ITMI20120121A1 - NEW ANTITUMOR COMPOUNDS - Google Patents

Description

DESCRIPTION

of the invention with the title: â € œNew anticancer compoundsâ €

Technical context

Currently, anticancer therapy is mainly based on the use of cytotoxic drugs with a low therapeutic index. Among these, for example, taxol (Paclitaxel, Taxol <®>) is able to inhibit tumor proliferation through a mechanism of interference with the dynamics of cellular microtubules and their irreversible stabilization, and is now recognized as one of the cytotoxic agents most effective for the treatment of different types of cancer including breast cancer, Kaposi's sarcoma and, in combination with cisplatin, advanced ovarian cancer and non-small cell lung cancer.

Unfortunately, in addition to cancer cells, other rapidly dividing cells, such as white blood cells and hair cells, are also attacked by the action of most cytotoxic drugs and their administration is consequently accompanied by a series of serious side effects such as suppression of the immune system (neutropenia), a significant reduction in the functioning of the sensory nerves (peripheral neuropathy), hair loss (alopecia), as well as blood and cardiac toxicity.

A recent approach to anticancer therapy involves delivering cytotoxic drugs to the receptors involved in tumor angiogenesis. Integrin alphaV receptors are overexpressed in activated endothelial cells and tumor cells, but are not in resting endothelial cells and in most healthy organs, and thus represent potential targets for targeted cytotoxic interventions.

X. Chen et al (J. Med. Chem. 2005, 48, 1098-1106) described and evaluated the in vitro cytotoxic efficacy of E [c (RGDyK)] 2-PTX, a conjugate of taxol with the dimer of a cyclic peptide RGD, ligand of the alphaV integrins.

A few years later, Q. Cao et al. (Eur. J. Nucl. Med. Mol. Imaging. 2008, 35, 1489â € “1498) evaluated the in vivo activity of the above conjugate on MDA-MB-435 breast cancer and were able to observe that this conjugate presents the drawback of having a very short half-life and of undergoing rapid clearance, also due to the instability of the ester bond between taxol and dimer.

The same conjugate has also been studied by C. Ryppa et al. (International Journal of Pharmaceutics 2009, 368, 89â € “97) who evaluated, among others, the antitumor activity in vivo on ovarian cancer OVCAR-3 xenografted in immunodeficient mice. These authors also confirmed that the half-life of the conjugate is very short and that consequently, after about 2 hours, half of the taxol is released. They also observed that, although it had provided promising results in vitro, said conjugate has no antitumor effect in vivo, with an efficacy therefore clearly worse than that obtained with direct administration of the taxol.

Recently, A. Ressurreiçà £ o, et al. (Chem. Eur. J. 2009, 15, 12184-12188) have described peptidomimetics-RGD containing bifunctional diketopiperazines, named cyclo- [DKP-1-RGD] and cyclo- [DKP-2-RGD], as potential ligands of integrins.

Aims of the invention

An object of the present invention is to provide anticancer compounds which are effective in vitro and in vivo.

Another object of the present invention is to provide antitumor compounds which have a high affinity for the overexpressed alphaV integrin receptors in some types of solid neoplasms.

The object of the invention is also to provide antitumor compounds which exhibit a high selectivity towards tumor cells.

Another purpose of the invention is to provide new compounds selective towards tumor cells, having a sufficient half-life to exert the antitumor action and a reduced systemic toxicity.

A further object of the invention is to provide new compounds having the characteristics indicated above, which can be obtained by means of simple synthesis processes and with good yields.

A further object of the invention is to provide a method for the treatment of tumors, in particular solid tumors, which includes the use of the compounds of the invention.

Description of the invention

Thus, according to one of its aspects, the invention relates to a compound of formula (I)

H2 N <NH>

H N

OR

OR

No.

H.

R2 O

N 6 H N

5

2

O 3 N

R1

NH O

NH

OR

OH

O (I)

in which:

- the carbon atoms of piperazine C3, C6 are respectively in the configurations R, S or S, R (trans configuration);

- R1 and R2 are independently selected from hydrogen, C1-C6-alkyl, C1-C6-alkoxy, benzyl and a group of formula (II)

-L-X-Z-Y-Ct (II)

where is it

- L and Z are independently chosen from the following groups:

n Ar

<n> O

n mm pn in which n and m are, each independently, an integer from 0 to 10, p is an integer from 1 to 10 and Ar is an aromatic or heteroaromatic group;

- X is a group chosen from the following

O H N H H

NNN N N N

H N

OR NOT

N H N

H H

S N N O O S O O N N N N H

O O O N

OR

OR

- Y is a group chosen from the following

O O SN

- Ct is a cytotoxic agent;

with the proviso that at least one of R1 and R2 is a group of formula (II);

and their pharmaceutically acceptable salts, solvates and hydrates.

According to the present invention, in the compounds of formula (I) when the carbon in position 3 of piperazine is R then the carbon in position 6 is S and vice versa, thus obtaining a relative trans configuration.

Furthermore, when R1 and R2 are both a sterically bulky group, for example one is a group of formula (II) and the other is a benzyl group, the compounds of formula (I) can exist as a mixture of two different conformers separable due to the impeded rotation of one ring around the other.

Each isomer, conformer or mixture of isomers and conformers as defined herein are the object of the present invention.

According to the present invention, the alkyl and alkoxy groups are intended to be linear or branched, saturated or unsaturated.

As indicated here, the term â € œaromatic or hetero aromatic groupâ € includes 1,2-phenylene, 1,3-phenylene, naphthylene, phenanthylene, anthracylene, indenylene, furylene, pyranylene, pyrrolylene, imidazolylene, pyridylene, pyrazilene, pyrimidinylene, indolylene, quinolinene, etc.

The variables m, n and p are preferably integers between 1 and 5, for example 1, 2 or 3; advantageously 1 or 2.

According to the present invention, “cytotoxic agent” is here intended to indicate a compound having cytotoxic activity, suitable for administration to humans and animals.

According to a preferred embodiment, the cytotoxic agent â € œCtâ € is selected from taxol, daunorubicin, doxorubicin, carminomycin, 4-epiadriamycin, 4-demethoxidaunomycin, 11-deoxidaunorubicin, 13-deoxidaunorubicin, 14-adriamycin adriamycin-14-octanoate, adriamycin-14-naphthalenacetate, vinblastine, vincristine, mitomycin C, N-methyl mitomycin C, bleomycin A2, dideazatetrahydrofolic acid, aminopteridine, methotrexate, camptothecin, colchicine and cisplatin.

According to a preferred embodiment, the cytotoxic agent â € œCtâ € is the taxol.

According to a preferred embodiment, the taxol is bound to the Y group through the hydroxyl group present in position 2â € ™.

According to a preferred embodiment, only one of R1e R2 is a group of formula (II), even more preferably one of R1e R2 is a group of formula (II) and the other is selected from hydrogen and a benzyl group , more particularly it is chosen as hydrogen.

According to a preferred embodiment, the group L is the group

n

m

where m = n = 1.

According to a preferred embodiment, group X is the group

OR

No.

H.

According to a preferred embodiment, the group Z is the group

n

where n = 2.

According to a preferred embodiment, the group Y is the group

OR

According to a preferred embodiment, in the compound of formula (I), L-X-Z-Y is the following group

OR

H.

No.

OR

A particularly preferred compound is the compound of formula (I) in which R1 = -L-X-Z-Y- Ã ̈

OR

H.

No.

OR

R2 = H, Ct is the taxol and the configuration of the carbon atoms 3 and 6 of piperazine is 3S, 6R.

In practice, the preferred compounds of formula (I) according to the invention, in general terms, can be considered as formed by the following covalently linked units:

- a cyclic peptidomimetic unit RGD consisting of a suitably functionalized diketopiperazine â € œscaffoldâ € and the amino acid sequence Arg-Gly-Asp and which has a high affinity for the alphaV integrin receptors,

- a unit linked to one of the endocyclic nitrogen atoms of diketopiperazine, ending with a primary amine, for example 4-aminomethyl-benzyl,

- a unit deriving from an alkyl chain dicarboxylic acid, for example succinic acid, or other acids capable of improving the pharmacological properties of the final molecule as regards stability and / or solubility,

- a unit corresponding to the cytotoxic agent, which is a molecule having cytotoxic activity, therefore able to inhibit and fight the development of tumors, for example taxol.

Representative and preferred compounds of the present invention are reported in the experimental section of the present description.

The cyclic peptidomimetic-RGD â € œscaffoldâ € that includes diketopiperazine can be prepared by substantially modifying what is described in the publication by A. Ressurreiçà £ o, et al. mentioned above, where two non-functionalized peptidomimetic-RGD cyclic compounds were synthesized by means of a synthesis strategy that presents significant differences. Specific examples of the new synthesis routes are provided in the experimental section of the present description, where the preparation of some representative structures â € œ-L-X-Z-Y-â € that connect the cytotoxic agent to the said â € œscaffoldâ € is also described.

The compounds of formula (I) showed a powerful antitumor action in in vitro and in vivo assays. In fact, in vitro cytotoxicity studies have been conducted on cell lines of different tumor types and in vivo studies of tumor regression measurement using the compounds of formula (I). These studies have shown that the compounds of formula (I) possess a surprising antitumor activity in vivo. In particular, the administration of some compounds representative of the invention in which Ct is the taxol, was able to exercise a higher activity than the activity exercised by the administration of an approximately double quantity of taxol, used as a control and therefore, in this case, not conjugated according to the compounds of formula (I).

For the compounds of formula (I) according to the present invention, or conjugates, as already mentioned above, experiments were conducted in vitro (to evaluate the cytotoxicity on cell lines of different tumor types) and in vivo (to evaluate the measurement of tumor regression). For example, with the conjugate Cyclo [DKP-f3-RGD] -PTX, whose structure is indicated below, a better Tumor Volume Inhibition (TVI) activity is observed than that observed using taxol alone. [TVI of the compound according to the invention = 85% vs. TVI of the taxol used as control = 76% on day 25] using about half (54.5%) of the active principle intended as a cytotoxic agent, that is, in this case taxol.

It therefore appears evident that the treatment with the conjugates described above has an evident therapeutic advantage.

In this case, in fact, the observed data is of double interest, both for the fact that the activity of the conjugates according to the present invention is superior to that of the taxol used alone as a reference compound, but also and above all for the the fact that said activity found to be higher is observed, in the case of the compounds of formula (I) of the invention, for quantities of taxol halved with respect to the quantity of taxol used as control. It is evident that this aspect represents a great advantage, as the possibility of obtaining interesting results about the anticancer activity with half the dose compared to what would be obtained with the control compound, allows to make available a class of products, according to the invention, capable of drastically reducing the side effects associated with the dosages of known compounds necessary to achieve the desired antitumor activity.

This result is decidedly unexpected and demonstrates that the compounds of formula (I) represent a notable technical progress in the field of anticancer therapy.

In fact, it is known that it is not possible to predict a priori whether the conjugation of a given pharmacological agent, in this case a cytotoxic, with a ligand of an overexpressed receptor where one would like the drug to act, such as the compounds of formula (I), allows to carry out the molecular recognition necessary for selective delivery and maintains a high therapeutic activity, in this case cytotoxic. Furthermore, it is not possible to predict whether in conjugated compounds, such as the compounds of formula (I), the bonds between the pharmacological agent and the ligand are sufficiently stable to provide the compound itself with a sufficient half-life to reach the site of action and exert the desired effect before splitting (as discussed for the compound referred to in the â € œTechnical contextâ € section of this description).

As said and as shown in the experimental section of the present description, the compounds of formula (I) proved to be particularly effective in vitro and in vivo, and some representative compounds containing the taxol showed an even higher antitumor activity, at significantly higher dosages. lower than that exerted by the taxol when administered alone, ie not conjugated to form the compounds of formula (I).

According to the present invention and as already mentioned above, the compounds of formula (I) are structurally characterized by the presence of a cytotoxic agent with an antitumor pharmacological action and of a cyclic peptidomimetic-RGD â € œscaffoldâ € which includes diketopiperazine and can be generally indicated with the term â € œconjugatesâ €, according to the present invention. The above described â € œscaffoldâ € suitably functionalized in association with the amino acid sequence Arg-Gly-Asp, acts as a carrier of the cytotoxic agent and is able to recognize the overexpressed integrin receptors in some types of solid neoplasms. The resulting â € œconjugateâ € surprisingly is able to selectively release the cytotoxic agent into cancer cells, reducing their systemic toxicity and improving their effectiveness. Furthermore, again in an unpredictable way, it has been seen that the functionalization of the diketopiperazine â € œscaffoldâ € and its conjugation with the cytotoxic agent do not compromise, but even maintain, the affinity towards the alphaV integrin receptors. This aspect represents a completely unpredictable and not at all obvious fact.

The above is exemplified here on the basis of the following data, which relate to in vitro â € œbindingâ € assays on the isolated alphaVbeta3 and alphaVbeta5 receptors (inhibition of the â € œbindingâ € of biotinylated vitronectin).

The data reported below refer to â € œbindingâ € assays carried out on some cyclic peptidomimetic-RGD (unconjugated) â € œscaffoldâ € (Table A), the structures of which are reported below, in comparison with the â € œbinding 'of some compounds according to the present invention, that is conjugated to the cytotoxic agent (Table B).

H2N <NH>

C Library of unconjugated ligands:

HN

cyclo [DKP-1-RGD]: 3S, 6S, R <1> = Bn, R <2> = H, n = 1 O cyclo [DKP-2-RGD]: 3R, 6S, R <1> = Bn , R <2> = H, n = 1

Ocyclo <[DKP-3-RGD]:> 3S, 6R, R <1> = Bn, R <2> = H, n = 1n N

H <cyclo [DKP-4-RGD]:> 6S, R <1> = H, R <2> = Bn, n = 1R <2> O3R,

N 6 HN

5cyclo [DKP-5-RGD]: 3R, 6S, R <1> = Bn, R <2> = Bn, n = 1 23N cyclo [DKP-6-RGD]: 3S, 6R, R <1> = H , R <2> = Bn, n = 1 O R <1>

cyclo <[DKP-7-RGD]:> 3S, 6R, R <1> = Bn, R <2> = Bn, n = 1NH O <cyclo [DKP-8-RGD]:> 3S, 6R, R < 1> = Bn, R <2> = H, n = 2

NH O OH

OR

where Bn = benzyl

TABLE A

 € œbindingâ € data related to some cyclic (unconjugated) peptidomimetic-RGD â € œscaffoldâ €

alfavbeta3 alfavbeta5 Compound

IC50 [nM] IC50 [nM] Cyclo [DKP-1-RGD] (cis) 3898 ± 418> 10 <4>

Cyclo [DKP-2-RGD] 3.2 ± 2.7 114 ± 99 (trans)

Cyclo [DKP-3-RGD]

4.5 ± 1.1 149 ± 25

(trans)

Cyclo [DKP-4-RGD] 7.6 ± 4.3 216 ± 5

(trans)

Cyclo [DKP-5-RGD] <a>

12.2 ± 5.0 131 ± 29

(trans)

Cyclo [DKP-6-RGD] 2.1 ± 0.6 79 ± 3

(trans)

Cyclo [DKP-7-RGD] -A <a>

220.2 ± 82.3> 10 <4>

(trans)

Cyclo [DKP-7-RGD] -B <a>

0.2 ± 0.09 109 ± 15

(trans)

Cyclo [DKP-8-RGD]

17.7 ± 0.1 420 ± 37

(trans)

Cyclo (RGDfV) <b> 3.2 ± 1.3 7.5 ± 4.8

ST1646 <c> 1.0 ± 0.5 1.4 ± 0.8

a) The compound can exist as a mixture of two different separable conformers (diastereoisomers) due to the impeded rotation of one ring around the other (the benzyl group of DKP cannot pass inside the macrolactam ring). In the case of Cyclo [DKP-5-RGD] only one diastereomer was isolated, while in the case of Cyclo [DKP-7-RGD] two diastereoisomers were isolated in the ratio A: B = 2: 1.

b) Cyclo (RGDfV) is a commercially available reference compound (H-2574 Bachem AG, Switzerland).

c) ST1646 is a reference compound, synthesized as described in L. Belvisi et al. (Bioorg. Med. Chem. 2006, 14, 169-180).

TABLE B

 € œbindingâ € data relating to some â € œconjugatesâ € of formula (I) according to the present invention.

alfavbeta3 alfavbeta5 Compound

IC50 [nM] IC50 [nM]

Cyclo [DKP-f2-RGD] -8.5 ± 0.8 518 ± 10 PTX

Cyclo [DKP-f3-RGD] -5.2 ± 2.3 219 ± 124 PTX

Cyclo [DKP-f4-RGD] -0.9 ± 0.6 76 ± 32

PTX

Cyclo [DKP-f6-RGD] -1.1 ± 0.1 22 ± 3

PTX

Cyclo (RGDfV) <a> 3.2 ± 1.3 7.5 ± 4.8

a) Cyclo (RGDfV) is a commercially available reference compound (H-2574 Bachem AG, Switzerland).

