HK1191021B - Beta-hairpin peptidomimetics as cxc4 antagonists - Google Patents

Description

Beta-hairpin peptidomimetics as CXC4 antagonists

The present invention provides β -hairpin peptidomimetics having CXCR4 antagonistic activity and covered by the general disclosure in WO2004/096840a1, but not specifically disclosed therein.

The β -hairpin peptidomimetics of the present invention are disulfide-bonded in Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof, Xaa³Is Ala, Tyr or Tyr (Me), Xaa as described below⁷Is as follows^DTyr、^DTyr (Me) or^DPro，Xaa⁸Is Dab or Orn (iPr), Xaa as described below¹³Is Gln or Glu, and Xaa¹⁴Is Lys (iPr) as described below.

Furthermore, the present invention provides efficient synthetic methods by which these compounds can be produced in a parallel library format, if desired. These β -hairpin peptidomimetics have advantageous pharmacological properties and, in addition, exhibit a suitable plasma protein binding and a suitable clearance rate. They can therefore be used in all kinds of pharmaceutical preparations, in particular extended-release pharmaceutical preparations, in very small amounts as active ingredients.

Many medically significant biological processes are mediated by signal transduction involving chemokines and their receptors in general and mesenchymal derived factor 1(SDF-1/CXCL12) and its receptor CXCR4 in particular.

CXCR4 and its ligand SDF-1 are involved in the trafficking of B cells, Hematopoietic Stem Cells (HSCs) and Hematopoietic Progenitor Cells (HPCs). For example, CXCR4 is in CD34⁺Is expressed on cells and has been implicated in CD34⁺Cell migration and homing processes (s.m. watt, s.p. forde, Vox sanguinis2008,94, 18-32). CXCR4 receptors have also been shown to play an important role in the release of stem and progenitor cells from bone marrow into peripheral blood (l.m.pelus, s.fukuda, leukamia 2008,22,466-. This activity of CXCR4 may be very important for efficient apheresis collection of peripheral blood stem cells. Autologous peripheral blood cells provide rapid and durable hematopoietic recovery after autograft following administration of large doses of chemotherapy or radiotherapy in patients with hematologic malignancies and solid tumors (w.c. liles et al, blood2003,102, 2728-2730).

Recently, SDF-1 has been shown to be locally upregulated in animal models of injury, including ischemic attack, global ischemia, myocardial infarction and hind limb ischemia, as well as involved in recovery following peripheral ischemia or liver, kidney or lung injury (A.E.Ting, R.W.Mays, M.R.Frey, W.Van't Hof, S.Medicetty, R.Deans, Critical reviews in Oncology/Hematology2008,65,81-93and littureacid tissue herein; F.Lin, K.Cordes, L.Li, L.Home, W.G.Coup, S.J.Shankland et al, J.Med.Soc.Nephrol.2003, 14, 1188-9; C.C.Dos, Int Sanve, 630. Car 619, 619). These results indicate that SDF-1 may be a chemotactic agent for CXCR4 positive stem cells for tissue and organ repair/regeneration (m.z. ratajczak, m.kucia, r.reca, m.majka, a.janowska-Wieczorek, j.ratajczak, leukamia 2004,18, 29-40). Thus, modulation of the SDF-1/CXCR4 axis by CXCR4 inhibitors should produce significant therapeutic benefits by modulating tissue repair through the use of released stem cells.

Recently, disruption of the CXCR4/SDF-1 axis by CXCR4 inhibitors has been shown to play a key role in differentially mobilizing progenitor cells such as HPCs, Endothelial Progenitor Cells (EPC) and mesenchymal progenitor cells (SPC) from bone marrow (s.c. pitchford, r.c. furze, c.p. jones, a.m. wegner, s.m. rankin, Cell Stem Cell2009,4, 62). In addition, bone marrow derived CXCR4⁺Very small embryonic-like stem cells (VSELs) mobilized in patients with acute myocardial infarction suggesting a putative compensatory mechanism (w.wojakowski, m.tendra, m.kucia, e.zuba-Surma, e.paczkowska, j.cisek, m.halasa, m.krol, m.kazmierski, p.buszman, a.ochala, j.ratajczak, b.machalini, m.z.ratajczak, j.am.col.cardiol.2009, 53.

1) The results of these studies can be used to provide effective stem cell therapy for tissue regeneration.

Mesenchymal Stem Cells (MSCs) are non-hematopoietic progenitor cells with the ability to differentiate into various tissues such as bone and cartilage (d.j. prockop, sciences 1997,276, 71). Since a small proportion of MSCs strongly express functionally active CXCR4, modulation of the CXCR4/SDF-1 axis can mediate specific migration and homing of these cells (r.f. wynn, c.a. hart, c.coradi-Perini, l.o' Neill, c.a. evans, j.e. wraith, l.j.fairbaim, i.bellatuo, blood2004,104, 2643).

There is increasing evidence that chemokines in general and SDF-1/CXCR4 in particular interactions play a key role in angiogenesis. Chemokines induce angiogenesis either directly by binding to cognate receptors on endothelial cells or indirectly by promoting infiltration of inflammatory cells that deliver other angiogenic stimuli. A number of pro-inflammatory chemokines, including interleukin 8(IL-8), growth-regulating oncogenes, stromal cell-derived factor 1(SDF-1), monocyte chemotactic protein 1(MCP-1), eosinophil chemokine 1, and 1-309, 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, J.Oppenheim, Microcirculation2003, (3-4), 359-) 370).

Recently obtained results show that CXCR4 receptor is involved in cancer cell chemotactic activities such as breast cancer metastasis or in metastasis of the following cancers: ovarian cancer (a.muller, b.homey, h.soto, n.ge, d.catron, m.e.buchan, t.mcclinahan, e.murphey, w.yuan, s.n.wagner, j.l.barrera, a.mohar, e.verastegui, a.zlotnik, Nature2001,50,410; j.m. hall, k.s.korach, Molecular Endocrinology2003,17,792-803), non-hodgkin lymphoma (f.bertolini, c.dell' Agnola, p.manusco, c.rabasco, a.burlini, s.monestri, a.gobbi, g.prune, g.martineli, Cancer Research2002,62,3106-3112), or lung Cancer (t.kijima, g.lautik, p.c.c.cancer, e.v.tibaldii, r.e.turner, b.rollins, m.satttt, b.e.johnson, r.salgia, Cancer Research, 2002,62,6304, melanoma, pre-cell lymphoma, kidney Cancer, renal carcinoma cell lymphoma, leukemia, melanoma, leukemia, melanoma-550-incorporated herein by reference, human lung Cancer introduction, reference documents, e.h, leuca, r.t.e.h, r.h, melanoma 6304, melanoma, leukemia, melanoma, leukemia, melanoma, leukemia, melanoma; z.wang, q.ma, q.liu, h.yu, l.zhao, s.shen, j.yao, british journal of Cancer2008,99,1695; b.sung, s.jhurani, k.s.ahn, y.mastuo, t.yi, s.guha, m.liu, b.aggarwal, Cancer res.2008,68,8938; liu, z.pan, a.li, s.fu, y.lei, h.sun, m.wu, w.zhou, Cellular and Molecular Immunology,2008,5, 373; rubie, o.kollmar, v.o.frick, m.wagner, b.britner, S.Schilling, Scandinavian journal of Immunology2008,68,635; s.gelminii, m.mangoni, f.castiglioe, c.beltrami, a.pierelli, k.l.andersson, m.fambri, g.i.taddie, m.serio, c.orlando, clin.exp.metastasis2009,26,261; gilbert, i.chandler, a.mclntyre, n.c.goddard, r.gabe, r.a.huddart, j.shipley, j.pathol.2009,217, 94). Blocking chemotactic activity with CXCR4 inhibitors should stop cancer cell migration and thus metastasis.

CXCR4 has also been implicated in the growth and proliferation of solid tumors and leukemias/lymphomas. Activation of the CXCR4 receptor has been shown to be critical for the growth of both malignant neuronal and glial tumors. In addition, systemic administration of the CXCR4 antagonist AMD3100 inhibited the growth of intracranial glioblastoma and medulloblastoma xenografts by increasing apoptosis and decreasing tumor cell proliferation (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 USA 2003,100(23), 13513-. CXCR4 inhibitors also show promising in vitro and in vivo efficacy in the following cancers: breast cancer, small cell lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, malignant melanoma, ovarian cancer, rhabdomyosarcoma, prostate cancer, as well as chronic lymphocytic Leukemia, acute myelocytic Leukemia, acute lymphocytic Leukemia, multiple myeloma, and non-hodgkin's lymphoma (j.a. burger, a.peled, leukamia 2009,23,43-52 and references cited therein).

Chemokines are well established to be involved in many inflammatory diseases and some of them show key roles in the regulation of osteoclast development. Immunostaining against SDF-1(CXCL12) on synovial and bone tissue biopsies from Rheumatoid Arthritis (RA) and Osteoarthritis (OA) samples has revealed strongly increased expression levels of chemokines under inflammatory conditions (F.Grassi, S.Cristino, S.Toneguzzi, A.Picentini, A.Facchini, G.Lisignoli, J.Physiol.2004; 199(2), 244-. CXCR4 receptors appear to play an important role in inflammatory diseases such as rheumatoid arthritis, asthma, multiple sclerosis, Alzheimer's disease, Parkinson's disease, atherosclerosis or eye diseases such as diabetic nephropathy and age-related macular degeneration (K.R. Shadii et al, Scandinavian journal of Immunology2003,57, 192-198; J.A. Gonzalo, J.Immunol.2000,165, 499-508; S.Hatse et al, FEBS letters2002,527,255-262 and cited references, A.T. Weerarata, A.Kalehua, I.Deleon, D.Bertak, G.Maher, M.S.Wade, A.Lustig, K.G.Becker, W.Wooya, D.G.Walker, T.G.Waach, D.D.D.313, Exub.313, Shikuni, Hakuni, Hakumi, Hazi et al, Haydi, E, Haydi, E, Haydi, E, Haydi, E. Mediated immune cell recruitment to the site of inflammation should be terminated by CXCR4 inhibitors.

