DOUBLE CHAIN PEPTIDE COMPOUNDS HAVING HEMOREGULATORY ACTIVITY

Abstract: There is disclosed dipeptide compounds, wherein the two peptide chains are joined together at a C alpha -atom of a non-terminal amino acid by a divalent bridging group -A-. The C alpha -atoms joined to group -A- are located in equivalent positions in each peptide chain and each lack their native alpha -side chain. Group -A- is as defined in claim 1. The bridged dipeptide compounds disclosed have a stimulating activity on cell division, especially for myelopoietic and bone marrow cells.

Description: DOUBLE CHAIN PEPTIDE COMPOUNDS HAVING HEMOREGULATORY ACTIVITY The present invention relates to the use of peptides having a stimulating effect on cell proliferation, and to novel peptides having specific and/or general cell stimulating effects. The mammalian body contains cells having enormously diverse structures and functions, and the mechanisms of differentiation and development have been the focus of much study. It is known that for systems of cells having a continuous turnover the mechanism commonly involves a reservoir of pluripotent stem cells which divide and constantly supply new cells to the system. While initially homogeneous the stem cells supplied from the "reservoir" soon become committed to one or other morphology and subsequently develop into the required functional cells. Examples of such stem cell systems are the haemopoietic system in bone marrow and the epithelial and epidermal systems. The manipulation or control of stem cell division is of great potential therapeutically and much research continues to be devoted to elucidating the mechanisms involved and the chemical messengers responsible. To date several biomolecules have been identified as possessing a role in cell production and differentiation either by the stimulation or inhibition of a step within the process. Myelopoiesis has been particularly well studied in this regard and molecules involved in its control include: colony-stimulating factors (CSF) such as granulocyte colony-stimulating factor (G-CSF) , macrophage colony-stimulating factor (M-CSF) , granulocyte-macrophage colony-stimulating factor (GM- CSF) , multi-lineage colony-stimulating factor (multi- CSF; IL-3) [see Metcalf, Science 229: 16 (1985)], interleukin 11 (IL-11) [see Paul et al Proc Natl Acad Sci USA 7 . : 7521 (1990)], Lactoferrin [see Broxmeyer et al Blood Cells J 1: 429 (1986)], prostoglandins [see Pelus et al J. Immunol 140: 479 (1988)], acidic (H- subunit) ferritin [see Broxmeyer et al Blood €__: 1257 (1986)], interferons ( , β and 7 ) [see Pelus et al supra, and Broxmeyer et al J. Immunol 131: 1300 (1983)], tumour necosis factors (α and β) [see Broxmeyer et al J Immunol 136: 4487 (1986)], transforming growth factor-/3 [see Ottman et al J Immunol 140: 2661 (1988)], and activin and inhibin [see Broxmeyer et al Proc Natl Acad Sci USA 86. 779 (1989)]. It has also been found that the haemoregulatory pentapeptide (pEEDCK) inhibits the proliferation of myelopoietic cells selectively [see Paukovits et al Z. Naturforsch 3__: 1297 (1982)] and other peptides corresponding to a narrow general formula were discovered to exert a similar inhibitory effect in hemopoiesis [see EP-A-112656 and WO90/02753]. Oxidation of the peptide monomers resulted in di eric molecules linked by a cysteine bridge and these dimeric molecules were found to stimulate myelopoiesis [see Laerum et al. Exp. Hematol __S: 274 (1988)]. The (pEEDCK) 2 dimer and other similar compounds are disclosed in WO-A-88/03535. Further dimeric peptide compounds are disclosed in EP-A- 408371 in which the disulphide bond has been replaced by a carbon or carbon/sulphur bridge linking the selected peptide chains. The bridge is thus relatively stable to hydrolysis but is itself inert and incapable of participating in receptor-dimer interactions. Whilst we do not wish to be bound by theoretical considerations, it is presently believed that such peptide compounds interact with stromal cells in vivo and that the stromal cells are responsible for stimulating or inhibiting cellular division via other soluble factors. The dimers are thus believed to induce or promote stromatic production of stimulatory cellular regulatory factor(s) whilst the monomeric peptides may either inhibit that process or cause the production of factors which prevent or hinder cell division. Thus, according to current thinking, the stromal cells may act to amplify the stimulatory or inhibitory effects of the dimeric and monomeric peptides respectively. There is a continuing need for dimeric peptide compounds capable of stimulating cell proliferation to a useful level in vivo. In this regard it should be noted that different degrees of stimulation may be more appropriate to certain clinical situations than to others and, in particular, selective stimulation of individual cell types is important. The present invention provides a peptide compound comprising two single-chain he oregulatory, for example haemopoesis-inhibiting, peptides joined together by a bridging group terminally attached to the Cα atoms of non-terminal amino acids in equivalent positions in each of said peptides, the native α-side chain being absent in each peptide, said bridging group being a divalent group -A-, where A is -(CH 2 ) y -(Y) z -B-(Y) 2 -(CH 2 ) y - (wherein each y is independently 0, 1 or 2; each z is independently 0 or 1; each Y is independently 0, S or NR, where R represents hydrogen or a C-attached organic group (e.g. an alkyl, aralkyl or aryl group) ; B represents carbocyclic or heterocyclic ring (e.g. a 5 or 6 membered aromatic ring) optionally containing one or two heteroatoms (for example oxygen, nitrogen, or sulphur) and optionally mono-, di- or tri-substituted by a group -OR A , -NR A R A , -C00R A or a halogen atom such as an iodine, chlorine, fluorine or bromine atom; and each R A independently represents a hydrogen atom, or alkyl, alkanoyl or alkoxyalkyl groups each of which may also be hydroxylated) . Where group B is di or tri-substituted by any of the groups mentioned above each substituent does not have to be identical to the others or of the same type. Where group R is a C-attached organic group it preferably contains 1 to 10 carbon atoms, especially 1 to 6 carbon atoms. Alkyl groups may be straight chained or branched and may be substituted by aryl groups having 6-10 carbon atoms (ie. forming an aralkyl group) , alkoxy, hydroxy, acyloxy, amino, acylamino or carboxy groups. Aryl groups include 5- or 6-membered heterocyclic aryl groups having one or more heteroatoms selected from O, N or S such as furyl, imidazolyl, pyrrolyl, pyridinyl and thienyl groups. Substituents which may be present on aryl groups include C,_ 6 alkyl groups, hydroxy and carboxy groups. Examples include methyl, ethyl, propyl, t-butyl, pentyl, carboxyethyl and benzyl groups. Group R A preferably contains 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, where it represents an alkyl, alkanoyl or alkoxyalkyl group. In one preferred embodiment the divalent bridging group -A- is -CH 2 CH 2 BzCH 2 CH 2 -, where Bz represents a benzene ring which is optionally mono-, di- or tri- substituted by a group -0R A , -NR A R A , -C00R A or a halogen atom such as an iodine, chlorine, fluorine or bromine atom and R A is as defined above. Any single chain peptide which exhibits a hemoregulatory effect is suitable as the peptide which is bridged in accordance with the invention. Alternatively expressed, the invention provides compounds according to the invention in which the said hemoregulatory peptide chains include those of formula: R a - R b - R c - R d - (R e ) n - R f (I) wherein R a represents R represents — NH - R c represents — NH— CH— CO- -NH— CH— CO- I I (CH 2 ) q CH 2 OH or COR 7 R represents - R e represents — NH— CH— CO — R9 R f represents -NH— CH — COR10 (wherein n and m independently represent 0 or 1; p, q and r independently represent 1 or 2; s represents 3 or 4; R 1 and R 2 are both hydrogen atoms or together represent an oxo group; R 3 and R 4 are both hydrogen atoms or together represent a carbon-carbon bond; R 5 is hydrogen or an acyl group; each R 6 and R 7 independently represent a hydroxy group or an amino group, but are preferably hydroxy groups, R 8 represents hydrogen; a C 2 . 