H, NH, excl. D2O);13C-NMR (75 MHz; DMSO-d6) 11.32, 22.6, 41.5, 47.3, 72.2, 105.7 (d, JCF = 32 Hz), 106.2 (d, JCF = 14 Hz), 114.1, 131.4, 144.3 (d, JCF = 14 Hz), 153.9, 160.4 (d, JCF = 305 Hz), 167.5, 170.2, 171.6. Element analysis Found (calculated) for C15H15FN4ABOUT4 (%): C 53.90 (53.89); H 4.65 (4.52); N 16.65 (16.76).

General procedure for the preparation of compounds A4a, b [0099] To a solution of A3a or A3b (0.49 mmol) in THF (14 ml) was added Lawesson's reagent (0.2 g; 0.49 mmol). The reaction mixture was heated at reflux for 2 hours, after which the solvent was removed under reduced pressure. The residue was chromatographed to afford the corresponding compounds A4a and A4b.

3- (4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (N-thioacetylaminomethyl) -oxazolidin-2-one (A4a): Yield (77 %); mp. 199,4-201,0 ° C; IR (Nujol) 3217 (NH), 1721 (NCO2), 1618 (thioamide) cm-1; 1HNMR (300 MHz; DMSO-d6) 2.47 (s, 3H, Me); 2.51 (s, 3H, CSMe); 3.95-4.03 (m, 3H, overlapping signals); 4.28-4.34 (m, 1H, C.4-H); 5.01-5.11 (m, 1H, C.5-H); 7.85 (d, 2H, J = 9.0 Hz, Ar); 8.18 (d, 2H, J = 9.0 Hz, Ar); 10.45 (bs, 1H, NH, ex. From D2ABOUT). Element analysis Found (calculated) for C15H16N4ABOUT3S (%): C 54.15 (54.20); H 4.85 (4.85); N 16.90 (16.86).

3- (3'-fluoro-4 '- (3-methyl-1,2,4-oxadiazol-5-yl) -phenyl) -5- (N-thioacetylaminomethyl) oxazolidin-2-one (A4b): Yield (93%); mp. 166,5-167,7 ° C; IR (Nujol) 3262 (NH), 1746 (NCO2), 1633 (thioamide) cm-1; 1 H-NMR (300 MHz; DMSO-d6) 2.48 (s, 3H, Me); 2.51 (s, 3H, CSMe); 3.94-4.00 (m, 3H, overlapping signals); 4.28-4.34 (m, 1H, C.4-H); 5.04-5.12 (m, 1H, C.5-H); 7.65 (dd, 1H, J1 = 9 Hz, J2 = 1.8 Hz, Ar); 7.78 (dd, 1H, J1 = 13.5 Hz, J2 = 1.8 Hz, Ar); 8.16-8.22 (m, 1H, Ar); 10.45 (bs, 1H, NH excl. D2ABOUT). Element analysis Found (calculated) for C15H14FN4ABOUT3S (%): C, 51.35 (51.42); H 4.30 (4.32); N, 16.05 (15.99).

General procedure for the preparation of compounds B1a, b [0102] In a glass tube, 1,2,3-triazole (0.124 g; 1.8 mmol) was added to 0.45 mmol of A1a or A1b. The mixture was heated until complete consumption of starting material as monitored by TLC. The residue was chromatographed to give the corresponding compounds B1a and B1b.

((3- (4- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) oxazolidin-2-one-5-yl) methyl) -4,5-dihydro-1H1 , 2,3-triazole (B1a): Yield (73%); mp. 208-210 ° C; IR (Nujol) 1751 cm-1; 1 H-NMR (300 MHz; CDCl 3) 2.46 (s, 3H), 4.03 (dd, J1 = 6.3 Hz, J2 = 9.3 Hz, 1H), 4.25 (dd, J1 = 9.3 Hz, J2 = 9.0 Hz, 1H), 4.82-4.83 (m, 2H), 5.08-5.14 (m, 1H), 7.59 (d, J = 9.0 Hz, 1H) , 7.75 (s, 1H), 7.80 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H); Element analysis Found (calculated) for C15H14N6ABOUT3 (%): C 55.30 (55.21); H 4.39 (4.32); N, 25.69 (25.75).

PZI6877IAG

[0104] ((3- (3-fluoro-4- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) oxazolidin-2-one-5-yl) methyl ) -4,5-dihydro 1 H-1,2,3-triazole (B1b): Yield (64%); mp. 176,2-177,8 ° C; IR (Nujol) 1751 cm-1; 1 H-NMR (300 MHz; CDCl 3) 2.48 (s, 3H), 4.03 (dd, J1 = 9.3 Hz, J2= 6.0 Hz, 1H), 4.25 (dd, J1 = 9.6 Hz, J2 = 9.0 Hz, 1H), 4.824,83 (m, 2H), 5.15-5.30 (m, 1H), 7.27 (dd, J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 7.56 (dd, J1 = 12.6 Hz, J2 = 1.8 Hz, 1H), 7.75 (s, 1H), 7.79 (s, 1H), 8.02 (t, J = 8.3 Hz, 1H); Element analysis Found (calculated) for C15H13FN6ABOUT3 (%): C 52.37 (52.33); H 3.85 (3.81); N 24.47 (24.41).

General procedure for the preparation of compounds B4a, b [0105] To a solution of 0.55 mmol of 6a or 6b in THF (5 mL) was added CH3NCS (0.041 mL; 0.60 mmol) and triethylamine (0.084 mL; 0.60 mmol). The solution was stirred for 3 hours at room temperature. Then, the solvent was removed under reduced pressure. The residue was chromatographed to afford the corresponding compounds B4a and B4b.