Available therapies to date for the treatment of HIV infection have resulted in significant improvement of symptoms and recovery from disease in infected people. Although highly active anti-retroviral therapy (HAART) involving reverse transcriptase/protease inhibitor combinations has greatly improved the clinical treatment of AIDS or HIV infected individuals, several serious problems remain, including multiple drug resistance, significant side effects and high cost. There is a particular need for anti-HIV agents that block HIV infection at an early stage of infection, such as at the time of viral entry. It has recently been recognized that the chemokine receptors CCR5 and CXCR4, as well as the major receptor CD4, are required by human immunodeficiency viruses for efficient entry into target cells (n.levy, engl.j.med.1996,335, 1528-1530). Thus, substances that can block CXCR4 chemokine receptors should prevent infection in healthy individuals and slow or stop viral progression in infected patients (j.cohen, sciences 1997,275, 1261-1264).

Among the different classes of CXCR4 inhibitors (M.Schwarz, T.N.C.wells, A.E.I.Proudfoot, Receptors and Channels2001,7,417-428; Y.Lavrovsky, Y.A.Ivannekov, K.V.Balakin, D.A.Medvewa, P.V.Ivacchtchenko, Mini.Rev.Med.Chem.2008, 11,1075-1087), a newly emerging class is based on naturally occurring cationic peptide analogs derived from limulus peptide II, which have antiparallel β -sheet structure and β -sheet maintained by two disulfide bonds (H.Nakashima, M.Masuda, T.Murakami, Y.Koyanagi, A.Sumatmoto, N.Yajii, N.Fumaentomoto, M.124, M.Masshima, T.1992, Y.Koyayaki, A.S.S.A.32, Massachima.K.K.K.K.J. Tokawa, Massach.K.K.K.K.K.K.K.K.K.K.H.K. 1087, Massach.K.K.3, Massach.K.K.K.K.M. 19, Massach.K. K. 19, Massach.K. K. 19, Massach.K. K. 19, S.19, Massach.K. K. A. K. 19, S. A. A.

The synthesis of structural analogues and structural studies by means of Nuclear Magnetic Resonance (NMR) spectroscopy have shown that these cationic peptides adopt a well-defined β -hairpin conformation due to the constraining effect of 1 or 2 disulfide bonds (h.tamamamura, m.sugioka, y.odagaki, a.omagari, y.kahn, s.oishi, h.nakashima, n.yamamoto, s.c.peiper, n.hamaraka, a.otaka, n.fujii, bioorg.med.chem.lett.2001, 359-362). These results show that the β -hairpin structure plays an important role in CXCR4 antagonistic activity.

Other structural studies have shown that this antagonistic activity may also be affected by modulation of the amphipathic structure and pharmacophore (H.Tamamura, A.Omagarai, 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-containing 1902, H.Tamamura, A.Omagarai, K.Hiramatsu, S.Oishi, H.Habashita, T.Kanamoto, K.Gotoh, N.Yamamoto, H.Nakashima, A.Otakan.Fujii, Bioorg.2002, Yamamoto.10, K.10-containing K.Gotoh, N.Yamato, H.10-Karmatto, H.S.10-K.K.K.1897, H.Okamokamoakoma, H.S.S.S.S.O.S.12, Nakayakoia, H.S.12, Nakayakoia, H.S.S.S.S.S.S.S.S.S.S.S.S.2002, H.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.A.A.S.7, H.S.S.S..

Disulfide bond at Cys⁴And Cys¹¹The inventive compound cyclo (-Tyr)¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) is a cyclic β -hairpin peptidomimetic which exhibits high CXCR4 antagonistic activity, is useful for efficient apheresis collection of mobilized peripheral blood stem cells and/or use of these mobilized cells to modulate tissue repair, and/or has anti-cancer, anti-inflammatory, and/or anti-HIV activity.

The cyclic β -hairpin conformation is formed by the D-amino acid residue Xaa⁷And D-amino acid residues^DPro¹⁵And (4) causing. Further stabilization of this hairpin conformation is achieved by the amino acid residue Cys at positions 4 and 11, which together form a disulfide bond.

Surprisingly, we have found that disulfide bonding at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) to position 14 of the amino acid sequence Lys (iPr) andthese properties, combined with a suitable plasma protein binding and a suitable clearance, lead to a pharmacological profile which allows these compounds to be used in low amounts as active ingredients in all kinds of pharmaceutical preparations, in particular extended-release pharmaceutical preparations.

The β -hairpin peptidomimetics of the present invention are disulfide-bonded in Cys⁴And Cys¹¹Cyclo (-Tyr) of the general formula¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶- (I) and pharmaceutically acceptable salts thereof,

wherein

Xaa³Is Ala, Tyr or Tyr (Me), the latter being (2S) -2-amino- (4-methoxyphenyl) -3-propionic acid,

Xaa⁷is that^DTyr、^DTyr (Me), i.e. (2R) -2-amino- (4-methoxyphenyl) -3-propanoic acid, or^DPro，

Xaa⁸Is Dab, i.e. (2S) -2, 4-diaminobutyric acid, or Orn (iPr), i.e. (2S) -N^ω-isopropyl-2, 5-diaminopentanoic acid,

Xaa¹³is a group of Gln or Glu,

Xaa¹⁴is Lys (iPr), i.e. (2S) -N^ω-isopropyl-2, 6-diaminohexanoic acid.

In a particular embodiment of the invention, the β -hairpin peptidomimetics are compounds of the general formula I and pharmaceutically acceptable salts thereof, wherein Xaa¹³Is Gln.

In another specific embodiment of the present invention, the β -hairpin peptidomimetic is a compound of formula I and pharmaceutically acceptable salts thereof, wherein Xaa is³Is Tyr or Tyr (Me), Xaa⁷Is that^DPro，Xaa⁸Is Orn (iPr) and Xaa¹³Is Gln.

In a preferred embodiment of the invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Ala³-Cys⁴-Ser⁵-Ala⁶-^DTyr⁷-Dab⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Tyr³-Cys⁴-Ser⁵-Ala⁶-^DPro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Tyr(Me)³-Cys⁴-Ser⁵-Ala⁶-^DPro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Ala³-Cys⁴-Ser⁵-Ala⁶-^DTyr(Me)⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

In another preferred embodiment of the invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Tyr³-Cys⁴-Ser⁵-Ala⁶-^DTyr⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

In still another preferred embodiment of the present invention, the compound is disulfide-bonded at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Tyr(Me)³-Cys⁴-Ser⁵-Ala⁶-^DTyr(Me)⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^DPro¹⁵-Pro¹⁶-) and pharmaceutically acceptable salts thereof.

According to the invention, these β -hairpin peptidomimetics can be prepared by a method comprising

(a) Coupling an appropriately functionalized solid support with an appropriate N-protected derivative of Pro at position 16 in the desired end product;

(b) removing the N-protecting group from the product thus obtained;

(c) with position 15 in the desired end product^DCoupling of an appropriate N-protected derivative of Pro to the product thus obtained;

(d) removing the N protecting group from the product obtained in step (c);

(e) performing steps substantially corresponding to steps (c) and (d) using an appropriate N-protected amino acid derivative wherein positions 14 to 1 are in the desired end product, any functional groups that may be present in said N-protected amino acid derivative being likewise suitably protected;

(f) if desired, forming a disulfide bond between the side chains of the Cys residues at positions 4 and 11; or alternatively, the aforementioned bond is formed after step (i), as described below;

(g) detaching the product thus obtained from the solid support;

(h) circularizing the product cleaved from the solid support;

(i) removing any protecting groups present on the functional groups of any modules of the chain of amino acid residues; and

(j) if desired, one or more isopropyl groups are attached

(k) Removing any protecting groups present on the functional groups of any module of the amino acid residue, if necessary; and

(l) If desired, the product thus obtained is converted into a pharmaceutically acceptable salt or the pharmaceutically acceptable or unacceptable salt thus obtained is converted into the corresponding free compound or into a different pharmaceutically acceptable salt.

The β -hairpin peptidomimetics of the invention can be generated, for example, by following a method comprising synthesizing a linear peptide on a resin, wherein the amino acid residues orn (ipr) or lys (ipr) carrying an isopropyl group are incorporated as commercially available or previously synthesized amino acid building blocks; or by following a method comprising synthesizing a linear peptide on a resin by employing an orthogonal protecting group strategy, wherein for example all amino acid residue side chains carrying an amino group which are not considered to be modified should be protected by ivDde or the like, such that amino acid residue side chains carrying an amino group which are protected by an acid labile protecting group suitable for Fmoc based solid phase peptide synthesis strategies can be derivatized in solution by coupling isopropyl groups at the very late stage of the synthesis cascade; or following a method comprising a suitable combination of the foregoing methods.

The proper selection of a functionalized solid support (i.e., a solid support plus a linker molecule) and cyclization sites play a key role in the synthesis of the β -hairpin peptidomimetics of the invention.

