NZ702470B2 - Beta-hairpin peptidomimetics - Google Patents

Abstract

Hairpin peptidomimetics of the general formula cyclo(-Tyr1-His2-Xaa3-Cys4-Ser5-Xaa6-DPro7-Xaa8-Arg9-Tyr10-Cys11-Tyr12-Xaa13-Xaa14-Xaa15-Pro16-), disulfide bond between Cys4 and Cys11, and pharmaceutically acceptable salts thereof, with Xaa3, Xaa6, Xaa8, Xaa13, Xaa14 and Xaa15 being amino acid residues of certain types which are defined in the description and the claims, have favorable pharmacological properties and can be used for preventing HIV infections in healthy individuals or for slowing and halting viral progression in infected patients; or where cancer is mediated or resulting from CXCR4 receptor activity; or where immunological diseases are mediated or resulting from CXCR4 receptor activity; or for treating immunosuppression; or during apheresis collections of peripheral blood stem cells and/or as agents to induce mobilization of stem cells to regulate tissue repair. These peptidomimetics can be manufactured by a process which is based on a mixed solid-and solution phase synthetic strategy. dues of certain types which are defined in the description and the claims, have favorable pharmacological properties and can be used for preventing HIV infections in healthy individuals or for slowing and halting viral progression in infected patients; or where cancer is mediated or resulting from CXCR4 receptor activity; or where immunological diseases are mediated or resulting from CXCR4 receptor activity; or for treating immunosuppression; or during apheresis collections of peripheral blood stem cells and/or as agents to induce mobilization of stem cells to regulate tissue repair. These peptidomimetics can be manufactured by a process which is based on a mixed solid-and solution phase synthetic strategy.

Description

BETA-HAIRPIN PEPTIDOMIMETICS The present invention provides B-hairpin peptidomimetics which are having CXCR4 antagonizing activity.

The B-hairpin peptidomimetics of the ion are cyclo(—Tyr1-HisZ-Xaas-Cys4-Ser5- Xaas—DPro7-Xaas—ArgQ—Tyrlo-Cysll-Tyrlz—Xaa13—Xaa14-Xaa15-Prole-), disulfide bond between Cys4 and Cysll, and ceutically acceptable salts f, with Xaa3 being Tyr, Tyr(Me) as described herein below or Tyr(CFg) as described herein below, Xaa6 being Ala or Acc as described herein below, Xaa8 being r) as described herein below, Xaa13 being Gln or Glu, Xaa14 being Lys(iPr) as described herein below, Xaa15 being DPro or DLys(iPr) as described herein below; with the proviso that if Xaa6 is Ala, then Xaa15 is DLys(iPr) as described herein below.

In addition, the t invention provides an efficient synthetic process by which these nds can, if desired, be made in parallel library-format. These B-hairpin peptidomimetics have favorable pharmacological properties and, in addition, show suitable plasma protein binding and appropriate clearance rates. Therefore they can be used as active ingredients in low amounts for all kind of drug formulations, in particular extended release drug formulations.

Many medically significant biological processes are mediated by signal transduction that involves ines and their receptors in general and stromal derived factor 1 (SDF-l/ CXCL12) and its receptor CXCR4 in ular.

CXCR4 and its ligand SDF-l are involved in trafficking of B—cells, hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC). For instance, CXCR4 is expressed on CD34+ cells and has been implicated in the process of CD34+ cell migration and homing (S.M. Watt, S.P. Forde, Vox sanguinis 2008, 94, 18-32). It has also been shown that the CXCR4 receptor plays an important role in the release of stem and progenitor W0 82240 cells from the bone marrow to the peripheral blood (L.M. Pelus, S. Fukuda, Leukemia 2008, 22, 466—473). This activity of CXCR4 could be very important for efficient sis collections of peripheral blood stem cells. Autologous peripheral blood cells provide a rapid and sustained hematopoietic recovery following auto-transplantation after the administration of high-dose herapy or radiotherapy in patients with haematological malignancies and solid tumors (W.C. Liles et al., Blood 2003, 102, 2728—2730).

Recently, it has been demonstrated that SDF-l is locally up-regulated in animal models of injury including focal ischemic stroke, global cerebral ischemia, myocardial infarction and hind limb ischemia as well as being involved in recovery after peripheral ischemia or following injury to the liver, kidney or lung (A.E. Ting, R.W.

Mays, M.R. Frey, W. Van’t Hof, S. Medicetty, R. Deans, Critical Reviews in Oncology/Hematology 2008, 65, 81—93 and ture cited herein; F. Lin, K. Cordes, L.

Li, L. Hood, W.G. , S.J. Shankland et al., J. Am. Soc. Nephrol. 2003, 14, 1188- 1199; CC. Dos Santos, Intensive Care Med. 2008, 34, 619-630). These results t that SDF-l may be a chemoattractant for CXCR4—positive stem cells for tissue and organ repair/regeneration (M.Z. zak, M. Kucia, R. Reca, M. Majka, A. Janowska- Wieczorek, J. Ratajczak, Leukemia 2004, 18, 29—40). Therefore, modulating the SDF-l/CXCR4 axis by CXCR4 inhibitors should result in a significant therapeutic t by using released stem cells to regulate tissue repair.

More recently, it has been shown that disrupting the CXCR4/SDF-1 axis by CXCR4 tors plays a crucial role in differential mobilization of progenitor cells like HPCs, endothelial (EPCs) and stromal progenitor cells (SPCs) from the bone marrow (S.C.

Pitchford, R.C. Furze, C.P. Jones, A.M. Wegner, S.M. Rankin, Cell Stem Cell 2009, 4, 62). In addition, bone —derived CXCR4+ Very Small Embryonic-Like Stem Cells (VSELs) were mobilized in patients with acute myocardial infarction indicating a hypothetical reparatory mechanism (W. Wojakowski, M. Tendra, M. Kucia, E. Zuba- Surma, E. Paczkowska, J. , M. Halasa, M. Krol, M. Kazmierski, P. Buszman, A.

W0 2013l182240 Ochala, J. Ratajczak, B. Machalinski, M.Z. Ratajczak, J. Am. Coll. l. 2009, 53, 1).

These findings may be exploited to provide efficacious stem cell therapy for tissue regeneration.

Mesenchymal stem cells (MSC) are nonhematopoietic progenitor cells having the capability of differentiating into tissues such as bone and cartilage (DJ. Prockop, Science 1997, 276, 71). As a small proportion of MSCs strongly expresses onally active CXCR4, modulation of the CXCR4/SDF-1 axis may mediate specific ion and homing of these cells (R.F. Wynn, C.A. Hart, C. Corradi-Perini, L. O’Neill, C.A.

Evans, J.E. Wraith, LJ. Fairbaim, I. Bellantuono, Blood 2004, 104, 2643).

There is increasing evidence suggesting that ines in general and the SDF- 1/CXCR4 interaction in particular play a l role in enesis. Chemokines induce angiogenesis directly by binding their cognate receptors on endothelial cells or indirectly by promoting inflammatory cell infiltrates, which deliver other angiogenic stimuli. A number of proinflammatory chemokines including interleukin 8 (IL-8), growth—regulated oncogene, stromal cell—derived factor 1 (SDF-l), te actic protein 1(MCP—1), eotaxin 1, and I-309 have been shown to act as direct inducers of angiogenesis (X. Chen, J.A. Beutler, T.G. McCloud, A. Loehfelm, L. Yang, H.F. Dong, O.Y. Chertov, R. Salcedo, J.J. Oppenheim, O.M. Howard. Clin. Cancer Res. 2003, 9(8), 3115-3123; R. Salcedo, JJ. Oppenheim, Microcircu/ation 2003, (3-4), 359- 370).

Recently obtained results show that the CXCR4 receptor is ed in the chemotactic activity of cancer cells, such as breast cancer metastasis or in metastasis of ovarian cancer (A. Muller, B. Homey, H. Soto, N. Ge, D. Catron, M.E. Buchanan, T.

Mc Clanahan, E. Murphey, W. Yuan, S.N. Wagner, J.L. Barrera, A. Mohar, E. egui, A. Zlotnik, Nature 2001, 50, 410; J.M. Hall, K.S. Korach, Molecular inology 2003, 17, 792-803), Non—Hodgin’s Lymphoma (F. Bertolini, C.

Dell’Agnola, P. Manusco, C. Rabascio, A. Burlini, S. Monestiroli, A. Gobbi, G. Pruneri, W0 2013l182240 G. Martinelli, Cancer Research 2002, 62, 112), or lung cancer (T. Kijima, G.

Maulik, P.C. Ma, E.V. Tibaldi, R.E. Turner, B. Rollins, M. Sattler, B.E. Johnson, R. Salgia, Cancer Research 2002, 62, 6304-6311), melanoma, prostate cancer, kidney , neuroblastomia, pancreatic cancer, multiple myeloma, chronic cytic leukemia, hepatocellular carcinoma, colorectal carcinoma, trial cancer and germ cell tumor (H. Tamamura et al., FEBS Letters 2003, 550, 79-83, cited ref.; Z. Wang, Q. Ma, Q. Liu, H.Yu, L. Zhao, S. Shen, J. Yao, British Journal of Cancer 2008, 99, 1695; B. Sung, S. Jhurani, K.S. Ahn, Y. Mastuo, T. Yi, S. Guha, M. Liu, B. Aggarwal, Cancer Res. 2008, 68, 8938; H. Liu, Z. Pan, A. Li, S. Fu, Y. Lei, H. Sun, M. Wu, W. Zhou, Cellular anal Molecular logy, 2008, 5, 373; C. Rubie, O. Kollmar, V.O. Frick, M. Wagner, B.

