ES2334547B1 - ANTIBACTERIAL PEPTIDIC COMPOUNDS. - Google Patents

Abstract

Antibacterial peptide compounds.

The compounds of formula (I), or retroenantiomers thereof not acylated at their end N-terminal and with amino acid configuration with the side chain R2 not inverted when R2 is -CH (CH 3) (OH), where: R 0 is CH 3 - (CH 2) m -, CH 3 -O- (CH 2 CH 2 O) 2 CH 2 - or phenyl- (CH 2) x -; m is an integer between 7 and 10; x is an integer between 1 and 3; R1, R3, R4, R7 and R8 are selected independently of the formula GF (CH 2) n - where n is an integer between 1 and 4; and GF is -NH2, -NH-C (= NH) -NH2 or 4 imidazolyl; R2 is -CH (CH3) (OH), -CH (CH 3) 2, -CH 2 NH 2 or -CH 2 OH; R 5 and R 6 are (C 1 -C 4) - linear alkyl or branched, - (CH 2) - R 10, -CH 2 -CH 2 -S-CH 3 or -CH 2 CH 2 S (= O) -CH 3; R 9 is H or Gly-sperm; and R 10 is phenyl, 3-indolyl, 4 imidazolyl, 4-hydroxyphenyl, alpha or beta-naphthyl or 2-, 3- or 4-pyridyl, have been found to be useful in the treatment of bacterial infections.

Description

Antibacterial peptide compounds.

The present invention is related to compounds that are active against bacteria Gram-positive and Gram-negative, its preparation procedure, pharmaceutical compositions that contain, and its use in the treatment of infections bacterial

State of the art

The fact that certain microorganisms pathogens have become resistant to therapies Antibiotics is a serious problem in public health. Part of This problem lies in the fact that certain bacteria and others infectious microorganisms are extraordinarily capable of Develop resistance to antibiotics. Another main cause is due to the deficient use of antibiotics in medicine, veterinary and agriculture

There is worldwide concern about the increasing prevalence of infections caused by multiresistant bacteria, such as methylphycin - resistant Staphylococcus aureus , vancomincin-resistant Enterococcus and certain Gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumanii and Klebsiella pneumoniae . Such infections are very difficult to control and a constant cause of disease and death. Conventional antibiotics usually act on one or more proteins or target receptors and genetic resistance appears at a frequency that depends on many factors, such as the number of said proteins or target receptors.

The continuous appearance of bacterial strains resistant to conventional antibiotics is leading to huge efforts aimed at the development of new drugs that act on the bacterial membrane, such as antimicrobial peptides ("anti-microbial peptides", AMP). Peptides Antimicrobials offer a new class of therapeutic agents to which bacteria are not able to develop resistance genetics, since they act primarily on the component lipid of cell membranes. Among said compounds, the Polymyxin B (PxB), which is approved for clinical use, is acquiring a new therapeutic relevance and is beginning to be considered as a representative of the class of antibiotics active against multi-resistant bacteria.

Polymyxins, in particular polymyxin B, constitute a family of antibiotics discovered in 1947 with high activity against Gram-negative bacteria. Polymyxin B is an antibiotic lipopeptide isolated from Bacillus polymyxa . Its basic structure consists of a polycationic peptide cycle from which a tripeptide linked to a fatty acid chain hangs. Polymyxin B has resurfaced in medical practice in recent years and its use will continue to increase due to the limited development of new antibiotics by pharmaceutical companies and the increasing worldwide prevalence of nosocomial infections caused by multiresistant Gram-negative bacteria ("multidrug resistant ", MDR). Polymyxin B and other members of the polymyxin family are drugs that are used as a last resort to treat infections caused by multiresistant bacteria and are sometimes the only active antibiotic available. In addition, resistance to polymyxin is rare and generally adaptive and therefore reversible. Polymyxin B is also capable of inhibiting the biological activity of bacterial lipopolysaccharide (LPS) through high affinity binding to lipid A, thus being the agent of choice for the treatment of septic shock induced by LPS. Unfortunately, polymyxin B has no activity against Gram-positive or anaerobic bacteria. In addition, polymyxins are of limited use because they have some nephrotoxicity and neurotoxicity.

Therefore, there is still a need to find new active antibacterial agents not only against Gram-negative bacteria but also against Gram-positive bacteria.

Explanation of the invention.

The inventors have found some compounds peptides with antibiotic activity that act on the component lipid of bacterial membranes and they are active both against Gram-positive bacteria as against Gram-negative These compounds are based on the natural polymyxin structure. However and unlike polymyxins, such compounds are active not only against Gram-negative bacteria but also in Gram-positive in the micromolar range. This is advantageous since they can act in response to infections caused by both types of bacteria.

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Thus, one aspect of the present invention is the provide compounds of formula (I),

one

or retroenantiomers thereof of formula (I '),

2

where

R_ {0} is a radical selected from CH 3 - (CH 2) m -, CH 3 -O- (CH 2 CH 2 O) 2 CH 2 -, Y

3

m is an integer between 7 and 10; x is an integer between 1 and 3; R 1, R 3, R 4, R 7, and R 8 are independently selected radicals that respond to the formula: GF- (CH 2) n -; n is an integer between 1 and 4; GF is a radical selected from
-NH2 (amino), -NH-C (= NH) -NH2 (guanidino) and 4-imidazolyl; R 2 is a radical selected from -CH (CH 3) (OH), -CH (CH 3) 2, -CH 2 NH 2 and -CH 2 OH ; R 5 and R 6 are radicals selected from: - (C 1 -C 4) - linear or branched alkyl; - (CH 2) - R 10, -CH 2 -CH 2 -S-CH 3, and -CH 2 -CH 2 -S (= O) -CH_ {3}, R 9 is H or Gly-NHCH 2 CH 2 CH 2 NHCH 2 CH 2 CH 2 CH 2 CH 2 NHCH 2
CH 2 CH 2 NH 2 (Gly-spermide); and R 10 is a radical selected from phenyl, 3-indolyl, 4-imidazolyl, 4-hydroxyphenyl, naphthyl and pyridyl; with the proviso that the compound of formula (I) is not one of the compounds of the following list, which have been previously described, but not its application as antibacterial agents against Gram-positive bacteria:

nonanoil-Dab-Thr-Dab- cycle (SS) [Cys-Dab-DPhe-Leu-Dab-Dab-Cys], (sp-B);

nonanoil-Dap-Thr-Dab- cycle (SS) [Cys-Dab-DPhe-Leu-Dap-Dab-Cys], (sP-D);

nonanoil-Dab-Thr-Dab- cycle (SS) [Cys-Dab-DPhe-Met-Dab-Dab-Cys], (sp-Met);

nonanoil-Dab-Thr-Dab- cycle (SS) [Cys-Dab-DPhe-Met (O) -Dab-Dab-Cys]; (sp-Met (O)); or

nonanoil-Dab-Thr-Dab- cycle (SS) [Cys-Dab-DTrp-Leu-Dab-Dab-Cys], (sp-Bw).