The functionalized solid support is conveniently derived from polystyrene crosslinked with preferably 1-5% divinylbenzene; polystyrene coated with polyethylene spacer(ii) a And polyacrylamide resins (D.Obrecht, J. -M.Villalgord, "Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries", Tetrahedron Organic Chemistry Series, Vol.17, Pergamon, Elsevier Science, 1998).

The solid support is functionalized by a linker, i.e., a bifunctional spacer molecule containing an anchor group on one end for attachment to the solid support and a selectively cleavable functional group on the other end for subsequent chemical conversion and cleavage processes. 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-. This type of linker forms an amide of the amino acid carboxyl group; examples of resins functionalized by such linker structures include 4- [ ((((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] PS resin, 4- [ (((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] -4-methyl-benzhydrylamine PS resin (Rink amide MBHA PS resin), and 4- [ ((((2, 4-dimethoxy-phenyl) Fmoc-aminomethyl) phenoxyacetamido) aminomethyl ] benzhydrylamine PS-resin (Rink amide BHA PS resin). Preferably, the support is derived from polystyrene cross-linked with most preferably 1-5% divinylbenzene and functionalized by means of a 4- (((2, 4-dimethoxyphenyl) Fmoc-aminomethyl) phenoxyacetamido) linker.

Type 2 linkers are designed to eventually release a carboxyl group under acidic conditions. This type of linker forms acid-labile esters with the carboxyl group of amino acids, typically acid-labile benzyl, benzhydryl and trityl esters; examples of such linker structures include 2-methoxy-4-hydroxymethylphenoxy (Sasrin)^RLinker), 4- (2, 4-dimethoxy-hydroxymethyl)) -phenoxy (Rink linker), 4- (4- (hydroxymethyl) -3-methoxyphenoxy) butanoic acid (HMPB linker), trityl and 2-chlorotrityl. Preferably, the support is derived from polystyrene cross-linked with most preferably 1-5% divinylbenzene and functionalized via a 2-chlorotrityl linker.

When implemented as a parallel array synthesis, the methods of the invention may advantageously be implemented as described below, but it will be immediately clear to the skilled person how these methods will have to be adapted in case one needs to synthesize one single compound of the invention.

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

The solvent to be used must be capable of swelling the resin and includes, but is not limited to, Dichloromethane (DCM), Dimethylformamide (DMF), N-methylpyrrolidinone (NMP), dioxane, toluene, Tetrahydrofuran (THF), ethanol (EtOH), Trifluoroethanol (TFE), isopropanol, and the like. Solvent mixtures containing a polar solvent as at least one component (e.g., 20% TFE/DCM, 35% THF/NMP) are useful for ensuring high reactivity and solvation of resin-bound peptide chains (G.B. fields, C.G. fields, J.Am.chem.Soc.1991,113, 4202-4207).

With the development of various linkers that release the C-terminal carboxylic acid group under mildly acidic conditions without affecting the functional groups in the side chain protected by acid labile groups, great progress has been made in the synthesis of protected peptide fragments. Linker derived from 2-methoxy-4-hydroxybenzyl alcohol(s) ((s))The linker, Mergler et al, Tetrahedron Lett.1988,294005-4008) is cleavable with dilute trifluoroacetic acid (0.5-1% TFA in DCM) and is stable to Fmoc deprotection conditions during peptide synthesis, with additional protecting groups based on Boc/tBu being compatible with this protection scheme. Other suitable linkers for the method of the invention includeAcid-labile 4- (2, 4-dimethoxy-hydroxymethyl) -phenoxy linker (Rink linker, h.rink, tetrahedron lett.1987,28, 3787-; 4- (4-hydroxymethyl-3-methoxyphenol) butanoic acid derived linker (HMPB-linker, Florsheimer and Riniker, Peptides1991,1990131) which was also cleaved with 1% TFA/DCM to generate peptide fragments containing all acid labile side chain protecting groups; and, in addition, a 2-chlorotrityl chloride linker (Barlos et al, Tetrahedron Lett.1989,30, 3943-.

Suitable protecting groups for amino acids and residues thereof are, for example, respectively

For amino groups (if present, for example also in the side chain of lysine or ornithine)

Cbz benzyloxycarbonyl

Boc tert-butyloxycarbonyl group

Fmoc 9-fluorenylmethoxycarbonyl

Alloc allyloxycarbonyl radical

Teoc trimethylsilylethoxycarbonyl group

Tcc Trichloroethoxycarbonyl

Nps o-nitrophenylsulfonyl;

trt trityl (triphenymethyl) or trityl (trityl)

ivDde (4, 4-dimethyl-2, 6-dioxan-1-ylidene) -3-methylbutyl

For carboxyl groups converted into esters with the alcohol component (if present, for example also in the side chain of glutamic acid)

tBu tert-butyl

Bn benzyl group

Me methyl group

Ph phenyl

Pac phenacyl

Allyl radical

Tse Trimethylsilylethyl group

Tce trichloroethyl;

ivDde (4, 4-dimethyl-2, 6-oxacyclohex-1-ylidene) -3-methylbutyl

For guanidino (if present, e.g. in the side chain of arginine)

Pmc2,2,5,7, 8-pentamethylbenzodihydropyran-6-sulfonyl group

Ts tosyl (i.e., p-toluenesulfonyl)

Cbz benzyloxycarbonyl

Pbf pentamethyl dihydrobenzofuran-5-sulfonyl

For hydroxy (if present, for example in the side chain of serine)

tBu tert-butyl

Bn benzyl group

Trt trityl radical

Alloc allyloxycarbonyl radical

And for thiol groups (if present, for example in the side chain of cysteine)

Acm acetylaminomethyl

tBu tert-butyl

Bn benzyl group

Trt trityl radical

Mtr 4-methoxytrityl.

9-fluorenylmethoxycarbonyl (Fmoc) -protected amino acid derivatives are preferably used as building blocks for the construction of the beta-hairpin loop mimetics of the invention. For deprotection, i.e. cleavage of the Fmoc group, 20% piperidine in DMF or 2% DBU/2% piperidine in DMF may be used.

It is known in the art that isopropyl groups are attached to the amino group bearing side chain of a 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acid derivative to form an isopropylated amino group bearing side chain of the (Fmoc) protected amino acid derivative. The procedure for introducing an isopropyl group can be accomplished, for example, by reductive alkylation, e.g., by treating the amino group of the amino acid-carrying side chain of an amino acid building block (e.g., Orn) with acetone in the presence of a suitable reducing agent, such as sodium triacetoxyborohydride. The protecting group for the amino-bearing side chain, e.g. Boc, suitable for isopropylation of an (Fmoc) -protected amino acid derivative can be introduced by subsequent reaction with di-tert-butyl dicarbonate in the presence of a base such as sodium bicarbonate.

The amount of reactant (i.e., amino acid derivative) is typically 1 to 20 equivalents (typically, 0.1 to 2.85meq/g for polystyrene resin) based on the milliequivalents per gram (meq/g) loading of functionalized solid support initially weighed into the reaction tube. Additional equivalents of reactants may be used, if desired, to drive the reaction to completion in a reasonable time. Preferred workstations, however, are, without limitation, the Labsource's Combi-chem workstation, the Protein Technologies ' Symphony and the MultiSynTech's-Syro synthesizer, which are additionally equipped with a transfer unit and a magazine during the process of detaching the fully protected linear peptide from the solid support. All synthesizers are capable of providing a controlled environment, for example, the reaction may be carried out at a temperature different from room temperature and, if desired, under an inert atmosphere.

When this activation is carried out with the aid of commonly used carbodiimides such as dicyclohexylcarbodiimide (DCC, Sheehan and Hess, J.Am.chem.Soc.1955,77,1067-1068) or diisopropylcarbodiimide (DIC, Saratakis et al biochem.Biophys.Res.Commun.1976,73,336-342), the produced dicyclohexylurea and the correspondingly produced diisopropylurea are insoluble and respectively soluble in the commonly used solvents, in a variant of the carbodiimide process, 1-hydroxybenzotriazole (HOBt,and Geiger, chem. Ber.1970,103,788-798) as additives to the coupling mixture. HOBt prevents dehydration, inhibits racemization of activated amino acids and acts as a catalyst to improve slow coupling reactions. Certain phosphonium reagents have been used as direct coupling reagents, such as benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP, Castro et al, Tetrahedron Lett.1975,14, 1219-; Synthesis1976,751-752), or benzotriazol-1-yl-oxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate (Py-BOP, Coste et al, Tetrahedron Lett.1990,31,205-208), or 2- (1H-benzotriazol-1-yl) 1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), or hexafluorophosphate (HBTU, Knorr et al, Tetrahedron Lett.1989,30, 1927-1930); these phosphonium reagents are also suitable for the in situ formation of HOBt esters with protected amino acid derivatives. More recently, diphenyl acid esters (DPPA) or O- (7-aza-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (TATU) or O- (7-aza-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU)/7-aza-1-hydroxybenzotriazole (HOAt, Carpino et al, tetrahedron Lett.1994,35,2279-, PosterPresentation, Gordon Conference February2002) as coupling reagent, and 1,1,3, 3-bis (methylene) chlorouronium hexafluorophosphate (PyCIU, used in particular for coupling N-methylated amino acids, j.coste, e.fr é rot, p.jouin, b.castro, Tetrahedron lett.1991,32,1967) or pentafluorophenyl bisphenyl phosphinate (s.chen, j.xu, Tetrahedron lett.1991,32,6711).