Furthermore, some cytotoxicity studies have been carried out on some of the conjugates according to the present invention, in comparison with the taxol and with one of the cyclic peptidomimetic-RGD â € œscaffoldâ €, obtaining the values indicated in Table C:

TABLE C

Cytotoxicity data on cell lines with different integrin expression

IC50 [nM] (exposure = 72 hours)

Compound IGROV-1 U2-OS SKOV3 PANC-1 MIA-PaCa2

IGROV-1 / Pt1 (HTB-96) (HTB-77) (CRL-1469) (CRL-1420) Cyclo [DKP-> 1200> 18000> 6300> 11600> 11600> 11600 f3-RGD]

Cyclo [DKP-f3-RGD] - 61.3 ± 19.1 4.9 ± 2.0 12.8 ± 0.1 1.2 ± 0.1 2.4 ± 0.8 2.3 ± 0.4

PTX

Cyclo [DKP-f2-RGD] - 17.7 ± 6.0 18.7 ± 6.0 2.2 ± 0.5 1.6 ± 1.0 5.8 ± 4.0 2.0 ± 0.7

PTX

Cyclo [DKP-f6-RGD] - 48.2 ± 2.2 2.4 ± 1.9 5.7 ± 4.4 2.4 ± 1.1 3.5 ± 0.1 2.5 ± 0.6

PTX

Cyclo [DKP-f4-RGD] - 34.4 ± 29.0 3.7 ± 2.0 6.8 ± 4.6 2.4 ± 0.9 3.2 ± 0.7 1.8 ± 0.6

PTX

Taxol 23.0 ± 0.8 2.2 ± 0.8 3.4 ± 0.4 2.7 ± 1.1 5.2 ± 1.9 7.2 ± 3.8

The IGROV-1 and IGROV-1 / Pt1 cell lines were obtained as indicated in Perego P., Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A., Zunino F. Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9: 1-8, 1998 and in Perego P., Giarola M., Righetti, S. C., Supino R., Caserini C., Delia D., Pierotti M. A., Miyashita T., Reed J. C., Zunino F. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems .. Cancer Res., 1996, 56, 556-562.

The other cell lines are subject to deposit at the authorized centers as indicated respectively (ATCC: U2OS HTB-96, SKOV3 HTB-77, PANC-1 CRL-1469, MIA Paca2 CRL-1420).

From in vivo data it is possible to carry out an analysis of the antitumor activity of the conjugate Cyclo [DKP-f3-RGD] -PTX (administration every 4 days for 4 times) which indicates its ability to inhibit the growth of the tumor IGROV- 1 / Pt1 significantly. The inhibition is dose-dependent as indicated by the comparison of the effect obtained by administering the conjugate Cyclo [DKP-f3-RGD] -PTX at doses of 30 mg / kg and 15 mg / kg. The antitumor activity of the conjugate 30 mg / kg is higher than that of taxol 30 mg / kg [for example, on day 25, Inhibition of Tumor Volume (TVI) = 76% for taxol and 85% for the conjugate]. However, since the amount of taxol administered to the animal treated with the conjugate 30 mg / kg is equal to 16.35 mg of taxol, the treatment with the conjugate has an evident therapeutic advantage. In practice, a better activity is observed with the conjugate than with taxol (TVI = 85% vs. TVI = 76% on day 25) with about half (54.5%) of the active principle.

The â € œconjugatesâ € of formula (I) of the invention have the further advantage of being obtainable through chemical synthesis reactions capable of giving high yields in the final product and have the further advantage of being able to be variously and easily functionalized at the level of the dichetopiperazine â € œscaffoldâ €. They also have the advantage of being stable in physiological solution, maintaining a purity higher than 99.5% after 4 days.

Thus, according to another of its aspects, the invention has as its object the use of the compounds of formula (I) in therapy, in particular in the treatment of tumors in humans and animals, in particular in mammals, for example solid tumors, such as breast cancer, ovarian cancer, Kaposi's sarcoma, and `` non-small cell '' lung cancer, whether metastatic or non-metastatic, possibly in combination with additional principles active, for example other cytotoxic agents such as, by way of illustration, cisplatin.

For their use as medicaments, the compounds of formula (I) can be administered in the form of dosage units. Said dosage units are preferably formulated in pharmaceutical compositions in which the compound of formula (I) is mixed with one or more pharmaceutical excipients.

Thus, according to another of its aspects, the present invention relates to pharmaceutical compositions which contain, as an active principle, a compound of formula (I) or one of its pharmaceutically acceptable salts, solvates and / or hydrates, possibly in combination with further principles active, for example one or more cytotoxic agents.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, transdermal, local or rectal administration, the active principles can be administered in the form of dosage units, in mixture with classical pharmaceutical supports, to animals. or to humans. Suitable unit forms of administration include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, topical administration forms, implants, forms of subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and rectal forms of administration.

The pharmaceutical compositions of the present invention can be prepared according to methods well known to those skilled in the art.

The associations of several active ingredients comprising the compounds of formula (I) as well as the kits comprising these associations constitute a further object of the present invention.

Finally, according to another of its aspects, the invention relates to a method for the treatment of tumors which comprises administering to a man or an animal an effective quantity of a compound of formula (I) as described herein or one of its pharmaceutically acceptable salts, solvates and / or hydrates, optionally in combination with further active ingredients, for example other cytotoxic agents.

The invention will now be illustrated by means of examples, for non-limiting purposes.

Brief description of the figures

Figure 1 shows the effects of the Cyclo [DKP-f3-RGD] -PTX conjugate administered intravenously in subcutaneously transplanted nude mice with IGROV-1 / Pt1 ovarian cancer using taxol as the reference drug.

Figure 2 shows the solubility and stability of Cycle [DKP-f3-RGD] -PTX verified by HPLC over time at 25 ° C.

Experimental section

Some representative compounds of the invention are here named as follows: cyclo [DKP-f2-RGD] -PTX: 3R, 6S, R1 = -L-X-Z-Y-PTX, R2 = H cyclo [DKP-f3-RGD] -PTX: 3S , 6R, R1 = -L-X-Z-Y-PTX, R2 = H cyclo [DKP-f4-RGD] -PTX: 3R, 6S, R1 = H, R2 = -L-X-Z-Y-PTX cyclo [DKP-f5a-RGD] -PTX: 3R , 6S, R1 = -L-X-Z-Y-PTX, R2 = Bn cyclo [DKP-f5b-RGD] -PTX: 3R, 6S, R1 = Bn, R2 = -L-X-Z-Y-PTX cyclo [DKP-f6-RGD] -PTX: 3S , 6R, R1 = H, R2 = -L-X-Z-Y-PTX cyclo [DKP-f7a-RGD] -PTX: 3S, 6R, R1 = -L-X-Z-Y-PTX, R2 = Bn cyclo [DKP-f7b-RGD] -PTX: 3S , 6R, R1 = Bn, R2 = -L-X-Z-Y-PTX where

the group L is the group

group X is the group

OR

No.

H.

group Z is the group

and group Y is the group

OR

to form then -L-X-Z-Y- which has the following formula

OR

H.

No.

OR

PXT is the taxol and Bn is a benzyl group.

These compounds constitute a further object of the invention.

Preparation of compounds representative of the invention

Materials and methods: All operations that required anhydrous conditions were carried out using previously torched glassware, with magnetic stirrer and under a nitrogen atmosphere. All commercially available reagents were used as received. The anhydride solvents were withdrawn from the bottles by means of a syringe, using a light pressure of nitrogen. The (S) - and the (R) -serine methyl ester hydrochloride, <(1)> the (2R) acid and (2S) -asparticβ-allyl ester hydrochloride, <(2)> the N-Boc acid (2R) -asparticβ-allyl ester, (S) - e (R) - N-Boc-serine methyl ester, <(3)> (S) - e (R) -3-azido-2- (tert- butoxycarbonylamino) methyl propanoate <(4)> the (S) -and- (R) -3-azido-2- (tert-butoxycarbonylamino) propanoic acid, <(4)> the (S) - e (R ) -dimethylaspartate hydrochloride, <(5)> the (S) - e (R) -N-benzyl dimethyl aspartate <(6)> and the N-Boc-glycine benzyl ester <(7)> were synthesized according to procedures present in literature and showed analytical data in agreement with the data already published. Reactions were monitored by analytical thin layer chromatography, using pre-coated glass plates of 0.25mm silica gel (DURASIL-25-UV254) and the compounds were detected by UV fluorescence and / or reaction with an aqueous solution of permanganate. of potassium or ninhydrin. Flash column chromatography was carried out according to the method developed by Still and collaborators <(8)> using Chromagel 60 ACC silica gel (40-63 Î1⁄4m). The melting points were obtained in an open capillary instrument and are not correct. The <1> H-NMR spectra were recorded on a spectrometer operating at 400.16 MHz. The chemical shifts of the proton are reported in ppm (Î ́) with the solvent reference, relative to tetramethylsilane (TMS), used as internal standard. The following abbreviations have been used to describe spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal, dd = doublet doublet. The <13> C-NMR spectra of the carbon were recorded on a spectrometer operating at 100.63 MHz, with total decoupling of the proton. The chemical shifts of the carbons are reported in ppm (Î ́) with the solvent reference, relative to tetramethylsilane (TMS), used as internal standard. The infrared spectra were recorded on a standard FT-IR instrument and the peaks are reported in cm <-1>. The optical rotation values were measured with an automatic polarimeter with a 1 dm cell operating on the sodium D line. The high resolution mass spectra (HRMS) were performed with an FT-ICR instrument equipped with an ESI source, available at the CIGA (Centro Interdepartimentale Grandi Apparecchiature c / o Università degli Studi di Milano). Low resolution (MS) mass spectra were acquired with a Waters Acquity UPLC-MS instrument (ESI source).

General procedure A for deprotection reactions: a quantity of TFA equal to half the volume of the solvent was added to the solution of the Boc-protected amino acid or peptide in CH2Cl2 (0.13 M) and the reaction was left under stirring at room temperature for 2 h. The solvent was then evaporated, the residue was dissolved in toluene and the solvent evaporated (procedure repeated twice); finally dissolved in ether and evaporated, thus obtaining the corresponding TFA salt of the amino acid or peptide.

General procedure B for the coupling reactions: HATU (1.2 eq.), HOAt (1.2 eq.) And DIPEA were added to the solution of the amino acid or N-protected peptide in DMF, under nitrogen atmosphere and at 0 ° C. (4 eq.). After 30 min. The DMF solution of the peptide TFA salt was added and the reaction mixture was stirred at 0 ° C for 1 h and at room temperature overnight. The mixture was then diluted with EtOAc washed with KHSO41 M (2x), aqueous solution of NaHCO3 (2x) and brine (2x); subsequently the organic phase was anhydrified with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product.

Preparation 1

(S) -benzyl 2- (2- (tert-butoxycarbonylamino) -5- (3- (4-methoxy-2,3,6-trimethylphenylsulfonyl) guanidino) pentanamido) acetate

(S) -Cbz-Arg (Mtr) -Gly-OBn

The protected amino acid Boc-Gly-OBn (1.6 mg, 6 mmol, 1.2 eq.) Was deprotected according to general procedure A. The corresponding trifluoroacetate salt was then coupled with the protected amino acid Boc-Arg (Mtr) (2.5g, 5.14 mmol, 1 eq.) According to the general procedure B. The residue was then purified by flash chromatography on silica gel (CH2Cl2 / MeOH; 97: 3) thus obtaining the desired product below form of white spongy solid (2.9 g, 90% yield).

Rf = 0.21 (CH 7: 3); <ï ›> Î ± <ï 20>

2Cl2 / MeOH 9D = -6.0 (c = 0.5 in CHCl3); <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.40 - 7.31 (m, 5H), 7.27 (br, 1H), 6.58 (s, 1H) , 6.19 (s, 2H), 6.00 (br, 1H), 5.50 (d, 1H, J = 6.6), 5.15 (s, 2H), 4.24 (m, 1H), 4.09 (dd, 1H, J = 17.5, 6.4), 3.99 (dd, 1H, J = 17.9, 5.7), 3.84 (s, 3H), 3.25 (m, 2H), 2.67 (s, 3H), 2.60 (s, 3H), 2.14 (s, 3H) , 1.84 (m, 2H), 1.61 (m, 2H), 1.43 (s, 9H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 172.9, 170.0, 158.5, 156.5, 156.0, 138.4, 136.5, 135.4 , 133.4, 128.5, 128.4, 128.2, 124.8, 111.7, 80.1, 67.1, 55.4, 50.5, 41.2, 40.4, 30.0, 28.0, 25.1, 23.8, 18.1, 11.6; IR (neat): Î1⁄2max3343, 2937, 1669, 1621, 1555, 1455, 1366, 1307, 1251, 1171, 1120; MS (ESI) m / z calc. for [C30H44N5O8S] <+>: 634.29 [M + H] <+>; found: 634.3.

Preparation 12

DKPf2 and DKPf3

4 - ((4-methoxy-2,3,6-trimethylphenylsulfonyl) aminomethyl) benzyl alcohol

A solution of (4- (aminomethyl) phenyl) methanol (1.3 g, 9.4 mmol, 1 eq.) And DIPEA (3.3 mL, 1.88 mmol, 2 eq.) In anhydrous THF (80 ml) was added dropwise drop, at 0 ° C and under nitrogen atmosphere, a solution of 4-methoxy-2,3,6-trimethylbenzene-1-sulfonyl chloride (2.57 g, 10.3 mmol, 1.1 eq.) in anhydrous THF (20 mL). The reaction was left under stirring for 30 min, then it was brought back to room temperature and left under stirring for 6 h. The formation of a white precipitate was observed. The latter was then filtered on a celite bed and the solvent was removed with the rotavapor, thus obtaining a pale yellow oil. Anhydrous DCM (5 mL) was then added and the resulting mixture was sonicated for a few minutes, until a precipitate was observed to form. The latter was removed with filtration on a buchner funnel, washed with a little cooled DCM and a little cooled Et2O and dried, thus obtaining the desired product in the form of white powder (2.79 g, 85% yield).

Rf = 0.40 (Hex / AcOEt 3: 7); <1> H NMR (400 MHz, CD3OD) Î ́ 7.22-7.14 (AB system, 4H), 6.76 (s, 1H), 6.63 (t, 1H, J = 6.3 Hz), 4.57 (d, 2H, J = 5.1 Hz), 4.10 (t, 1H, J = 5.1 Hz), 4.05 (d, 2H, J = 6.3 Hz), 3.88 (s, 1H), 2.66 (s , 3H), 2.55 (s, 3H), 2.1 (s, 3H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 159.9, 142.4, 139.4, 139.3, 137.2, 131.2, 128.5, 127.2, 127.1, 125.4, 113.0, 64.3, 56.0, 46.8, 24.4, 18.1, 12.0; m.p .: 157-158 ° C; MS (ESI) m / z calc. for [C18H24NO4S] <+>: 350.14 [M + H] <+>; found: 350.1.

Preparation 3

4 - ((4-methoxy-2,3,6-trimethylphenylsulfonyl) aminomethyl) benzaldehyde

To a solution in anhydrous THF (200 ml) of 4 - ((4-methoxy-2,3,6-trimethylphenylsulfonyl) aminomethyl) benzyl alcohol (2.79 g, 8.0 mmol, 1 eq.) Was added, at room temperature , MnO2 activated (7.65 g, 88 mmol, 11 eq.). The mixture was left under stirring overnight, then filtered on a celite bed. The solvent was removed in the rotavapor thus obtaining a white solid. (2.76 g, quantitative yield).

Rf = 0.60 (Hexane / AcOEt 1: 1); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 9.97 (s, 1H), 7.76 (d, 2H, J = 8.2 Hz), 7.43 (d , 2H, J = 8.2 Hz), 6.89 (t, 1H, J = 6.5 Hz), 6.73 (s, 1H), 4.21 (d, 2H, J = 6.5 Hz), 3.86 (s, 3H), 2.64 (s , 3H), 2.55 (s, 3H) .; <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 192.4, 160.0, 145.7, 139.4, 139.3, 136.6, 130.1, 129.1, 129.0, 125.5, 113.0, 56.0, 46.8, 24.4, 18.1, 12.0; m.p .: browns at 95 ° C and melts with decomposition at 145 ° C; MS (ESI) m / z calc. for [C18H22NO4S] <+>: 348.13 [M + H] <+>; found: 348.2.

Preparation 4

(S) -methyl 3-hydroxy-2- (4 - ((4-methoxy-2,3,6trimethylphenylsulfonamido) methyl) benzylamino) propanoate

(R) -methyl 3-hydroxy-2- (4 - ((4-methoxy-2,3,6trimethylphenylsulfonamido) methyl) benzylamino) propanoate

To a suspension in anhydrous THF (100 ml) of (S) - or (R) -serine methyl ester hydrochloride (1.1 g, 7.0 mmol, 1.2 eq.), 4 - ((4-methoxy-2,3,6-trimethylphenylsulfonyl ) aminomethyl) benzaldehyde (2.0 g, 5.7 mmol, 1 eq.) and triethylamine (1.2 mL, 8.6 mmol, 1.5 eq) NaBH (OAc) 3 (3.6 g, 17.0 mmol, 3 eq.) was added in small portions , under a nitrogen atmosphere and at 25 ° C. The mixture was left under stirring for 5 h. An aqueous solution of NaHCO3 was then added and the resulting mixture was extracted with EtOAc (3x60 ml). The organic phases were combined, washed with brine and dried with Na2SO4; the solvent was removed in the rotavapor thus obtaining the desired product in the form of a white solid (2.5 g, 95% yield).

R <0>

f = 0.30 (AcOEt 100%); <ï ›> Î ± <ï 2>

D for the enantiomer (R) = 19.0 (c = 1.0 in CH2Cl2); <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.21 (d, 2H, J = 8.2 Hz), 7.10 (d, 2H, J = 8.2 Hz), 6.61 (s, 1H), 4.82 (t, 1H, J = 6.3 Hz), 4.01 (d, 2H, J = 6.3 Hz), 3.85 (s, 3H), 3.81 (d, 1H, J = 13.2 Hz), 3.73-3.70 (m, 4H), 3.65 (d, 1H, J = 13.2 Hz), 3.57 (dd, 1H, J = 6.3, 10.5 Hz), 3.57 (dd, 1H, J = 4.6, 6.3 Hz), 2.65 (s, 3H), 2.51 (s, 3H), 2.11 (s, 3H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 173.7, 159.8, 139.7, 139.3, 139.0, 136.1, 129.3, 128.7, 128.3, 125.7, 112.6, 62.8, 62.3, 55.9, 52.4, 51.9, 46.9, 24.4, 18.1, 12.1; m.p .: 105-106 ° C; MS (ESI) m / z calc. for [C22H31N2O6S] <+>: 451.18 [M + H] <+>; found: 451.2.