Brittner, S. Graber, M.K. Schilling, Scandinavian Journal oflmmunology 2008, 68, 635; S. Gelmini, M. Mangoni, F. Castiglioe, C. Beltrami, A. Pieralli, K.L. Andersson, M. ni, G.|. Taddie, M. Serio, C. Orlando, Clin. Exp. Metastasis 2009, 26, 261; DC Gilbert, |. Chandler, A. McIntyre, N.C. Goddard, R. Gabe, R.A. Huddart, J. y, J.

Pathol. 2009, 217, 94). ng the chemotactic activity with a CXCR4 inhibitor should stop the migration of cancer cells and thus metastasis.

CXCR4 has also been implicated in the growth and eration of solid tumors and leukemia/lymphoma. It was shown that activation of the CXCR4 receptor was critical for the growth of both malignant neuronal and glial . Moreover, systemic stration of the CXCR4 antagonist AMD3100 ts growth of intracranial glioblastoma and medulloblastoma xenografts by increasing apoptosis and decreasing the proliferation of tumor cells (J.B. Rubin, A.L Kung, R.S Klein, J.A. Chan, Y. Sun, K.

Schmidt, M.W. Kieran, A.D. Luster, R.A. Segal, Proc Natl Acad Sci U 5 A. 2003, 100(23),13513-13518; S. Barbero, R. Bonavia, A. Bajetto, C. Porcile, P. Pirani, J.L.

Ravetti, G.L. Zona, R. Spaziante, T. Florio, G. Schettini, Cancer Res. 2003, 63(8), 1969- 1974; T. Kijima, G. Maulik, P.C. Ma, E.V. Tibaldi, R.E. Turner, B. Rollins, M. Sattler, B.E. n, R. Salgia. Cancer Res. 2002, 62(21), 6304-6311). CXCR4 inhibitors also showed promising in vitro and in vivo efficacies in breast cancer, small cell lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, malignant melanoma, W0 82240 ovarian cancer, rhabdomyo-sarcoma, prostate cancer as well as chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma and Non-Hodgkin’s lymphoma (J.A. Burger, A. Peled, Leukemia 2009, 23, 43-52 and literature cited herein).

It is well established that ines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Immunostaining for SDF-l (CXCL12) on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples have revealed strong increases in the expression levels of chemokines under inflammatory conditions (F. Grassi, S. Cristino, S. Toneguzzi, A. Piacentini, A. Facchini, G. Lisignoli, J.

Cell Physiol. 2004; 199(2), 244—251). It seems likely that the CXCR4 receptor plays an ant role in inflammatory diseases such as rheumatoid tis, asthma, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, atherosclerosis, or eye diseases such as diabetic retinopathy and age related macular degeneration (K.R.

Shadidi et al., Scandinavian Journal of Immunology 2003, 57, 192-198; J.A. Gonzalo, J.

Immunol. 2000, 165, 499-508; S. Hatse et al., FEBS s 2002, 527, 255—262 and cited references, A.T. Weeraratna, A. Kalehua, |. DeLeon, D. Bertak, G. Maher, M.S.

Wade, A. Lustig, K.G. Becker, W. Wood, D.G. , T.G. Beach, D.D. Taub, Exp. Cell Res. 2007, 313, 450; M. Shimoji, F. Pagan, E.B. Healton, I. Mocchetti, Neurotox. Res. 2009, 16, 318; A. Zernecke, E. Shagdarsuren, C. Weber, Arteriosc/er. Thromb. Vasc.

Biol. 2008, 28, 1897; R. Lima e Silva, J. Shen, S.F. Hackett, S. Kachi, H. Akiyama et al., FASEB 2007, 21, 3219). The mediation of recruitment of immune cells to sites of inflammation should be stopped by a CXCR4 inhibitor.

To date the available therapies for the treatment of HIV infections have been leading to a able ement in symptoms and recovery from disease in infected people. gh the highly active anti-retroviral therapy ) which involves a combination of e transcriptase/ protease-inhibitor has ically improved the clinical treatment of individuals with AIDS or HIV infection, there have still remained several serious ms including multi drug resistance, significant adverse W0 2013!182240 effects and high costs. Particularly desired are IV agents that block the HIV infection at an early stage ofthe infection, such as the viral entry. It has recently been recognized that for efficient entry into target cells, human immunodeficiency viruses require the ine receptors CCR5 and CXCR4 as well as the primary receptor CD4 (N. Levy, Engl. J. Med. 1996, 335, 1528—1530). Accordingly, an agent which could block the CXCR4 chemokine receptors should prevent infections in healthy individuals and slow or halt viral progression in infected patients (J. Cohen, e 1997, 275, 1261-1264).

Among the ent types of CXCR4 inhibitors (M. z, T.N.C. Wells, A.E.|. oot, Receptors and Channels 2001, 7, 417-428; Y. Lavrovsky, Y.A. Ivanenkov, K.V. Balakin, D.A. Medvedewa, P.V. Ivachtchenko, Mini Rev. Med. Chem. 2008, 11, 1075-1087), one emerging class is based on lly occurring cationic peptide analogues derived from Polyphemusin II which have an antiparallel B-sheet structure, and a B-hairpin that is maintained by two disulfide bridges (H. Nakashima, M.

Masuda, T. Murakami, Y. Koyanagi, A. Matsumoto, N. Fujii, N. Yamamoto, crobial Agents and Chemoth. 1992, 36, 1249—1255; H. Tamamura, M. Kuroda, M. Masuda, A. Otaka, S. Funakoshi, H. Nakashima, N. to, M. Waki, A.

Matsumotu, J.M. Lancelin, D. Kohda, S. Tate, F. Inagaki, N. Fujii, Biochim. Biophys.

Acta 1993, 209, 1163; WO 95/10534 A1).

Synthesis of structural analogs and structural studies by nuclear magnetic nce (NMR) spectroscopy have shown that the cationic peptides adopt well defined B- hairpin conformations, due to the constraining effect of one or two disulfide bridges (H. Tamamura, M. Sugioka, Y. Odagaki, A. Omagari, Y. Kahn, S. Oishi, H. Nakashima, N.

Yamamoto, S.C. Peiper, N. Hamanaka, A. Otaka, N. Fujii, Bioorg. Med. Chem. Lett. 2001, 359-362). These results show that the B-hairpin structure plays an ant role in CXCR4 antagonizing activity. onal structural studies have indicated that the antagonizing activity can also be influenced by modulating amphiphilic structure and the pharmacophore (H.

W0 82240 ra, A. Omagari, K. Hiramatsu, K. Gotoh, T. Kanamoto, Y. Xu, E. Kodama, M.

Matsuoka, T. Hattori, N. Yamamoto, H. Nakashima, A. Otaka, N. Fujii, Bioorg. Med.

Chem. Lett. 2001, 11, 1897-1902; H. ra, A. Omagari, K. Hiramatsu, S. Oishi, H.

Habashita, T. Kanamoto, K. Gotoh, N. Yamamoto, H. Nakashima, A. Otaka N. Fujii, Bioorg. Med. Chem. 2002, 10, 1417—1426; H. Tamamura, K. Hiramatsu, K. Miyamoto, A. Omagari, S. Oishi, H. Nakashima, N. Yamamoto, Y. Kuroda, T. Nakagawa, A. Otaki, N. Fujii, Bioorg. Med. Chem. Letters 2002, 12, 923-928).

The compounds cyclo(—Tyr1-HisZ-Xaa3-Cys4-Ser5-Xaa6-DPro7-Xaa8-Arg9—Tyr10-Cys11- TyrlZ-Xaa13-Xaa14-Xaa15—Pr016—), disulfide bond between Cys4 and Cys“, of the invention are cyclic B-hairpin omimetics exhibiting high CXCR4 antagonizing activity, being useful for efficient apheresis collections of mobilized peripheral blood stem cells and/or using these mobilized cells to regulate tissue repair, and/or having anti-cancer activity, nflammatory activity and/or anti-HIV activity.

The cyclic B-hairpin conformation is d by the D-amino acid residue DPro7 and the D—amino acid residue Xaa15. Further stabilization of the hairpin conformation is achieved by the amino acid es Cys at positions 4 and 11, which, taken together, form a disulfide bridge.

Surprisingly we have found that the introduction of the basic amino acid residues Orn(iPr) at position 8, Lys(iPr) at position 14, supported by an optional introduction of DLys(iPr) at position 15 of cyclo(—Tyr1—HisZ-Xaa3-Cys4—SerS-Xaae-DPro7—Xaa8-Arg9- TyrlO-Cysll-Tyrlz-Xaa13-Xaa14—Xaa15-Pr016-), disulfide bond between Cys4 and Cys“, result in pin peptidomimetics which have favorable pharmacological properties.

These properties, combined with suitable plasma protein binding and riate clearance rates form a pharmacological e which allows these compounds to be used as active ingredients in low amounts for all kind of drug formulations, in particular extended release drug formulations.