Due to the fact that nearly quantitative coupling reactions are important, experimental evidence with reaction termination is needed. Ninhydrin tests (Kaiser et al, anal. biochemistry1970,34,595) can be easily and rapidly performed after each coupling step, where a positive colorimetric reaction against an aliquot of the resin-bound peptide qualitatively indicates the presence of a primary amine. Fmoc chemistry allows spectrophotometric detection of Fmoc chromophores when released with base (Meienhofer et al, int.j.peptide Protein res.1979,13, 35-42).

The resin bound intermediate inside each reaction vessel was washed free of excess carry over reagents, solvents and by-products by repeated exposure to pure solvent using one of two methods:

1) the reaction vessel is filled with solvent (preferably 5mL), stirred for 5 to 300 minutes, preferably 15 minutes, and drained to drive off the solvent;

2) the reaction vessel is filled with a solvent (preferably 5mL) and discharged 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), and the efficiency of removing reagents, solvents and by-products is monitored by various methods such as TLC, GC or visual inspection of the eluate.

The above process of reacting the resin-bound compound with the reagent inside the reaction tube is repeated with the removal of excess reagent, by-products and solvent, with continuous conversion, until the resin-bound fully protected final linear peptide has been obtained.

In Cys before such fully protected linear peptide is detached from the solid support⁴And Cys¹¹Disulfide bonds between them may be formed.

To form disulfide bonds, preferably, 10 equivalents of iodine solution is applied in DMF or in CH₂Cl₂In a/MeOH mixture for 1.5 h, wherein the iodine solution is filtered off and fresh iodine solution is used, the preceding procedure is repeated for a further 3h, or applied to a mixture of DMSO and acetic acid solution, wherein the mixture is treated with 5% NaHCO₃Buffered to pH5-6 for 4 hours, or applied by stirring to water adjusted to pH8 with ammonium hydroxide solution for 24 hours, or applied to a solution of NMP and tri-n-butylphosphine (preferably 50 equivalents).

Alternatively, in Cys⁴And Cys¹¹The formation of disulfide bonds therebetween can be carried out after process step 2) as described below by:the fully deprotected and cyclized crude peptide was stirred in water containing up to 15% DMSO by volume with 5% NaHCO for 24 h₃Buffered to a pH of 5-6, or buffered to a pH of 7-8 with ammonium acetate or adjusted to a pH of 8 with ammonium hydroxide. In the evaporation ring (-Tyr)¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-5Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) to dryness to obtain Cys⁴And Cys¹¹Disulfide bonds between as end products.

Detachment of the fully protected linear peptide from the solid support is achieved by exposing the loaded resin to a solution of a reagent for cleavage (preferably 3 to 5 mL). Temperature control, stirring and reaction monitoring were performed as described above. By means of a transfer device, the reaction vessel is connected to a cartridge containing a reservoir for efficient collection of the solution of the cleaved products. The resin remaining in the reaction vessel is then washed 2 to 5 times with 3 to 5mL of a suitable solvent as described above to extract (elute) as much of the liberated product as possible. The product solutions thus obtained were combined, taking care to avoid cross-mixing. Each solution/extract was then manipulated as necessary to isolate the final compound. Common manipulations include, but are not limited to, evaporation, concentration, liquid/liquid extraction, acidification, basification, neutralization, or additional reactions in solution.

The solution containing the fully protected linear peptide derivative which has been cleaved from the solid support and neutralized with a base is evaporated. Cyclization is then effected in solution using solvents such as DCM, DMF, dioxane, THF, and the like. A variety of coupling reagents as mentioned hereinbefore may be used for the cyclisation. The duration of the cyclization is about 6 to 48 hours, preferably about 16 hours. The progress of the reaction is followed, for example, by RP-HPLC (reverse phase high performance liquid chromatography). The solvent is then removed by evaporation, the fully protected cyclic peptide derivative is dissolved in a water-immiscible solvent (such as DCM) and the solution is extracted with water or a water-miscible solvent mixture to remove any excess coupling reagent.

Finally, the fully protected peptide derivative was treated with 95% TFA, 2.5% H₂O, 2.5% TIS, or another scavenger combination treatment to effect cleavage of the protecting group. The cleavage reaction time is typically 30 minutes to 12 hours, preferably about 2.5 hours.

Alternatively, detachment and complete deprotection of a fully protected peptide from a solid support can be achieved manually in a glass container.

After complete deprotection, for example, the following methods can be used for further processing steps:

1) the volatiles were evaporated to dryness and the crude peptide was dissolved in 20% AcOH in water and extracted with isopropyl ether or other suitable solvent. The aqueous layer was collected and evaporated to dryness and fully deprotected peptides were obtained, disulfide bond at Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) as the final product;

2) the deprotection mixture was concentrated under vacuum. After precipitation of the fully deprotected peptide in diethyl ether, preferably at 0 ℃, the solid is washed up to about 10 times, preferably 3 times, dried, and if already at Cys on a solid support as described herein above⁴And Cys¹¹Form disulfide bond therebetween, to obtain fully deprotected peptide, i.e. disulfide bond in Cys⁴And Cys¹¹Cyclo (-Tyr) between¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) as the final product.

If the above-mentioned orthogonal protecting group strategy of introducing one or more isopropyl groups in solution has been followed, all amino groups of the amino acid residue side chains are still protected by non-acid labile protecting groups, whereas amino acid residues previously protected by acid labile protecting groups have been released at this stage of the synthesis cascade. Thus, isopropyl groups may be coupled if desired. Preferably, ivDde et al are acid stable protecting groups for the side chain carrying an amino group, which remains unmodified during the coupling of the isopropyl group with the released amino group. This coupling can be accomplished using, for example, reductive alkylation using acetone in the presence of a suitable reducing agent such as sodium cyanoborohydride. Thus, for example, the peptide is dissolved in MeOH (4.4mM) containing acetic acid (0.2M). After addition of excess acetone (780 equivalents), the reaction mixture was completed with a solution of sodium cyanoborohydride in MeOH (0.6M; 1.3 equivalents/isopropyl group to be introduced) and shaken vigorously at room temperature. After the conversion was complete as monitored by LC-MS, water was added and the solvent was evaporated. The residual solid containing the peptide was dissolved in DMF (0.01M) and a solution of 5% hydrazine in DMF was used to finally remove the ivDde protecting group.

As mentioned earlier, if desired, the fully deprotected cyclic product thus obtained may thereafter be converted into a pharmaceutically acceptable salt or the pharmaceutically acceptable or unacceptable salt thus obtained may be converted into the corresponding free compound or into a different pharmaceutically acceptable salt. Any of these operations may be performed by methods well known in the art.

The β -hairpin peptidomimetics of the invention can be used in a wide variety of applications in order to prevent HIV infection in healthy individuals or to delay and stop viral progression in infected patients; or in situations where cancer is mediated or resulting from CXCR4 receptor activity; or in situations where the immunological disorder is mediated or results from CXCR4 receptor activity; or these β -hairpin peptidomimetics can be used to treat immunosuppression; or they may be used during apheresis collection of peripheral blood stem cells and/or as agents that induce stem cell mobilization to regulate tissue repair.

The β -hairpin peptidomimetics of the invention may be administered as such or may be administered as a suitable formulation together with carriers, diluents or excipients well known in the art.

When used to treat or prevent HIV infection or cancer such as breast cancer, brain cancer, prostate cancer, hepatocellular cancer, colorectal cancer, lung cancer, kidney cancer, neuroblastoma, ovarian cancer, endometrial cancer, germ cell tumors, ocular cancers, multiple myeloma, pancreatic cancer, gastric cancer, rhabdomyosarcoma, melanoma, chronic lymphocytic leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, multiple myeloma, and non-hodgkin's lymphoma; metastasis, angiogenesis and hematopoietic tissues; or inflammatory diseases such as asthma, allergic rhinitis, hypersensitivity lung disease, hypersensitivity pneumonitis, eosinophilic pneumonia, delayed hypersensitivity, Interstitial Lung Disease (ILD), idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, systemic anaphylaxis or hypersensitivity responses, drug allergies, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, alzheimer's disease, parkinson's disease, atherosclerosis, myasthenia gravis, juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis, transplant rejection including allograft rejection or graft-versus-host disease, inflammatory bowel disease and inflammatory skin diseases; or for the treatment of eye diseases such as glaucoma, diabetic retinopathy and age-related macular degeneration; or for the treatment of ischemic stroke, global cerebral ischemia, myocardial infarction, hindlimb ischemia or peripheral ischemia; or for treating liver, kidney or lung injury; or for the treatment of immunosuppression, including immunosuppression caused by chemotherapy, radiation therapy or graft/transplant rejection, the beta-hairpin peptidomimetics of the invention may be administered alone, as a mixture of several beta-hairpin peptidomimetics, in combination with other anti-HIV agents or antimicrobial agents or anti-cancer or anti-inflammatory agents, or in combination with other pharmaceutically active substances. The β -hairpin peptidomimetics of the invention can be administered as such or as a pharmaceutical composition.

Pharmaceutical compositions comprising the β -hairpin peptidomimetics of the invention may be manufactured by conventional mixing, dissolving, granulating, tablet-making, comminuting, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or excipients that facilitate processing of the active beta-hairpin peptidomimetic into a preparation, which can be used pharmaceutically. The correct formulation depends on the chosen method of administration.

