LT3614B - Peptide compounds, method for the preparation and use thereof - Google Patents

Abstract

The invention reveals dipeptide compounds, where two peptide chains are jointly connected to C atom of a non-rear amino acid through two-valent group -A- connecting by means of the bridge. C α-atoms connected to group -A- are laid out in equivalent positions in every mentioned peptide chain and every atom has no native α sided chain.The revealed bridge dipeptide compound has a stimulation effect for cell division, in particular for the cells of myelopoesis and bone marrow.

Description

The present invention relates to the use of peptides that stimulate cell proliferation and to novel peptides having a specific and / or general cell-stimulating effect.

The mammalian body is made up of cells that are very different in structure and function, and the mechanisms of cell differentiation and development have been the subject of much research. It is known that a system of continuously regenerating cells usually has a reservoir of pluripotent stem cells that divide and continuously supply new cells to the system. Although the stem cells supplied from the reservoir are initially identical, they soon undergo some form of morphological alteration and subsequently develop into cells with the required function.

Examples of these stem cell systems are the bone marrow hematopoietic system and the epithelial and epidermal systems.

The management or regulation of stem cell division is a very promising field of therapy, and so much of the research remains focused on elucidating the mechanisms and finding specific chemical mediators. To date, several molecules that are truly involved in cell production and differentiation have been identified that stimulate or inhibit a particular process chain. In this regard, myelopoiesis has been particularly well studied and its molecules have been identified: colony stimulating factor (CSF), such as granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor ( GM-CSF), multi-origin colony stimulating factor (multi-CSF; IL-3)

11th

[see Metcalf, Science 2_29: 16 (1985) 1, interleukin (IL-11) [cf. Paul et. al., Proc. Natl. Acad Sci.,

USA, 87: 7521 (1990)], lactoferrin [cf. Broxmeyer et al., Blood Cells, 11: 429 (1989)], prostaglandins [cf. Pelus et al., J. Immunol. 140: 479 (1988)], acidic (H-subunit) ferritin [cf. Broxmeyer et al., Blood 68: 1257 (1986)], interferons (α, β and γ) [cf.

Pelus et al., Supra, and Broxmeyer et al., J. Inununol., 131: 1300 (1983)], tumor necrosis factors (a and β) [cf. Broxmeyer et al., J. Immunol. 136: 4487 (1986)], β-transforming growth factor [cf. Ottrnan et al., J. Immunol., 140: 2661 (1988)], and activin and inhibin [cf. Broxmeyer et al., Proc. Natl. Acad. Sci., USA, 86: 779 (1989)].

Hemorrhagic pentapeptide (pEEDCK) has also been found to selectively inhibit the proliferation of myelopoietic cells [cf. Paukovits et al., Z. Naturforsch 37: 1297 (1982)], and other peptides corresponding to a narrow general formula have been found to have a similar inhibitory effect in haemopoiesis [see, e.g. EP-A-112656 and WO90 / 02753]. Oxidation of peptide monomers gave dimeric molecules linked by a cysteine bridge and those dimeric molecules were found to stimulate myelopoiesis. Laerum et. al., Exp. Heinatol., 16: 274 (1988)].

(pEEDCK) 2 dimer and other related compounds are disclosed in WO-A-88/03535. Other dimeric peptide compounds are disclosed in EP-A-408371 in which the disulfide bond has been replaced by a carbon or a carbon / sulfur bridge linking selected peptide chains. The bridge is thus relatively resistant to hydrolysis but is itself inert and cannot participate in receptor dimer interactions.

Although we do not wish to be bound by theoretical considerations, it is now believed that such peptide compounds interact with stromal cells in vivo and that stromal ί ι t

cells are responsible for stimulating or inhibiting cell division by other elucidating factors. Thus, dimers are believed to induce or accelerate the production of stimulatory cellular regulatory factors in the stroma, whereas monomeric peptides may either inhibit this process or induce the production of factors that prevent or interfere with cell division. Thus, in this approach, stromal cells may potentiate the stimulatory or inhibitory effects of dimers and monomeric peptides, respectively.

There is a continuing need for dimeric peptide compounds capable of stimulating cell proliferation to normal levels in vivo. In this regard, it should be noted that different degrees of stimulation may be more appropriate for certain clinical situations than others, and selective stimulation of individual cell types is particularly important.

The present invention provides a peptide compound consisting of two single-chain hemoregulatory peptides, in particular inhibitors of haemopoiesis, linked together by non-amino acids at Ca equivalent at each of said peptides via a carbon-carbon bond or a terminal divalent bridging group -A-, the native α-side chain is absent at said Ca atoms, where A is a Ο _χ _ ₆ carbon chain that is

1) a) mono- or poly-substituted with -R ^A , -OR ^A , -S ^{A A} , -NR R ^a or -COOR ^A or with a halogen atom such as fluorine, chlorine, bromine or iodine (where each R ^A independently represents a hydrogen atom or Ο ₇ _ ₆ alkyl, or alkanoyl -alkoksialkilo group which may be mono- or poly-hydroxylated) and / or

(b) containing one or more double or triple carbon-carbon bonds and / or

c) interrupted by one or more groups -Z-, where each -Z- is independently -0-, -CO-, - NR ^B, or -NR ^b - (wherein each R B is independently vandeilio atom or a C, _ ₆ alkyl or C ₆ _ ₁₀ aryl group), wherein one or more substituents or unsaturated carbon-carbon links are the group -A-, -Z- may also be -S-or ii); (CH _; ) ', wherein y is 1, 5 or 6.

Preferably, the total number of atoms in the bridging group -A- is not greater than 6.

Where the group R ^A represents an alkyl, alkoxy or alkanoyl group, each may be a linear or branched chain. Suitable alkyl groups include methyl, ethyl, 2-hydroxyethyl, propyl, butyl, 1,3-dihydroxybutyl, 2-methyl-3-hydroxybutyl, and pentyl. Methoxy, ethoxy, propoxy and 2-hydroxypropoxy are all suitable for the alkoxy group R ^A , while the alkanoyl group may, for example, be formyl, acetyl, propionyl.

For R, as the alkyl group, the alkyl carbon chain may be linear or branched, and the groups mentioned above for R ' ¹ are also suitable herein.

Suitable group A skeletal structures (which may be further modified as above) thus include:

-CH ₂ -ch ₂ ch ₂ ch ₂ ch ₂ ch ₂ LT 3614 B

-ch ₂ ch ₂ ch ₂ ch ₂ ch ₂ ch ₂ -HC = CH-HC = C = CH-ch ₂ -ch = ch-ch ₂ —ch ₂ ^_ c = c — ch ₂ -

-ch ₂ -ch = ch-ch = ch-ch ₂ -ch ₂ -ch ₂ -c = c-ch ₂ -ch ₂ -ch ₂ -c = cc = c-ch ₂ ch ₃ ch ₃

I I

-ch-ch = ch-ch ₂ -chOH OH

II ~ CH-CH = CH-CH-CH ₂ -O-CH ₂ -CH ₂ -NH-CH ₂ CH ₂ CH ₂ CH ₂ -CO-CH ₂ CH ₂

-CH-CH = CH-CHI I och ₃ och ₃

-ch ₂ -ch = ch-ch ₂ -s-ch ₂ -CH ₂ -CsC-CH ₂ -NH-CH ₂ CH ₂ - or

-CH ₂ -CH = CH-CH (COOH) ch ₂ Any single chain peptide that exhibits hemoregulatory activity is suitable as a peptide that is bridged according to the present invention.

Alternatively, the present invention provides compounds of the present invention wherein said hemoregulatory peptide chains include the following formulas:

R ^a - R ^b - R ^c - R ^d - (P ^e ) _n - R ^f where R ^a stands for

arbe r nh - c :: - co.

<ch> i ² p con aroa

CH --- Coo

I

CH OH

R stands for • NH -CH -

CO cch ^>

² 'IA COR. NU-CH - CO.

I

CH. OH or

R stands for. ni: —-- ch -. co tCH>

ί V

COR. NH - CH - CO

I cn, on or

R stands for

- NH

CH ~ - CO

-NH ·

CH-CO

I

CH.

NH

CH SR Arbi

CH CO -

I

CH OH

R ^e means

-NH- CH- COF

R * stands for

GOK

I

CH,,>

-NH · or

- CH

I (CH J

CCS

NH CH-CCR ⁱ⁰

NH

NH ai'ba

NH

NH (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 or together represent an oxo group;

R ³ and R ^{4 are} both hydrogen or taken together to represent a carbon-carbon bond;

R ⁵ is hydrogen or acyl;

each R ⁶ and R ⁷ independently represents a hydroxyl group or an amino group, but hydroxyl groups are more preferred;

J I;

g

R ⁸ represents hydrogen, C ₂ ,. Alkyl group of ₆ , C ₇ . ₂ o aralkyl group which may carry one or more hydroxy, amino or methoxy substituents; or a metabolically labile S-protecting group;

R represents hydrogen or a methyl group; and

R ^J ° represents a hydroxyl or amino group, an amino acid glutamine residue or a peptide containing an N-terminal glutamine moiety).

All of the above amino acid residues may be in either the D or L form. However, the L form of amino acids is more preferred.

Where the N-terminal protecting group R 0 is present, it may be, as noted above, an acyl group having 1-20 carbon atoms, e.g., a lower alkanoyl group having 1 to 5 carbon atoms such as an acetyl group, or an aroyl or the aralkanoyl group having from 7 to 20 carbon atoms flows as a benzoyl or phenylacetyl group.

R ⁵ may also be an acyl group derived from an amino acid or peptide chain. Specifically, 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 consecutive N-terminal amino acids:

Lys-Ile-Ile-His-Glu-Asp-Gly-Tyr-Ser.

It is preferred that the terminal amino group of the peptide of general formula (I) is protected, for example, by acylation with an alkanoyl, aralkanoyl or aroyl group.

Where R ⁸ is a C ₂ -C ₆ alkyl group, it may be, for example, ethyl, butyl or hek? o the group. When R is an aralkyl group, it may be convenient; be an arylmethyl group such i

such as benzyl, diphenylmethyl or triphenylmethyl. Where R ⁸ is a metabolically labile group, it may, for example, be an arylthio group having 5 to 10 carbon atoms, such as pyridylthio or acyl group as defined above.

More preferred compounds of the present invention are pentapeptides, i.e. n is better 0.

Preferably, the residue R ^{a of the} cyclic group has five members, i.e. m is better 0.

Although any of the peptides defined by Formula (I) above have low or negligible hemoregulatory activity, they may nonetheless be an effective bridge form of the present invention in stimulating cell proliferation.

In dimeric peptides consisting of peptide chains as depicted in formula (I), a linking chain point is desirable at R ^d .

Particularly suitable peptide compounds of the present invention are those compounds of formula (II)

R ^a - R ^b - R ^c - NH - CH - CO - (R ^e ) _n - R ^f

I

A (II),

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 as defined above , and the group -NH-CH-CO- is a derivative of R ^d which is attached to the bridging group -A- such that it does not have its own side chain.

n

One particularly suitable peptide compound of formula (II) is pGlu-Glu-Asp-NH-CH-CO-LysOH

I

A (II),

I pGlu-Glu-Asp-NH-CH-CO-LysOH wherein -A- is a divalent bridging group as discussed above.

The present invention is particularly suitable for stimulating myelopoiesis in patients suffering from diminished myelopoietic activity, including bone marrow injury, agranulocytosis, and aplastic anemia. This includes treatment of patients with reduced bone marrow function due to immunosuppressive therapy to suppress tissue response - i.e. in bone marrow transplant surgery.

The compounds may also be used to accelerate bone marrow regeneration following cytostatic chemotherapy and radiation therapy for neoplastic and viral diseases.

In addition, novel compounds can be of great value where patients have serious infections due to lack of immunoreaction after bone marrow injury.

Another clinical application will be in combination with the appropriate monomers or related myelopoiesis inhibitors as disclosed in EP-A-112656. or WO-A-90/02753, induce variable high and low activity peaks in bone marrow cells, thereby enhancing the natural circadian rhythm of hemopoiesis. Thus, cytostatic therapy can be used during periods of low bone marrow activity, thereby reducing the risk of bone marrow damage

while regeneration will be accelerated by successive activity peaks.

In general, to achieve a stimulatory effect, the peptides of the present invention may be administered to a patient by the oral route or by injection at a daily dose of 0.001-100 mg, e.g. 1-5 mg per 70 kg body weight. For intravenous or subcutaneous administration, the dose may be in the range of 1-10 mg for 70 kg body weight per day, for example about 6 mg, for up to 10 days. Nasal, topical (transdermal), or rectal prescription is also, of course, flexible. Basically, it is necessary to produce peptide concentrations of about 10 M to 10 M in the patient's extracellular fluid.

Further aspects of the present invention provide pharmaceutical compositions comprising, as active ingredient, one or more compounds of formula (I) as hereinbefore described, or a physiologically compatible salt thereof, in a pharmaceutically acceptable carrier or excipient. The compositions of the present invention may be presented, e.g., in a form suitable for oral, nasal, parenteral or rectal administration.

As used herein, the term pharmaceutical includes veterinary applications of the present invention.

The compounds of the present invention may be in the form of conventional dosage forms such as tablets, coated tablets, nasal aerosols, solutions, emulsions, powders, capsules or sustained release formulations. Conventional pharmaceutical excipients, as well as conventional manufacturing techniques, can be used to prepare these forms. The tablets may be made, for example, by mixing the active component or components with known excipients such as diluents such as calcium carbonate, calcium phosphate or lactose, disintegrating agents such as cereal starch or alginic acid, binders,

II like starch or gelatin, smear! 3c s magnesium stearate or Ico masonry with a poly (i) n, a c o r t i o n, or a polyvinylacetate.

such as carbohydrate

The tablets may consist of several layers, if necessary. Coated tablets may be made by coating the middle, similar to tablets, with agents commonly used to coat tablets, such as polyvinyl pyrrolidone or belac, gum arabic, talcum powder, titanium dioxide or sugar. The middle may consist of several layers in order to obtain sustained release or to avoid incompatibility. The tablet coating may also consist of several layers to provide sustained release, in which case the above-mentioned tablet fillers may be employed.

