IT201600098005A1 - Analogs and derivatives of dicarboxylic amino acids as antibacterials - Google Patents

Description

Description of the patent application for Industrial Invention entitled: "Analogues and derivatives of dicarboxylic amino acids as antibacterials"

Technical field

The present invention relates to analogues and derivatives of dicarboxylic amino acids which can be used as antibacterials. The compounds of the invention can be used in particular as antibiotics in monotherapy or in combination in the treatment of bacterial infections sustained by both Gram-negative and Gram-positive (or mixed) bacteria, especially to counteract antibiotic resistance, but also as disinfectants and antiseptics.

Known art

Antibiotics are widely used in the medical field to eradicate bacterial infections of varying severity and localization in the human body. However, thanks to their genomic plasticity, bacteria are able to adapt quickly and often to develop resistance to the action (bacteriostatic or bactericidal) of these molecules, thus remaining immune to treatment.

The World Health Organization (WHO) has issued an alert regarding the often improper use of antibiotics and the consequent recent development in bacterial populations of dangerous resistances or multi-resistances to the molecules currently used in therapy (http: // www. Agenziafarmaco.gov.it/it/content/nuovo-rapportodell%E2%80%99oms-evidenzia-che-la-resistenza-agli-antibiotics-%C3%A8-ancora-una-minaccia-l) in particular for Gramnegative bacteria such as multidrug-resistant Escherichia coli. The need to develop and bring to the market new molecules that have a different mechanism of action from most antibiotics currently in use and that are also effective on bacteria that have now developed multi-resistance is therefore increasingly strongly recognized.

Α-aminophosphinic acids have important pharmaceutical properties. For example they are antimicrobial agents in vitro at low concentrations (0.8-50 pg / ml) going to inhibit the growth in minimal medium (consisting of salts and a carbon source) of pathogenic bacteria for humans such as Escherichia coli, and other enterobacteria. , Pseudomonas aerugìnosa and, among the yeasts, Candida albicane and Candida tropicalis [US Patent 4,147,780].

Furthermore, peptides containing a-aminophosphinic acids at the C-terminal end also show antimicrobial properties. For example, they are effective at low concentrations (0.001 - 50 pg / ml, depending on the bacterial species) in inhibiting the growth in minimal in vitro medium of pathogenic bacteria, both Gram-negative and Gram-positive, such as E. coli , Proteus vulgaris, Providencia rettgeri, Salmonella typhimurium and other enterobacteria, Pseudomonas aeruginosa and pathogenic yeasts, such as C. albicans and C. tropicalis.

[US Patent 4,213,969].

The compound L-Glu-y-Pii is a chemically known compound and its chemical synthesis has been described [Khomutov et al., 2016 and cited bibliographies]. In addition to that L-Glu-γ-ΡΗ can be isolated from Streptomyces viridochromogenes and Streptomyces hygroscopicus grown under particular conditions or mutated, leading to its accumulation [Seto H et al., (1983) J Antibiot (Tokyo) 36: 96- 98; Imai S. et al. (1985) J Antibiot (Tokyo) 38: 687-690].

An L-Glu-y-PH-L-Ala-L-Ala tripeptide (consisting of L-Glu-γ-ΡΗ and two L-alanine residues) is a chemically known compound and has been isolated from microbial sources [US patent 4,466,913].

However, neither for L-Glu-γ-Ριι nor for the tripeptide L-Glu-y-PH-A-Ala-L-Ala, mentioned above, the antibiotic activity has never been described in the scientific literature or in patents.

Summary of the invention

The present invention relates to both known and unknown compounds of Formula 1 to be used as disinfectants and antiseptics, in the treatment of bacterial infections. The compounds of Formula 1 are particularly suitable as antimicrobial agents for use against Gram-positive and Gram-negative pathogenic bacteria, in particular against pathogens resistant to common antibiotics, as highlighted in E. coli (Table 1).

The compounds in Formula 1 have been shown to possess antibiotic properties and are proposed as active ingredients in formulations against pathogens and for the treatment of pathologies associated with bacterial infections. They can be used alone or in combination with other pharmacologically active compounds and can optionally be formulated in kits for combined, sequential or delayed administration, alone or in combination with other active ingredients.

Therefore, the object of the present invention are the compounds in Formula 1 to be used in the medical field as antibiotics, antiseptics and as disinfectants, in particular:

Antibiotics for the treatment of infections caused by Gram-positive and Gram-negative bacteria, including those resistant to antibiotics currently in clinical use

Antibacterials for the deactivation or inhibition of bacterial growth on surfaces and inert objects (disinfectants) or on the skin and tissues (antiseptics)

Further objects will become evident from the detailed description that follows.

Detailed description of the invention

The present invention relates to analogues and derivatives of dicarboxylic amino acids which can be used as antibacterials, both in the treatment of bacterial infections and to counteract antibiotic resistance.

Amino acids are organic molecules which in their structure carry both the amino functional group (-NH2) and the carboxylic group (-COOH). In biochemistry, the term "amino acid" refers more often to α-amino acids, of the generic formula NH2CHRCOOH, ie those whose amino group and carboxylic group are bonded to the same carbon atom (called carbon a). The natural amino acids included in the present invention are in general those present in nature, known to the skilled in the art, which are synthesized through the specific biosynthetic pathways by living organisms (be they bacteria, archaea, plants, animals), while more specifically the proteinogenic amino acids, again within the scope of the present invention and known to the skilled in the art, are 23 and are those that are incorporated into proteins and for which therefore there is a specific codon that dictates their incorporation into the chains polypeptides during protein synthesis. The latter are all in L configuration among the natural α-amino acids. Therefore, apart from the 23 L a-proteinogenic amino acids, among the natural non-proteinogenic amino acids, the D α-amino acids as well as other compounds such as GABA (a neurotransmitter) fall within the scope of the present invention and known to the skilled in the art. alanine, omithin, canavanine, penicillamines and cephalosporins.

The term "derivatives" also includes the transamination (a-ketoglutarate-γ-Ρκ) and decarboxylation (GABA-PH) products.

The compounds of the invention have the following Formula 1 and the invention also relates to the synthesis of new derivatives that have been found to have antibacterial activity and an absence of toxicity in vivo. The compounds in Formula 1 are characterized by the presence of two pharmacophores, considered by the inventors to be responsible for their biological activity, namely the α-aminocarboxylic unit (meaning the amino unit and / or the carboxyl unit) and the phosphine group.

The present invention describes in its broadest sense the use of the compounds of Formula 1 against pathogenic microorganisms.

which includes isomers and diasteroisomers in zwitterionic form, and corresponding salts with both organic and inorganic acids and bases, in which:

n is a positive integer between 1 and 4

R = hydrogen or deuterium

Ri hydrogen, or deuterium, or Ri and R2 together form a double bond of the carbonyl type C = 0;

R2 is hydrogen, 0 deuterium, 0 an amino group, not

substituted 0 substituted with C (0) -CH (NH2) -R4,

where R4 is the side chain of a natural amino acid, preferably proteinogenic, in particular but not exclusively L-Leu, 0 L-Ala, or a di-, tri-, i-im-peptide residue

derived from proteinogenic amino acids;

R3 is hydrogen, or deuterium, 0 a carboxylic group, 0 C (0) -Rs

where R5 is the side chain of a natural amino acid, preferably proteinogenic, in particular but not exclusively L-Leu, or L-Ala, preferably a residue of-, tri-,

tetra-peptide derived from proteinogenic amino acids.