1 - ((3- (4- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) oxazolidin-2-one-5-yl) methyl) -3-methylthiourea (B4a) : Efficiency (80%); mp. 189,4-191,8 ° C; IR (Nujol) 3364, 1732 cm-1; 1 H-NMR (300 MHz; CDCl3) 2.39 (s, 3H), 2.82 (bs, 3H), 3.82-4.00 (m, 3H), 4.20 (dd, J1 = 8.7 Hz, J2 = 6.0 Hz, 1H), 4.91 (bs, 1H), 7.77-7.80 (m, 3H), 8.09 (d, J = 6.9 Hz, 2H); Element analysis Found (calculated) for C15H17N5ABOUT3S (%): C, 51.91 (51.86); H, 5.00 (4.93); N 20.20 (20.16).

1 - ((3- (3-fluoro-4- (3-methyl-1,2,4-oxadiazol-5-yl) phenyl) oxazolidin-2-one-5-yl) methyl) -3-methylthiourea (B4b): Yield (88%); 170,7-172,4 ° C; IR (Nujol) 3370, 1739 cm-1; 1 H-NMR (300 MHz, DMSO) 2.48 (s, 3H), 2.89 (bs, 3H), 3.89-4.07 (m, 3H), 4.24-4.30 (m, 1H), 4.89 (bs, 1H), 7.74 (s, 1H), 7.79 (dd, J1 = 13.5 Hz, J2= 2.1 Hz, 2H), 8.20 (t, J = 9.0 Hz, 2H); Element analysis Found (calculated) for C15H16FN5ABOUT3S (%): C, 49.21 (49.31); H 4.35 (4.41); N 19.10 (19.17).

LIST OF REFERENCES [0108] [1] S. Tsiodras, HS Gold, G. Sakoulas, GM Eliopoulos, C. Wennersten, L. Venkataraman, RC Moellering, MJ Ferraro, Lancet 2001,358, 207-208.

[2] C. Auckland, L. Teare, F. Cooke, ME Kaufmann, M. Warner, G. Jones, K. Bamford, H. Ayles, AP Johnson, J. Antimicrob. Chemother. 2002, 50, 743-746.

[3] J. Seedat, G. Zick, I. Klare, C. Konstabel, N. Weiler, H. Sahly, Antimicrob. Ag. Chemother. 2006, 50, 4217-4219.

[4] S. Kelly, J. Collins, M. Maguire, C. Gowing, M. Flanagan, M. Donnelly, PG Murphy, J. Antimicrob. Chemother. 2008, 61, 901-907.

[5] JVN Vara Prasad, Curr. Op. Microbiol. 2007, 10, 454-460.

[6] C. Farrerons Gallemi, 2005, US Patent 2005I0014806.

[7] LB Snyder, Z. Meng, R. Mate, SV D'Andrea, A. Marinier, et al; Bioorg. Med. Chem. Lett., 2004, 14, 4735-4739.

[8] A. Palumbo Piccionello, R. Musumeci, C. Cocuzza, CG Fortuna, A. Guarcello, P. Pierro, A. Pace, Eur. J. Med. Chem. 2012, 50, 441-448.

[9] A. Pace, P. Pierro, Org. Biomol. Chem. 2009, 7, 4337-4348.

PZI6877IAG

[10] S. Buscemi, A. Pace, R. Calabrese, N. Vivona, P. Metrangolo, Tetrahedron 2001,57, 5865-5871.

[11] S. Buscemi, A. Pace, A. Palumbo Piccionello, I. Pibiri, N. Vivona, Heterocycles 2004, 63, 16191628.

[12] A. Palumbo Piccionello, A. Pace, I. Pibiri, S. Buscemi, N. Vivona, Tetrahedron 2006, 62, 87928797.

[13] A. Palumbo Piccionello, A. Pace, P. Pierro, I. Pibiri, S. Buscemi, N. Vivona, Tetrahedron 2009, 65, 119-127.

[14] KC Grega, MR Barbachyn, SJ Brickner, SA Mizsak, J. Org. Chem. 1995, 60, 5255-5261.

[15] H. Biswajit Das, H. Sonali Rudra, A. Songita Songita, P. Mohammad Salman, H. Ashok Rattan, 2008, US Patent 2008/0188470.

[16] Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - ed. 9. 2011, M07-A9, 32, Wayne, Pennsylvenia.

[17] EC Pirtle, amended by J. Vet. Res. 1966, 27, 747-749.

[18] DP Aden, A. Fogel, S. Plotkin, I. Damjanov, BB Knowles, Nature 1979, 282, 615-616.

[19] G. Pozzi, M. Guidi, F. Laudicina, M. Marazzi, L. Falcone, R. Betti, C. Crosti, E. Muller, GE Di Mattia, V. Locatelli, A. Torsello, J. Endocrinol . Invest. 2004, 27, 142-149.

[20] A. Bulbarelli, E. Lonati, E. Cazzaniga, M. Gregori, M. Masserini, Mol. Cell. Neurosci. 2009, 42, 7580.

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2014059896	International Bureau of the World Intellectual Property Organization (WIPO)	W	2014-03-17
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Novel 1, 2, 4-oxadiazol compounds active against gram-positive pathogens

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EP 2 970 244 B1

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EP 2 970 244 B1

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EP 2 970 244 B1

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PZJ6877IAG

PZI6877IAG

EP 2 970 244 B1

PZI6877IAG

EP 2 970 244 B1

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PZI6877IAG

VP / 6877 / AG

EP 2 970 244 B1

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