Organic specific carrier systems may also be utilized.

Injection solutions may be prepared, for example, by conventional means such as the addition of preservative agents such as hydroxybenzoates or stabilizers flowing as EDTA. The solutions are then filled into injection vials or ampoules.

Non-gaseous aerosols may be similarly formulated in aqueous solution and packaged in spray containers, either with an aerosol ejector or with hand-held means. Capsules containing one or more active components may be prepared, for example, by mixing the active components with inert carriers such as lactose or sorbitol and filling the mixture into gelatin capsules.

Suitable suppositories may, for example, be prepared by mixing the active ingredient or combinations of active ingredients with conventional carriers for this purpose, such as natural fat or polyethylene glycol or derivatives thereof.

Suitable dosage units containing the compounds of the present invention contain from 0.1 to 10 mg, for example 1-5 mg of the peptide of formula (I) or a salt thereof.

Thus, the present invention provides peptide compounds and mixtures as described above for use in stimulating cell division. The use of peptide compounds of the present invention in the manufacture of a medicament for stimulating cell division is a further aspect of the present invention. Myelopoiesis and bone marrow regeneration are of special importance.

However, another major use of the new peptides is the production of materials for immunoassay techniques. yeah. The peptide can then be covalently attached to a suitable large molecular carrier such as albumin, polylysine or polyproline for introduction into antibody producing animals (e.g. rabbits, guinea pigs, or goats). In vitro immunization techniques may also be employed. High specificity antisera are prepared using well-known absorption techniques using a large molecular carrier. by introducing radioactivity ( ³ H, ¹²⁵ I, ¹⁴ C, ³⁵ S) into a peptide molecule, radioimmunoassay can be designed and used to detect the peptide in various biological fluids such as serum (plasma), urine, and cerebrospinal fluid.

The peptides of the present invention can be synthesized in any convenient manner. Suitable methods for forming amino acid units are described, for example, in Synthesis of Optically

II .1.

Active α-Amino Acids (Synthesis of Optically Active α-Amino Acids), Robert M. Williams (Pergamon Press, 1989). In general, the reactive side chain groups (amino, thiol and / or carboxyl) will be protected throughout the fusion coupling reactions, but some side chain groups may be left unprotected (hydroxyl groups, imidazole groups, primary amide groups, cyclic amino acids such as pyroGlu). ) throughout the synthetic procedure.

Thus, the final step will be the blocking of a fully protected or partially protected peptide derivative of general formula I, and these processes form a further aspect of the present invention.

Schollkopf et al. described the preparation of various amino acids by metallization of bis-lactim ethers followed by alkylation (see, e.g., Tetrahedron 39: 2085 (1983) and Topics

Curr. CHEM., 109: 65 (1983). Application of this method has proven to be particularly useful in the preparation of the bridged amino acids which form the basis of the present invention. Specifically, the bis-lactim ether derived from the valinoglycine dipeptide forms a useful starting compound in the bridging reaction which can be summarized as follows:

OCH ,,

NH.

NH ^

I

CH

I

CH.

-Α-Χ

HG --- Λ

COOH

COOH

-78 ° C

CH ..

CH

The ι ι

II, ι

(wherein X is a leaving group such as a halogen atom such as bromine).

The bridged (S, S) -α, α'-diamino acids can be obtained by this method if D-valine is initially used to form the bis-lactim ether. Similarly, (R, R) -α, α'-diamino acids can be formed using Lvaline.

An alternative technique useful for the preparation of the α, α'-diamino bridges which form the basis of the bridged dipeptide compounds of the present invention is the application of the method developed by Vederas et al. (J. Am. Chem. Soc. 107: 7105 (1985) and J. Am. Chem. Soc. 110: 2237 (1988). Based on this alternative procedure based on ring opening reactions of β-lactones derived from serine, β-lactones can be subjected to a series of delicate nucleophiles which cause degradation of the lactone alkyl-oxygen bond. Aza-substituted bridging amino acids comprising oxa and thia analogs may be synthesized according to the procedure illustrated in the following scheme:

II f

For the titanium dipeptide compounds of the present invention, wherein -A- is -CH ₂ -, reference may be made to Belokon et al. Okay. Nauk. USSR. Ser Khim, (1987), 852 (Chemi na. Abstracts 108: 132255v).

In this respect, the present invention also provides a process for preparing a peptide compound comprising partially or completely blocking its derivative.

In another aspect, the present invention provides a process for preparing a peptide compound which comprises:

a) bis-lactation ether metallization and sequential alkylation · to form the bis-lactim dipeptide

-ether;

b) hydrolyzing the bis-lactim ether of step a) to obtain the α, α'-dramic acid bridged bridge;

introducing into the peptide chains the remaining amino acids; and

(d) deploying any protecting group.

bis-lactim dipeptide ethers and bridging acid © c ,, ct '' - a diamino acid prepared by this method constitute a further aspect of the present invention.

Once the bridge dipeptides are formed, the remaining amino acids in the peptide chain can be introduced by conventional methods.

For peptide chains, one can start from either the C-terminus or the N-terminus, although only the C-terminal procedure is generally used.

, ι

Thus, the reaction of a properly protected C-terminus, such as lysine, with a properly protected cysteine derivative can be initiated. The lysine derivative will have the free α-amino group, while the other reagent will have either the free or activated carboxyl group and the protected amino group. After coupling, the intermediates can be purified, e.g., by chromatography, and then selectively N-protected to add a free or activated amino acid residue. This procedure is continued until the required amino acid sequence is formed.

Carboxylic acid activating substituents which may, for example, be used include symmetric or mixed anhydrides or activated esters such as p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, N-hydroxybenzotriazole ester (OBt). ), N-hydroxysuccinimidyl ester (OSu) or pentafluoro-phenyl ester (OPFP).

The coupling of the free amino and carboxyl groups may, for example, be accomplished using dicyclohexylcarbodiimide (DCC). Another coupling agent that may be used, for example, is N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

In general, coupling reactions at low temperatures, e.g., -20 ° C to room temperature, conveniently in a suitable solvent system such as tetrahydrofuran, dioxane, dimethylformamide, methylene chloride, or a mixture of these solvents are convenient.

Synthesis on a solid-phase resin carrier may be more convenient. Chloromethylated polystyrene (stitched with 1% divinylbenzene) is one useful type of carrier; in which case, the synthesis will begin at the C-terminus, e.g., by attaching the N-protected lysine to the carrier.

r

A number of suitable solid phase methods are described by Eric Atherton, Christopher J. Logan, and Robert C. Sheppard, J. Chem. Soc. Perkin I, 1981, 533-46; James P. Tam,

Foe S. Tjoeng, and R.3. Merrifield, J. Am. Chem. Soc., 102, 6117-27 (1980); James P. Tam, Richard D. Dimarchi and R.3. Merrifield, Int. J. Peptide Prctein Res 16, 412-25. (1980); .Manfred Mutter and Dieter Bellof, Helvetica Chimica Act, 67, 2009-16 (1984).

The coupling reactions can also be performed in solution.

A wide variety of amino acid protecting groups are known and exemplified by Schroder, E. and Lubke, 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 Organic 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 Francisco, London; Solid Phase Peptide Synthesis 2nd ed., John M. Stewart, Janis D. Young, Pierce Chemical Company.

Thus, for example, amino protecting groups which may be employed include protecting groups such as carbobenzoxy (Z-), t-butoxycarbonyl (Boc-), 4-methoxy-2,3,6-trimethylbenzene sulfonyl (Mtr-). ) and 9-fluorenylmethoxycarbonyl (Fmoc-).

It will be understood that when the peptide is composed of a C-terminus, the amino protecting group will be in the α-amino group of each new residue added and will need to be selectively removed before the next coupling step. For solid phase systems, one particularly useful group for such a temporary period

I amine protectors are an Fmoc group that can be removed selectively by treatment with piperidine in an organic solvent. For the synthesis in solution, Boc- is a more suitable protecting group which may be introduced and removed by conventional means.

Amino acids or peptides often require silylation before protection, for example by adding Fmoc to improve their solubility in organic solvents. Silylation and Fmoc protection reactions are summarized below

COOH

A ^CGH 3 ^> 3 ^{S 1 HN} - ^CH -GOOS1 <CH,) ₃

A -—

I CCHiSICl v>

H ₂ N-CH-COOH

Pmoc-Cl <li> H ^ O

FmocN-CH-COOH

FmocN-CH-COOH

Carboxyl protecting groups which may be used, for example, include cleaved ester groups such as benzyl (-OBZI), p-nitrobenzyl (-ONB) or t-butyl (-tOBu) as well as linkages on a solid support, e.g. methyl groups attached to polystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt), and acetamidomethyl (Acm).

It will be appreciated that there are many other groups such as those described in detail in the above liτ

T

T

II, and the use of all such groups in the processes described hereinabove is within the scope of the present invention.

There are many procedures for removing amino and carboxyl protecting groups. However, they must be compatible with the fusion strategy used. The side-chain protecting groups must be resistant to the conditions used to remove the temporary γ-amino protecting groups before the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups such as toBu can be removed simultaneously by treatment with an acid such as trifluoroacetic acid. Thiol protecting groups such as Trt can be removed selectively by utilization

oxidizing agent, such as j skins. Cysteine having peptides can be synthesized indeed, described in the text, of all protecting groups

removal, including thiolc protecting groups as the final step in the synthesis.

The following examples are provided for illustrative purposes only.

EXAMPLE (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) - (E) oct-4-eno-1,8-dicarboxylic acid

a) 1,4-Bis ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (E) -but-2-ene to stirred (2R) - A solution of 2,5-dihydro-3,6-dimethoxy-1-zopropylpyrazine (5.53 g, 30 mmol) in THE (100 mL) at

-78 ~ C, 1.55 M butyl lithium (19.62 ml,

TT ί ι

II mmol) in hexane and stirring was continued for 1 hour at -78 ° C. A solution of (E) -1,4-dibromobut-2-ene (3.21 g; 15 mmol) in THF (20 mL) was then added and stirring was continued overnight. The solvent was removed under reduced pressure and the residue was dissolved in diethyl ether and extracted with water. The organic layer was dried over magnesium sulfate, the ether evaporated and the residue purified by flash chromatography (ethyl acetate / hexane 1/4; silica gel).

Yield 4.73 g (75%), a pale yellow liquid.

¹ H NMR (CDCl ₃ ): δ

0.67 (d, 6H), 1.04 (d, 6H), 2.1-2.4 (m, 2H), 2.48 (dd, 4H), 3.67 (s, 6H), 3.68 (s, 6H), 3.93 (dd, 2H) , 4.03 (dd, 2H), 5.35 (dd, 2H).

¹³ C NMR (CDCl ₃ ): δ

16.52, 19.07, 31.65, 37.09, 52.16, 52.23, 55.62, 60.60, 127.50

C ₂₂ H ₃₅ N ₄ O ₄ (420)

Final%: C: 62.86; H: 8.57 N: 13.33

Found: C: 62.65; H: 8.62 N: 12.91

b) Dimethyl ester of (S, S) -2,7-diamino- (E) -oct-4-eno-1,8-dicarboxylic acid

To 1,4-bis ((2R, 5S) -2,5-dihydroxy-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (E) -but-2-ene (4.02 g, 9.9 mmol) and 0.5 N HCl (40 mL, 20 mmol) in 40 mL dioxane were added and the solution was stirred at room temperature for 4 hours. It was extracted with diethyl ether, and aqueous ammonia was added to the aqueous solution until

pH was reached 9. The aqueous phase was then extracted with chloroform and the organic layer was dried over magnesium sulfate. After removal of the solvent, the solution was redissolved at 30-40 ° C (0.04 mbar), separating the valine methyl ester. The non-distilled title compound was used without further purification.

Yield: 1.92 g (84.3%), yellow oil.

¹ H NMR (CDCl ₃ ): δ

1.70 (s, 4H), 2.20 - 2.60 (m, 4H), 3.51 (dd, 2H), 3.69 (s, 6H), 5.46 (dd, 2H).

¹³ C NMR (CDCl ₃ ): 6

37.99, 52.00, 54.11, 128.84, 175.32

c) (S, S) -2,7-Diamino- (E).-oct-4-eno-1,8-dicarboxylic acid dihydrochloride (S, S) -2,7-Diamino- (E) -oct-4 The dimethyl ester of -eno-1,8-carboxylic acid (1.65 g, 7.17 mmol) was refluxed with 6 N HCl (10 mL, 60 mmol) for two hours. The solvent was then evaporated, the residue dissolved in water (10 mL), and ethanol (100 mL) was added. The white crystals were filtered off and dried in vacuo at 40 ° C.

Yield 1.32 g (67%); white crystals.

¹ H NMR (D ₂ O): 5

2.53 (dd, 4H), 3.93 (dd, 2H), 5.49 (dd, 2H).

¹³ C NMR (ϋ ₂ Ο): δ ι

.11.

I

33.57, 53.07, 128.49, 171.49

C ₈ H ₁₆ N ₂ O ₄ C 1 ₂ (275)

Shit. %: C: 34.91; H: 5.81; N: 10.18; Cl: : 25.78 % Found C: 36.58; H: 5.80; N: 9.70; Cl: : 26.01

FAB-MS signal at m / z

405.3 (11), 203.2 (100), 157.1 (19), 130.1 (7), 93.0 (18),

73.9 (13).

d) (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) -E) oct-4-eno-1,8-dicarboxylic acid

Acid Hydrochloride - (S, S) -2,7-Diamino-E-oct-4eno-1,8-dicarboxylic acid dihydrochloride (1.59 g, 5.8 mmol) was suspended in hexamethyldisilazane (20 mL) and 1 mL of trimethylsilyl chloride was added. The suspension is then refluxed overnight. The solvent was removed under reduced pressure and the residue was dissolved in anhydrous methylene chloride. This solution was cooled to 0 ° C and a solution of 9-fluorenylmethyl chloroformate (3.10 g, 12 mmol) in methylene chloride was added. The solution was stirred for 1 hour before being removed from the cooling bath. The next morning, the solvent was evaporated and the residue was dissolved in THF. The mixture was then quenched with 1N aqueous HCl and the solution was stirred for 2 hours. The organic solvent was removed and the aqueous phase was extracted with chloroform. The organic layer was dried over magnesium sulfate and evaporated. The residue was dissolved in methylene chloride and precipitated with ether.