Formula 1 includes the following compounds; the known

Compound R Ri Ri and are:

2-Amino-4- (hydroxyphosphinyl) butyric acid;

R = Rt = H; R2 = NH2; /?.,= COOH

G1U-Y-PH

L-2-Amino-4- (hydroxyphosphinyl) butyric acid;

L-Glu-y-PHn = 2; R = Rt = K; NH2; K, = COOH

CAS: 126204-83-9 58,1Ds: //asischem.com/asis-0069.htmI

CAS: 85178-62-7 (for free amino acid)

n = 2; R = R

N- [L-2- Amino-4- (hydroxyphosphinyl) butyry 1] - {= H; R2 = NH2; R3 = C (0) -R5

R -, ieira-peptides formed by L- Alany 1-L-Alanine; s = di-, tri residues

natural amino acids, especially proteinogenic, L-Glu-Y-PH-L-Ala-L-Ala

preferably L-Ala-L-Ala

4- (Hydroxyphosphinyl) -2-oxobutyric acid;

n = 2; R = H; RI + R2 => 0; R3 = COOH

a-ketoglutarate-Y-PH; a-KG-Y-PH

3-Aminopropylphosphinic acid;

n = 2; R = RI = R2 = H; R3 = NH2

GABA-PHhttD: //www.chemicalbook.com/ProdSuDDlierGW EN.asD CAS: 103680-47-3 x? CBNumber = CB71339083 & ProvID = 1001

2-Amino-3- (hydroxyphosphinyl) propionic acid;

η = 1; Λ = Λ, = Η; Λ2 = ΝΗ

Asp-3-Ρπ2; «, = COOH

3- (hydroxyphosphinyl) propionic acid;

n = 2; R = Rr = R2 = H; R3 = COOH

Succinate-PH

The known compounds were prepared as described in:

Glu-y-PH: Maier L, Rist G. (1983) Phosph. Sulfur., 17, 21-28; Baylis et al. (1984)

J. Chem. Soc. Perkin Trans. I., 2845-2853; Khomutov et al. (2016) Rus. J.

Bioorg. Chem., 42 (6) (In Press).

L-Glu-γ-Ρκ: Selvam et al. (2007) J. Med. Chem., 50, 4656-4664.

L-Glu-y-Pn-l-Ala-L-Ala: Seto et al. (1983) J. Antibiot., 36, 96-98.

a-KG-Y-PH: Jpn. Kokai Tokkyo Koho (1983), JP 58219192; Jpn. Kokai Tokkyo Koho

(1989), JP 01268695.

GABA-PH: Dingwall et al. (1989) Tetrahedron., 45 (12), 3787-3808; Queffelec et al.

(2008) J. Org. Chem., 73, 8987-8991; Khomutov et al. (2016) Rus. J.

Bioorg. Chem., 42 (6) (In Press).

Α8ρ-β-ΡΗ: Dingwall et al. (1989) Tetrahedron., 45 (12), 3787-3808; Khomutov et al.

(1996) Rus. Chem. Bull., 45 (8), 1961-1964.

Succinate-PH: Prishchenko et al. (1994) Zh. Obshch. Khim., 64 (8), 1311-1312; Boyd

et al. (1990) Tetrahedron Lett., 31 (20), 2933-2936.

Formula 1 includes the following NOT known compounds:

Compound R Ri Rz and R \ are

D-2-Amino-4- (hydroxyphosphinyl) butyric acid;

n = 2; /? = /?, = H; /? 2 = NH2; RI = COOH

Z) -G1U-Y-P „(example 2)

P-Deutero- [2-amino-4- (hydroxyphosphinyl) butyric] acid; n = 2; /? = D; Rt = H; fl2 = NH2; R2 = C OOH GIu-γ-Ρη (example 3)

P-Deutero-L- [2-amino-4- (hydroxyphosphinyl) butyric] acid; n = 2; R = D; «, = H; R2 = NH2; R2 = COOH .L-G1U-Y-PD (example 3)

P-Deutero- D- [2-amino-4- (hydroxyphosphinyl) butyric] acid; n = 2; /? = D; Λ, = Η; R2 = NH2; R2 = COOH Z) -G1U-Y-PD (example 3)

7V- (L-Leucyl) -2-amino-4- n = 2; /? = /?, = H; R2 = NH-C (O) -CH (NH2) - "4; R <= (hydroxyphosphinyl) butyric acid; COOH; /? 4 = side chain of a proteinogenic L-Leu-Glu-y-PH amino acid (example 4), preferably L-Leu, L-Ala iV-L-Leucyl- [2-Amino-4- n = 2; /? = /?] = H; R2 = NH-C (O) -CH (NH2) - / 4; R2 = (hydroxyphosphinyl) butyryl] -L-Alanyl-L- C (0) -Rs; /? 4 = side chain of an Alanine amino acid; proteinogenic, preferably L-Leu; Rs = di-, tri-, teira-peptide residues formed by amino acids L-Leu-L-Glu-y-PM-L-Ala-L-Ala (example 5)

proteinogenic, preferably L-Ala-L-Ala P- £) and Miero- (3-aminopropylphosphinic) acid;

n = 2; R = D; R2 = R2 = H; R2 = NH2

GABA-PD (example 3)

n = 2; R = Ri = R2 = H; /?, = NH-C (O) -CH (NH2) - "4; A- (L-Leucyl) -3-aminopropylphosphinic acid; / f4 = side chain of a natural amino acid, in L-Leu-GABA-PH (example 6) particular proteinogenic, preferably L-Leu, or L-Ala

P-Deutero- 2-Amino-3 - n = 1; R = D; Λ, = Η; /? 2 = NH2; 3⁄4 = COOH (hydroxyphosphinyl) propionic acid;

Asp-β-Ρ ,, (example 3)

TV- (L-Leucyl) -2-amino-3- n = 1; /? = /?, = H; R2 = NH-C (0) -CH (NH2) - /? 4; "3 =

COOH; Rx = side chain of an amino acid (hydroxyphosphinyl) propionic acid;

natural, in particular proteinogenic,

L-Leu-Asp-β-Ρπ (example 7) preferably L- Leu, or L-Ala

2-Amino-5- (hydroxyphosphinyl) valeric acid;

n = 3; R = R \ = H; /? 2 = NH2; 3⁄4 = COOH AACÌ-S-PH (example 8)

L-2-Amino-5- (hydroxyphosphinyl) valeric acid;

n = 3; R = Ri = H; fl2 = NH2; Λ., = COOH

L-AACÌ-5-PH (example 9)

iV- [L-2-amino-3- (hydroxyphosphinyl) propionyl] n = 1; R = R {= H; ff2 = NH2; R3 = C (0) - /? 5; Rs = di-, tri-, tetra-peptide residue consisting of L-Alanyl-L-Alanine;

natural amino acids, in particular

Asp-P-PH-I <-Ala-.L-Ala (example 10)

proteinogenic, preferably L-Ala-L-Ala

Particularly preferred compounds are:

or

HJ? HJI H ^ H

COOH; , P - \ / \ XOOH

HO '* HO Η0' <Ρ'ν / '> Ύ COOH

NH2NH2NH2

2-Amino-4- (hydroxyphosphinyl) butyric acid (Glu-y-PH)

L-GIu-y-PHA ^ -GIu-γ-ΡHD-GIu-γ-Ρκ

COOH

<H>>

HO or NH2

2-Amino-3- (hydroxyphosphinyl) propionic acid (Asp-β-Ρπ)

O COOH

.H

OH

NH, O

yV- (L-Leucyl) -2-amino-4- (hydroxyphosphinyl) butyric acid (L-Leu-rac-Glu-y-PH)

The inventors also perfected the synthesis of compounds

new (i.e. NOT known):

Peptide derivatives of Formula 1

The syntheses of the peptide derivatives of the general Formula 1 are

were performed starting from unprotected aminophosphinates; or Glu-γ

By being already incorporated into di-, tri-, or tetrapeptides having free carboxy and amino groups. The elongation or construction of the peptide chain was obtained using an activated derivative of the N-protected amino acid capable of reacting in organic-water mixtures. The activations used are those commonly used in peptide chemistry, preferably the activated esters, for example the N-hydroxysuccinimide esters. [Anderson et al. (1964) J. Am. Chem. Soc. 86, 1839-1842, and Houben-Weyl Volume 15/2, page 149 et seq.]. The N-protecting groups used are those commonly used in peptide chemistry, for example the benzyloxycarbonyl group (Cbz) and the iert-butoxycarbonyl group (Boc). The reactions were carried out in an aqueous organic solvent mixture, such as ethanol / water, 1,4-dioxane / water, tetrahydrofuran / water or 1,2-dimethoxyethane / water, at temperatures between 0 ° C and 50 ° C, preferably at room temperature. Suitable auxiliary bases in aqueous organic systems are alkali metal carbonates and bicarbonates, or equivalent quantities of alkali metal hydroxides, such as sodium hydroxide, preferably sodium and potassium carbonate / bicarbonate; or tertiary amines such as triethylamine. The protecting groups which remain in the molecule after the peptide chain has been constructed can be cleaved by methods known in the literature, for example the Cbz group for hydrogenolysis, the Boc group for acidolysis and the ester groups for acid or alkaline hydrolysis. Depending on the nature and sequence of the removal of the protecting groups, the peptides of Formula 1 according to the invention are obtained in free form or as salts. The salts can be converted into free peptides by methods known in literature (for example by reacting the hydrochlorides / hydrobromides with propylene oxide) or with ion exchange resins, preferably Dowex 50 in the H <+> form. Salts are obtained from free peptides by titration with an equivalent of acid or base and evaporating the aqueous solution.

