WO2010029196A1

WO2010029196A1 - Antibacterial peptide compounds

Info

Publication number: WO2010029196A1
Application number: PCT/ES2009/000445
Authority: WO
Inventors: Francesc Rabanal Anglada; Yolanda Cajal Visa; Maria Garcia Subirats; Montserrat RODRÍGUEZ NUÑEZ
Original assignee: Universidad De Barcelona
Priority date: 2008-09-10
Filing date: 2009-09-09
Publication date: 2010-03-18
Also published as: ES2334547B1; ES2334547A1

Abstract

Compounds of formula (I), or retroenantiomers thereof unacylated at their N-terminal end and having the configuration of the aminoacid with the R₂ side chain non-inverted when R₂ is -CH(CH₃)(OH), wherein R₀ is CH₃-(CH₂)_m-, CH₃-O-(CH₂CH₂O)₂CH₂- or phenyl-(CH₂)_x-; m is an integer between 7 and 10; x is an integer between 1 and 3; R₁, R₃, R₄, R₇ and R₈ are independently selected from the formula GF-(CH₂)_n-, wherein 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(CH₃)₂, -CH₂NH₂ or -CH₂OH; R₅ and R₆ are -(C₁-C₄-linear or branched alkyl, -(CH₂)-R₁₀, -CH₂-CH₂-S-CH₃ or -CH₂-CH₂-S(=O)-CH₃; R₉is H or Gly-spermide; and R₁₀ is phenyl, 3-indolyl, 4-imidazolyl, 4-hydroxyphenyl, α- or β-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 relates to compounds that are active against Gram-positive and Gram-negative bacteria, their preparation process, pharmaceutical compositions containing them, and their use in the treatment of bacterial infections.

STATE OF THE TECHNIQUE

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

There is a worldwide concern about the increasing prevalence of infections caused by multiresistant bacteria, such as, for example, methicine-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 the 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 enormous efforts aimed at the development of new drugs that act on the bacterial membrane, such as antimicrobial peptides ("anti-microbial peptides", AMP). Antimicrobial peptides offer a new class of therapeutic agents to which bacteria are not able to develop genetic resistance, since they act primarily on the lipid component of cell membranes. Among these compounds, polymyxin B (PxB), which is approved for clinical use, is acquiring a new relevance therapeutic and is beginning to be considered as a representative of the class of active antibiotics against multiresistant bacteria.

Polymyxins, in particular Ia polymyxin B, constitute a family of antibiotics discovered in 1947 with a high activity against Gram-negative bacteria. Polymyxin B is an antibiotic lipopeptide isolated from Bacillus polvmvxa. 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 the 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 in general, adaptive and, therefore, reversible. Polymyxin B is also capable of inhibiting the biological activity of bacterial lipopolysaccharide (LPS) by means of 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 peptide compounds with antibiotic activity that act on the lipid component of bacterial membranes and that are active against both Gram-positive and Gram-negative bacteria. These compounds are based on the structure of the natural polymyxin. However, unlike polymyxins, these 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.

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

(i)

or retroenantiomers thereof of formula (V),

R «

(! ') where R ₀ is a radical selected from CH ₃ - (CH ₂ ) _m -, CH ₃ -O- (CH ₂ CH ₂ O) ₂ CH ₂ -, and

m is an integer between 7 and 10; x is an integer between 1 and 3; Ri, R ₃ , R ₄ , R ₇ , and Re 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 -NH ₂ (amino), -NH-C (= NH) -NH ₂ (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 Re are radicals selected from: - (Ci-C ₄ ) -a 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

GIy-NHCH ₂ CH ₂ CH ₂ NHCH 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 condition 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 (S-S) rCvs-Dab-DPhe-Leu-Dab-Dab-Cvsl, (sp-B); nonanoil-Dap-Thr-Dab-cycle (S-S) rCvs-Dab-DPhe-Leu-Dap-Dab-Cvsl, (sP-D); nonanoil-Dab-Thr-Dab-cclo (S-S) rCvs-Dab-DPhe-Met-Dab-Dab-Cvsl.

(sp-Met); nonanoil-Dab-Thr-Dab-cycle (S-S) [Cvs-Dab-DPhe-Met (O) -Dab-Dab-Cvs1: (sp-

Me to)); or nonanoil-Dab-Thr-Dab-cycle (S-S) rCvs-Dab-DTrp-Leu-Dab-Dab-Cvs1, (sp-Bw).

The term "cycle (S-S)" 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 retroenantiomers of certain cyclic peptides and depsypeptides maintain the 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 an analog peptide consisting of the reverse amino acid sequence of the compound (I) with simultaneous inversion of the configuration of all chiral centers except that of the amino acid with the side chain R ₂ when said R ₂ is threonine, and that is not necessarily acylated at its N-terminal end.

In a preferred embodiment, the compounds of formula (I) or (I ') are those mentioned above where R ₂ is -CH (CHs) (OH), that is, R ₂ is the threonine side chain.