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The term " cycle (SS) " means a macrocycle formed by a disulfide bridge between the two cysteines.

A retroenantiomer of a peptide is a peptide analogue consisting of the reverse amino acid sequence with respect to the original peptide with simultaneous inversion of the configuration of the stereocenters, in particular those of the alpha carbon of each amino acid. It is known that the retroenantiomers of certain cyclic peptides and depsypeptides maintain biological activity despite the fact that the direction of the amide bond (defined in the direction of the carbonyl carbon atom to the nitrogen atom) that binds the residues has been reversed (cf. M. Goodman et al ., "On the concept of linear modified retro-peptide structures", Acc. Chem. Res . 1979 vol. 12, pp. 1-7). The biological activity is attributed to the three-dimensional orientation of the side chains and not to the peptide skeleton.

The retroenantiomeric compound (I ') is a analog peptide consisting of the reverse amino acid sequence of compound (I) with simultaneous inversion of the configuration of all chiral centers except the amino acid chain lateral R2 when said R2 is threonine, and that no it is necessarily acylated at its end N-terminal

In a preferred embodiment, the compounds of formula (I) or (I ') are those mentioned above where R2 is -CH (CH3) (OH), that is, R2 is the string threonine side.

Preferred compounds of formula (I) are those selected from the following list:

nonanoil-Arg-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys], (sp-2Arg);

nonanoil-Arg-Thr-Arg- cycle (SS) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys], (sp-3Arg);

nonanoil-Arg (NO 2) - Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg (NO 2) - Dab-Cys], (sp-2Arg (NO 2)) ;

nonanoil-Arg-Thr-Arg- cycle (SS) [Cys-Arg-DPhe-Leu-Arg-Arg-Cys], (sp-5Arg);

nonanoil-Dap [C (NH) -NH2] - Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap [C (NH) - NH2] - Dab-Cys], (sP-2Dap
guanidylated); Y

nonanoil-Arg-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys] -Gly-NHCH_2CH2CH2CH2 NHCH2CH2 CH_ {2}
CH 2 NHCH 2 CH 2 CH 2 NH 2, (sP-2Arg-Gly-spermide).

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Preferred compounds of formula (I ') are those selected from the following list:

cycle (SS ) [DCys-DDab-DArg-DLeu-Phe-DDab-DCys] -DDab-DThr-DArg-octylamide, (sP-2Arg retroenantium); Y

cycle (SS ) [DCys-DDab-DDab-DLeu-Phe-DDab-DCys] -DDab-DThr-DDab-octylamide, (sP-B retroenantium).

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The compounds of the present invention can be prepared by Fmoc solid phase synthesis. The preparation process for each amino acid may comprise the following steps: (i) several washings of the resin with N , N- dimethylformamide (DMF); (ii) treatment with 20% piperidine / DMF; (iii) washing with DMF several times; (iv) acylation with the amino acid protected Fmoc and N , N' -diisopropylcarbodiimide / 1-hydroxybenzotriazole (DIPCDI / HOBt) in DMF; (v) washing with DMF several times and then in dichloromethane (CH 2 Cl 2) several times; (vi) Kaiser test (with a sample of peptide resin); and (vii) washing with DMF several times.

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They can also be prepared by solid phase synthesis Boc. In this case, the preparation procedure for each amino acid can comprise the following steps: (i) washing the resin with CH 2 Cl 2 several times; (ii) treatment with 40% trifluoroacetic acid (TFA) / CH 2 Cl 2; (iii) washing the resin with CH 2 Cl 2 several times; (iv) treatment with 5% DIEA / CH 2 Cl 2 several times; (v) washing the resin with CH2Cl2 several times; (vi) washing the resin with DMF several times; (vii) acylation with the amino acid protected Boc and benzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate (PyBOP) and N , N- diisopropylethylamine (DIEA) hexafluorophosphate in the minimum amount of DMF; (viii) washed with DMF several times and CH 2 Cl 2 several times; and (ix) Kaiser test (with a sample of peptide resin). In step (vii) instead of PyBOP and DIEA, DIPCDI / HOBt can be used.

The peptides obtained by the procedures above can be purified by preparative HPLC, by which can be obtained more than 99% purity.

Thus, the compounds of the present invention will be easily prepared by chemical synthesis according to the procedures mentioned above while commercial polymyxin is obtained by fermentation.

Another aspect of the present invention are the compounds of formula (I) or (I '), as well as any of the following compounds:

nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dab-Dab-Cys], (sp-B);

nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Dab-Leu-Dab-Cys], (sp-C);

nonanoil-Dap-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap-Dab-Cys], (sP-D);

nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met-Dab-Dab-Cys], (sp-Met);

nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met (0) -Dab-Dab-Cys], (sp-Met (O)); or

nonanoil-Dab-Thr-Dab- cycle (SS ) fCys-Dab-DTrp-Leu-Dab-Dab-Cys], (sp-Bw);

for use as antibacterial agents against Gram-positive bacteria. Among the medically relevant Gram-positive bacteria are several of the well-known genus such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Mycoplasma and Actinobacteria .

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In a preferred embodiment, Gram-positive bacteria are selected from Micobacterium phlei, Staphylococcus aureus and Bacillus cereus var. Mycoides In a particular embodiment, Gram-positive bacteria are selected from Staphylococcus aureus 6538 and Bacillus cereus var. mycoides 11778 .

This aspect of the invention can be formulated also as the use of a compound as defined above, including known compounds mentioned for the preparation of a medicine for the treatment of a bacterial infection caused by bacteria Gram-positive in a mammal, including a human.

The invention is also related to a method for the treatment and / or prophylaxis of a mammal, including a human, who suffers or is susceptible to infections Bacterial caused by Gram-positive bacteria, in particular to the infections mentioned above, said method comprising administration to said patient of a therapeutically effective amount of compounds of the present invention, including the known compounds mentioned above, together with pharmaceutically excipients or carriers acceptable.

Compounds of formula (I) or (I ') as defined above for use as antibacterial agents against Gram-negative bacteria are also part of the invention. Medically relevant Gram-negative bacteria include Escherichia coli, Salmonella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Legionella, Hemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens and Acinetobacter baumanii .

In a preferred embodiment, Gram-negative bacteria are selected from Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli . In a particular embodiment, Gram-negative bacteria are selected from Salmonella typhimurium 14028 , Pseudomonas aeruginosa 9027 and Escherichia coli 8739 . This aspect of the invention can also be formulated as the use of a compound of formula (I) or (I ') as defined above for the preparation of a medicament for the treatment of a bacterial infection caused by Gram-negative bacteria in A mammal, including a human.