W0 82240 The B-hairpin peptidomimetics of the present invention are compounds of the general formula cyclo(—Tyrl-HisZ-Xaa3—Cys4-Ser5-Xaa6-DPro7-Xaa8- Argg-Tyrlo-Cysll-Tyrn—Xaa13-Xaa14- XaalS-Pr016-), disulfide bond between Cys4 and Cysll, and pharmaceutically acceptable salts thereof, wherein Xaa3 is Tyr, Tyr(Me), i.e. (2$)amino-(4-methoxyphenyl)propionic acid, or Tyr(CF3), i.e. (2$)amino-(4—trifluoromethoxyphenyl)propionic acid, Xaa6 is Ala or Acc, the latter being l-aminocyclopropane-carboxylic acid, Xaa8 is r), i.e. °’-isopropyl-2,5-diaminopentanoic acid, Xaa13 is Gln or Glu, Xaa14 is Lys(iPr), i.e. (2$)-N“’-isopropyl-2,6-diaminohexanoic acid, Xaa15 is DPro, or DLys(iPr), i.e. (2R)-N‘”—isopropyl-2,6-diaminohexanoic acid, with the proviso that if Xaa6 is Ala, then Xaa15 is DLys(iPr).

In a particular ment of the present invention the compound is cyclo(—Tyrl-HisZ-Tyr3-Cys4-Ser5-Ala6-DP ro7-O rn(iPr)8-Arg9-Tyrlo-Cysll—Tyrlz-G|n13- Lys(iPr)14-DLys(iPr)15—Pr016-), disulfide bond between Cys4 and Cysll, and pharmaceutically acceptable salts thereof.

W0 2013!182240 In r particular embodiment of the present invention the compound is cyclo(—Tyrl-HisZ—Tyrg—Cys4-SerS—Accs—DP ro7-Orn(iPr)8—Arg9-Tyr10—Cysll—Tyr12-G|n13- Lys(iPr) 14-DProl‘r’-Pr016—), disulfide bond n Cys4 and Cys“, and pharmaceutically acceptable salts thereof.

In accordance with the present invention these B-hairpin omimetics can be prepared by a process which comprises (a) coupling an appropriately onalized solid support with an appropriately N-protected derivative of Pro which in the desired end-product is in position 16; removing the N-protecting group from the product thus obtained; coupling the product thus obtained with an appropriately N—protected derivative of that amino acid which in the desired end-product is in position , any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; removing the N-protecting group from the product obtained in step (c); effecting steps ntially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the d oduct are in positions 14 to 1, any functional group(s) which may be present in said ected amino acid derivatives being likewise appropriately protected; if desired, forming a disulfide bridge between the side—chains of the Cys residues at position 4 and position 11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; detaching the product thus obtained from the solid support; cyclizing the product cleaved from the solid support; removing any protecting groups present on functional groups of any s of the chain of amino acid e; and if desired, attaching one or several isopropyl groups W0 2013!182240 (k) if d, converting the product thus obtained into a pharmaceutically able salt or ting a pharmaceutically acceptable, or unacceptable, salt thus ed into the corresponding free compound or into a different, pharmaceutically acceptable, salt.

The B-hairpin peptidomimetics of this invention can be produced, for example, by following a procedure comprising the synthesis of the linear e on resin whereas the isopropyl bearing amino acid residue(s) Orn(iPr), Lys(iPr) or DLys(iPr) will be incorporated as amino acid building block(s) being commercially available or synthesized hand; or a procedure comprising the synthesis of a linear peptide on resin and the derivatisation of the amino group—bearing side chains of amino acid residues protected by acid labile protecting groups suitable to the Fmoc—based solid phase peptide synthesis strategy by coupling isopropyl groups in solution at a very late stage of the synthesis cascade; or following a ure sing a suitable combination of the procedures described before.

The proper choice of the functionalized solid-support (i.e. solid support plus linker molecule) and the site of cyclization play key roles in the synthesis process of the B- n peptidomimetics of the invention.

The functionalized solid support is conveniently derived from polystyrene crosslinked with, preferably 1-5%, divinylbenzene; polystyrene coated with polyethyleneglycol spacers (Tentagel®),' and polyacrylamide resins (see also D. Obrecht, J.-M. Villalgordo, ”Solid— Supported Combinatorial and Parallel Synthesis of Small—Molecular-Weight Compound Libraries”, Tetrahedron Organic Chemistry Series, Vol. 17, on, Elsevier Science, 1998).

The solid support is onalized by means of a linker, Le. a bifunctional spacer molecule which contains on one end an anchoring group for attachment to the solid support and on the other end a selectively cleavable functional group used for the W0 2013!182240 subsequent chemical transformations and cleavage procedures. For the purposes of the present invention two types of linkers are used: Type 1 linkers are designed to release the amide group under acidic conditions (H.

Rink, Tetrahedron Lett. 1987, 28, 3783-3790). Linkers of this kind form amides of the carboxyl group of the amino acids; examples of resins functionalized by such linker structures include 4-[(((2,4-dimethoxy-phenyl)Fmoc-aminomethyl)phenoxyacet amido) ethyl] PS resin, 4-[(((2,4-dimethoxyphenyl)Fmoc-aminomethyl) y-acetamido) aminomethyl] —4-methyl-benzydrylamine PS resin (Rink amide MBHA PS Resin), and 2,4-dimethoxyphenyl)Fmoc-aminomethyl) phenoxyacetamido) aminomethyl] benzhydrylamine PS-resin (Rink amide BHA PS resin). Preferably, the support is derived from polystyrene crosslinked with, most preferably 1—5%, divinylbenzene and functionalized by means of the 4-(((2,4-dimethoxy-phenyl)Fmoc-aminomethyl)phenoxyacetamido) linker.

Type 2 linkers are designed to eventually release the carboxyl group under acidic conditions. Linkers of this kind form acid-labile esters with the carboxyl group of the amino acids, usually acid-labile benzyl, benzhydryl and trityl ; examples of such linker structures include 2-methoxyhydroxymethylphenoxy (SasrinR linker), 4-(2,4—dimethoxyphenyl-hydroxymethyl)-phenoxy (Rink linker), 4—(4-hydroxymethyl- 3-methoxyphenoxy)butyric acid (HMPB linker), trityl and 2-chlorotrityl. Preferably, the support is derived from yrene crosslinked with, most preferably 1—5%, lbenzene and functionalized by means of the 2—chlorotrityl linker.

When carried out as parallel array syntheses the processes of the invention can be advantageously carried out as bed herein below but it will be immediately nt to those skilled in the art how these procedures will have to be modified in case it is desired to synthesize one single compound of the ion.

A number of reaction vessels equal to the total number of compounds to be synthesized by the parallel method are loaded with 25 to 1000 mg, preferably 60 mg, W0 2013!182240 of the appropriate functionalized solid support, preferably 1 to 3% cross-linked polystyrene or Tentagel resin.

The solvent to be used must be e of swelling the resin and includes, but is not limited to, dichloromethane (DCM), ylformamide (DMF), ylpyrrolidone (NMP), dioxane, toluene, tetrahydrofuran (THF), ethanol (EtOH), trifluoroethanol (TFE), isopropylalcohol and the like. Solvent mixtures containing as at least one component a polar solvent (e. g. 20% TFE/DCM, 35% THF/NMP) are beneficial for ensuring high reactivity and solvation of the resin-bound peptide chains (G.B. Fields, C.G. Fields, J. Am. Chem. SOC. 1991, 113, 4202-4207).

With the pment of various linkers that release the C-terminal carboxylic acid group under mild acidic conditions, not affecting acid—labile groups protecting functional groups in the side chain(s), considerable sses have been made in the sis of protected peptide nts. The 2—methoxyhydroxybenzylalcohol- derived linker n® linker, Mergler et al., Tetrahedron Lett. 1988, 29 4005-4008) is cleavable with diluted trifluoroacetic acid (0.5-1% TFA in DCM) and is stable to Fmoc deprotection conditions during the peptide synthesis, Boc/tBu—based additional protecting groups being compatible with this protection scheme. Other linkers which are suitable for the process of the invention include the super acid labile -dimethoxyphenyl-hydroxymethyl)-phenoxy linker (Rink linker, H. Rink, Tetrahedron Lett. 1987, 28, 3787-3790), where the removal of the peptide requires % acetic acid in DCM or 0.2% trifluoroacetic acid in DCM; the 4-(4-hydroxymethyl-3—methoxyphenoxy)butyric acid-derived linker (HMPB-linker, Fl'orsheimer & Riniker, Peptides 1991, 1990 131) which is also cleaved with 1% TFA/DCM in order to yield a peptide fragment ning all acid labile hain protective groups; and, in addition, the 2-chlorotritylchloride linker (Barlos et al., Tetrahedron Lett. 1989, 30, 3943-3946), which allows the peptide detachment using a mixture ofglacial acetic acid/trifluoroethanol/DCM (12:7) for 30 min.

