WO2008105759A1

WO2008105759A1 - Methods for synthesis of modified peptides

Info

Publication number: WO2008105759A1
Application number: PCT/US2007/004975
Authority: WO
Inventors: Justin O. Brower
Original assignee: Argolyn Bioscience, Inc.
Priority date: 2007-02-27
Filing date: 2007-02-27
Publication date: 2008-09-04

Abstract

Methods for the synthesis of peptides and peptide analogs containing aminoacid residues bearing substituted amino- and guanido- sidechains, such as alkyl lysine and arginine residues, are provided.

Description

METHODS FOR SYNTHESIS OF MODIFIED PEPTIDES

Field of the Invention

The field of the invention is methods for the synthesis of peptides and peptide analogs containing sidechains comprising amino and guanido groups, including substituted derivatives, from precursor peptides, both in solution and bonded to a polymeric support.

Background Peptides and peptide analogs containing aminoacids bearing basic nitrogenous sidechains, such as lysine (Lys), ornithine (Om), and arginine (Arg), and derivatives of these aminoacids, are of great potential importance as medicinal compounds and research reagents. For example, a highly cationic peptide sequence in HIV Tat-1 comprising multiple arginine residues has been found to facilitate cellular uptake of proteins. Cellular uptake is vital for the action of many medicinal compounds. Also, many bioactive natural peptides, synthetic peptide analogs, and fragments of biologically important proteins contain nitrogenous aminoacid sidechain residues. Therefore, synthetic methods for the preparation of materials of this class, particularly if they enable the preparation of series of analogs, are a matter of broad interest.

As peptides are linear polyamides comprising a great variety of structural sequence variability but a relatively small number of commonly encountered monomelic units (aminoacids, or for peptide analogs, aminoacid analogs), various standardized methods have been developed for their synthesis. Among these, solid phase peptide synthesis methods are well-known in the art, wherein a precursor molecule, such as the carboxy-terminal aminoacid of a desired sequence, is anchored in blocked form to an insoluble substrate such as a polymer bead (the "solid phase"), then through an iterative sequence of deblocking and coupling steps a protected version of the target peptide, anchored to the insoluble substrate, is prepared. Subsequent cleavage from the substrate and deblocking yields the target peptide, sometimes in reasonable yield and purity.

Once a target peptide is fully deblocked, further structural modifications are usually very difficult to carry out in any kind of selective manner; the multiple functional groups present on the amino acid sidechains are likely to interfere. Selective deblocking, while possible, complicates synthetic schemes.

To address the issue of synthesizing modified peptides such as those containing aminoacid analogs using solid-phase techniques, two different general approaches have been taken. One is to use in the solid phase synthesis a blocked, aminoacid analog precursor containing the desired modification of the sidechain, so that the aminoacid analog residue is built into the growing peptide chain during the solid phase synthesis process and after deblocking and cleavage provides the target structure. The other is to synthesize a peptide with a precursor residue at the position in the peptide analog where the modified aminoacid residue is desired to be located, then to modify that residue with an appropriate reagent while the blocked peptide is still immobilized on the solid phase ("on-resin"), or after cleavage from the resin ("off-resin").

There are significant advantages to the second approach, notably that a single immobilized blocked peptide analog bearing the precursor residue can be prepared in a single solid phase synthesis, then prior to deblocking and cleavage the solid phase beads can be partitioned into a plurality of reactions wherein different reagents are allowed to react with the precursor to provide a diverse set of products. Alternatively, if the conversion takes place after cleavage, with or without deblocking, the cleaved product can likewise be divided into batches and reacted with various reagents to yield a set of analog structures. In contrast, using the first approach, a different blocked analog aminoacid would be needed to be incorporated into each of a plurality of separate solid phase syntheses to achieve the same result. An alternative general approach to the synthesis of peptides and their analogs is solution-phase synthesis. This is most often used when shorter peptide sequences are targeted or there is a need for larger quantities of product. Here, the synthesis is carried out in the solution phase as in a typical organic synthesis. Using a solution phase approach to prepare peptides with analog aminoacids, an at least partially blocked precursor peptide can be allowed to react with a reagent to carry out the desired conversion of a sidechain precursor moiety to the desired analog. Li the solution phase approach, the same two techniques of either using directly a reagent containing the desired sidechain group in blocked form which is then freed at the end of the synthesis, or using a reagent containing a precursor moiety that can then be treated, usually at the end of the synthetic procedure, with one or several reagents that yield the desired product or set of products are available.