The invention is also related to a method for the treatment and / or prophylaxis of a mammal, including a human who suffers or is susceptible to infections Bacterial caused by Gram-negative bacteria, in particular to any of the infections mentioned above, the method comprising administration to said patient of a therapeutically effective amount of the compounds of formula (I) or (I ') as defined above, together with excipients or pharmaceutically acceptable carriers.

The compounds of the present invention are may use similarly to other antibacterial agents known. These can be used alone or in combination with other appropriate bioactive compounds. In one embodiment In particular, the compounds of formula (I) or (I ') can be used in the treatment of bacteremias and / or septicemia consequence of Gram-negative bacteria infections, administered alone or in combination with antibiotics conventional. In a preferred embodiment, the compounds of Formula (I) or (I ') of the present invention are used topically, or orally for the decontamination of the digestive tract prior to surgery

As mentioned above, these compounds have the advantage that they can act on a broad spectrum of bacteria and because apparently it is believed that act on the membrane, may be more effective than other antibiotics that act on a receptor or enzyme against Antibiotic resistance

A further aspect of the present invention is related to a pharmaceutical composition comprising a therapeutically effective amount of the compounds of formula (I) or (I '), together with appropriate amounts of excipients or carriers pharmaceutically acceptable.

Pharmaceutical compositions can be prepare by combination of the compounds of formula (I) or (I ') of the present invention with solid excipients or carriers or pharmaceutically acceptable liquids, following practices Standard pharmaceuticals

The pharmaceutical compositions herein invention can be administered suitably for the disease to be treated, for example orally, parenteral, inhalation, rectal, transdermal or topical. In using therapeutic to treat or fight bacterial infections in patients like humans and other animals that could be diagnosed with bacterial infections, said compounds or pharmaceutical compositions are preferably administered at a dose to obtain or maintain a concentration, that is, a amount or level in blood of the active component in the patient that Follow the treatment that is effective as an antibacterial. Generally, said amount or dose that is effective as antibacterial will be in the approximate range of 0.1 to 100 mg / kg, more preferably around 3.0 to 50 mg / kg of weight body / day. It is understood that the doses may vary depending of the patient's requirements, the severity of the infection bacterial to be treated and the particular compound used.

In addition, the present invention covers all possible combinations of particular groups mentioned previously.

Throughout the description and the claims, the word "comprises" and its variations do not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects,  advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

Abbreviations: Acm, acetamidomethyl; AcOH, acetic acid; Boc, tert-butoxycarbonyl; Bn, benzyl; Dab: 2,4-diaminobutyric acid; DMAP, N , N- dimethylaminopyridine; DIEA, N , N -diisopropylethylamine; DIPCDI, N , N'- diisopropylcarbodiimide; DMF, N , N- dimethylformamide; ES, electrospray; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HPLC, high efficiency liquid chromatography; MALDI-TOF, ionization by laser desorption assisted by matrix-time of flight; MBHA, 4-methylbenzydrylamine resin; MIC, minimum inhibitory concentration; Pmc, 2,2,5,7,8-pentamethyl-chromane-6-sulfonyl; PyBOP, benzotriazol-1-yloxytris (pyrrolidine) phosphonium hexafluorophosphate; TES: triethylsilane; TFA, trifluoroacetic acid; TIS: triisopropylsilane; TMS-Cl, trimethylsilyl chloride; Trt, trityl; DAB, 2,4-diaminobutyric acid.

General methods

Two methods have been used to prepare Compounds of the examples: Fmoc solid phase synthesis and synthesis solid phase Boc.

Fmoc synthesis protocol : for each amino acid cycle it consists of the following steps: (i) washing the resin with DMF (5 x 30 s); (ii) treatment with 20% piperidine / DMF (1 x 1 min. + 2 x 10 min., Fmoc deprotection); (iii) washing with DMF (5 x 30 s); (iv) acylation with the amino acid Fmoc protected (3 times excess) and DIPCDI / HOBt (3 times excess) in the minimum amount of DMF; (v) washing with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s); (vi) Kaiser test (with a sample of peptide resin); (vii) DMF wash (5 x 30 s).

Synthesis protocol Boc : for each amino acid cycle it consists of the following steps: (i) washing the resin with CH 2 Cl 2 (5 x 30 s); (ii) treatment with 40% trifluoroacetic acid / CH 2 Cl 2 (1 x 1 min. + 2 x 10 min., Boc elimination); (iii) washing the resin with CH2Cl2 several times; (iv) treatment with 5% DIEA / CH 2 Cl 2 several times; (v) washing the resin with CH 2 Cl 2 (5 x 30 s); (vi) washing the resin with DMF (5 x 30 s); (vii) acylation with the amino acid protected Boc (3 times in excess) and PyBOP (3 times in excess) and DIEA (9 times in excess) in the minimum amount of DMF; (viii) washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s); (ix) Kaiser test (with a sample of peptide resin). In step (vii) instead of PyBOP and DIEA, alternatively, DIPCDI / HOBt can be used.

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Purification was carried out by HPLC preparative The Waters Delta Prep equipment was used in the Examples 3000 system with a Phenomenex C18 column (2) (250 x 10 mm, 5 um) and eluted in gradient with mixtures of H 2 O-acetonitrile-0.1% TFA and UV detection at 220 nm.

Peptides were characterized by analysis of amino acids with a Beckman 6300 analyzer and by spectrometry of MALDI-TOF masses in a mass spectrometer VOYAGER-DE-RP (Applied Biosystems) or in an electrospray mass spectrometer (ESI +) ZQ-Micromass (Waters) or mass spectrometer ESI + LC / MSD-TOF (Agilent Technologies). The homogeneity of purified peptides was checked by HPLC analytical using Nucleosil C18 reverse phase columns (4 x 250 mm, 5 µm of particle diameter and a pore measurement of 120 \ ring {A}). Elution was carried out at 1 ml • -1 with mixtures of H2O-0.045% TFA and acetonitrile-0.036% TFA and UV detection at 220 nm.