W0 2013l182240 Suitable protecting groups for amino acids and, respectively, for their residues are, for example, - for the amino group (as is t e.g. also in the side-chain of lysine or ornithine) Cbz be nzyloxyca rbonyl Boc tert-butyloxyca rbonyl Fmoc renylmethoxycarbonyl Alloc allyloxycarbonyl Teoc trimethylsilylethoxyca rbonyl ch trichloroethoxycarbonyl Nps o-nitrophenylsulfonyl; Trt triphenymethyl or trityl idee (4,4-dimethyl-2,6-dioxocyclohexylidene)—3- methylbutyl - for the carboxyl group (as is present e. g. also in the side-chain of glutamic acid) by conversion into esters with the alcohol components tBu tert-butyl Bn benzyl Me methyl Ph phenyl Pac phenacyl allyl Tse trimethylsilylethyl Tce trichloroethyl; idee (4,4-dimethyl—2,6-dioxocyclohexylidene)—3- methylbutyl W0 2013!182240 - for the guanidino group (as is present e. g. in the hain of arginine) Pmc 2,2,5,7,8—pentamethylchromansulfonyl Ts tosyl (i. e. p-toluenesulfonyl) Cbz be nzyloxyca rbonyl be pentamethyldihydrobenzofuran-S-sulfonyl - for the hydroxy group (as is t e. g. in the side-chain of serine) tBu tert-butyl Bn benzyl Trt trityl Alloc allyloxycarbonyl - and for the mercapto group (as is present e. g. in the side-chain of cysteine) Acm acetamidomethyl tBu tert-butyl Bn benzyl Trt trityl Mtr 4-methoxytrityl.

The 9-fluorenylmethoxycarbonyl (Fmoc) —protected amino acid derivatives are ably used as the building blocks for the uction of the B-hairpin loop mimetics of the invention. For the deprotection, i. e. cleaving off of the Fmoc group, % piperidine in DMF or 2% DBU/2% piperidine in DMF can be used.

The linkage of isopropyl groups to amino group—bearing side chains of 9-fluorenylmethoxycarbonyl (Fmoc) -protected amino acid derivatives to form W0 2013!182240 isopropylated amino group—bearing side chains of (Fmoc) -protected amino acid derivatives is known in the art. The procedure for introducing an isopropyl group can be accomplished e.g. by reductive alkylation e.g. treatment ofthe amino group ofthe amino group—bearing side chain of an amino acid building block like e.g. Orn with acetone in the presence of a suitable reducing agent like e.g. sodium triacetoxyborohydride. Protecting groups like e.g Boc suitable for ispropylated amino group—bearing side chains of (Fmoc) -protected amino acid derivatives can be introduced by subsequent reaction with di-tert-butyl onate in the presence of a base such as sodium bicarbonate.

The quantity of the reactant, i.e. of the amino acid derivative, is usually 1 to 20 equivalents based on the milliequivalents per gram (meq/g) loading of the functionalized solid support (typically 0.1 to 2.85 meq/g for polystyrene resins) originally weighed into the on tube. Additional lents of reactants can be used, if ed, to drive the reaction to completion in a reasonable time. The preferred workstations (without, however, being limited thereto) are rce's Combi—chem station, Protein Technologies’ Symphony and MultiSyn Tech's—Syro sizer, the latter additionally equipped with a transfer unit and a reservoir box during the process of detachment of the fully ted linear peptide from the solid support. All synthesizers are able to provide a controlled environment, for example, reactions can be accomplished at temperatures different from room temperature as well as under inert gas here, if desired.

Amide bond formation requires the activation of the oc-carboxyl group for the ion step. When this activation is being carried out by means of the commonly used carbodiimides such as dicyclohexylcarbodiimide (DCC, Sheehan & Hess, J. Am.

Chem. Soc. 1955, 77, 1067-1068) or diisopropylcarbodiimide (DIC, Sarantakis et al m. Biophys. Res. Commun. 1976, 73, 336-342), the resulting dicyclohexylurea and, tively, ropylurea is insoluble and, respectively, soluble in the solvents generally used. In a variation of the carbodiimide method 1-hydroxybenzotriazole (HOBt, Konig & Geiger, Chem. Ber. 1970, 103, 788-798) is W0 82240 included as an additive to the coupling mixture. HOBt prevents dehydration, suppresses racemization of the activated amino acids and acts as a catalyst to improve the sluggish coupling reactions. Certain phosphonium reagents have been used as direct coupling reagents, such as benzotriazol-l-yl—oxy-tris—(dimethyl- amino)—phosphonium uorophosphate (BOP, Castro et al., Tetrahedron Lett. 1975, 14, 222; Synthesis 1976, 751-752), or benzotriazol-l-yl- oxy-tris—pyrrolidino-phosphonium hexaflurophoshate (Py—BOP, Coste et al., Tetrahedron Lett. 1990, 31, 205-208), or 2-(1H-benzotriazol1—yl-) 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), or hexafluorophosphate (HBTU, Knorr et al., Tetrahedron Lett. 1989, 30, 930); these phosphonium reagents are also suitable for in situ formation of HOBt esters with the protected amino acid derivatives. More recently diphenoxyphosphoryl azide (DPPA) or O-(7-aza-benzotriazol—l-yl)-N,N,N’,N'—tetramethyluronium tetrafluoroborate (TATU) or O—(7-aza—benzotriazol-l—yl)—N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU)/7-azahydroxy benzotriazole (HOAt, o et al., Tetrahedron Lett. 1994, , 2279-2281) or -(6-Ch|oro-1H-benzotriazol-l-yl-)-N,N,N’,N’-1,1,3,3-tetramethyl- uronium tetrafluoroborate (TCTU), or hexafluorophosphate (HCTU, Marder, Shivo and Albericio: HCTU and TCTU: New Coupling Reagents: pment and Industrial Applications, Poster Presentation, Gordon Conference February 2002) have also been used as coupling ts as well as 1,1,3,3-bis(tetramethylene)chlorouronium hexafluoro-phosphate (PyClU) ally for coupling N-methylated amino acids (J.

Coste, E. Frérot, P. Jouin, B. Castro, Tetrahedron Lett. 1991, 32, 1967) or pentafluorophenyl diphenyl—phosphinate (S. Chen, J. Xu, Tetrahedron Lett. 1991, 32, 6711) Due to the fact that near-quantitative coupling ons are essential, it is desirable to have experimental evidence for completion of the reactions. The ninhydrin test (Kaiser et al., Anal. Biochemistry 1970, 34, 595), where a positive colorimetric response to an aliquot of bound peptide indicates qualitatively the presence of the y amine, can easily and quickly be performed after each coupling step.

W0 2013!182240 Fmoc try allows the ophotometric detection of the Fmoc phore when it is released with the base (Meienhofer et al., Int. J. Peptide Protein Res. 1979, 13, 35—42).

The bound intermediate within each on vessel is washed free of excess of retained ts, of solvents, and of by-products by repetitive exposure to pure solvent(s) by one of the two following methods: 1) The on vessels are filled with solvent (preferably 5 mL), agitated for 5 to 300 minutes, preferably 15 minutes, and drained to expel the solvent; 2) The reaction vessels are filled with solvent (preferably 5 mL) and drained into a receiving vessel such as a test tube or vial.

Both of the above washing procedures are repeated up to about 50 times (preferably about 10 times), monitoring the efficiency of reagent, solvent, and by-product removal by methods such as TLC, GC, or inspection ofthe washings.

The above described procedure of reacting the resin-bound compound with ts within the reaction tubes followed by removal of excess reagents, by-products, and solvents is repeated with each successive transformation until the final resin-bound fully protected linear peptide has been obtained.

Before this fully protected linear e is detached from the solid support, a disulfide bridge between Cys4 and Cys11 can be formed.

For the formation of a disulfide bridge preferably a solution of 10 lents of iodine solution is applied in DMF or in a mixture of CHZCIZ/MeOH for 1.5 h which is repeated for another 3 h with a fresh iodine solution after filtering of the iodine solution, or in a mixture of DMSO and acetic acid solution, buffered with 5% NaHC03 to pH 5—6 for 4h, or in water after adjusting to pH 8 with ammonium hydroxide solution by stirring for 24 h, or in a solution of NMP and tri—n-butylphosphine (preferably 50 eq.).

W0 2013!182240 Alternatively, the formation of the disulfide bridge between Cys4 and Cys11 can be carried out subsequent to the work—up method 2), as described herein below, by stirring the crude fully deprotected and cyclized peptide for 24h in water containing DMSO up to 15% by volume, buffered with 5% NaHC03 to pH 5—6, or buffered with ammonium acetate to pH 7—8, or adjusted with ammonium ide to pH 8.

Following evaporation to dryness cyclo(-Tyr1-HisZ—Xaa3-Cys4-Ser5-Xaa6-DPro7- Xaa8—ArgQ-Tyrlo—Cysll—Tyrlz-Xaal3—Xaal4—Xaa15—Pr016—), disulfide bond between Cys4 and Cysll, is obtained as end-product.

Detachment of the fully protected linear peptide from the solid support is achieved by exposing the loaded resin with a solution of the reagent used for cleavage (preferably 3 to 5 mL). Temperature control, agitation, and reaction monitoring are implemented as described above. Via a transfer-unit the reaction vessels are connected with a reservoir box containing reservoir tubes to ently collect the cleaved product solutions. The resins remaining in the on vessels are then washed 2 to 5 times as above with 3 to 5 mL of an riate solvent to extract (wash out) as much of the detached products as possible. The t solutions thus obtained are combined, taking care to avoid cross-mixing. The individual solutions/extracts are then manipulated as needed to isolate the final compounds.

Typical manipulations include, but are not limited to, evaporation, tration, /liquid extraction, acidification, basification, neutralization or additional reactions in solution.