6 alkyl group; a C 7.20 aralkyl group, which may carry one or more hydroxy, amino or methoxy substituents; or a metabolically labile S-protecting group; R 9 represents hydrogen or a methyl group; and R 10 represents a hydroxy or amino group, the residue of the amino acid glutamine or a peptide having an N- terminal glutamine unit) . All the said amino acid residues may be in either the D or the L form. The L-form of the amino acids is, however, preferred. Where an N-terminal protecting group R 5 is present this may, as indicated above, be an acyl group having 1- 20 carbon atoms, e.g. a lower alkanoyl group having 1-5 carbon atoms such as the acetyl group, or an aroyl or aralkanoyl group having 7 to 20 carbon atoms such as the benzoyl or phenylacetyle group. R 5 may also be an acyl group derived from an amino acid or a peptide chain. In particular, R 5 may be an acyl group derived from serine or any of the peptides derived from the following amino acid sequence by removal of successive N-terminal amino acids: Lys-Ile-Ile-His-Glu-Asp-Gly-Tyr-Ser. The terminal amino group of the overall peptide of formula (I) is preferably protected, e.g. by acylation with an alkanoyl, aralkanoyl or aroyl group. Where R 8 is a C 2 . 6 alkyl group this may, for example, be an ethyl, butyl or hexyl group. When R 8 is an aralkyl group, this may conveniently be an arylmethyl group such as benzyl, diphenylmethyl or triphenylmethyl. Where R 8 is a metabolically labile group this may, for example, be an arylthio group having 5 to 10 carbon atoms, e.g. the pyridyl thio group, or an acyl group as defined above. The compounds of the invention are preferably pentapeptides in each chain, that is n is preferably 0. The cyclic groups in the R a residue are preferably five-membered, that is m is preferably 0. Insofar as any of the peptides defined by formula I above are of low or negligible haemoregulatory activity, they may nevertheless be effective, in the bridge form according to the invention, in stimulating cell proliferation. In dimeric peptides formed from peptide chains as described in formula I the bridging point of the chain is desirably at R d . Particularly preferred peptide compounds according to the present invention are those of formula II R a - R D - R c - NH - CH - CO ~(R e ) n - R f R a - R b - R c - NH - CH - CO -(R e ) n - R f wherein R a , R b , R c , R e , R f , A and n are all defined as above and the group -NH-CH-CO- is the derivatized form of R d which is attached to the bridging group -A- in such a manner that it's native side chain is absent. One especially preferred peptide compound of formula II is pGlu-Glu-Asp-NH-CH-CO-LysOH pGlu-Glu-Asp-NH-CH-CO-LysOH where group -A- is the divalent bridging group as discussed above. The invention is of particular application in stimulating myelopoiesis in patients suffering from reduced myelopoietic activity, including bone marrow damage, agranulocytosis and aplastic anaemia. This includes treatment of patients having depressed bone marrow function due to immunosuppressive treatment to suppress tissue reactions, e.g. in bone marrow transplant surgery. The compounds may also be used to promote more rapid regeneration of bone marrow after cytostatic chemotherapy and radiation therapy for neoplastic and viral diseases. In addition, the new compounds may be of particular value where patients have serious infections due to lack of immune response following bone marrow failure. Another clinical application will be in combination with the corresponding monomers or related myelopoiesis inhibitors as disclosed in EP-A-112656 or WO-A-90/02753 to induce alternating peaks of high and low activity in the bone marrow cells, thus augmenting the natural circadian rhythm of haemopoiesis. In this way, cytostatic therapy can be given at periods of low bone marrow activity, thus reducing the risk of bone marrow damage, while regeneration will be promoted by the succeeding peak of activity. In general, in order to exert a stimulatory effect, the peptides of the invention may be administered to human patients orally or by injection in the dose range 0.001-100 mg, for example 1-5 mg, per 70 kg body weight per day. If administered intravenously or subcutaneously, the dose may be in the range 1-10 mg per 70 kg body weight per day, for example about 6 mg, for up to ten days. Nasal, topical (transdermal) or rectal administration is, of course, also feasible. In principle it is desirable to produce a concentration of the peptide of about 10 ~13 M to 10 ~5 M in the extracellular fluid of the patient. According to a further feature of the present invention there are provided pharmaceutical compositions comprising as active ingredient one or more peptide compounds according to the invention, especially those compounds of formula (I) above, or physiologically compatible salts thereof, in association with a pharmaceutical carrier or excipient. The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral or rectal administration. As used herein, the term "pharmaceutical" includes veterinary applications of the invention. The compounds according to the invention may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Tablets may be produced, for example, by mixing the active ingredient or ingredients with known excipients, such as for example with diluents, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as magnesium stearate or talcum, and/or agents for obtaining sustained release, such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate. The tablets may if desired consist of several layers. Coated tablets may be produced by coating cores, obtained in a similar manner to the tablets, with agents commonly used for tablet coatings, for example, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar. In order to obtain sustained release or to avoid incompatibilities, the core may consist of several layers too. The tablet-coat may also consist of several layers in order to obtain sustained release, in which case the excipients mentioned above for tablets may be used. Organ specific carrier systems may also be used. Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules. Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression. Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatin capsules . Suitable suppositories may, for example, be produced by mixing the active ingredient or active ingredient combinations with the conventional carriers envisaged for this purpose, such as natural fats or polyethyleneglycol or derivatives thereof. Dosage units containing the compounds of this invention preferably contain O.l-lOmg, for example l-5mg of the peptide of formula (I) or salt thereof. According to a still further feature of the present invention there is provided a method of stimulation of cell division, especially myelopoiesis, which comprises administering an effective amount of a pharmaceutical composition as hereinbefore defined to a subject. A further major use of the new peptides, however, is in the production of material for immunological assay techniques. The peptide may then be covalently attached to a suitable high molecular carrier such as albumin, polylysine or polyproline in order to be injected into antibody-producing animals (e.g. rabbits, guinea pigs or goats) . .In vitro immunisation techniques may also be used. High specificity antisera are obtained by use of well known absorption techniques, using the high molecular carrier. By introducing radioactivity ( 3H, --,*■ i4 35 '"I, C, S) into the peptide molecule, a radioimmuno assay can be designed and used for determining the peptide in the different biological fluids such as serum (plasma) , urine and cerebrospinal fluid. The peptides of the invention may be synthesised in any convenient way. Suitable methods for forming the amino acid units are described in, for example, "Synthesis of Optically Active α-Amino Acids" by Robert M. Williams (Perga on Press, 1989). In general, the reactive side chain groups present (amino, thiol and/or carboxyl) will be protected during the coupling reactions of the overall synthesis but it is possible to leave some side chain groups unprotected (hydroxy groups, imidazole groups, primary amide groups, amide groups in cyclic amino acids like pyroGlu) during the entire synthetic procedure. The final step will thus be the deprotection of a fully protected or a partly protected derivative of a peptide of the general formula I and such processes form a further aspect of the invention. Schόllkopf et al have described the preparation of a variety of amino acids by the metallation and subsequent alkylation of bis-lactim ethers (see, for example, Tetrahedron 3__: 2085 (1983) and Topics Curr Chem 109: 65 (1983)). An adaptation of this method has proved particularly useful for the preparation of the bridged amino acids which form the basis of the present invention. In particular, a bis-lactim ether derived from a valine-glycine dipeptide forms a useful starting compound for the bridging reaction which may be summarized as follows: (wherein X is a leaving group such as a halogen atom, for example bromine) . Bridged (Ξ,S) -a , a'-diamino acids can be prepared by this method if D-valine is initially used to form the