Specifically regarding peptides containing aminoacids with nitrogenous sidechains, two overall conversions are of interest, namely the introduction of an amino group (as is found in lysine and ornithine) to a sidechain and the introduction of a guanido group (as is found in arginine) to a sidechain.

An approach has been reported with respect to the introduction of a methylated lysine residue into a peptide wherein the o>amino group has been replaced by a methyl group. M. Kyle Hadden, et al. (2005),

Neuropharmacology, 49(8), 1149-1159 discuss the preparation of neurotensin analogs wherein a methylated lysine residue is introduced by reaction of an aminoacid derivative bearing a chloroalkylglycine group with methylamine, dimethylamine and trimethylamine, followed by incorporation into a peptide. Peptides containing modified lysine sidechains of this type are also discussed in pending PCT patent applications, numbers PCT/US2005/021580, filed Jun 17, 2005 and published as WO 2006/009902A2, and PCT/US2006/047860, filed Dec 15, 2006.

Several approaches to this problem with respect to the introduction of a guanido group to a peptide sidechain have been described in the literature.

P. Theobald, et al. (1991), J. Med. Chem., 34, 2395-2402, describe an on- resin synthetic method for the introduction of cyano-guanidine groups wherein a sidechain aminoalkyl group is derivatized with diphenyl cyanocarbonimidate:

followed by reaction with an alkylamine, to yield a blocked, immobilized peptide with sidechain -NH-C(=NCN)-NHR. By this method, only one nitrogen atom of the guanido group could bear a substituent. No method was provided for removal of the cyano group

Y. F. Yong, et al. (1996), J. Org. Chem., 62, 1540-1542, describe an on- resin conversion of aminoalkyl sidechains to guanido groups using the reagent N,N'-bis(Boc)thiourea and 2-chloro-N-methylpyridinium iodide. While this reagent introduces guanido groups, no substituted guaπido groups are accessible by this method.

Y.F. Yong, et al. (1998), Tetrahedron Lett., 40, 53-56, describe an on- resin conversion of aminoalkyl sidechains with the reagents:

B^ocH

oc , wherein X = H, NO₂. Again, substituted guanido groups are inaccessible using this reagent.

Z. Szekely, et al. (1999), Tetrahedron Lett., 40, 4439-4442, describe the on-resin use of the reagent

followed by a mono- or dialkylamine to provide guanido peptide derivatives wherein the free amino group of the guanidine moiety may be mono-or disubstituted.

P. Wender, et al. (2000), Proc. Nat'l Acad. ScL US, 97(24), 13003-13008, describe a solution-phase (following cleavage of the precursor peptide from the solid-phase resin) guanidylation procedure using pyrazole-1-carboxyamidine. Only unsubstituted guanido groups are introduced by this method.

Y. Zhang and A.J. Kennan (2001), Organic Letters, 3(15), 2341-2344, describe an on-resin method for conversion of lysine and of 2-aminoethylglycine residues into their corresponding guanido derivatives by condensation of the deblocked sidechain amino group with the reagent:

This reagent similarly does not allow for the introduction of substituted guanidine groups, so only unsubstituted guanido derivatives (e.g., arginine and its homologs) can be prepared by this method.

M. Ito, et al. (2003), Tetrahedron Letters, 44, 7949-7952, describe a solution-phase conversion of an amino group of a macrocyclic peptoid to a guanido group via reaction with N,N'-bis(Boc)-S-methylthiourea. J.C. Califano, et al. (2005), Tetrahedron, 61, 8821-8829, describe a process for a solution-phase guanidylation conversion using pyrazole-1- carboxyamidine, as in Wender's above-cited work.

For the purposes of synthesizing peptides with substituted guanido sidechain, there is a need for a method allowing for greater structural flexibility.

The preparation of an amino-containing sidechain of a constituent aminoacid of a target peptide via a synthetic conversion carried out on a peptide substrate is similarly desirable in that not only can the usual primary amino groups of aminoacid sidechains (lysine, ornithine) be prepared from precursor aminoacid residues, but substituted sidechain amines as well.

Therefore, there is an ongoing need for a method of guanidylation and for a method of animation whereby products with substituted guanido or amino groups can be obtained, particularly in the field of peptide synthesis.