Yield: 2.50 g (67%); white crystals.

Ti

II

NMR (DMSO): δ 2.2-2.4 (s, 4H), 3.6 (s, 2H), 1 5.9-4 i. 5 (m, 8H), 5.55 (s, 2H), 7. 2-8.1 (m, 16H). ¹³ C NMR (DMSO): δ 33.98, 46.56, 53.93, 65.43, 119. 66, 124.81, 126. .61, 127.17, 127.98, 140.19, 143.28, 155 .43 , 172.59

^ 33 ^ 34 ^ 2θε (646)

Shit. %: C: 70.59; H, 5.26; N: 4.33

Found: C: 70.17; H: 5.58; N: 4.08

FAB-MS signal at m / z

661.3 (3), 647.4 (3), 191.2 (17), 179.2 (100), 165.1 (18),

78.9 (31).

EXAMPLE (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) - (Z) oct-4-eno-1,8-dicarboxylic acid

a) 1,4-Bis ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (Z) -but-2-ene (2R) - (- ) -2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (1.3 mL, 6.4 mmol) was dissolved in dry THF (20 mL) and cooled to -78 ° C. BuLi (1.6 M in hexane, 3.9 mL, 6.4 mmol) was added dropwise and the solution was stirred for 30 min. Cis-1,4-dichloro-2-butene (0.32 mL, 3 mmol) was dissolved in dry THF (10 mL) and added all at once at -78 ° C. The reaction mixture was stirred overnight under N ₂ . The reaction was quenched with ammonium chlo-. (3 mL), the mixture was evaporated. The residue was extracted ι

1!

diethyl ether, and the organic phase was washed twice with water. It was then dried over MgSO ₄ and evaporated. Purification is carried out on silica gel (heptane: EtOAc / 2: 1).

Yield: 0.81 g (60%), yellowish oil.

PSG (thin layer chromatography) (heptane: EtOAc / 2/1): Rf = 0.39

HPLC (high performance liquid chromatography) (50-100% MeOH, 10 minutes): Rt = 6.75 minutes.

¹ H NMR (CDCl ₃ ): δ

0.66 (d, 6H), 1.02 (d, 6H), 2.55 (m, 2H), 2.66 (m, 4H), 3.66 (s, 6H), 3.70 (s, 6H), 3.90 (t, 2H), 4.01 (m, 2H), 5.39 (t, 2H)

b) Dimethyl ester of (S, S) -2,7-diamino- (Z) -oct-4-eno-1,8-dicarboxylic acid

1,4-Bis ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (Z) -but-2-ene (0.58 g, 1.23 mmol) was dissolved in dioxane ( 8 mL) and 0.25 M HCl (8 mL, 2.5 mmol) was added and stirred overnight at room temperature. PSG (thin layer chromatography) (MeCN: MeOH: H ₂ O / 4: 1: 1). The solution was extracted with diethyl ether and the aqueous phase evaporated. The residue was dissolved in a small amount of water and the aqueous ammonia solution was added until pH 9 was reached. The aqueous phase was extracted twice with diethyl ether and the organic phase was dried over MgSO ₄ and evaporated.

Yield: 0.6 g, yellow oil

I

T ι

i.

c) (S, S) -2,7-Diamino- (Z) -oct-4-eno-18-carboxylic acid dihydrochloride

To the mixture of (S, S) -2,7-diamino- (Z) -oct-4-eno-1,8-carboxylic acid dimethyl ester (0.6 g, 1.3 mmol) was added almost. HCl (1 mL) and dioxane (10 mL) were added and the solution was refluxed for 2 hours then evaporated.

Yield 0.6 g, white solid.

NMR (D ₂ O): 3

0.88 (2d, 6H), 2.15 (m, 1H), 2.57 (2d, 4H), 3.68 (m,

1H), 3.86 (m, 2H), 5.51 (m, 2H).

d) (S, S) -2,7-Bis (9-Fluoro-phenylmethyloxycarbonylanino) (Z) -oct-4-eno-1,1'-dicarboxylic acid

The acid hydrochloride from step c) (0.6 g, 2 mmol) was suspended in hexamethyldisilazane (HMDS) (12 mL) and trimethylsilyl chloride. The (TMS-Cl) suspension was refluxed overnight under nitrogen. Excess HMDS and TMS-Cl were distilled in vacuo. The residue was dissolved in dry methyl chloride and cooled to 0 ° C. A solution of fluorenylmethyl chloroformate (712 mg, 2.8 mmol) in dry methyl chloride was added and the mixture was stirred for 2 hours then evaporated. The residue was dissolved in THF quenched with M HCl and stirred for 2 hours. The organic solvent was removed and the aqueous phase was extracted with chloroform. The organic layer was dried over MgSO ₄ and evaporated. Purification was carried out on silica (heptane: EtOAc: AcOH / 3: 6: 1).

HPLC (40-70% MeCN 0.1% TFA, 10 min): R t = 4.75 min.

¹ H NMR (CDCl ₃ ): δ

I I

2.4-2.7 (s, 4H), 3.7 (s, 2H), 4.0-4.5 (m, 8H), 5.5 (s, 2H), 7.1-7.8 (m, 16H).

EXAMPLE (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylanino) -oct-4-amino-1,8-dicarboxylic acid

a) 1,4-Bis ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) -but-2-one (2R) - (-) - 2, 5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (2.76 g, 15 mmol) was dissolved in 100 mL of dry THF and cooled to -78 ° C, then 9.5 mL of 1.6 M BuLi (15 mmol) was added dropwise with stirring. . After one hour, 0.92 g (7.5 mmol) of 1,4-dichloro-2,3-butine was slowly added. The reaction mixture PSC (thin layer chromatography) and DC (gas chromatography) after 1 hour showed substantial amounts of unreacted 2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine and monoalkylated product. The reaction was allowed to continue overnight and gradually reached room temperature. The THF was evaporated under reduced pressure and the residue partitioned between ether and water. The aqueous phase was extracted twice with ether, dried (MgSO ₄ ) and evaporated. Kugelrohr distillation of the reaction mixture gave 2.3 g (77%) of the pure product.

³ H NMR (CDCl ₃ ): 8

4.03 (m, 4H, ring H's), 3.68 (m, 12H, 4 x OCH ₃ ), 2.63 (pi.AB q, 4H, 2 x CH ₂ ), 2.27 (m, 2H, 2 x CH (CH ₃ )). ₂ ), 1.05 (d, J = 6.8Hz, 6H, 2 x CH ₃ ), 0.64 (d, J = 6.8Hz, 6H, 2 x CH ₃ ).

¹³ C NMR (CDCl ₃ ): δ τ

τ π

II

I i:

163.9, 161.0, 77.4, 60.6,

52.2, 31.6 (31.20), 25.5 (6.9).

(60.68), 54.6 (25.6), 19.3 (54.2), 52.3, (19.5), 16.7

MS (CI) 419 (M ⁺ +, 55), 403 (4), 391 (7), 279 (15), 236 (17), 183 (44), 167 (34), 149 (100), 141 ( 41), 113 (44).

b) (S, S) -2,7-Diamino-oct-4-yn-1,8-carboxylic acid dimethyl ester

To the product of step a) was added 20 mL of 1.0 M HCl and 60 mL of MeOH to give 0.25 M HCl H ₂ O / MeOH. The reaction mixture was stirred at room temperature overnight, and when the starting material was found to be gone, all solvent was removed. The residue was dissolved in water, extracted with ether and the aqueous phase was neutralized to pH 8. NH-OH. The solution was washed with ether and extracted several times with EtOAc until all product had passed into the EtOAc phase. It was then dried, the solvent removed and the valine methyl ester separated from the solution by Kulgelrohr distillation to give 0.97 g (78%) of the methyl ester title compound.

* H NMR (D ₂ O): δ

4.0 (m, 2H, HC-α), 3.6 (d, 6H, 2 x OCH ₃ ), 2.6 (m, 4H, 2 x CH ₂ ).

¹³ C NMR (D ₂ O): δ

171, 81.6, 61.6, 56.6, 20.5

c) (S, S) -2,7-Diaxnino-oct-4-yno-1,8-dicarboxylic acid

Sufficient MeOH to dissolve the product of step b) and ml of 6 M HCl was stirred and stirred at room temperature

II

I peratur overnight. PSC utilizing

1: 1: 1: 1 / nBuOH: EtOAc: MeOH: H, O, and ninhydrin nebulizer, showed the presence of methyl ester. The reaction mixture was refluxed at 60 ° C for 24 hours until all methyl ester was hydrolyzed. The solution was evaporated to dryness, the residue was dried under high vacuum and used without further purification.

¹³ C NMR (D ₂ O): 8

172.3, 81.1, 55.0, 24.3

d) (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) oct-4-yno-1,8-dicarboxylic acid in 1 ml HMDS and 1 ml TMSC1 were added to the product from above and the mixture was refluxed at 120 ° C. Under nitrogen atmosphere until a clear solution was obtained after approximately 24 hours. Excess HMDS was distilled off and the product was dissolved in 50 mL · dry CH ₂ Cl ₂ and cooled to 0 ° C. While stirring, 3 g of FMOC-C1 dissolved in 20 ml of dry CH ₂ Cl ₂ were added gradually and the reaction was carried out overnight. The solvent was removed under reduced pressure and the product was partitioned between saturated aqueous NaHCO ₃ solution and ether to remove unreacted FMOC-C1 and other FMOC decomposition products. The aqueous solution was cooled to 0 ° C and acidified with 2M HCl to pH 3, and the product was extracted 5 times with EtOAc, then washed, dried and evaporated. Chromatography using CHCl ₃ : MeOH / 96: 4 gave 2.35 g (66%) of the title compound. Although PSC showed it to be pure, reverse phase HPLC showed the presence of impurities, which almost coincided with the peak of the parent product. The product dissolved in 40% MeCN with 0.1% TFA was purified by preparative HPLC using a Beckman ODC C-18 column and 0.1% TFA in H ₂ O, and 0.1% TFA in MeCN as eluent.

T

T

T

³ H NMR (DMSO): δ 30th 7.9 (d, J 7.3 Hz, 4H, arom. H), 7.7 (d, J = 7Hz, 4H, aroma. H), 7.3 (d triplet, J 7.2 Hz, 8H, arom. H), 4.26 (m, 8H, OCH _2, 2 x CH), 2.5 (m, 4H, CH ₂ ).

¹³ C NMR (DMSO): δ

170.4, 154.5, 142.5, 139.4, 126.5, 125.9, 124.2, 119.0,

77.6, 65.5, 53.0, 46.5, 21.7.

EXAMPLE (S, S) -2,4-Bis (9-Fluorenylmethyloxycarbonylanino) - (E) pentano-1,5-dicarboxylic acid

a) 1,1-Di ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (E) -methane

To a stirred solution of (2R) -2,5-dihydro-3,6-dimethoxy-isopropylpyrazine (5.53 g, 30 mmol) in THF (100 mL) at -78 ° C was added 1.55 M butyl lithium (19.62 mL, 31 mL). mmol) in hexane and stirring is continued for 1 hour at -78 ° C. A solution of (E) -bromochloromethane (15 mmol) in THF (20 mL) was then added and stirring continued overnight. The solvent is removed under reduced pressure and the residue is dissolved in diethyl ether and extracted with water. The organic layer was dried over magnesium sulfate, the ether evaporated and the residue purified by flash chromatography (ethyl acetate / hexane 1/4; silica gel).

b) (S, S) -2,4-Diamino- (E) -pentane-1,5-dicarboxylic acid ester

To 1,1-di ((2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl) - (E) -methane (4.02 g, 9.9 mmol) and 0.5 N HCl

T '(40 mL, 20 mmol) was added 40 mL of dioxane and the solution was stirred at room temperature for 4 hours. It is then extracted with diethyl ether and the aqueous ammonia is added to the aqueous solution until pH 9 is reached. The aqueous phase is then extracted with chloroform and the organic layer is dried over magnesium sulfate. After removal of the solvent, the valine methyl ester is removed by distillation from flask to flask at 30-40 ° C (0.04 mbar).

The non-distilled title compound is used without further purification.

c) Dimethyl ester of (S, S) -2,4-Diamino (E) -pentane-1,5-dicarboxylic acid (S, S) -2,4-Diamino- (E) -pentano-1,5-dicarboxylic acid (1.65 g, 7.17 mmol) was refluxed with 6 N HCl (10 mL, 60 mmol) for 2 hours. The solvent is then evaporated and the residue is dissolved in water (10 ml) and 100 ml of ethanol are added. The white crystals were filtered off and dried in vacuo at 40 ° C.

d) (S, S) -2,4-Bis (9-Fluorenylmethyloxycarbonylamino) (E) -pentane-1,5-dicarboxylic acid

Acid hydrochloride - (S, S) -2,4-Diamino- (E) -pentane-1,5-dicarboxylic acid dihydrochloride (1.59 g, 5.8 mmol) is suspended in hexamethyldisilazane (20 mL) and 1 mL of trimethylsilyl chloride is added. The suspension is then deflegmated overnight. The solvent is removed under reduced pressure and the residue is dissolved in anhydrous methylene chloride. The solution was cooled to 0 ° C and a solution of 9-fluorenylmethyl chloroformate (3.10 g, 12 mmol) in methylene chloride was added. Stir the solution for 1 hour before removing the freeze bath. The next morning, the solvent was evaporated and the residue was dissolved in THF. The mixture is then treated with 1 N aqueous HCl and the solution is stirred

T somas for 2 hours. The organic solvent is removed and the aqueous phase is extracted with chloroform. The organic layer was dried over magnesium sulfate and evaporated. The residue is dissolved in methylene chloride and precipitated with ether.