bis-lactim ether. Equally, bridged (R,R)-α,_'-diamino acids may be formed by the use of L-valine. We have also found that aryl (including heteroaryl) diglycine derivatives can be prepared from corresponding di-(halocarbonylmet yl) aryl compounds. The latter compounds may be reacted with a chiral oxazolidone to form a carboximide enolate which is then reacted with an azide forming reagent such as 2,4,6-triisopropyl benzenesulphonylazide to introduce azido groups, followed by hydrogenolysis eg. with 10% palladium/ charcoal, to produce the desired diglycine. Our procedure is an adaption of methodology described by Evans et al., in J. Am. Chem. Soc. 109 6881 (1987) and J. Am. Chem. Soc. Ill 1063 (1987). Alternatively, a suitable electrophilic amino nitrogen can be found in an azodicarboxylate ester in which case the corresponding dihydrazino acid is an intermediate for hydrogenolysis to the amino acid. This methodology is illustrated by the reaction scheme given below: Thus, the present invention also provides a process for producing a peptide compound comprising deprotecting a partially or fully protected derivative thereof. Further the invention also provides a process for producing a peptide compound, said process comprising a) metallating and subsequently alkylating a bis- lactim ether to form a bis-lactim dipeptide ether; b) hydrolysing the bis-lactim dipeptide ether of step (a) to form a bridged α,α'-diamino acid; c) introducing the remaining amino acids in the peptide chains; and d) deprotecting any protected group. The bis-lactim dipeptide ethers and bridged acid α, α' diamino acid produced by this technique form a further aspect of the present invention. Once the bridged dipeptide has been formed, then the remaining amino acids in the peptide chain can be introduced using conventional techniques. In building up the peptide chains, one can in principle start either at the C-terminal or the N-terminal although only the C-terminal starting procedure is in common use. Thus, one can start at the C-terminal by reaction of a suitably protected derivative of, for example lysine with a suitable protected derivative of cysteine. The lysine derivative will have a free α-amino group while the other reactant will have either a free or activated carboxyl group and a protected amino group. After coupling, the intermediate may be purified for example by chromatography, and then selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid residue. This procedure is continued until the required amino acid sequence is completed. Carboxylic acid activating substituents which may, for example, be employed include symmetrical or mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2 ,4,5,trichlorophenylester, N-bydroxybenzotriazole ester (OBt) , N-hydroxy- succinimidylester (OSu) or pentafluorophenylester (OPFP) . The coupling of free amino and carboxyl groups may, for example, be effected using dicyclohexylcarbodi- imide (DCC) . Another coupling agent which may, for example, be employed is N-ethoxycarbonyl-2- ethoxy-l,2-dihydroquinoline (EEDQ) . In general it is convenient to effect the coupling reactions at low temperatures, for example, -20"C up to ambient temperature, conveniently in a suitable solvent system, for example, tetrahydrofuran, dioxan, dimethylformamide, methylene chloride or a mixture of these solvents. It may be more convenient to carry out the synthesis on a solid phase resin support. Chloro- methylated polystyrene (cross-linked with 1% divinyl benzene) is one useful type of support; in this case the synthesis will start the C-terminal, for example by coupling N-protected lysine to the support. A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logan, and Robert C. Sheppard, J. Chem. Soc. Perkin I, 538-46 (1981) ; James P. Tarn, Foe S. Tjoeng, and R. B, Merrifield J. Am. Chem. Soc. 102. 6117-27 (1980) ; James P. Tam, Richard D. Dimarchi and R. B. Merrifield Int. J. Peptide Protein Res 1_5 412-25 (1980) ; Manfred Mutter and Dieter Bellof, Helvetica Chimica Acta __Z 2009-16 (1984) . It is also possible for the coupling reactions to be performed in solution. A wide choice of protecting groups for amino acids are known and are exemplified in Schroder, E. , and Llbke, K. , The Peptides, Vols. 1 and 2, Academic Press, New York and London, 1965 and 1966; Pettit, G.R . Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970, 1971, 1975 and 1976; Houben-Weyl, Methoden der Organischen Chemie, Synthese von Peptiden, Band 15, Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides, Analysis, synthesis, biology 1-7, Ed: Erhard Gross, Johannes Meienhofer, Academic Press, NY, San Fransisco, London; Solid phase peptide synthesis 2nd ed. , John M. Stewart, Janis D. Young, Pierce Chemical Company. Thus, for example a ine protecting groups which may be employed include protecting groups such as carbobenzoxy (Z-) , t-butoxycarbonyl (Boc-) , 4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr-) , and 9-fluorenylmethoxycarbonyl (Fmoc-) . It will be appreciated that when the peptide is built up from the C-terminal end, an amine protecting group will be present on the α-amino group of each new residue added and will need to be removed selectively prior to the next coupling step. For solid phase systems one particularly useful group for such temporary amine protection is the Fmoc group which can be removed selectively by treatment with piperidine in an organic solvent. For synthesis in solution, Boc- is a preferred protecting group, which can be introduced and removed in a conventional manner. The amino acids or peptides often require to be silylated prior to protection eg. by addition of Fmoc in order to improve their solubility in organic solvents. Silylation and Fmoc protection reactions are summarized below: H 2 N—CH—COOH (CH 3 ) 3 SiHN—CH-COOSi (CH 3 ) 3 HN(Si(CH 3 ) 3 2 I A H N—CH-COOH (CH 3 )SiCl ( (CCHH 3 )), 3 SSiiHHNN——CCHH--CCOOOOSSii (CH 3 ) Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (-OBZ1) , p-nitrobenzyl (-0NB) , or t-butyl (-tOBu) as well as the coupling on solid supports, for example methyl groups linked to polystyrene. Thiol protecting groups include p-methoxybenzyl (Mob) , trityl (Trt) and acetamidomethyl (Acm) . It will be appreciated that a wide range of other such groups exists as, for example, detailed in the above-mentioned literature references, and the use of all such groups in the hereinbefore described processes fall within the scope of the present invention. A wide range of procedures exists for removing amine- and carboxyl-protecting groups. These must, however, be consistent with the synthetic strategy employed. The side chain protecting groups must be stable to the conditions used to remove the temporary α-amino protecting groups prior to the next coupling step. Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be removed simultan¬ eously by acid treatment, for example with trifluoro acetic acid. Thiol protecting groups such as Trt may be removed selectively using an oxidation agent such as iodine. The cystein containing peptides may be synthesised by the methods described in the text with removal of all protecting groups including the thiol protecting groups as the last synthetic step. The following Examples are given by way of illustration only. HOBt = Hydroxybenzotriazole Pfp = Pentafluorophenyl Fmoc = 9-Fluorenylmethoxycarbonyl Boc = t-Butoxycarbonyl DCC = Dicyclohexylcarbodiimide (E)-DHs = (S,S)-3^'-Diamino-l^-diethylbenzene-?,^- dicarboxylic acid EXAMPLE 1 (S.S) -a, '-Diamino-1, 3-benzenedipropionic acid a) _,-'-Bisf (2R, 5S^ -2 , 5-dihvdro-3 , 6-dimethoxy-2- isopropyl-5-pyrazinyl1-m-xylene n-Butyllithium in hexane (1.6 M, 9.4 ml, 15 mmol) was added to a solution of (2R)-(-)-2,5-dihydro-3,6- dimethoxy-2-isopropylpyrazine (2.8 g, 15 mmol) in dry THF (5 ml) cooled to -78°C. The mixture was stirred for 15 minutes. A solution of α,α'-dibromo-m-xylene (2.0 g, 7.5 mmol) in dry THF (5 ml) was added. The reaction mixture was allowed to come to ambient temperature overnight. The solvent was evaporated and the residue was taken up in diethyl ether. The ether solution was washed with water, dried (MgSO and evaporated. The product was purified by flash chromatography (silica gel, hexane/EtOAc 3:1) . Yield 2.8 g (80%) , yellow-white, semi-solid. 