Summary of the Invention

The present invention provides a method of preparation of a compound of Formula (I): R³

(I), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, O(Ci-C6alkyl), 0(Ce-Ci₀ aryl), NR₂, an aminoacid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an aminoacid residue, or a peptide residue; or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer; R is independently at each occurrence H, alkyl, aryl, heterocyclyl, heteroaryl, a nitrogen protecting group, or a nitrogen capping group, or two R groups together with the nitrogen atom to which they are bound form a 5-7 membered heterocyclic ring that can further comprise 1-3 additional heteroatoms N, O or S;

L is a saturated or unsaturated, linear, branched or cyclic hydrocarbyl or a combination thereof, comprising n carbon atoms, wherein any carbon atom can be substituted or unsubstituted, wherein 0-3 heteroatoms O, S, or NR can be substituted for a carbon atom of L; n is 1 to about 10,

R¹, R² and R³ are independently hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl, or heteroaryl, wherein one of R¹, R² or R³ may be absent, wherein any alkyl can contain 0-3 heteroatoms comprising N, O, or S, wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted; the method comprising contacting a compound of Formula (II):

(II), wherein Y is a leaving group; with an amine of the formula R¹R²R³N, in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (I).

The present invention further provides a method for the preparation of a compound of Formula (IET):

(in), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(C₁-C₆ alkyl), 0(C₆-Ci₀ aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an amino^" acid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer;

R is independently at each occurrence H, alkyl, aryl, heterocyclyl, heteroaryl, a nitrogen protecting group, or a nitrogen capping group, or two R groups together with the nitrogen atom to which they are bound form a 5-7 membered heterocyclic ring that can further comprise 1-3 additional heteroatoms N, O or S;

L is a saturated or unsaturated, linear, branched or cyclic hydrocarbyl or a combination thereof, comprising n carbon atoms, wherein any carbon atom can be substituted or unsubstituted, wherein 0-3 heteroatoms O, S, or NR can be substituted for a carbon atom of L; n is 1 to about 10;

R⁴ and R⁵ are independently at each occurrence H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein any alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl residue may be unsubstituted or substituted; provided that R⁴ or R⁵ is not a Boc group; the method comprising contacting a compound of Formula (V):

(V) wherein R¹ is hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocycles, or heteroaryl; wherein any alkyl can further contain 0-3 heteroatoms comprising N, O, or S, wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted; with a compound of Formula (IV):

SR⁶

R⁵L ^^ _R4

R⁵

(IV), wherein R⁶ is substituted or unsubstituted alkyl or aryl, or a salt thereof; in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (III).

The invention further provides a method of preparation of a compound of formula (III):

(πi), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(Ci-Ce alkyl), 0(Ce-CiO aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an amino acid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer;

R at each occurrence is independently H, alkyl, aryl, heterocyclyl, heteroaryl, a nitrogen protecting group, or a nitrogen capping group, or two R groups together with the nitrogen atom to which they are bound form a 5-7 membered heterocyclic ring that can further comprise 1-3 additional heteroatoms N, O or S;

R¹ is hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocycles, or heteroaryl; wherein any alkyl can contain 0-3 heteroatoms comprising N, O, or S, wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted;

R⁴ and R⁵ are at each occurrence independently H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein any alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl residue may be unsubstituted or substituted; provided that R⁴ or R⁵ is not a Boc group; from a compound of formula (II):

(II), wherein Y is a leaving group; the method comprising contacting the compound of formula (II) with an amine of the formula R¹NH₂, in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (V):

(V), then, contacting the compound of formula (V) with a compound of Formula (IV):

SR⁶

^R5\ ^^ _^R⁴ R»

Detailed Description of the Invention Definitions

As used herein, an "aminoacid" refers to an organic molecule including an amino group and a carboxylic acid group, which can be an o>aminoacid, a β- aminoacid, a ribosomal aminoacid, a non-natural aminoacid, a D-aminoacid, an L-aminoacid, a racemic amino acid or any other organic molecule comprising an amino group and a carboxylic acid group in free or blocked form. It also includes an organic molecule that resembles a natural aminoacid, that is, an aminoacid analog. For example, the term "aminoacid," includes natural (ribosomal) amino acids (e.g. Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, HyI, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI) and also includes the D as well as the L form, as well as non-natural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and non-natural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and non-natural amino acids protected at the carboxy teiminus (e.g. as a (Ci-C6)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, Greene, T.W.; Wutz, P.G.M. Protecting Groups In Organic Synthesis, 2^nd edition, John Wiley & Sons, Inc., New York (1991) and references cited therein).