D-2-Amino-4- (hydroxyphosfinyl) butyric acid (D-Glu-γ-ΡΗ) The compound ZJ-Glu-γ-ΡΗ was enzymatically prepared and isolated from the corresponding racemic mixture (rac-Glu-γ-Ρκ ) which was initially subjected to a decarboxylation reaction with the enzyme glutamate decarboxylase (GadB) of Escherichia coli until complete exhaustion of the L isomer, substrate of GadB, leaving intact the D isomer, which was then isolated by chromatography a ion exchange from the decarboxylation product of L-Glu-γ-ΡΗ (GAB A- Pausing a Dowex 50Wx8 ion exchange resin.

L-2-amino-5- (hydroxyphosfinyl) valeric and racemic acid- (L-AAd-δ-Ρκ and AAd-δ-Ριι)

AAd-δ-ΡΗ and L-AAC1-5-PH were prepared starting respectively from the corresponding racemic mixture and the L isomer of the allylglycine compound protected by known methods of peptide chemistry; for example, lower alkyl groups and benzyl groups are suitable for protecting the acid groups, while the N-protecting groups used were those commonly used in peptide chemistry, for example the benzyloxycarbonyl (Cbz) group. Thus, preferably methyl- N- (benzyloxycarbonyl) allylgbcine, followed by the radical (anti-Markonikov) addition of hypophosphorous acid in water-alcohol mixtures, preferably methanol / water at temperatures between 40 ° C and 100 ° C, preferably between 50 ° C and 80 ° C, and subsequent deprotection and isolation of the target compound by ion exchange chromatography.

P-Deutero - derivatives of the compound of Formula 1

The compound of Formula 1 containing the phosphorus-hydrogen bond was dissolved in a solution of volatile deuterated strong acids in deuterium oxide (D2O), preferably deuterium chloride (DC1) in D2O, and incubated at a temperature between 10 ° C and 80 ° ° C, preferably between 20 ° C and 50 ° C, until no proton signal> P-H has been detected in the Ή-NMR spectra. It is subsequently neutralized with proton-free organic bases, or with propylene oxide and then crystallized to obtain the corresponding desired> P-D derivatives with high yields.

Other deuterated derivatives

When Ri or R2 in the compound of Formula 1 are deuterium, this can also be introduced by deuteration of a compound of Formula 1 where Ri or R2 is hydrogen, or by deuteration of suitable precursors of the compound of Formula 1, by means of processes known to those skilled in the art. .

4- (hydroxyphosfinyl) -2-oxobutyric acid (a-ketoglutarate-γ-PH; a-KG-γ-Ριι)

a-KG-γ-ΡΗ is a known compound and its synthesis is described in two patents [Kokai Tokkyo Koho (1989), JP 01268695 and Jpn. Kokai Tokkyo Koho (1983), JP 58219192]. The first suggests reacting H0PH (0) CH2CH2C02R (R = Ci-β alkyl) with di (2-ethylhexyl) oxalate in toluene in the presence of sodium ethylate at temp. room for 24 h. the subsequent hydrolysis (20% HC1, reflux, 8 hr) allows to obtain H0PH (0) CH2CH2C0C02H (CL-KG-Y-PH) in high yields. This protocol is very similar to that used to prepare the α-ketoglutaric acid itself [Organic Syntheses, Coll. Vol. 5, p.687 (1973); Vol 44, p.67 (1964). D01: 10.15227 / orgsyn.044.0067]. However, according to our knowledge, a-KG-γ-ΡΗ is unstable at reflux in 20% HCl (unpublished data). The second patent used nonenzymatic transamination (G1U-Y-PH and glyoxalic acid in the presence of Ni <2+> ions in water-pyridine solution) and isolation of Q-KG-Y-PH using subsequent chromatography on Amberlite IR120B (H <+>), DEAE-Sephadex A-25 (Cl ·) and Sephadex G-10. This allowed to obtain the target compound a-KG-γ-ΡΗ with a yield of 39.6%. In the present invention (Example 10) we used pyridoxal for the non-enzymatic transamination of Glu-γ-PH in the presence of Al <3+> ions. This allowed to simplify the isolation of a-KG-γ-ΡΗ using a single chromatography on Dowex 50Wx8 resin (form H <+>) which led to obtain the target compound a-KG-γ-ΡΗ with an overall yield of 45 %.

In order to investigate its antibacterial potential and the possibilities of use in the treatment of infections due to bacteria, both Gram-negative and Gram-positive even in the presence of multiresistances developed towards other antibiotics, the most representative compounds among those listed here and above are indicated Structural formulas were analyzed for their ability to influence the growth of E. coli K12 strain MG1655. To test the effectiveness of these molecules also on bacteria resistant to commonly used antibiotics, experiments were conducted on E. coli K12 MG 1655 transformed in order to carry the genes for antibiotic resistance Ampicillin (Amp) and Kanamycin (Kan).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were analyzed in minimal medium (minimum E containing glucose) [Vogel and Bonner, 1955, J. Biol. Chem. 218, 97-106].

Table 1. MIC90 of Table Compounds (pg / mL) in E. coli MIC90 Compound (ug / mL) Amino Acids

AL-Glu-y-Pn 7 AL-Glu-y-Ps * 1800 (MIC20) L-Glu-γ-Ρπ 6-7 L-Glu-y-Pn 6-7 in resistant Kan Amp strain

D-Glu-y-PH128 (MIC10) D.L-Asp-P-Pn 1800 (MIC50) Dipeptides

L-Leu-Z), L-Glu-y-Pn 0.1-1 Antibiotics

Kanamycin ** 16 Ampicillin ** 8

* the phosphonic compound (D.L-Glu-y-Ps) is exclusively reported for comparison

** from: Chiang et al (2011) J. Exp. Microbiol. Immunol., 15, 59-63.

The experiments whose results are reported in Table 1 are detailed in the examples and were conducted both on bacteria of the strain of E. coli MG1655 and on bacteria of the same strain modified with techniques known to the skilled in the art, in order to present a double resistance to Kanamycin and Ampicillin (Kan Amp).

The compound L-Glu-γ-ΡΗ is the active isomer in the racemic mixture and exerts a bacteriostatic effect in the low micromolar range. The presence of the phosphine group is essential for the molecule to enter the cell and for effective intracellular activity, as evidenced by the significant difference in the MIC90 value between L-Glu-γ-ΡΗ and its phosphonic derivative (-PO3H2, P5 ) L-Glu-γ-Ρδ (reported in Table 1 for comparison only). Furthermore Γ L-Glu-γ-Ριι is found to be much more active than its carbon-lacking analog, ASP-6-PH.

An unaltered MIC of L-Glu-γ-ΡΗ on a strain resistant to two known bactericidal antibiotics, Ampicillin (a 6-lactam antibiotic) and Kanamycin (an aminoglycoside antibiotic), suggests that L-Glu-γ- ΡΗ it acts by interfering with processes totally different from the target ones of the two antibiotics and that its penetration and effectiveness in the cell is not altered by the presence of these antibiotics.