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Example 1 Preparation of nonanoil-Arg-Thr-Dab-cycle (S-S) [Cys-Dab-DPhe-Leu-Arg-Dab-Cysl (compound of formula (I) with R 0 = CH 3 (CH 2) 7 -, R 1 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (chain side of Arg), R 4 = -CH 2 (CH 3) OH, R 3 = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (Dab side chain), R 5 = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH (CH 3) 2 (Leu side chain), R 7 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (side chain of Arg), and R 8 = -CH 2 CH 2 NH 2 (Dab side chain); sp-2Arq)

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Leu-OH,
Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The spacer ("linker") Fmoc-Rink (319 mg, 0.59 mmol, 3 excess equiv.) anchored to the resin 4-MBHA-polystyrene (201 mg, f = 0.98 mmol / g) by activation with DIPCDI (92 µL, 0.59 mmol, 3 excess equivalents). The amino acids in the sequence are introduced according to a standard solid phase synthesis protocol Fmoc / t Bu as described above. Once the sequence was completed, nonanoic acid was coupled (103.6 µl, 0.59 mmol, 3 times excess) with DIPCDI / HOBt in the amount DMF minimum. Upon completion of the reaction as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide was completed, the disulfide bond was formed by oxidation with iodine (200.3 mg, 4 equiv.) in DMF for two hours. The resin was washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL)

De-anchoring and deprotection of the peptide was performed by acidolysis with TFA: triethylsilane: water (95: 3: 2, v / v / v, 5 mL) for 1.5 hours at room temperature. Then, the acidolytic mixture was evaporated with N2 stream and the oily residue was precipitated with dry ethyl ether. The peptide crude was recovered by centrifugation and decantation of the ether phase. The weight of the peptide crude was 200 mg (yield 76%, purity 80%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1335.32
([M + H] +, 100%), 1357.31 ([M + Na] +, 16.8%).

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Example 2 Preparation of nonanoil-Arg-Thr-Arg-cycle (S-S) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys] (Compound (I) with R_ {0} = CH 3 (CH 2) 7 -, R 1 = -CH 2 CH 2 CH, NHC (= NH) -NH 2 (chain side of Arg), R2 = -CH (CH3) OH, R3 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (side chain of Arq), R 4 = -CH 2 CH 2 NH 2 (side chain of Dab), R 5 = -CH 2 Ph (side chain of DPhe R 6 = -CH 2 CH (CH 3) 2 chain side of Leu), R_ {7} = -CH 2 CH 2 CH 2 NHC (NH) -NH 2 (side chain of Arg), and R 8 = -CH 2 CH 2 NH 2 (Dab side chain); sp-3Arg)

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Leu-OH,
Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The first amino acid in the sequence, Fmoc-Cys (Acm) -OH, was anchored directly to the resin 4-MBHA-polystyrene (151.4 mg, f = 0.98 mmol / g). The amino acids of the sequence were introduced according to a standard solid phase synthesis protocol Fmoc / t Bu as described above. Once the sequence was completed, nonanoic acid was coupled (78.0 µl, 0.45 mmol, 3 times excess) with DIPCDI / HOBt in the amount DMF minimum. Upon completion of the reaction as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide was completed, the disulfide bond was formed by oxidation with iodine (150.6 mg ,, 4 equiv.) in DMF for two hours. The resin was washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL)

De-anchoring and deprotection of the peptide is performed by acidolysis with TFA: TMS-Cl: HBr (33% in AcOH): triethylsilane (3.21 ml TFA, 1.29 ml TMS-Cl, 0.25 ml TES, 0.25 ml HBr, total volume 5 ml) for 3 hours at room temperature. Then the acidolytic mixture is evaporated with a stream of N 2 and the oily residue precipitated with dry ethyl ether. The peptide crude was recovered by centrifugation and decantation of the etheric phase. Crude oil weight Peptide was 75.1 mg (yield 82%, purity 80%). Characterization  of the purified peptide: Homogeneity (by integration of the area of HPLC trace)> 99%; MALDI-TOF: m / z 1391.73 ([M + H] +, 32.9%), 1413.70 ([M + Na] +, 100%)

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Example 3 Preparation of nonanoil-Arg (NO 2) - Thr-Dab-cycle (S-S) [Cys-Dab-DPhe-Leu-Arg (NO 2) - Dab-Cys] (Compound (I) with R_ {0} = CH 3 (CH 2) 7 -, R 1 = -CH 2 CH 2 CH 2 NHC (= NH) -NH-NO 2 (side chain of Arq (NO2)), R2 = - CH (CH 3) OH, R 3 = CH 2 CH 2 NH 2, (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (chain side of Dab), R 5 = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH (CH 3) 2 (side chain of Leu) R_ {7} = -CH 2 CH 2 CH 2 NHC (= NH) -NH-NO 2  (side chain of Arg (NO_ {2})) and R_ {8} = -CH 2 CH 2 NH 2 (Dab side chain); sp-2Arg (NO2)

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Arg (NO2) - OH, Fmoc-Leu-OH,
Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The spacer ("linker") Fmoc-Rink (329 mg, 0.61 mmol, 3 excess equiv.) anchored to the resin 4-MBHA-polystyrene (209 mg, f = 0.98 mmol / g) by activation with DIPCDI (95 µL, 0.61 mmol, 3 equiv. excess). The amino acids of the sequence were introduced according to a standard solid phase synthesis protocol Fmoc / t Bu as described above Once the sequence is complete, it coupled nonanoic acid (107.4 µL, 0.61 mmol, 3 times of excess) with DIPCDI / HOBt in the minimum amount of DMF. One time the reaction concluded as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide is completed, the disulfide bond was formed by oxidation with iodine (207.6 mg, 4 equiv.) In DMF for two hours. The resin was washed with  DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL).

De-anchoring and deprotection of the peptide is performed by acidolysis with TFA: triethylsilane: water (95: 3: 2, v / v / v, 5 mL) for 1.5 hours at room temperature. Then the Acidolytic mixture was evaporated with N2 stream and the residue Oily precipitated with dry ethyl ether. The peptide crude is recovered by centrifugation and decantation of the etheric phase. The weight of the peptide crude was 245 mg (yield 84%, purity 85%). Characterization of the purified peptide: Homogeneity (by HPLC trace area integration)> 99%; ESI +: m / z 476.5 ([M + 3H] 3 + / 3, 100%), 714.0 ([M + 2H] 2 + / 2, 73%)

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Example 4 Preparation of nonanoil-Arg-Thr-Arg-cycle (S-S) [Cys-Arg-DPhe-Leu-Arg-Arq-Cys] (Compound (I) with R 0 = CH 3 (CH 2) 7, R 1 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (Arg side chain), R2 = -CH (CH3) OH, R 3 = -CH 2 CH 2 CH 2 NHC (= NH) -NH2 (side chain of Arg), R4 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (chain side of Arg), R 5 = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH (CH 3) 2 (side chain of Leu), R_ {7} = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (chain side of Arg), and R_ {8} = -CH 2, CH 2 NHC (= NH) -NH 2 (side chain from Arg); sp-5Arg)

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Leu-OH, Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The first amino acid in the sequence, Fmoc-Cys (Acm) -OH, was anchored directly to the resin 4-MBHA-polystyrene (151.4 mg, f = 0.98 mmol / g). The amino acids of the sequence were introduced according to a standard solid phase synthesis protocol Fmoc / t Bu as described above. Once the sequence was completed, nonanoic acid was coupled (78.0 µl, 0.45 mmol, 3 times excess) with DIPCDI / HOBt in the amount DMF minimum. Upon completion of the reaction as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide was completed, the disulfide bond was formed by oxidation with iodine (150.6 mg, 4 equiv.) in DMF for two hours. The resin was washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL)