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

nonanoil-Arq-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-Arg-Dab-Cvsl. (sp-2Arg); nonano¡l-Arq-Thr-Arg-cclo (SS) rCvs-Dab-DPhe-Leu-Arg-Dab-Cys1, (sp-3Arg); nonano¡l-Arg (NO ₂ ) -Thr-Dab-cjcJρiS _I S) ICys-Dab-DPhe-Leu-Arg (NO ₂ ) -Dab- Cys], (sp-2Arg (NO ₂ ); nonanoil-Arq- Thr-Arq-c¡clo (SS) fCvs-Arq-DPhe-Leu-Arg-Arq-Cvs1, (sp-5Arg); nonano¡l-DaprC (NH) -NH _? 1-Thr-Dab-c¡clo (SS) rCvs-Dab-DPhe-Leu-DaprC (NH) -

NH ₂ ] -Dab-Cys], (guanidylated sP-2Dap); and nonanoil-Arq-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-Arq-Dab-Cvs1-Glv- NHCH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ NH ₂ , (sP-2Arg-Gly-spermide).

Preferred compounds of formula (I ¹ ) are those selected from the following list:

cycle (SS) fDCvs-DDab-DArg-DLeu-Phe-DDab-DCys1-DDab-DThr-DArq-octylamide, (sP-2Arg retroenantium); Y cycle (SS) rDCvs-DDab-DDab-DLeu-Phe-DDab-DCvs1-DDab-DThr-DDab-octylamide, (sP-B retroenantium).

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

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 CI ₂ several times; (ii) treatment with 40% trifluoroacetic acid (TFA) / CH ₂ CI ₂ ; (ii) washing the resin with CH ₂ CI ₂ several times; (iv) treatment with 5% DIEA / CH ₂ CI ₂ several times; (v) washing the resin with CH ₂ CI ₂ 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 NN-diisopropylethylamine (DIEA) hexafluorophosphate in the minimum amount of DMF; (viii) washed with DMF several times and CH ₂ CI 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 above procedures can be purified by preparative HPLC, whereby a purity greater than 99% can be obtained.

Thus, the compounds of the present invention are 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 ¹ ), as well as any of the following compounds: nonano¡l-Dab-Thr-Dab-cclo (SS) ÍCvs-Dab-DPhe-Leu-Dab-Dab-Cvs1, (sp-B); nonanoil-Dab-Thr-Dab-cicloofS-SHCvs-Dab-DPhe-Dab-Leu-Dab-Cvsl, (sp-C); nonanoil-Dap-Thr-Dab-cclo (SS) rCvs-Dab-DPhe-Leu-Dap-Dab-Cvsl, (sP-D); nonano¡l-Dab-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Met-Dab-Dab-Cvsl, (sp-Met); nonanoil-Dab-Thr-Dab-cyclo (SS) rCvs-Dab-DPhe-Met (O) -Dab-Dab-Cvs1, (sp-Met (O)); or nonanoil-Dab-Thr-Dab-cycle (S-SHCvs-Dab-DTrp-Leu-Dab-Dab-Cvs1, (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, Mvcoplasma and Actinobacteria.

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

This aspect of the invention can also be formulated as the use of a compound as defined above, including the known compounds mentioned for the preparation of a medicament for the treatment of a bacterial infection caused by Gram-positive 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, that suffers or is susceptible to bacterial infections caused by Gram-positive bacteria, in particular to the aforementioned infections, said method comprising The administration to said patient of a therapeutically effective amount of compounds of the present invention, including the known compounds mentioned above, together with pharmaceutically acceptable excipients or carriers.

It is also part of the invention the compounds of formula (I) or (I ') as defined above for use as antibacterial agents against Gram-negative bacteria 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 baumanü.

In a preferred embodiment, Gram-negative bacteria are selected from Salmonella typhimuríum, Pseudomonas aeruginosa and Escherichia coli. In a particular embodiment, Gram-negative bacteria are selected from Salmonella tvphimurium 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 ¹ ) 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 the prophylaxis of a mammal, including a human who suffers or is susceptible to bacterial infections caused by Gram-negative bacteria, in particular to any of the infections mentioned above, the method the administration to said patient comprising a therapeutically effective amount of the compounds of formula (I) or (I ¹ ) as defined above, together with pharmaceutically acceptable excipients or carriers.

The compounds of the present invention can be used analogously to other known antibacterial agents. These can be used alone or in combination with other appropriate bioactive compounds. In a particular embodiment, the compounds of formula (I) or (I ¹ ) can be used in the treatment of bacteremias and / or septicemia resulting from infections by Gram-negative bacteria, administered alone or in combination with conventional antibiotics. In a preferred embodiment, the compounds of formula (I) or (I ¹ ) 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 Since it is apparently believed that they act on the membrane, they 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 ¹ ), together with appropriate amounts of pharmaceutically acceptable excipients or carriers.

The pharmaceutical compositions can be prepared by combining the compounds of formula (I) or (I ¹ ) of the present invention with pharmaceutically acceptable solid or liquid carriers or excipients, following standard pharmaceutical practices.