When the L-Glu-γ-ΡΗ compound is functionalized with other amino acids, as in the case reported in Table 1 for the dipeptide L-Leu-AÌ'-Glu-y-PH, there is a MIC at least 10 times lower than the L-Glu compound. -γ-ΡΗ as such, suggesting that the molecule performs better its bacteriostatic activity as a dipeptide, probably because it enters the target cell more easily by means of dipeptide transporters, showing promise for further study.

Preliminary metabolomics studies (data not shown) on E. coli MG1655 cells treated with G1U-Y-PH indicate that GABA-PH, Succinate-PH and CL-KG-PH are present. To indicate that compounds that also fall into formula 1 are its metabolites and therefore L-Glu-γ-ΡΗ is in effect a prodrug molecule, that is, a precursor of the active ingredient.

Finally, the inventors verified the cytotoxic and antiproliferative effects of the compounds according to the invention on HeLa (ATCC® CCL-2 ™) and Caco2 (ATCC® HTB-37 ™) cell lines grown in microplates and treated with MTT, a dye that is converted only in live cells. In both cases even using Glu-γ-Ριι at a concentration of 1 mM no significant differences (data not shown) are observed between treated and untreated cells, meaning that the Glu-γ-ΡΗ compound is neither toxic nor has antiproliferative effects.

This is also confirmed by the limited toxicity in animal models (LD50 in male and female mice were respectively: 2.7 and 2.9 g / kg, per os; 1.1 and 1.3 g / kg after i.p injection; in rats> 5.0 g / kg; Yonaga T . et al. (1982) Yakuri to Chiryo 10 (11), 43-51 (in Japanese)).

The compounds of the invention can be administered, alone or in combination with other antimicrobial agents. An advantageous aspect of the compounds is that their use in combination with antibiotics currently on the market such as, for example, not limiting: penicilins, cephalosporins, fluoroquinolones, aminoglycosides, monobactams, carbapenems, macrolides and related mixtures, or still in the clinical trial phase (such as e.g. β-lactams, also in combination with their inhibitors, carbapenems, aminoglycosides, quinoions, macrolides, glycopeptides, sulfamides, tetracyclines, oxozolidinones, fusidic acids, pleurimutilins, cephalosporins, membrane-acting agents, such as peptides, antimicrobials peptide deformylase, fatty acid synthesis inhibitors, antituberculosis, nitroimidazoles, and any combination thereof, antibiotics and any combination thereof) is more effective than any of the compounds used alone. The compounds of the invention have low toxicity to mammals (LD50 in male and female mice respectively: 2.7 and 2.9 g / kg, per os; 1.1 and 1.3 g / kg after i.p injection; in rats> 5.0 g / kg) and can be used for the treatment of diseases in animals, especially mammals, and in humans, and can be used as antiseptic agents for the protection, in addition to living beings, also of materials from microbial contamination.

Consequently, the invention provides therapeutic compositions comprising a pharmaceutically effective amount of a compound of formula 1 and at least one vehicle or a pharmaceutically acceptable excipient among those known to the skilled in the art, such as a solid support or a liquid diluent.

The pharmaceutical compositions according to the invention contain at least one compound of general formula 1 as an active substance together with a conventional pharmaceutical carrier. The type of carrier that can be used depends largely on the specific use, that is, for example, to disinfect healthy skin, disinfect wounds, in the treatment of dermatitis and mucosal diseases caused by bacteria and in diseases of the uro-genital system, Formulations such as ointments, powders, sprays, tinctures, and bandages are used in particular.

Formulations for gargling or concentrates for their preparation, and tablets for slow dissolution in the mouth, are suitable for the disinfection of the oral cavity and throat.

Solid dosage units, especially tablets, dragees (dragees) and capsules, are convenient for use in intestinal disinfection.

In all forms of administration the compounds of the general formula 1 can be present as single active ingredients or can also be combined with other ingredients or can also be combined with other known pharmaceutically active compounds, and above all antibacterial and / or antifungal and / anti-inflammatory or other antimicrobial active substances, for example to broaden the range of applications.

The invention also provides a method for protecting an organic / inorganic material, for example an artifact, including surfaces, vessels, instruments, equipment and other medical equipment, susceptible to bacterial attack. The method comprises the step of treating the material with a formulation comprising the compound of Formula 1. The organic material can be a synthetic natural or polymeric material, a protein or carbohydrate-based surface, or a natural or synthetic fiber or textile material. .

The compounds of the present invention can be administered to a patient, (human or animal) in pharmaceutical compositions in which they are mixed with suitable solid or liquid pharmaceutically acceptable carriers or excipients suitable at doses capable of improving or treating a series of conditions. These compositions may also contain, in addition to the compounds of the invention and the carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers and other components well known in the art. The term "pharmaceutically acceptable" means non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.

The techniques for the formulation and administration of the compounds of the invention can be easily found [Gennaro, A. R. (ed) (2000) Remington: the Science and Practice of Pharmacy, 20 <th> edition. Lippincott Williams and Wilkins, Baltimore, MA; Gennaro, A. R. (ed) (2000) Remington's Pharmaceutical Sciences, 20 <th> edition. Mack Publishing Co., Easton, PA]. Further, a therapeutically effective dose refers to that amount of compound sufficient for the improvement of symptoms, for example, treatment, cure, prevention or improvement of the related medical status.

Suitable routes of administration may, for example, include oral, rectal, vaginal, transmucosal, or intestinal ingestion; parenteral, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. The administration of the compounds of the present invention which can be used in the pharmaceutical compositions or in the treatment methods according to standard protocols, can be carried out in a variety of conventional routes, such as oral ingestion, inhalation, topical or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous application.

The inhalation and topical routes are preferred administration routes for the purposes of the invention because they are easy to use. For use by inhalation, the compounds are diluted in a suitable solvent, generally aqueous, and sprayed in the form of aerosols on the mucous surfaces of the body cavities accessible from the outside. For topical use, the compounds are mixed with appropriate vehicles, obtaining creams, gels, oils, emulsions spread on the skin surface. Another possible topical application is that which uses transdermal therapeutic systems (patches and the like) to be applied to the epidermis so that the active ingredient is released and absorbed by the skin continuously and almost constantly over time.

Another type of formulation is the liquid one as a disinfectant composition with the active ingredient combined with the usual liquid formulations, within the reach of the person skilled in the art.

The compounds of formula 1 are able to inhibit bacterial growth in a dose-dependent manner with MIC90 in minimum medium <10, for example between 10 and 0.1.

The compounds of formula 1 are able to inhibit bacterial growth even in the presence of multi-resistances and the deuterated compounds are also active.

The compounds of formula 1 are effective against pathogenic bacteria such as: Escherichia coli, Proteus vulgaris, Providencia rettgeri, Salmonella typhimurium and other enterobacteria such as Clostridium difficile, as well as against Pseudomonas aeruginosa and yeasts such as Candida albicans and Candida tropicalis, also including pathogens that show resistance to common antibiotics such as methicillin-resistant Staphylococcus aureus.

The following examples are given to illustrate the invention and are not to be considered as limiting its scope.

EXAMPLES

Example 1

Thin layer chromatography was performed on Cellulose F254 plates (Merck) in the system: i-PrOH-25% NH4OH-H2O, 7: 1: 2 (A). The aminophosphinates were revealed on the plates with ammonium molybdate reagent or with ninhydrin staining. The products were purified on Dowex 50Wx8 (BioRad) resin (100-200 mesh) in H <+> form (column dimensions and eluents are indicated in the text); while the intermediates were purified on Kieselgel (Merck) 0.040-0.063 mm (column dimensions and eluents are indicated in the text).