An "aminoacid residue" is an aminoacid or an aminoacid analog incorporated into a polymeric chain, for example a polyamide chain. More specifically, the polyamide chain can be a peptide chain, blocked or unblocked, on-resin or off-resin. As used herein a "peptide" includes a linear or a cyclic polyamide comprising natural or non-natural aminoacids bonded to each other via amide linkages between their respective amino groups and carboxyl groups, but may also include other moieties. For example, a peptide as defined herein may include a sugar group, a phosphate group, a polysaccharide group, a nucleoside group, a fluorescent reporter group, an antigenic group, or another complex organic structure, covalently bonded to the linear polyamide. A "peptide" as the term is used herein also includes what is frequently termed a "peptoid," that is, an organic structure that contains natural or non-natural aminoacids bonded to each other via amide linkages but also can include linkages other than amide bonds between aminoacids, and can be linear or cyclic, i.e., peptide analogs. For example, a cyclic depsipeptide is a "peptide" within the meaning herein, as are structures resembling peptides wherein isosteric replacements of amide linkages, such as hydroxyethyl linkages and the like, are present. Thus, an "aminoacid residue" can be a ribosomal aminoacid incorporated into a polyamide chain wherein the polyamide is a peptide, or it can be a non-natural amino acid such as a β-aminoacid or a D-aminoacid incorporated into a peptide analog.

When the aminoacid is internal to the chain, that is, it is bonded to an adjacent amino group via the aminoacid's carboxyl group (forming an amide bond) and to an adjacent carboxyl group via the aminoacid residue's amino group (forming another amide bond) the term "residue" refers to the anhydro form of the aminoacid, wherein the elements of water have been removed from the aminoacid through the formation of the two amide or peptide bonds. When the aminoacid residue is at an end of a polyamide chain, for example a C- terminal aminoacid residue, it is understood to include a group attached to the carbonyl of the aminoacid residue's carboxyl group such as a hydroxyl group, an alkoxyl or aryloxyl group, and amino group, an alkylamino group, an arylamino group or the like, to occupy the valence. When the aminoacid residue is, for example, an N-terminal aminoacid residue of a peptide, it is understood to include a free amino group, a substituted amino group, or a blocked or capped amino group, or the like, to occupy the valence.

A "peptide residue" as used herein refers to a group containing two or more aminoacid residues, incorporated into a polymeric chain, for example a polyamide chain such as a peptide, similarly as described above for an aminoacid residue. It is understood that when the peptide residue is disposed at the end of a peptide chain, all valances are filled, for example the carboxyl group can have a free OH group, or an alkoxy, aryloxy, amino, alkylamino, arylamino, or the like bonded to the carbonyl carbon atom as with the aminoacid residue. The term "peptide residue" as used herein includes blocked and capped sequences, that is, sequences either on-resin or off-resin which include sidechain, N-terminal, or C-terminal blocking or capping groups.

The methods of the invention can be carried out on either "free" peptides, or "immobilized" peptides. Free peptides refer to peptides, including blocked derivatives, that are in solution, such as solution in water, alcohol, or organic solvents. Immobilized peptides refer to peptides that are bound to a polymer such as polystyrene, polyester, and the like (including porous glass). For example, the polymer may be formed into a polymer bead such as is used in solid phase peptide synthesis, or the polymer may be for the purposes of controlled release of the peptide wherein the peptide-polymer bond is slowly broken when implanted in a patient. As is well known in the art, derivatized polystyrene beads are suitable for reversible covalent attachment of aminoacids and peptides for the purposes of synthesizing longer peptide chains. The methods of the present invention can be carried out either on molecules in solution, or on molecules that are immobilized in this manner.

A "hydrocarbyl" as the term is used herein refers to a carbon-based molecular entity that can be bonded to other atoms or groups, that is, which may have one, two, or more unfilled valances available for bonding. A hydrocarbyl can be a linear hydrocarbon chain, a branched hydrocarbon chain, a cyclic hydrocarbon, or any combination thereof, to which other atoms or groups may be bonded covalently. A hydrocarbyl can be an alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, cycloalkenylalkyl, cycloalkynylalkyl, cycloalkylalkenyl, cycloalkenylalkenyl, cycloalkynylalkenyl, cycloalkylalkynyl, cycloalkenylalkynyl, cycloalkynylalkynyl, aryl including monocyclic and polycyclic aryls and aryls fused with cycloalkyls, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, and the like. A hydrocarbyl can be unsubstituted (i.e., bearing only hydrogen atoms) or can be substituted, for example with alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfmyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR", wherein each R^x and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. A hydrocarbyl can also contain covalently bonded heteroatoms, for example O, S, N or P. "Alkyl" refers to a C l -C 18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, -CH3), ethyl (Et, - CH2CH3), I -propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, - CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (i- Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2- methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -

CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (- CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1-butyl (-CH2CH2CH(CH3)2), 2-methyl-l- butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3», 2- methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (- CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (- CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl

(-C(CH₃)2CH(CH₃)2), 3,3-dimethyl-2-butyl (-CH(CH₃)C(CH₃)₃. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., alkylene).