The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated, for example by oral, parenteral, inhalation, rectal, transdermal or topical route. In the therapeutic use to treat or combat bacterial infections in patients such as humans and other animals that could be diagnosed with bacterial infections, said pharmaceutical compounds or 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 following the treatment that is effective as an antibacterial. Generally, said amount or dose that is effective as an antibacterial will be in the approximate range of 0.1 to 100 mg / kg, more preferably about 3.0 to 50 mg / kg body weight / day. It is understood that the doses may vary depending on the requirements of the patient, the severity of the bacterial infection to be treated and the particular compound used.

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

Throughout the description and the claims, the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be detached in part from 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-dmethylaminopyridine; DIEA, N.N-diisopropylethylamine; DIPCDI, N.N'-düsopropylcarbodiimide; 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-CI, trimethylsilyl chloride; Trt, trityl; DAB, 2,4-diaminobutyric acid.

General methods

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

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 in excess) and DIPCDI / HOBt (3 times in excess) in the minimum amount of DMF; (v) washing with DMF (5 x 30 s) and CH ₂ CI ₂ (5 x 30 s); (vi) Kaiser test (with a sample of peptide resin); (vii) DMF wash (5 x 30 s).

Boc synthesis protocol: for each amino acid cycle it consists of the following steps: (i) washing the resin with CH ₂ CI ₂ (5 x 30 s); (ii) treatment with 40% trifluoroacetic acid / CH ₂ CI ₂ (1 x 1 min. + 2 x 10 min., Boc elimination); (iii) washing the resin with CH ₂ CI ₂ several times; (iv) treatment with 5% DlEA / CH ₂ Cb several times; (v) washing the resin with CH ₂ CI 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 CI 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.

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

The peptides were characterized by amino acid analysis with a Beckman 6300 analyzer and by MALDI-TOF mass spectrometry in a VOYAGER-DE-RP (Applied Biosystems) mass spectrometer or in an Electrospray (ESI +) ZQ-Micromass (Waters mass spectrometer) ) or ESI + LC / MSD-TOF (Agilent Technologies) mass spectrometer. The homogeneity of the purified peptides was checked by analytical HPLC using Nucleosil C18 reverse phase columns (4 x 250 mm, 5 μm particle diameter and a pore measurement of 120 A). Elution was carried out at 1 ml-min ^"1 with mixtures of H ₂ O- 0.045% TFA and acetonitrile-0.036% TFA and UV detection at 220 nm.

Example 1: Preparation of nonanoyl-Arq-Thr-Dab-cycle (SS) Ívs-Dab-DPhe-Leu-Arq-Dab-Cysi (compound of formula (I) with Rn = CHa (CHg) ₇ -, Ri = - CH? CH? CH? NHC (= NH) -NH? (Am side chain). R? = -CH (CH ₃₎ OH, R _n = -CH7CH9NH7 (Dab side chain), R A.dbd ? CH? NH? (Dab side chain), R _n = -CH? Ph (DPhe side chain), RR = -CH? CH (CH _g Leu side chain). R _z = -CH _? CH _? CH _? NHC (= NH) -NH? (Am side chain) v RR = -CHpCH ₂ NH? (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 Fmoc / Bu protocol in polypropylene syringes with a polyethylene filter. The spacer ("linker") Fmoc-Rink (319 mg, 0.59 mmol, 3 equiv. Excess) was anchored to the 4-MBHA-polystyrene resin (201 mg, f = 0.98 mmol / g) by activation with DIPCDI (92 μl_, 0.59 mmol , 3 excess equivalents). The amino acids of the sequence were introduced according to a standard Fmoc / Bu solid phase synthesis protocol as described above. Once the sequence was completed, the nonanoic acid (103.6 μl, 0.59 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. Once the reaction was 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 Cb (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 CI ₂ (5 x 5 mL).

De-anchoring and deprotection of the peptide was performed by acidolysis with TFA: triethylsilic: water (95: 3: 2, v / v / v, 5 mL) for 1.5 hours at room temperature. Then, the acidolytic mixture was evaporated with a stream of N ₂ 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%).

Example 2: Preparation of nonanoyl-Aro-Thr-Arg-cyclo-S-SKCvs-Dab-DPhe-Leu-Arg-Dab-Cvsi (Compound (I) with R ₀ = CH ₃ (CHg)? -. Ri =

-CH ₂ CH _Z CH _? NHC (= NH) -NH ₂ (Arg side chain), R? = -CH (CHg) OH, Ra = -CH ₂ CH ₂ CH ₂ NHC (= NH) -NH ₂ (Arg side chain), R ₄ = -CH ₂ CH ₂ NH? (Dab side chain). Rg = -CH ₂ Ph (DPhe side chain), Rg = -CH ₂ CH (CH ₃ )? (Leu side chain), R ₇ = -CH _? CH ₂ CH ₂ NHC (= NH) -NH ₂ (Aro side chain), v Rq = -CH ₂ CH ₂ NH ₂ (Dab side chain): sp- 3Arc

Manual synthesis was carried out according to a standard Fmoc / Bu protocol in polypropylene syringes with a polyethylene filter. The first amino acid of The sequence, Fmoc-Cys (Acm) -OH, was directly anchored to the 4-MBHA-polystyrene resin (151.4 mg, f = 0.98 mmol / g). The amino acids of the sequence were introduced according to a standard Fmoc / Bu solid phase synthesis protocol as described above. Once the sequence was completed, the nonanoic acid (78.0 μl, 0.45 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. Once the reaction was 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 CI 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 CI ₂ (5 x 5 mL).