De-anchoring and deprotection of the peptide is performed by acidolysis with TFA: TMS-CI: HBr (33% in AcOH): triethylsilane (3.21 ml TFA, 1.29 ml TMS-Cl, 0.25 ml TES, 0.25 ml HBr, total volume 5 ml) for 3 hours at room temperature. Then the acidolytic mixture is evaporated with a stream of N 2 and the oily residue precipitated with dry ethyl ether. The peptide crude was recovered by centrifugation and decantation of the etheric phase. Crude oil weight Peptide was 97.9 mg (yield 80%, purity 80%). Characterization  of the purified peptide: Homogeneity (by integration of the area of HPLC trace)> 99%; MALDI-TOF: m / z 1503'63 ([M + H] +, 29.3%), 1525.64 ([M + Na] +, 100%)

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Example 5 Preparation of nonanoil-Dap [(C = NH) -NH2 -Thr-Dab-cycle (S-S) [Cys-Dab-DPhe-Leu-Dap [(C = NH) -NH2)] - Dab- Cys] (Compound (I) with R_ {0} = CH 3 (CH 2) 7 -, R 1 = -CH 2 NH (C = NH) -NH 2 (side chain of Dap [(C = NH) -NH2)]; Guanidylated dap; version short of Arq, with two less methylenes], R2 = -CH (CH 3) OH, R 3 = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (side chain of Dab), R 5 = -CH 2 Ph (side chain of DPhe), R 6 = -CH 2 CH (CH 3) 2 (Leu side chain), R_ {7} = -CH2 NH (C = NH) -NH2 (side chain of Dap [(C = NH) -NH2], guanidylated Dap), and R 8 = -CH 2 CH 2, NH 2 (Dab side chain); guanidylated sP-2Dap)

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Z) -OH, Fmoc-Dap (Boc) -OH, Fmoc-Leu-OH, Fmoc-DPhe-OH, Fmoc-Dab (Z) -OH, Fmoc-Thr (Bzl) -OH, Fmoc-Dap (Boc) -OH.

Manual synthesis was carried out according to a standard Fmoc / t Bu protocol in polypropylene syringes with a polyethylene filter. The first amino acid in the sequence, Fmoc-Cys (Acm) -OH, was directly anchored to the 4-MBHA-polystyrene resin (126.8 mg, f = 0.98 mmol / g). The rest of the amino acids in the sequence were introduced according to a standard Fmoc / t Bu solid phase synthesis protocol as described above: Fmoc-Dab (Z) -OH, Fmoc-Dap (Boc) -OH, Fmoc-Leu-OH, Fmoc-DPhe-OH, Fmoc-Dab (Z) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Dab (Z) -OH, Fmoc-Thr (Bzl) -OH, Fmoc- Dap (Boc) -OH. Finally, nonanoic acid (65.3 µL, 0.37 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. After completion of the reaction as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). The Boc groups of the Dap residues were removed by acidolysis with TFA following steps ii) to iv) of the Boc synthesis protocol. The free amino groups of the Dap amino acids were guanidylated with N , N' -di-Boc- N -trifluoromethanesulfonyl guanidine (486.3 mg, 1.24 mmol, 5 equiv. Excess per NH2) in DMF for five days. Disulfide bond cyclization was achieved by oxidation with iodine (126.2 mg, 4 equiv.) In DMF for two hours. The resin was washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL).

De-anchoring and deprotection of the peptide was performed by acidolysis with TFA: TMS-Cl: HBr (33% in AcOH): triethyl-
silane (3.4 mLTFA, 0.2 mL HBr, 0.4 mL total anisole volume 4 mL) for 4 hours at 35 ° C. The acidolytic mixture was evaporated with N2 stream and the oily residue was precipitated with dry ether. The peptide crude was recovered by centrifugation and decantation of the ether phase. The weight of the peptide crude was 34.3 mg (yield 22%, purity 50%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>95%; MALDI-TOF: m / z 1279.54 ([M + H] +, 100%), 1301.29 ([M + Na] +, 50.3%), 1317.25 ([M + K] +, 5.5%)

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Example 6 Preparation of cycle (S-S) [DCys-DDab-DArq-DLeu-Phe-DDab-DCys] DDab DThr-DArg-octylamide of sP-retroenantium 2Arg (Compound of formula (I ') with R 0 = CH 3 CH 2) 7 -, R 1 = -CH 2 CH 2 CH 2, NHC (= NH) -NH 2 (chain side of DArq), R2 = -CH (CH3) OH, R3 = -CH 2 CH 2 NH 2 (DDab side chain), R 4 = -CH 2 CH 2 NH 2 (DDab side chain) R 5 = -CH 2 Ph (Phe side chain), R 6 = -CH 2 CH (CH 3) 2 (DLeu side chain), R_ {7} = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (chain side of DArg), and R 8 = -CH 2 CH 2 NH 2 (chain DDab side); sP-retroenantio 2Arg)

Protected amino acids: Boc-DArg (Z2) - OH, Boc-DThr (Bzl) -OH, Boc-DDab (Z) -OH Boc-DCys (Acm) -OH,
Fmoc-Phe-OH, Boc-DLeu-OH.

Manual synthesis was carried out according to a standard Boc protocol in polypropylene syringes with a polyethylene filter. A reference amino acid (Gly) was introduced first. Fmoc-Gly-OH (174 mg, 0.59 mmol, 3 equiv. Excess), was anchored to the 4-MBHA-polystyrene resin (200 mg, f = 0.98 mmol / g) by reaction with DIPCDI (91 µl, 0.59 mmol, 3 times excess) and HOBt (91 mg, 0.59 mmol, 3 equiv. Excess). The Fmoc group was removed by treatment with 20% piperidine solution in DMF (2 x 10 min). The resin was washed with DMF (5 x 30 s). Next, the spacer ("linker") 4-hydroxyphenylpropionic acid (97.7 mg, 0.59 mmol, 3 equiv. Excess) was introduced into the aminoacrylic resin by reaction with DIPCDI (91 µL, 0.59 mmol, 3 times excess ) and HOBt (91 mg, 0.59 mmol, 3 excess equiv) in the minimum amount of DMF. The resin was left overnight with 20% piperidine solution in DMF and subsequently washed with DMF (5 x 30s). The first amino acid in the sequence, Boc-DArg (Z2) -OH (319 mg, 0.59 mmol, 3 equiv. Excess) was esterified to the spacer ("linker") with DIPCDI (91 µl, 0.59 mmol, 3 equiv. excess) and DMAP (7.18 mg, 0.059 mmol, 0.3 equiv.). This stage was repeated twice. The rest of the amino acids of the sequence from Thr to Cys, were introduced according to a standard Boc synthesis protocol as described above. Once the sequence was completed, the disulfide bond was formed by oxidation with iodine (198.98 mg, 0.78 mmol, 4 equiv.) In DMF for two hours. The resin was washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL). The peptide was removed from the resin by aminolysis with octylamine (28.4 µL, 0.19 mmol, 1 equiv.) In DMF overnight. The solution was filtered and the solvent was removed by rotary evaporation. The oily residue was triturated with acetonitrile. A
white solid