The alkyl can optionally be substituted with one or more suitable substituents including alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR^X, wherein each R^x and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. The alkyl can optionally be interrupted with one or more non-peroxide oxy (-O-), thio (-S-), imino (-N(H)-), methylene dioxy (-OCH₂O-), carbonyl (- C(=O)-), carboxy (-C(=O)O-), carbonyldioxy (-OC(=O)O-), carboxylato (- OC(=O)-), imine (C=NH), sulfinyl (SO) or sulfonyl (SO₂). Additionally, the alkyl can optionally be at least partially unsaturated, thereby providing an alkenyl. In all cases where a group is "substituted", it is recognized that a suitable substituent is a substituent that is stable to the reaction conditions of a method herein as applied.

The term "alkoxy" refers to the groups alkyl-O-, where alkyl is defined herein. Preferred alkoxy groups include, e.g., methoxy, ethoxy, n-propoxy, iso- propoxy, w-butoxy, terf-butoxy, sec-butoxy, «-pentoxy, n-hexoxy, 1,2- dimethylbutoxy, and the like. The term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferred aryls include phenyl, naphthyl and the like. The aryl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR", wherein each R^x and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. The term

"cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

The cycloalkyl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR", wherein each R^x and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy. The cycloalkyl can optionally be at least partially unsaturated, thereby providing a cycloalkenyl.

The term "halo" refers to fluoro, chloro, bromo, and iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

"Haloalkyl" refers to alkyl as defined herein substituted by 1-4 halo groups as defined herein, which may be the same or different. Representative haloalkyl groups include, by way of example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl, 2-bromooctyl, 3-bromo-6-chloroheptyl, and the like.

The term "heteroaryl" is defined herein as a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, 4n//-carbazolyl, acridinyl, benzo[ό]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnaolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, naptho[2,3-Z>], oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from the group non- peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl. In another embodiment heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, or tetramethylene diradical thereto. The heteroaryl can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, benzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR^X, wherein each R* and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.

The term "heterocycle" refers to a saturated or partially unsaturated ring system, containing at least one heteroatom selected from the group oxygen, nitrogen, and sulfur, and optionally substituted with alkyl or C(=O)OR^b, wherein R is hydrogen or alkyl. Typically heterocycle is a monocyclic, bicyclic, or tricyclic group containing one or more heteroatoms selected from the group oxygen, nitrogen, and sulfur. A heterocycle group also can contain an oxo group (=O) attached to the ring. Non-limiting examples of heterocycle groups include 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2//-pyran, 2- pyrazoline, 4//-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuclidine, and thiomoφholine. The heterocycle can optionally be substituted with one or more alkyl, alkenyl, alkylidenyl, alkenylidenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido, benzenesulfinyl, benzenesulfonamido, beπzenesulfonyl, benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl, benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino, thiosulfo, NR^xR^y and/or COOR^X, wherein each R^x and R^y are independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl or hydroxy.

Examples of nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, moφholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles. In one specific embodiment of the invention, the nitrogen heterocycle can be 3-methyl-5,6-dihydro-4H-pyrazino[3,2,l- jk]carbazol-3-ium iodide.