De-anchoring and deprotection of the peptide was performed by acidolysis with TFA: TMS-CI: HBr (33% n AcOH): triethylsilane (3.21 ml TFA, 1.29 ml TMS-CI, 0.25 ml TES, 0, 25 ml HBr, total volume 5 ml) for 3 hours at room temperature. Then, the acidolytic mixture was evaporated with a stream of N ₂ 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 75.1 mg (yield 82%, purity 80%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1391.73 ([M + H] ⁺ , 32.9%), 1413.70 ([M + Naf, 100%).

Example 3: Preparation of nonanoyl ~ Arq (NO ₂ ) -Thr-Dab-cycle (SS ^' ) ljCvs-Dab- DPhe-Leu-Arq (NO ₂ ) -Dab-Cvs] (Compound (I) with Rn = CHg ( CH?)? -, Ri = -CH ₂ CH ₂ CH ₂ NHCf = NH) -NH-NO ₂ (side chain of Am (NOp)), R ₂ = -CH (CHg) QH, R ₃ = CH ₃ CHpNH ₂ (Dab side chain), R ₄ = -CHaCHgNH ₂ (Dab side chain), R _n = -CH ₂ Ph (DPhe side chain), Rs = -CH ₂ CH (CH _ñ Leu side chain), R ₇ = ~ CH ₂ CH ₂ CH ₂ NHC (= NH) -NH-NO ₂ (Am side chain (NO ₂ )) and

R _n = -CH ₂ CH ₂ NH ₂ (Dab side chain): sp-2Arα (NO _? )

Protected amino acids: Fmoc-Cys (Acm) -OH, Fmoc-Dab (Boc) -OH, Fmoc-Arg (NO ₂ ) ~ OH, Fmoc-Leu-OH, Fmoc-DPhe-OH, Fmoc-Thr (tBu) - OH

Manual synthesis was carried out according to a standard Fmoc / Bu protocol in polypropylene syringes with a polyethylene filter. The spacer ("linker") Fmoc-Rink (329 mg, 0.61 mmol, 3 equiv. Excess) was anchored to the 4-MBHA-polystyrene resin (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 Fmoc / Bu solid phase synthesis protocol as described above. Once the sequence was completed, the nonanoic acid (107.4 μl, 0.61 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. Once the reaction was 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 CI2 (5 x 30 s). Once the lipopeptide was 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 CI 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 a stream of N ₂ 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 245 mg (yield 84%, purity 85%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; ESI +: m / z 476.5 ([M + 3H] ³⁺ / 3, 100%), 714.0 ([M + 2H] ²⁺ / 2, 73%).

Example 4: Preparation of nonanoyl-Arg-Thr-Arg-cycle (S-S) fCvs-Arg-DPhe-

Leu-Arq-Arq-Cvsi (Compound (I) with Rn = CHj (CH?) ?, Ri = -CH _Z CH _Z CH _Z NHC (= NH) -NH _Z (Aro side chain),

R? = -CH (CH _n ) OH,

Rg = -CH ₂ CH ₂ CHgNHCf-NH) -NH ₂ (Aro side chain).

R ¿= -CH _Z CH _Z CH? NHC (= NH) -NH _Z (Aro side chain). Rg = -CHpPh

(DPhe side chain), Rg = -CH? CH (CHgM Leu side chain),

R _n = -CH _Z CH _Z CH? NHC (= NH) -NH _Z (Ara side chain); sp-5Arg)

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

De-anchoring and deprotection of the peptide was carried out by acidolysis with TFAiTMS-ChHBr (33% in AcOH): triethylsilane (3.21 mi TFA, 1.29 mi TMS-CI, 0.25 mi TES, 0.25 mi HBr, volume total 5 mi) for 3 hours at room temperature. Then, the acidolytic mixture was evaporated with a stream of N ₂ 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 97.9 mg (yield 80%, purity 80%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1503'63 ([M + H] \ 29.3%), 1525.64 ([M + Na] \ 100%).