The deprotection of the side chains is performed by acidolysis with TFA: HBr (33% in AcOH): thioanisole (85: 5: 10) for 3 hours at 35 ° C. The acidolytic mixture was evaporated with a stream of N 2 and the oily residue precipitated with dry ether The peptide crude was recovered by centrifugation and decantation of the etheric phase. The weight of the peptide crude was 90 mg (yield 35%, purity 50% by presence of thioanisole residues and of the benzyl sulphonium salt of thioanisole). Characterization of purified peptide: Homogeneity (by integration of the area of the HPLC trace)> 99%; MALDI-TOF: m / z 1308.21 ([M + H] +, 100%), 1330.20 ([M + Na] +, 23%).

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Example 7 Preparation of cycle (S-S) [DCys-DDab-DDab-DLeu-Phe-DDab-DCys] DDab-DThr-DDab-octylamide (Compound of formula (I ') with R_ {0} = CH 3 CH 2) 7 -, R 1 = CH 2 CH 2 NH 2 (DDab side chain), R2 = -CH (CH2 3) OH, R 3 = -CH 2 CH 2 NH 2 (DDab side chain), R 4 = -CH 2 CH 2 NH 2 (DDab side chain), R 5 = -CH 2 Ph (Phe side chain), R 6 = -CH 2 CH (CH 3) 2 (DLeu side chain), R 7 = -CH 2 CH 2 NH 2 (DDab side chain), and R 8 = -CH 2 CH 2 NH 2 (DDab side chain); sP-B retroenantium)

Protected amino acids: Boc-DDab (Z) -OH, Boc-DThr (Bzl) -OH, Boc-DCys (Acm) -OH, Fmoc-Phe-OH, Boc-
DLeu-OH.

Manual synthesis was carried out according to a Standard Boc protocol in polypropylene syringes with a filter of polyethylene as described in Example 6, starting from 4-MBHA-polystyrene resin (200 mg, f = 0.98 mmol / g). The weight of the peptide crude was 190 mg (yield 80%, purity 40% by presence of thioanisole residues and salt of thioanisole benzyl sulphonium). Peptide Characterization purified: Homogeneity (by integration of the trace area of HPLC)> 95%; MALDI-TOF: m / z 1196.15 ([M + H] +, 100%), 1218.07 ([M + Na] +, 87.2%).

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Example 8 Preparation of nonanoil-Arg-Thr-Dab-cycle (S-S) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys] -Gly-NHCH 2 CH 2 CH 2 NH 2 CH 2 CH 2 CH 2 NHCH 2 CH 2 CH 2 NH 2 (Compound of formula (I) with R_ {0} = CH 3 (CH 2) 7 -, R 1 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (chain side of Arg), R2 = -CH (CH3) OH, R3 = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (Dab side chain), R 5 = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH (CH 3) 2 (Leu side chain), R 7 = -CH 2 CH 2 CH 2 NHC (= NH) -NH 2 (side chain of Arg), and R 8 = -CH 2 CH 2 NH 2 (Dab side chain); sP-2Arg-spermide)

Protected amino acids: Fmoc-Ala-OH, Boc-Gly-OH, Boc-Cys (Acm) -OH, Boc-Dab (Z) -OH, Boc-Arg (Z2) - OH, Boc-Leu-OH, Boc-DPhe-OH.

An amino acid of first was introduced reference (Wing). Fmoc-Ala-OH (167 mg, 0.54 mmol, 3 equiv. excess), was anchored to the resin 4-MBHA-polystyrene (113 mg, f = 0.98 mmol / g) by reaction with DIPCDI (83.5 µL, 0.54 mmol, 5 times of excess) and HOBt (82.5 mg, 0.54 mmol, 5 equiv. excess). The group Fmoc was removed by treatment with 20% piperidine solution in DMF (2 x 10 min.). The resin was washed with DMF (5 x 30 s). TO then the acid spacer ("linker") was introduced 4-hydroxyphenylpropionic (97.7 mg, 0.59 mmol, 3 equiv. excess) in the aminoacrylic resin by reaction with DIPCDI (50 µL, 0.32 mmol, 3 times excess) and HOBt (49.5 mg, 0.32 mmoles, 3 equiv. excess) in the minimum amount of DMF. The resin is left overnight with 20% piperidine solution in DMF. The first sequence amino acid, Boc-Gly-OH (188.86 mg, 1.08 mmol, 10 equiv. excess) the spacer ("linker") was esterified with DIPCDI (167 µL, 1.08 mmol, 10 equiv. Excess) and DMAP (13.16 mg, 0.108 mmol, 1 equiv.). The rest of the amino acids in the sequence from Cys to Arg, were introduced according to a standard protocol of Boc synthesis as described above. Finally I know coupled nonanoic acid (94.3 µl, 0.53 mmol, 5 times of excess) with DIPCDI / HOBt in the minimum amount of DMF. Once the sequence was completed, the disulfide bond was formed by oxidation with iodine (99 mg, 4 equiv.) in DMF for two hours. The resin is washed with DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL). The peptide is resin stripped by spermine aminolysis (259.5 µL, 0.32 mmole, 3 equiv. excess) in DMF overnight. Leaked The solution and solvent were removed by rotary evaporation. The residue Oily was triturated with acetonitrile. A solid was obtained White.

The deprotection of the side chains is performed by acidolysis with TFA: HBr (33% in AcOH): anisole (85: 5: 10) for 3 hours at 35 ° C. The acidolytic mixture was evaporated with stream of N2 and the oily residue precipitated with ether dry. The peptide crude was recovered by centrifugation and decantation of the etheric phase. The weight of the peptide crude was 191 mg (quantitative yield, 50% purity). Characterization of purified peptide: Homogeneity (by integration of the area of the HPLC trace)> 98%; MALDI-TOF: m / z 1578.18 ([M + H] +, 100%), 1600.16 ([M + Na] +, 37%).