1 H NMR (200 MHz, CDCl 3 ) : _ 0.60 (6H, d) , 0.94 (6H, d, CH 3 ) , 2.1 (2H, m) , 3.0 (4H, m) , 3.3 (2H, m) , 3.66 (6H, s) 3.71 (6H, s) , 4.3 (2H, dd) , 6.8-7.1 (4H, m) ; 13 C NMR (50 MHz, CDCI 3 ) ; _ 18.0, 20.6, 32.6, 41.5, 53.2, 53.4, 57.9, 61.3, 127.5, 128.1, 131.9, 137.1, 162.5, 163.7. MS (IP 70 eV) : m/z 470 (0.2) , 288 (50), 183 (47), 182 (20) , 141 (100) , 105 (40) . C 26 H 38 N 4 ° 4 Calc: c 66.34 ; H 8.14 ; N 11.91 ; Found: C 66.48; H 8.07; N 11.85. b) Dimethyl fS ,S) -_._ '-diamino-1, 3-benzene-dipropionate , '-Bis[ (2R,5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl- 5-pyrazinyl]-m-xylene (2.00 g, 4.25 mmol) was dissolved in methanol (10 ml) and 0.25 M hydrochloric acid (68 ml, 17 mmol) was added. The mixture was stirred at ambient temperature overnight. The water/methanol solution was extracted with diethyl ether and then the pH was adjusted to 10 by addition of ammonia. The products were extracted into ether. The ether solution was dried (MgSO ) , filtered and evaporated. D-Valine methylester was distilled off in a Kugelrohr apparatus (50-60"C/0.1 mbar) . The residue was used in the next step without further purification. Yield 1.10 g (92%) . 1 H NMR (200 MHz, CDC1 3 ) : . 1.4 (4H, br s) , 2.83 (2H, dd, 2 J 13.5Hz, 3 J 7.9 Hz) , 3.05 (2H, dd, 2 J 13.5Hz, 3 J 5.1 Hz), 3.7 (2H, dd) , 3.71 (6H, s) , 7.0-7.3 (4H, m) . c ) (S.S) -α,α'-Diamino-1, 3-benzene-dipropionic acid ' 2HCl A solution of the dimethyl (S,S)-_,_'-diamino-1,3- benzene-dipropionate (1.0 g, 3.6 mmol) in 6 M hydrochloric acid (10 ml) was stirred at 50"C overnight. The hydrochloric acid was removed on a rotavapor and the resulting white solid was dried in a vacuum oven at 40°C. Yield 1.1 g (95%) . 1 H NMR (200 MHz, H.,0) : _ 3.18 (2H, dd, 2 J 14.5Hz, 3 J 7.4 Hz) , 3.29 (2H, dd, 2 J 14.5Hz, 3 J 5.7 Hz) , 4.28 (2H, dd, 3 J 5.7, 7.4 Hz) , 7.1-7.4 (4H, m) ; 13 C NMR (50 MHz, H-O) : δ 39.2), 57.7, 131.8, 132.8, 133.0, 137.7, 173.7. C 12 H 18 C1 2 N 2 0 4 Calc: C 44.32; H 5.58; N 8.61; Found: C 44.78; H 5.81; N 8.73. d) (S.S) -α,α'-Bis(9-fluorenylmethoxycarbonylamino) - 1,3-benzene-dipropionic acid A suspension of (S,S) -_,_'-diamino-1, 3-benzene- dipropionic acid dihydrochloride (1.05 g, 3.23 mmol) in a mixture of hexamethyldisilazane (15 ml) and trimethylchlorosilane (1.5 ml) was stirred under nitrogen at 100"C overnight. The clear reaction mixture was cooled and excess silylating reagents were removed in vacuo. The residue was dissolved in dry dichloromethane (10 ml) and cooled in an ice/water bath. A solution of 9-fluorenylmethoxycarbonyl chloride (1.75 g, 6.76 mmol) in dry dichloromethane (5 ml) was added and the reaction mixture was stirred under nitrogen for 3 hours. Dichloromethane was removed on a rotavapor and the residue was dissolved in a mixture of THF (8 ml) and water (1 ml) . This mixture was stirred for 30 minutes and then evaporated. The residue was taken up in ethyl acetate and washed several times with water. The organic phase was dried (MgS0 4 ) , filtered and concentrated. The product crystallized upon addition of hexane. The crystals were washed with hexane and the product was purified by column chromatography (silica, hexane/ethyl acetate acetic acid 3:6:1), white solid, mpt 143-144 °C. Yield 1.85 g (82%) . 1 H NMR (200 MHz, CDC1 3 ) : _ 2.8-3.1 (4H, m) , 4.0-4.4 (8H, m) , 7.0-8.0 (22H, m) ; 13 C NMR (50 MHz, CDC1 3 ) ; _ 37.5, 47.6, 55.3, 66.4, 120.2, 125.3, 127.1, 127.3, 127.7, 128.1, 129.9, 137.8, 140.6, 143.6, 155.7, 172.9. FAB-MS signal at m/z 719.1(2) , 539.1(6) , 271.0(6) and 179.1(100) . EXAMPLE 2 (S,S)-α. '-Bis(9-fluorenylmethoxycarbonylamino) -1.4- benzene-dipropionic acid a) _,_'-Bisr (2R,5S) -2 ,5-dihvdro-3.6-dimethoxy-2- isopropypyl-5-pyrazinyl]-p-xylene The title compound was prepared as above from (2R)-(-)- (2,5-dihydro-3 , 6-dimethoxy-2-isopropylpyrazine (18 mmol) and α,α'-dibromo-p-xylene (9 mmol). The crude product was purified by flash chromatography on silica gel (CHC1 3 ) . Yield 76%. 1 H NMR (CDC1 3 ) : δ 0.6-0.9 (2d, 12H) , 2.05-2.20 ( , 2H) , 3.05 (d, 4H) , 3.25 (s, 2H) , 3.60 (s, 6H) , 3.70 (s, 6H) , 6.9 (s, 4H) . TLC (heptane:Et0Ac; 12:1), Rf 0.32. HPLC (50-100% MeOH, 10 min), RT 7.87 min. b) Dimethyl (S,S -α._'-diamino-1.4-benzene- dipropionate The product from the preceding step (3.4 mmol) was cleaved to the title compound using 0.25 M HCl at ambient temperature as described for the m-isomer above. TLC (MeCN:MeOH:H 2 0, 4:1:1), Rf 0.36. ~ ) (S,S) -α.Q. ' -Diamino-1,4-benzene-dipropionic acid dihydrochloride The methyl ester group in the preceding product was hydrolyzed by heating with cone. HCl as described for the m-isomer above. TLC (MeCN:MeOH:H 2 0, 4:1:1). 1 H NMR (D 2 0) : 6 0.8-0.9 (2d, 6H) , 2.10 (m, 1H) , 3.00 (m, 2 H9 ) , 3 . 45 ( d , 1H) , 3 . 81 ( t , 2H) , 7 . 13 ( ε , 4H) . d) (S.Sl-a.α 1 -Bis(9-fluorenylmethoxycarbonylamino-1,4- benzene-dipropionic acid The title compound was prepared from the above amino acid and 9-fluorenylmethoxycarbonyl chloride as described for the m-isomer above. TLC (Heptane:EtOAc:AcOH, 3:6:1), Rf 0.58. HPLC (40-70% MeCN in 0.1% TFA, 10 Min.) , RT 4.86 min. 1 H NMR (DMSO-d 6 ) : _ 2.79-3.11 ( , 6H) , 4.09-4.21 (br. s, 6H) , 7.13-7.92 (m, 20H) . FAB-MS: (M+H) 697. EXAMPLE 3 (S.S)-a.tt'-Diamino-1.2-benzene-dipropionic acid a ) α.α'-Bisr (2R.5S) -2.5-dihvdro-3 ,6-dimethoxy-2- isopropyl-5-pyrazinyl1-o-xylene (R) -2 ,5-Dihydro-3, 6-dimethoxy-2-isopropylpyrazine (3.50 g, 19.00 mmol) was dissolved in dry THF (8 ml) . The solution was cooled (-78°C) and BuLi (13 ml, 19.50 mmol, 1.5 M in hexane) was added dropwise under nitrogen. The electrophile, α,_'-dichloro-o-xylene (1.66 g, 9.50 mmol) in THF (5 ml) was added dropwise (-78°C) . The reaction mixture was allowed to warm to ambient temperature overnight. The solvent was removed and crude product was purified by flash chromatography using hexane/ethyl acetate 4:1 as eluent. Pale yellow crystalls, 3.512 g (79%) . Mp. 109-112°C. H NMR (300 MHz, CDC1 3 ) δ : 0.59 (6H, d, J 6.7 Hz, 2 X CH 3 ) , 0.94 (6H, d, J 6.8 Hz, 2 x CH 3 ) , 2.13 (2H, , 2 X CH) , 2.95, 3.45 (4H, m, 2 X CH 2 ) , 3.28 (2H, t, J 3.3 Hz, 2 X CH) , 3.62 (6H, s, OCH 3 ) , 3.71 (6H, s, 0CH 3 ) , 4.26 (2H, m, 2 x CH) , 7.05 (4H, m, 4 X CH) . 13 C NMR (75 MHz, CDC1 3 ) _: 16.38, 18.98, 30.96, 36.49, 52.10, 52.20, 57.14, 59.98, 125.86, 130.41, 136.75, 162.85, 163.61. C 2 6 H 38 N A ° 4 Calc - : c ' 66.35; H, 8.14. Found: C, 66.03; H, 8.04. b) Dimethyl (S,S) -α.α'-diamino-1,2-benzene-dipropionate To <_,_ ■■ -Bis[ (2R,5S) -2,5-dihydro-3,6-dimethoxy-2- isopropyl-5-pyrazinyl]-o-xylene (1.155 g, 2.45 mmol) was added 0.5M HCl (20 ml, 10.00 mmol) and dioxane (20 ml). This mixture was stired for 7 hours at ambient temperature. Most of the solvents were removed under reduced pressure, water (15 ml) and chloroform (15 ml was added) . The chloroform layer was discharged, and the water layer was made alkaline using cone. NH 3 (pH 10) . The water was extracted using chloroform (3 x 20 ml) and the combined chloroform layers were dried (MgSO ) and concentrated. The valine methyl ester was removed by bulb to bulb distillation, (0.3 mm Hg, 50°C) for 1.5 hours. The residue is a yellowish oil, 0.550 g (80%). 1 H NMR (200 MHz, CDC1 3 ) δ: 1.63 (4H, s, NH 2 ) , 3.02 (4H, m, 2 x CH 2 ) , 3.71 (6H, s, OCH.) , 3.74 (2H, m, 2 x CH) , 7.18 (4H, , 4 x CH) . 13 C NMR (50 MHz, CDC1 3 ) <S: 38.26, 52.47, 56.10, 127.48, 130.80, 136.73, 175.92. C 1 H 18 N 2 0 A Calc: C, 59.98; H, 7.19. Found: C, 59.54; H, 7.04. c) CS.S)-α,α'-Diamino-1.2-benzene-dipropionic acid«2HCl Dimethyl (S,S) -α, a ~ -diamino-1,2-benzene-dipropionate (270 mg, 0.963 mmol) was treated with 6N HCl (20 ml) at 50°C for 20 hours. Water was removed in vacuo. benzene was added and evaporated (2 x 20 ml) to leave a colourless solid, 0.313 g. NMR (300 MHz, D 2 0) _: 3.18 (4H, m, 2 X CH 2 ) , 4.12 (2H, m, CH) , 4.64 (6H, s, NH 3 ) , 7.23 (4H, m, 4xCH) . 13 C NMR (75 MHz, D 2 0) _: 32.56, 53.85, 128.62, 130.81, 133.27, 171.36. d) (S,S) -α.o:'-Bis(9-fluorenylmethoxycarbonylamino) - 1.2-benzene-dipropionic acid (S,S)-α,α'-Diamino-l,2-benzene-dipropionic acid dihydrochloride (130 mg, 0.40 mmol) was suspended in hexamethyldisilazane (7 ml) and trimethylsilyl chloride (0.5 ml) and refluxed under nitrogen overnight. Solvents were removed in vacuo. the residue dissolved in dry dichloromethane (10 ml) . The solution was cooled (0°C) and 9-fluorenylmethoxycarbonyl chloride (260 mg, 1.00 mmol) in dry dichloromethane (5 ml) was added dropwise. The cooling bath was removed after 1 hour and the reaction mixture was stirred overnight under nitrogen. IN HCl (5 ml) was added. The solvents were removed and the residue was washed with IM HCl (5 ml) in dioxane (5 ml) and dried. Yield: 100 mg (40%) . 1 H NMR (200 MHz, DMSO-d 6 ) <S: 2.49-3.54 (14H, m, 4xCH 2 , 4xCH, 2 X NH) , 7.29-7.84 (20H, m, 20xCH) . 