Example 5: Preparation of nonanoyl-Dapr (C = NH) -NH?) 1-Thr-Dab-cycle (S-S) rCvs-Dab-DPhe-Leu-Dapr (C = NH) -NH _? ) l-Dab-Cvs1 (Compound (I) with Rn = CHs (CH ₂ )? -, R ₁ = -CH ₂ NH (C = NH) -NH? (Dap side chain [(C = NH) -NHg ) I: Guanidylated Dap: short version of Arq, with two methylene less than R? = -CH (CHa) OH, Ra = -CH ₂ CH ₂ NH? (Dab side chain). R ₄ = -CH ₂ CH ₂ NH ? (Dab side chain), Rs = -CH ₂ Ph (DPheV RR side chain = -CHgCHfCHa Leu side chain), R _z = -CH ₂ NH (C = NH) -NH ₂

(DaPf side chain (C = NH) -NH?) !, Guanidylated Dap), v R «= -CH ₂ CH ₂ NH? (Dab side chain): quanidylated 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 / Bu protocol in polypropylene syringes with a polyethylene filter. The first amino acid of 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 of the sequence were introduced according to a standard Fmoc / '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. Once the reaction was 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 CI 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 amino acids Dap were guanidylated with N, N '-di-Boc- H "-trifluoromethanesulfonyl guanidine (486.3 mg, 1.24 mmol, 5 equiv. Excess by NH ₂ ) in DMF for five days. disulfide 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 CI ₂ (5 x 5 mL).

De-anchoring and deprotection of the peptide was carried out by acidolysis with TFA: TMS-CI: HBr (33% in AcOH): triethylsilane (3.4 mLTFA, 0.2 mL HBr, 0.4 mL total anisole volume 4 mL) for 4 hours at 35 ⁰ C. The acidolytic mixture was evaporated with N ₂ 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%).

Example 6: Preparation of cyclo-SWDCvs-DDab-DArq-DLeu-Phe-DDab-DCvs] DDab pThr-DArq-octylamide of sP-retroenanthium 2Ara (Compound of formula (! ') With R ₀ = CHa (CH ₂ )? -, R ₁ = -CH ₂ CH ₂ CH ₂ NHCf = NH) -NH? (DArq side chain), R ₂ = -CH (CHa) OH, Rg = -CH ₂ CHgNH ₂ (DDab side chain), R _£ = -CH ₂ CH ₂ NH ₂ (DDab side chain), R _fi = -CH ₂ Ph (Phei side chain. R _R = -CH ₂ CH (CHrQ? (DLeu side chain), R ₇ = -CH ₂ CH ₂ CH? NHC (= NHV NH? (DArq side chain), and Ra = -CH7CH7NH2 (DDab side chain); sP-retroenantio 2Arg)

Protected amino acids: Boc-DArg (Z ₂ ) ~ 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 (GIy) was first introduced. Fmoc-Gly-OH (174 mg, 0.59 mmol, 3 equiv. Excess), 4-MBHA-polystyrene resin (200 mg, f = 0.98 mmol / g) was anchored 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 (2x10 min). The resin was washed with DMF (5 x 3Os). Then, 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 3Os). The first amino acid of the sequence, Boc-DArg (∑ ₂ ) -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 CI ₂ (5 x 5 mL). The peptide was removed from the resin by aminolysis with octylamine (28.4μL, 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 was obtained.

The deprotection of the side chains was performed by acidolysis with TFA: HBr (33% in AcOH): thioanisole (85: 5: 10) for 3 hours at 35 ⁰ C. The acidolytic mixture was evaporated with N ₂ stream and the residue Oily precipitated with dry ether. The peptide crude was recovered by centrifugation and decantation of the ether phase. The weight of the peptide crude was 90 mg (35% yield, 50% purity due to the presence of thioanisole residues and the thioanisole benzyl sulphonium salt). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1308.21 ([M + H] ⁺ , 100%), 1330.20 ([M + Na] ⁺ , 23%).

Example 7: Preparation of cycle (SS) rDCys-DDab-DDab-DLeu-Phe-DDab-DCyslDDab-DThr-DDab-octylamide (Compound of formula (V) with Rn = CHa (CH ₂ )? - _. , Rj = CH ₂ CH ₂ NH? (DDab side chain), R ₂ = -CH (CH ₃ ) OH, Ra = -CH ₂ CH ₂ NH ₂ (DDab side chain), R ₄ = -CH ₂ CH ₂ NH ₂ ( DDab side chain), R _n = -CH ₂ Ph (Phe side chain), R _n = -CH ₂ CH (CHa)? (DLeu side chain), R? = -CH ₂ CH ₂ NH ₂ (chain DDab side), and RR = -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 polyethylene filter 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% due to the presence of thioanisole residues and the benzyl sulphonium salt of thioanisole). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>95%; MALDI-TOF: m / z 1196.15 ([M + H] ⁺ , 100%), 1218.07 ([M + Na] ⁺ , 87.2%).