For topical administration, the β -hairpin peptidomimetics of the invention may be formulated as solutions, gels, ointments, creams, suspensions, powders, and the like, as is well known in the art.

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

For injections, the β -hairpin peptidomimetics of the invention can be formulated in a sufficient solution, preferably in a physiologically compatible buffer such as Hink solution, Ringer solution or physiological saline. The solution may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the β -hairpin peptidomimetics of the invention may be in powdered form for combination with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

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

For oral administration, the compounds can be readily formulated by combining the active β -hairpin peptidomimetics of the invention with pharmaceutically acceptable carriers well known in the art. Such carriers make it possible for the beta-hairpin peptidomimetics of the invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, ointments, suspensions, powders, and the like, for oral ingestion by the patient to be treated. For oral formulations, such as powders, capsules and tablets, suitable excipients include fillers such as sugars, for example lactose, sucrose, mannose and sorbose; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agent and adhesive. If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate. The solid dosage forms may be sugar coated or enteric coated, if desired, using standard techniques.

For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols and the like. In addition, a flavoring agent, a preservative, a coloring agent, and the like may be added.

For buccal administration, the compositions may take the form of tablets, lozenges, and the like, formulated as conventional.

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

In addition to the formulations described above, the β -hairpin peptidomimetics of the invention can also be formulated as long acting formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. To make such long acting formulations, the β -hairpin peptidomimetics of the invention may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble salts.

In addition, other drug delivery systems may be used, such as liposomes and emulsions well known in the art. Certain organic solvents such as dimethyl sulfoxide may also be used. Additionally, the β -hairpin peptidomimetics of the invention can be delivered using a sustained release system, such as a semipermeable matrix of a solid polymer containing the therapeutic agent (e.g., for a coated stent). Various sustained release materials are established and are well known to those skilled in the art. Depending on their chemical nature, sustained release capsules can release the compound for several weeks up to over 100 days. Depending on the chemical nature and biological stability of the therapeutic agent, additional protein stabilization strategies may be employed.

Because the β -hairpin peptidomimetics of the invention contain charged residues, they can be included in any of the foregoing formulations, either as such or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free form. Particularly suitable pharmaceutically acceptable salts include salts with the following acids: carboxylic, phosphonic, sulfonic and sulfamic acids, for example acetic, propionic, octanoic, decanoic, dodecanoic, glycolic, lactic, fumaric, succinic, adipic, pimelic, suberic, azelaic, malic, tartaric, citric, amino acids, such as glutamic or aspartic acid, maleic, hydroxymaleic, methylmaleic, cyclohexanecarboxylic, adamantanecarboxylic, benzoic, salicylic, 4-aminosalicylic, phthalic, phenylacetic, mandelic, cinnamic, methanesulfonic or ethanesulfonic, 2-hydroxyethanesulfonic, ethane-1, 2-disulfonic, benzenesulfonic, 2-benzenesulfonic, 1, 5-naphthalenedisulfonic, 2-, 3-or 4-methylbenzenesulfonic, methylsulfuric, ethylsulfuric, dodecylsulfuric, N-cyclohexylsulfamic, acid, N-methyl-, N-ethyl-or N-propylsulfamic acid and other organic protic acids, such as ascorbic acid. Suitable inorganic acids are, for example, hydrohalic acids (e.g. hydrochloric acid), sulfuric acid and phosphoric acid.

The β -hairpin peptidomimetics of the invention or compositions thereof will generally be used in an amount effective to achieve the intended purpose. It will be appreciated that the amount used will depend on the particular application.

For topical administration to treat or prevent HIV infection, a therapeutically effective dose can be determined using, for example, the in vitro assays provided in the examples. Treatment may be applied when HIV infection is evident, or even when it is not. One of ordinary skill will be able to determine, without undue experimentation, a therapeutically effective amount for treating a topical HIV infection.

For systemic administration, the therapeutically effective dose can be initially estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve an IC that includes as determined in cell culture₅₀Such information can be used to more accurately determine the dosage for use in humans.

Initial doses can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data. The number of doses administered alone as an anti-HIV agent can be adjusted to provide plasma levels of the β -hairpin peptidomimetics of the invention sufficient to maintain the therapeutic effect. Therapeutically effective serum levels can be achieved by administering multiple doses per day.

In the case of topical administration or selective ingestion, the effective local concentration of the β -hairpin peptidomimetics of the invention may not be correlated with plasma concentrations. One of ordinary skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

Of course, the amount of β -hairpin peptidomimetic administered will depend on the subject being treated, the weight of the subject, the severity of the condition, the mode of administration, and the judgment of the prescribing physician.

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

Normally, a therapeutically effective dose of the β -hairpin peptidomimetics described herein will provide therapeutic benefit without causing significant toxicity the β -hairpin peptidomimetics of the invention can be determined for toxicity by standard pharmaceutical procedures in cell culture or experimental animals, e.g., by determining the LD₅₀(dose lethal to 50% of the population) and LD₁₀₀The data obtained from these cell culture assays and animal studies can be used to create a range of doses that are non-toxic for use in humansWithin a range of circulating concentrations that include effective doses under conditions of low or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. The exact formulation, route of administration and dosage can be selected by The individual physician In view of The patient's condition (see, e.g., Fingl et al 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

The invention may also include compounds that are disulfide-bonded at Cys⁴And Cys¹¹A compound of the general formula-cyclo (-Tyr)¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) are identical, differing in that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature, e.g.²H(D)、³H、¹¹C、¹⁴C、¹²⁹I, and the like. These isotopic analogs and their pharmaceutically acceptable salts and formulations are contemplated as useful materials in therapy and/or diagnosis, for example, but not limited to, situations in which fine tuning of the in vivo half-life may result in an optimized dosage regimen.

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

Examples

1. Peptide synthesis

Coupling of the first protected amino acid residue to the resin

1g (1.4mMol) 2-chlorotrityl chloride resin (1.4 mMol/g; 100-. Suspending the resin in CH₂Cl₂(5mL) and allowed to swell at room temperature under constant shaking for 30 minutesA clock. Addition of a first appropriately protected amino acid residue in CH mixed with 960. mu.l (4 equivalents) Diisopropylethylamine (DIEA)₂Cl₂(5mL) in 0.98mMol (0.7 equiv.) solution (see below). After shaking the reaction mixture at 25 ℃ for 4 hours, the resin was filtered off and washed with CH₂Cl₂(1x), DMF (1x) and CH₂Cl₂(1x) washing sequentially. Will CH₂Cl₂A solution of/MeOH/DIEA (17/2/1, 10 mL) was added to the resin and the suspension was shaken for 30 min. After filtration, the resin was treated with CH in the following order₂Cl₂(1x)、DMF(1x)、CH₂Cl₂(1x)、MeOH(1x)、CH₂Cl₂(1x)、MeOH(1x)、CH₂Cl₂(2x)、Et₂O (2 ×) washed and dried under vacuum for 6 hours.

The loading is generally 0.6-0.7 mMol/g.

The following pre-loaded resins were prepared: Fmoc-Pro-2-chlorotrityl resin.

The synthesis was performed using a Syro-peptide synthesizer (MultiSynTech) using 24-96 reaction vessels. In each vessel, 0.04mMol or more of resin was placed and the resin was allowed to stand in CH₂Cl₂And DMF for 15 min, respectively. The following reaction cycles were programmed and performed:

repeat step 41 time.

Unless otherwise indicated, the final coupling of the amino acids was followed by Fmoc deprotection by using steps 1-3 of the reaction cycle described above.

Amino acid building block synthesis

Synthesis of Fmoc-Orn (iPr, Boc) -OH

By suspending 15.2g Fmoc-Orn-OH HCl in 150mL THF (0.26M) followed by 375mL acetone (132 equivalents) and 20.6g triacetoxyborohydrideSodium (2.5 equiv.) is dissolved to complete (2S) -N^αfluorenylmethoxycarbonyl-N^ω,N^ωSynthesis of-tert-butyloxycarbonyl-isopropyl-2, 5-diaminopentanoic acid. The reaction mixture was stirred for 2 hours and then the reaction was completed (monitored by LC-MS). 120mL of saturated Na was added₂CO₃Solution and 10.2g Boc₂O (1.2 equiv.). After stirring overnight, saturated Na was added depending on the remaining starting material₂CO₃Solution and Boc₂O is again added in two portions. After completing the Boc introduction, hexane was added 2 times, separated, and the aqueous layer was washed with 5NHCl_aq(pH =1) acidified and thereafter extracted 3 times with ethyl acetate. Finally, the combined organic layers were washed with Na₂SO₄Dried and evaporated to give the product as a white foam.

The amino acid building block Fmoc-Lys (iPr, Boc) -OH can be synthesized accordingly or can be obtained commercially.

Amino acid structural units Fmoc-Tyr (Me) -OH and Fmoc-^DTyr (Me) -OH is also commercially available.

Cyclization and preparation of backbone cyclized peptides

Cleavage of well-protected peptide fragments

After completion of the synthesis, the resin (0.04mMol) was suspended in 1mL (0.13mMol, 3.4 equiv.) of CH₂Cl₂1% TFA (v/v) for 3 min, filtered, and the filtrate was taken up in 1mL (0.58mMol, 14.6 equiv.) in CH₂Cl₂DIEA (v/v) of 10% of (1). This process was repeated 3 times to ensure completion of the lysis. The filtrate was evaporated to dryness and a sample of the product was fully deprotected by using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% Triisopropylsilane (TIS) and analyzed by reverse phase-HPLC (C18 column) and ESI-MS to monitor the efficiency of linear peptide synthesis.