Preparation 5

(S) -4-allyl 1 - ((R) -3-methoxy-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) -3-oxopropyl) 2- (tert -butoxycarbonylamino) succinate

(R) -4-allyl 1 - ((S) -3-methoxy-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) -3-oxopropyl) 2- (tert -butoxycarbonylamino) succinate

To a solution of N-Boc- (2R or 2S) -aspartic acid β-allyl ester (1.9 g, 7.1 mmol, 1.3 eq.) In anhydrous DMF (50 ml), were added, at 0 ° C and under atmosphere of nitrogen, HATU (2.7 g, 7.1 mmol, 1.3 eq.), HOAt (0.97 g, 7.1 mmol, 1.3 eq.) and DIPEA (1.9 mL, 11.0 mmol, 2 eq.). The mixture was left under stirring for 30 min and then (S) - or (R) -methyl 3-hydroxy-2- (4 - ((4-methoxy-2,3,6 trimethylphenylsulfonamide) was added methyl) benzylamino) propanoate (2.5 g, 5.5 mmol, 1 eq.). The reaction mixture was allowed to react at 0 ° C for 1 h and at room temperature overnight. The mixture was then diluted with AcOEt (250 ml) and washed with KHSO41M (2x50 ml), aqueous solution of NaHCO3 (2x50 ml) and brine (2x50 ml); It was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the raw product. The latter was purified by flash chromatography on silica gel (Hex / AcOEt from 7: 3 to 5: 5) thus obtaining the desired product in the form of transparent oil (3.2 g, 82% yield).

Rf = 0.30 (Hexane / AcOEt 6: 4); ï ›Î ± ï <20>

D = + 11.4 (c = 1.0 in CH2Cl2) for (S) -4-allyl 1 - ((R) -3-methoxy-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl ) benzylamino) -3-oxopropyl) 2- (tert-butoxycarbonylamino) succinate; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.21 (d, 2H, J = 8.1 Hz), 7.09 (d, 2H, J = 8.1 Hz), 6.61 (s, 1H), 5.88 (ddt, 1H, J = 5.7, 10.5, 17.2 Hz), 5.40 (d, 1H, J = 8.2 Hz), 5.28 (dq, 1H, J = 1.3, 17.2 Hz), 5.21 (dq, 1H, J = 1.3, 10.4 Hz), 4.87 (t, 1H, J = 6.2 Hz), 4.59-4.48 (m, 3H), 4.35 (dd, 1H, J = 4.7, 10.9 Hz ), 4.28 (dd, 1H, J = 4.7, 10.9 Hz), 4.01 (d, 2H, J = 6.3 Hz), 3.85 (s, 3H), 3.81 (d, 1H, J = 13.3 Hz), 3.70 (s , 3H), 3.63 (d, 1H, J = 13.3 Hz), 3.46 (t, 1H, J = 4.7 Hz), 2.92 (dd, 1H, J = 4.7, 16.9 Hz), 2.80 (dd, 1H, J = 4.7, 16.9 Hz), 2.65 (s, 3H), 2.51 (s, 3H), 2.11 (s, 3H), 1.41 (s, 9H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 172.7, 171.0 , 170.8, 159.8, 155.5, 139.7, 139.3, 139.0, 136.0, 132.3, 129.2, 128.7, 128.3, 125.6, 118.5, 112.5, 80.3, 66.2, 65.9, 59.5, 55.9, 52.4, 51.7, 50.3, 46.9, 37.0, 28.4 , 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C34H48N3O11S] <+>: 706.30 [M + H] <+>; found: 706.3.

Preparation 6

Allyl-2 - ((2S, 5R) -5- (hydroxymethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl )acetate

Allyl-2 - ((2R, 5S) -5- (hydroxymethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl )acetate

(S) -4-allyl 1 - ((R) -3-methoxy-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) -3-oxopropyl) 2- (tert -butoxycarbonylamino) succinate or (R) -4-allyl 1 - ((S) -3-methoxy-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) -3-oxopropyl ) 2- (tert-butoxycarbonylamino) succinate (3.0 g, 4.3 mmol, 1 eq.) Was deprotected according to general procedure A. The corresponding trifluoroacetate salt was dissolved in AcOEt (200 ml) and was then addition of aqueous solution of NaHCO3 (100 ml). The mixture was extracted with AcOEt (2x100 ml). The organic phases were combined and the solvent was removed in the rotavapor thus obtaining a transparent and viscous oil. This compound was dissolved in i-PrOH (250 ml) and the mixture was left under stirring for 18 h at room temperature. The solution was then concentrated in the rotavapor and the residue was purified by flash chromatography on silica gel (Hex / AcOEt, 75:25) thus obtaining the desired product in the form of a white spongy solid (2.3 g, 93% surrender).

Rf = 0.30 (AcOEt 100%); <ï ›> Î ± <ï 20>

D = + 26.9 (c = 1.0 in CH3OH) for Allyl-2 - ((2S, 5R) -5- (hydroxymethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) -3,6-dioxopiperazin-2-yl) acetate; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.15 (second order system A2, 4H), 6.99 (s, 1H), 6.62 (s, 1H), 5.90 (ddt, 1H, J = 5.7, 10.5, 17.2 Hz), 5.30 (dq, 1H, J = 1.3, 17.2 Hz), 5.22 (dq, 1H, J = 1.3, 10.5 Hz), 5.14-5.10 (m, 2H), 4.59 (dt, 2H, J = 1.3, 5.7 Hz), 4.56 (dd, 1H, J = 3.9, 8.0 Hz), 4.07 (d, 1H, J = 15.2 Hz), 3.99 (d, 2H, J = 6.3), 3.92 (1H, dd, J = 3.5, 10.5), 3.87-3.77 (m, 4H), 3.73 (br t, 1H), 3.39 (br t, 1H), 3.14 (dd, 1H , J = 3.9, 17.4), 2.79 (dd, 1H, J = 8.0, 17.4), 2.63 (s, 3H), 2.51 (s, 3H), 2.12 (s, 3H); <13> C NMR (101 MHz , CD2Cl2) Î ́ 171.1, 168.5, 166.9, 159.8, 139.3, 139.1, 137.0, 135.8, 132.2, 129.0, 128.7, 128.5, 125.6, 118.7, 112.5, 66.1, 62.3, 62.0, 55.9, 51.4, 47.5, 46.7, 37.3 , 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C28H36N3O8S] <+>: 574.22 [M + H] <+>; found: 574.2.

Preparation 6

Preparation of hydrazoic acid

In a three-necked flask 3 g of NaN3 were dissolved in 3 ml of H2O. Then 20 ml of toluene were added and the flask, under vigorous stirring, was placed in an ice bath. Subsequently, at 0 ° C, 1.2 ml of concentrated H2SO4 were dropped very slowly, making sure that the temperature never exceeded 10 ° C. Subsequently the reaction was carried out for 1 hour at 0 ° C and subsequently the white solid formed was filtered on cotton, washing it twice with a little toluene. The solution of HN3 in toluene was titrated by diluting 1 ml of the solution of HN3 in 50 ml of distilled water, adding a few drops of phenophthalein as an indicator and using a 0.1M NaOH solution as a titrant.

Preparation 7

Allyl 2 - ((2S, 5R) -5- (azidomethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl) acetate

Allyl 2 - ((2R, 5S) - 5- (azidomethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl) acetate

To a solution of allyl-2 - ((2S, 5R) or (2R, 5S) -5- (hydroxymethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) -3,6-dioxopiperazin-2-yl) acetate (1.4 g, 2.44 mmol, 1 eq.) In CH2Cl2 / toluene (18 ml / 30 ml) PPh3 was added under nitrogen atmosphere and at -20 ° C (850 mg, 3.2 mmol, 1.33 eq.) And the mixture was left under stirring until complete solubilization. Then HN3 (1.5 M in toluene, 10 ml, 15 mmol, 6.25 eq.) Was added and, drop by drop, DIAD (0.7 ml, 3.5 mmol, 1.4 eq.) And the reaction was left under stirring. overnight at -25 ° C. The reaction mixture was then purified directly, without evaporating the solvent, by flash chromatography on silica gel (Hex / AcOEt, from 5: 5 to 4: 6) thus obtaining the desired product in the form of a white spongy solid ( 1.25 g, 86% yield).

Rf = 0.3 (Hexane / AcOEt 5: 5); <ï ›Î ± ï 20>

D = -29.1 (c = 1.0 in CH2Cl2) for allyl 2 - ((2S, 5R) -5- (azidomethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl ) -3,6-dioxopiperazin-2-yl) acetate; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.16 (second order system A2, 4H), 6.62 (br s, 2H), 5.92 (ddt, 1H, J = 5.7, 10.5, 17.2 Hz), 5.36-5.30 (m, 1H, superimposed on solvent peaks), 5.25 (dq, 1H, J = 1.3, 10.5 Hz), 5.05 (d, 1H, J = 15.2 Hz), 4.86 (br t, 1H, J = 6.3 Hz), 4.62 (dt, 2H, J = 1.3, 5.7 Hz), 4.56 (dd, 1H, J = 3.4, 8.9 Hz), 4.16 (d, 1H, J = 15.2 Hz), 4.01 (d, 2H, J = 6.3), 3.88-3.82 (m, 5H), 3.64 (dd, 1H, J = 3.3, 12.7 Hz), 3.21 (dd, 1H, J = 3.6, 17.5 Hz), 2.79 (dd, 1H, J = 8.8, 17.5), 2.64 (s, 3H), 2.53 (s, 3H), 2.12 (s, 3H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 170.9, 166.3, 166.2, 159.8, 139.3, 139.0, 137.2, 135.6, 132.1, 129.0, 128.7, 128.5, 125.7, 118.8, 112.5, 66.2, 59.7, 55.9, 52.2, 51.4, 47.7, 46.7, 37.5, 24.4, 18.1 , 12.1; MS (ESI) m / z calc. for [C28H35N5O7S] <+>: 599.23 [M + H] <+>; found: 598.3.

Preparation 8

Boc-DKPf2-OAllile

Boc-DKPf3-OAllile

To a solution of allyl 2 - ((2S, 5R) -5- (azidomethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin- 2-yl) acetate or allyl 2- (2R, 5S) -5- (azidomethyl) -4- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazine -2-yl) acetate (1.2 g, 2 mmol, 1 eq.) In THF (50 ml), Me3P (5.0 ml of a 1 M solution in THF, 5 mmol, 2.5 eq.) and 2- (t-butoxycarbonyloximino) -2-phenylacetonitrile (Boc-ON, 1.2 g, 0.83 mmol, 2.5 eq.). The reaction was left under stirring for 5 h at room temperature, then CH2Cl2 (200 ml) was added and the solution was washed with H2O (3x50 ml) and brine. The organic phase was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, from 99: 1 to 95: 5) thus obtaining the desired product in the form of a spongy white solid (1.2 g, 89% yield).

Rf = 0.25 (DCM / MeOH 97: 3); <ï ›> Î ± <ï 20>

D = + 26.1 (c = 1.00 in CH2Cl2) for DKP-f2-OAllile; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.14 (system AB, 4H), 6.82 (br s, 1H) 6.62 (br s , 1H), 5.90 (ddt, 1H, J = 5.7, 10.5, 17.2 Hz), 5.31 (dq, 1H, superimposed on solvent peaks, J = 1.3, 17.2 Hz), 5.23 (dq, 1H, J = 1.3, 10.5 Hz), 5.1 (br s, 1H), 4.96 (t, 1H, J = 6.3 Hz), 4.60 (dt, 2H, J = 1.3, 5.7 Hz), 4.42 (dd, 1H, J = 3.6, 8.5 Hz ), 4.03-3.99 (m, 3H), 3.85 (s, 3H), 3.73-3.62 (m, 2H), 3.47 (ddd, 1H, J = 2.5, 6.2, 14.4), 3.20 (dd, 1H, J = 3.6, 17.5 Hz), 2.80 (dd, 1H, J = 8.5, 17.5), 2.64 (s, 3H), 2.52 (s, 3H), 2.11 (s, 3H); <13> C NMR (101 MHz, CD2Cl2 ) Î ́ 171.0, 167.4, 165.5, 159.8, 156.2, 139.3, 139.0, 136.9, 135.8, 132.1, 129.0, 128.7, 125.6, 118.8, 112.5, 80.3, 66.1, 60.0, 55.9, 51.2, 47.2, 46.7, 41.1, 37.8 , 28.3, 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C33H45N4O9S] <+>: 673.29 [M + H] <+>; found: 673.3.

Preparation 9

Boc-DKPf2-OH

Boc-DKPf3-OH

COOH O Mtr

HN N N H O NH-Boc

Pyrrolidine (0.28 ml) was added to a solution of Boc-DKPf2-OAllyl or Boc-DKPf3-OAllyl (1.2 g, 1.78 mmol, 1 eq.) In CH2Cl2 (40 ml). , 3.56 mmol, 2 eq), PPh3 (220 mg, 0.89 mmol, 0.5 eq) and [Pd (PPh3) 4] (170 mg, 0.147 mmol, 0.08 eq). The solution was left under stirring for 1 h at 0 ° C, then EtOAc (200 ml) was added and the solution was acidified to pH = 2 with aqueous KHSO4 (1M, 100 ml). The organic phase was separated and the aqueous phase was extracted with EtOAc (2x50 ml). The organic phases were combined, anhydrified with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified from PPh3 by flash chromatography on a small bed of silica gel (CH2Cl2100% up to CH2Cl2 / MeOH 9: 1) thus obtaining the desired product in the form of a spongy white solid (1.1 g, 98% yield), which was subsequently used without further purification.

Preparation 10

DKPf2-RGD

Boc-DKPf2-Arg (Mtr) -Gly-OBn

H H N N

Mtr

NH O H

BnOOC N

N O H O Mtr

HN N

H.

NO

Boc-HN

Boc-Arg (Mtr) -Gly-OBn (480 mg, 0.76 mmol, 1.2 eq.) Peptide was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Boc-DKPf2-OH (400 mg, 0.63 mmol, 1 eq.) according to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 93: 7) thus obtaining the desired product in the form of a spongy white solid (505 mg, 70% yield).

Rf = 0.4 (CH2Cl2 / MeOH 9: 1); ï ›Î ± ï <20>

D = + 34.7 (c = 1.0 in CH2Cl2); <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.79 (br s, 2H), 7.39 (br s, 1H), 7.32-7.24 (m, 5H), 7.15 (system AB, 4H), 6.60 (s, 1H), 6.50 (s, 1H), 6.17-5.9 (m, 4H), 5.71 (br s, 1H), 5.28 (d overlapped to solvent signal, 1H, J = 14.3 Hz), 5.07 (s, 2H), 4.55 (br s, 1H), 4.42 (br s, 1H), 4.01-3.83 (m, 9H), 3.77 (s, 3H), 3.74-3.61 (m, 1H), 3.50-3.38 (m, 1H), 3.15-2.81 (m, 4H), 2.61 (s, 3H), 2.60 (s, 3H), 2.53 (s, 3H), 2.50 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 1.80-1.69 (m, 1H), 1.63-1.52 (m, 1H), 1.51-1.30 (m, 11H); <13> C NMR (101 MHz, CD2Cl2 ) Î ́ 173.0, 170.9, 170.4, 168.1, 167.1, 159.7, 158.6,156.8, 156.5, 139.1, 138.8, 137.5, 136.8, 135.8, 135.5, 134.0, 129.3, 128.9, 128.7, 128.6, 128.4, 125.6, 125.1, 112.5 , 112.1, 80.1, 67.5, 60.5, 55.9, 55.8, 53.151.2, 47.5, 46.3, 41.7, 41.2, 40.8, 37.728.3, 24.5, 24.2,18.5, 18.1, 12.1, 12.0; MS (ESI) m / z calc. for [C55H74N9O14S2] <+>: 1148.48 [M + H] <+>; found: 1148.6.

Preparation 11

Cbz-Asp (OtBu) -DKPf2-Arg (Mtr) -Gly-OBn

Boc-DKPf2-Arg (Mtr) -Gly-OBn (500 mg, 0.32 mmol, 1 eq.) Was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Cbz-Asp (OtBu ) -OH (155 mg, 0.48 mmol, 1.5 eq.) According to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product below white spongy solid form (385 mg, 88% yield).

Rf = 0.35 (CH2Cl2 / MeOH 9: 1); ï ›Î ± ï <20 1>

D = + 8.0 (c = 1.0 in CH3OH); H NMR (400 MHz, (CD3) 2CO) Î ́ 8.00 (t, 1H, J = 6.2 Hz), 7.84 (t, 1H, J = 6.1 Hz), 7.65 (d, 1H, J = 7.8 Hz), 7.52 (s, 1H), 7.38-7.29 (m, 10H), 7.18 (system AB, 4H), 6.76-6.74 (m, 2H), 6.67-6.64 (m, 2H), 6.49 (br s, 2H), 6.25 (br s, 1H), 5.25 (d, 1H, J = 15.4 Hz), 5.14-5.05 (m, 4H), 4.67 (t, 1H, J = 5.8 Hz), 4.59-4.50 (m, 2H), 4.10 (d, 2H, J = 15.4 Hz), 4.04-3.93 (m, 4H), 3.88 (s, 3H), 3.85-3.82 (m, 4H), 3.59 (dt, 1H, J = 4.7, 13.9 Hz), 3.26-3.12 (m, 2H), 3.02 (dd, 1H, J = 15.4, 5.6 Hz), 2.84-2.72 (m superimposed on water peaks, 2H), 2.67 (s, 3H), 2.64 (s, 3H), 2.63-2.57 (m, 4H), 2.55 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.96-1.86 (m, 1H), 1.69-1.52 (m, 3H) , 1.39 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.8, 172.5, 171.3, 170.6, 170.5, 167.8, 167.7, 160.0, 159.0, 157.6, 157.5, 139.5, 139.4, 139.2, 138.2, 137.9, 137.1, 136.7, 136.0, 131.2, 129.34, 129.26, 129.03, 128.96, 128.95, 128.86, 128.82, 128.73, 125.5, 124.9, 113.1, 112.5, 81.3, 67.3, 67.1, 60.4, 56.1, 55.9, 53.4, 53.0, 5 2.1, 47.9, 46.8, 41.8, 41.1, 40.4, 38.36, 38.28, 28.3, 26.4, 24.4, 24.3, 18.7, 18.2, 12.16, 12.13; MS (ESI) m / z calc. for [C66H85N10O17S2] <+>: 1353.56 [M + H] <+>; found: 1353.6.