Example 8: Preparation of nonanoyl-Arq-Thr-Dab-cycle (SS) fCvs-Dab-DPhe-Leu-Arg-Dab-Cvsl-Glv-NHCH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ NH ₂ (Compound of formula (I) with Rn = CHa (CH ₂ )? -, Rj = -CH ₂ CH ₂ CH ₂ NHCf = NH) -NH ₂ (side chain of Arq), R ₂ = - CH (CHa) OH. R _n = -CH ₂ CH ₂ NH ₂ (Dab side chain), R ₄ = -CH ₂ CH ₂ NH ₂ (Dab side chain), R _n = -CH ₂ Ph (DPhe side chain), Rg = -CH ₂ CH (CHaM Leu side chain), R ₇ = -CH ₂ CH ₂ CH ₂ NHC (= NH) -NH? (side chain of Arg), and Rs = -CH ₂ CH ₂ NH ₂ (side chain of Dab): sP-2Arg-spermide)

Protected amino acids: Fmoc-Ala-OH, Boc-Gly-OH, Boc-Cys (Acm) -OH, Boc-Dab (Z) -OH, Boc-Arg (Z ₂ ) -OH, Boc-Leu-OH, Boc -DPhe-OH. A reference amino acid (Ala) was introduced first. Fmoc-Ala-OH (167 mg, 0.54 mmol, 3 excess equiv.), 4-MBHA-polystyrene resin (113 mg, f = 0.98 mmol / g) was anchored by reaction with DIPCDI (83.5 μl, 0 , 54 mmol, 5 times excess) and HOBt (82.5 mg, 0.54 mmol, 5 equiv. Excess). The Fmoc group was removed by treatment with 20% piperidine solution in DMF (2x10 min.). The resin was washed with DMF (5 x 3Os). Then, 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 (50 µl, 0.32 mmol, 3 times excess) and HOBt (49.5 mg, 0.32 mmol, 3 excess equiv.) in the minimum amount of DMF. The resin was left overnight with 20% piperidine solution in DMF. The first amino acid of the sequence, Boc-Gly-OH (188.86 mg, 1.08 mmol, 10 equiv. Excess) was esterified to the spacer ("linker") with DIPCDI (167μl, 1.08 mmol, 10 equiv. Excess) and DMAP (13.16 mg, 0.108 mmol, 1 equiv.). The rest of the amino acids of the sequence of Cys to Arg, were introduced according to a standard Boc synthesis protocol as described above. Finally, nonanoic acid (94.3 µl, 0.53 mmol, 5 times excess) was coupled 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 was washed with DMF (8 x 5 mL) and CH ₂ CI ₂ (5 x 5 mL). The peptide was removed from the resin by aminolysis with spermine (259.5μL, 0.32 mmol, 3 equiv. Excess) 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 was obtained.

The deprotection of the side chains was performed by acidolysis with TFA: HBr (33% in AcOH): anisole (85: 5: 10) for 3 hours at 35 ⁰ C. The acidolytic mixture was evaporated with N ₂ stream and the residue Oily precipitated with dry ether. The peptide crude was recovered by centrifugation and decantation of the ether phase. The weight of the peptide crude was 191 mg (quantitative yield, 50% purity). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>98%; MALDI-TOF: m / z 1578.18 ([M + H] ⁺ , 100%), 1600.16 ([M + Na] ⁺ , 37%).

The following peptides: Nonanoil-Dab-Thr-Dab-cycle (S-8) rCvs-Dab- (D) Phe-Leu-Dab-Dab-Cvsl (Compound of formula (I) with R ₀ = CH ₃ (CH ₂ )? -, Ri = -CH ₂ CH ₂ NH ₂ (side chain of f Dab), R _z = -CH (CHg) OH, R ₃ = -CH ₂ CH ₂ NH ₂ (side chain of Dab), R ₄ = -CH ₂ CH ₂ NH ₂ (Dab side chain), R ₅ = -CH ₂ Ph (DPhe side chain), R ₆ = -CH ₂ CH (CHs) 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) rCvs-Dab- (D) Phe-Dab-Leu-Dab-Cvs1 (Compound of formula (I) with R ₀ = CH ₃ (CH ₂ ) _/ -, Ri = -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-cclo (S-S) rCvs-Dab- (D) Phe-Leu-Dap-Dab-Cvsl

(Compound of formula (I) with Ro = CH ₃ (CH ₂ ) ₇ , Ri = CH ₂ NH ₂ (Dap 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 (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. Phvs. Chem B, 2007, vol. 111, pp. 551-563.

Nonanoil-Dab-Thr-Dab-cycle (8-S) rCys-Dab-DPhe-Met-Dab-Dab-Cvs (Compound of formula (I) with R ₀ = CH ₃ (CH ₂ ) ₇ -, Ri = - 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 (chain side of Dab), and R ₈ = -CH ₂ CH ₂ NH ₂ (side chain of Dab]; sp-Met) is also known and can be prepared as described in Example 9. 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 Fmoc / Bu protocol in polypropylene syringes with a polyethylene filter. The first amino acid of the sequence, Fmoc-Cys (Acm) -OH, was directly anchored to the 4-MBHA-polystyrene resin (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 Fmoc / Bu solid phase synthesis protocol as described above. Once the sequence was completed, the nonanoic acid (58.5 μl, 0.33 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. Once the reaction was completed as verified by the Kaiser test (with a sample of peptide resin), the resin was washed with DMF (5 x 30 s) and CH2CI2 (5 x 30 s). Once the lipopeptide was 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 CI ₂ (5 x 5 mL).