Cyclization of linear peptides

The fully protected linear peptide (0.04mMol) was dissolved in DMF (4. mu. Mol/mL). 30.4mg (0.08mMol, 2 equiv.) of HATU, 10.9mg (0.08mMol, 2 equiv.) of HOAt were then addedAnd 28 μ l (0.16mMol, 4 equiv.) of DIEA, and the mixture was vortexed at 25 ℃ for 16 hours and then concentrated under high vacuum. The residue is in CH₂Cl₂And H₂O/CH₃And CN (90/10: v/v). Will CH₂Cl₂The phases are evaporated to yield fully protected cyclic peptide.

Adequate deprotection of cyclic peptides

The cyclic peptide obtained was dissolved in 3mL of a cleavage mixture containing 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% Ethanedithiol (EDT). The mixture was allowed to stand at 25 ℃ for 2.5 hours and then concentrated under vacuum. In diethyl ether (Et) at 0 deg.C₂O) precipitation of the fully deprotected cyclic peptide, the solid is then treated with Et₂O washed twice and dried.

Disulfide/beta chain bond formation and purification

After sufficient deprotection, the crude peptide was dissolved in 0.1M ammonium acetate buffer (1mg/1mL, pH = 7-8). DMSO (up to 5% by volume) was added and the solution was shaken overnight. After evaporation, the residue was purified by preparative reverse phase hplc.

After lyophilization, the product was obtained as a white powder and analyzed by means of the following analytical method: using an Ascentisoxpress C18 column, 50X3.0mm, (code 53811-U-Supelco), the following solvent A (H)₂O +0.1% TFA) and B (CH)₃CN +0.1% TFA) and gradient: 0-0.05 min: 97% A, 3% B; 3.4 minutes: 33% a, 67% B; 3.41-3.65 minutes: 3% a, 97% B; 3.66-3.7 minutes: 97% A, 3% B; flow rate (gas) =1.3 ml/min; UV _ Vis =220nm determines the analytical HPLC residence time (RT in minutes).

Example 1:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Finally grafting to the resin at position 15^DPro. The synthesis on a solid support is carried out according to the procedure described above in the following orderA linear peptide: resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^DPro⁷-Ala⁶-Ser⁵-Cys⁴-Tyr³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ after preparative HPLC ]: 95%; RT: 1.56; [ M +3H ]/3= 685.7).

Example 2:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Finally grafting to the resin at position 15^DPro. The linear peptide was synthesized on a solid support according to the procedure described above in the following order: resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^DPro⁷-Ala⁶-Ser⁵-Cys⁴-Tyr(Me)³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ after preparative HPLC ]: 95%; RT: 1.7; [ M +3H ]/3= 690.4).

Example 3:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Most preferablyFinal grafting to the resin at position 15^DPro. The linear peptide was synthesized on a solid support according to the procedure described above in the following order:

resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Dab⁸-^DTyr⁷-Ala⁶-Ser⁵-Cys⁴-Ala³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ after preparative HPLC ]: 95%; RT: 1.57; [ M +3H ]/3= 658.3).

Example 4:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Finally grafting to the resin at position 15^DPro. The linear peptide was synthesized on a solid support according to the procedure described above in the following order: resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^DTyr(Me)⁷-Ala⁶-Ser⁵-Cys⁴-Ala³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ post preparative HPLC ]: 95%; RT: 1.70; [ M +3H ]/3= 681.7).

Example 5:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Finally grafting to the resin at position 15^DPro. The linear peptide was synthesized on a solid support according to the procedure described above in the following order: resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^DTyr⁷-Ala⁶-Ser⁵-Cys⁴-Tyr³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ after preparative HPLC ]: 95%; RT: 1.60; [ M +3H ]/3= 707.4).

Example 6:

the starting resin was Fmoc-Pro-O-2-chlorotrityl resin prepared as described above. Finally grafting to the resin at position 15^DPro. The linear peptide was synthesized on a solid support according to the procedure described above in the following order: resin-Pro¹⁶-^DPro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^DTyr(Me)⁷-Ala⁶-Ser⁵-Cys⁴-Tyr(Me)³-His²-Tyr¹After final deprotection of Fmoc as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as shown above after formation of disulfide β -chain bond as described above.

HPLC residence time (min) was determined using analytical methods as described above (UV-purity [ after preparative HPLC ]: 95%; RT: 1.83; [ M +3H ]/3= 717.0).

2. Biological method

2.1 preparation of peptides

The lyophilized peptides were weighed on a microbalance (Mettler MT5) and dissolved in DMSO to a final concentration of 10 mM. The mother liquor was stored at +4 ℃ protected from light. Unless otherwise indicated, biological assays were performed under assay conditions with less than 1% DMSO.

2.2 cell culture

Namalwa cells (nonadherent cells naturally expressing CXCR4, ATCC CRL-1432) were cultured in RPMI1640 with the addition of 10% FBS and penicillin/streptomycin. HELA cells were maintained in RPMI1640 supplemented with 10% FBS, penicillin/streptomycin and 2mM L-glutamine. Cos-7 cells were grown in DMEM medium containing 4500mg/ml glucose, supplemented with 10% FCS, penicillin/streptomycin and 2mM L-glutamine. All cell lines were at 37 ℃ in 5% CO₂Cell culture medium, medium supplement, PBS-buffer, Hepes, antibiotics/antifungals, penicillin/streptomycin, non-essential amino acids, L-glutamine, β -mercaptoethanol, and serum were purchased from Gibco (Pailsey, UK.) all fine chemicals were supplied by MERCK (Darmstadt, Germany).

2.3 chemotaxis assay (cell migration assay)

The chemotactic response of Namalwa cells (ATCC CRL-1432) to a gradient of stromal cell-derived factor 1 α (SDF-1) was measured using a modified Boyden chamber chemotactic system (ChemoTx; Neuroprobe). In this system, the upper chamber of each well was separated from the lower chamber containing the chemotactic agent SDF-1 by a polycarbonate membrane (5 μm pore size). The circular area of the membrane in the area that fully covered each lower well was surrounded by a hydrophobic hood to retain the cell suspension inside this area the system was prepared by filling the bottom well with an aliquot of 30 μ l of chemotactic medium (RPMI 1640 without phenol red +0.5% BSA) containing a suitable serial dilution of peptide or no peptide at all, combined or not with SDF-1(0.9 nM). The membrane was placed over the bottom well and the chemotactic medium was placed either above the bottom well and the membrane was placed in the same positionAliquots of 50. mu.l Namalwa cell suspension (3.6X 10) in the lysis medium⁶Individual cells/ml) is delivered onto each hydrophobic confinement region of the upper surface of the membrane, wherein the chemotactic medium is pre-mixed with a suitable serial dilution comprising the peptide or chemotactic medium without the peptide at all. Cells were allowed to grow at 37 ℃ in 5% CO₂Travel down to the bottom chamber for 5 hours. After this time, the membrane was removed and its top side carefully wiped and washed with PBS to eliminate non-migrated cells. Using a "funnel" adapter, migrated cells were transferred to a 96-well receiver plate and based on measurement of cellular DNA content by fluorescent dye binding, by using CyQuant^TMThe NF cell proliferation assay (Invitrogen) determines cell number. Following the manufacturer's instructions, 50. mu.l CyQuant^TMDye reagent/HBSS buffer (1/500[ v/v ]]) Added to each well of the 96-well receiving plate mentioned above. After incubation for 0.5 hours at room temperature, the plates were sealed and incubated by using Wallac1420VICTOR^2TMThe fluorescence intensity of each sample was measured with a plate reader (PerkinElmer) with excitation at 485nm and emission detection at 535 nm. Finally, data were normalized by using controls and GraphPad Prism was used^TM(GraphPad) IC was determined by fitting a logarithmic curve to the averaged data points₅₀The value is obtained.

2.4. Cytotoxicity assays

The cytotoxicity of the peptides on 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). In short, the process proceeds as follows: 4000 HELA cells/well and 3400 COS-7 cells/well were seeded in 96-well microtiter plates and incubated at 37 ℃ in 5% CO₂Incubation was carried out for 24 hours. Thereafter, time zero (Tz) was determined by MTT reduction (see below). The supernatant of the remaining wells was discarded and fresh medium and compounds from serial dilutions (12.5, 25 and 50 μ M, triplicates; 0 μ M, blank) were aspirated into the wells. At 37 ℃ in 5% CO₂After 48 hours of incubation of the cells, the supernatant was discarded again and 100. mu.l MTT reagent (0.5 mg/ml in RPMI1640 and DMEM, respectively) was added per well.After incubation at 37 ℃ for 2-4 hours, the medium was aspirated and internal standards (100. mu.l isopropanol/well) were added to the cells. Absorbance (OD) of dissolved formazan was measured at 595nm₅₉₅Peptides). For each concentration, the average was calculated from three replicates. The percentage of growth was calculated as follows: (OD)₅₉₅peptide-OD₅₉₅Tz)/(OD₅₉₅blank-OD₅₉₅Tz) x 100%. By using the trend line function (= trend (C) for each concentration (50, 25, 12.5, and 0 μ M), concentration, corresponding percentage, and value 50₅₀:C₀,%₅₀:%₀50), calculating the GI of each peptide₅₀(growth inhibition).