EXAMPLE (S, S) -2,9-Diamindecandicarboxylic acid

a) 1,6-Bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] hexane (R) -2,5-Dihydro-3,6-dimethoxy -2-Isopropylpyrazine (4.12 g, 22.4 mmol) was dissolved in anhydrous THF and the solution was cooled to -78 ° C. BuLi solution in hexane (22.42 mmol, 14.0 mL) was added. After 30 minutes at -78 ° C, a solution of 1.6-dibromohexane (11.2 mmol, 2.73 g) in THF was added dropwise and the solution was allowed to reach room temperature overnight. After hydrolysis with phosphate buffer (pH = 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO ₄ ), the solvent was evaporated and the residue was purified by flash chromatography (hexane / ethyl acetate 9/1).

Yield: 2.57 g (51%).

¹ H NMR (CDCl ₃ ): δ

0.67 (d, 6H), 1.04 (d, 6H), 1.10-1.30 (m, 8H), 1. 60- 1.85 (m, 4H), 2.15-2.35 (m, 2H), 3.66 (s, 6H), 3.67 (s, 6H), 3.91 (dd, 2H), 3.95 -4.05 (m, 2H). ¹³ C NMR (CDCl ₃ ): δ

“T ---— · r · i

I 1

16.54, 19.08, 24.44, 29.46, 31.63, 34.15, 52.27,

52.28, 55.47, 60.68, 163.41, 163.96.

FAB-MS signals at m / z 451.2 (80), 407.2 (33),

141.0 (100). C ₂₄ H ₄₂ N ₄ O ₄ (450).

b) dimethyl diester of (S, S) -2,9-Diamindecandicarboxylic acid

A solution of 1,6-bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -hexane in 40 mL of dioxane and 22.8 mmol of HCl (1.90 mL) in 36 mL water was stirred at room temperature for 6 hours, the solvent removed and the solution extracted with diethyl ether. Aqueous ammonia solution was added until pH 9 was reached and then the solution was extracted with chloroform. The organic layer was dried (MgSO ₄ ). Valine methyl ester was distilled in vacuo (0.03 torr, room temperature).

Yield: 1.48 g (100%).

¹ H NMR (CDCl ₃ ): δ

1.20-1.70 (m, 16H), 3.36 (dd, 2H), 3.65 (s, 6H).

¹³ C NMR (CDCl ₃ ): 6

25.40, 29.06, 34.77, 51.76, 54.30, 176.51.

c) (S, S) -2,9-Diaminodecandicarboxylic acid · 2HCl

The dimethyl diester from step b) (1.40 g, 5.38 mmol) was dissolved in 6 N HCl (40 mL, 240 mmol) and stirred under N ₂ at 60 ° C for 12 h. The solvent was evaporated, the residue was dissolved in EtOH and crystallized by addition of diethyl ether. The crystals were filtered off.

Yield: 0.700 g, white crystals (42.7%).

^: H NMR (D ₂ O): δ

1.07-1.37 (m, 8H), 1.60-1.90 (m, 4H), 3.85 (m, 2H).

¹³ C NMR (D ₂ O): 6

23.81, 27.65, 29.61, 53.09, 172.55.

FAB-MS signals at m / z 439.3 (15), 340.3 (100),

233.2 (11).

C ₁₀ H ₂₂ N ₂ Cl ₂ O ₄ (450): Final%: N, 9.18.

Found,%: N, 9.13.

d) (S, S) -2,9-Bis (9-Fluorenylmethyloxycarbonylamino) -decandicarboxylic acid (S, S) -2,6-Diaminodecandicarboxylic acid dihydrochloride (0.50 g, 1.64 mmol) suspended in HMDS and TMSC1 and deprotected under nitrogen. overnight. The solvents were removed and the residue was dissolved in anhydrous CH ₂ Cl ₂ . The solution was cooled to 0 ° C and a solution of FmocCl (1.69 g, 6.56 mmol) in CH ₂ Cl _{2 was added} . The solution was stirred overnight, the solution removed and the residue dissolved in THF. After addition of 5 ml of 0.5 M HCl, the mixture was stirred for 2 hours, extracted with chloroform and dried (MgSO ₄ ). The residue was purified by flash chromatography (heptane / ethyl acetate / acetic acid 4/6/1).

Yield: 0.580 g (52.5%).

¹ H NMR (DMSO): δ

1.00-1.44 (m, 8H), 1.44-1.80 (m, 4H), 3.90 (s, 2H),

4.13-4.43 (m, 4H); 7.25-8.05 (m, 16H);

¹³ C NMR (DMSO): δ

21.29, 25.63, 28.62, 31.08, 46.86, 54.17, 65.70, 120.25, 125.44, 127.20, 127.77, 140.86, 143.96, 144.04, 156.21, 174.28

FAB-MS signals at m / z 699.5 (6), 677.4 (2), 603.6 (4),

409.3 (4), 339.3 (4); 179.1 (55), 72.9 (100).

EXAMPLE (S, S) -2,8-Diaminonone-dicarboxylic acid

a) 1,5-Bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -pentane

To a solution of (R) -2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (3.68 g, 20 mmol) in THF (68 mL) was added dropwise (10 min) a solution of 1.6 M n-BuLi in hexane (12.8 mL, 20.4 mmol) and stirring was continued at -78 ° C for 1 hour. A solution of 1,5-dibromopentane (2.3 g, 10 mmol) in THF (13.2 mL) was then added dropwise and the mixture was allowed to come to room temperature overnight. The reaction was quenched by the addition of 50 mL of NaHCO ₃ and the mixture was partitioned between ether (50 mL) and water (20 mL). After separation, the aqueous layer was extracted twice with ether (50 mL) and the combined organic layers were dried (MgSO ₄ ), filtered, concentrated, and purified by flash chromatography (silica gel, hexane: ethyl acetate = 12: 1).

Yield: 3.68 g (8.43 mmol, 84.3%).

¹ H NMR (300 MHz, CDCl ₃ ): δ

4.02 (s 2H, J 2.1 Hz), 3.92 (t, 2H, J 3.3 Hz), 3.68 (s, 12H), 2.28 (d sept, 2H, J 3.3, 6.8 Hz), 1.71 (m,

II

J

4H), 1.21 (m, 6H), 1.05 (d, 6H, J 6.8), 0.68 (d, 6H, J 6.8);

¹³ C NMR (75 MHz, CDCl ₃ ): δ

163.89, 163.31, 60.62, 55.40, 52.22. 52.19, 34.02,

34.56, 29.31, 24.33, 19.01, 16.48.

FAB-MS m / z 437 (M + 1).

C ₂₃ H ₄₀ N ₄ O ₄ (436.59): Final%: C, 63.28; H, 9.23; N, 12.83.

Found: C, 62.95; H, 9.01; N, 129.91

b) dimethyl diester of (S, S) -2,8-diaminononandicarboxylic acid

1,5-Bis [(1S, 4R) -3,6-4 -isopropyl-2,5-dihydropyrazinyl] pentane (2.8 g, 6.4 mmol) was dissolved in dioxane (51.2 mL) and 0.5 M HCl (51.2 mL) and stirred for 4 hours at room temperature. This mixture was washed with diethyl ether (100 ml) and made pH 9 by adding 25% ammonia water, and quickly extracted CHC'l ₃ (3 x 75 ml). The organic layer was dried over MgSO ₄ , filtered, and concentrated. Methyl valinate was removed by Kulgelrohr distillation (25-50 ° C, 0.01 torr) to give 1.505 g (0.11 mmol, 95.5%).

¹ H NMR (300 MHz, CDCl ₃ ): δ

3.71 (s, 6H), 3.43 (t, 2H, J 6.0 Hz), 1.70 (m, 2H)

1.57-1.34 (m, 8H).

¹³ C NMR (75 MHz, CDC.U): δ

176.40, 54.33, 51.86, 34.79, 29.08, 25.42.

I): ιι

c) (S, S) -2,8-Diaminononandicarboxylic acid

The ester from step b) was stirred under refluxing in 6 M HCl (7.2 mL) for 2 hours under nitrogen. The volatile compounds were evaporated. The residue was dissolved in water (5 mL), ethanol (20 mL) was added and the slow addition of ether gave a white, crystalline precipitate. This mixture was kept in a refrigerator for 12 hours, filtered, rinsed with ether and vacuum dried.

Yield 1.37 g (4.71 mmol, 77) .4%). ¹ H NMR (300 MHz, CDCl ₃ ): δ 3.74 (m, 2H), i.80-1.63 (m, 4H), 1.38- -1.15 (m, 6H). ¹³ C NMR (75 MHz, CDCl ₃ ): δ 173.35, 53.62, 29.71, 27.65, 23.65. FAB-MS m / z 437 (M ⁺ -72). C ₉ H ₂₀ Cl ₁₂ N ₂ O ₄ (291.17): Last, % C, 37.13; H, 6.92 Found % C, 37.90; H, 7.21

d) (S, S) -2,8-Bis (9-Fluorenylmethyloxycarbonylamino) -nonandicarboxylic acid (S, S) -2,8-Diaminononanoic acid (291 mg, 1 mmol) TMSC1 (260 mg; 2.4 mmol) and HMDS ( 3.5 ml) was refluxed under nitrogen overnight. After cooling to room temperature, the volatiles were removed (to 50 ° C, 0.01 Torr), the residue dissolved in CH ₂ Cl ₂ (6.6 mL), cooled to 0 ° C, and FmocCl (905 mg, 3.5 mmol) in CH ₂ Cl _{2 was} added dropwise. (6.6 mL). The mixture was allowed to reach room temperature overnight. The solvent was evaporated and the residue was dissolved in THF (7.5 mL) and 1M HCl (7.5 mL) was added.

I III

The mixture was stirred for 4 hours at room temperature. Chloroform (50 mL) and brine (40 mL) were then added and the phases separated. The aqueous layer was extracted with 2 x CHCl ₃ (50 mL). The combined organic layers were dried (MgSO ₄ ), concentrated, and purified by flash chromatography (silica gel, hexane: ethyl acetate: acetic acid = 50: 50: 5). The product-containing fractions were concentrated and lyophilized to yield 615 mg (0.928 mmol, 92.8%).

NMR (300 MHz, d ₆ -DMSO): δ

7.86 (d, 4H, J 7.2 Hz), 7.70 (d, 4H, J 7.5 Hz), 7.41- 7.27 (m, 8H), 4.24 (m, 6H), 3.86 (sq, 2K, 0 '4.5 Hz), 1.62 (m, 4H), 1.24 (m, 6H).

¹³ C NMR (75 MHz, CDCl ₃ ): δ

174.40, 172.39, 156.30, 144.31, 144.22, 141.10, 129.31, 127.99, 127.68, 127.44, 125.67, 121.77, 120.46, 65.91, 54.66, 47.12, 31.40, 28. 68, 25.57, 21.53 FAB-MS m / z 663 (M ⁺ +1); C ₃₉ H ₃₈ N ₂ O ₈ (662.73). Last, %: C, 70.68; H, 5.78; N, 4.23. Found, %: C, 71.20; H, 5.62; N, 4.07.

EXAMPLE

2,3-Diaminoglyceric acid

a) 2,3-Bis (benzylamino) succinic acid

Benzylamine (85 g, 0.8 mol) was slowly added at room temperature to a mechanically stirred solution of meso-2,3-dibromo-succinic acid (27.6 g, 0.1 mol) in alcohol (200 mL). After adding everything up, the mixture deflates overnight.

t. A heavy salt precipitate appeared. The mixture was cooled to 50 ° C and concentrated hydrochloric acid was added until the pH reached 4-5. The precipitate was filtered off, washed several times with water and alcohol and dried.

Yield 24.5 g (75%).

NMR (300 MHz, D ₂ O / NaOD); δ

3.08 (2H, s), 3.30 (2H, d, J 13 Hz), 3.55 (2H, d, J 13 Hz), 7.2 (10H, m).

¹³ C NMR (75 MHz, D ₂ O / NaOD); δ

50.9, 64.7, 127.1, 128.5 (br), 139.2, 179.0.

b) Meso-2,3-diaminogutaric acid

Meso-2,3-bis (benzylamino) succinic acid (9.5 g, 0.029 mol) was dissolved in a mixture of glacial acetic acid (50 mL) and concentrated hydrochloric acid (50 mL). The hydro is carried out at room temperature in 10% palladium on charcoal (1.0 g) as a catalyst. The reaction continued until the NMR showed no further unreacted starting material. Water (100 mL) was added and the catalyst was removed by filtration. The filtrate was concentrated (rotary evaporator) and the residue was dissolved in dilute aqueous sodium hydroxide. The pH was adjusted to about 5 by the addition of glacial acetic acid. The crystals were filtered off and washed.

Yield 3.8 g (88%) as a white solid.

¹ H NMR (300 MHz, D ₂ O / NaOD):?

3.21 (s).

II C H ³³ C NMR (75 MHz, D ₂ O / NaOD):?

60.3, 180.0.

C ₄ H ₈ N ₂ O ₄

Shit. %: C, 32.44; H,

Found: C, 31.51; H,

5.44; N, 18.91.

5.41; N, 18.32.

(c) Di (trimethylsilyl) ester of meso-2,3-bis (trimethylsilylamino) succinic acid

The reaction was carried out under a nitrogen atmosphere. A suspension of meso-2,3-diaminogenic acid (296 mg, 2.00 mmol) in hexamethyldisilazane (10 mL) and trimethylchlorosilane (1 mL) was stirred at 100 ° C overnight to give a clear solution. The excess silylating reagents were distilled off under reduced pressure. The solid white residue was directly used in the acylline reaction.

² H NMR (300 MHz, CDCl ₃ ): δ

0.03 (18H, s), 0.28 (18H, s), 3.5 (2H, m).