De-anchoring and deprotection of the peptide was performed by acidolysis with TFA: TMS-CI: triethylsilane (2.77 mL TFA, 1.03mL TMS-CI, 0.2 mL TES, total volume 4 mL) for 3 hours at room temperature. Then, the acidolytic mixture was evaporated with a stream of N ₂ 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 109.7 mg (yield 79.3%, purity 75%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1241, 62 ([M + H] \ 59.4%), 1263.68 ([M + Na] ⁺ , 100%).

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 (O) -OH, Fmoc-DPhe-OH, Fmoc-Thr (tBu ) -OH.

Manual synthesis was carried out according to a standard Fmoc / Bu protocol in polypropylene syringes with a polyethylene filter. The first amino acid of The sequence, Fmoc-Cys (Acm) -OH, was directly anchored to the 4-MBHA-polystyrene resin (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 Fmoc / Bu solid phase synthesis protocol as described above. Once the sequence was completed, the nonanoic acid (58.0μl, 0.33 mmol, 3 times excess) was coupled with DIPCDI / HOBt in the minimum amount of DMF. Once the reaction was 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 CI ₂ (5 x 30 s). Once the lipopeptide was 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 CI ₂ (5 x 5 mL).

The de-anchoring and deprotection of the peptide was carried out by acidolysis with TFA: triethylsilane (95: 5, v / v, 4 mL) for 3 hours at 45 ⁰ C. Then, the acidolytic mixture was evaporated with N ₂ stream and the residue Oily precipitated with dry ether. The peptide crude was recovered by centrifugation and decantation of the ether phase. The weight of the peptide crude was 43.5 mg (yield 31'4%, purity 80%). Characterization of the purified peptide: Homogeneity (by integration of the HPLC trace area)>99%; MALDI-TOF: m / z 1257.84 ([M + H] ⁺ , 100%), 1279.84 ([M + Naf, 95.0%).

Example 11: antibacterial activity test

The antibacterial activity of synthetic lipopeptides was determined in sterile 96-well plates (Corning Costar 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 colony forming units / mL in culture medium (MH, Muller Hinton Broth, Difco, USA) at pH 7.4, were 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 at pH 7.4 (Jorgensen & Turnide, 2003). Inhibition of bacterial growth was determined from the absorbance at 492 nm in a Microplate Absorbance reader ELx 800 (Bio-tek Instruments) instrument after incubation at 37 ⁰ C for 18-20 h. The antibacterial activity was expressed as MIC, the concentration at which no growth is detected after 18-20 h of incubation. Organisms were cultured in Tryptycase Soy Broth (Pronadisa, Barcelona), incubating at 37 ⁰ C to observe bacterial growth. Then, they plated on Trypticase Soy Agar (Pronadisa, Barcelona) and incubated at 37 ⁰ C until observing the formation of colonies. The microorganisms were stored in cryoballs (EAS laboratoire, France) at -20 ⁰ C.

The strains of the bacteria used to carry out 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 tvphimurium ATCC14028

Staphylococcus aureus ATCC 6538 Bacillus cereus var. mycoides ATCC11778 Mvcobacterium phlei ATCC41423

The results are shown in Table 1 and Table 2.

Table 1: Antibacterial activity (MIC) in Gram positive expressed in μg / ml

Table 2: Antibacterial activity (MIC) in Gram negative expressed in μg / ml

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

These results demonstrate that compounds (I) and (I ') show antibacterial activity at the micromolar level both against Gram-positive bacteria and against Gram-negative bacteria (in the latter case, the MIC is slightly higher than that of the natural polymyxin, but polymyxin does not show activity against Gram-positive bacteria). Consequently, the new compounds have a higher spectrum of activity since the available peptide antibiotics (natural polymyxin and daptomycin) are only active against a type of bacteria, Gram-negative or Gram-positive, respectively.

Claims

1. Compound of formula (I),

(Or or a retroenantiomer thereof of formula (I ¹ ),

R,

(i ¹ ) where:

Ro is a radical selected from the group consisting of: CH3- (CH2) nrr>

CH ₃ -O- (CH ₂ CH ₂ O) ₂ CH ₂ -, and

m is an integer between 7 and 10;

x is an integer between 1 and 3;

Ri, R ₃ , R ₄ , R ₇ , and Rs are independently selected radicals having 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) -NH ₂ and 4-imidazolyl;

R ₂ is a radical selected from the group consisting of -CH (CHs) (OH), -CH (CHs) ₂ , -CH ₂ NH ₂ and -CH ₂ OH;

R ₅ and RQ are radicals independently selected from the group consisting of - (Ci-C ₄ ) -linear or branched alkyl, - (CH ₂ ) -Ri ₀ , -CH ₂ -CH ₂ -S-CH ₃ and -CH ₂ -CH ₂ -S (= O) -CH ₃ ;