13 C NMR (75 MHZ, DMSO-d 6 ) δ : 33.41, 46.94, 54.75, 66.14, 120.46, 125.64, 126.79, 127.48, 128.07, 130.21, 136.42, 141.02, 144.05, 156.37, 173.64. EXAMPLE 4 (S , S ) - . '-Diamino-2 , 5-thiophene-dipropionic acid a) 2.5-Bis( T2R.5S) - ( -) -2 ,5-dihvdro-3 , 6-dimethoxy-2- isopropylpyrazinyllmethyl) -thiophene (R) -2 , 5-Dihydro-3 , 6-dimethoxy-2-isopropylpyrazine (5.00 g, 27.14 mmol) was dissolved in dry THF (15 ml) . The solution was cooled (-78°C) and BuLi (19 ml, 30.4 mmol, 1.5 M in hexane) was added dropwise under nitrogen. After 10 minutes, the electrophile, 2,5- bis(chloromethyl) thiophene (2.50 g, 13.80 mmol) in THF (10 ml) was added dropwise. The reaction mixture was allowed to reach ambient temperature overnight, under nitrogen. The solvent was removed in vacuo and the crude product was purified by flash chromatography, hexane/ethyl acetate 4:1. Yield: 4.87 g (75%) of white crystals, 2.3% of wrong diastereomer (which can be removed by flash chromatography or recrystallisation from acetonitrile) . Mp. 7 ° C (MeCN) . C 24 H 36 N 4 0 4 S Calc: C, 60.47, H, 7.61 Found: C, 60.45, H, 7.68 1 H NMR (300 MHz, CDC1 3 ) δ : 0.63 (6H, d, J 6.9 Hz, CH 3 ) , 0.96 (6H, d, J 6.9 Hz, CH 3 ) , 2.16 (2H, m, CH) , 3.58 (2H, t, J 3.5 Hz, CH) , 3.65 (6H, s, OCH 3 ) , 3.71 (6H, s, OCH 3 ) , 4.23 (2H, dd, CH) , 6.48 (2H, s, H-3, H-4). 13 C NMR (75 MHZ, CDC1 3 ) δ : 16.56, 18.85, 31.46, 34.59, 51.94, 52.41, 56.04, 60.48, 125.80, 137.65, 161.57, 164.43. b) Dimethyl (S.S) -α,α' -diamino-2 ,5-thiophene- dipropionate 2,5-Bis( [ (2R, 5S (-(-) -2 , 5-dihydro-3,6-dimethoxy-2- isopropyl-5-pyrazinyl]methyl)thiophene (1.007 g, 2.113 mmol) was suspended in a mixture of dioxane (20 ml) and HCl (0.7 ml, 8.5 mmol, 12 M) and water (20 ml) . After 4 hours at ambient temperature diethyl ether (30 ml) was added. The ether phase was discharged, and the water phase made alkaline using cone. NH 3 (pH 10) . This was extracted with chloroform (3x30 ml) . The combined organic layers were dried (MgS0 4 ) , and the solvents were removed. The valine methyl ester was removed by bulb to bulb distillation (0.5 mmHg, 50°C, 1.5 h) . Yield 0.555 g (92%) , of a yellowish oil. C 12 H 16 N 2 0 4 S Calc: C, 50.33; H, 6.34 Found: C, 50.79; H, 6.53. 1 H NMR (300 MHz, CDC1 3 ) <S: 1.60 (4H, bs, NH 2 ) , 3.16 (4H, m, CH 2 ) , 3.70 (2H, m, CH) , 3.73 (6H, s, OCH 3 ) , 6.68 (2H, s, H-3, H-4) . 3 C NMR (75 MHz, CDC1 3 ) δ : 35.26, 52.02, 55.52, 126.32, 138.19, 174.85. c) (S.S) -α.c.'-Diamino-2.5-thiophene-dipropionic acid«2HCl Dimethyl (S,S) - , '-diamino-2 ,5-thiophene-dipropionate (531 mg. 1.854 mmol) was treated with 6N HCl (30 ml) at 70°C for 20 hours. The solution was evaporated, and remaining water azeotroped with benzene (2x30 ml) . Slightly brownish crystalls, 613 mg (100%) . 1 H NMR (300 MHz, D 2 0) δ : 3.35 (4H, d, J 5.9, CH 2 ) , 4.16 (2H, t, J 5.8 Hz, CH) , 4.65 (8H, S, NH 3 ) , 6.78 (2H, s, H- 3, H-4). 13 C NMR (75 MHz, D 2 0) δ : 29.96, 54.07, 128.61, 135.89, 171.35. d ) (S.S)-a.α'-Bis (9-fluorenylmethoxycarbonylamino) - 2.5-thiophenedipropionoic acid (S,S) -α, ~ -Diamino-2, 5-thiophenedipropionic acid dihydrochloride (590 mg, 1.78 mmol) was suspended in hexamethyldisilazane (15 ml) and trimethylsilyl chloride (1 ml) . The mixture was refluxed overnight. The solution was evaporated under reduced pressure, and the residue was dissolved in dry dichloromethane (15 ml) . The solution was cooled to 0°C and 9-fluorenylmethyloxy- carbonyl chloride (1.055 g, 4.08 mmol) in dichloro¬ methane (5 ml) was added dropwise under nitrogen. The cooling bath was removed after 1 hour, and the mixture stirred overnight at ambient temperature under nitrogen. The solvent was removed, and the compound was dissolved in THF (5 ml) , and IN HCl (5 ml) was added. The water phase was removed after 2 hours and washed with chloroform (3x20 ml) . The combined organic layers were dried (MgS0 4 ) and the solvents were removed. The crude product was purified by flash chromatography using dichloro ethane/methanol/acetic acid 10:1:0.1 as eluent. Yellow crystalls: 0.557 g (44%). Mp. 249°C (decomp.) . 'H NMR (200 MHz, DMSO-d 6 ) <S 2.90-4.20 (14H, m, CH 2 x4, CHX4, NHX2), 6.69 (2H, s, H-3 , H-4) , 7.20-8.00 (16H, m, CHX16) . 13 C NMR (50 MHz, DMSO-d 6 ) δ: 21.47, 31.50, 109.50, 120.20, 121.56, 127.50, 129.15, 137.71, 139.83, 143.00, 172.00. EXAMPLE 5 (S,S) - .α'-Diamino-cis-1.2-cvclopropane-dipropionic acid a ) cis-l.2-Bis(bromomethyl)cyclopropane To a cooled (0°C) suspension of triphenylphosphine (10.8 g, 41.16 mmol) in acetonitrile (70 ml) bromine (6.6g, 41.16 mmol) was slowly added. The cooling bath was removed and a solution of cis-l, 2-bis(hydroxymethyl) cyclopropane (2.0g, 19.6 mmol) in acetonitrile (5 ml) was added dropwise. This mixture was stirred at ambient temperature for 1 hour, the precipitate was filtered off and the filtrate was concentrated in vacuo. The residue was dissolved in ether (70 ml) , filtered, concentrated and subjected to vacuum distillation (12 bar, bp 135-155°C) . Yield: 3.463 g (71%) . 1 H NMR (CDC1 3 ) δ : 0.44(q, J 5.8 Hz, 1H) 1.18(m,lH), 1.66(m,2H) , 3.50(m,4H) 13 C NMR (CDC1 3 ) δ: 16.66, 22.68, 33.46 b) cis-l.2-Bisf f2R.5S)-2,5-αihvdro-3 , 6-diethoxy-2- isopropyl-5-pyrazinyllmethyl-cyclopropane n-Butyl lithium in hexane (1.6N, 5.483 ml, 8.774 mmol) was added slowly to a cooled (-78°C) solution of (2R) -2 , 5-dihydro-3 , 6-diethoxy-2-isopropylpyrazine (1.862 g, 8.774 mmol) in THF (30 ml) and stirring was continued for 30 minutes. After this period a solution of cis-l,2-bis-(bromomethyl)cyclopropane in anhydrous THF (10 mL) was added slowly and the solution was allowed to come to ambient temperature overnight. The reaction was quenched by addition of phosphate buffer pH 7 (30 ml) and the mixture was extracted twice with ether (30 ml each) . The combined organic layers were dried (MgS0 4 ) , evaporated to dryness and the product was isolated by flash chromatography (hexane/ethyl ether 8/1) . Yield: 0.609 g, 28%. NMR (CDC1 3 ) δ : -0.15(q, J 5.85Hz,lH), 0.5(m,lH), 0.65(d,6H) , 0.99(d,6H), 1.22 (dt, 12H) , 1.51(m,2H), 1.92(m,2H), 2.23(m,2H), 3.83(m,2H), 3.98-4.19(m,12H) 13 C NMR (CDC1 3 ) δ : 10.51, 10.90, 11.08, 14.37, 16.55, 17.03, 19.02, 19.12, 31.41, 31.59, 32.54, 33.31, 33.70, 46.78, 55.83, 56.15, 60.26, 60.38, 60.52, 60.55- 60.68, 61.03, 162.87, 163.03, 163.26, 163.38. c) Diethyl (S.S) -a , a '-diaminoamino-cis-1.2- cvclopropane-dipropionate Hydrochloric acid (IN, 3.51 ml) was added to a solution of cis-l,2-bis[ (2R,5S) -2,5-dihydro-3,6-diethoxy-2- isopropyl-5-pyrazinyl]methyl-cyclopropane (430 mg, 0.876 mmol) in dioxane (3.5 ml). The mixture was stirred at ambient temperature for 1 hour followed by removal of the dioxane in vacuo. The residue was extracted twice with chloroform (15 ml each) after addition of 1.5 ml of concentrated ammonia, the combined extracts were dried (MgSQ 4 ) , evaporated and the valine ester was removed by vacuum distillation (55°C, 0.03 mbar) . The oily residue consists of pure title compound. Yield: 238 mg, 99.7%. 1 H NMR (CDC1 3 /D 2 0) δ : -0.15 (q, 1H) , 0.68 (m, 1H) , 0.79 (m, 2H) , 1.22 (t, J 7.2 Hz, 6H) , 1.36 (m, 2H) , 1.78 (m, 2H), 3.41 (m, 2H) , 4.10 (q, J 7.2 Hz, 4H) . 13 C NMR (CDC1 3 /D 2 0) δ : 10.72, 11.37, 11.44, 13.88, 33.74, 54.56, 54.79, 60.36, 60.38, 175.39, 175.672 d) Diethyl (S.Sl-α.a 1 -Bis(9-fluorenylmethyloxycarbonyl- amino) -cis-l.2-cvclopropane-dipropionate Fmoc-chloride (678 mg, 2.622 mmol) and IN NaHCO-.- solution (3 ml) were added in turn at ambient temperature to a solution of diethyl(S,S) -α, '-diaminoamino-cis-1,2- cyclopropane-dipropionate (238 mg, 0.874 mmol) in dioxane (4 ml) . Removal of the dioxane after stirring for 30 minutes was followed by extraction with chloroform (30 ml) and flash chromatography of the residue left after evaporation of the CHC1 3 (hexane/ethyl acetate 4/1 to 2/1) gave the title compound. Yield: 483 mg, 77.1%. 'H NMR (CDC1 3 ) δ : -0.10(br Ξ,1H) , 0.77(br s,3H) , 1.28(t,6H), 1.56(m,2H), 2.03(m,2H), 4.23(m,6H), 4.41(m,6H), 5.46(m,2H) , 7.25-7.77 (m, 16H) 13 C NMR (CDC1 3 ) <S: 11.05, 11.24, 14.13, 31.77, 31.82, 47.16, 54.11, 54.28, 61.49, 66.93, 119.93, 125.01, 126.99, 12'/.65, 141.26, 143.72, 143.86, 155.69, 172.11, 172.28. FAB-MS: MH+ 717.4 e) (S,S) -α. '-Bis(9-fluorenylmethyloxycarbonylamino) - cis-l .2-cvclopropane-dipropionic acid A solution of diethyl (S,S) -α,α'-bis(9-fluorenyl- methyloxycarbonylamino-cis-1, 2-cyclopropane- dipropionate (497 mg, 0.693 mmol) in dioxane (5 ml) was heated to 90°C with 6N HCl (3 ml) for 26 hours. The dioxane was removed in vacuo and the aqueous residue was extracted twice with chloroform (20 ml each) . The solvent was evaporated after drying (MgSO and the residue was purified by flash chromatography (hexane/ethyl acetate/acetic acid 10/10/1) . Fractions containing the product were pooled, concentrated to a small volume in vacuo and lyophilized to give the title compound a white powder. Yield: 280 mg, 61.2%. 1 H NMR (DMSO-d 6 /D 2 0) _: -0.04(m,lH) , 0.56(m, 1H) , 0.82(m,2H), 1.41(m,lH) , 1.60(m,lH), 1.74(m,lH) , 1.88(m,lH) , 3.97(m,2H) , 4.25(m,6H) , 7.27-7.88 (m, 16H) . 13 C NMR (DMSO-d 6 /D 2 0) δ : 10.88, 12.35, 13.13, 30.15, 30.59, 47.02, 54.84, 66.02, 120.43, 125.63, 127.44, 128.02, 136.68, 141.04, 144.10, 144.15, 156.43, 156.49, 174.00, 174.12. FAB-MS: MH+ 661.3 EXAMPLE 6 (S.S) -α.c.'