Preparation 12

H-Asp (OtBu) -DKPf2-Arg (Mtr) -Gly-OH

H H

N N

Mtr

NH H O HOOC N N O H O Mtr

HN N N H O HN

H2N

O O O

The compound Cbz-Asp (OtBu) -DKPf2-Arg (Mtr) -Gly-OBn (380 mg, 0.28 mmol, 1 eq.) Was dissolved in a 1: 1 mixture of THF / H2O (100 ml) and à 10% Pd / C was added (30 mg, 0.028 mmol, 0.1 eq.). The reaction flask was placed under a hydrogen atmosphere and the reaction was carried out, at room temperature and under stirring, for the whole night. The mixture was then filtered on a celite bed, washing the latter with 1: 1 THF / H2O. The filtered solution was then concentrated in the rotavapor, thus obtaining the crude product

(320 mg, 100% yield) which was used without further purification.

Preparation 13

cyclo [Asp (OtBu) -DKPf2-Arg (Mtr) -Gly]

To a solution of H-Asp (OtBu) -DKPf2-Arg (Mtr) -Gly-OH (200 mg, 0.18 mmol, 1 eq.) In DMF (130 ml) were added, under nitrogen atmosphere and at 0 ° C , HATU (273 mg, 0.72 mmol, 4 eq.), HOAt (98 mg, 0.72 mmol, 4 eq.) And DIPEA (0.180 ml, 1.06 mmol, 6 eq). The temperature was allowed to rise to room temperature and the reaction was left under stirring overnight. The DMF was then removed in the rotavapor and the residue was dissolved in EtOAc (200 ml) and washed with KHSO41M (2x30 ml), aqueous NaHCO3 (2x30 ml) and brine (2x30 ml). The organic phase was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product in the form of a floccular solid (110mg, 57% yield).

Rf = 0.34 (CH2Cl2 / MeOH, 9: 1); ï ›Î ± ï <20>

D = + 7.5 (c = 1.00 in CH2Cl2) <1> H NMR (400 MHz, (CD3) 2SO) Î ́ 8.59 (br s, 1H), 8.49 (d, 1H, J = 7.0 Hz), 8.13 (s , 1H), 8.05 (d, 1H, J = 8.0 Hz), 7.89-7.83 (m, 2H), 7.14 (system AB, 4H), 6.76 (s, 1H), 6.68 (s, 1H), 5.06 (d , 1H, J = 15.0 Hz), 4.53-4.45 (m, 2H), 4.28-4.17 (m, 1H), 4.00-3.84 (m, 5H), 3.82 (s, 3H), 3.79 (s, 3H) , 3.62 (d, 1H, J = 5.4 Hz), 3.49 (br d, 1H, J = 13.6 Hz), 3.09-3.0 (m, 2H), 2.76-2.67 (m, 2H), 2.60 (s, 3H) , 2.56 (s, 3H), 2.52 (s, 3H), 2.45 (s, 3H), 2.27 (dd, 1H, J = 12.6, 3.0 Hz), 2.05 (s, 6H), 1.72-1.63 (m, 1H ), 1.53-1.46 (m, 3H), 1.37 (s, 9H); <13> C NMR (101 MHz, (CD3) 2SO) Î ́ 171.6, 171.1, 170.7, 169.0, 168.5, 168.2, 167.1, 158.4, 157.4, 156.1, 138.2, 137.73, 137.57, 137.4, 135.5, 135.1, 134.6, 130.0, 127.9, 127.4, 124.0, 123.5, 112.2, 111.7, 80.2, 58.6, 55.61, 55.43, 54.1, 51.4, 49.8, 45.9, 45.0, 41.6, 39.8, 39.0, 38.1, 37.0, 28.9, 27.6, 25.9, 23.73, 23.54, 17.9, 17.6, 11.71, 11.69; MS (ESI) m / z calc. for [C51H71N10O14S2] <+>: 1111.46 [M + H] <+>; found: 1111.7.

Preparation 14

cyclo [Asp-DKPf2-Arg-Gly] (trifluoroacetate salt)

The cyclo compound [Asp (OtBu) -DKPf2-Arg (Mtr) -Gly] (110 mg, 0.10 mmol) was dissolved in TFA (10 ml) in the presence of ion scavengers: thioanisole (1.5 ml), ethanedithiol (0.75 ml), phenol (150 mg). The mixture was cooled to 0 ° C and put under nitrogen flow. Then trimethylsilylbromide (2ml) was added. The reaction was left under stirring until it reached room temperature and then left under stirring for 2 h. The TFA was then removed in the rotavapor, and the crude product was dissolved in a 1: 1 mixture of water and diisopropyl ether (30 ml). The aqueous phase was then washed several times with diisopropyl ether and subsequently concentrated in the rotavapor, thus obtaining the crude product, which was finally purified by semipreparative HPLC (Water's Atlantis column 21 mm x 10 cm, gradient: 100% H2O 0.1% TFA up to 70% H2O 0.1% TFA / 30% acetonitrile), thus obtaining the desired product (as trifluoroacetate salt) in the form of a white solid (63 mg, 75% yield).

<1> H NMR (400 MHz, D2O) Î ́ 7.47 (d, 2H, J = 8.1 Hz), 7.39 (d, 1H, J = 8.1 Hz), 5.13 (d, 1H, J = 15.6 Hz), 4.89 (t, 1H, J = 7.1 Hz), 4.61 (dd, 1H, J = 7.8, 5.8 Hz), 4.33 (d, 1H, J = 17.0 Hz), 4.26-4.18 (m, 5H), 4.02 (d, 1H, J = 14.9 Hz), 3.71-3.61 (m, 2H), 3.25 (t, 2H, J = 6.8 Hz), 2.99-2.90 (m, 2H), 2.81 (dd, 1H, J = 7.0, 17.0 Hz ), 2.69 (dd, 1H, J = 5.4, 14.0 Hz), 1.83 (ddt, 1H, J = 14.3, 9.7, 4.7 Hz), 1.83 (ddt, 1H, J = 14.3, 9.7, 4.7 Hz), 1.74- 1.61 (m, 2H); <13> C NMR (101 MHz, D2O) Î ́ 174.7, 174.5, 173.7, 173.5, 171.5, 170.7, 157.4, 136.6, 133.1, 130.1, 128.9, 60.1, 54.6, 52.8, 50.1, 48.3, 43.3, 43.2, 41.3, 39.9, 38.7, 35.3, 26.5, 25.3; MS (ESI) m / z calc. for [C27H39N10O8] <+>: 631.30 [M + H] <+>; found: 631.4

Preparation 15

DKPf3-RGD

Boc-DKPf3-Arg (Mtr) -Gly-OBn

Boc-Arg (Mtr) -Gly-OBn (1.5 g, 2.37 mmol, 1.25 eq.) Peptide was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Boc-DKPf3-OH (1.2 g, 1.90 mmol, 1 eq.) according to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 93: 7) thus obtaining the desired product in the form of a white spongy solid (1.5 g, 69% yield).

Rf = 0.4 (CH2Cl2 / MeOH 9: 1); <ï ›Î ± ï 20>

D = -29.0 (c = 1.0 in CH2Cl2); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.86 (t, 1H, J = 6.0 Hz), 7.61 (d, 2H, J = 11.0 Hz ), 7.39-7.29 (m, 5H), 7.18 (AB system, 4H), 6.76 (s, 1H), 6.70-6.66 (m, 2H), 6.49-6.44 (m, 3H), 5.35 (d, 1H, J = 15.5 Hz), 5.14 (s, 2H), 4.60-4.50 (m, 2H), 4.04-3.91 (m, 5H), 3.88 (s, 3H), 3.82 (s, 3H), 3.76-3.70 (m , 2H), 3.55-3.45 (m, 1H), 3.24-3.11 (m, 2H), 3.07 (dd, 1H, J = 15.6, 4.6 Hz), 2.78 (dd, 1H, J = 15.6, 6.8 Hz), 2.67 (s, 3H), 2.64 (s, 3H), 2.62 (s, 3H), 2.55 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H) 1.92-1.83 (m, 1H), 1.66-1.58 (m, 3H), 1.40 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 173.0, 171.1, 170.4, 167.9, 167.4, 160.0, 159.0, 157.6, 156.9, 139.49, 139.31, 139.16, 138.2, 137.14, 137.03, 136.5, 135.8, 131.2, 129.3, 129.0, 128.7, 125.5, 124.9, 113.1, 112.5, 79.6, 67.2, 60.8, 56.1, 55.9, 53.3, 52.3, 47.5, 46.7, 41.8, 41.5, 41.1, 38.8, 30.3, 29.8, 28.7, 26.1, 24.42, 24.30, 18.7, 18.2, 12.15, 12.14; MS (ESI) m / z calc. for [C55H74N9O14S2] <+>: 1148.48 [M + H] <+>; found: 1148.5.

Preparation 16

Cbz-Asp (OtBu) -DKPf3-Arg (Mtr) -Gly-OBn

H H N N

Mtr

NH HO

BnOOC N

N O H O Mtr

HN N

H.

NO

H HN N

Cbz O

O O

Boc-DKPf3-Arg (Mtr) -Gly-OBn (1.4 g, 1.22 mmol, 1 eq.) Compound was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Cbz-Asp (OtBu ) -OH (455 mg, 0.48 mmol, 1.2 eq.) According to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product below form of white spongy solid (1.35 g, 82% yield).

R <0>

f = 0.31 (CH2Cl2 / MeOH 9: 1); <ï ›Î ± ï 2>

D = -15.5 (c = 1 in CH2Cl2); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.83 (t, J = 6.1 Hz, 1H), 7.79 (t, 1H, J = 6.2 Hz ), 7.59 (m, 2H), 7.38-7.28 (m, 10H), 7.19 (system AB, 4H), 6.75 (m, 2H), 6.66 (d, 2H, J = 6.4 Hz), 6.48 (br s, 2H), 5.28 (d, 1H, J = 15.5 Hz), 5.14-5.00 (m, 4H), 4.63-4.51 (m, 3H), 4.09 (d, 1H, J = 15.5 Hz), 4.03 (d, 2H , J = 6.3 Hz), 3.97 (dd, 2H, J = 5.8, 3.5 Hz), 3.90-3.81 (m, 8H), 3.63 (ddd, 1H, J = 13.5, 5.8, 3.5 Hz), 3.17 (m, 2H), 3.05 (dd, 1H, J = 15.7, 4.7 Hz), 2.85-2.80 (m, 1H), 2.79-2.76 (m, 1H), 2.67 (s, 3H), 2.65 (m, 4H), 2.62 (s, 3H), 2.55 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.93-1.84 (m, 1H), 1.65-1.55 (m, 3H), 1.39 (s, 9H ); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.9, 172.5, 171.1, 170.7, 170.4, 167.63, 167.44, 160.0, 159.0, 157.5, 157.2, 139.51, 139.33, 139.17, 138.1, 137.9, 137.13, 137.06, 136.6, 135.9, 131.2, 129.33, 129.26, 129.02, 128.99, 128.75, 128.73, 125.5, 124.9, 113.1, 112.5, 81.3, 67.30, 67.15, 60.3, 56.1, 55.9, 53.2, 53.0, 52.2, 47.7, 46.8, 41.8, 41.1, 40.4, 38.6, 38.2, 30.4, 28.3, 26.2, 24.44, 24.31, 18.7, 18.2, 12.16, 12.14; MS (ESI) m / z calc. for [C66H85N10O17S2] <+>: 1353.56 [M + H] <+>; found: 1353.6.

Preparation 17

H-Asp (OtBu) -DKPf3-Arg (Mtr) -Gly-OH

The compound Cbz-Asp (OtBu) -DKPf3-Arg (Mtr) -Gly-OBn (700 mg, 0.52 mmol, 1 eq.) Was dissolved in a 1: 1 mixture of THF / H2O (200 ml) and à 10% Pd / C was added (100 mg, 0.10 mmol, 0.2 eq.). The reaction flask was placed under a hydrogen atmosphere and the reaction was carried out, at room temperature and under stirring, for the whole night. The mixture was then filtered on a celite bed, washing the latter with 1: 1 THF / H2O. The filtered solution was then concentrated in the rotavapor, thus obtaining the crude product

(580 mg, 100% yield) which was used without further purification.

Preparation 18

cyclo [Asp (OtBu) -DKPf3-Arg (Mtr) -Gly]

To a solution of H-Asp (OtBu) -DKPf3-Arg (Mtr) -Gly-OH (580 mg, 0.52 mmol, 1 eq.) In DMF (440 ml) were added, under nitrogen atmosphere and at 0 ° C , HATU (900 mg, 2.36 mmol, 4.5 eq.), HOAt (323 mg, 2.36 mmol, 4.5 eq.) And DIPEA (0.630 ml, 3.70 mmol, 7 eq.). The temperature was allowed to rise to room temperature and the reaction was left under stirring overnight. The DMF was then removed in the rotavapor and the residue was dissolved in EtOAc (300 ml) and washed with KHSO41M (2x40 ml), aqueous NaHCO3 (2x40 ml) and brine (2x40 ml). The organic phase was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product in the form of a floccular solid (470 mg, 81% yield).

Rf = 0.34 (CH2Cl2 / MeOH, 9: 1); <ï ›Î ± ï 20 1>

D = -41.6 (c = 1.00 in CH3OH); H NMR (400 MHz, (CD3) 2CO) Î ́ 8.63 (d, 1H, J = 6.7 Hz), 8.01 (dd, 1H, J = 9.3, 2.4 Hz), 7.47 (d, 1H, J = 8.8 Hz) , 7.22 (AB system, 4H), 6.92 (s, 1H), 6.76-6.73 (m, 2H), 6.68 (s, 1H), 6.53 (s, 2H), 5.06 (d, 1H, J = 14.9 Hz) , 4.88 (td, 1H, J = 8.9, 6.3 Hz), 4.44 (td, 2H, J = 9.7, 7.4 Hz), 4.14 (d, 1H, J = 14.9 Hz), 4.08 (d, 2H, J = 6.3 Hz), 4.02 (dd, 1H, J = 13.9, 7.2 Hz), 3.90-3.80 (m, 8H), 3.50 (dt, 1H, J = 13.9, 6.8 Hz), 3.35 (dd, 1H, J = 16.9, 2.4 Hz), 3.25 (m, 2H), 2.91 (dd, 1H, J = 16.2, 9.0 Hz), 2.81-2.78 (m superimposed on water peaks, 1H), 2.68 (s, 3H), 2.63 ( s, 6H), 2.55-2.50 (m, 4H), 2.43 (dd, 1H, J = 16.2, 6.1 Hz), 2.20-2.10 (m, 7H), 1.99-1.93 (m, 1H), 1.72-1.51 ( m, 2H), 1.41 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 173.1, 172.7, 171.47, 171.30, 171.10, 170.8, 170.3, 159.9, 158.9, 157.4, 139.46, 139.29, 139.12, 138.7, 137.0, 135.9, 131.2, 129.3, 129.0, 125.5, 124.8, 113.0, 112.5, 81.0, 60.3, 56.10, 56.02, 55.88, 52.8, 49.6, 47.8, 46.7, 43.2, 39.2, 38.6, 36.7, 28.2, 27 .4, 26.9, 24.41, 24.26, 18.7, 18.2, 12.1; MS (ESI) m / z calc. for [C51H71N10O14S2] <+>: 1111.46 [M + H] <+>; found: 1111.6.

Preparation 19

cyclo [Asp-DKPf3-Arg-Gly] (trifluoroacetate salt)

The cyclo compound [Asp (OtBu) -DKPf3-Arg (Mtr) -Gly] (200 mg, 0.18 mmol) was dissolved in TFA (20 ml) in the presence of ion scavengers: thioanisole (3 ml), ethanedithiol (1.5 ml), phenol (300 mg). The mixture was cooled to 0 ° C and put under nitrogen flow. Then trimethylsilylbromide (4ml) was added. The reaction was left under stirring until it reached room temperature and then left under stirring for 2 h. The TFA was then removed in the rotavapor, and the crude product was dissolved in a 1: 1 mixture of water and diisopropyl ether (50 ml). The aqueous phase was then washed several times with diisopropyl ether and subsequently concentrated in the rotavapor, thus obtaining the crude product, which was finally purified by semipreparative HPLC (Water's Atlantis column 21 mm x 10 cm, gradient: 100% H2O 0.1% TFA up to 70% H2O 0.1% TFA / 30% acetonitrile), thus obtaining the desired product (as trifluoroacetate salt) in the form of a white solid (127 mg, 75% yield).