The solutions containing fully protected linear peptide tives which have been cleaved off from the solid support and lized with a base, are evaporated.

Cyclization is then effected in solution using solvents such as DCM, DMF, dioxane, THF and the like. Various coupling reagents which were mentioned earlier can be used for the cyclization. The duration of the cyclization is about 6—48h, ably about 16h.

The progress of the reaction is ed, e. g. by RP-HPLC se Phase High Performance Liquid Chromatography). Then the solvent is removed by evaporation, the fully protected cyclic e derivative is dissolved in a solvent which is not W0 2013!182240 2012/060766 miscible with water, such as DCM, and the solution is extracted with water or a mixture of water-miscible solvents, in order to remove any excess of the coupling reagent.

Finally, the fully protected e derivative is treated with 95% TFA, 2.5% H20, 2.5% TIS or another combination of gers for effecting the cleavage of protecting groups. The cleavage reaction time is commonly 30 minutes to 12 h, preferably about 2.5 h.

Alternatively, the detachment and complete deprotection of the fully protected peptide from the solid support can be achieved ly in glass vessels.

After full ection, for example, the ing methods can be used for further work—up: l) The volatiles are evaporated to dryness and the crude peptide is dissolved in % AcOH in water and extracted with isopropyl ether or other solvents which are suitable therefor. The aqueous layer is collected and evaporated to dryness, and the fully deprotected cyclic peptide, cyclo(—Tyr1—HisZ-Xaa3-Cys4—SerS-Xaae-DPro7—Xaa8-Arg9- TyrlO—Cysll-Tyrlz-Xaa13-Xaa14—Xaa15-Pr016-), disulfide bond between Cys4 and Cysll,is obtained as final t; 2) The deprotection mixture is concentrated under vacuum. Following precipitation of the fully deprotected peptide in diethylether at preferably 0 °C the solid is washed up to about 10 times, preferably 3 times, dried, and the fully deprotected cyclic peptide, cyclo(-Tyr1-HisZ-Xaa3-Cys4-Ser5-Xaa6-DPro7-Xaa8-Arg9- TyrlO-Cysll-Tyrlz-Xaa13-Xaa14—Xaa15-Pr016-), disulfide bond n Cys4 and Cysll, is obtained as final product, if a disulfide bond between Cys4 and Cys11 has been formed on solid support as described herein above.

W0 2013!182240 2012/060766 If the above mentioned orthogonal protecting group strategy for introducing one or more isopropyl groups in on has been followed, then all amino groups of amino acid residues formerly protected by acid labile protecting groups have been liberated at this stage of the synthesis cascade. Thus, it is possible, if desired, to couple an isopropyl group. This ng can be accomplished by applying e.g. a reductive alkylation using acetone in the presence of a suitable reducing agent like e.g. sodium cyano borhydride. Thus, for example, the peptide is dissolved in MeOH (4.4 mM) containing acetic acid (0.2 M). After adding an excess of acetone (780 eq) the reaction mixture is completed with a solution of sodium cyano borhydride in MeOH (0.6 M; 1.3 eq per isopropyl group desired to be introduced) and vigorously shaken at room ature. Following completion of the conversion monitored by LC-MS, water is added and the solvents are evaporated.

As mentioned earlier, it is thereafter possible, if desired, to convert the fully ected cyclic product thus ed into a pharmaceutically acceptable salt or to convert a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound, or into a different, pharmaceutically acceptable, salt. Any of these ions can be carried out by methods well known in the art.

The B-hairpin peptidomimetics of the invention can be used in a wide range of applications in order to prevent HIV infections in healthy individuals and slow or halt viral progression in infected ts, or where cancer is mediated or resulting from the CXCR4 or activity, or where immunological diseases are mediated or resulting from CXCR4 receptor activity; or these B-hairpin peptidomimetics can be used to treat immunosuppression, or they can be used during apheresis collections of peripheral blood stem cells and/or as agents to induce zation of stem cells to regulate tissue repair.

W0 2013!182240 The B-hairpin peptidomimetics of the invention may be stered per se or may be applied as an appropriate ation together with rs, diluents or excipients well known in the art.

When used to treat or prevent HIV infections or cancer such as breast cancer, brain cancer, prostate cancer, heptatocellular carcinoma, colorectal cancer, lung cancer, kidney cancer, neuroblastoma, ovarian cancer, endometrial cancer, germ cell tumor, eye cancer, multiple myeloma, pancreatic cancer, gastric cancer, rhabdomyo-sarcoma, melanoma, chronic lyphomphocytic leukemia, acute myelogenous leukemia, acute blastic leukemia, multiple myeloma and Non-Hodgkin’s lymphoma; metastasis, angiogenesis, and haematopoetic tissues; or inflammatory ers such as asthma, allergic rhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis, eosinophilic pneumonias, delayed-type hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing itis, systemic sclerosis, Sjogren‘s me, systemic laxis or hypersensitivity responses, drug allergies, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, atherosclerosis, myasthenia , juvenile onset diabetes, glomerulonephritis, autoimmune throiditis, graft ion, including allograft rejection or graft-versus-host disease, inflammatory bowel diseases and inflammatory dermatoses; or to treat eye diseases like ma, diabethic pathy and age related macular degeneration; or to treat focal ischemic stroke, global cerebral ischemia, myocardial infarction, hind limb ischemia or peripheral ischemia; or to treat injury of the liver, kidney or lung; or to treat immunosuppression, including immunosuppression induced by chemotherapy, ion therapy or graft/transplantation rejection, the B-hairpin peptidomimetics of the invention can be administered , as mixtures of several B-hairpin peptidomimetics, in combination with other anti-HIV agents, or antimicrobial agents or anti-cancer agents or anti-inflammatory agents, or in combination with other W0 2013!182240 pharmaceutically active . The B-hairpin peptidomimetics of the invention can be administered per se or as pharmaceutical compositions.

Pharmaceutical compositions comprising B—hairpin peptidomimetics of the invention may be manufactured by means of conventional mixing, dissolving, granulating, coated tablet-making, levigating, emulsifying, encapsulating, entrapping or |yophi|izing processes. Pharmaceutical compositions may be formulated in tional manner using one or more logically acceptable carriers, diluents, ents or auxilliaries which facilitate processing of the active B-hairpin peptidomimetics into preparations which can be used pharmaceutically. Proper formulation s upon the method of administration chosen.

For topical administration the B-hairpin omimetics of the invention may be formulated as solutions, gels, ointments, creams, suspensions, powders etc. as are well-known in the art.

Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal ion, as well as those designed for transdermal, transmucosal, oral or ary administration.

For injections, the B—hairpin peptidomimetics of the invention may be formulated in adequate ons, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s on, or physiological saline buffer. The solutions may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, the B-hairpin peptidomimetics of the invention may be in powder form for combination with a suitable vehicle, e.g., sterile pyrogen—free water, before use.

For transmucosal administration, penetrants appropriate to the r to be permeated are used in the formulation as known in the art.

W0 2013!182240 For oral administration, the compounds can be readily formulated by combining the active B—hairpin peptidomimetics of the invention with pharmaceutically acceptable carriers well known in the art. Such carriers enable the B-hairpin peptidomimetics of the invention to be formulated as tablets, pills, dragees, es, liquids, gels, syrups, slurries, suspensions, powders etc., for oral ingestion by a patient to be treated. For oral formulations such as, for e, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato , gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, egrating agents may be added, such as cross—linked polyvinylpyrrolidones, agar, or alginic acid or a salt thereof, such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard ques.

For oral liquid preparations such as, for example, sions, elixirs and solutions, suitable carriers, ents or ts include water, glycols, oils, alcohols, etc. In addition, flavoring agents, preservatives, coloring agents and the like may be added.

For buccal stration, the composition may take the form of tablets, lozenges, etc. formulated as usual.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories together with appropriate suppository bases such as cocoa butter or other glycerides.

In addition to the formulations bed above, the B-hairpin peptidomimetics of the invention may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For the manufacture of such depot W0 82240 preparations the B-hairpin peptidomimetics of the invention may be formulated with suitable polymeric or hydrophobic materials (e.g. as an on in an acceptable oil) or ion exchange resins, or as sparingly e salts.

In addition, other pharmaceutical delivery systems may be employed such as liposomes and emulsions well known in the art. Certain organic ts such as dimethylsulfoxide may also be employed. Additionally, the B-hairpin peptidomimetics of the ion may be delivered using a sustained—release system, such as semipermeable matrices of solid polymers containing the therapeutic agent (e.g. for coated stents). Various sustained-release materials have been ished and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of the therapeutic agent, additional strategies for protein stabilization may be employed.

As the pin peptidomimetics ofthe ion contain charged residues, they may be included in any of the above described formulations as such or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free forms.

Particluarly suitable pharmaceutically acceptable salts include salts with carboxylic, onic, sulfonic and sulfamic acids, e.g. acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, maleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, lic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or -sulfonic acid, 2-hydroxyethanesulfonic acid, ethane—1,2-disulfonic acid, benzenesulfonic acid, 2—naphthalenesulfonic acid, 1,5- naphthalenedisulfonic acid, 2-, 3- or 4-methyl-benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N—methy|—, N- ethyl- or yl-sulfamic acid, and other organic protonic acids, such as ascorbic W0 2013!182240 acid. Suitable inorganic acids are for e hydrohalic acids, such as hydrochloric acid, sulfuric acid and phosphoric acid.