¹³ C NMR (75 MHz, CDCl ₃ ): δ

-0.3, 0.03, 60.9, 174.1.

d) Meso-2,3-bis [(9-fluorenylmethoxycarbonyl) amino) succinic acid

The reaction is carried out under a nitrogen atmosphere. The silylated draminic succinic acid obtained in step c) was dissolved in dry dichloromethane (10 mL) and cooled to 0 ° C. A solution of 9-fluorenylmethyl chloroformate (1.14 g, 4.4 mmol) in dry dichloromethane (4 mL) was added. The reaction mixture was stirred overnight and then concentrated. The residue was dissolved in THF (10 mL) and water (approximately 1 mL) was added. The mixture was stirred for 10 minutes and then concentrated. The product was extracted with ethyl acetate, the solution was washed with water, dried (MgSO ₄ ) and filtered. Hexane was added and the precipitate was filtered off and washed with hexane. Finally, the product was purified by column chromatography (silica, hexane / ethyl acetate / acetic acid 1: 3: 1).

Yield 0.85 g (72%), white crystals.

² H NMR (300 MHz DMSO-d ₆ ): δ

4.1-4.3 (6H, m), 4.5 (2H, d), 7.2-7.4 (12H, m), 7.6-7.7 (4H, 7.8-7.9 (4H, m)).

¹³ C NMR (75 MHz DMSO-d ₆ ): δ

47.0, 56.1, 66.5, 120.5, 125.9, 127.5, 128.0, 141.0,

144.1, 156.4, 171.1.

FAB-MS signals at m / z 637.2 (3), 631.1 (5), 615.2 (21),

593.2, 435.1 (9), 326.3 (15), 179.1 (100).

(e) Racemic-2,3-bis [(9-fluorenylmethoxycarbonyl) amino] succinic anhydride

A suspension of meso-2,3-bis [(9-fluorenylmethoxycarbonyl) -amino] succinic acid (150 mg, 0.25 mmol) in acetic anhydride was stirred at 120 ° C until all material was dissolved (1-2 minutes). The excess anhydride was separated in vacuo. The crude product was used in peptide synthesis without further purification.

¹ H NMR (300 MHz DMSO-d ₆ ): δ

4.25 (2H, t), 4.39 (4H, d), 4.80 (2H, d), 7.2-7.4 (8H, m), 7.66 (4H, d), 7.88 (4H, d), 8.30 (2H, d) .

¹³ C NMR (75 MHz DMSO-d ₆ ): δ

46.8, 55.6, 66.9, 120.6, 125.5, 127.5, 128.1, 141.1,

143.9, 156.4, 168.2.

EXAMPLE (S, S) -2,7-Diamino-E-oct-4-endicarboxylic acid

a) 1,4-Bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -E-but-2-ene (2R) -2,5- Dihydro-3,6-dimethoxy-2-isopropylpyrazine (5.53 g, 30 mmol) was dissolved in anhydrous THF and the solution cooled to -78 ° C. A solution of BuLi in hexane (19.62 mL, 31.0 mmol) was added. After 60 minutes at -78 ° C, a solution of E1, 4-dibromobut2-ene (3.21 g, 15 mmol) in 30 mL of THF was added dropwise and the solution was brought to room temperature overnight. After hydrolysis with phosphate buffer (pH 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO ₄ ), the solvent was evaporated and the residue was purified by flash chromatography (hexane / ethyl acetate 4/1).

Yield 4.73 (75%).

³ H NMR (CDCl ₃ ): δ

0.67 (d, 6H), 1.04 (d, 6H), 2.1-2.4 (m, 2H), 2.48 (dd, 4H), 3.67 (s, 6H), 3.68 (s, 6H), 3. 93 (dd, 2H), 4.03 (dd, 2H), 5.35 (dd, 2H). ¹³ C - • (CDC1 _3): 6 16.52, 7.07 pm, 31.61 , 37.09, 52.16, 52 .23, 55. 62, 60.60, 128. 50

I!

.i j:

C ₃₃ H ₃₆ N, O (420):

Found,%: C, 62.86; H, 8.57.

Found: C, 62.65; H, 8.62.

b) Dimethyl diester of (S, S) -2,7-diamino-E-oct-4-enodicarboxylic acid

A solution of 1,4-bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -E-but-2-ene (4.02 g, 9.9 mmol) in 40 mL dioxane and 20.0 mmol HCl (1.67 mL) in 38 mL of water were stirred at room temperature for 4 hours, after which the solution was extracted with diethyl ether. Aqueous ammonia solution was added until pH 9 was reached and the solution was extracted with chloroform. The organic layer was dried (MgSO ₄ ). Valine methyl ester was distilled in vacuo (0.04 torr, room temperature).

Yield: 192 g (84.3%).

³ H NMR (CDCl ₃ ): δ

1.70 (s, 4H), 2.2-2.6 (m, 4H), 3.51 (dd, 2H), 3.69 (s, 6H), 5.46 (dd, 2H).

¹³ C - (CDCl ₃ ): 5

37.99, 52.00, 54.11, 128.84, 175.32.

c) (S, S) -2,7-Diamino-E-oct-4-enodicarboxylic acid · 2 HCl

The dimethyl diester from step b) (1.65 g, 7.17 mmol) was dissolved in 6N HCl (10 mL, 60 mmol) and the reaction mixture was stirred under nitrogen for 2 hours. The solvent was evaporated and the residue was dissolved in 10 mL of water and crystallized by adding EtOH (100 mL). The crystals were filtered off.

Yield 132 g = 67%.

ιι

NMR (D ₂ O): δ

2.53 (dd, 4H), 3.97 (dd, 2H), 5.52 (dd, 2H).

¹³ C - (D ₂ O): 6

33.57, 53.07, 128.85, 171.49.

FAB-MS signals at m / z 405.3 (11), 203.2 (100),

157. (19), 130.1 (7), 93.0 (18), 73.9 (13).

C ₈ H ₁₆ N ₂ O ₄ C 1 ₂ (275):

Shit. %: C, 34.91; H, 5.81; N, 10.18; Cl, 25.78.

Found: C, 36.58; H, 5.80; N, 9.70; Cl, 26.01.

d) (S, S) -2,7-Bis (9'-Fluorenylethylethyloxycarbonylamino) -Ect-4-enodicarboxylic acid (S, S) -2,7-Diamino-E-oct-4-enodicarboxylic acid dihydrochloride (1.59 g) , 5.8 mmol) in HMDS (20 mL) and TMSC1 suspended and refluxed overnight. The solvents were removed and the residue was dissolved in anhydrous dichloromethane. This solution was cooled to 0 ° C and a solution of FmocCl (3.10 g, 12 mmol) in dichloromethane was added. The solution was stirred for 1 hour at this temperature and overnight at room temperature. The solvent was evaporated and the residue was dissolved in THF. After addition of 1 N aqueous HCl, the solution was stirred for two hours, extracted with chloroform and dried (MgSO ₄ ). The residue was dissolved in dichloromethane and crystallized with diethyl ether.

Yield: 2.50 g (66.8%).

³ H - (DMSO): δ

Ml.G.

2.2-2.4 (s, 4H), 3.6 (s, 2H), 3.9-4.5 (m, 8H), 5.55 (s, 2H), 7.2-8.1 (m, 16H).

C - (DMSO): δ

33.98, 46.56, 53.93, 65.49, 119.66, 124.81, 126.61,

127.17, 127.98, 140.19, 143.28, 155.43, 172.59 ^ 38 ^ 34 ^ 2 ^ 8 (646):

Shit.

C, 70.59; N, 5.26; N, 4.33.

Found: C, 70.17; N, 5.58; N, 4.08.

FAB-MS signal at m / z 661.3 (3), 647.4 (3), 191.2 (17), 179.2 (100), 165.1 (18), 78.9 (31).

EXAMPLE (S, S) -2,7-Diamino-Z-oct-4-enodicarboxylic acid

a) 1,4-Bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -Z-but-2-ene (2R) -2,5- Dihydro-3,6-dimethoxy-2-isopropylpyrazine (7.3 g, 39.2 mmol) was dissolved in anhydrous THF and the solution was cooled to -78 ° C. BuLi solution in hexane (24.5 mL, 39.2 mmol) was added. After 15 minutes at -78 ° C, 16.9 ml of 1,3-dimethyl-2-imidazolindicarboxylic acid (156 mmol) was added, and after 5 minutes Z-1,4-dichlorobut-2-ene (2.45 g,

19.6 mmol) in 10 mL of THF and the solution stirred for 6 h and stored at -20 ° C overnight. After hydrolysis with phosphate buffer (pH = 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO ₄ ), the solvent was evaporated and the residue was purified by flash chromatography (hexane / ethyl acetate 9/1).

Yield: 710 mg (10.2%).

1 H NMR (CDCl ₃ ): δ

0.67 (d, 6H), 1.03 (d, 6H), 2.25 (m, 2H), 2.55 (m, 4H), 3.66 (s, 6H), 3.68 (s, 6H), 3.91 (dd, 2H), 4.07 (m,

2H), 5.38 (dd, 2H).

¹³ C NMR (CDCl ₃ ): δ

16.50, 19.01, 31.57, 32.01, 52.22, 52.31, 55.46, 60.67, 127.30, 163.19, 163.66.

b) Dimethyl diester of (S, S) -2,7-Dianinc-Z-oct-4-endicarboxylic acid

A solution of 1,4-bis [(1S, 4R) -3,6-dimethoxy-4-isopropyl-2,5-dihydro-5-pyrazinyl] -Z-but-2-ene (0.23 g, 0.55 mmol) in 30 mL dioxane and 2.2 mmol HCl (0.183 mL) in 10 mL of water were stirred at room temperature for 12 hours. The solution was extracted with diethyl ether. Aqueous ammonia solution was added until pH 9 was reached and the solution was extracted with chloroform. The organic layer was dried (MgSO ₄ ). Valine methyl ester was distilled off (0.02 torr, room temperature).

Yield: 0.127 g (85%).

³ H NMR (CDCl ₃ ): 6

1.54 (s, 4H), 2.3-2.6 (m, 4H), 3.52 (dd, 2H), 3.71 (s,

6H), 5.54 (m, 2H).

¹³ C NMR (CDCl ₃ ): 0

32.55, 52.02, 54.17, 127.94, 175.67.

c) (S, S) -2,7-Diamino-Z-oct-4-endicarboxylic acid · HCl

To a solution of dimethyl diester from step b) (0.108 g, 0.47 mmol) in 0.7 mL of methanol was added 2N LiOH in water (1.41 mmol, 0.71 mL). The mixture was stirred at room temperature overnight then acidified by the dropwise addition of 1 N HCl. The crystals were filtered off.

Yield: 50 mg (38.7%).

³ H NMR (ϋ ₂ 0): δ

2.58 (m, 4H), 3.99 (m, 2H), 5.51 (m, 2H).

¹³ C NMR (ϋ ₂ Ο): δ

27.59, 52.20, 126.84, 171.10.

d) (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) -Zoct-4-endicarboxylic acid (S, S) -2,7-Diamino-Z-oct-4-endicarboxylic acid (0.050 g, .0.25 mmol ) and Na ₂ CO ₃ (0.21 g, 20.0 mmol) were dissolved in 3 mL of water and 2 mL of dioxane. The solution was cooled to 0 ° C and a solution of FmocCl (0.26 g, 1.0 mmol) in 1 mL of dioxane was added. The mixture was stirred at 0 ° C for 3 hours and gained room temperature overnight. After extraction with diethyl ether, it was acidified by the addition of hydrochloric acid. The solution was extracted with chloroform and the amino acid crystallized from chloroform / hexane. The compound was further purified by flash chromatography (chloroform / acetic acid 9/1).

Yield: 90.0 mg (55.9%).

¹ H NMR (DMSO): δ

I!

2.12-2.64 (m, 4H), 3.88 (s, 2H), 4.04-4.37 (m, 6H),

5.42 (s, 2H), 7.02 (s, 2H>, 7.20-8.00 (m, 16H).

¹³ C NMR (DMSO): δ

29.77, 46.88, 55.53, 65.66, 120.23, 125.43, 127.23,

127.74, 129.08, 140.84, 144.11, 155.59, 174.10.

FAB-MS signals at m / z 676.2 (2), 587.5 (5), 447.4 (10), 419.3 (45), 391.3 (100), 363.3 (38), 261.2 (45), 233.2 (29).

EXAMPLE (S, S) -2,7-Diamino-oct-4-indicator carboxylic acid

a) 1,4-Bis - {(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) but-2-one

To a solution of (2R) -2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (4.605 g, 25 mmol) and 1,3-dimethyl-2-imidazolidinone (11.4 g, 102 mmol) in THF (48 mL) drip added

1.6 M n-BuLi (16 mL, 25.5 mmol). After 1 hour, a solution of 1,4-dichloro-2-butine (1.54 g, 12.5 mmol) in THF was added dropwise. The mixture was allowed to reach room temperature overnight and quenched with 1 M phosphate buffer (pH 7). The mixture was extracted with diethyl ether (150 mL) and water (40 mL), and the layers were separated. The aqueous layer was extracted twice with ether (2 x 150 mL), and the combined organic layers were dried (MgSO ₄ ), filtered, concentrated and purified by flash chromatography (silica gel, hexanes: ether = 7: 1).

Yield 3.325 g (7.94 mmol, 63.6%).

¹ H NMR (300 MHz, CDCl ₃ ): δ

4.05-3.99 (m, 4H), 3.68 (s, 6H), 3.67 (s, 6H), 2.70 (dd, 2H, J -14.1, 3.0 Hz), 2.57 (dd, 2H, J -14.1, 3.0

Hz), 2.26 (d Sept, 2H, J 6.9, 3.0 Hz), 1.05 (d, 6H, J

6.9 Hz), 0.64 (d, 6H, J 6.9 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ): δ

164.56, 161.53, 77.52, 60.51, 54.46, 52.34, 52.22,

31.43, 25.26, 19.07, 16.40.

FAB-MS m / z 419 (M ⁺ +1).

C ₂₂ H ₃₄ N ₄ O ₄ (418.53):

Shit. %: C, 63.14; H, 8.19; N, 13.39.

Found: C, 63.27; H, 8.12; N, 13.54.

b) Dimethyl diester of (S, S) -2,7-Diamino-oct-4-indoic acid

1,4-Bis - ((2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) but-2-one (920 mg, 2.2 mmol) was dissolved in dioxane (18 mL) and 0.5 M HCl (18 mL) and stirred at room temperature for 12 hours. The mixture was washed with diethyl ether (30 mL), 25% ammonia in water was added, the pH of the aqueous layer was adjusted to 9, and rapidly extracted with CHCl ₃ (3 x 75 mL). The organic layer was dried over MgSO ₄ , filtered, and concentrated. Methyl valinate was removed by Kugelrohr distillation (25-50 ° C, 0.01 tor).