<1> H NMR (400 MHz, D2O) Î ́ 7.43 (system AB, 4H), 5.12 (d, 1H, J = 15.6 Hz), 4.89 (t, 1H, J = 7.1 Hz), 4.61 (dd, 1H , J = 7.8, 5.8 Hz), 4.33 (d, 1H, J = 17.0 Hz), 4.26-4.19 (m, 5H), 4.02 (d, 1H, J = 14.9 Hz), 3.71-3.61 (m, 2H) , 3.25 (t, 2H, J = 6.8 Hz), 2.99-2.90 (m, 2H), 2.81 (dd, 1H, J = 17.0, 7.0 Hz), 2.69 (dd, 1H, J = 14.0, 5.4 Hz), 2.02 (ddt, 1H, J = 14.2, 9.9, 4.9 Hz), 1.83 (ddt, 1H, J = 14.3, 9.7, 4.7 Hz), 1.75-1.59 (m, 2H); <13> C NMR (101 MHz, D2O) Î ́ 174.74, 174.55, 173.67, 173.51, 171.5, 170.7, 169.3, 157.4, 136.6, 133.1, 130.1, 128.9, 60.1, 54.6, 52.8, 50.1, 48.3, 43.34, 43.17, 41.3, 39.9, 38.7, 35.3, 26.5, 25.3; MS (ESI) m / z calc. for [C27H39N10O8] <+>: 631.30 [M + H] <+>; found: 631.5.

Preparation 20

DKPf4 and DKPf6

(R) -dimethyl-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) succinate

(S) - dimethyl-2- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzylamino) succinate

To a solution of (S) - or (R) -dimethylaspartate hydrochloride (490 mg, 2.5 mmol, 1 eq.) And sodium cyanoborohydride (160 mg, 2.5 mmol, 1 eq.) In methanol (12 ml), was added , under vigorous stirring, at room temperature and under nitrogen, the 4 - ((4-methoxy-2,3,6-trimethylphenylsulfonyl) aminomethyl) benzaldehyde (870 mg, 2.5 mmol, 1 eq.) in a single portion. The mixture was left under stirring for 4 h, then it was cooled to 0 ° C and the pH was adjusted to approximately 1 with 37% HCl. The temperature was allowed to rise to room temperature and the reaction was left under stirring for 2 h. The solvent was then removed on the rotavapor. The residue was suspended in a little water and the pH was increased to about 7 with a saturated aqueous solution of NaHCO3. EtOAc was then added and the mixture was stirred for a few minutes. The emulsion formed was filtered on a celite bed, obtaining a separable mixture. After separation, the aqueous phase was extracted 3 times with EtOAc; the organic phases were combined and anhydrified with Na2SO4. The solvent was removed in the rotavapor, thus obtaining a clear oil. The latter was dissolved in THF (50 ml) and an excess of activated MnO2 was added: in this way the by-product formed (N- (4- (hydroxymethyl) benzyl) -4-methoxy-2,3, 6-trimethylbenzenesulfonamide) has been reoxidized to aldehyde, making it less polar and more easily separable on a chromatographic column. The mixture was left under stirring for 4 h, then it was filtered on a celite bed and the solvent was removed in a rotavapor. The crude product (brown oil) was purified by flash chromatography on silica gel (Hex / EtOAc, 1: 1) thus obtaining the desired product in the form of transparent viscous oil (800 mg, 66% yield).

<20> Rf = 0.45 (Hexane / AcOEt, 1: 1); ï ›Î ± ï D = -20.5 (c = 1.0 in CH3OH); <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.18 (AB system , 4H), 6.66 (s, 1H), 4.88 (t, 1H, J = 5.6 Hz), 4.06 (d, 2H, J = 6.3 Hz), 3.90 (s, 3H), 3.85 (d, 1H, J = 13.9 Hz), 3.75 (s, 3H), 3.69-3.61 (m, 5H), 2.77-2.64 (m, 5H), 2.56 (s, 3H), 2.16 (s, 3H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 174.2, 171.5, 159.7, 139.9, 139.2, 139.0, 135.9, 129.2, 128.6, 128.2, 125.6, 112.5, 57.4, 55.9, 52.3, 52.0, 51.8, 46.9, 38.3, 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C24H33N2O7S] <+>: 493.20 [M + H] <+>; found: 493.2.

Preparation 21

(S) -dimethyl-2 - ((R) -3-azido-2- (tert-butoxycarbonylamino) -N- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) propanamido) succinate

(R) -dimethyl-2 - ((S) -3-azido-2- (tert-butoxycarbonylamino) -N- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) propanamido) succinate

The (S) -o- (R) -3-azido-2- (tert-butoxycarbonylamino) propanoic acid (1.84 g, 8.0 mmol, 5 eq.) Was dissolved in THF (120 ml) and the DCC (826 g, 4 mmol, 2.5 eq.) Was added in a single portion. Immediately a white precipitate (DCU) was formed and the solution was left under stirring for 1 h at room temperature. The mixture was then filtered on cotton to remove the DCU, which was washed with a little cold CH2Cl2 and the filtrate was concentrated in the rotavapor, keeping the bath at room temperature. The residue was left to pump, thus obtaining the symmetrical anhydride in the form of a pale yellow sponge, which was used without further purification. (S) - or (R) -dimethyl-2- (4 - ((4-methoxy-2,3,6- trimethylphenylsulfonamido) methyl) benzylamino) succinate (780 mg, 1.59 mmol, 1 eq.) Was dissolved in CH2Cl2 (12 ml) and a solution of symmetrical anhydride in CH2Cl2 (8 ml) was dropped very slowly and at 0 ° C to the solution. The reaction was left under stirring until it reached room temperature and allowed to react further overnight. The solvent was removed with the rotavapor and the crude product was purified by flash chromatography on silica gel (Hex / EtOAc, 6: 4) thus obtaining the desired product in the form of transparent viscous oil (440 mg, 40% surrender).

Rf = 0.43 (Hex / AcOEt 1: 1); ï ›Î ± ï <20>

D = -31.6 (c = 0.5 in CH2Cl2) for (S) -dimethyl-2 - ((R) -3-azido-2- (tert-butoxycarbonylamino) -N- (4 - ((4-methoxy-2, 3,6-trimethylphenylsulfonamido) methyl) benzyl) propanamido) succinate; <1> H NMR (400 MHz, CD2Cl2) (ratio of rotamers in CD2Cl2A / B = 4: 1) Î ́ 7.34 â € “7.17 (m, 4HA) , 7.12 â € “7.01 (m, 4HB), 6.66 â €“ 6.60 (m, 1HA + 1HB), 5.49 (d, 1HB, J = 8.2 Hz), 5.26 (d, 1HA, J = 8.5 Hz), 5.14 â € “5.07 (m, 1HB), 4.99 â €“ 4.92 (m, 1HB), 4.78 (t, 1HA, J = 6.3 Hz), 4.74 â € “4.60 (m, 3HA + 1HB), 4.36 (t, 1HA, J = 6.4 Hz), 4.29 (d, 1HB, J = 15.9 Hz), 4.05 - 3.96 (m, 2HA + 2HB), 3.85 (s, 3HA + 3HB), 3.71 - 3.53 (m, 6HA + 8HB), 3.45 (dd, 1HA, J = 12.3, 6.3 Hz), 3.34 (dd, 1HA, J = 12.3, 6.1 Hz), 3.22 (dd, 1HA, J = 16.9, 7.3 Hz), 2.98 (dd, 1HB, J = 17.3, 7.0 Hz), 2.72 - 2.61 (m, 3HA + 4HB), 2.60 - 2.45 (m, 4HA + 3HB), 2.14 (s, 3HA + 3HB), 1.50 - 1.35 (m, 9HA + 9HB); <13> C NMR (101 MHz, CD2Cl2) Î ́ 171.6, 171.0, 170.0, 159.8, 155.2, 139.3, 139.0, 137.3, 135.6, 129.4, 128.8, 128.2, 127.8, 125.7, 112.5, 80.7, 57.8, 56.7, 55.9, 53.1, 52.8, 52.4, 52.2, 51.1, 50.7, 47.5, 46.7, 35.0, 34.6, 28.3, 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C32H45N6O10S] <+>: 705.29 [M + H] <+>; found: 705.4.

Preparation 22

Methyl-2 - ((2S, 5R) -5- (azidomethyl) -1- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl ) acetate Methyl-2 - ((2R, 5S) -5- (azidomethyl) -1- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2 -yl) acetate

(S) -dimethyl-2 - ((R) -3-azido-2- (tert-butoxycarbonylamino) -N- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) propanamido) succinate or (R) -dimethyl-2 - ((S) -3-azido-2- (tert-butoxycarbonylamino) -N- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) propanamido) succinate (400 mg, 0.57 mmol, 1 eq.) was deprotected according to general procedure A, with the addition of Et3SiH (0.23 ml, 1.43 mmol, 2.5 eq.) as ion scavenger. The corresponding trifluoroacetate salt was dissolved in iPrOH (8 ml) and, at room temperature, DIPEA (0.4 ml, 2.28 mmol, 4 eq.) Was added. The reaction was kept under stirring for 5 h at room temperature, then the solvent was removed in a rotavapor and the residue was purified by flash chromatography on silica gel (Hex / EtOAc, 2: 8) thus obtaining the desired product in the form of a spongy white solid (300 mg, 92% yield).

Rf = 0.49 (AcOEt); <ï ›Î ± ï 20>

D = + 15.6 (c = 0.5 in CH2Cl2) for Methyl-2 - ((2S, 5R) -5- (azidomethyl) -1- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamide) methyl) benzyl) -3,6-dioxopiperazin-2-yl) acetate; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.17 â € “7.10 (m, 4H), 6.62 (s, 1H), 6.59 (s, 1H), 5.08 (d, 1H, J = 15.3 Hz), 4.92 (t, 1H, J = 6.3 Hz), 4.46 - 4.40 (m, 1H), 4.08 (d, 1H, J = 15.3 Hz), 4.04 - 3.97 (m, 3H), 3.90 - 3.82 (m, 4H), 3.79 (dd, 1H, J = 12.6, 3.5 Hz), 3.60 (s, 3H), 3.00 (dd, 1H, J = 17.5, 3.4 Hz), 2.80 (dd, 1H, J = 17.5, 5.0 Hz), 2.64 (s, 3H), 2.52 (s, 3H), 2.12 (s, 3H); <13> C NMR (101 MHz , CD2Cl2) Î ́ 170.7, 167.7, 164.9, 159.8, 139.3, 139.0, 137.1, 135.5, 128.8, 128.3, 125.7, 112.5, 56.2, 55.9, 54.6, 53.7, 52.4, 47.3, 46.7, 34.9, 24.4, 18.1, 12.1 ; MS (ESI) m / z calc. for [C26H33N6O7S] <+>: 573.21 [M + H] <+>; found: 573.5.

Preparation 23

H-DKPf4-OMe

H-DKPf6-OMe

Methyl-2 - ((2S, 5R) -5- (azidomethyl) -1- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin-2-yl ) acetate or Methyl-2 - ((2R, 5S) - 5- (azidomethyl) -1- (4 - ((4-methoxy-2,3,6-trimethylphenylsulfonamido) methyl) benzyl) -3,6-dioxopiperazin- 2-yl) acetate (300 mg, 0.52 mmol, 1 eq.) Was dissolved in THF (45 ml) and 10% Pd / C (56 mg, 0.052 mmol, 0.1 eq.) Was added. The flask was placed under a hydrogen atmosphere and left under stirring, at room temperature, for 4 h. afterwards the mixture was filtered on a celite bed, washing it thoroughly with THF. The filtrate was concentrated in the rotavapor, thus obtaining the desired product in the form of a spongy white solid (280 mg, 99% yield), which was used without further purification.

Preparation 24

Boc-DKPf4-OMe

Boc-DKPf6-OMe

DIPEA (0.17 ml, 1.0 mmol, 2 eq.) and Boc2O (132 mg, 0.6 mmol, 1.2 eq.). The reaction was kept under stirring for 6 h at room temperature, then the solvent was removed in a rotavapor and the residue was purified by flash chromatography on a small bed of silica gel (EtOAc, 100%) thus obtaining ¬ the desired product in the form of a white spongy solid (320 mg, 96% yield).

Rf = 0.59 (AcOEt); ï ›Î ± ï <20>

D = + 60.0 (c = 1.00 in CH2Cl2) for Boc-DKPf4-OMe; <1> H NMR (400 MHz, CD2Cl2) Î ́ 7.17 â € “7.06 (m, 5H), 6.62 (s, 1H), 5.39 (t, 1H, J = 6.0 Hz), 5.27 (t, 1H, J = 5.9 Hz), 4.97 (d, 1H, J = 15.3 Hz), 4.29 (t, 1H, J = 4.4 Hz), 4.14 (d , 1H, J = 15.3 Hz), 4.03 - 3.96 (m, 3H, J = 6.1 Hz), 3.85 (s, 3H), 3.66 (ddd, 1H, J = 14.2, 6.6, 4.3 Hz), 3.59 ( s, 3H), 3.57 - 3.48 (m, 1H), 2.92 (dd, 1H, J = 17.1, 4.1 Hz), 2.76 (dd, 1H, J = 17.1, 5.1 Hz), 2.63 (s, 3H) , 2.52 (s, 3H), 2.11 (s, 3H), 1.41 (s, 9H); <13> C NMR (101 MHz, CD2Cl2) Î ́ 170.7, 167.8, 166.5, 159.7, 157.2, 139.3, 139.1, 137.1 , 135.7, 128.7, 128.1, 125.6, 112.5, 80.1, 56.8, 56.0, 55.9, 52.4, 47.6, 46.6, 42.7, 35.3, 28.4, 24.4, 18.1, 12.1; MS (ESI) m / z calc. for [C31H43N4O9S] <+>: 647.27 [M + H] <+>; found: 647.4 Prep 25

Boc-DKPf4-OH

Boc-DKPf6-OH

Boc-DKPf4-OMe or Boc-DKPf6-OMe (320 mg, 0.5 mmol, 1 eq.) Was dissolved in THF (20 ml) and at 0 ° C a solution of LiOH H2O (52 mg , 1.24 mmol, 2.5 eq.) In H2O (10 ml). The resulting solution was left to react for 1 h at 0 ° C; then the mixture was acidified, at 0 ° C, to pH1-2 with KHSO41M. The solution was extracted with CH2Cl2 (4x), the organic phases were combined and dried with Na2SO4. The solvent was removed in the rotavapor, thus obtaining the desired product in the form of a spongy white solid (310 mg, 100%) which was used without further purification.

Preparation 26

DKPf4-RGD

Boc-DKPf4-Arg (Mtr) -Gly-OBn

Boc-Arg (Mtr) -Gly-OBn (280 mg, 0.44 mmol, 1.2 eq.) Peptide was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Boc-DKPf4-OH (230 mg, 0.37 mmol, 1 eq.) according to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 93: 7) thus obtaining the desired product in the form of a white spongy solid (270 mg, 64%).

Rf = 0.36 (CH2Cl2 / MeOH 9: 1); <ï ›> Î ± <ï 20>

D = + 29.7 (c = 1.0 in CH2Cl2); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.84 (t, 1H, J = 5.9 Hz), 7.49 (d, 1H, J = 7.3 Hz ), 7.40-7.32 (m, 5H), 7.19-7.15 (A2 system, 4H), 6.76 (s, 1H), 6.71-6.67 (m, 2H), 6.51 (br s, 2H), 6.16 (t, 1H , J = 4.7 Hz), 5.15 (s, 2H), 5.06 (d, 1H, J = 15.2 Hz), 4.52-4.47 (m, 1H), 4.33 (t, 1H, J = 4.9 Hz), 4.20 (d , 1H, J = 15.2 Hz), 4.07-4.02 (m, 3H), 4.00 (d, 2H, J = 6.8 Hz), 3.88 (s, 3H), 3.84 (s, 3H), 3.70 (ddd, 1H, J = 14.0, 5.9, 4.6 Hz), 3.57-3.51 (m, 1H), 3.27-3.11 (m, 2H), 2.92-2.82 (m superimposed on water peaks, 2H), 2.67 (s, 3H) , 2.63 (s, 3H), 2.62 (s 3H), 2.57 (s, 3H), 2.10 (s, 6H), 1.87-1.82 (m, 1H), 1.63-1.52 (m, 4H), 1.41 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.6, 170.3, 169.9, 169.1, 167.0, 160.0, 159.0, 157.57, 157.53, 139.51, 139.32, 139.17, 138.2, 137.12, 137.08, 136.7 , 135.8, 131.2, 129.3, 129.12, 128.99, 128.97, 128.6, 125.5, 124.9, 113.0, 112.5, 79.6, 67.1, 58.0, 56.14, 56.10, 55.91, 53.3, 47.6, 46.8, 43.2, 41.8, 41.1, 37.5, 30.2 , 28.6, 26.3, 24.42, 24.28, 18.7, 18.2, 12.1; MS (ESI) m / z calc. for [C55H74N9O14S2] <+>: 1148.48 [M + H] <+>; found: 1148.6.

Preparation 27

Cbz-Asp (OtBu) -DKPf4-Arg (Mtr) -Gly-OBn

Boc-DKPf4-Arg (Mtr) -Gly-OBn (270 mg, 0.235 mmol, 1 eq.) Was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Cbz-Asp (OtBu ) -OH (91 mg, 0.28 mmol, 1.2 eq.) According to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product below form of white spongy solid (210 mg, 66% yield).