The B-hairpin peptidomimetics of the invention, or compositions f, will generally be used in an amount effective to achieve the intended purpose. It is to be understood that the amount used will depend on a particular ation.

For topical stration to treat or prevent HIV infections a therapeutically effective dose can be determined using, for example, the in vitro assays provided in the examples. The ent may be applied while the HIV infection is visible, or even when it is not visible. An ordinary skilled expert will be able to determine therapeutically effective amounts to treat topical HIV infections without undue experimentation.

For systemic administration, a eutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating B—hairpin peptidomimetic concentration range that includes the IC50 as determined in the cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be determined from in vivo data, e.g. animal , using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

Dosage amounts for applications as anti-HIV agents may be adjusted individually to provide plasma levels of the B-hairpin peptidomimetics of the ion which are sufficient to maintain the therapeutic effect. Therapeutically effective serum levels may be achieved by administering multiple doses each day.

In cases of local administration or selective , the effective local concentration of the B-hairpin peptidomimetics of the invention may not be related to plasma W0 2013!182240 concentration. One having the ordinary skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

The amount of B-hairpin peptidomimetics stered will, of course, be dependent on the subject being d, on the subject’s weight, the ty of the affliction, the manner of administration and the judgement ofthe prescribing ian.

The anti-HIV therapy may be repeated intermittently while infections are detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs, such as for example other anti-HIV agents or anti- cancer agents, or other antimicrobial agents.

Normally, a therapeutically effective dose ofthe B-hairpin peptidomimetics described herein will provide therapeutic benefit without causing ntial toxicity.

Toxicity of the B-hairpin peptidomimetics of the invention can be determined by standard ceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio n toxic and therapeutic effect is the therapeutic index. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in humans. The dosage of the B—hairpin omimetics of the ion lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within the range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dose can be chosen by the individual physician in view of the patient's condition (see, e.g. Fingl et al. 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.1).

The present invention may also include nds, which are identical to the compounds of the general formula cyclo(—Tyr1—HisZ—Xaa3—Cys4-Ser5—Xaa6—DPro7—Xaa8— W0 2013!182240 Arg9-Tyrlo-Cysll-Tyrlz-Xaa13-Xaa14-Xaa15-Prol6-), disulfide bond n Cys4 and Cysll, except that one or more atoms are replaced by an atom having an atomic mass number or mass different from the atomic mass number or mass usually found in nature, e.g. compounds enriched in 2H (D), 3H, 11C, 14C, 129| etc. These isotopic analogs and their pharmaceutical salts and ations are considered useful agents in the therapy and/or stic, for example, but not limited to, where a fine-tuning of in vivo half—life time could lead to an optimized dosage regimen.

The following Examples illustrate the present invention but are not to be construed as limiting its scope in any way.

W0 82240 Examples 1. Peptide Synthesis Coupling of the first protected amino acid residue to the resin 1 g (1.4 mMol) 2—chlorotritylchloride resin (1.4 mMol/g; 100 — 200 mesh, copoly(styrene-1% DVB) polymer matrix; Barlos et al. Tetrahedron Lett. 1989, 30, 946) was filled into a dried flask. The resin was suspended in CHZCIZ (5 mL) and allowed to swell at room temperature under constant shaking for 30 min. A solution of 0.98 mMol (0.7 eq) of the first suitably protected amino acid residue (see below) in CHZCIZ (5 mL) mixed with 960 pl (4 eq) of ropylethylamine (DIEA) was added.

After shaking the reaction mixture for 4 h at 25 °C, the resin was filtered off and washed successively with CHZCIZ (1x), DMF (1x) and CHZCIZ (1x). A solution of CHZClz/MeOH/DIEA (17/2/1, 10 mL) was added to the resin and the sion was shaken for 30 min. After filtration the resin was washed in the following order with CH2C|2(1x), DMF (1x), CH2C|2(1x), MeOH (1x), CHZCIZ (1x), MeOH (1x), CHZCIZ (2x), EtZO (2x) and dried under vacuum for 6 hours.

Loading was typically 0.6-0.7 mMol/g.

The following preloaded resins was prepared: Fmoc—Pro-O-Z—chlorotrityl resin.

The synthesis was carried out employing a Syro-peptide sizer (MultiSynTech) using 24-96 reaction vessels. In each vessel 0.04 mMol of the above resin was placed and the resin was swollen in CHZCIZ and DMF for 15 min, respectively. The following on cycles were programmed and carried out: W0 2013!182240 Step Reagent Time 1 DMF, wash 2x1 min 2 20% piperidine/DMF 1x5 min, 1x15 min 3 DMF, wash 5x1 min 4 5 eq Fmoc amino MF +5 eq /DMF, 10 eq DIEA/DMF 1x60 min DMF, wash 3x1 min Step 4 was repeated once.

Unless indicated otherwise, the final coupling of an amino acid was followed by Fmoc deprotection by applying steps 1-3 ofthe above described reaction cycle.

Amino acid building block syntheses Synthesis of Fmoc-Orn(iPr,Boc)-OH The synthesis of (25)-N°‘-fluorenylmethoxylcarbonyl-N‘”,Nw-tert-butyloxycarbonyl- isopropyl-2,5—diaminopentanoic acid was lished by suspending 15.2 g Fmoc- Orn-OH*HC| in 150 mLTHF (0.26 M) ed by adding 375 mL acetone (132 eq) and .6 g sodium triacetoxyborohydride (2.5 eq). The reaction mixture was stirred for 2 h and uent to completion of the reaction (monitored by LC—MS) 120 mL of sat.

Na2C03-solution and 10.2 g Boczo (1.2 eq) were added. After stirring overnight sat.

Na2C03-solution and BocZO were added again twice in portions according to the remaining starting material. Following completion of the Boc-introduction hexane was added twice, separated, and the aqueous layer was acidified with 5 N HCIaq (pH = 1) and extracted thrice with ethyl acetate thereafter. Finally, the combined organic layers were dried with NaZSO4 and evaporated to obtain the product as white foam.

W0 2013i182240 The amino acid building blocks Fmoc-DLys(iPr,Boc)-OH and Fmoc-Lys(iPr,Boc)-OH could be synthesized accordingly; the latter is also commercially available. ation and work up of backbone cyclized peptides Cleavage of the fully ted peptide nt After completion of the synthesis, the resin (0.04 mMol) was suspended in 1 mL (0.13 mMol, 3.4 eq) of 1% TFA in CHZCIZ (v/v) for 3 minutes, filtered, and the filtrate was neutralized with 1 mL (0.58 mMol, 14.6 eq) of 10% DIEA in CHZCIZ (v/v). This procedure was repeated three times to ensure completion of the cleavage. The filtrate was evaporated to dryness and a sample ofthe t was fully deprotected by using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) to be analyzed by reverse phase-HPLC (C18 column) and ESI-MS to monitor the efficiency of the linear peptide synthesis.

Cyclization of the fully protected linear peptide The fully protected linear peptide (0.04 mMol) was dissolved in DMF (4 uMol/mL).

Then 30.4 mg (0.08 mMol, 2 eq) of HATU, 10.9 mg (0.08 mMol, 2 eq) of HOAt and 28 pl (0.16 mMol, 4 eq) DIEA were added, and the mixture was vortexed at 25 °C for 16 hours and subsequently concentrated under high vacuum. The residue was partitioned between CHZCIZ and H20/CH3CN (90/10: v/v). The CHZCIZ phase was ated to yield the fully ted cyclic peptide.

W0 2013!182240 Full deprotection of the cyclic peptide The fully protected cyclic peptide obtained was ved in 3 mL of the cleavage mixture containing 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% ethanedithiole (EDT). The e was allowed to stand at 25 °C for 2.5 hours and thereafter concentrated under vacuum. After precipitation of the cyclic fully ected peptide in diethylether (EtZO) at 0 °C the solid was washed twice with EtZO and dried.

Formation of disu/fide ,B-strand linkage and purification Afterfull deprotection, the crude peptide was dissolved in 0.1 M ammonium acetate buffer (1 mg/ 1 mL, pH = 7—8). DMSO (up to 5% by volume) was added and the solution was shaken overnight. ing evaporation the residue was ed by preparative reverse phase HPLC.

After lyophilisation the products were obtained as white powders and analysed by the following analytical method: Analytical HPLC retention times (RT, in minutes) were determined using a Ascentis Express C18 column, 50 x 3.0 mm, (cod. 53811-U- Supelco) with the following solvents A (H20 + 0.1% TFA) and B (CH3CN + 0.1% TFA) and the gradient: 0-0.05 min: 97% A, 3% B; 3.4 min: 33% A 67% B; 3.41-3.65 min: 3% A, 97% B; 3.66—3.7 min: 97% A, 3% B. Flow rate = 1.3 mL/min; UV_Vis = 220 nm.

Example 1: Starting resin was Fmoc-Pro-O-Z-chlorotrityl resin, which was prepared as described above. To that resin Pr), y at position 15, was grafted. The linear peptide was sized on solid support according to the procedure described above in the following sequence: Resin—Prole-DLys(iPr)15-Lys(iPr)14-Gln13-Tyrlz—Cysll-Tyrlo-Argg—Orn(iPr)8-DPro7-Ala6-Ser5- Cys4-Tyr3-Hisz-Tyr1. Following a final Fmoc deprotection as described above, the W0 2013!182240 peptide was cleaved from the resin, cyclized, deprotected and, after formation ofthe disulfide B-strand linkage as described above, purified as indicated above.