2.5. Hemolysis of blood

The peptides were tested for their hemolytic activity on human erythrocytes (hRBC). Fresh hrbcs were washed 4 times with Phosphate Buffered Saline (PBS) and centrifuged at 3000x g for 10 minutes. The compound (100. mu.M) was incubated with 20% hRBC (v/v) for 1 hour at 37 ℃ and shaken at 300 rpm. The final concentration of red blood cells was about 0.9x10⁹Individual cells/ml. By containing H in each case₂hRBC were incubated in the presence of 0.001% acetic acid in O and 2.5% Triton X-100 in PBS to determine the values for 0% and 100% cell lysis, respectively. The samples were centrifuged, the supernatant diluted 8-fold in PBS buffer and the Optical Density (OD) measured at 540 nm. 100% dissolution value (OD)₅₄₀H₂O) yields an OD of about 0.5-1.0₅₄₀. Percent hemolysis was calculated as follows: (OD)₅₄₀peptide/OD₅₄₀H₂O)x100%。

2.6 plasma stability

The stability of the peptides in human and mouse plasma was determined by performing the following method: separately, 346.5. mu.L/deep well of thawed fresh human plasma (Basler Blutspende-diene) and mouse plasma (Harlan Sera-Lab, UK) were dissolved in DMSO/H3.5. mu.L/well₂O(90/10[v/v]1mM, three replicates) and incubated at 37 ℃. At t =0, 15, 30, 60, 120, 240 and 1440 minutes, 50 μ Ι _ aliquots were transferred to the wells of the filter plate containing 150 μ Ι _ per well of 2% formic acid in acetonitrile. After shaking for 2 minutes, the emerging suspension was filtered by vacuum. Transfer 100. mu.l of each filtrate to propylene microdropletsFixing the plate and in N₂And (5) drying. The residual solid was rehydrated by addition of 100 μ L/well of water/acetonitrile, 95/5(v/v) +0.2% formic acid and analyzed by LC/MS as follows: column: waters, XBridge C18, mobile phase: (A) water +0.1% formic acid and (B) acetonitrile/water, 95/5(v/v) +0.1% formic acid, gradient: 5% -100%; (B) within 1.8 minutes, electrospray ionization, MRM detection (triple charged quadrupole). Peak areas were determined and triplicate values were averaged. Stability is expressed as a percentage of the initial value at t =0 (tx/t0x 100). By using the trend function of EXCEL (Microsoft Office2003), T is determined_1/2。

2.7 plasma protein binding

Aliquots of 495. mu.L of human plasma (Basler Blutspendedestinst) and aliquots of 495. mu.L of PBS were placed in respective wells of a polypropylene plate (Greiner) and each was internally labeled with 5. mu.L of a solution of 1mM peptide in 90% DMSO. After shaking the plate at 600 rpm for 2 minutes, 150 μ L aliquots of the plasma/peptide mixture were transferred in triplicate to polypropylene filter plates (10kDa, Millipore), while 150 μ L aliquots of the PBS/peptide mixture were transferred either to individual wells of the filter plates (filtered control) or directly to individual wells of a medium receiver plate (Greiner) (unfiltered control). The plate stack consisting of the filter plate and the receiver plate was incubated at 37 ℃ for 1 hour and subsequently centrifuged at 3220g at 15 ℃ for 2 hours. The filtrate in the receiving plate was analyzed by LC/MS as follows: column: waters, XBridge C18, mobile phase: (A) water +0.1% formic acid and (B) acetonitrile/water, 95/5(v/v) +0.1% formic acid, gradient: 5% -100%; (B) within 1.8 minutes, electrospray ionization, MRM detection (triple charged quadrupole). Peak areas were determined and triplicate values were averaged. The percentage of binding to the filtered and unfiltered controls is expressed by the following formula: 100- (100 xT)_1h/T_ctr). Finally, the average of these values is calculated.

The results of the experiments described at 2.3-2.7 are shown in tables 1,2, 3and 4 below.

2.8. Pharmacokinetic study (PK)

Pharmacokinetic studies were performed after intravenous (i.v.) administration for the compounds of experiment 1, experiment 2, experiment 3, experiment 4, experiment 5 and experiment 6.

Male CD-1 mice of 30 grams (+ -20%) body weight obtained from Charles River Laboratories Deutschland GmbH were used. Vehicle (phosphate buffered saline) was added to give a final concentration of 0.5mg/ml of compound. The volume was 2mL/kg and the compound was injected to produce a final intravenous dose of 1 mg/kg. Approximately 300-400 μ L of blood was removed by cardiac puncture under mild isoflurane anesthesia at predetermined time intervals (5, 15, 30 minutes and 1,2, 3,4 hours) and added to heparinized tubes. Plasma was separated from the pelleted cells upon centrifugation and frozen at-80 ℃ prior to HPLC-MS analysis.

Preparation of plasma correction samples and plasma study samples

A 50 μ l aliquot of mouse plasma from untreated animals ("blank" mouse plasma) was used with known amounts of compound, internal standard experiment 1, experiment 2, experiment 3, experiment 4, experiment 5 and experiment 6, respectively, to obtain 10 plasma calibration samples for each compound in the range 1-4000 ng/ml. Aliquots of 50 μ l of each mouse plasma from untreated animals were used as plasma study samples.

Extraction of plasma correction samples and plasma study samples

All plasma samples were extracted with an appropriate internal standard and acetonitrile containing 2% formic acid. The supernatant was evaporated to dryness under nitrogen and the remaining solid was rehydrated in water/acetonitrile 95/5(v/v) +0.2% formic acid.

LC-MS/MS-analysis

The extracts were then analysed by reverse phase chromatography using the following conditions (Acquity BEHC18, 100 × 2.1mm, 1.7 μm column, Waters for experiment 1 and Acquity HSS C18SB, 100 × 2.1mm, 1.8 μm column, Waters for experiment 2, experiment 3, experiment 4, experiment 5 and experiment 6): experiment 1, mobile phase: (A) water/acetonitrile 95/5(v/v) +0.1% formic acid, (B) acetonitrile/water 95/5(v/v) +0.1% formic acid, gradient: 0-0.1 min for experiment 11% (B), 0.1-2.5 min for 15% (B), and 0-0.1 min for 1% (B), 0.1-2.5 min for experiment 2, experiment 3, experiment 4, experiment 5, and experiment 6 for 40% (B). Detection and quantification was performed by mass spectrometry, the electrospray interface was in positive ion mode and the analyte was selectively fragmented (4000QTrap mass spectrometer, AB Sciex).

Pharmacokinetic evaluation

Analysis by WinNonLin Using Single Chamber model^TMVersion 5.3 of the software (Pharsight-Acertara)^TMCompany, Moutain View, CA94041USA) calculates PK parameters. PK parameters were determined by least squares fitting of the model to experimental data.

The results of the experiment described in 2.8 are shown in tables 5a and 5b below.

2.9. Drug load calculation with maintenance dose rate (infusion rate)

Following the rationale in Pharmacokinetics, the drug loading of implants containing the peptides of the invention was calculated (see also J.Gabrielsson, D.Weiner, "Pharmaceutical and Pharmaceutical co-dynamic Data Analysis: Concepts and Applications", 4 th edition, Swedish Pharmaceutical Press, Stockholm, Sweden,2006) to maintain the dose rate (infusion rate, R.R._in) Can be defined as the rate at which the drug will be administered to achieve a steady state of a dose in plasma. Maintenance dose rate can express the usage dependence R_in[g/(h*kg)]=CL_iv[L/(h*kg)]x C_ss,_eff[g/l]In which CL is_ivIs clearance (intravenous administration) and C_ss,_effIs the effective concentration of drug at steady state in plasma considering the residual efficacy a: c_ss,_eff[g/L]=A x(IC₅₀/f_u)x MW[(mol/L)*(g/mol)]。

Thus, the total amount of a drug loaded into an implant (which provides a constant effective concentration of the drug in plasma for a certain period of time in a subject with a certain body weight) can be calculated by taking the following correlation:

medicine_{Capacity of carrying capacity}[ g/subject]=R_in[g/(h*kg)]x duration [ h ]]x BW [ kg/subject]。

The results of the calculations described in 2.9 are shown in table 6 below and are based on the data given in tables 1, 4 and 5 b. Further provisos are a residual efficacy a =3, a study duration of 672 hours (28 days) and a human subject weight of 70 kg. Glomerular Filtration Rate (GFR), which primarily affects peptide clearance, is highly species dependent. Generally, human GFR averages 107mL/(h × kg) compared to 840mL/(h × kg) mouse GFR. Therefore, before using in the above correlation, the CL shown in Table 5b_ivMouse values were normalized in a manner of differential growth (allometric scaling) to 107mL/(h x kg)/840mL/(h x kg) = 0.127.

3.0. HSC mobilization in mice

For the compounds of experiment 1 and experiment 2, a HSC mobilization study was performed, consisting of a time-response study and a subsequent dose-response study evaluating the maximum mobilization time after dosing.

Time-response study

Male C57BI/6 mice (Janvier, france; n =5 for experiment 1; n =3 for experiment 2) received intraperitoneal bolus injections (5mg/kg) of experiment 1 and experiment 2, respectively, dissolved in 10 μ l of water containing 0.9% NaCl per gram of mouse body weight. Blood was drawn from the buccal pouch into EDTA-coated tubes at time points 0, 0.5, 1,2, 4,6 and 8 hours post-application. Colony forming units in culture counts (CFU-C counts) were determined by performing the CFU-C assay as described below. The results of the time-response studies of experiment 1 and experiment 2 are shown in tables 7a and 7 b.