In general, "substituted" refers to an organic group as defined (e.g., alkyl, aryl, cycloalkyl, aralkyl, heterocyclyl, heteroaryl, etc.) in which one or more bonds to a hydrogen atom contained therein is replaced by a bond to a non- hydrogen atom such as, but not limited to: a halogen (F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups that can be free or can be blocked as with a hydroxyl protecting group such as a silyl ether, in ethers such as alkoxy or aryloxy groups, aryloxy groups, and aralkyloxy groups, in acyloxy groups such as carboxy esters, carbamyl esters, carbonate esters and the like, and in inorganic esters such as boronate, phosphate, phosphonate, phosphinate, sulfenate, sulfonate, or sulfonate esters; a carbon atom in groups such as cyano, carboxyl, acyl, ester, amide and the like; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, alkoxy- or aryloxy-sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as nitro, amines, hydroxylamines, N-oxides, hydrazides, azides, and enamines; and other covalently bonded heteroatoms, such as phosphorus in groups such as phosphonates and phosphinates. The organic group as defined can also be substituted with groups wherein more than one bond to hydrogen atoms on a carbon atom are replaced by two or more distinct bonds to two or three heteroatoms atoms of a single substituent group, or alternatively including double or triple bonds to a heteroatom such as, but not limited to: oxygen in carbonyl (oxo), two oxygens as in cyclic acetals, hemiacetals, ketals, and hemiketals; three oxygens as in ortho-esters, an oxygen and a nitrogen as in cyclic aminals and hemiaminals; nitrogen as in imines, hydroxyimines, oximes, hydrazones, and nitriles; sulfur such as in thiocarbonyls; and phosphorus as in phosphorus ylidene compounds. Substituted ring groups such as substituted aryl, heterocyclyl and heteroaryl groups also include rings and fused ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with alkyl, alkenyl, and alkynyl groups as defined herein. The term "heteroatoms" as used herein refers to non-carbon and non- hydrogen atoms, and is not otherwise limited. Typical heteroatoms are N, O, and S. When sulfur (S) is referred to, it is understood that the sulfur can be in any of the oxidation states in which it is found, thus including sulfoxides (R- S(O)-R') and sulfones (R-S(O)₂-R'), unless the oxidation state is specified; thus, the term "sulfone" encompasses only the sulfone form of sulfur; the term "sulfide" encompasses only the sulfide (R-S-R¹) form of sulfur. When the phrases such as "heteroatoms selected from the group consisting of O, NH, NR' and S," or "[variable] is O, S . . ." are used, they are understood to encompass all of the sulfide, sulfoxide and sulfone oxidation states of sulfur, wherein sulfur is also bonded to two carbon atoms.

A nitrogen protecting group or an amine (or amino) protecting group, as the terms are used herein, refers to a functional group that, when attached to the nitrogen atom of an amino group, renders the amino group much less nucleophilic and thus less prone to form bonds with reactants with which it would normally react in a free state. A protecting group also has the property of being removable under relatively mild reaction conditions, such that the amino group can be exposed when further reaction at the group is desired. Examples of nitrogen protecting groups include amides, carbamates (urethanes) and the like. Common protecting groups include formyl, acetyl, benzyloxycarbonyl (Cbz), t- butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), and the like. A nitrogen capping group or an amine (or amino) capping group is similarly a group that when attached to the nitrogen atom of an amino group, renders the amino group much less nucleophilic and thus less prone to form bonds with reactants with which it would normally react in a free state.

However, it need not possess the property of being readily removable. Typical capping groups include acetyl, benzoyl, alkylsulfonyl, arylsulfonyl groups and the like.

Detailed Description

(I), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, O(Cj-C₆ alkyl), 0(C₆-Cio aryl), NR₂, an aminoacid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

R¹ , R² and R³ are independently hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl, or heteroaryl, wherein one of R¹, R² or R³ may be absent, wherein any alkyl can contain 0-3 heteroatoms comprising N, O, or S, wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted; the method comprising contacting a compound of Formula (II):

(π), wherein Y is a leaving group; with an amine of the formula R¹R²R³N, in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (T).

The invention also provides a method for the preparation of a compound of Formula (III):

(HI), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(Cj-C₆ alkyl), 0(C₆-Ci₀ aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

(IV), wherein R⁶ is substituted or unsubstituted alkyl or aryl, or a salt thereof; in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (III) .

The invention provides a method of preparation of a compound of formula (III):

R⁵

(πi), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, O(Ci-C6 alkyl), 0(C6-Cio aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

R at each occurrence is independently H, alkyl, aryl, heterocyclyl, heteroaryl, a nitrogen protecting group, or a nitrogen capping group, or two R groups together with the nitrogen atom to which they are bound form a 5-7 membered heterocyclic ring that can further comprise 1 -3 additional heteroatoms N, O or S;

(H), wherein Y is a leaving group; the method comprising contacting the compound of formula (II) with an amine of the formula R¹NH₂, in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (V):