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The following peptides:

Nonanoil-Dab-Thr-Dab- cycle (SS ) fCys-Dab- (D) Phe-Leu-Dab-Dab-Cys] (Compound of formula (I) with R_ {0} = CH_ {3} (CH_ {2 ) 7 -, R 1 = -CH 2 CH 2 NH 2 (side chain of f Dab), R 2 = -CH (CH 3) OH , R_ {3} = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (Dab side chain), R_ {5} = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH (CH 3) 2 (Leu side chain), R 7 = -CH 2 CH 2 NH 2 (Dab side chain), R 8 = -CH 2 CH 2 NH 2 (Dab side chain) (spB);

Nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab- (D) Phe-Dab-Leu-Dab-Cys] (Compound of formula (I) with R_ {0} = CH_ {3} (CH_ { 2) 7 -, R 1 = -CH 2 CH 2 NH 2 (Dab side chain), R 2 = -CH (CH 3) OH, R_ {3} = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = CH 2 CH 2 NH 2 (Dab side chain), R_ { 5} = -CH 2 Ph (DPhe side chain), R 6 = -CH 2 CH 2 NH 2 (Dab side chain), R 7 = CH 2 CH (CH 3) 2 (Leu side chain), and R 8 = CH 2 CH 2 NH 2 (Dab side chain) (sp-C); and

Nonanoil-Dap-Thr-Dab- cycle (SS ) [Cys-Dab- (D) Phe-Leu-Dap-Dab-Cys] (Compound of formula (I) with R_ {0} = CH_ {3} (CH_ { 2) 7, R 1 = CH 2 NH 2 (Dap side chain), R 2 = -CH (CH 3) OH, R 3 = -CH_ {2} CH2 NH2 (Dab side chain), R4 = CH2CH2 NH2 (Dab side chain), R5 = CH2 Ph (DPhe side chain), R 6 = CH 2 CH (CH 3) 2 (Leu side chain), R 7 = -CH 2 NH 2 (Dap side chain), and R 8 = -CH 2 CH 2 NH 2 (Dab side chain) sP-D);

they are known and can be prepared as described in the following scientific publication: Adriá Clausell et al ., "Gram negative bacteria outer and inner membrane models: Insertion of cyclic cationic peptides" J. Phys. Chem B , 2007, vol. 111, pp. 551-563.

Nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met-Dab-Dab-Cys (Compound of formula (I) with R 0 = CH 3 (CH 2) _ {7} -, R 1 = -CH 2 CH 2 NH 2 (Dab side chain), R 2 = -CH (CH 3) OH, R 3 = -CH 2 CH 2 NH 2 (Dab side chain), R 4 = -CH 2 CH 2 NH 2 (Dab side chain), R 5 = -CH 2 Ph (DPhe side chain), R 6 = CH 2 CH 2 SCH 3 (Met side chain), R 7 = -CH 2 CH 2 NH 2 (Dab side chain), and R 8 = -CH 2 CH 2 NH 2 (Dab side chain]; sp-Met) is also known and can be prepared as It is described in Example 9.

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Example 9 Preparation of the sP-Met peptide

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Dap (Boc) -OH, Fmoc-Met-OH,
Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The first amino acid in the sequence, Fmoc-Cys (Acm) -OH, was anchored directly to the resin 4-MBHA-polystyrene (113.7 mg, f = 0.98 mmol / g) by activation with DIPCDI (52 µL, 0.33 mmol, 3 equiv. excess). The amino acids of the sequence were introduced according to a standard solid phase synthesis protocol Fmoc / t Bu  as described above. Once the sequence is completed, nonanoic acid (58.5 µL, 0.33 mmol, 3) was coupled excess times) with DIPCDI / HOBt in the minimum amount of DMF. A once the reaction is completed as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide is completed, the disulfide bond was formed by oxidation with iodine (113.1 mg, 4 equiv.) In DMF for two hours. The resin was washed with  DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL).

De-anchoring and deprotection of the peptide is performed by acidolysis with TFA: TMS-Cl: triethylsilane (2.77 mL TFA, 1.03 mL TMS-Cl, 0.2 mL TES, volume total 4 mL) for 3 hours at room temperature. Then, the acidolytic mixture was evaporated with N2 stream and the Oily residue was precipitated with dry ether. The peptide crude is recovered by centrifugation and decantation of the etheric phase. He Peptide crude weight was 109.7 mg (yield 79.3%, purity 75%) Characterization of the purified peptide: Homogeneity (by HPLC trace area integration)> 99%; MALDI-TOF: m / z 1241.62 ([M + H] +, 59.4%), 1263.68 ([M + Na] +, 100%).

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Example 10 Preparation of the sP-Met (O) peptide

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Dap (Boc) -OH, Fmoc-Met (0) -OH, Fmoc-DPhe-OH, Fmoc-Thr (tBu) -OH.

Manual synthesis was carried out according to a standard protocol Fmoc / t Bu in polypropylene syringes with a polyethylene filter The first amino acid in the sequence, Fmoc-Cys (Acm) -OH, was anchored directly to the resin 4-MBHA-polystyrene (112.7 mg, f = 0.98 mmol / g) by activation with DIPCDI (51.3 µL, 0.33 mmol, 3 equiv. excess). The amino acids of the sequence were introduced according to a standard solid phase synthesis protocol Fmoc / t Bu  as described above. Once the sequence is completed, nonanoic acid was coupled (58.0, 0.33 mmol, 3 excess times) with DIPCDI / HOBt in the minimum amount of DMF. A once the reaction is completed as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH 2 Cl 2 (5 x 30 s). Once the lipopeptide is completed, the disulfide bond was formed by oxidation with iodine (112.1 mg, 4 equiv.) In DMF for two hours. The resin was washed with  DMF (8 x 5 mL) and CH 2 Cl 2 (5 x 5 mL).

De-anchoring and deprotection of the peptide is performed by acidolysis with TFA: triethylsilane (95: 5, v / v, 4 mL) for 3 hours at 45 ° C. Then the acidolytic mixture is evaporated with a stream of N 2 and the oily residue precipitated with dry ether. The peptide crude was recovered by centrifugation and decantation of the etheric phase. The weight of the peptide crude was 43.5 mg (yield 31.4%, purity 80%). Peptide Characterization purified: Homogeneity (by integration of the trace area of HPLC)> 99%; MALDI-TOF: m / z 1257.84 ([M + H] +, 100%), 1279.84 ([M + Na] +, 95.0%).

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Example 11 Antibacterial activity test

The antibacterial activity of the lipopeptides Synthetic was determined in sterile 96-well plates (Corning Cost 3598 microtiter plates) with a final volume of 200 µL as follows: aliquots (100 µL) of a suspension of bacteria at a concentration of 10 5 units colony forming / mL in culture medium (MH, Muller Hinton Broth, Difco, USA) at pH 7.4, was added to 100 µL of lipopeptide solution prepared from a stock solution in water of 1 mg / mL, in serial dilutions at double dilution in MH a pH 7.4 (Jorgensen & Turnide, 2003). Inhibition of bacterial growth was determined from absorbance at 492 nm on an Absorbance Microplate reader ELx 800 instrument (Bio-tek Instruments) after incubation at 37 ° C for 18-20 h. The antibacterial activity was expressed as CMI, the concentration at which no growth is detected after 18-20 h incubation.