Yield: 486 mg (2.13 mmol, 96.8%) of a yellow oil.

¹ H NMR (300 MHz, CDCl ₃ ): δ

3.74 (s, 6H), 3.59 (t, 2H, J 5.4 Hz), 2.60 (d, 4H, J

5.4 Hz).

ⁿ c NMR (75 MHz, CDC1 ₃₎ δ

174.46, 78.31, 53.31, 52.22, 25.15.

c) (S, S) -2,7-Diainino-Z-oct-4-indole-carboxylic acid · 2 HCl

A solution of dimethyl diester from step b) (450 mg, 1.97 mmol) was heated with 6 M HCl (7.2 mL) to 50-60 ° C for 12 h under a nitrogen atmosphere. Volatile compounds evaporated (rotary evaporator, water bath 40 ° C). The residue was dissolved in water (1 mL, cloudy), ethanol (20 mL) was added and a slow addition of ether gave a white crystalline precipitate. This mixture was kept in a refrigerator for 12 hours, filtered, rinsed with ether and vacuum dried.

Yield 399 mg (1.46 mmol, 74.2%) ³ H NMR (300 MHz, D ₂ O / DC 1): 0

4.18 (t, 2H, J 4.8 Hz), 2.85 (m, ΑΑ'Χ, 4H);

¹³ C NMR (75 MHz, D ₂ O / DC 1): δ

172.62, 79.88, 53.75, 22.60.

FAB-MS m / z 201 (M ⁺ -72).

d) (S, S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) oct-4-indole-carboxylic acid (S, S) -2,7-Diamino-Z-oct-4-indo-carboxylic acid · 2 HCl (400 mg, 1.46) solution of dioxane and 1 M Na ₂ CO ₃ solution (10.22 mL, 10.22 mmol) was cooled to 0 ° C and a solution of FmocCl (1.14 g, 4.39 mmol) in 4 mL of dioxane was added. The mixture was stirred at 0 ° C for 1 h and allowed to come to room temperature over

ι.ι.

at night. After extraction with diethyl ether, (2 x 50 mL) the aqueous layer was acidified with dilute HCl. The solution was extracted with chloroform (3 x 50 mL), dried (MgSO ₄ ), and concentrated. The residue was purified by flash chromatography (silica gel, hexane: ethyl acetate: acetic acid = 10: 10: 1).

Yield 376 mg (0:58 mmol, 39%) ^J 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 (d, 4H, J 7.5 Hz), 4.26 (m, 8H) , 2.52 (m, 4H).

¹³ C (DMSO): 6

170.43, 154.62, 124.35, 119.12,

FAB-MS m / z 645 (M + + -38-32-32-8-8 (644.67):

Shit. %: C, 70.80;

Found: C, 69.20;

152.58, 142.32. 139.47, 77.68, 65.45, 53.08, 46 + 1) ·

H, 5.00; N, 4.35.

H, 5.34; N, 4.13.

126.22, 125.94, 57, 21.77.

EXAMPLE (2S, 4R, 5R, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid

a) (2R, 3R,) - 1,4-Bis (2R, 5S) -3,6-Dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) -2,3-dihydroxy-2,3-0 , 0isopropylidenebutane (2R) -2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (7.36 g, 40 mmol) was dissolved in aqueous THF, and the melt 3636B was cooled to ~ 76 ° C. A solution of BuLi in hexane (40.0 mmol, 25 mL) was added. After 20 minutes at -78 ° C, a solution of (2R, 3R) -1,4-dibromo-2,3-dihydroxy-2,3-O-isopropylidenobutane (3.78 g, 13.1 mmol) in 10 mL of THF was added dropwise , and the solution is kept at 4 ° C for 36 hours. After hydrolysis with phosphate buffer (pH 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO ₄ ), the solvent was evaporated and the residue was purified by recrystallization from acetonitrile.

Yield: 3.70 g (57%).

³ C NMR (CDC1 _3): δ 0.68 (d, 6H), 1.03 (d, 6H), 1.34 (s, 6H), 1.95-2. .15 (m, 4H), 2 '. 24 (m, 2H), 3.65 (s, 6H), 3.67 (s, 6H), 3. . 94 (m, 2H), 4. 0 4 (m, 211), 4.11 (m, 2H). ¹³ C NMR (CDCI3) : δ 16.91, 19.05, 27.39 , 31.73, 38.32, 52 .27, 52.36, 52. .70, 60.60, 77.89, 108.1 9, 163.66 , 163.70. FAB-MS m / z sig nalas at 495 .4 (100).

C ₂₅ H ₄₂ N ₄ O (494):

Shit. %: C, 60.71; H, 8.56; N, 11.33.

Found: C, 60.76; H, 8.32; N, 11.36.

b) Dimethyl diester of (2S, 4R, 5R, 7S) -2,7-diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid (2R, 3R,) - 1,4-Bis ((2R , 5S) -3,6-Dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) -2,3-dihydroxy-2,3-0,0-isopropylidenobutane (1.22 g, 2.47 mmol) was dissolved in 10 mL dioxane and 20 mL of MeOH. 9.88 mmol HCl was added

II. It is dissolved in water, and the solution is stirred at room temperature overnight. The solvents were removed, the residue was dissolved in water and extracted with diethyl ester, ammonia was added to the aqueous layer until pH 9 and this layer was extracted with chloroform. After drying (MgSO ₄ ), the solvent was removed and the valine methyl ester was distilled (30-40 ° C, 0.04 torr).

Yield: 0.67 g (89.3%).

⁷ H NMR (CDCl ₃ ): δ

1.34 (s, 6H), 1.64 (pi s, 4H), 1.72-1.84 (m, 2H), 1.992.08 (m, 2H), 3.65 (m, 2H), 3.71 (s, 6H), 3.80 (m) , 2H).

¹³ C NMR (CDCl ₃ ): 6

27.17, 37.54, 52.04, 52.74, 78.78, 108.99, 175.51.

c) (2S, 4R, 5R, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid

The dimethyl diester from step b) (0.62 g, 2.04 mmol) was dissolved in 2.5 mL of dioxane and 2.1 mL of 2N LiOH in water (4.1 mmol) was added and the solution was stirred under argon overnight. PSC showed quantitative formation of the title compound. The crude reaction solution was used in the next step.

d) (2S, 4R, 5R, 7S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino) -4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid

The crude solution of (2S, 4R, 5R, 7S) -2,7-diamino-4,5-dihydroxy-4,535, O-isopropylideneoctanedicarboxylic acid (2.04 mmol) was cooled to 0 ° C and 10 mL of dioxane and 1N NaOH (10 mmol) were added. ) solution. Then 2.07 g was added

K ι, .ui ·.

FmocCl (8.0 mmol) was stirred at 0 ° C overnight. The mixture was small portions and the mixture was extracted with diethyl ether for 5 hours at room temperature and acidified by addition of KHSO ₄ .

The extraction C'HC1 ₃ and the organic layer was dried (MgSO ₄₎ and solvent removed. The excess unreacted FmocCl was isolated by filtration on silica gel with acetone / MeOH and the residue was purified by precipitation from CHCl ₃ / EtOEt.

Yield: 0.49 g (33.6%)

NMR (DMSO / D ₂ O) δ:

1.20 (s, 6H), 1.73 (m, 2H), 1.99 (m, 2H), 3.87 (m, 2H), 4.14-4.2Φ (m, 6H), 7.23-7.82 m, 16H).

¹³ C NMR (DMSO) δ:

27.78, 36.21, 47.27, 53.75, 66.19, 78.82, 107.89, 120.67, 125.81, 127.76, 128.32, 141.24, 144.39, 156.12, 175.96.

FAB-MS signal at m / z 563.2 (2), 179.1 (100)

C ₄₁ H ₄₀ N ₂ O ₁₀ (720): Last: N, 3.89

Found: N, 3.81.

EXAMPLE (2S, 4S, 5S, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid

a) (2S, 3S) -1,4-Bis ((2R, 5S) -3,6-dimethoxy-2-isopropyl)

2,5-Dihydro-5-pyrazinyl) -2,3-dihydroxy-2,3-O, O-isopropylidene butane ιι .11 (2R) -2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine ( 9.59 g, 52.1 mmol) was dissolved in anhydrous THF and the solution was cooled to -78 ° C. BuLi solution in hexane (52 mmol, 32.5 mL) was added. After 60 minutes at -78 ° C, a solution of (2S, 3S) -1,4-dibromo-2,3-dihydroxy-2,3-O, O-isopropylidene-butane (6.0 g, 20.83 mmol) was added dropwise 10 and the solution reached room temperature overnight and stirred for an additional 24 hours. After hydrolysis with phosphate buffer (pH 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO ₄ ), the solvent was evaporated and the residue was purified by recrystallization from MeCN.

Yield: 3.43 g (33.3%)

^J H NMR (CDCl ₃ ) 5: 0.70 (d, 6H), 1.03 (d, 6H), 1.44 (s, 6H), 2.15-2.30 (m, 4Η), 3.66 (s, 6H), 3.69 (s, 6H), 3.92 (m, 2H), 4.04 (m, 2H), 4.09-4.20 (m, 4H). ¹³ C NMR (CDCl ₃ ) δ: 16.78, 19.11, 27.67, 31.88, 38.38, 52 .37, 52.44, 52. . 83, 60.84, 77.65, 108. 57, 163.15, 163.88

FAB-MS signal at m / z 495.4 (53), 253.2 (40),

197.2 (58), 141.1 (100)

C ₂₅ H ₄₂ N ₄ O (494):

Shit. %: C, 60.71; H, 8.56; N, 11.33

Found: C, 60.89; H, 8.42; N, 11.45.

I!. IHJ ·

b) (2S, 3S) -1,4-Bis ((2R, 4S, 5S, 7S) -2,7-Dramino-4,5-dihydroxy-4,5-0,0-isopropylideneoctanedicarboxylic acid dimethyl diester) 5S) -3,6-Dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) -2,3-dihydroxy-2,3-0,0-isopropylidenebutane (2.5 g, 5.06 mmol) was dissolved in 40 mL of dioxane . 20.0 mmol of HCl solution in 40 mL of water was added and the solution was stirred at room temperature overnight. The solvents were removed, the residue was dissolved in water and extracted with diethyl ether. ammonia was added to the aqueous layer until pH 9 was reached and this layer was extracted with chloroform. After drying (MgSO4, solvent removed and valine methyl ester distilled (30-40 ° C, 0.04 torr).

Yield: 1.507 g (98.0%).

hl NMR (CBC1 ₃₎ δ:

1.32 (s, 6H), 1.53 (br s, 4H) 1.60-1.68 (m, 2H), 1.85-1.94 (m, 2H), 3.63 (m, 2H), 3.67 (s, 6H), 3.80 (m, 2H).

¹³ C NMR (CDCl ₃ ) δ:

27.25, 37.20, 51.96, 52.74, 77.58, 108.80, 176.10.

c) (2S, 4S, 5S, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-0,0-isopropylideno-octanedicarboxylic acid

The dimethyl diester from steps b) (1.51 g, 4.96 mmol) was dissolved in 5 mL of dioxane, 4.95 mL of 2N LiOH in water (9.91 mmol) was added and the solution was stirred under argon overnight. PSC showed quantitative formation of (2S, 4S, 5S, 7S) -2,7-diamino-4,5-dihydroxy-4,50,0-isopropylideneoctandicarboxylic acid. The crude solution was used in the next step.

.11

d) (2S, 4S, 5S, 7S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino-4,5-dihydroxy-4,5-O, O-isopropylideneoctandicarboxylic acid (2S, 4S, 5S, 7S) -2.7 The crude solution of -diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandioic acid (4.96 mmol) was cooled to 0 ° C and 16 mL of 1 N NaOH solution (16 mmol) was added, followed by small portions of 3.89 g of FmocCl. (15.0 mmol) in 16 mL of dioxane and the mixture was stirred at room temperature for 90 min. The solution was acidified by addition of KHSO _4. After extraction with CHCl ₃ , the organic layer was dried (MgSO ₄ ), the solvent removed and the residue purified by flash chromatography (hexane / ethyl acetate / acetic acid 5/5/1).

Yield: 1.69 (46%).

¹ H NMR (DMSO / D ₂ O) δ:

1.23 (s, 6H), 1.7-2.0 (m, 4H), 3.62 (m, 2H), 4.02 (m, 2H),

4.1-4.35 (m, 6H), 7.2-7.9 (m, 16H).

¹³ C NMR (DMSO) δ:

27.94, 34.59, 47.39, 52.29, 66.45, 77.49, 109.13, 120.90, 125.91, 127.97, 128.59, 141.44, 144.44, 157.02, 174.75.

FAB-MS signal at m / z 743.3 (28), 563.2 (58),

502.2 (72).

C ₄₁ H ₄₀ N ₂ O ₁₀ (720):

Shit. %: N, 3.89.

Found: N, 4.09.

I L ium

EXAMPLE (2S, 4R, 5S, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-isopropylideneoctandicarboxylic acid

a) (2R, 3S) -1,4-Bis [(2R, 5S) -3,6-dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl] -2,3-dihydroxy-2,3-0 , 0-Isopropylidenobutane (2R) -2,5-dihydro-3,5-dimethoxy-2-isopropylpyrazine (6.08 g, 33.0 mmol) was dissolved in anhydrous THF and the solution was cooled to -78 ° C. BuLi solution in hexane (33.0 mmol, 20.6 mL) was added. After 45 minutes at -78 ° C, a solution of (2R, 3S) -1,4-dibromo-2,3-dihydroxy-2,3-O-isopropylidenobutane (4.52 g, 15.7 mmol) in 10 mL of THF was added dropwise. The solution was brought to room temperature overnight and stirred for an additional 24 hours. After hydrolysis with phosphate buffer (pH 7), the mixture was extracted with diethyl ether and the organic layer was washed with water and brine. After drying (MgSO 4), the solvent was evaporated and the residue was purified by flash chromatography (hexane / ethyl acetate 9/1).