The HPLC-retention time (minutes) was determined using the analytical method as described above (UV-purity [after preparative HPLC]: 95%; RT: 1.54; [M+3H]/3 = 709.9).

Example 2: Starting resin was Fmoc—Pro-O—2—chlorotrityl resin, which was prepared as described above. To that resin DPro, finally at position 15, was grafted. The linear e was synthesized on solid support according to the procedure described above in the following sequence: Resin-Prole-DProls-LysfiPr)14-G|n13-Tyrlz-Cysfl-Tyrlo-Argg-Orn(iPr)8-DPro7-Acc6-Ser5- Cys4—Tyr3-Hisz—Tyr1. Following a final Fmoc deprotection as bed above, the peptide was cleaved from the resin, cyclized, deprotected and, after formation ofthe disulfide B-strand e as described above, purified as ted above.

The HPLC-retention time (minutes) was determined using the ical method as described above rity [after preparative HPLC]: 95%; RT: 1.58; [M+3H]/3 = 689.3).

W0 2013!182240 2. Biological methods 2.1. Preparation of the peptides Lyophilized peptides were d on a Microbalance (Mettler MT5) and dissolved in DMSO to a final concentration of 10 mM. Stock solutions were kept at +4 °C, light protected. The biological assays were carried out under assay conditions having less than 1% DMSO unlike ted otherwise. 2.2. Cell culture Namalwa cells (CXCR4 ly sing non-adherent cells, ATCC 32) were cultured in RPM|1640 plus 10% FBS, and pen/strept. HELA cells were maintained in RPMI164O plus 10% FBS, pen/strept and 2 mM L-glutamine. Cos-7 cells were grown in DMEM medium with 4500 mg/mL glucose supplemented with 10% FCS, pen/strept and 2 mM L—glutamine. All cell lines were grown at 37 °C at 5% C02. Cell media, media supplements, ffer, HEPES, antibiotic/antimycotic, pen/strept, non essential amino acid, L—glutamine, B—mercaptoethanol and sera were purchased from Gibco (Pailsey, UK). All fine chemicals were supplied by Merck (Darmstadt, Germany). 2.3. Chemotactic Assay (Cell migration assay) The Chemotactic response of Namalwa cells (ATCC CRL-1432) to a gradient of stromal cell-derived factor 1d (SDF-l) was measured using a modified Boyden chamber axis system (ChemoTx; Neuroprobe). In this system, the upper chamber of each well is separated from the lower chamber containing the chemoattractant SDF-l by a polycarbonate membrane (5pm pore size). A circular area of that membrane in the region that covers each lower well is enclosed by a hydrophobic mask to retain W0 2013!182240 the cell suspension within this area. The system was prepared by loading the bottom wells with aliquots of 30 pL of chemotaxis medium (RPMI 1640 without Phenol red + 0.5% BSA) comprising either appropriate serial dilutions of peptides or no peptide at all in combination with SDF-l (0.9 nM) or without the chemoattractant. The membrane was placed over the bottom wells, and aliquots of 50 uL of a suspension of Namalwa cells (3.6 x 106 cells/mL) in chemotaxis , premixed with chemotaxis medium comprising either appropriate serial dilutions of peptides or no peptide at all, was delivered onto each of the hydrophobically limited regions of the upper surface ofthe membrane. The cells were allowed to migrate into the bottom chamber for 5 h at 37 °C in 5% C02. After this period, the membrane was d and its topside was carefully wiped and washed with PBS to eliminate non-migrated cells. Migrated cells were transferred using a ”funnel” adaptor to a receiving 96-well plate and the cell number was determined by using the CyO.uantTM NF cell proliferation assay (lnvitrogen) based on the measurement of cellular DNA content via scent dye binding. Following the manufacturer’s directions, 50 uL of CyQuantTM dye t/HBSS buffer (1/500 [v/v]) were added to each well of the above ned receiving 96—well plate. After incubation for 0.5 h at room temperature the plate was sealed and the fluorescence ity of each sample was measured by using a Wallac 1420 VICTOR2TM plate reader (PerkinElmer) with excitation at 485 nm and emission ion at 535 nm. Finally, the data were normalized by using the controls and |C50- values were determined using GraphPad M (GraphPad) by fitting a logarithmic curve to the averaged datapoints. 2.4. Cytotoxicity assay The cytotoxicity of the es to HELA cells (Acc57) and COS-7 cells (CRL—1651) was determined using the MTT reduction assay (T. Mossman, J. Immunol. Meth. 1983, 65, 55-63; M.V. Berridge, A.S. Tan, Arch. Biochem. Biophys. 1993, 303, 474-482). y, the method was as follows: 4000 HELA cells/well and 3400 COS-7 cells/well were W0 2013!182240 seeded and grown in 96-well microtiter plates for 24 h at 37 °C at 5% C02. Thereafter, time zero (T2) was determined by MTT ion (see below). The supernatant ofthe remaining wells was ded, and fresh medium and compounds in serial dilutions (12.5, 25 and 50 (1M, triplicates; 0 uM, blank) were pipetted into the wells. After incubation of the cells for 48 h at 37 °C at 5% C02 the supernatant was discarded again and 100 pL MTT reagent (0.5 mg/mL in RPMI1640 and DMEM, respectively)/well was added. Following incubation at 37 °C for 2—4 h the media were aspirated and the cells were spiked (100 pL isopropanol/well). The absorbance ofthe solubilized formazan was measured at 595 nm (OD595peptide). For each tration averages were calculated from triplicates. The tage of growth was calculated as follows: (OD595peptide-OD595Tz)/(OD595blank-OD595Tz) x 100%. The Glso (Growth Inhibition) concentrations were calculated for each peptide by using a trend line function for the trations (50, 25, 12.5 and 0 uM), the corresponding percentages and the value 50, (=TREND (C502C0,%50:%0,50). 2.5. Hemolysis The peptides were tested for their hemolytic activity against human red blood cells (hRBC). Fresh hRBC were washed four times with phosphate buffered saline (PBS) and centrifuged for 10 min at 3000 x g. Compounds (100 uM) were incubated with 20% hRBC (v/v) for 1 h at 37 °C and shaking at 300 rpm. The final erythrocyte concentration was approximately 0.9 x 109 cells/mL. A value of 0% and 100% cell lysis, respectively, was determined by incubation of hRBC in the ce of PBS ning 0.001% acetic acid and 2.5% Triton X-100 in H20, respectively. The samples were centrifuged, the supernatants were 8-fold diluted in PBS buffer and the optical densities (OD) were measured at 540 nm. The 100% lyses value (OD540H20) gave an OD540 of approximately 0.5-1.0.

Percent hemolysis was calculated as follows: (OD540peptide/OD540HZO) x 100%.

W0 2013!182240 2.6. Plasma stability The stability ofthe peptides in human and mouse plasma was determined by applying the following : 346.5 p well of freshly thawed human plasma (Basler BIutspende-dienst) and mouse plasma (Harlan Sera-Lab, UK), tively, were spiked with 3.5 uL/well of compound dissolved in DMSO/HZO (90/10 [v/v], 1 mM, cate) and incubated at 37° C. At t = 0, 15, 30, 60, 120, 240 and 1440 min aliquots of 50 (LL were transferred to filtration plate wells containing 150 l of 2% formic acid in acetonitrile. Following shaking for 2 min the occurred suspensions were filtrated by vacuum. 100 uL of each filtrate were transferred to a propylene microtiter plate and dried under N2. The residual solids were reconstituted by adding 100 l of water/acetonitrile, 95/5 (v/v) + 0.2% formic acid and analyzed by LC/MS as follows: Column: , XBridge C18, mobile phases: (A) water + 0.1% formic acid and (B) acetonitriIe/water, 95/5 (v/v) + 0.1% formic acid, gradient: 5%-100% (B) in 1.8 minutes, electrospray ionization, MRM detection (triple quadrupole). The peak areas were determined and triplicate values are averaged. The stability is expressed in t of the initial value at t = 0. (tx/tO x 100). By using the TREND function of EXCEL (Microsoft Office 2003) T1/2 were determined. 2.7. Plasma Protein Binding 495 uL aliquots of human plasma (Basler Blutspendedienst) as well as 495 uL aliquots of PBS were placed in individual deepwells of a polypropylene plate (Greiner) and spiked each with 5 (1L of 1 mM solutions of peptides in 90% DMSO. After shaking the plate for 2 min at 600 rpm 150 uL aliquots of the plasma/peptide mixtures were transferred in triplicates to the polypropylene filter plate (10 kDa, Millipore) whereas 150 uL ts of the PBS/peptide mixtures were transferred either to the individual wells of the filter plate (filtered controls) or directly into the individual wells of the W0 2013!182240 receiving plate (Greiner) (non-filtered controls). The plate sandwich consisting of filter and ing plate was incubated for 1 h at 37 °C and subsequently centrifuged at 3220 g for 2h at 15 °C. The filtrates in the receiving plate were analysed by LC/MS as follows: Column: Waters, XBridge C18, mobile phases: (A) water + 0.1% formic acid and (B) acetonitrile/water, 95/5 (v/v) + 0.1% formic acid, gradient: 5%-100% (B) in 1.8 minutes, electrospray ionization, MRM detection (triple quadrupole). The peak areas were determined and cate values are averaged. The g is expressed in t of the filtered and non-filtered controls by 100—(100x Tlh/Tctr)- Finally the average of these values is calculated.