The NMR spectra were recorded in D2O on a Bruker Avance AMX III 400 (Germany) spectrometer with a working frequency of 400.1 MHz for Ή NMR (sodium 3-trimethyl-l-propane sulfonate as internal standard) 100.1 MHz for <13> C ( with carbon-proton decoupling, sodium 3-trimethyl-1-propanesulfonate as internal standard) and 162 MHz for <31> P (with phosphorus-proton decoupling, if not specified, 85% H3PO4 as internal standard); Chemical shifts are given in ppm.

High resolution mass spectra (HRMS) were recorded on a Bruker Daltonics micrOTOF-Q II instrument using electrospray ionization (ESI). The measurements were acquired in positive ion mode with the following parameters: voltage at the capillary interface 4500 V; mass range from m / z 50 to 3000; external calibration (electrospray Calibrant Solution, Fluka); atomization pressure 0.4 bar; flow rate 3 ml / min; nitrogen was applied as a dry gas (4 L / min); the interface temperature was set at 200 ° C.

Melting points were determined in open capillary tubes on Electrothermals Mel-Temp 1202D instrument and are incorrect.

Example 2

Z> -2-Amino-4- (hydroxyphosfinyl) butyric acid; (D-GIU-Y-PH) The compound D-Glu-γ-ΡΗ was prepared enzymatically and isolated from the corresponding racemic mixture (rac-Glu-γ-Ρκ) which was initially subjected to a decarboxylation reaction with the Escherichia coli glutamate decarboxylase (GadB) enzyme until complete exhaustion of the L isomer, substrate of GadB, leaving intact the D isomer, which was then isolated by ion exchange chromatography Dowex 50. In short, 167 mg of the racemic mixture D, L-G1U-Y * PH (equal to 1 mmol of compound) were weighed and dissolved in a final volume of 1.25 ml of 0.2 M Pyridine-HCl buffer, pH 4.6, containing 1 mM PLP and 0.1 mM DTT . This solution was mixed with an equal volume (1.25 ml) of GadB of E. coli (concentrated 2 mg / ml) dissolved in the same buffer in order to have a final concentration of D, L-Glu-γ-PH of 0 , 4 M and GadB of 1 mg / ml.

The reaction was periodically monitored to evaluate the pH and, when necessary, correct it with additions of HC1 IN in order to keep it constant at values between pH 4.6 and pH 5.5, thus ensuring that the enzyme was always active (> 50% of the maximum activity). At intervals of times, 15 µl / 2500 µl were taken and transferred to an eppendorf tube containing 10 µl of 250 mM NaOH in order to instantly stop the reaction. The resulting 25 µl were transferred into NMR sample tubes after dilution in 600 µl of D2O (dilution 1:25) and analyzed on a Bruker 500 UltraShieldTM instrument. For simplicity and for the relative quantization of the substances present in the reaction mixture, the kinetics was followed as 31P NMR, but the same samples were also analyzed in 1H NMR. The 31P signal in the D, L-G1U-Y-PH (28.98 ppm) gradually decreases until it reaches about 50% of the starting value. At this point the reaction was stopped as all the L isomer was converted into its decarboxylation product.

Then 7.8ml of MilliQ water was added to 2.2ml of filtrate from the bioconversion reaction to give a total volume of 10ml, which was applied to a Dowex 50 column (20ml; 1.7 x 9 cm) thus allowing the sample to enter the column at a rate of 1 ml / min.

Chromatography was started with 400ml of MilliQ water. The D-Glu-γ-ΡΗ isomer, which did not react in the bioconversion reaction, was then eluted by initially applying 40 ml collected in a flask, while the subsequent ones were collected in 22 fractions (n ° 1 → 20) of 5 mi each and then another 30 fractions (from n ° 21 to n ° 50) from 0 mi each. At the end of this elution a TLC was carried out to check the elution of the D-Glu-γ-ΡΗ. The fractions from n ° 6 to n ° 31 were then combined and their total content evaporated in a rotary evaporator until the Z <)> - G1U-Y-PH powder was completely dried, which was recrystallized from an ethanol / water mixture to give 62 mg (yield of 37%, calculated from TOC-GIU-Y-PH), mp 222-223 ° C, dee., [a] D <20> -20.4 ° ( 1% in water), Rf 0.24 (A). Ή-NMR (D20) δ: 6.98 (dt, IH, VHP523 Hz, <3> JHH1.5 Hz, P-H), 4.09 (t, IH, <3> JHH6.1 Hz, CH), 2.22-2.07 (m , 2H, CH2P), 1.83-1.61 (m, 2H, CIfcCH). 'C-NMR (D20) δ: 174.91 s, 56.15 d (<3> JCp16.2 Hz), 29.18 d (VCP88.8 Hz), 25.08 s. s <i> p-NMR (D20) δ: 29.10 s.

Example 3

P-Deuterated aminophosphinic acids (Glu-γ-Ρϋ, D-GIU-Y-PD, L-Glu-y-PD, GABA-PD, Asp-β-Ρϋ). General synthesis procedure.

A solution of 167 mg (1 mmol) of Glu-γ-ΡΗin 37% DC1 (5 mL) was incubated at 20 ° C for 4 hours, evaporated to dryness in vacuo, coevaporated in vacuo with D20 (2 x 5 mL) and then incubated in 37% DC1 (5 mL) for further 4 hours which according to NMR [<31> P-NMR (35% DC1, uncoupled protons) δ: 35.87 (t, VDP88.4 Hz), <31> P- NMR (35% DC1, coupled protons) δ: 35.87 (t, VDP88.4 Hz), <31> P-NMR (35% DC1, deuter-decoupled) δ: 35.87 (s)] showed the formation of compound with phosphorus-deuterium bond and the absence of the phosphorus-hydrogen compound. The reaction mixture was co-evaporated in vacuo with D20 (2 x 5 mL) and the residue was dissolved in the minimum volume of methanol-^. The solution thus obtained was neutralized with propylene oxide, diluted with an equal volume of isopropanol and kept at -20 ° C until the end of the precipitation which allowed to obtain, after drying in vacuo on P2O5 HO mg (66%) of Glu -γ-Ρϋ. Ή-ΝΜβ (D2O) δ: 4.06 (t, IH, <3> JHH6.0 Hz, CH), 2.18-2.01 (m, 2H, CH2P), 1.78-1.57 (m, 2H, CH2CH).

Using the same protocol, L-Glu-γ-Ρο, GABA-PD and Asp-β-Ρϋ can be obtained with a high yield.

Example 4

IV- (L-Leucyl) -2-amino-4- (hydroxyphosfinyl) butyric acid (L-Leu-Glu-γ-ΡΗ).