The microorganisms were grown in Tryptycase I am Broth (Pronadisa, Barcelona), incubating at 37ºC until observing Bacterial growth. They were then sown in Trypticase I am Agar (Pronadisa, Barcelona) and they were incubated at 37ºC until Observe colony formation. The microorganisms are they kept in cryoballs (EAS laboratoire, France) at -20ºC.

Strains of bacteria used to carry After the antibacterial activity test were obtained from: the American Type Culture Collection (ATCC, Rockville, MD, USA):

Escherichia coli ATCC 8739

Pseudomonas aeruginosa ATCC 9027

Salmonella typhimurium ATCC14028

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Staphylococcus aureus ATCC 6538

Bacillus cereus var. mycoides ATCC11778

Mycobacterium phlei ATCC41423

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The results are shown in Table 1 and in the Table 2.

TABLE 1 Antibacterial activity (MIC) in Gram positive expressed in \ mug / ml

4

TABLE 2 Antibacterial activity (MIC) in Gram negative expressed in \ mug / ml

5

Control (polymyxin B): (S) -6-Methyloctanoyl-Dab-Thr-Dab- cycle [Dab-Dab-DPhe-Leu-Dab-Dab-Dab].

These results demonstrate that the compounds (I) and (I ') show antibacterial activity at micromolar level both against Gram-positive bacteria and against Gram-negative bacteria (in the latter case, the MIC is slightly higher than that of natural polymyxin, but the polymyxin shows no activity against bacteria Gram-positive). Consequently, the new compounds have a higher spectrum of activity since the available peptide antibiotics (natural polymyxin and daptomycin) are only active against one type of bacteria, Gram-negative or Gram-positive, respectively.

Claims (15)

1. Compound of formula (I),

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6

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or a retroenantiomer thereof of formula (I '),

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7

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where: R_ {0} is a radical selected from the group consisting of: CH 3 - (CH 2) m -, CH 3 -O- (CH 2 CH 2 O) 2 CH 2 -, Y 8 m is an integer between 7 and 10; x is an integer between 1 and 3; R 1, R 3, R 4, R 7, and R 8 they are independently selected radicals that have the following formula: GF- (CH 2) n -; where n is an integer between 1 and 4; and GF is a radical selected from the group consisting of -NH_ {2}, -NH-C (= NH) -NH2 and 4-imidazolyl; R2 is a radical selected from the group consisting of -CH (CH3) (OH), -CH (CH 3) 2, -CH 2 NH 2 and -CH 2 OH; R 5 and R 6 are selected radicals independently among the group consisting of - (C 1 -C 4) - linear alkyl or branched, - (CH 2) - R 10, -CH 2 -CH 2 -S-CH 3 Y -CH 2 -CH 2 -S (= O) -CH 3; R 9 is H or Gly-NHCH 2 CH 2 CH 2 NHCH 2 CH 2 CH 2 CH 2 NHCH 2 CH 2 CH 2 CH 2 NH 2; Y R_ {10} is a radical selected from the group consisting of phenyl, 3-indolyl, 4-imidazolyl, 4-hydroxyphenyl, α or β-naphthyl and 2-, 3- or 4-pyridyl; with the proviso that the compound of formula (I) is not one of the following compounds: nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dab-Dab-Cys]; nonanoil-Dap-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met-Dab-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met (0) -Dab-Dab-Cys]; or nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DTrp-Leu-Dab-Dab-Cys].

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2. Compound according to claim 1, wherein R2 is -CH (CH3) (OH).

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3. Compound according to any of the claims 1-2, which is the compound of formula (I).

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4. Compound according to claim 3, which is select from the following: nonanoil-Arg-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys]; nonanoil-Arg-Thr-Arg- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys]; nonanoil-Arg (NO2) -Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg (NO2) - Dab-Cys]; nonanoil-Arg-Thr-Arg- cycle (SS ) [Cys-Arg-DPhe-Leu-Arg-Arg-Cys]; nonanoil-Dap [(C = NH) -NH2] - Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap [(C = NH) -NH2] - Dab-Cys ]; Y nonanoil-Arg-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Arg-Dab-Cys] -Gly-NHCH_2CH2CH2CH2 NHCH2CH2 CH 2 CH 2
NHCH 2 CH 2 CH 2 NH 2.

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5. Compound according to any of the claims 1-2, which is the compound of formula (I ').

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6. Compound according to claim 5, which is select from the following: cycle (SS ) [DCys-DDab-DArg-DLeu-Phe-DDab-DCys] DDab-DThr-DArg-octylamide; Y cycle (SS ) [DCys-DDab-DDab-DLeu-Phe-DDab-DCys] DDab-DThr-DDab-octylamide.

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7. Compound according to any of the claims 1-6, or one of the following compounds: nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dab-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Dab-Leu-Dab-Cys]; nonanoil-Dap-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met-Dab-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met (0) -Dab-Dab-Cys]; or nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DTrp-Leu-Dab-Dab-Cys]; for use as an antibacterial agent against Gram-positive bacteria.

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8. A compound according to claim 7, wherein the Gram-positive bacteria are selected from the group consisting of Micobacterium phlei, Staphylococcus aureus and Bacillus cereus var. mycoides 9. Compound according to any of the claims 1-6, for use as an agent antibacterial against Gram-negative bacteria. 10. A compound according to claim 9, wherein the Gram-negative bacteria are selected from the group consisting of Salmonella tvphimurium, Pseudomonas aeruginosa and Escherichia coli . 11. Use of a comma compound has been defined in any of claims 1-6, or one of the following compounds, nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dab-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Dab-Leu-Dab-Cys]; nonanoil-Dap-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Leu-Dap-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met-Dab-Dab-Cys]; nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DPhe-Met (0) -Dab-Dab-Cys]; Y nonanoil-Dab-Thr-Dab- cycle (SS ) [Cys-Dab-DTrp-Leu-Dab-Dab-Cys]; for the preparation of a medicine for the treatment of a bacterial infection caused by bacteria Gram-positive in a mammal, including a human.

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12. Use according to claim 11, wherein the Gram-positive bacteria are selected from the group consisting of Micobacterium phlei , Staphylococcus aureus and Bacillus cereus var. mycoides 13. Use of a compound as defined in any of claims 1-6, for the preparation of a medicine for the treatment of an infection bacterial caused by Gram-negative bacteria in A mammal, including a human. 14. Use according to claim 13, wherein the Gram-negative bacteria are selected from the group consisting of Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli . 15. Pharmaceutical composition comprising a therapeutically effective amount of a compound as has been defined in any of claims 1-6, together with pharmaceutically acceptable excipients or carriers.