Rf = 0.45 (CH2Cl2 / MeOH 9: 1); <ï ›> Î ± <ï 20>

D = + 22.1 (c = 1 in CH2Cl2); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.85 (t, 1H, J = 5.5 Hz), 7.74 (t, 1H, J = 5.8 Hz ), 7.47 (d, 1H, J = 7.9 Hz), 7.40-7.28 (m, 10H), 7.17 (A2 system, 4H), 6.76 (m, 2H), 6.68 (m, 2H), 6.51 (br s, 2H), 5.14-5.01 (m, 5H), 4.54 (td, 1H, J = 7.8, 6.0 Hz), 4.47 (m, 1H), 4.41 (t, 1H, J = 5.4 Hz), 4.22 (d, 1H , J = 15.1 Hz), 4.06 (t, 1H, J = 5.3 Hz), 4.02 (d, 2H, J = 6.3), 3.99 (d, 2H, J = 6.6), 3.90-3.84 (m, 4H), 3.83 (s, 3H), 3.61 (dt, J = 14.0, 6.6 Hz, 1H), 3.25-3.12 (m, 2H), 2.91-2.85 (m, 2H), 2.81-2.79 (m superimposed on peaks of water, 1H), 2.69-2.65 (m, 4H), 2.64 (s, 3H), 2.62 (s, 3H), 2.55 (s, 3H), 2.10 (s, 6H), 1.87-1.81 (m, 1H) , 1.62-1.51 (m, 3H), 1.40 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.8, 172.5, 170.7, 170.3, 169.8, 169.0, 166.9, 160.0, 158.9 , 157.5, 157.0, 139.51, 139.34, 139.17, 138.07, 137.88, 137.12, 137.08, 136.8, 135.8, 129.32, 129.23, 129.14, 129.09, 128.99, 128.95, 128.7, 125.5, 124.8, 113.0, 112.5, 81.4, 67.21, 67.11 , 58.1, 56.08, 55.88, 5 5.2, 53.3, 52.9, 47.6, 46.8, 42.3, 41.8, 38.4, 37.5, 30.3, 28.2, 26.2, 24.43, 24.29, 18.7, 18.2, 12.1; MS (ESI) m / z calc. for [C66H85N10O17S2] <+>: 1353.56 [M + H] <+>; found: 1353.6.

Preparation 28

H-Asp (OtBu) -DKPf4-Arg (Mtr) -Gly-OH

H H

N N

Mtr

NH

OR

H.

N COOH

N H O O N

Mtr-HN NH

O NH NH

O2

O O

The compound Cbz-Asp (OtBu) -DKPf4-Arg (Mtr) -Gly-OBn (200 mg, 0.148 mmol, 1 eq.) Was dissolved in a 1: 1 mixture of THF / H2O (50 ml) and à 10% Pd / C was added (16 mg, 0.015 mmol, 0.1 eq.). The reaction flask was placed under a hydrogen atmosphere and the reaction was carried out, at room temperature and under stirring, for the whole night. The mixture was then filtered on a celite bed, washing the latter with 1: 1 THF / H2O. The filtered solution was then concentrated in the rotavapor, thus obtaining the crude product

(166 mg, 100% yield) which was used without further purification.

Preparation 29

cyclo [Asp (OtBu) -DKPf4-Arg (Mtr) -Gly]

H H

N N

Mtr

NH

OR

OR

N H O N HN H N NH

Mtr O

NH O NH O O

OR

To a solution of H-Asp (OtBu) -DKPf4-Arg (Mtr) -Gly-OH (166 mg, 0.148 mmol, 1 eq.) In DMF (110 ml) were added, under nitrogen atmosphere and at 0 ° C , HATU (224 mg, 0.593 mmol, 4 eq.), HOAt (mg, 0.592 mmol, 4 eq.) And DIPEA (0.160 ml, 0.89 mmol, 6 eq.). The temperature was allowed to rise to room temperature and the reaction was left under stirring overnight. The DMF was then removed in the rotavapor and the residue was dissolved in EtOAc (150 ml) and washed with KHSO41M (2x20 ml), aqueous NaHCO3 (2x20 ml) and brine (2x30 ml). The organic phase was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product in the form of a floccular solid (110 mg, 67% yield).

Rf = 0.4 (CH2Cl2 / MeOH 9: 1); <ï ›Î ± ï 20>

D = -42.0 (c = 1.00 in DMSO); <1> H NMR (400 MHz, (CD3) 2SO) Î ́ 8.88 (br s, 1H), 8.25 (m, 2H), 8.03 (br s, 1H) , 7.85 (t, 1H, J = 6.2 Hz), 7.41 (br s, 1H), 7.12 (system AB, 4H), 6.75 (s, 1H), 6.68 (s, 1H), 6.41 (br s, 1H) , 5.18 (d, 1H, J = 14.1 Hz), 4.26-4.18 (m, 2H), 4.02-3.97 (m, 1H), 3.92-3.87 (m, 3H), 3.82 (s, 3H), 3.80-3.74 (m, 4H), 3.70-3.56 (m, 2H), 3.34 (m superimposed on water peaks, 1H), 3.08-2.95 (m, 3H), 2.93-2.83 (m, 1H), 2.64-2.60 (m, 4H), 2.54 (m, 7H), 2.50 (m superimposed on solvent peaks, 1H), 2.44 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.73-1.61 ( m, 1H), 1.55-1.40 (m, 12H); <13> C NMR (101 MHz, (CD3) 2SO) Î ́ 171.2, 170.3, 169.8, 169.1, 168.4, 167.6, 158.5, 157.5, 156.1, 138.2, 137.79, 137.65, 137.3, 135.6, 135.0, 130.0, 128.0, 127.5, 124.0, 123.5, 112.2, 111.7, 80.5, 55.67, 55.50, 52.19, 52.05, 51.98, 45.10, 45.00, 42.1, 39.9, 39.7, 36.1, 35.6, 28.1, 27.7, 25.8, 23.81, 23.64, 18.0, 17.7, 11.8; MS (ESI) m / z calc. for [C51H71N10O14S2] <+>: 1111.46 [M + H] <+>; found: 1111.7.

Preparation 30

cyclo [Asp-DKPf4-Arg-Gly] (trifluoroacetate salt)

The cyclo compound [Asp (OtBu) -DKPf4-Arg (Mtr) -Gly] (90 mg, 0.081 mmol) was dissolved in TFA (10 ml) in the presence of ion scavengers: thioanisole (1.5 ml), ethanedithiol (0.75 ml), phenol (150 mg). The mixture was cooled to 0 ° C and put under nitrogen flow. Then trimethylsilylbromide (2ml) was added. The reaction was left under stirring until it reached room temperature and then left under stirring for 2 h. The TFA was then removed in the rotavapor, and the crude product was dissolved in a 1: 1 mixture of water and diisopropyl ether (50 ml). The aqueous phase was then washed several times with diisopropyl ether and subsequently concentrated in the rotavapor, thus obtaining the crude product, which was finally purified by semipreparative HPLC (Water's Atlantis column 21 mm x 10 cm, gradient: 100% H2O 0.1% TFA up to 70% H2O 0.1% TFA / 30% acetonitrile), thus obtaining the desired product (as trifluoroacetate salt) in the form of a white solid (49 mg, 70% yield).

<1> H NMR (400 MHz, D2O) Î ́ 7.40 (AB system, 4H), 5.35-5.18 (m, 1H), 4.44 (br s, 1H), 4.35 (dd, 1H, J = 9.1, 5.0 Hz ), 4.28 (br s, 1H), 4.22-4.12 (m, 3H), 4.08-3.94 (m, 2H), 3.78-3.63 (m, 2H), 3.43 (br s, 1H), 3.22 (t, 2H , J = 6.6 Hz), 3.10-3.00 (m, 1H), 2.962.83 (m, 3H), 1.93-1.76 (m, 2H), 1.73-1.61 (m, 2H); <13> C NMR (101 MHz, D2O,) Î ́ 174.5, 173.9, 173.4, 171.3, 169.1, 157.4, 136.7, 132.9, 130.0, 128.6, 58.2, 54.2, 53.6, 53.0, 47.3, 43.3, 43.1, 41.05, 41.02, 36.9, 35.2, 28.3 , 25.2; MS (ESI) m / z calc. for [C27H39N10O8] <+>: 631.30 [M + H] <+>; found: 631.4

Preparation 31

DKPf6-RGD

Boc-DKPf6-Arg (Mtr) -Gly-OBn

Boc-Arg (Mtr) -Gly-OBn (460 mg, 0.73 mmol, 1.1 eq.) Peptide was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Boc-DKPf6-OH (420 mg, 0.66 mmol, 1 eq.) according to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 93: 7) thus obtaining the desired product in the form of a spongy white solid (508 mg, 67% yield).

Rf = 0.47 (DCM / MeOH 9: 1); <ï ›> Î ± <ï 20>

D = -34.8 (c = 1.0 in CH2Cl2); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.97 (br s, 1H, J = 5.3 Hz), 7.73 (d, 1H, J = 7.4 Hz), 7.49 (s, 1H), 7.40 - 7.27 (m, 5H), 7.23 - 7.13 (m, 4H), 6.80 - 6.73 (m, 2H), 6.67 (s, 1H), 6.58 (br s, 2H), 6.25 (t, 1H, J = 5.0 Hz), 5.25 (d, 1H, J = 15.1 Hz), 5.13 (s, 2H), 4.68 - 4.58 (m, 1H, J = 4.4 Hz), 4.31 (br s, 1H, J = 3.8 Hz), 4.14 - 3.91 (m, 6H), 3.87 (s, 3H), 3.82 (s, 3H), 3.74 - 3.63 (m , 1H), 3.62 â € “3.52 (m, 1H), 3.15 (m, 2H), 3.02 (dd, 2H), 2.89 (d, 1H, J = 12.7 Hz), 2.68 (s, 3H), 2.66 â € “2.60 (m, 6H), 2.55 (s, 3H), 2.09 (s, 6H), 1.89 - 1.77 (m, 1H), 1.66 - 1.46 (m, 3H), 1.40 (s, 9H) ); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.9, 170.4, 170.0, 169.0, 167.3, 159.9, 158.9, 157.6, 157.4, 139.4, 139.3, 139.2, 138.3, 137.1, 136.9, 136.1, 135.6, 131.0, 129.3, 129.1, 129.0, 128.8, 125.4, 124.9, 113.0, 112.5, 79.6, 67.2, 57.4, 56.2, 56.1, 55.9, 52.9, 46.9, 46.6, 43.3, 41.7, 40.9, 37.3, 30.7, 28.6, 25.9, 24.4, 24.3, 18.7, 18.2, 12.1; MS (ESI) m / z calc. for [C55H74N9O14S2] <+>: 1148.48 [M + H] <+>; found: 1148.7

Preparation 32

Cbz-Asp (OtBu) -DKPf6-Arg (Mtr) -Gly-OBn

Boc-DKPf6-Arg (Mtr) -Gly-OBn (420 mg, 0.37 mmol, 1 eq.) Was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Cbz-Asp (OtBu ) -OH (141.6 mg, 0.44 mmol, 1.2 eq.) According to the general procedure B. The crude product was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product below white spongy solid form (334 mg, 67% yield).

Rf = 0.49 (DCM / MeOH 9: 1); <ï ›> Î ± <ï 20>

D = -27.6 (c = 0.5 in MeOH); <1> H NMR (400 MHz, (CD3) 2CO) Î ́ 7.97 (t, 1H, J = 5.5 Hz), 7.83 (br s, 1H), 7.70 ( d, 1H, J = 6.8 Hz), 7.48 (s, 1H), 7.40 - 7.24 (m, 10H), 7.24 - 7.14 (m, 4H), 6.84 (d, 1H, J = 8.2 Hz) , 6.79 â € “6.71 (m, 2H), 6.66 (s, 1H), 6.58 (br s, 2H), 5.21 (d, 1H, J = 15.0 Hz), 5.16 â €“ 5.06 (m, 3H), 5.00 (d, 1H, J = 12.5 Hz), 4.65 - 4.55 (m, 2H), 4.38 (t, 1H, J = 4.8 Hz), 4.17 - 3.92 (m, 6H), 3.90 - 3.77 (m, 7H), 3.75 - 3.65 (m, 1H), 3.28 - 3.09 (m, 2H), 3.04 - 2.95 (m, 1H), 2.94 - 2.86 (m, 1H) , 2.85 - 2.76 (m, 1H), 2.75 - 2.59 (m, 10H), 2.55 (s, 3H), 2.17 - 2.05 (m, 6H), 1.88 - 1.76 (m, 1H) ), 1.66 - 1.46 (m, 3H), 1.38 (s, 9H); <13> C NMR (101 MHz, (CD3) 2CO) Î ́ 172.9, 170.7, 170.4, 170.0, 168.9, 167.1, 159.9, 158.9, 157.6, 157.1, 139.3, 139.2, 138.2, 137.8, 137.1, 136.9, 136.2, 135.7, 131.0, 129.2, 129.0, 128.7, 125.4, 124.9, 113.0, 112.5, 81.4, 67.2, 57.5, 56.1, 55.9, 55.4, 53.0, 52.9, 47.1, 46.7, 42.3, 41.8, 41.0, 38.4, 37.3, 30.7, 28.2, 26.1, 24 .4, 18.7, 18.2, 12.1; MS (ESI) m / z calc. for [C66H85N10O17S2] <+>: 1353.55 [M + H] <+>; found: 1353.8.

Preparation 33

H-Asp (OtBu) -DKPf6-Arg (Mtr) -Gly-OH

H H

N N

Mtr

NH

OR

H.

N COOH

N H O O N

Mtr-HN NH

O NH NH

O2

O O

The compound Cbz-Asp (OtBu) -DKPf6-Arg (Mtr) -Gly-OBn (317 mg, 0.23 mmol, 1 eq.) Was dissolved in a 1: 1 mixture of THF / H2O (80 ml) and à 10% Pd / C was added (25 mg, 0.023 mmol, 0.1 eq.). The reaction flask was placed under a hydrogen atmosphere and the reaction was carried out, at room temperature and under stirring, for the whole night. The mixture was then filtered on a celite bed, washing the latter with 1: 1 THF / H2O. The filtered solution was then concentrated in the rotavapor, thus obtaining the crude product

(260 mg, 100% yield) which was used without further purification.

Preparation 34

cyclo [Asp (OtBu) -DKPf6-Arg (Mtr) -Gly]

To a solution of H-Asp (OtBu) -DKPf6-Arg (Mtr) -Gly-OH (260 mg, 0.23 mmol, 1 eq.) In DMF (165 ml) were added, under nitrogen atmosphere and at 0 ° C , HATU (350 mg, 0.92 mmol, 4 eq.), HOAt (125 mg, 0.92 mmol, 4 eq.) And DIPEA (0.236 ml, 1.38 mmol, 6 eq.). The temperature was allowed to rise to room temperature and the reaction was left under stirring overnight, then the DMF was removed in the rotavapor and the residue was dissolved in EtOAc (200 ml) and washed. with KHSO41M (2x30 ml), aqueous NaHCO3 (2x30 ml) and brine (2x30 ml). The organic phase was then dried with Na2SO4 and the solvent was removed with the rotavapor, thus obtaining the crude product. The latter was purified by flash chromatography on silica gel (CH2Cl2 / MeOH, 9: 1) thus obtaining the desired product in the form of a floccular solid (170 mg, 68% yield).

Rf = 0.35 (DCM / MeOH 9: 1); <ï ›> Î ± <ï 20>

D = -38.0 (c = 0.5 in DMSO); <1> H NMR (400 MHz, CD3OD) Î ́ 7.19 â € “7.06 (m, 4H), 6.67 (s, 1H), 6.65 (s, 1H), 5.18 (d, 1H, J = 14.7 Hz), 4.50 - 4.39 (m, 2H), 4.12 - 3.95 (m, 5H), 3.92 - 3.81 (m, 7H), 3.78 - 3.65 (m, 2H), 3.61 (dd, 1H, J = 13.7, 3.6 Hz), 3.29 - 3.08 (m, 2H), 2.99 (dd, 1H, J = 16.6, 7.0 Hz), 2.75 - 2.69 (m, 2H), 2.66 (s, 3H), 2.64 - 2.56 (m, 7H), 2.47 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 1.83 - 1.71 (m, 1H), 1.69 - 1.49 (m, 3H), 1.45 (s, 9H); <13> C NMR (101 MHz, CD3OD) Î ́ 174.2, 174.0, 173.1, 172.3, 171.8, 171.3, 160.7 , 159.9, 158.2, 139.9, 139.5, 138.6, 137.9, 136.3, 134.7, 131.0, 129.5, 129.0, 126.1, 125.7, 113.2, 112.8, 82.3, 56.2, 56.0, 54.5, 52.2, 47.8, 46.9, 44.0, 42.5, 36.6 , 36.1, 28.3, 24.5, 24.4, 18.9, 18.2, 12.1; MS (ESI) m / z calc. for [C51H71N10O14S2] <+>: 1111.46 [M + H] <+>; found: 1111.6

Preparation 35

cyclo [Asp-DKPf6-Arg-Gly] (trifluoroacetate salt)

H.

N NH2

NH CF3COOH

O O N H O N HN

H2N NH

OR CF3COOH

NH O NH O OH O

The cyclo compound [Asp (OtBu) -DKPf4-Arg (Mtr) -Gly] (90 mg, 0.081 mmol) was dissolved in TFA (10 ml) in the presence of ion scavengers: thioanisole (1.5 ml), ethanedithiol (0.75 ml), phenol (150 mg). The mixture was cooled to 0 ° C and put under nitrogen flow. Then trimethylsilylbromide (2ml) was added. The reaction was left under stirring until it reached room temperature and then left under stirring for 2 h. The TFA was then removed in the rotavapor, and the crude product was dissolved in a 1: 1 mixture of water and diisopropyl ether (50 ml). The aqueous phase was then washed several times with diisopropyl ether and subsequently concentrated in the rotavapor, thus obtaining the crude product, which was finally purified by semipreparative HPLC (Water's Atlantis column 21 mm x 10 cm, gradient: 100% H2O 0.1% TFA up to 70% H2O 0.1% TFA / 30% acetonitrile), thus obtaining the desired product (as trifluoroacetate salt) in the form of a white solid (50 mg, 71% yield).