Dose-response study

Male C57BI/6 mice (Janvier, france; n =5 per dose group for experiment 1; n =3 per dose group for experiment 2) received intraperitoneal bolus injections of experiment 1 and experiment 2 (compound dissolved in 10 μ l of water containing 0.9% NaCl per gram of mouse body weight) at doses of 0.5, 1.5, 5 and 15mg/kg, respectively. Blood was collected at the maximum mobilization for experiment 1(4 hours) and experiment 2(2 hours), respectively, as described above. The results of the dose-response studies of experiment 1 and experiment 2 are shown in tables 8a and 8 b.

CFU-C assay

CFU-C counts were determined by culturing aliquots of lysed peripheral blood in standard semi-solid progenitor cell culture medium. Briefly, a defined volume of blood was treated with PBS buffer containing 0.5% bovine serum albuminWashing followed by a hypotonic NH₄Erythrocytes were lysed in Cl buffer (Sigma) and a second washing step. Resuspending the cell pellet in DMEM containing 10% FCSIn (b), suspended in 2mL of a commercially available cytokine-filled methyl fiber medium for murine cells (Cell Systems, USA), and plated in triplicate in 35mm Cell culture dishes. Under standard conditions (20% O)₂Saturated humidity, 5% CO₂CFU-C was assessed by incubation at 37 ℃ for 7-8 days. Peripheral blood cell properties were analyzed using an automated blood counter (DrewScientific).

Log₁₀Dose-response curves and ED₅₀

Shown in FIG. 1 are the logs of experiment 1 and experiment 2 based on CFU/mL values at doses of 1.5, 5 and 15mg/kg, as shown in tables 8a and 8b, respectively₁₀Dose-response curves and the curves were fitted using a sigmoidal dose-response fitting function in GraphPad Prism version 5.03. Considering the curve-advancing of both compounds, the dose-response was limited to a maximal response of 4000 CFU/mL. ED shown in Table 9₅₀The value thus corresponds to a reaction of 2000 CFU/mL. TABLE 1

Experiment of	IC50[nM]+ -SD, CXCR4 receptor
		1	0.42±0.1
2	0.69±0.43
		3	0.11±0.01
4	0.18±0.09
		5	0.43±0.24
6	0.09±0.09

TABLE 2

TABLE 3

TABLE 4

Experiment of	Plasma protein binding [% ]]	Unbound moiety, fu
			1	30	0.7
2	48	0.52
			3	48	0.52
4	56	0.44
			5	54	0.46
6	63	0.37

TABLE 5a

TABLE 5b

Intravenous route	Experiment 1	Experiment 2	Experiment 3	Experiment 4	Experiment 5	Experiment 6
							Dosage [ mg/kg]	1	1	1	1	1	1
Vdis[mL/kg]	547	635	762	607	1039	2975
							CL[mL/h/kg]	868	659	1113	744	836	2446
AUC0-∞[ng*h/mL]	1151	1518	898	1345	1196	409
							Cmax[ng/ml]	1829	1575	1313	1615	1313	686
Half life time (hour)]	0.4	0.7	0.5	0.6	0.9	0.8

TABLE 6

TABLE 7a

TABLE 7b

TABLE 8a

TABLE 8b

TABLE 9

	ED50[mg/kg]	95% confidence interval
			Experiment 1	1.76	0.57-5.39
Experiment 2	2.38	1.15-4.89

Claims (17)

1. A backbone cyclized peptide compound of 16 amino acid residues having the formula,

cyclo (-Tyr)¹-His²-Xaa³-Cys⁴-Ser⁵-Ala⁶-Xaa⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-^DPro¹⁵-Pro¹⁶-) (I)，

Wherein

Xaa³Is Ala; tyr or Tyr (Me),

tyr (Me) is (2S) -2-amino- (4-methoxyphenyl) -3-propionic acid,

Xaa⁷is that^DTyr；^DTyr (Me); or^DPro，

^DTyr (Me) is (2R) -2-amino- (4-methoxyphenyl) -3-propionic acid, or,

Xaa⁸is Dab or Orn (iPr),

dab is (2S) -2, 4-diaminobutyric acid,

orn (iPr) is (2S) -N^ω-isopropyl-2, 5-diaminopentanoic acid,

Xaa¹³is a group of Gln or Glu,

Xaa¹⁴is a group of Lys (iPr),

lys (iPr) is (2S) -N^ω-isopropyl-2, 6-diaminohexanoic acid,

all amino acid residues not specifically designated as D-amino acid residues are L-amino acid residues, and

two L-cysteine residues Cys⁴And Cys¹¹two-SH groups in (a) are replaced by 1-S-group.

2. A compound of formula I according to claim 1, wherein Xaa is in free form or in pharmaceutically acceptable salt form¹³Is Gln.

3. A compound according to claim 1 or claim 2 of formula I, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Tyr or Tyr (Me), Xaa⁷Is that^DPro，Xaa⁸Is Orn (iPr) and Xaa¹³Is Gln.

4. A compound according to claim 1 or claim 2 of formula I, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Ala, Xaa⁷Is that^DTyr，Xaa⁸Is Dab, and Xaa¹³Is Gln.

5. A compound of formula I according to claim 1 or 2, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Tyr, Xaa⁷Is that^DPro，Xaa⁸Is Orn (iPr) and Xaa¹³Is Gln.

6. A compound of formula I according to claim 1 or 2, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Tyr (Me), Xaa⁷Is that^DPro，Xaa⁸Is Orn (iPr) and Xaa¹³Is Gln.

7. A compound according to claim 1 or claim 2 of formula I, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Ala, Xaa⁷Is that^DTyr(Me)，Xaa⁸Is Orn (iPr), and Xaa¹³Is Gln.

8. A compound according to claim 1 or claim 2 of formula I, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Tyr, Xaa⁷Is that^DTyr，Xaa⁸Is Orn (iPr), and Xaa¹³Is Gln.

9. A compound according to claim 1 or claim 2 of formula I, wherein Xaa is in free form or in pharmaceutically acceptable salt form³Is Tyr (Me), Xaa⁷Is that^DTyr(Me)，Xaa⁸Is Orn (iPr), and Xaa¹³Is Gln.

10. A compound of formula I as defined in claim 1 or 2, in free form or in pharmaceutically acceptable salt form, for use as a pharmaceutically active substance.

11. A compound of formula I as defined in claim 1 or 2, in free form or in pharmaceutically acceptable salt form, for use as a substance having CXCR4 antagonism, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity.

12. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 11 in free form or in pharmaceutically acceptable salt form and a pharmaceutically inert carrier.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in a form suitable for oral, topical, transdermal, injection, buccal or transmucosal administration.

14. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in the form of a tablet, lozenge, capsule, solution, liquid, gel, paste, cream, ointment, syrup, slurry, suspension, powder, or suppository.

15. Use of a compound according to any one of claims 1 to 11 for the manufacture of a medicament having CXCR4 antagonism, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilization activity.

16. Use according to claim 15, wherein the medicament is for the prevention of HIV infection in healthy individuals; for delaying or halting viral progression in an HIV-infected patient; for use in the treatment or prevention of a cancer or immune disorder mediated by or resulting from CXCR4 receptor activity; for use in the treatment of immunosuppression; for apheresis collection with peripheral blood stem cells; or for inducing mobilization of stem cells to regulate tissue repair.

17. A process for the manufacture of a compound of formula I as defined in any one of claims 1 to 11, comprising the steps of

(a) Coupling the functionalized solid support with an N-protected amino acid Pro, said Pro forming the basis of an amino acid residue in position 16 of the compound of formula I;

(b) removing the N protecting group from the product of step (a);

(c) amino acids protected with N^DPro is coupled to the product of step (b), said^DPro forms the basis of an amino acid residue in position 15 of the compound of formula I;

(d) removing the N protecting group from the product of step (c);

(e) adding each desired amino acid residue to the free amino group of the amino acid residue in each case at the free end of the growing peptide chain coupled to a solid support, one after the other in positions 14 to 1 of the compound of formula I, in a manner analogous to that described in steps (c) and (d), the desired amino acid used in the coupling step analogous to step (c) being N-protected in each case and any functional groups present in the desired amino acid being protected in addition to the carboxyl group of the alpha amino moiety;

(f) replacing the two-SH groups in the two Cys residues in the product of step (e) with 1-S-group, unless this-S-group is formed in step (j);

(g) detaching the sixteen peptides from the solid support;

(h) coupling the amino acid residue in position 1 of the sixteen peptide with the amino acid residue in position 16 of the sixteen peptide;

(i) deprotecting any protected functional groups present in the product of step (h);

(j) (ii) replacing the two-SH groups in the two Cys residues in the product of step (i) with 1-S-group, unless this-S-group is formed in step (f);

(k) if desired, attaching one or more isopropyl substituents to the product of step (j);

(l) Deprotecting any protected functional groups present in the product of step (k); and is

(m) if the desired product of the process is a compound of formula I in the form of a pharmaceutically acceptable salt, converting the compound of formula I in free form to the corresponding compound of formula I in the form of a pharmaceutically acceptable salt, or if the desired product of the process is a compound of formula I in free form, converting the compound of formula I in the form of a pharmaceutically acceptable salt to the corresponding compound of formula I in free form, or if the desired product of the process is a compound of formula I in a different pharmaceutically acceptable salt, converting the compound of formula I in the form of a pharmaceutically acceptable salt to the corresponding compound of formula I in a different pharmaceutically acceptable salt.