-Diamino-cis-1.2-cvclobutane-diprc ' ionate acid a) cis-l, 2-Bis-(bromomethyl)cyclobutane To a cooled (0°C) suspension of triphenylphosphine (2.664 g, 10.16 mmol) in acetonitrile (15 mL) bromine (1.623g, 10.16mmol) was slowly added. The cooling bath was removed and a solution of cis-l, 2-bis(hydroxymethyl) cyclobutane (562 mg, 4.838 mmol) in acetonitrile (5 ml) was added dropwise. This mixture was stirred at ambient temperature for 1 hour, followed by removal of the solvent in vacuo and trituration of the residue with ether (30 ml) . The precipitate was removed by filtration, the filtrate was concentrated and subjected to vacuum distillation (Kugelrohr, 0.3 mbar, bp 110°C) . Yield: 628 mg, 53.6%. η H NMR (CDC1 3 ) <S: 1.71(s,2H) , 2.10(s,2H) , 2.83(s,2H) , 3.44(m,2H) , 3.58(m,2H) 13 C NMR (CDC1 3 ) _: 23.72, 33.69, 39.45 b) cis-l.2-BisC (2R.5S)-2.5-dihydro-3 ,6-diethoxy-2- isopropyl-5-dihydropyrazinyllmethyl-cvclobutane n-Butyl lithium in hexane (1.6N, 4.92 ml, 7.87 mmol) was added slowly to a cooled (-78 β C) solution of (2R) -2 , 5-dihydro-3 , 6-dimethoxy-2-isopropylpyrazine (1.449 g, 7.865 mmol) in THF (30 ml) and stirring was continued for 30 minutes. After this period a solution of cis-l,2-bis-bromomethyl-cyclobutane (906 mg, 3.745 mmol) in anhydrous THF (4 ml) was added slowly and the solution was allowed to come to ambient temperature overnight. The reaction was quenched by addition of phosphate buffer pH 7 (30 ml) and the mixture was extracted twice with ether (30 ml each) . The combined organic layers were dried (MgSO , evaporated to dryness and the product was isolated by flash chromatography (hexane/ethyl ether 8/1) . Yield: 517 mg, 30%. 1 H NMR (CDCI 3 ) δ : 0.60(d,6H) , 0.98(d,6H), 1.63(m,4H) , 1.85(m,4H), 2.17(m,2H), 2.45(m,2H), 3.57-3.67 (m, 12H) , 3.80-3.94(m,4H) . 13 C NMR (CDC1 3 ) δ : 16.35, 16.40, 19.02, 24.84, 25.54, 31.30, 31.36, 33.87, 34.22, 34.72, 34.81, 51.96, 52.04, 52.16, 54.22, 54.75, 60.23, 60.36, 162.73, 163.20, 164.02, 164.19 c) Diethyl (S . S ) - . a '-diaminoamino—cis-l, 2-cyclobutane -dipropionate Hydrochloric acid (IN, 4.61 ml) was added to a solution of cis-l, 2-bis[ (2R, 5S) -2 , 5-dihydro-3 , 6-diethoxy-2- isopropyl-5-dihydropyrazinyl]methyl-cyclobutane (517 mg, 1.152 mmol) in dioxane (5 L) , the mixture was stirred at ambient temperature for 1 hour followed by removal of the dioxane in vacuo. The residue was extracted twice with chloroform (15 mL each) after addition of 1.5 mL of concentrated ammonia, the combined extracts were dried (MgS0 4 ) , evaporated and the valine ester was removed by vacuum distillation (55°C, 0.03 mbar) . The oily residue consists of pure title compound. Yield: 293 mg, 98.5%. Η NMR (CDC1 3 ) δ : 1.36(br s,4H) , 1.50(m,4H), 1.74(m,2H) , 1.91(m,2H), 2.43(m,2H) ,3.24(m,2H) , 3.59(m, 6H) 13 C NMR (CDC1 3 ) δ : 24.02, 24.80, 33.54, 33.86, 35.07, 35.67, 51.56, 51.68, 52.47, 53.03, 176.25, 176.59 d) Diethyl (S . S ) -a , a'-Bis (9-fluorenylmethyloxycarbonyl- amino) -cis-l.2-cyclobutane-dipropionate Fmoc-chloride (880 mg, 3.402 mmol) and IN NaHC0 3 -solution (3 ml) were added in turn at ambient temperature to a solution of diethyl (S,S)-α,α*-diaminoamino-cis-l,2- cyclobutane-dipropionate (293 mg, 1.134 mmol) in dioxane (4 ml) . Removal of the dioxane after stirring for 30 minutes and extraction with chloroform (30 ml) was followed by flash chromatography of the residue left after evaporation of the CHC1 3 (hexane/ethyl acetate 4/1 to 2/1) to give the title compound. Yield: 572 g, 71.8%. Η NMR (CDC1 3 ) δ : 1.77(m,4H), 2.00(m,6H) , 2.25(m,2H), 3.74(m,6H) , 4.10-4.50(m,6H) , 5.10-5.20(m,2H) , 7.27-7.77 (m,16H) 13 C NMR (CDC1 3 ) δ : 24.38, 24.95, 33.01, 33.18, 33.68, 33.78, 47.15, 52.19, 52.30, 52.40, 52.65, 66.93, 119.92, 125.01, 126.98, 127.65, 141.25, 143.67, 143.79, 155.63, 155.91, 172.84, 173.16 e) (S.S)-tt.- 1 -Bis(9-fluorenylmethyloxycarbonylamino) - cis-l.2-cvclobutane-dipropionic acid A solution of diethyl (S,S) - , ' -bis(9-fluorenyl- methyloxycarbonylamino)-cis-l, 2-cyclobutane-dipropionate (572 mg, 0.814 mmol) in dioxane (5 ml) was heated to 65°C with 6N HCl (3 mL) for 20 hours. The dioxane was removed in vacuo and the aqueous residue was extracted twice with chloroform (20 mL each) . The solvent was evaporated after drying (MgS0 4 ) and the residue was purified by flash chromatography (hexane/ethyl acetate/acetic acid 10/10/1) . Fractions containing the product were pooled, concentrated to a small volume in vacuo and lyophilized to give the title compound as a white powder. Yield: 316 mg, 57.5%. 'H NMR (DMSO-d 6 /D 2 0) δ : 1.4-2.0(m,6H) , 2.40(m,2H) , 3.6(br, 2H) , 3.82(m,2H), 4.25(m,6H) , 7.26-7.90 ( , 16H) 13 C NMR (DMSO-d 6 /D 2 0) <5: 23.58, 24.77, 31.00, 32.12, 33.62, 46.98, 51.84, 53.08, 65.83, 120.37, 125.55, 127.36, 127.94, 141.00, 144.01, 144.05, 144.11, 156.27, 156.43, 174.13, 174.55. EXAMPLE 7 fS.S) - . '-Diamino-trans-1.4-cyclohexane-diacetic acid a) trans-1.4-Bis ( (2R.5S)-2.5-dihvdro-3.6-dimethoxy-2- isopropyl-5-pyrazinyl)-cyclohexane To a solution of (2R) -2, 5-dihydro-3 , 6-dimethoxy-2- isopropylpyrazine (2.76 g, 15 mmol) and 1, 3-dimethyl-2- imidazolidinone (6.87 g, 60 mmol) in THF (48 ml) 1.6 M n-BuLi (9.6 ml, 15.3 mmol) was added dropwise. After 1 hour a solution of 1, 4-trans-dibromocyclohexane (1.82 g, 7.5 mmol) in THF (5 ml) was added dropwise. The mixture was allowed to come to ambient temperature overnight and quenched with 1 M phosphate buffer (pH=7) . The mixture was diluted with diethyl. ether (100 ml) and water (20 ml) and the layers separated. The aqueous layer was extracted twice more with ether (2 x 150 ml) and the combined organic layers dried (MgS0 4 ) , filtered, concentrated and purified by flash chromatography (silica gel, hexanes : ether =12 : 1). The product from the combined fractions was recrystallized from MeOH (100ml). Yield: 2.774 g (6.18 mmol, 82.4 %) . 1 H NMR (300 MHz, CDC1 3 ) : δ 3.91 (t, 2H, J 3.6 Hz) , 3.86 (q, 2H, J 3.0 Hz) , 3.69 (s, 6H) , 3.68 (s, 6H) , 2.67 (d sept, 2H, J 3.0, 6.9 Hz) , 1.78 - 1.54 (m, 8H) , 1.05 (d, 6H, J 6.9 Hz) , 0.97 (m, 2H) , 0.66 (d, 6H) ; 13 C NMR (75 MHZ, CDC1 3 ) : _ 163.23, 163.15, 60.30, 59.95, 52.24, 41.30, 31.40, 28.91, 26.17, 19.03, 16.38. FAB-MS m/Z 449 (M + + 1) . C 24 H AO N 4 0 4 (448.60) . Calc: C, 64.26; H, 8.98; N, 12.49. Found: C, 64.20; H, 9.04; N, 12.37. b) Dimethyl (S,S) - . '-diamino-trans-1.4-cvclohexane- diacetic acid The compound from step (a) (1.00 g, 2.23 mmol) was dissolved in dioxane (17.8 ml) and 0.5 M HCl (17.8 ml, 8.92 mmol) was added and the mixture was stirred at ambient temperature for 12 hours. The dioxane was removed at aspirator vacuum, the residue diluted with water and extracted with ether (3 x 30 ml) . The combined CHCl 3 -layer was dried (MgS0 4 ) , filtered and concentrated. The residue was brought into a Kugelrohr apparatus and and the methyl valinate was removed at 0.1 torr at 25 - 50°C. Yield: 530 mg (2.05 mmol, 92 %) . 1 H NMR (300 MHz, CDC1 3 ) : δ 3.68 (9s, 6H) , 3.26 (d, 2H, J 5.1 Hz) , 1.72-1.54 (m, 6H) , 1.26 - 1.19 ( , 2H) , 1.13-1.06 (m, 2H) ; 13 C NMR (75 MHz, CDC1 3 ) : δ 175.83, 59.22, 51.74, 41.64, 29.10, 27.17. c) (S.S) - .α'-Diamino-trans-1,4-cvclohexane-diacetic acid A solution of the above compound (442 mg, 1.71 mmol) in 6 M HCl (10 ml) was heated at 50°C for 14 hours. After the mixture was allowed to come to ambient temperature the volatile compounds were evaporated at the rotavap and the residue dissolved (not fully) in water Ethanol was added and the mixture left overnight in the refrigerator. The precipitate was then filtered, washed with ether and dried. Yield: 378 mg (1.25 mmol, 72.9 %) . 1 H NMR (300 MHz, D 2 0, DC1): _ 3.63 (m, 2H) , 1.68 - 1.43 (m, 6H) , 1.00 (dd, 2H) , 0.89 (dd, 2H) . 13 C NMR (75 MHz, D 2 0/DC1) : δ H O . 77 , 56.96, 37.30, 26.90, 26.71. FAB-MS m/z 231 (M + - 72); C 10 H 22 N 2 O 4 (258.31). d) (S.Sl-tt.tt 1 -Bis-9-fluorenylmethyloxycarbonylamino)- trans-1.4-cvclohexane-diacetic acid To a solution of the amino acid from step (c) (420 mg,1.38 mmol) in water (4 ml) and dioxane (4 ml) a solution of FmocCl (1.43g, 5.54 mmol) in dioxane (3 ml) followed by 1 M NaOH (8.31 ml, 8.31 mmol) was added. The mixture was stirred overnight. The resulting slurry was extracted with diethyl ether (2 x 30 ml) . The aqueous layer was acidified with 6 M HCl and extracted with CHC1 3 (3 x 30 ml) . The CHCl 3 -phase was concentrated and purified by flash chromatography (silica gel, hexanes:ethyl acetate: acetic acid = 10 : 10 : 1) . Yield 648 mg (0.96 mmol, 69.5%) . 'H NMR (300 MHz, DMSO) : δ 7.87 (d, 4H, J 7.5 Hz) , 7.73 (d, 4H, J 7.5 Hz), 7.41 (t, 4H, J 7.5 Hz) , 7.33 (t, 4H, J 7.5 Hz) , 4.30 - 4.20 (m, 6H) , 3.86 (t, 2H) , 1.75 - 155 (m, 6H) , 1.25 - 0.95 (m, 4H) . 