127 mg (1.5 mmol) of NaHCOe, 158 mg ( 1.5 mmol) of NaHC03e glima (1 mL) followed by a 1.08 g (3 mmol) solution of the N-benzyloxycarbonyl-L-Leucine IV-hydroxysuccinimide ester in glyme (5 mL) freshly recrystallized (from isopropanol ). The practically clear solution thus obtained was left for 16 hours at 20 ° C. Glime was removed in vacuo, water (10 mL) was added, and the resulting solution was extracted with ethyl acetate (2 x 5 mL), acidified to pH 1 with 37% hydrochloric acid and extracted with acetate of ethyl (3 x 7 mL). The last extracts in ethyl acetate were washed with water (3 mL), brine (5 mL) and dried over magnesium sulfate. The ethyl acetate solution was concentrated in vacuo and acetic acid (3 mL), anisole (0.2 mL), and 35% HBr / AcOH (2.2 mL) were added to the residue. After 1.5 hours at 20 ° C the reaction mixture was combined in ethyl ether (50 ml) and left for 5 hours at -20 ° C. From the separated oil (after washing it with ethyl ether) the L-Leu-Glu-γ-ΡΗ target was isolated by column chromatography of Dowex 50Wx8 resin (100-200 mesh) in H <+> form (column volume 12 mL, elution with water). The fractions containing L-Leu-Glu-γ-ΡΗ were evaporated to dryness in vacuo and then vacuum dried over P2O5 / KOH to obtain 0.64 g (76% for two steps) of L-Leu-Glu-γ-ΡΗ as a colorless solid. . Rf 0.46 (A). Ή NMR (D2O): δ: 6.89 (dt, IH, VHP 514.3 Hz, <3> JHH 1.8 Hz, H-P), 4.43-4.35 (m, IH, CH-COOH), 4.00 (dd, IH, 3⁄4 /HHa7.5 & 7.4 Hz, <3> JHHb7.4 & 6.7 Hz, CH-NH2), 2.11-1.99 (m, IH, CHa-P), 1.95-1.82 (m, IH, CHb-P), 1.77 -1.50 (m, 5H, CH2-CH2-P, CH2-CH-NH2, CH- (CH3) 2), 0.93-0.89 (m, 6H, CH- (CH3) 2). <13> C NMR (D20 ): δ: 177.79 & 177.30 (2xs, COOH), 173.30 & 173.18 (2xs, CONH), 56.51 & 56.25 (2xd, <3> JCP16.6 & 16.3 Hz, CH-COOH), 54.78 & 54.61 (2xs, CH -NH2), 42.58 (s, CH2-CH-NH2), 30.26 & 30.04 (2xd, VCP 89.3 Hz, CH2-P), 26.61 (d, VCP 20.8 Hz, CH2-CH2-P), 25.82 & 25.78 (2xs , CH- (CH3) 2), 24.39 & 24.35 & 23.94 & 23.91 (4xs, CH3). <3i> p NMR (D20): δ: 29.34 & 29.17 (2xs). The signs and "x" indicate differences for the same signals and constants of two diastereomers (L, L- and LJ) -). HRMS (ESI-MS): Calculated for C10H21N2O5P [M + H] <+> 281.1261, found 281.1271.

Example 5

iV-L-Leucyl- {L- [2-Amino-4- (hydroxyphosfinyl) butyryl]} - L-Alanyl-L-Alanine (L-Leu-L-Glu-y-Pii-L-Ala-L-Ala ) Prepared as described in Example 4 starting from 78 mg (0.25 mmol) of AML- [2-amino-4- (hydroxyphosphinyl) butyryl]} - L-Alanyl-L-Alanine (L-Glu-y-PH- £ -Ala-L-Ala) and 100 mg (0.28 mmol) of the ester of the Af-benzyloxycarbonyl-L-Leucine N-hydroxysuccinimide ester freshly recrystallized (from isopropanol) which after removal of the benzyloxycarbonyl group with 35% HBr / AcOH and isolation of L-Leu-L-Glu-y-PH - ^ - Ala-L-Ala by chromatography column with Dowex 50Wx8 resin (100-200 mesh) in H <+> form (column volume 2 mL, elution with water) gave 68 mg (68% for two stages) of L-Leu-L-Glu-y-PH-L-Ala-L-Ala as a colorless solid. Ή-NMR (D2O) δ: 6.95 (d, IH, VHP520 Hz, H-P), 4.22-4.05 (m, 2H, 2x CH-CH3), 3.95 (t, IH, <3> JHH7.4 Hz, CH- NH2), 1.92-1.82 (m, 2H, CH2-P), 1.80-1.55 (m, 5H, CH2-CH2-P, CH2-CH-NH2, CH- (CH3) 2), 1.18 (d, 3H, <3> JHH7.2 Hz, CH3), 1.12 (d, <3> JHH7.2 Hz, 3H, CH3), 0.92-0.88 (m, 6H, CH- (CH3) 2). <31> P-NMR (D2O) δ: 29.14 (s). HRMS (ESI-MS): calculated for CI6H3IN407P [M + H] <+> 423.2003, found 423.2008.

Example 6

IV- (L-Leucyl) -3-aminopropylphosphinic acid (L-Leu-GABA-PH)

Prepared as described in Example 4 starting from 123 mg (1 mmol) of 3-aminopropylphosphinic acid (GABA-PH) and 397 mg (1.1 mmol) of the ester of A-benzyloxycarbonyl-L-Leucine N-hydroxysuccinimide (freshly recrystallized from isopropanol) and after removal of the benzyloxycarbonyl group with 35% HBr / AcOH and isolation of L-Leu-GABA-PH by chromatographic column with Dowex 50Wx8 resin (100-200 mesh) in H <+> form (column volume 5 mL , elution first with water and then with 1 mM HCl) gave L-Leu-GABA-PH hydrochloride, which was converted into zwitterionic form using propylene oxide (similarly to what is described in Example 3) which after drying in vacuo on P2O5 / KOH provided 137 mg (87% for two stages) of L-Leu-GABA-PH as a colorless solid. Rf 0.71 (A). Ή-NMR (D2O) δ: 6.88 (dt, IH, VHP 509.5 Hz, <3> JHH 1.6 Hz, H-P), 3.89 (t, IH, <3> J 7.3 Hz, CH-NH2), 3.31-3.19 ( m, 1H, CH2-NH), 1.66-1.46 (m, 7H, CH (CH3) 2, CH2-CH (CH3) 2, CH2-P, CH2-CH2-P), 0.91 (d, 3H, <3 > JHH 6.1 Hz, CH3), 0.90 (d, 3H, <3> JHH 6.1 Hz, CH3). <13> C-NMR (DaO) 8: 172.97 (s, C (O) NH), 54.90 (s, CH-C (O) NH), 42.85 (d, * JCp17.4 Hz, CH2-NH), 42.63 (s, CH2-CH-NH2), 31.06 (d, VCP 89.7 Hz, CH2-P), 26.71 ( s, CH- (CH3) 2), 24.16 (d, <2> JCP 32.1 Hz, CH2-CH2-P), 23.44 & 23.42 (2xs, CH3). <3i> p.

NMR (D2O) δ: 30.62 s. HRMS (ESI-MS): calculated for C9H21N2O3P [M + H] + 237.1363, found 237.1364.

Example 7

IV- (L-Leucyl) -2-amino-3- (hydroxyphosfinyl) propionic acid (L-Leu-Asp-β-ΡΗ)

Prepared as described in Example 4 starting from 306 mg (2 mmol) of rac-Asp-β-Ριι and 795 mg (2.2 mmol) of the ester of N-benzyloxycarbonyl-L-Leucine iV-hydroxysuccinimide (freshly recrystallized from isopropanol ) and after removal of the benzyloxycarbonyl group with 35% HBr / AcOH and isolation of L-Leu-Asp-8-PH by means of a chromatographic column with Dowex 50Wx8 resin (100-200 mesh) in H <+> form (column volume 10 mL , elution with water) and vacuum drying over P2O5 / KOH yielded 383 mg (total yield 72% for two steps) of L-Leu-Asp-B-PH as a colorless solid. <!> H-NMR (D2O) δ: 7.05 (d, 1H, VHP516 Hz, H-P), 4.48-4.39 (m, IH, CH-COOH), 4.02-3.98 (m, IH, CH-NH2), 2.23-2.10 (m, IH, CHa-P), 2.01-1.89 (m, 1H, CHb-P), 1.78-1.57 (m, 3H, CH2-CH-NH2, CH- (CH3) 2), 0.94-0.89 (m, 6H, CH- (CH3) 2) . <31> P-NMR (D20) 6: 21.9. HRMS (ESI-MS): calculated for C9H19N2O5P [M + H] <+> 267.1104, found 267.1107.