<1> H NMR (400 MHz, D2O) Î ́ 8.06 (t, 1H, J = 6.1 Hz), 7.43 (system AB, 4H), 5.16 (d, 1H, J = 15.7 Hz), 4.53 (t, 1H , J = 7.1 Hz), 4.39 (dd, 1H, J = 9.3, 5.7 Hz), 4.36 - 4.29 (m, 2H), 4.20 (s, 2H), 4.05 (t, 1H, J = 6.2 Hz) , 3.89 (d, 1H, J = 15.4 Hz), 3.82 (d, 1H, J = 15.4 Hz), 3.77 - 3.71 (m, 2H), 3.21 (t, 2H, J = 6.8 Hz), 3.11 ( dd, 1H, J = 17.0, 6.8 Hz), 2.92 - 2.76 (m, 3H), 1.92 - 1.54 (m, 4H); <13> C NMR (101 MHz, D2O) Î ́ 175.2, 173.9 , 173.3, 172.5, 171.6, 168.8, 167.4, 157.4, 136.9, 132.9, 130.0, 128.8, 58.9, 55.4, 54.2, 51.6, 48.3, 43.4, 43.4, 41.7, 41.0, 35.9, 34.4, 28.4, 25.1; MS (ESI) m / z calc. for [C27H39N10O8] <+>: 631.29 [M + H] <+>; found: 631.5

Preparation 36

2â € ²-Succinyl-Taxol

Pyridine (1.90 ml, 23.4 mmol, 100 eq.) In CH2Cl2anhydrous (1 ml) was added to a solution of Tassol (200 mg, 0.234 mmol, 1 eq.) And succinic anhydride (28.0 mg, 0.281 mmol, 1.2 eq.) .). The resulting solution was left to react, under an argon atmosphere, for 3 h at 0 ° C and then left under stirring until it reached room temperature. After 20 h the reaction was concentrated in the rotavapor, the resulting solid was dissolved in water / acetonitrile / acetic acid (2 ml, 1: 1: 1) and purified by reverse phase chromatography (biotage) eluting with water and acetonitrile, thus obtaining the product in the form of a white solid (200 mg, 90% yield).

<1> H NMR (400 MHz, CD3OD): Î ́ 8.14 (d, 2H, J = 7.2 Hz), 7.85 (d, 2H, J = 7.2 Hz), 7.42-7.72 (m, 10H), 7.30 (t , 1H, J = 7.5 Hz), 6.47 (s, 1H), 6.10 (m, 1H), 5.86 (d, 1H, J = 6.4 Hz), 5.66 (d, 1H, J = 6.4 Hz), 5.50 (d , 1H, J = 6.4 Hz), 5.02 (d, 1H, J = 9.2 Hz), 4.36 (m, 1H), 4.21 (s, 2H), 3.84 (d, 1H, J = 6.8 Hz), 2.64-2.74 (m, 4H), 2.49 (m, 1H), 2.42 (s, 3H), 2.15-2.30 (m, 4H), 1.94 (s, 3H), 1.86 (m, 1H), 1.81 (m, 1H), 1.68 (s, 3H), 1.16 (s, 6H; <13> C NMR (101 MHz, CD3OD): Î ́ 206.1, 177.0, 174.4, 172.5, 172.2, 171.5, 171.3, 168.6, 143.4, 139.3, 136.5, 135.7 , 135.5, 133.7, 132.3, 132.1, 130.9, 130.6, 130.4, 129.5, 86.8, 83.1, 79.9, 78.3, 77.7, 77.1, 76.8, 73.8, 73.1, 60.1, 56.1, 48.8, 45.4, 38.4, 37.3, 30.3, 27.8 , 24.1, 23.2, 21.6, 15.8, 11.2; MS (ESI) m / z calc. For [C51H56NO17] <+> 954.36 [M + H] <+>; found: 954.2

EXAMPLE 1

2â € ²-cyclo [Asp-DKPf-Arg-Gly] -succinamyl) -Tassolo.

General procedure:

To a solution of 2â € ²-succinyl-Taxol (49 mg, 0.0513 mmol, 1.25 eq.) And N-hydroxysulfosuccinimide sodium salt (13.94 mg, 0.0642 mmol, 1.55 eq.) In anhydrous DMF (2.0 ml) was added diisopropylcarbodiimide (11.93 Î1⁄4L, 9.72 mg, 0.077 mmol, 1.9 eq.). The resulting solution was allowed to react under argon at room temperature for 24 h. The reaction was then concentrated under vacuum, obtaining a whitish solid. The latter was dissolved in acetonitrile (2 ml) and to this solution was added a solution of cyclo [Asp-DKP-Arg-Gly] (35 mg, 0.0414 mmol) in an aqueous solution of phosphate buffer at pH 7.0 (0.5 M, 1.0 ml), then adjusting the pH to 7 with the addition of a few drops of 0.2 M NaOH. The resulting solution was rapidly cooled to 0 ° C, then left to react for 10 h at 0 ° C and then left under stirring until it reaches room temperature. After 18 h the solution was concentrated in the rotavapor. The crude product was purified by semipreparative HPLC (19 mm x 10 cm Xbridge column, gradient: 90% H2O 0.1% formic acid / 10% acetonitrile up to 30% H2O 0.1% formic acid / 70% acetonitrile), thus obtaining the desired product (after lyophilization) in the form of a white solid (60% yield).

EXAMPLE 2

2â € ²-cyclo [Asp-DKPf2-Arg-Gly] -succinamyl) -Tassolo.

By operating as described in example 1, the compound of the title is obtained.

<1> H NMR (400 MHz, CD3OD) Î ́ 8.09 (dd, 2H, J = 8.4, 1.2 Hz), 7.84 (dd, 2H, J = 7.1, 1.6 Hz), 7.74 (tt, 1H, J = 6.8 , 1.6 Hz), 7.64 (t, 2H, J = 7.6 Hz), 7.59-7.54 (m, 1H), 7.49-7.44 (m, 6H), 7.28-7.25 (m, 4H), 7.22 (tt, 1H, J = 5.8, 2.8 Hz), 6.43 (s, 1H), 5.98 (t, 1H, J = 9.1 Hz), 5.69 (d, 1H, J = 7.9 Hz), 5.61 (d, 1H, J = 7.0 Hz) , 5.45 (d, 1H, J = 7.9 Hz), 5.24 (d, 1H, J = 15.1 Hz), 5.10 (dd, 1H, J = 9.7, 1.6 Hz), 4.77 (m superimposed on water peaks, 1H), 4.65 (m superimposed on water peaks, 1H), 4.40-4.30 (m, 4H), 4.21 (m, 3H), 4.08 (d, 2H, J = 15.7 Hz), 3.85 (d, 1H , J = 4.6 Hz), 3.78 (d, 1H, J = 7.0 Hz), 3.64 (d, 1H, J = 16.3 Hz), 3.28 (m superimposed on solvent peaks, 1H), 3.22 (t, 2H, J = 6.7 Hz), 2.98 (dd, 1H, J = 13.2, 7.4 Hz), 2.84-2.75 (m, 2H), 2.71-2.68 (m, 2H), 2.64-2.50 (m, 3H), 2.37-2.34 ( m, 4H), 2.18 (s, 3H), 2.01 (m, 1H), 1.89-1.79 (m, 5H), 1.75-1.59 (m, 7H), 1.14 (s, 3H), 1.11 (s, 3H) ; <13> C NMR (101 MHz, CD3OD) Î ́ 205.7, 175.8, 175.0, 174.5, 174.17, 174. 02, 173.7, 172.09, 171.96, 171.1, 170.74, 170.54, 169.8, 168.0, 158.3, 142.3, 139.6, 137.9, 135.5, 135.04, 134.95, 134.7, 133.2, 131.1, 130.8, 130.2, 129.94, 129.82, 129.71, 129.3, 129.0, 128.64, 128.46, 85.9, 82.0, 79.1, 77.5, 76.7, 76.14, 76.06, 72.9, 72.1, 60.3, 59.1, 55.8, 55.4, 53.2, 51.3, 48.1, 47.8, 44.3, 43.7, 43.0, 40.7, 39.9, 37.32, 37.21, 36.0, 31.0, 29.9, 28.2, 26.8, 26.2, 23.4, 22.3, 20.9, 15.0, 10.6; MS (ESI) m / z calc. for [C78H92N11O24] <+>: 1566.63 [M + H] <+>; found: 1566.6.

EXAMPLE 3

2â € ²-cyclo [Asp-DKPf3-Arg-Gly] -succinamyl) -Tassolo.

By operating as described in example 1, the compound of the title is obtained.

<1> H NMR (400 MHz, CD3OD) Î ́ 8.12 (dd, 2H, J = 8.5, 1.4 Hz), 7.83 (dd, 2H, J = 8.5, 1.4 Hz), 7.71-7.66 (m, 1H), 7.60 (t, 2H, J = 7.5 Hz), 7.56-7.52 (m, 1H), 7.50-7.42 (m, 6H), 7.30 (s, 4H), 7.25 (tt, 1H, J = 7.1, 1.6 Hz) , 6.45 (s, 1H), 6.05 (td, 1H, J = 9.1, 1.0 Hz), 5.79 (d, 1H, J = 6.5 Hz), 5.64 (d, 1H, J = 7.2 Hz), 5.44 (d, 1H, J = 6.5 Hz), 5.13 (d, 1H, J = 14.9 Hz), 5.03 (dd, 1H, J = 9.4, 1.6 Hz), 4.91-3.86 (m, 1H), 4.75 (dd, 1H, J = 6.5, 4.7 Hz), 4.44-4.36 (m, 3H), 4.30-4.22 (m, 2H), 4.20 (br s, 2H, J = 4.2 Hz), 4.16 (ddd, 1H, J = 12.0, 8.7, 3.6 Hz), 4.09-4.08 (m, 2H), 3.90 (d, 1H, J = 6.0 Hz), 3.82 (d, 1H, J = 7.1 Hz), 3.74-3.68 (m, 2H), 3.61 (d, 1H, J = 17.2 Hz), 3.54 (dt, 1H, J = 11.7, 2.8 Hz), 3.42 (dd, 1H, J = 14.6, 6.4), 3.27-3.16 (m, 2H), 2.80-2.75 (m, 2H), 2.72-2.51 (m, 7H), 2.37 (s, 3H), 2.18-2.12 (m, 4H), 2.09-2.01 (m, 1H), 1.92 (s, 3H), 1.86-1.76 (m, 3H), 1.68-1.63 (m, 5H), 1.14 (s, 3H), 1.13 (s, 3H); <13> C NMR (101 MHz, CD3OD) Î ́ 205.5, 174.0, 173.60, 173.46, 173.0, 1 71.63, 171.54, 171.46, 171.2, 170.5, 167.7, 142.4, 140.2, 138.4, 135.5, 134.80, 134.63, 132.9, 131.39, 131.22, 130.1, 129.72, 129.60, 129.56, 129.3, 128.6, 100.0, 85.9, 82.3, 79.0, 77.5, 76.9, 76.2, 75.9, 72.9, 72.4, 61.6, 60.6, 59.2, 55.4, 54.4, 53.2, 50.5, 48.0, 44.6, 43.76, 43.69, 42.2, 39.9, 37.6, 36.49, 36.36, 31.1, 29.8, 27.7, 26.9, 26.5, 25.1, 23.3, 22.3, 20.8, 15.1, 10.5; MS (ESI) m / z calc. for [C78H92N11O24] <+>: 1566.63 [M + H] <+>; found: 1566.6.

EXAMPLE 4

2â € ²-cyclo [Asp-DKPf4-Arg-Gly] -succinamyl) -Tassolo.

By operating as described in example 1, the compound of the title is obtained.

<1> H NMR (400 MHz, (CD3) 2SO) Î ́ 9.22 (d, 1H, J = 8.5 Hz), 8.95 (s, 1H), 8.79 (s, 1H), 8.44-8.40 (m, 1H) , 8.35 (t, 1H, J = 5.7 Hz), 8.20 (s, 1H), 7.98 (dd, 2H, J = 7.1, 1.3 Hz), 7.86 (dd, 2H, J = 7.2, 1.3 Hz), 7.76- 7.69 (m, 1H, J = 1.5 Hz), 7.69-7.63 (m, 2H), 7.59-7.53 (m, 1H), 7.49 (d, 1H, J = 7.6 Hz), 7.46-7.42 (m, 5H) , 7.23-7.17 (m, 5H), 6.30 (s, 1H), 5.83 (t, 1H, J = 8.9 Hz), 5.54 (t, 1H, J = 8.7 Hz), 5.42 (d, 1H, J = 7.1 Hz), 5.36 (d, 1H, J = 8.9 Hz), 5.21 (d, 1H, J = 14.4 Hz), 4.92 (d, 2H, J = 10.6 Hz), 4.62 (s, 1H), 4.27-4.07 ( m, 5H), 4.04-3.99 (m, 3H), 3.94-3.87 (m, 1H), 3.83-3.79 (m, 1H), 3.70 (br s, 2H), 3.58 (d, 1H, J = 7.1 Hz ), 3.43-3.26 (m superimposed on water peaks, 2H), 3.07 (br s, 2H), 2.89 (br s, 2H), 2.69-2.56 (m, 3H), 2.45 (t, 2H, J = 6.8 Hz), 2.38-2.30 (m, 2H), 2.24-2.20 (m, 4H), 2.10 (s, 3H), 1.84-1.76 (m, 5H), 1.64 (t, 1H, J = 12.4 Hz) , 1.54-1.41 (m, 7H), 1.02 (s, 3H), 1.00 (s, 3H); <13> C NMR (101 MHz, (CD3) 2SO) Î ́ 202.3, 173.9, 172.13, 171.94, 170.8, 170.2 , 169.63, 169.50, 169.1, 168.84, 168.74, 168.3, 167.9, 166.4, 165.2, 157.3, 139.4, 138.7, 137.3, 134.8, 134.3, 133.45, 133.33, 132.7, 131.4, 129.95, 129.93, 129.56, 129.54, 128.73, 128.65 , 128.55, 128.29, 128.14, 127.83, 127.71, 127.65, 127.4, 83.6, 80.2, 76.7, 75.3, 74.68, 74.54, 74.50, 72.5, 70.7, 70.4, 57.4, 56.1, 53.98, 53.86, 52.17, 52.13, 46.1, 44.9 , 42.9, 42.1, 41.9, 40.1, 39.9, 38.2, 36.5, 35.7, 34.4, 29.5, 28.7, 27.6, 26.3, 25.3, 22.5, 21.4, 20.63, 20.52, 13.9, 9.7; MS (ESI) m / z calc. for [C78H92N11O24] <+>: 1566.63 [M + H] <+>; found: 1566.6

EXAMPLE 5

2â € ²-cyclo [Asp-DKPf6-Arg-Gly] -succinamyl) -Tassolo.

By operating as described in example 1, the compound of the title is obtained.

<1> H NMR (400 MHz, (CD3) 2SO) Î ́ 9.42 (s, 1H), 9.25 (d, 1H, J = 8.5 Hz), 8.74 (s, 1H), 8.62 (s, 1H), 8.45 (s, 1H), 8.37 (t, 1H, J = 5.7 Hz), 7.99-7.97 (m, 2H), 7.87-7.84 (m, 3H), 7.73 (t, 1H, J = 7.3 Hz), 7.66 ( t, 2H, J = 7.4 Hz), 7.56 (tt, 1H, J = 7.3, 2.0 Hz), 7.50-7.44 (m, 7H), 7.25-7.15 (m, 5H), 6.30 (s, 1H), 5.83 (t, 1H, J = 8.8 Hz), 5.53 (t, 1H, J = 8.7 Hz), 5.41 (d, 1H, J = 7.2 Hz), 5.35 (d, 1H, J = 9.0 Hz), 5.11 (d , 1H, J = 14.9 Hz), 4.98-4.90 (t, 2H), 4.64 (s, 1H), 4.29-4.20 (m, 3H), 4.17-4.08 (m, 2H), 4.04-3.95 (m, 3H ), 3.93-3.79 (m, 3H), 3.74-3.64 (m, 1H), 3.59 (d, 1H, J = 6.8 Hz), 3.53-3.43 (m, 1H), 3.26-3.19 (m, 1H), 3.07 (br s, 1H), 2.97 (br s, 1H), 2.72-2.58 (m, 4H), 2.56-2.52 (m, 1H), 2.45 (t, 2H, J = 6.8 Hz), 2.40-2.28 ( m, 2H), 2.23 (s, 3H), 2.10 (s, 3H), 1.82-1.60 (m, 7H), 1.52-1.46 (m, 6H), 1.02 (s, 3H), 1.00 (s, 3H) ; <13> C NMR (101 MHz, (CD3) 2SO) Î ́ 202.7, 172.0, 171.8, 170.3, 169.72, 169.57, 169.2, 168.79, 168.72, 166.8, 166.4, 165.2, 157.3, 139.5, 137.4, 135.2, 13 4.3, 133.5, 133.32, 133.28, 132.7, 131.5, 129.9, 129.6, 128.7, 128.38, 128.21, 128.0, 127.7, 127.5, 83.6, 80.3, 76.7, 75.3, 74.69, 74.62, 74.49, 70.7, 70.4, 57.2, 54.4, 54.1, 51.98, 51.92, 46.10, 45.97, 43.0, 41.9, 40.9, 39.7, 37.8, 36.6, 34.4, 29.5, 28.7, 28.0, 26.4, 24.4, 22.6, 21.4, 20.7, 14.0, 9.8; MS (ESI) m / z calc. for [C78H92N11O24] <+>: 1566.63 [M + H] <+>; found: 1566.6.