13 C(DMSO) δ 173.60, 156.64, 144.29, 144.17, 141.10, 128.02, 127.45, 125.77, 120.48, 66.05, 59.35, 47.10, 29.14, 27.90, 21.51. FAB-MS m/z 677 (M + + 1) . C 40 H 40 N 2 O 8 (676.76) . Calc: C, 70.99; H, 5.96; N, 4.14. Found: C, 68.9; H, 6.08; N, 3.99. EXAMPLE 8 (S.S) -α._'-Bis(9-fluorenylmethoxycarbonylamino) -1.4- benzenediacetic acid a) (S. S) -β. β '-Dioxo-β. β ' -bisf4-benzyl-2-oxo-3- oxazolidinyl) -1, 4-diethylbenzene A solution of (4S) -4-benzyl-2-oxazolidone (1.08? g, 6.13 mmol) and triphenyl-methane (indicator) (2.5 mg) in dry THF (10 ml) was stirred at -78°C under argon and treated dropwise with 1.6 M nBuLi (3.83 ml, 6.13 mmol) until an orange colour persisted. The solution was kept between -65°C and -78°C during the addition. After recooling to -78°C, the solution was treated with 1,4- benzenediacetyl chloride (0.75 g, 3.25 mmol) over a 2 minute period. The resulting mixture was warmed to 25°C, treated with 10 ml of saturated aqueous sodium bicarbonate and stirred at 25°C for 30 minutes. The mixture was extracted with three portions of dichloromethane. The dichloromethane extracts were combined, washed with 5% aqueous sodium carbonate, washed with brine, dried (MgSO ) and evaporated. The crude product was purified by flash chromatography eluting with hexane: ethyl acetate = 1 : 2. Yield (white solid) : 698 mg (45%) . 1 H NMR (200 MHz, CDC1 3 ) : δ 2.77 (dd, J 9.4, 13.3 Hz, CHHPh) , 3.28 (dd, J 3.2, 13.3 Hz, CHHPh), 4.18-4.22 ( , CH 2 0) , 4.31 (Ξ, CH 2 C0) , 4.64-4.72 (m, CHN) , 7.35-7.13 (m, 14H, aromatics) . 13 C NMR (50 MHz, CDCl 3 ) : <5 37.7, 41.2, 55.2, 66.0, 126.9, 128.5, 129.0, 132.1, 134.7, 152.9, 170 . 5 . b) (S.S.S.Sϊ-α.c,'-Diazido-β. β'-dioxo-g. β'-bis(4- benzyl-2-oxo-3-oxazolidinyl) -1,4-diethylbenzene A precooled (-78°C) solution of (S,S) -β , β'-dioxo-3,/3'- bis(4-benzyl-2-oxo-3-oxazolidinyl)-l,4-diethylbenzene (1.4 g, 2.73 mmol) in dry THF (25 ml) was added via cannula to a precooled (-78°C) solution of 0.5 M potassium bis(trimethylsilyl)amide (12 ml, 6.01 mmol) in toluene diluted with dry THF (30 ml). After 30 minutes, the resulting solution of the potassium enolate was treated, via cannula, with a -78°C solution of 2,4,6- triisopropylbenzenesulfonyl azide (trisyl azide) (2.02 g, 6.55 mmol) dissolved in dry THF (15 ml). After stirring the solution for 2 minutes, the reaction was quenched by addition of glacial acetic acid (0.983 g, 16.38 mmol). The slurry was immediately warmed to 25°C with a water bath and was stirred at this temperature for 1.5 hours. The reaction slurry was then partitioned between 200 ml brine and 250 ml ethyl acetate, and the aqueous phase was washed twice with 100 ml ethyl acetate. The combined organic extracts were washed with dilute aqueous sodium bicarbonate, dried over sodium sulfate, and concentrated in vacuo. The crude product was purified by flash chromatography eluting first with hexane : ethyl acetate = 2 : 1 then ethyl acetate. The product was purified further by flash chromatography eluting first with hexane : ethyl acetate = 2 : 1 then hexane : ethyl acetate = 1 : 2. Yield (pale yellow glass foam) : 698 mg (43%) . 1 H NMR (200 MHz, CDC1 3 ) : <S 2.88 (dd, J 9.4, 13.3 Hz, CHHPh) , 3.41 (dd, J 3.2, 13.3 Hz, CHHPh) , 4.17-4.20 (m, CH 2 0) , 4.64-4.73 (m, CHN), 6.13 (s, CHN 3 ) , 7.21-7.42 (m, 10H, aromatics) , 7.50 (s, 4H, aromatics) . 13 C NMR (50 MHz, CDC1 3 ) : δ 37.9, 55.8, 63.1, 66.6, 127.2, 128.7, 128.9, 129.0, 133.7, 134.1, 152.0, 168.2. c) (S,S) - ,α'-Diazido-1.4-benzenediacetic acid A solution of LiOH H 2 0 (0.222 g, 5.29 mmol) in H 2 0 (8 ml) was added dropwise at 0°C to a solution of (S,S,S,S)-α- α'-diazido-jS, β '-dioxo-3 , β ~ -bis(4-benzyl-2-oxo-3- oxazolidinyl) -1,4-diethylbenzene (0.683 g, 1.15 mmol) in THF (16 ml) . The solution was stirred for 1 hour at 0°C. A dilute solution of aqueous sodium carbonate was added and the oxazolidone was extracted out with ethyl acetate. The organic extract was washed twice with a dilute solution of aqueous sodium carbonate. The combined aqueous extracts were acidified with an aqueous solution of potassium hydrogen sulfate, and the product was extracted out with ethyl acetate (2x) . The solution was dried over sodium sulfate and evaporated. The residue was used in the next step without further purification. Yield: 206 mg (65%) . 1 H NMR (200 MHz, CDCl 3 /DMSO-d 6 ) : . 4.93 (s, CH) , 7.40 (s, Ph) . 13 C NMR (50 MHz, CDCl 3 /DMSO-d 6 ) : _ 67.5, 126.6, 134.0, 170.1. d) (S .S) - .a'-Diamino-1.4-benzenediacetic acid 10% Pd/C (70 mg) was added to a solution of (S,S)-c.,α'- diazido-l,4-benzenediacetic acid (180 mg, 0.65 mmol) in ethanol : acetic acid = 8 : 1 (12 ml) . The resulting reaction slurry was hydrogenated under 15 psi H 2 for 4 hours. The reaction slurry was then filtered through Celite, and the Celite filter cake was washed with several portions of ethanol. The solvent was removed in vacuo. and the acetic acid was removed by azeotropic destination with toluene. The white solid was spinned in ethyl acetate, filtered and dried in vacuo. Yield: 99 mg (68%) . 1 H NMR (200 MHz, D 2 0) : _ 5.16 (s, CH) , 7.48 (s, aromatics) . e) (S,S) -α.α' -Bis (9-fluorenylmethoxycarbonylamino) - 1.4-benzenediacetic acid A suspension of α, _'-diaraino-l,4-benzenediacetic acid (45 mg, 0.2 mmol) in a mixture of hexamethyldisilazane (5 ml) and trimethylchlorosilane (0.5 ml) was stirred under argon at 100°C overnight. The reaction mixture was cooled and excess silylating reagents were removed in vacuo. The residue was dissolved in dry dichloromethane (5 ml) and cooled in an ice/water bath. A solution of 9-fluorenylmethoxycarbonyl chloride (109 mg, 0.42 mmol) in dry dichloromethane (2 ml) was added and the reaction mixture was stirred under argon for 3 hours. Dichloromethane was evaporated and the residue was dissolved in a mixture of THF (4 ml) and water (0.5 ml) . The mixture was stired for 30 minutes and then evaporated. The residue was taken up in ethyl acetate and washed several times with water. The organic phase was dried (MgS0 4 ) , filtered and evaporated. The crude product was purified by flash chromatography eluting with ethyl acetate : acetic acid = 95 : 5. Yield (pale yellow solid) : 48 mg (40%) . 1 H NMR (200 MHz, DMSO-d 6 ) : δ 4.20-4.50 ( , CH and CH 2 ) , 4.88 (s, CHN) , 7.20-8.0 (m, 20H, aromatics) . 13 C NMR (50 MHz, DMSO-d 6 ) : δ 46.8, 65.5, 77.7, 119.9, 127.0, 127.8, 128.1, 128.5, 136.7, 140.5, 143.7, 155.9, 172.8. EXAMPLE 9 Peptide synthesis: (pGlu-Glu-Asp)_-r (m) -Xyll (Lvs)- The peptide is synthesized using a Labortec Peptide Synthesizer. Fmoc-Lys(Boc) -Sasrin polymer (1.0 g, 0.6 mmol; Bachem A.G. ; substitution 0.6 mmol/g) is charged into a 100 ml reaction flask. Fmoc-[ (m) -Xyl] (160 mg, 0.23 mmol) , DCC (290 mg, 1.4 mmol) and HOBt (211 mg, 1.4 mmol) in DMF (20 ml) are added to the polymer and the reaction allowed to proceed for 9 hours. Additional DCC (1.4 mmol) and HOBt (1.4 mmol) are added and the reaction allowed to proceed for another 2 hours. The polymer is then washed with CH 2 C1 2 , with 30% MeOH in CH 2 C1 2 and with DMF. Free amino groups on the polymer are acetylated using 10% Ac 2 0 in DMF (3x over 1 hour; negative Kaiser test) . The rema. ing synthesis is carried out by standard protocol using Fmoc-Asp(OtBu) -Opfp (1.33 g, 2.3 mmol), Fmoc-Glu(OtBu)-Opfp (1.35 g, 2.3 mmol) and pGlu- pentachlorophenyl ester (0.86 g, 2.3 mmol). HOBt (350 g, 2.3 mmol) is added in each coupling step which is allowed to proceed for 1 hour. Completion of the coupling is ascertained by negative Kaiser test. After the coupling with the Fmoc-amino acid the polymer is washed with DMF, the protecting group cleaved off by 20% piperidine in DMF, and the polymer again washed with DMF. After the final coupling the polymer is washed with MeOH/CH 2 Cl 2 and CH 2 C1 2 . The weight of dried polymer- peptide is 1 g. The peptide is cleaved from the polymer by TFA:CH 2 C1 2 1:1, the solution freeze-dried, the residue dissolved in water, filtered (0.45 μ) and the filtrate freeze-dried. For further purification 100 mg of this product is dissolved in water (0.5 ml) and subjected to preparative HPLC using 2.1 x 15 cm Beckman Ultrasphere ODS and solutions A 0.1% TFA in H 2 0, B 0.1% TFA in MeCN:H 2 0 40:60. The gradient is 0-15% B, 120 minutes, flow rate 5 ml/minute. 19 mg of pure peptide (> 95%) is obtained after freeze-drying of the homogenous fractions collected. EXAMPLE 10 Solid-phase synthesis of peptides Solid-phase peptide synthesis was carried out essentially according to the principles of the fluorenylmethoxycarbonyl (Fmoc)-polyamide strategy (Atherton & Sheppard, Solid phase peptide synthesis: a practical approach. Oxford: IRL Press at Oxford University Press, 1989) . Commercially available synthesis resins were used; for batch synthesis either manually or using a semi-automatic instrument (Labortec Peptide Synthesizer 5P 650) these were of polystyrene with acid-labile (Wang, J. Am. Chem. Soc, 95. 