(IV), wherein R⁶ is substituted or unsubstituted alkyl or aryl, or a salt thereof; in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (HI). In the method of preparation of a compound of formula (I) from a compound of formula (II), of in the method of preparation of a compound of formula (V) from a compound of formula (II) as an intermediate in the method of preparation of a compound of formula (111), the compound of formula (II) comprises Y being a leaving group, which can include Y = halo, for example, Y = chloro, bromo or iodo, as well as Y = a sulfonate ester, for example alkylsulfonyl (e.g., methylsulfonyl), haloalkylsulfonyl (e.g., trifluoromethylsulfonyl) or arylsulfonyl (e.g., p-toluenesulfonyl). Y, as a leaving group, undergoes nucleophilic displacement by NR¹R²R³ (in the preparation of formula (I)), or by R¹NH₂ (in the preparation of formula (V)), at the carbon center to which Y is attached. As is known in the art, certain groups are better leaving groups or have better "leaving ability", so it is within the knowledge of a skilled artisan to select without undue experimentation an appropriate leaving group based upon the degree of reactivity of the reagent amine. When an amine that is more sterically hindered, or electronically less nucleophilic (e.g., more electron-withdrawn) is used, a leaving group Y of greater leaving ability will be selected in order to achieve a reasonable reaction rate such that the product is obtained in good yield in a relatively short time period and with minimal side reactions. For example, an amine like trimethylamine is both highly nucleophilic and unhindered sterically. Therefore a poorer leaving group Y such as chloro may be adequate for the practice of the method of the invention. Conversely, a less reactive amine, for example an aniline derivative, may require the selection of a better leaving group Y, such as trifiate or bromo, by the skilled artisan.

The compound of formula (II) can be a dipeptide, a polypeptide, a free peptide or an immobilized polypeptide, a peptide analog (for example, having a hydrogen atom or an alkyl group other than a methyl group in place of the Qt- amino group), all containing a precursor sidechain -(CH₂),,₊! Y, wherein n is 1 to about 10. A compound of formula(H), for example, a compound including the types of sidechains found on ribosomal aminoacids, can have those sidechains in blocked form (e.g., amines substituted with Boc groups, Cbz groups, and the like) when the reaction with the reagent amine (NR¹R²R³ or R¹NH₂) is carried out, or, alternatively, the sidechains can all be unblocked as long as there is no sidechain that can react with the reagent amine at a higher rate than does the precursor sidechain -(CH2)_n+i Y- When selecting a sidechain blocking group as are well known in the art for use with the assorted sidechain functionalities as are found in natural aminoacids (carboxylates, phenols, thiols, carboxamides, and the like), the sidechain should not be protected in such as way as to activate it for nucleophilic displacement by the reagent amine. Such protecting groups are well known in the art, and a suitable blocking group can be selected without undue experimentation by the skilled artisan. However, none of the sidechains found in natural (ribosomal) aminoacids interfere with the reaction of the invention, therefore sidechain protected is unneeded for the purpose of carrying out the reaction of the invention.

In the case where unnatural aminoacid residues make up a peptide of formula (II), any sidechain functionalities that are prone to nucleophilic displacement by the amine NR¹R²R³ should be suitably protected unless it is the intention that those functionalities undergo the displacement reaction. For example, if there are two hydroxyl-containing sidechain functionalities in a peptide of formula (II), and it is only desired that one of the hydroxyl-containing sidechains should be converted to a compound of formula (I), an amine, for instance via conversion to a sulfonate ester followed by displacement with the reagent amine, the other hydroxyl-containing sidechain should be suitably protected prior to carrying out the method of the invention. The selection of blocking groups when needed to protect sidechain functionalities is readily made by a person of ordinary skill without undue experimentation based on well- known blocking groups for the kinds of sidechain functionalities that may be encountered.

The substituents of the sidechain amino group in the compound of formula (T), R¹, R² and R³, are independently hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl, or heteroaryl, wherein one of R¹, R² or R³ may be absent, wherein any alkyl can contain 0-3 heteroatoms comprising N, O, or S, and wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted. For example, compounds of formula (T) that may be prepared by a method of the invention include N-methyllysine, N,N- dimethyllysine, and the quarternary ammonium metho salt of N,N- dimethyllysine. Other N^-alkyllysine derivatives such as N-benzyllysine, N- picolyllysine, and the like, can also be prepared. By N-alkyl or N-aryl lysine derivatives is meant herein lysine derivatives bearing the substituent on the sidechain (c) amino group.