Yield: 0.77 g (10%).

¹ H NMR (CDCl ₃ ) δ:

0.68 (m, 6H), 1.03 (m, 6H), 1.32 (s, 3H), 1.45 (s, 3H),

1.30-1.55 (m, 1H), 1.85-2.30 (m, 5H), 3.62 (s, 3H), 3.66 (s, 3H), 3.68 (s, 6H), 3.92 (m, 2H), 4.06-4.26 ( m, 3H),

4.45 (m, 1H).

¹³ C NMR (CDCl ₃ ) δ:

16.62, 16.77, 19.04, 19.08, 26.20, 28.49, 31.71, 31.91, 33.69, 35.07, 52.21, 52.27, 52.32, 52.35, 52.40, 52.98, "!, E

60.58, 60.80, 74.31, 74.66, 107.16, 163.03, 163.40,

163.91, 164.30.

b) Dimethyl diester of (2S, 4R, 5S, 7S) -2,7-diamino-4,5-dihydroxy-4,5-0,0-isopropylidenectanodicarboxylic acid (2R, 3S) -1,4-Bis ((2R, 5S) -3,6-Dimethoxy-2-isopropyl-2,5-dihydro-5-pyrazinyl) -2,3-dihydroxy-2,3-0,0-isopropylidenebutane (0.77 g, 1.55 mmol) was dissolved in 12 mL of dioxane . 6.0 mmol HCl in 12 mL water was added and the solution was stirred at room temperature for 5 hours. Ammonia was added until pH 9 was reached and the solution was extracted with chloroform. After drying (MgSO ₄ ), the solvent was removed and the valine methyl ester was removed

was distilled (30-40 ° C, 0.05 torus). Yield 0.36 g (77 g) %). ^y H NMR (CDC1 ₃₎ δ: 1.30 (s, 3H), 1.41 (s, 3H), 1.40-1.48 (m, 1H), 1.72 (s, 4H), 1.75-2.00 (m, 3H), 4.40 (m, 2H). 3.60-3.67 (m, 2H) , 3.70 (s, 6H) , 4.20- ¹³ C NMR (CDCl 3) δ: 25.81, 28.26, 34. 69, 34.89, 51.55, 52.00, 52.02, 52.66,

74.26, 75.20, 108.23, 175.43, 176.51.

c) (2S, 4R, 5S, 7S) -2,7-Diamino-4,5-dihydroxy-4,5-0,0-isopropylideneoctandicarboxylic acid

The dimethyl diester from step b) (0.36 g, 1.19 mmol) was dissolved in 5 mL of dioxane, and 1.19 mL of 2 N was added.

LiOH solution in water (2.38 mmol) and the solution was stirred under argon overnight. PSC control showed quantitative (2S, 4R, 5S, 7S) -2,7-diamino-4,5-dihydroxy-4,5I L 1111 ·.

0, O-isopropylideneoctandioic acid. The crude reaction solution was used in the next step.

d) (2S, 4R, 5S, 7S) -2,7-Bis (9-Fluorenylmethyloxycarbonylamino-4,5-dihydroxy-4,5-O, O-isopropylideneoctandicarboxylic acid

The crude solution of (2S, 4R, 5S, 7S) -2,7-diamino-4,5-dihydroxy-4,50, O-isopropylideneoctanedicarboxylic acid (1.19 mmol) was cooled to 0 ° C and 4.0 mL of 1 N NaHCO _{3 was} added ( 4.0 mmol). Then 1.03 g of a solution of FmocCl (4.0 mmol) in 4 ml of dioxane was added in small portions and the mixture was stirred at room temperature for 60 minutes. The solution was acidified by addition of KHSO ₄ . After extraction with CHCl ₃ , the organic layer was dried (MgSO ₄ ) and the solvent removed. The residue was purified by flash chromatography (hexane / ethyl acetate / acetic acid 5/5/1).

Yield: 0.566 (66%).

² H NMR (DMSO, D ₂ O) δ:

1.16 (s, 3H), 1.30 (s, 3H), 1.50-2.05 (m, 4H), 3.80-4.40 (m, 8H), 7.15-8.0 (m, 16H).

NMR (DMSO, D ₂ O) δ: 63, 29.05, 31.50, 31 -94, 47.43, 51.52, 52.19, , 66.50, 10, 75.10, 108.36, 120. 88, 125.93, 127.98, 128.60, .48, 144.39, 144.76, 156 ..82, 157.12, 174.11, 175.11.

u

EXAMPLE (S, S) -2,6-Diamino-4-oxaheptandicarboxylic acid

a) Bis [(2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl] methyl ether

Butylitis in hexane (1.6 M, 6.25 mL, 10 mmol) was added to a solution of (2R) -2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (1.84 g, 10.0 mmol) in dry THF (8 mL) in the presence of - 78 ° C. The mixture is stirred for 15 minutes. A solution of bis (chloromethyl) ether (0.67 g, 5.0 mmol) in dry THF (2 mL) was added. The reaction mixture was allowed to reach room temperature overnight. The solvent was evaporated and the residue was taken up in diethyl ether. The ether solution was washed with water, dried (MgSO4 and evaporated. The product was purified by chromatography (silica gel, hexane / ethyl acetate 2: 1)).

Yield: 0.65 g (32%), the yellow ^J H NMR (300 MHz, CDC1 ₃₎ δ:

s oil.

0.63 (6H, d), 1.05 (6H, d), dd), 3.63 (6H, s), 3.64 (6H, (2H, t), 3. , 98 (6 H, m). ¹³ C NMR (75 MHz, CDCl ₃ ) δ:

2.25 (2H, m), 3.58 (2H,

s), 3.76 (2H, dd), 3.83

16.4, 19.1, 30.1, 52.1, 52.2,

165.0.

56.9, 60.3, 72.5, 161.0,

b) Dimethyl diester of (S, S) -2,6-diamino-4-oxaheptandicarboxylic acid

Bis [(2R, 5S) -2,5-dihydro-3,6-dimethoxy-2-isopropyl-5-pyrazinyl] methyl ether (0.65 g, 1.49 mmol) was dissolved

1 L methanol (5 mL) and 0.25 M hydrochloric acid (23.8 mL, 5.96 mmol) was added. The mixture was stirred at room temperature overnight. The water / methanol solution was extracted with diethyl ether. The pH was then adjusted to pH 10 by the addition of ammonia and the D-valine methyl ester extracted with ether. Most of the water is evaporated. The residue was used in the next step without further purification.

NMR (300 MHz, DMSO-d ₆₎ δ:

3.72 (6H, s), 3.85 (4H, m), 4.23 (2H, t).

¹³ C NMR (75 MHz, CDCl ₃ ) δ:

52.8, 53.2, 68.9, 168.4.

c) (S, S) -2,6-Diarttino ~ 4-oxa-hepcandicarboxylic acid · 2HCl

The dimethyl ester described above is deflegmated in 6M hydrochloric acid (5 mL) for 3 hours. The solution was evaporated and freeze-dried.

Yield: 420 mg of white solid.

³ K NMR (300 MHz, D ₂ O) δ:

3.87 (2H, dd, J 10.8 Hz, J 3.2 Hz), 3.95 (2H, dd, J

10.8 Hz J 4.6 Hz), 4.13 (2H, dd J 3.2 and 4.6 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ) δ:

53.4, 68.40, 170.3.

u

JI!

d) (S, S) -2,6-Bis [(9-fluorenylmethoxycarbonyl) amino] -4-oxaheptanedicarboxylic acid (5 mL). Tir9-fluorenylReaction was carried out under a nitrogen atmosphere. A suspension of (S, S) -2,6-diamino-4-oxa-heptanedioic acid dihydrochloride (200 μς, 0.75 mmol) in hexamethyldisilazane (10 mL) and chlorotrimethylsilane (1 mL) was heated at 120 ° C for three hours to give a clear solution. The excess of silylating reagents was removed in vacuo and the residue was dissolved in dry dichloromethane, cooled to 0 ° C, and a solution of chloroformate (425 mg, 1.65 mmol) in dry dichloromethane (2 mL) was added. The mixture was stirred for 2 hours and then evaporated. The residue was taken up in THF (5 mL) and 1 mL of 0.1 M hydrochloric acid was added. The mixture was stirred for 15 minutes and then evaporated. The residue was taken up in ethyl acetate. The organic phase was washed with water, dried (MgSO ₄ ) and evaporated. The product was purified by column chromatography (silica, chloroform / methanol / acetic acid 85: 10: 5).

Yield: 310 mg (65%) as a white solid.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ:

3.7 (4H, m), 4.1 (2H, m), 4.25 (6H, m), 7.2-7.4 (10H,

m), 7.7 (4H, d), 7.8 (4H, d).

EXAMPLE (S, S) -2,7-Diamino-4-azaoctandicarboxylic acid

a) A solution of (S, S) -2,7-Bis (tert-butyloxycarbonylamino) -4-azaoctandicarboxylic acid (S) -4-amino-2-tert-butyloxycarbonylamino butyric acid (1.03 g, 4.70 mmol) in acetonitrile ( 50 ml) was added to a .11 (S) -boc-serine-p-lactone (0.8 g, 27.4 mmol) in acetonitrile (50 ml) and degassed solution of H _z O. the reaction medium pH was adjusted to 5.5 dropwise with 1 M NaHCO ₃ and stirring. The mixture was stirred for 5 days at room temperature. The pH was adjusted by addition of NaHCO ₃ and the reaction was controlled by PSC. Nudistiliuoti solvents, the residue is pulverized with chloroform (5 ml), to which acetic acid was added to obtain pH 5. The chloroform solution was evaporated and the product was separated by chromatography on silica gel ((CHCl _3: MeOH 2: 1).

Yield: 0.92 g (53%) of a white solid.

NMR (DMSO-d _6, 300 MHz): 6

7.33 (d, J 6.3 Hz; 1H), 6.27 (d, J 5.7 Hz, 1H), 4.42 (d, J 7.5 Hz, 1 K), 4.15 (dd, J i 10.5 Hz, J ₂ 3.9 Hz, 1H) ), 4.01 (d, J 6.3 Hz; 1H), 3.87 (pi s; 1H), 2.78 (pi s; 1H), 1.78-2.21 (m; 4H), 1.36 and 1.35 (2 s; 18 H) .

13C NMR {DMSO-d _6, 75.43 MHz): δ

174.95, 155.74, 155.25, 79.58, 78.22, 78.04, 62.97,

56.42, 56.00, 51.17, 48.99, 38.14, 28.62, 28.46.

C ₁₇ H ₃₁ N ₃ O _s (405.45).

b) (S, S) -2,7-Bis (tert-Butyloxycarbonylamino) -4- (benzyloxycarbonyl) -4-azaoctandicarboxylic acid

M NaHCO ₃ (5.9 mL, 5.92 mmol) and benzyloxycarbonyl chloride (0.5 g, 0.42 mL, 2.96 mmol) were added to (S, S) 2,7-bis (tert-butyloxycarbonylamino) -4-azaoctanedicarboxylic acid (0.6 g, 1.48 mmol) ) in dioxane (40 mL) and H ₂ O (30 mL) at 0 ° C. The mixture was stirred at 0 ° C for 3 hours at room temperature for 6 hours, evaporated, and the residue triturated with chloroform (5 ml) and acetic acid to pH 5. The chloroform solution was evaporated and the product was separated by chromatography on silica gel. (CHCl ₃ : MeOH 4: 1).

Yield: 0.39 g (50%) of a white solid.

¹ H NMR (DMSO-d ₆ , 300 MHz): δ

-7.37 (m, 5H), 7.18 (d , J 7.8 Hz; 1 H), 6.23 (d, J Hz; 1 H), 4.98 (s, 2H) , 4.43 {dd, J 10th 8 Hz, J 3.3 1 H), 4.09 {dd, J 10.2 Hz, J 6.3 Hz; 1H) , 3.88-4.02 2H), 3.01 {d, J 5.7 Hz; 2H), 1.7 4-1 .88 (m, 1H), -1.72 (m, 1H), 1.35 and 1.33 (2d, 18H)

¹³ C NMR (DMSO-d ₆ , 75.43 MHz): δ

177.42, 177.38, 161.20, 160.63, 160.16, 142.20, 133.48, 132.91, 132.78, 83.55, 83.14, 70.76, 70.40, 59.39,

56.44, 53.61, 42.30, 36.01, 33.28.

C25H37N3O10 (539.58).

c) (S, S) -2,7-Diamino-4-benzyloxycarbonyl-4-azaoctanedicarboxylic acid (S, S) -2,7-Bis (tert-butyloxycarbonyl) -4-benzoloxycarbonyl-4-azaoctandicarboxylic acid (0.29 g, 0.37 g) mmol) dissolved in THF (10 mL), the solution was stirred at room temperature for 2 hours and TFA was distilled off to give the title compound which was used in the next step without further purification.

d) (S, S) -Benzyloxycarbonyl-2,7-bis (9-fluorenyl-methoxycarbonyl) -4-azaoctandicarboxylic acid (S, S) -2,7-Diamino-4-benzyloxycarbonyl-4-azaoctandicarboxylic acid ( 0.2 g, 0.37 mmol) dissolved in dioxane (10 mL) and H ₂ O (10 mL) were added 9-fluorenylmethyl chloroformate (0.29 g,

1.11 mmol) and 1M NaHCO ₃ (2.96 mL, 2.96 mmol) were stirred at 0 ° C and stirring was continued for 10 hours at room temperature, after which the solvents were evaporated. The remaining solid was triturated with chloroform (5 mL) and the pH was maintained at about 5 by the addition of acetic acid. The solution was then evaporated and the product was separated by chromatography on silica gel (AcOEt: hexane: AcOH 10: 10: 1).

Yield: 0.1 g (35%) as a white solid.