R ₉ is H or GIy-NHCH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ NH ₂ ; Y

R-io 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: nonanoyl-Dab-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-Dab-Dab-Cvsl: nonanoil-Dap- Thr-Dab-cycle (SS) fCvs-Dab-DPhe-Leu-Dap-Dab-Cvsl: nonanoil-Dab-Thr-Dab-cycle (SS) rCys-Dab-DPhe-Met-Dab-Dab-Cys1: nonanoil-Dab-Thr-Dab-cycle (SS) | ^' Cys-Dab-DPhe-Met (Q) -Dab-Dab-Cys]: or nonanoil-Dab-Thr-Dab-cycle (S-SMCys-Dab-DTrp-Leu-Dab-Dab-Cys].

2. Compound according to claim 1, wherein R ₂ is -CH (CH ₃ ) (OH).

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

4. Compound according to claim 3, which is selected from the following:

nonanoil-Arq-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-Arq-Dab-Cvs1; nonano¡l-Arg-Thr-Arg-cclo (SS) fCvs-Dab-DPhe-Leu-Arg-Dab-Cys]; nonano¡l-Arg ( ^" Nθ7) -Thr-Dab-cclo (SS) fCvs-Dab-DPhe-Leu-Arg (NO?) - Dab-

Cy ₈ ]; nonanoil-Arg-Thr-Arg-cyclo (SS) Ívs-Arg-DPhe-Leu-Arg-Arq-Cvs1; nonanoil-Dapr (C = NH) -NH _? 1-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-

Dap [(C = NH) -NH ₂ ] -Dab-Cys]; and nonanoil-Arg-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Leu-Arg-Dab-Cvs1-Glv-

NHCH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ NH ₂ .

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

6. Compound according to claim 5, which is selected from the following:

cycle (S-S) rDCvs-DDab-DArg-DLeu-Phe-DDab-DCvsiDDab-DThr-DArg-octylamide; and cycle (S-S) fDCvs-DDab-DDab-DLeu-Phe-DDab-DCvsiDDab-DThr-DDab-octylamide.

7. Compound according to any of claims 1-6, or one of the following compounds:

nonanoil-Dab-Thr-Dab-cycle (SS) fCvs-Dab-DPhe-Leu-Dab-Dab-Cys1: nonano¡l-Dab-Thr-Dab-cicloofS-S) fCys-Dab-DPhe-Dab-Leu-Dab-Cvs1; nonanoií-DaD-Thr-Dab-cycle ( ^r SS) rCvs-Dab-DPhe-Leu-Dap-Dab-Cvs1: nonano¡l-Dab-Thr-Dab-cycle (SS) rCvs-Dab-DPhe-Met-Dab -Dab-Cvs1: nonanoil-Dab-Thr-Dab-cyclois-S) fCvs-Dab-DPhe-Met (O) -Dab-Dab-Cvsl; or nonanoil-Dab-Thr-Dab-cycle ('SS) rCvs-Dab-DTrp-Leu-Dab-Dab-Cvsl;

for use as an antibacterial agent against Gram-positive bacteria.

8. Compound according to claim 7, wherein the Gram-positive bacteria are selected from the group consisting of Micobacterium phlei,

Staphylococcus aureus v Bacillus cereus var. mvcoides.

9. Compound according to any of claims 1-6, for use as an antibacterial agent against Gram-negative bacteria.

10. Compound according to claim 9, wherein the Gram-negative bacteria are selected from the group consisting of Salmonella typhimurium, Pseudomonas aeruqinosa and Escherichia coli.

11. Use of a compound as defined in any of claims 1-6, or one of the following compounds,

nonanoil-Dab-Thr-Dab-cycle (S-S) rCvs-Dab-DPhe-Leu-Dab-Dab-Cvsl; nonanoil-Dab-Thr-Dab-cycle (S-S) fCvs-Dab-DPhe-Pab-Leu-Dab-Cvs1; nonano¡l-Dap-Thr-Dab-cycle (S-S) rCvs-Dab-DPhe-Leu-Dap-Dab-Cvs1; nonanoil-Dab-Thr-Dab-cycle (S-S) fCvs-Dab-DPhe-Met-Dab-Dab-Cvs1; nonanoil-Dab-Thr-Dab-cyclo (S-S) fCvs-Dab-DPhe-Met (O) -Pab-Dab-Cys1; and nonanoil-Dab-Thr-Dab-cycle (S-S) rCvs-Dab-DTrp-Leu-Pab-Dab-Cvs1;

for the preparation of a medicament for the treatment of a bacterial infection caused by Gram-positive bacteria in a mammal, including a human.

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. mvcoides.

13. Use of a compound as defined in any of claims 1-6, for the preparation of a medicament for the treatment of a bacterial infection 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 tvphimurium, Pseudomonas aeruginosa and Escherichia coli.

15. Pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in any of claims 1-6, together with pharmaceutically acceptable carriers or carriers.