Example 8

2-Amino-5- (hydroxyphosphmyl) valeric acid (AAd-δ-ΡΗ) A solution of 790 mg (3 mmol) of methyl iv-benzyloxycarbonylallylglycine (from rac-allylglycine), 2.2 g 50% acid aq. hypophosphorous (26 mmol) and 41 mg (0.25 mmol) of α, α, '- azoisobutyronitrile in methanol (20 mL) was refluxed under Argon atmosphere for 5 hr and by addition of water (15 mL) the methanol was removed in vacuo. The residue was extracted with ethyl acetate (2 x 10 mL), the organic extracts were washed with water (3 mL), brine (5 mL) and dried over magnesium sulfate. The ethyl acetate solution was concentrated in vacuo and methyl N-benzyloxycarbonyl-AAd- □ -PH SU column Kieselgel (3.5 x 12 cm) was isolated from the residue, using dioxane-25% NH4OH, 85/15 for elution. The fractions containing methyl ZV-benzyloxycarbonyl-AAd-δ-ΡΗ were evaporated to dryness in vacuo, 20% HCl (35 mL) was added to the residue and refluxed in an Argon atmosphere for 3 hr. the reaction mixture was evaporated to dryness in vacuo AAd-Π-Ρΐί was isolated on Dowex 50Wx8 resin (100-200 mesh) in H <+> form (column volume 30 mL, elution with water). The fractions containing AAd-δ-ΡΗ were evaporated to dryness in vacuo and then dried in vacuo over P2O5 / KOH to yield 109 mg (20% as calculated for allylglycine, i.e. for 4 stages) of AAd-δ-ΡΗ as a colorless solid; Rf 0.26 (Al ^ H-NMR (D20) 6: 7.01 (d, IH, VHP513 Hz, P-H), 3.98 (t, IH, 3⁄47HH5.9 Hz, CH), 2.12-1.98 (m, IH, CHa -P), 1.96 1.80 (m, 1H, CHb-P), 1.73-1.45 (m, 4H, CH-CH2-CH2). <31> P-NMR (D20) δ: 32.3. HRMS (ESI-MS) : calculated for C5H12NO4P [M + H] <+> 182.0577, found 182.0580.

Example 9

L-2-Amino-5- (hydroxyphosfinyl) valeric acid (L-AAd-δ-ΡΗ) Prepared as described in Example 8 starting from 920 mg (3.5 mmol) of methyl iV-benzyloxycarbonyl-L-allylglycine (from L -allylglycine), which yielded 115 mg (18% as calculated for L-allylglycine, i.e. for 4 stages) of L-AAd-δ-ΡΗ as a colorless solid. The Rf value and the <1> H- and <31> P-NMR spectra are identical to those of the compound described in Example 8. HRMS (ESI-MS): calculated for C5H12NO4P [M + H] <+> 182.0577, found 182.0579.

Example 10

4- (hydroxyphosphmyl) -2-oxobutyric acid (a-ketoglutarate-γ-PH; O-KG-Y-PH)

A solution of 240 mg (1.44 mmol) of rac-Glu-γ-ΡΗ, 422 mg (2.07 mmol) of pyridoxal hydrochloride and 50 mg (0.21 mmol) of aluminum chloride hexahydrate in water (120 mL) were brought to pH 6.8 with diluted ammonium hydroxide, then refluxed for 1.5 hr and evaporated in vacuo to 15 mL. Aketoglutarate-γ-ΡΗ was isolated from this solution by means of a chromatographic column on Dowex 50Wx8 resin (100-200 mesh) in H <+> form (40 mL column volume, elution with water), collecting fractions of "2,4-dinitrophenyl hydrazine positive ”, which were combined, evaporated to dryness in vacuo, dissolved in water and then lyophilized to give 108 mg (45%) of CL-KG-Y-PH as a semi-waxy solid. a-KG-γ-ΡΗ exists in solution as an equilibrium of the Keto- and Diol forms. Keto-form:

Ή-NMR (D20, pH 1.8): δ: 7.05 (dt, IH, <I> JHP 546.9 Hz, 3⁄4 / HH 2.0 Hz, H-P), 3.10 (ddd, 2H, <3> </ΗΗ37.7 Hz, 3⁄4 / ΗΙ3⁄4 7.8 Hz, 3⁄4 / HP 13.9 Hz, CH2-C (O)), 2.12-1.89 (m, 2H, CH2-P). <13> C-NMR (D20, pH 1.8): δ: 196.30 (d, 3⁄4 / CP 12.4 Hz, C = 0), 163.05 (s, COOH), 30.77 (s, CH2-C (0)), 22.80 (d, VCP 92.8 Hz, CH2-P). <3i> p-NMR (D20, pH 1.8): 8: 33.42 (s).

Rung-shape: Ή-NMR (D20, pH 1.8): 8: 7.00 (dt, IH, VHP 544.7 Hz, <3> JHH 1.8 Hz, H-P), 2.12-1.68 (m, 4H, CH2-P & CH2- C (OH) 2). <13> C-NMR (D20, pH 1.8): 8: 174.04 (s, COOH), 94.2 (d, <3> JCP 17.9 Hz, C (OH) 2), 29.92 (s , CH2-C (OH) 2), 23.58 (d, VCP 91.7 Hz, CH2-P). <3i> p. NMR (D20, pH 1.8): 8: 34.43 (s).

Growth media composition

Minimum EG p H 7:

Minimum E medium at concentration IX pH 7 [Vogel and Bonner, 1955, J. Biol. Chem. 218, 97-106].

D-glucose 0.4%

Growth and treatment of bacteria in medium EG minimum pH 7 The bacterial strain E. coli K12 MG1655 is taken from stock in glycerol (-80 ° C), the strain is streaked on LB-Agar plate and incubated at 37 ° C when light ( ON). The following day a second passage is carried out on LB-Agar plate with subsequent incubation at 37 ° C ON. The next day a pre-inoculum is carried out (taking about 1 cm from the plate with a sterile loop) in 2 mL of LB and nightmare at 37 ° C ON. Then the ON culture is centrifuged at 3500 rpm for 15 minutes and the pellet is resuspended in 2 mL of saline (0.9% NaCl). The optical density (O.D.) at 600 nm is corrected with physiological solution to obtain an O.D. 600 nm = 1.

One proceeds by carrying out a 1:25 inoculum in 3 mL of minimum EG pH 7 medium and incubating the culture at 37 ° C under moderate stirring. As soon as the culture reaches (within the day) Ο.Ό.βοο between 0.35-0.4 (approximately 7-8 hours from inoculation), a new dilution 1:25 is made in 3 mL of Minimum EG pH 7 medium and proceeds by inoculating 1:10 on a 96-well microplate previously prepared with suitable dilutions of the compound under study in Minimo EG pH 7 medium. 1OD is then recorded at time 0 by means of a special microplate reader and finally incubated at 37 ° C.

From the 13th hour, for each hour, the O.D is recorded. inoculation on a microplate in order to obtain a growth curve over time.

The negative control (white) is represented by the same medium in which, however, the bacteria have not been inoculated, while the positive control is represented by the bacterial strain {E. coli K12 MG1655) grown on a microplate in the same medium but in the absence of molecules in table 1.

For the experiments with E. coli MG1655 Amp Kan resistant strain the strain is streaked on LB-Agar plate, cultured in LB and then cultured exactly as above except that in all cases AmplOO pg / ml and Kan 25 pg / are always present. ml.

Live cell count and MIC and MBC calculation

In order to derive the starting colony forming units (CFU / mL), 100 ul of the 1:25 dilution in 3 mL of medium EG pH 7 are diluted 1:10 in 1 ml of physiological solution in a sequential manner (dilution method serial) until a final dilution 1: 1000 is obtained. 10 ul of this dilution are plated on LB-Agar (in triplicate) and the plates incubated at 37 ° C ON.

The next day we proceed with the count of live cells from which the CFU / mL value is then obtained.

The MIC is calculated starting from the values of the optical densities (O.D.

595 nm) obtained from the microplate reading at the 20th hour of incubation by applying the following formula:

% inhibition = [1- (O.D. well / O. D. negative control)] * 100 The negative control is the same culture in the absence of the compound.

The MBC is obtained by plating on LB-Agar 10 and 100 ul coming from the well containing a concentration of the compound under study 4 times higher than the MIC. The plates are finally incubated at 37 ° C ON.

Cellular toxicity test

The experiments on HeLa cells were conducted in standard medium for the growth of these cells, which is DMEM with a high concentration of glucose (Life Technologies) and containing 10% Fetal Bovine Serum (FBS; Sigma) on a 96-well microplate, with 1.2 x 10 <4> cells per well - kept in incubator at 37 ° C, humidity 95%, 5% CO2.