The results of the experiments described under 2.3 — 2.7 are indicated in the Tables 1, 2, 3 and 4 herein below. 2.8. Pharmacokinetic study (PK) For the compounds of Ex. 1 and Ex. 2 pharmacokinetic studies after intravenous (iv) administration were performed. grams (i 20%) male CD-1 mice obtained from Charles River Laboratories Deutschland GmbH were used. The e, phosphate buffered , was added to give a final concentration of0.5 mg/mL of the compound. The volume was 2 mL/kg and the compound was injected to give a final intravenous dose of 1 mg/kg.

Approximately 300-400 pL of blood was removed under light isoflurane anesthesia by cardiac puncture at ermined time intervals (5, 15, 30 min and 1, 2, 3, 4, hours) and added to heparinized tubes. Plasma was d from pelleted cells upon centrifugation and frozen at —80 °C prior to HPLC-MS analysis.

Preparation of plasma calibration- and plasma study-samples Aliquots of 50 (LL each of mouse plasma of untreated s (”blank” mouse plasma) were spiked with known s of the compounds Ex. 1 and Ex. 2 in order to obtain plasma calibration samples for each compound in the range 1 — 4000 ng/mL.

Aliquots of 50 pL each of mouse plasma from treated animals were used as plasma study samples.

Extraction ma calibration- and plasma study-samples All plasma samples were spiked with an riate internal standard and extracted with acetonitrile containing 2% formic acid. Supernatants were evaporated to dryness under nitrogen and the remaining solids reconstituted in water + 0.2% formic cetonitrile 95/5 (v/v).

LC—MS/MS-analysis Extracts were then analyzed by reverse—phase chromatography (Acquity HSS C18 SB, 100 x 2.1 mm, 1.8 pm column, ), using the following conditions: mobile phases: (A) water + 0.1% formic acid/acetonitrile 95/5 (v/v), (B) aceto-nitrile/water + 0.1% formic acid 95/5 (v/v), gradient: 1% (B) 001 min, 40% (B) 01-25. The detection and quantification was performed by mass spectrometry, with electrospray interface in positive mode and selective fragmentation of analytes (4000 Q Trap mass spectrometer, AB Sciex). cokinetic evaluation PK parameters were calculated by WinNonLinTM software version 5.3 (Pharsight— A CertaraTM Company, Moutain View, CA 94041 USA) using a one-compartmental model is. PK parameters were determined by least-square fitting of the model to the experimental data.

The results of the experiments bed in 2.8 are indicated in Tables 5a and 5b herein below. 2.9. Drug loading calculations via maintainance dose rate (rate of infusion) The drug load for an implant comprising a peptide of the ion was calculated following the basic principles in pharmacokinetics (see also J. Gabrielsson, D. Weiner, ”Pharmacokinetics and Pharmaco-dynamics Data is: Concepts and ations”, 4th edition, Swedish Pharmaceutical Press, Stockholm, Sweden, 2006) whereby the maintainance dose rate (rate of infusion, Rm) can be defined as the rate at which a drug is to be administered to reach a steady state of a certain dose in the plasma. The maintainance dose rate can be expressed using the correlation Rm [g/(h*kg)] = CLiV [L/(h*kg)] x Css’eff [g/L], wherein CLiV is the clearance (i.v. — admin.) and Cssfiff the effective concentration of the drug in the plasma at steady state considering an efficacy margin A: Cssfiff [g/L] = A x (IC50/fu) x MW [(Mol/L)*(g/Mol)].

Therefore, the total amount of a drug loaded into an implant providing for a constant effective tration of that drug in the plasma for a certain period of time in a subject of a certain body weight can be calculated by ng the following correlation: Drugload [g/subject] = Rm [g/(h*kg)] x on [h] x BW [kg/subject].

The results of the calculations bed in 2.9 are indicated in Table 6 herein below and based on the data given in Tables 1, 4 and 5b. Further pre-conditions are an efficacy margin of A = 3, a study duration of 672 h (28 days) and a body weight of a human suject of 70 kg. The glomerular filtration rate (GFR) which mainly influences the clearance of the peptide is highly dependent on the species. In general, the GFR of humans is averaged to be 107 mL/(h*kg) compared to the GFR of mouse being 840 mL/(h*kg). Therefore, the CLiV-mouse values indicated in Table 5b were allometrically scaled by 107 kg)/840 mL/(h*kg) = 0.127 before employed in the above described correlations. 2012/060766 Table 1 m leo [nM] i SD, CXCR4 receptor 0.17 i 0.1 Table 2 _Hemolysis Hela Cells Cos—7 Cells at GI50 [HM] GI50 [MM] 100 PM Table 3 a Plasma stability human pl. human pl. mouse pl. mouse pl.

Tl/z [min] cpd left at Tl/z [min] cpd left at 1440 min 1440 min [%] [%] 1440 1440 n 1440 1440 Table 4 E Plasma protein binding [%] Fraction unbound, fu 2012/060766 Table 5a Ex. 1 Ex. 2 i.v. route i.v. route Dose: 1 mg/kg Dose: 1 mg/kg < LoQ below Limit of Quantification Molecular Weight CLiV, human (salt free), (allometric MW [g/Mol] scaled) 1 212753 E 206640

Claims (15)

1. A compound of the general formula cyclo(-Tyr 1-His 2-Xaa 3-Cys 4-Ser 5-Xaa 6-DPro 7-Xaa 8- 5 Arg 9-Tyr 10 -Cys 11 -Tyr 12 -Xaa 13 -Xaa 14 -Xaa 15 -Pro 16 -), disulfide bond between Cys4 and Cys11 , or a pharmaceutically able salt thereof, wherein Xaa 3 is Tyr; Tyr(Me); or Tyr(CF Tyr(Me) is (2S)amino-(4-methoxyphenyl)propionic acid, 10 Tyr(CF 3) is (2S)amino-(4-trifluoromethoxyphenyl)propionic acid, Xaa 6 is Ala; or Acc Acc is 1-aminocyclopropane-carboxylic acid, Xaa 8 is Orn(iPr), r) is (2S)-Nω-isopropyl-2,5-diaminopentanoic acid, 15 Xaa 13 is Gln; or Glu, Xaa 14 is Lys(iPr), Lys(iPr) is (2S)-Nω-isopropyl-2,6-diaminohexanoic acid, Xaa 15 is DLys(iPr), DLys(iPr) is (2R)-Nω-isopropyl-2,6-diaminohexanoic acid, 20 with the proviso that if Xaa6 is Ala, then Xaa15 is DLys(iPr).

2. cyclo(-Tyr1-His 2-Tyr 3-Cys 4-Ser 5-Ala 6-DPro 7-Orn(iPr) 8-Arg 9-Tyr 10 -Cys 11 -Tyr 12 -Gln 13 - Lys(iPr) 14 -DLys(iPr) 15 -Pro 16 -), disulfide bond n Cys4 and Cys11 , or a ceutically acceptable salt thereof.

3. A compound according to claim 1 or claim 2 for use as a therapeutically active substance.

4. A compound according to claim 3, for use as a therapeutically active substance 30 having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity.

5. A ceutical composition comprising a compound according to any one of claims 1 to 3 and a pharmaceutically inert carrier.

6. The pharmaceutical composition according to claim 5, in a form suitable for 5 oral, topical, transdermal, injection, buccal or transmucosal administration.

7. The pharmaceutical composition according to claim 6, in the form of a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, nt, syrup, slurry, suspension, powder or suppository.

8. The use of a compound according to any one of claims 1 to 3 for the cture of a medicament having CXCR4 antagonizing, anti-cancer ty and/or anti-inflammatory activity and/or stem cell mobilizing activity. 15

9. Use ing to claim 8, wherein the ment is for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV ed patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of eral blood 20 stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.

10. A process for the manufacture of compounds according to any one of claims 1-3 which process comprises (a) ng an appropriately functionalized solid support with an appropriately 25 N-protected derivative of Pro which in the desired end-product is in position (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 15, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately ted; (d) removing the N-protecting group from the t obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using 5 appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n-2 to 1, any onal group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if desired, forming a disulfide bridge between the side-chains of the Cys 10 residues at P4 and P11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on onal groups of any members 15 of the chain of amino acid e; and (j) if desired, attaching one or several isopropyl groups (k) if desired, ting the product thus obtained into a pharmaceutically acceptable salt or ting a ceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound or into a different, 20 pharmaceutically acceptable, salt.

11. The compound according to claim 1, substantially as herein described with reference to any one of the Examples thereof. 25

12. The compound according to claim 2, substantially as herein bed with reference to any one of the Examples thereof.

13. The pharmaceutical composition according to claim 5, wherein the compound is substantially as herein described with reference to any one of the Examples 30 thereof.

14. The use according to claim 8, wherein the compound is substantially as herein described with reference to any one of the Examples thereof.

15. The process according to claim 10, substantially as herein described with 5 nce to any one of the Examples thereof.