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Solubility and cycle stability [DKP-f3-RGD] -PTX

[DKP-f3-RGD] -PTX Cycle solubility and stability were verified by HPLC over time at 25 ° C (see Figure 2). A solution of Cycle [DKP-f3-RGD] -PTX of 3.0 mg / ml (1 ml solvent = 0.05 ml Cremophor EL 0.05 ml Ethanol 0.90 ml physiological solution) was prepared. The HPLC purity is> 99.5%. The purity of the compound remained constant (> 99.5%) over a period of 96 hours (4 days). Initially the solution was oversaturated (3.0 mg / mL) and part of the compound precipitated. After 42 h the precipitate was filtered and the concentration of the solution (2.0 mg / mL) was stable over time up to 96 h. However, the precipitate had a high purity (> 99.5%).

BIOLOGICAL ASSAYS

Material and methods

Binding assays on isolated receptors: The purified receptors Î ± vβ3e Î ± vβ5 (Chemicon Internatinal, Inc., Temecula, CA, USA) were diluted in a concentration of 0.5Î1⁄4g / ml in a coating buffer containing Tris -HCl 20mM (pH 7.4), NaCl 150mM, MnCl21mM, CaCl22mM and MgCl21mM. An aliquot of diluted receptors (100 Î1⁄4l / well) was added to the 96-well micro titration plates (NUNC MW 96F MEDISORP STRAIGHT) and incubated overnight at 4 ° C. The plates were then incubated with a blocking solution (coating buffer solution with the addition of 1% bovine serum albumin) for an additional 2 hours at room temperature to block non-specific binding, followed by 3 hours of incubation at room temperature with different concentrations (10 <-12> - 10 <-5> M) of the compounds to be tested, in the presence of 1 Î1⁄4g / ml of biotinylated vitronectin. Biotinylation was conducted using the EZ-Link Sulfo-NHS-Biotynilation kit (Pierce, Rockford, IL). After washing, the plates were incubated for 1 hour at room temperature with the biotinylated streptavidin-peroxidase complex (Amersham Biosciences, Uppsala, Sweden), followed by a 30-minute incubation with 100 Î1⁄4l of Substrate Reagent Solution (R&D Systems , Minneapolis, MN), before stopping the reaction with the addition of 50 Î1⁄4l of H2SO42N. The absorbance was then measured at 415 nm with the Synergy <TM> HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point reported is the result of the average of three plates and was analyzed by non-linear regression analysis using the Prism GraphPad program.

Cell pharmacology studies.

Cell lines and growth conditions. Ovarian cancer cell lines SKOV3 (ATCC), IGROV-1 [Perego P. et al., 1998], cisplatin-resistant variant IGROV-1 / Pt1 [Perego et al., 1996], pancreatic cancer lines MIA Paca2 (ATCC) and PANC1 (ATCC) were maintained in RPMI-1640 medium; the U2-OS human osteosarcoma lineage (ATCC) was grown in Mc Coy's 5A medium. The medium was completed with the addition of glutamine (1%) and fetal bovine serum (10%).

Cellular sensitivity to drugs. Cell sensitivity to drugs was tested using the cell count-based growth inhibition assay. In summary, cells were seeded in duplicate in 12-well plates (Corning Costar, Euroclone) and exposed to drugs after 24 h. Taxol and study drugs were dissolved in dimethyl sulfoxide and then added to the culture medium at different concentrations starting from 200x solutions. To some wells only the vehicle was added, which at these concentrations (0.005%) was not toxic. After 72 h of drug incubation, cells were collected and counted with an automatic counter. The IC50 is defined as the drug concentration that reduces cell growth by 50%.

Integrin level analysis. The expression of the integrins was examined by indirect immunofluorescence using the flow cytometer, after suitable optimization of the antibody concentration. Exponentially growing cells were harvested and incubated for 30 min at 4 ° C with antibodies to human integrinsï ¡vï ¢ 3, ï ¡5ï ¢ 1 (Millipore, Temecula, CA) ï ¡vï ¢ 5, Chemicon International) or with isotypic controls. Cells were washed in buffered saline and samples were immediately analyzed by flow cytometry (FACScan, Becton-Dickinson). Integrin expression was expressed as the ratio of the mean fluorescence intensity obtained in isolated cells incubated with anti-integrin antibodies divided by that of cells incubated with isotypic controls.

Study of antitumor activity in vivo

Evaluation of anticancer activity. The experiments were conducted using female Athymic Swiss naked mice (age 8-10 weeks, Charles River, Calco, Italy). The animals were housed in laminar flow rooms at constant temperature and humidity. The mice had free access to food and water. The experiments were approved by the Ethics Committee for Animal Experimentation of the National Cancer Institute of Milan, in accordance with institutional and international guidelines.

The study was carried out using the IGROV-1 / Pt1 ovarian cancer model. Cells from in vitro monolayer cultures were inoculated subcutaneously into healthy mice. The in vivo tumor line was maintained by serial passages in animals. For the purposes of the experiment, tumor fragments were implanted subcutaneously in the two flanks of the animal. Each experimental group consisted of 4 animals. Three days after transplantation, when the tumor mass was not yet measurable, the animals were randomized for treatment, administering the compounds intravenously (iv) every 4 days for 4 times. The drugs, dissolved in a mixture of ethanol-Cremophor EL in equal parts (10% of the final volume) and diluted in physiological solution, were administered in a volume of 10 ml / kg of body weight. Tumor growth was followed by measuring the diameters of the tumors with a caliper twice a week; tumor volume (TV) was calculated according to the formula TV = d <2> xD / 2 (d = minor diameter, D = major diameter). Animals were eliminated when the mean tumor volume exceeded 1000 mm <3>. The efficacy of the treatment was evaluated in terms of tumor volume inhibition [TVI% = 100- (mean VT of treated mice / mean VT of control mice x 100)]. The animals were weighed twice a week and the differences in weight in percentage (body weight loss, BWL%) compared to the start of treatment were calculated. To assess the toxicity of the treatment, the maximum induced weight loss was considered. The treated animals, who died before the first check, were considered dead from toxic effects (lethal toxicity). For the statistical analysis of the results, the tumor volumes of the treated animals with respect to the controls were compared using the Student t test (two tailed); P <0.05 was considered significant.

Histological analysis. Animals carrying IGROV-1 / Pt1 ovarian cancer were treated iv every 4 days twice with the conjugate (30 mg / kg), its ligand (dose corresponding to 30 mg / kg) or taxol (30 mg / kg). . Treatment began when VT reached 250-300 mm <3>. Twenty-four hours after the second treatment, the animals were sacrificed and the tumor masses were collected and kept in buffered formalin for 48 hours. The samples were transferred to 70% ethanol and subsequently processed for immunohistochemistry. To evaluate the micro vascular density (MVD), the samples fixed in the presence of zinc were immunodecorated with a mouse anti-mouse CD31 monoclonal antibody. The reaction was developed by incubating the sections with 3,3â € ²-diaminobenzidine and the sections were counterstained with hematoxylin. MVD was evaluated under the microscope (Ã — 400) in 3-4 sections examined in blind to calculate the mean value of each group.

Results

Cell pharmacology studies. Cellular sensitivity to the different conjugates was examined using cell lines of different tumor types, characterized by a variable expression of integrins (Table 1). In the U2-OS osteosarcoma line, the basal expression of ï ¡vï ¢ 5 was more marked than that of the other cell lines, while the cell line with the highest levels of integrin ï ¡vï ¢ 3 was the cisplatin- variant. resistant IGROV-1 / Pt1. Ovarian cancer cell lines (IGROV-1, IGROV-1 / Pt1 and SKOV3) had elevated levels of ï ¡5ï ¢ 1. The evaluation of cellular sensitivity to conjugates, examined by proliferation inhibition test, indicated that the conjugates had a high cytotoxic power, comparable to that of taxol (Table 2). The observed proliferation inhibition was not attributable to the ligand (Table 2) which did not inhibit proliferation.

Studies of anticancer activity

The effects of the Cyclo [DKP-f3-RGD] -PTX conjugate administered intravenously (iv) were evaluated in subcutaneously transplanted nude mice with IGROV-1 / Pt1 ovarian cancer using taxol as the reference drug. The results are presented in Table 3 and Figure 1. Using two doses of Cyclo [DKP-f3-RGD] -PTX (15 mg / kg and 30 mg / kg), a dose-dependent antitumor activity was observed. When taxol and Cyclo [DKP-f3-RGD] -PTX were compared at the same dose, the conjugate showed superiority in terms of tumor volume inhibition (TVI = 85% vs. TVI = 76%). Furthermore, 2 out of 8 tumors in the group receiving Cyclo [DKP-f3-RGD] -PTX 30 mg / kg disappeared without evidence of regrowth at the end of the experiment. The treatment was well tolerated as no toxic deaths or significant weight losses were observed by the treated animals.

Results

Cell pharmacology studies in models characterized by a heterogeneous expression profile of the integrins of interest indicate that the synthesized conjugates maintain excellent cytotoxic potency when their effect is tested with cell proliferation inhibition tests. An analysis of the conjugateâ € ™ s anticancer activity highlights its ability to significantly inhibit the growth of the IGROV-1 / Pt1 tumor. The inhibition is dose-dependent as evidenced by the comparison of the effect obtained by administering the conjugate Cyclo [DKP-f3-RGD] -PTX at doses of 30 mg / kg and 15 mg / kg. The antitumor activity of the conjugate 30 mg / kg is higher than that of taxol 30 mg / kg [for example, on day 25, Inhibition of Tumor Volume (TVI) = 76% for taxol and 85% for the conjugate]. However, since the amount of taxol administered to the animal treated with the conjugate 30 mg / kg is equal to 16.35 mg of taxol, the treatment with the conjugate has an evident therapeutic advantage. In practice, a better activity is observed with the conjugate than with taxol with approximately half (54.5%) of the active principle. The therapeutic advantage is also supported by the failure to regrow two out of eight tumors at the end of the experiment.

Table 1 - Flow cytometric analysis of integrin levels ï ¡vï ¢ 3, ï ¡vï ¢ 5, eï ¡5ï ¢ 1 in cell lines of different tumor types <[a]>

Average fluorescence intensity <a> ___________________________________________________

Cellular line ï ¡vï ¢ 3ï ¡vï ¢ 5ï ¡5ï ¢ 1

IGROV-1 4.8 ± 1.9 3.4 ± 0.9 47.6 ± 11.2

IGROV-1 / Pt1 23.3 ± 5.0 3.3 ± 0.5 23.9 ± 2.4

SKOV-3 6.4 ± 0.05 4.4 ± 0.5 20.3 ± 2.1

U2-OS 1.8 ± 0.6 27.4 ± 0.1 38.2 ± 5.7

PANC-1 7.9 ± 2.8 25.7 ± 6.5 10.7 ± 2.6

MIA-PaCa2 1.2 ± 0.1 5.6 ± 0.9 4.8 ± 0.2

<[a]> Ratio between the mean fluorescence intensity of cells incubated with the primary antibody and those incubated with isotype control

Table 2 - Cellular sensitivity of cell lines of different tumor types to Cyclo [DKP-f2-RGD] -PTX, Cyclo [DKP-f3-RGD] -PTX, Cyclo [DKP-f4-RGD] -PTX and Cyclo conjugates [DKP-f6-RGD] -PTX <[a]>

IC50 [nM] (exposure = 72 hours)

Compound U2-OS SKOV3 PANC-1 MIA-PaCa2

IGROV-1

IGROV-1 (HTB-96) (HTB-77) (CRL-1469) (CRL-1420)

[b] / Pt1 <[b]>

[c] [c] [c] [c]

Cyclo [DKP-> 1200> 18000> 6300> 11600> 11600> 11600 f3-RGD]

Cyclo [DKP-f3-RGD] - 61.3 ± 19.1 4.9 ± 2.0 12.8 ± 0.1 1.2 ± 0.1 2.4 ± 0.8 2.3 ± 0.4 PTX

Cyclo [DKP-f2-RGD] - 17.7 ± 6.0 18.7 ± 6.0 2.2 ± 0.5 1.6 ± 1.0 5.8 ± 4.0 2.0 ± 0.7 PTX

Cyclo [DKP-f6-RGD] - 48.2 ± 2.2 2.4 ± 1.9 5.7 ± 4.4 2.4 ± 1.1 3.5 ± 0.1 2.5 ± 0.6 PTX

Cyclo [DKP-f4-RGD] - 34.4 ± 29.0 3.7 ± 2.0 6.8 ± 4.6 2.4 ± 0.9 3.2 ± 0.7 1.8 ± 0.6 PTX

Taxol 23.0 ± 0.8 2.2 ± 0.8 3.4 ± 0.4 2.7 ± 1.1 5.2 ± 1.9 7.2 ± 3.8

<[a]> Evaluated with cell growth inhibition assay after 72 h of drug exposure.

<[b]> The IGROV-1 and IGROV-1 / Pt1 cell lines were obtained as indicated in Perego P., Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A. , Zunino F. Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9: 1-8, 1998 and in Perego P., Giarola M., Righetti, S. C., Supino R., Caserini C., Delia D., Pierotti M. A., Miyashita T., Reed J. C., Zunino F. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems .. Cancer Res., 1996, 56, 556-562.

<[c]> The cell lines (ATCC: U2OS HTB-96, SKOV3 HTB-77, PANC-1 CRL-1469, MIA PaCa2 CRL-1420) are subject to deposit at the authorized centers as indicated respectively.

Table 3 - Antitumor activity of Cyclo [DKP-f3-RGD] -PTX conjugate and taxol on IGROV-1 / Pt1 ovarian carcinoma xenograft subcutaneously in naked mice

Drug Dose (mg / kg) TVI% <[1]> CR <[2]> NED <[3]> BWL% <[4]>

Taxol 30 76 3/8 0/8 4

Cyclo 15 64 0/8 - 0

[DKP-f3-RGD] -PTX

Cyclo 30 85 2/8 2/8 3

[DKP-f3-RGD] -PTX

<[1]> Inhibition of tumor volume% in treated mice compared to control mice, evaluated 10 days after the last treatment.

<[2]> Complete responses, disappearance of the tumor lasting at least 10 days.

<[3]> Failure to regrow the tumor at the end of the experiment (day 66).

<[4]> Treatment-induced weight loss-%, the largest difference is reported.

No significant weight loss was observed during treatment, as weight loss is less than 10%. Furthermore, no deaths were found in any of the experimental groups. Therefore the conjugate according to the invention is well tolerated.

Bibliography

Perego P., Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A., Zunino F. Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9: 1-8, 1998.

Perego P., Giarola M., Righetti, S. C., Supino R., Caserini C., Delia D., Pierotti M. A., Miyashita T., Reed J. C., Zunino F. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems .. Cancer Res., 56, 556-562, 1996.

Claims (12)

CLAIMS 1. Compound of formula (I) H2 N <NH> H N OR OR No. H. R2 O N 6 H N 5 2 3 No. Or R1 NH O NH OR OH O (I) in which: - the carbon atoms of piperazine C3, C6 are in the configurations R, S or S, R; - R1 and R2 are independently selected from hydrogen, C1-C6-alkyl, C1-C6-alkoxy, benzyl and a group of formula (II) -L-X-Z-Y-Ct (II) where is it - L and Z are independently chosen from the following groups: n Ar <n> O n mm pn where n and m are, each independently, an integer from 0 to 10, p is an integer from 1 to 10 and Ar is an aromatic or hetero aromatic group; - X is a group chosen from the following N H H NNN N N N H N OR NOT N H N H H S N N O O S O O N N N N H O O O N O OR - Y is a group chosen from the following O O SN - Ct is a cytotoxic agent; with the proviso that at least one of R1 and R2 is a group of formula (II); and their pharmaceutically acceptable salts, solvates and hydrates. 2. Compound of formula (I) according to claim 1, wherein Ct is selected from taxol, daunorubicin, doxorubicin, carminomycin, 4-epiadriamycin, 4-demethoxy-daunomycin, 11-deoxidaunorubicin, 13-deoxidaunorubicin, adriamycin-14- benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalenacetate, vinblastine, vincristine, mitomycin C, n-methyl mitomycin C, bleomycin A2, dideazatetrahydrofolic acid, aminopteridine, methotrexate, camptothecin, colchatis and cycline. Compound of formula (I) according to claim 2, wherein Ct is the taxol. Compound of formula (I) according to any one of claims 1 to 3, wherein m, n and p are 1 or 2. 5. Compound of formula (I) according to any one of claims 1 to 4 wherein only one of R1 and R2 is a group of formula (II) and the other is hydrogen. 6. Compound of formula (I) according to any one of claims 1 to 5, wherein L-X-Z-Y is the following group OR H. No. OR 7. Compound of formula (I) according to claim 1, wherein R1 = -L-X-Z-Y- Ã OR H. No. OR R2 = H, Ct is the taxol and the configuration of the carbon atoms 3 and 6 of piperazine is 3S, 6R. Compound of formula (I) according to claim 1, wherein the configuration of the carbon atoms 3 and 6 of piperazine, R1 and R2 are as follows: 3R, 6S, R1 = -L-X-Z-Y-taxol, R2 = H. 3R, 6S, R1 = H, R2 = -L-X-Z-Y-taxol 3R, 6S, R1 = -L-X-Z-Y-, R2 = benzyl 3R, 6S, R1 = benzyl, R2 = -L-X-Z-Y-taxol 3S, 6R, R1 = H, R2 = -L-X-Z-Y-taxol 3S, 6R, R1 = -L-X-Z-Y-taxol, R2 = benzyl 3S, 6R, R1 = benzyl, R2 = -L-X-Z-Y-taxol and where -L-X-Z-Y- is OR H. No. OR 9. Compound of formula (I) according to any one of claims 1 to 8, for use in therapy. 10. Compound of formula (I) according to any one of claims 1 to 8, for use in antitumor therapy. Compound for use according to claim 10, for the treatment of breast carcinoma, ovarian carcinoma, Kaposi's sarcoma and lung carcinoma of the "non-small cell" type. Pharmaceutical composition comprising, as active principle, a compound of formula (I) according to any one of claims 1 to 8, together with at least one pharmaceutically acceptable carrier or excipient.