The experiments on Caco2 cells were conducted in standard medium for the growth of these cells and which is DMEM with a low concentration of glucose (Life Technologies) and containing 20% Fetal Bovine Serum (FBS; Sigma) on a 96-well microplate, with 1.5 x 10 <4> cells per well - kept in incubator at 37 ° C, humidity 95%, 5% CO2.

The cells were then treated with MTT, a dye that is converted only in living cells [Mosmann, 1983. Journal of Immunological Methods. 65: 55-63]. In both cases D, L-Glu-y-PH up to a concentration of 1 mM was used to evaluate the possible toxic or antiproliferative effects.

Claims (1)

CLAIMS 1. Compounds for pharmaceutical use as antimicrobial agents against Gram-positive and Gram-negative pathogenic bacteria, in particular against pathogens resistant to antibiotic cornymes, said compounds having the following Formula 1: OR R P- (CH2) n-C-R3 I HAVE Re Formula 1 which includes isomers and diasteroisomers in zwitterionic form, and corresponding salts with both organic and inorganic acids and bases, in which: n is a positive integer between 1 and 4 R = hydrogen or deuterium Ri is hydrogen, or deuterium, or Ri and R2 together form a double bond of the carbonyl type C = 0; R2 is hydrogen, 0 deuterium, 0 an amino group, unsubstituted 0 substituted with C (0) -CH (NH2) -R4, where R4 is the side chain of a preferably proteinogenic natural amino acid, in particular but not exclusively L-Leu, or L-Ala, preferably a di-, tri-, iira-peptide residue derived from proteinogenic amino acids; R3 is hydrogen, 0 deuterium, 0 a carboxylic group, 0 C (0) -R5 where Rs is the side chain of a natural amino acid, preferably proteinogenic, in particular but not exclusively L-Leu, 0 L-Ala, preferably a residue di-, tri-, ieira-peptide derived from proteinogenic amino acids, 2. Compounds according to claim 1 which are prodrugs. 3. Compounds according to claims 1-2 wherein at least one of R, R1, R2and R3 is deuterium. Compounds according to any one of claims 1-3 for use as antibiotics and as active ingredients in formulations against pathogens and for use in the treatment of pathologies associated with bacterial infections. 5. Compounds according to any one of claims 1-4 for use alone or in combination with other pharmacologically active compounds, preferably formulated in kits for combined, sequential or delayed administration. 6. Compounds according to any one of claims 1-5 for use in the medical field as antibiotics, antiseptics and as disinfectants, in particular: as antibiotics for the treatment of infections caused by Gram-positive and Gram-negative bacteria, including those resistant to antibiotics currently in clinical use; as antibacterials for the deactivation or inhibition of bacterial growth on surfaces and inert objects (disinfectants) or on the skin and tissues (antiseptics). 7. Compound according to Formula 1 of claim 1 for medical use selected from: 2-Amino-4- (hydroxyphosfinyl) butyric acid; Glu-γ-ΡΗ, L-2-Amino-4- (hydroxyphosfinyl) butyric acid; L-Glu-γ-ΡΗ IV- [L-2-Amino-4- (hydroxyphosfinyl) butyryl] -L-Alanyl-L-Alanine; L-Glu- ^ PH-L-Ala-L-Ala 4- (hydroxyphosfinyl) -2-oxobutyric acid; a-ketoglutarate-γ-ΡΗ; a-KG-γ-ΡΗ 3-Aminopropylphosphine acid; GABA-PH 2-Amino-3- (hydroxyphosfinyl) propionic acid; Asp-β-ΡΗ 3- (hydroxyphosfinyl) propionic acid; Succinate-PH 8. Compound according to Formula 1 of claim 1 selected from: D-2-Amino-4- (hydroxyphosfinyl) butyric acid; D-Glu-γ-ΡΗ P-Deuther- [2-amino-4- (hydroxyphosphine) butyric acid]; Glu-γ-Ρϋacid P- Deutero-L- [2-amino-4- (hydroxyphosfinyl) butyric]; L-Glu-γ-PD P-Deutero-Ό- [2-amino-4- (hydroxyphosphmyl) butyric acid]; D-Glu-γ-PD ! V- (L-Leucyl) -2-amimno-4- (hydroxyphosfinyl) butyric acid; L-Leu-Glu-γ-ΡΗ LV-L-Leucyl- [2-Amino-4- (hydroxyphosphinyl) butyryl] -L-Alanyl-L-Alanine; L-Leu-L-Glu-y-PH-L-Ala-L-Ala P- Deutero- (3-aminopropylphosphinic) acid; GABA-PD LV- (L-Leucyl) -3-aminopropylphosphine acid; L-Leu-GABA-PH P-Z <)> euiero-2-Amxnino-3- (hydroxyphosfinyl) propionic acid; Asp-β-Ρο iV- (L-Leucyl) -2-amino-3- (hydroxyphosphinyl) propionic acid; L- Leu-Asp-β-ΡΗ 2-Amino-5- (hydroxyphosfinyl) valeric acid; AAd-δ-ΡΗ L-2-Amino-5- (hydroxyphosfinyl) valeric acid; L-AAd-δ-ΡΗ iV- [L-2-amino-3- (idiOxyphosfmyl) propionyl] L-Alanyl-L-Alanine; Asp ^ -PH-L-Ala-L-Ala IV- (L-Leucyl) -2-aniinino-4- (hydroxyphosfinyl) butyric acid; L-Leurac-Glu-γ-ΡΗ and among the compounds in which at least one of R, Ri, R2and R3 is deuterium Compounds according to any one of claims 1-8 for use in combination with β-lactams, in particular penicilins, cephalosporins, monobactams, carbapenems, also in combination with β-lactamase inhibitors, as well as fluoroquinolones, aminoglycosides, macrolides and their mixtures 10. Compounds according to any one of claims 1-9 to be used for the treatment of diseases in humans and animals, especially mammals. Pharmaceutical compositions comprising a compound according to any one of claims 1-10 and at least one pharmaceutically acceptable carrier or excipient, such as for example a solid support or a liquid diluent. 12. Compositions according to claim 11 for use as disinfectants, in particular for disinfecting healthy skin or for disinfecting wounds and in the treatment of dermatitis and affections of the mucous membranes caused by bacteria, for disinfection of the oral cavity, throat and as intestinal disinfectants and of the uro-genital system. 13. Compositions according to any one of claims 11-12 formulated as creams, gels, oils, ointments, powders, sprays, tinctures, and bandages, liquids or concentrates for gargling, tablets, for example tablets for slow dissolution in the mouth, dosage units solids, in particular tablets, dragees and capsules. 14. Compositions according to any of claims 11-13 to be used as disinfectants and antibacterials to be applied on organic / inorganic materials, for example manufactured articles, including surfaces, containers, instruments, equipment and medical equipment, the organic material being able to be a natural material o synthetic polymer, a protein or carbohydrate-based surface, or a natural or synthetic fiber or textile material. 15. Compositions according to any one of claims 11-14 formulated for oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, inhalation, topical or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous application. 16. Compositions according to claim 15 formulated for the inhalation and topical routes with the active principle diluted in a liquid suitable to be sprayed in the form of aerosol on the mucous surfaces of the body cavities accessible from the outside; or with the active principle mixed with suitable vehicles to obtain emulsions that can be spread on the skin surface. 17. Compositions according to claim 15 formulated for transdermal therapeutic systems such as patches to be applied on the epidermis so that the active principle is released and absorbed by the skin in a continuous and almost constant manner over time. 18. Use of the compounds according to claims 1-10 as antibiotics, alone or in combination with antibacterial compounds against Gram-positive and Gram-negative bacteria, in particular against strains of Escherichia coli, Proteus vulgaris, Providencia rettgeri, Salmonella typhimurium and others enterobacteria such as Clostridium difficile, as well as against Pseudomonas aeruginosa and yeasts such as Candida albicans and Candida tropicalis, and to counter Γ antibiotic resistance in diseases caused by bacterial strains resistant to common antibiotics, such as those caused by methicillin-resistant Staphylococcus aureus.