1328- 1333. 1973) or acid hyperlabile linkage agents (Merger et al., Tetrahedron Letters 29, 4005-4008, 1988). Alternatively, peptides were assembled in fully automatic mode on flow resins (Atherton et al., J. Che . Soc.. Chem. Commun.. 1151-2, 1981) using an LKB Biolynx 4170 Automated Peptide Synthesizer. Synthesis resins were purchased which already contained the protected desired C-terminal Fmoc-amino acid residue. Chain elongation was achieved variously with side-chain protected Fmoc-amino acid pentafluorophenyl esters (Kisfaludy & Schoen, Synthesis. 325-327, 1983) , using activation with dicyclohexylcarbodiimide (DCC)/1- hydroxybenzotriazole (HOBt) (Koenig & Geiger, Che . Ber. 103, 2034-2040, 1970) or using the coupling reagent PyBOP (Coste et al. , Tetrahedron Lett.. 31. 205-208, 1990) . The lysine side-chain amino group was protected with the t-butyloxycarbonyl function, the side-chain carboxyl groups of glutamic and aspartic acid were protected as the t-butyl esters. Synthesis resin bearing the desired N-deprotected C- terminal residue was acylated with one half equivalent of Fmoc-protected diaminodicarboxylic acid with the aid of DCC and HOBt. After completion of the reaction excess reagents were washed off. The peptidyl resin was then treated once more with DCC/HOBt in order to anchor carboxyl groups which may have remained free. After this step the resin was washed with methanol to deactivate any carboxyl groups unable to be linked with resin-bound amino groups. Finally excess amino groups were capped by acetylation. The peptide synthesis was then continued as usual. After complete solid-phase assembly of the desired sequences, the peptides were cleaved from the synthesis resins with concomitant side-chain deprotection using trifluoroacetic acid to which suitable scavenger chemicals (King et al., Int. J. Peptide Protein Res.. 36 255-268, 1990) had been added. After evaporation, the peptides were isolated by precipitation with diethyl ether and drying. Purification was by preparative reversed phase high performance liquid chromatography. Table 1 Analytical data for peptides with the general structure (Pyr-Glu-Asp) 2 -bridge residue-(Lys-OH) 2 Bridge Residue Type 3 HPLC Method" Purity 0 FAB-MS (Retention time, min) (%) [M+ H] ' 1,2-Benzenedipropionate 1,3-Benzenedipropionate 1,4-Benzenedipropionate 2,5-Thiophenedipropionate cis-l,2-Cyclopropane- dipropionate cis-l,2-Cyclobutane dipropionate trans-l,4-Cyclohexane- 0-30-20 (14) 99 1197.8 dipropionate 1,4-Benzenediacetate a Refer to Examples b The methods are expressed as gradients of mobile phase B in A over time, eg. 10-40-20 refers to a gradient starting at 10 and finishing at 40 % B over 20 minutes. Mobile phases: A) 0.1% TFA B) 0.1% TFA in 40% MeCN. Column: Vydac TP54,C18, 0.46x25 cm,5μm particles,lOOA pore; flow ImL/min c Refers to integration of HPLC chromatogram peaks (λ = 215 nm) Table 2 Amino acid analysis data for peptides with the general structure (Pyr-Glu-Asp) 2 -bridge residue-(Lys-OH) 2 Bridge Residue Type £ 1,2-Benzenedipropionate 1,3-Benzenedipropionate 1,4-Benzenedipropionate 1.0(1) 2.16(2) 0.93(1) 2,5-Thiophenedipropionate ciε-1,2-Cyclopropane- dipropionate cis-l,2-Cyclobutane dipropionate trans-l,4-Cyclohexane- 1-0(1) 2.12(2) 0.81(1) dipropionate 1,4-Benzenediacetate Refer to Examples

Claims: Claims 1. Peptide compounds comprising two single-chain hemoregulatory peptides joined together by a divalent bridging group -A- which is terminally attached to the Cα atoms of amino acids in non-terminal equivalent positions in each of said peptides wherein the native α- si e chains of the Cα atoms attached to group -A- are absent, wherein said divalent bridging group -A- is -(CH 2 ) y -(Y) z -B-(Y) z -(CH 2 ) y - (wherein each y is independently 0, 1 or 2; each z is independently 0 or 1; each Y is independently 0, S or NR, where R represents hydrogen or a C-attached organic group; B represents carbocyclic or heterocyclic ring optionally containing one or two heteroatoms and optionally mono-, di- or tri-substituted by a group -OR , -NR A R A , -C00R A or a halogen atom; and each R A independently represents a hydrogen atom, or alkyl, alkanoyl or alkoxyalkyl groups each of which may also be hydroxylated) . 2. Peptide compounds as claimed in claim 1 wherein said divalent bridging group -A- is -CH 2 CH 2 BzCH 2 CH 2 - (wherein Bz represents a benzene ring which is optionally mono-, di- or tri-substituted by a group -OR A , -NR A R A , -COOR A or a halogen atom, where R A is as defined in claim 1) . 3. Peptide compounds as claimed in either of claims 1 and 2 wherein said single-chain hemoregulatory peptides are of formula I R a - R b - R c - R d - (R e ) n - R (I) wherein R a represents - R represents -NH— CH— CO — -NH— CH— C0- I I CH 2 OH (CH 2 ) r or COR 6 R c represents COR 7 R d represents R e represents R f represents -NH —NH—CH 2 —COR 10 (wherein n and m independently represent 0 or 1; p, q and r independently represent 1 or 2; s represents 3 or 4; R 1 and R 2 are both hydrogen atoms or together represent an oxo group; R 3 and R 4 are both hydrogen atoms or together represent a carbon-carbon bond; R 5 is hydrogen or an acyl group; each R 6 and R 7 independently represent a hydroxy group or an amino group, but are preferably hydroxy groups, R 8 represents hydrogen; a C 2.6 alkyl group; a C 7 . 20 aralkyl group, which may carry one or more hydroxy, amino or methoxy substituents; or a metabolically labile S-protecting group; R 9 represents hydrogen or a methyl group; and R 10 represents a hydroxy or amino group, the residue of the amino acid glutamine or a peptide having an N- terminal glutamine unit) . 4. Peptide compounds as claimed in claim 3 wherein in a peptide chain of formula I n represents 0. 5. Peptide compounds as claimed in either of claims 3 and 4 wherein in a peptide chain of formula I m represents 0. 6. Peptide compounds as claimed in any one of claims 3 to 5 wherein said bridging group -A- is attached to the Cα atoms of amino acids R d of formula I. 7. Peptide compounds as claimed in any one of claims 3 to 6 of formula II l a - R - R c - NH - CH - CO ~(R e ) n - R f R a - R b - R c - NH - CH - CO ~(R e ) n - R f wherein -A- is as defined in claim 1 and R a , R b , R c , R e , R f and n are as defined in claim 3. 8. Peptide compounds as claimed in any one of claims 1 to 7 of formula pGlu-Glu-Asp-NH-CH-CO-LysOH pGlu-Glu-Asp-NH-CH-CO-LysOH wherein -A- is as defined in claim 1. 9. A pharmaceutical composition comprising a peptide compound as claimed in any one of claims 1 to 8 together with a pharmaceutical carrier or excipient. 10. Peptide compounds as claimed in any one of claims 1 to 8 for use in stimulating cell division. 11. Peptide compounds as claimed in any one of claims 1 to 8 for use in stimulating myelopoiesis or regeneration of bone marrow. 12. Use of peptide compounds as claimed in any one of claims 1 to 8 in the manufacture of a medicament to stimulate cell division. 13. Use as claimed in claim 12 in the manufacture of a medicament to stimulate myelopoiesis or regeneration of bone marrow. 14. Use of peptide compound as claiimed in any one of claims 1 to 8 for the stimulation of cell division. 15. Use as claimed in claim 14 for the stimulation of myelopoiesis or regeneration of bone marrow. 16. Method of stimulating cell division in a patient, said method comprising administering to said patient an effective amount of a composition as claimed in claim 9. 17. Method as claimed in claim 16 wherein division of myelopoietic or bone marrow cells is stimulated. 18. Process for producing a peptide compound as claimed in any of claims l to 8 comprising deprotecting a partially or fully protected derivative thereof. 19. Process for producing a peptide compound as claimed in any one of claims 1 to 8, said process comprising a) metallating and subsequently alkylating a bis- lactim ether to form a bis-lactim dipeptide ether; b) hydrolysing a bis-lactim dipeptide ether of step (a) to form a bridged α,α'-diamino acid; c) introducing the remaining amino acids in the peptide chains; and d) deprotecting any protected group. 20. Bridged _,_'-diamino acids of formula NH, NH, HC - A - CH HOOC COOH wherein -A- is as defined in claim 1. 21. Bis-lactim dipeptide ethers of formula wherein -A- is as defined in claim 1 ,

Inventors: SOLBAKKEN MAGNE, UNDHEIM KJELL, SOLBAKKEN, MAGNE, UNDHEIM, KJELL
Applicants: HAFSLUND NYCOMED AS, HOLMES MICHAEL JOHN, SOLBAKKEN MAGNE, UNDHEIM KJELL, HOLMES, MICHAEL, JOHN
Application year: 1993
Main classification: C07K7
IPC codes: A61K38, A61P7, A61P43, C07C229, C07D241, C07D333, C07K1, C07K7