² H NMR (DMSO-d 6, 300 MHz): δ

7.21-7.44, 7.61-7.91 (m, 22H), 6.88 (d, J 6.3 Hz, 1H),

4.96 (s, 2H), 4.56 '(dd, J 10.8 Hz, J 3.3 Hz; 1H), 3.944.28 (m, 8H), 3.08 (d, J 5.7 Hz, 2H), 1.86-1.98 (m,

1H)., 1.69-1.82 (m; 1H).

^13C NMR (DMSO ₆ , 75.43 MHz): δ 172.98, 172.49, 156.49, 156.04, 144.32, 144.24, 144.17, 142.97, 141.07, 139.81, 137.82, 137.55, 12:30 pm, 128.66, 128.06, 127.99, 127.66, 127.46, 125.68, 121.75, 120.39, 110.06, 66.22, 65.60, 55.26, 52.20, 47.08, 47.04,

36.63, 37.74.

C ₄₅ H ₄₁ N ₃ O ₁₀ (783.83).

II

: .. 11

Solid phase peptide synthesis

Solid phase peptide synthesis is performed essentially according to the principles of fluorenylmethoxycarbonyl (Fmoc)-polyamide synthesis (Atherton and Sheppard, Solid phase peptide synthesis: a practical approach, Oxford: IRL Press at Oxford University Press, 1989). Commercially available synthetic resins were used; they were polystyrene with acid labile (Wang, J. Amer. Chem. Soc., 95, 1328-1333, 1973) or acid hyperlabile coupling agents (Merger) by periodic synthesis either manually or using a semi-automatic device (Labortec Peptide Synthesizer 5P 650). et al., Tetrahedron Letters 29, 4005-4008, 1988). Alternatively, peptides are constructed completely automatically on fluid resins (Atherton et al., J. Chem. Soc. Chem. Commun., 1151-2, 1981) using an LKB Biolynx 4170 automated peptide synthesizer. Resins that already had the protected required C-terminal Fmoc-amino acid residue were purchased. Chain elongation was achieved in various ways with side chain protected Fmoc-amino acid pentafluorenyl esters (Kisfaludy and Schoen, Synthesis. 325-327).

1983) using activation with dicyclohexylcarbodiimide (DDC) / 1-hydroxybenzotriazole (HOBt) (Koenig and Geiger, Chem Ber. 103, 2034-2040, 1970) or using the coupling reagent PyBOP (Coste et <1, Tetrahedron Lett). ., 31, 205-208, 1990). The amino group of the lysine side chain was protected as t-butyloxycarbonyl, the carboxyl groups of the glutamine or aspartic acid side chain were protected as t-butyl esters.

Synthetic resins carrying the required N-deprotected C-terminal residue were acylated with 1/2 equivalent of Fmocoprotected diaminocarboxylic acid by addition of DCC and

KGBt, After the reaction was completed, excess reagent was washed off. Peptidyl resin exposed again to DCC / HOBt, if f. IU · to attach carboxyl groups which may have remained free. After this step, the resin was washed with methanol to deactivate any carboxyl groups that cannot bind to the resin-bound amino groups. Finally, the excess amino groups are acetylated. Peptide synthesis was then continued as usual.

After complete formation of the required sequences on the solid phase, 10 peptides are cleaved from the synthesis resins with concurrent side-chain deprotection using trifluoroacetic acid to which the appropriate cleaning chemicals have been added (King et al., Int. J.Peptide Protein Res. 35,

255-268, 1990). After evaporation, the peptides were separated by precipitation with diethyl ether and drying. Purification was by preparative reverse-phase liquid chromatography

Table

Analysis of peptides of general structure (Pyr-Glu-Asp) _2- bridging residue (Lys-OH) ₂

Bridge Remnant Type ^a AVSC Method ^b (Delay Time, Minutes) Purity ^c (%) FAB-MS [M + H] ⁺ 1 2 3 4 C-6 10-40-20 (16) 95 1199.5 C-5 0-30-20 (17) 95 1185 C-l C-0 (mezo) 0-20-20 (19) 98 1115.4 C-0 (1 isomer) 0-20-20 (19) 98 1115.4 C-0 (2 isomers) 0-20-20 (19) 97 1115.4 C-4-trans-en 0-30-20 (12) 98 1169.4

JI H

Table I (continued)

1 2 3 4 C-4-cis-ene 10-40-20 (13) 100 1170.6 C-4-in 0-30-20 (10) 98 1167.5 C-4-vic-glycol 0-30-20 (14) 98 1203.2 (1 isomer) C-4-vic-glycol 0-30-20 (13) 98 1203.4

(Isomer 2) C-4-vic-glycol (Isomer 3) C-3-Amine

C-3-Ether ^a Specifies examples ^b Methods are expressed as gradients of mobile phase BA 5 over time, e.g. 10-40-20 refers to a gradient beginning at 10 and ending at 40% B in .20 minutes. Mobile phases: A) 0.1% TFA B) 0.1% TFA% MeCN. Column: Vydac TP54, O.x25 cm, 5 μιη particles, 100A pair; flow rate of 1 ml / min.

^c Indicates HPLC peak integration (λ = 215 nm)

II

I

1UJ ·

Table

Amino acid analysis data for the general structure peptide (Pyr-Glu-Asp) _2- bridging residue (Lys-OH) ₂

Bridge Remnant Type Asp found (theor.) Glu found (theor.) Lys found (theor.) C-6 1.0 (1) 2.15 (2) 0.93 (1) C-5 1.0 (1) 2.18 (2) 0.94 (i) C-l C-0 (mezo) 1.0 (1) 2.15 (2) 0.93 (1) C-0 (1 isomer) 1.0 (1) 2.18 (2) 0.93 (1) C-0 (2 isomers) C-4-trans-en 1.0 (1) 2.15 (2) 0.93 (1) C-4-cis-ene 1.0 (1) 2.17 (2) 0.93 (1) C-4-in 1.0 (1) 2.17 (2) 0.95 (1) C-4-vic-glycol (1 isomer) C-4-vic-gi icol (2 isomers) C-4-vic-glycol (3 isomers) C-3-Amine C-3-Ether 1.0 (1) 2.15 (2) 0.93 (1)

^a Specifies examples

EXAMPLE

Peptide Synthesis: (pGlu-Glu-Asp) ₂ - (E) -DHs (Lys) ₂

The peptide was synthesized using the Labortec peptide synthesizer. Fmoc-Lys (Boc) -Sasrin polymer (1.0 g, 0.6 mmol; BAchem AG; substitution 0.6 mmol / g) was placed in a 100 mL reaction flask. Fmoc-DHs (150 mg, 0.23 mmol), DCC (290 mg, 1.4 mmol) and HOBt (211 mg, 1.4 mmol) in DMF (20 mL) were added to the polymer and allowed to react for 9 hours. Additional DCC (1.4 mmol) and HOBt (1.4 mmol) were added and the reaction allowed to proceed for a further 2 hours. The polymer was then washed with CH ₂ Cl ₂ , 30% MeOH / CH ₂ Cl _2, and DMF. The free amino groups in the polymer were acetylated using 10% Ac ₂ O / DMF (3x in 1 hour; Kaiser negative test).

The remainder of the synthesis was carried out according to the standard procedure using Fmoc-Asp (OtBu) -Opfp (1.35 g, 2.3 mmol), Fmoc-Glu (OtBu) -Opfp (1.35 g, 2.3 mmol) and pGlupentachlorophenyl ester (0.86 g, 2.3 mmol). HOBt (35 mg,

2.3 mmol) was added at each coupling step which was allowed to proceed for 1 hour. The end of the connection was determined by a negative Kaiser test. After coupling with Fmocamic acid, the polymer was washed with DMF, the protecting group was removed with 20% piperidine in DMF, and the polymer was again washed with DMF. After final coupling, the polymer was washed with MeOH / CH ₂ Cl ₂ and CH ₂ Cl ₂ . The weight of the dried polymer-peptide was 1 g. The peptide was separated from the polymer by TFA: CH ₂ Cl ₂ 1: 1, the solution was dried in water, filtered by freezing; Yield of mg of this product was directed by freeze-drying, the residue was dissolved (0.45 μ) and the filtrate was dried

430 mg. For further purification, 100 was dissolved in water (0.5 mL) and a rotary AVSC using a 2.1x15 cm Beckman Ultrasphere ODS column and solutions A 0.1% TFA H ₂ O, B 0.1%

TFA MeCN: H ₂ O 40:60. The gradient was 0-15% B, 120 miII

I

Flow rate, 5 ml / min. 19 mg of pure peptide (> 95%) was obtained after freeze-drying. FAB-MS:

[M + H] 1169.3.

HOBt = Hydroxybenzotriazole

Pfp = Pentafluorophenyl

Fmoc = 9-Fluorenylmethoxycarbonyl

Boc = t-Butoxycarbonyl

DCC = Dicyclohexylcarbodiimide (E) -DHs = (S, S) -2,7-Diamino- (E) -oct-4-eno-1,8-dicarboxylic acid

JUK

Claims (8)

DEFINITION OF INVENTION 1. A peptide compound comprising two single-chain haemoregulatory peptides linked together by an amino acid incapable of C atoms in equivalent positions in each of said peptides via a carbon-carbon bond or through a divalent linking group bridged -A-, where A is C ₁ _ ₆ carbon chain which is i) a) mono- or poly-substituted with -R ^A , -OR ^A , -S ^{A A} , -NR ^a R ^a or -COOR ^A , or with a halogen atom such as fluorine, chlorine, bromine or iodine (where each R ^A independently represents a hydrogen atom or a C ₁ _ ₆ alkyl, or alkanoyl -alkoksialkilo group which may be mono- or polyhydroxylated) and / or (b) containing one or more carbon-carbon double or triple bonds, and / or c) to which is added one or more groups -Z-, wherein each -Z- is independently -0-, -CO-, J > C = NR or -NR - (wherein each R is independently a hydrogen atom or a Ci-g alkyl or _{C 6} _ aryl group) or where one or more substituents or unsaturated anglisanglis the interconnections group -A-, -Z - may also be -Or (ii) (CH ₂ ) _y , where y is 1, 5 or 6, II There is no n-chain attached to each of the above Ca atoms. A peptide compound according to claim 1, characterized in that said linker -A- is selected from -ch ₂ -ch ₂ ch ₂ ck ₂ ch ₂ ch ₂ 10 -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CK ₂ -HC = CH-HC = C = CH ~ -CH ₂ -CH = CH-CH ₂ -ch ₂ -c = c-ch ₂ 15 Clb-CH-CH- '' H-CH-CH ·? ” -CH-CH ₂ -CsC-CH ₂ -CH ₂ -ch ₂ -c = cc = c-ch ₂ ch ₃ ch ₃ ! ί 20 -CH-CH = CH-CH ₂ -CHOH OH I I -CH-CH = CH-CH25 -CH ₂ -O-CH ₂ -CH ₂ -NH-CH ₂ CH ₂ -ch ₂ ch ₂ -co-ch ₂ ch ₂ -CH-C K = CH-CH 30! | och ₃ och _{3 is a} native alpha group -ch ₂ -ch = ch-ch ₂ -s-ch ₂ 1Š1J -CH ₂ -CsC-CH ₂ - NH-CH ₂ CH ₂ - or -CH ₂ -CH = CH-CH (COOH) CH ₂ -. 3. The peptide compound of claim 2, wherein said bridging group -A- is selected from (E) -buten-2-enylene, (Z) -buten-2-enylene, buten-2-inylene, methylene, n-pentylene and hexylene. Peptide compounds according to any one of claims 1 to 3, characterized in that said single-chain homoregulatory peptides are represented by formula I R ^a - R ^b - R ^c - R ^d - (R ^e ) _n -R ^f (I) in which R ^a represents li'Nii - CH - CO I <Cii i R NH or Clf - CO i CI1..OH R stands for --NH- CH co-- nu- CH-- COCCH> CH, OH I Y COR or II I BB R stands for - NH CH - co, vol t'CH _> I V COR ' R stands for. NH ----- Cil - CO · I CH SP? ' or R stands for R stands for • Nil CH —— COR I C1I> I ^s NH_. NH - CH - CO ~ I CH, OH ----- Ni i CH- CO I Cil. - Nll ------- CH --- _co - Alba ch ₂ oh Oj. nh -— ch - co · R ⁹ . NH - CH - COR I -3 NH ai'ba. NH - CIU-COR NH NH (Where n and m independently represent 0 or II jtm p, q, and r independently represent 1 or 2; s represents 3 or 4; R ¹ and R ^{2 are} both hydrogen or together represent 5 oxo; R ³ and R ^{4 are} both hydrogen or taken together to represent a carbon-carbon bond; R ⁵ is hydrogen or acyl; each R ⁶ and R ⁷ independently represents a hydroxyl group or an amino group, but preferably a hydroxyl group; R ⁸ represents hydrogen; C ₂ _ ₆ alkyl group; C ₇ _ ₂₀ -aralkilo group which may have one or more hydroxy, amino or methoxy substituents; or a metabolically labile S-protecting group; R ⁹ represents hydrogen or methyl; and R ¹⁰ represents a hydroxyl or amino group, an amino acid glutamine residue, or a peptide having an N-terminal glutamine moiety. 5. Peptide compounds according to claim 4, characterized in that they are represented by formula II R ^a - R ^b - R ^c - NH - CH - CO - (R ^e ) _n - R ^f 30 | A (II), 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 4. it Pharmaceutical composition comprising a peptide compound together with a pharmaceutical carrier or excipient, characterized in that the peptide compound is a compound according to any one of claims 1 to 5. A peptide compound according to any one of claims 1 to 5 for use in stimulating myelopoiesis or bone marrow regeneration. 8. A process for the preparation of a peptide compound according to any one of claims 1-5, characterized in that a) bis-lactim ether is metallized and then alkylated to form the bis-lactim dipeptide ether; b) hydrolyzing the bis-lactim dipeptide ether from step a) to form the α, α'-diamino bridged bridge; C) Remaining Amino Acids Introduces into peptide chains; and (d) deploys any protected group. 9. The bridged α, α'-diamino acids of the formula NH_ NH _ i ~ 1 ~ HG-A -GH 1 I, λ HOOG GOGU wherein -A- is as defined in point 1. 35 10. Dipeptide ethers of bis- lactim according to the formula II .IIJHI