X can be OH, O(Ci-C₆alkyl), 0(C₆-Cio aryl), NR₂, an aminoacid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; and Z can be H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an aminoacid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that when Z is NHR, X is an amino acid residue or a peptide residue, or is an aminoacid residue or a peptide residue respectively covalently bonded to a polymer. For example Z can be hydrogen or alkyl. Z and X cannot both be bonded to a polymer; furthermore, at least one of Z and X must comprise an aminoacid residue, a peptide residue, or an aminoacid residue or a peptide residue respectively bonded to a polymer. The ratio of the reagent amine to the substrate precursor peptide can vary; preferably an excess of reagent amine is used. Molar ratios can vary between about 1 :1 up to about 100:1 of the reagent amine to the precursor peptide. Higher ratios of reagent amine are advantageously used to suppress possible unwanted side reactions, for example, if a precursor group is present in the same molecule with another nucleophilic sidechain group, such as a thiol (cysteine), an amine (lysine), or a non-natural group of high nucleophilicity, use of a large excess of reagent amine is favored in reducing the amount of intra- or intermolecular coupling. Larger excesses of the reagent amine also serve to increase the reaction rate and to drive the reaction to completion due to the bimolecular kinetics of the nucleophilic displacement reaction.

The conversion of a compound of formula (II) to a compound of formula (I) or a compound of formula (V) can be carried out in any suitable solvent. Examples are water, methanol, ethanol, or a mixture thereof. Other solvents, such as dipolar aprotic solvents that are capable of dissolving the substrates and which may enhance the rate of nucleophilic displacement of Y by the reagent amine can also be used. Examples are DMSO, DMF, DMAc, NMP, and the like. When the peptidyl reagents have sufficient solubility in nonpolar solvents, suitable nonpolar solvents such as chloroform and dichloromethane can also be used.

The reaction time and temperature can be varied depending upon the specific reaction in question to provide a reasonable yield of the desired product within a relatively short period of time. For example, the reaction can be carried out at about 20-80⁰C, for a time period of about 1 hr to about 7 days. If necessary, reaction temperatures can be as high as about 100⁰C or 120⁰C using a solvent with a reflux temperature that exceeds the given temperature.

In solution phase reactions, the progress of the reaction towards completion is readily monitored by a person of ordinary skill in the art using techniques such as high performance liquid chromatography (HPLC), by which means the disappearance of precursor and the appearance of product are readily viewed. In solid phase reactions, for the monitoring of reaction progress it may be necessary to remove small aliquots of the polymeric material and cleave the product from them to ascertain the degree of reaction using HPLC. Other methods such as nuclear magnetic resonance (NMR) may also be used to follow the reaction progress. It is within the knowledge of a person of ordinary skill in the art to determine suitable reaction conditions for a particular conversion using standard analytical techniques that are available. The conversion of a compound of formula (V) to a compound of formula

(III) via contact of the compound of formula (V) with a compound of formula

(IV) takes place by addition of the NHR¹ group of the compound of formula (V) to the compound of formula (IV) with elimination of the SR⁶ group. The compound of formula (V) can be a dipeptide, a polypeptide, a free peptide or an immobilized polypeptide, a peptide analog (for example, having a hydrogen atom or an alkyl group such as a methyl group in place of the α-amino group), all containing a sidechain amino group bearing at least a single hydrogen atom. This amino group bears an R¹ group, wherein R¹ is hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocycles, or heteroaryl; wherein any alkyl can further contain 0- 3 heteroatoms comprising N, O, or S, and wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted.

A compound of formula (V), for example, which can be a compound including the types of sidechains found on ribosomal aminoacids, can have those sidechains in blocked form (e.g., amines substituted with Boc groups, Z groups, and the like) when the reaction with the compound of formula (IV) is carried out, or, alternatively, the sidechains can all be unblocked as long as there is no sidechain that can react with a compound of formula (IV) at a higher rate than does the precursor sidechain bearing the NHR¹ group. When selecting a sidechain blocking group as are well known in the art for use with the assorted sidechain functionalities as are found in natural aminoacids (carboxylates, phenols, thiols, carboxamides, and the like), the sidechain should not be protected in such as way as to activate it for nucleophilic attack on a compound of formula (IV). Such protecting groups are well known in the art, and a suitable blocking group can be selected without undue experimentation by the skilled artisan. For example, carboxylates and phenols can respectively be blocked as esters and ethers, such as benzyl ethers. However, none of the sidechains found in natural (ribosomal) aminoacids, except for amino groups undergo reaction with the thiouronium compound, therefore sidechain protected is generally unneeded for the purpose of carrying out the reaction of the invention. In the case where unnatural aminoacid residues make up a compound of formula (V), any sidechain functionalities that are prone to nucleophilic addition to the compound of formula (FV) should be suitably protected unless it is the intention that those functionalities undergo the reaction. For example, if there are two amine-containing sidechain functionalities in a compound of formula (V), and it is only desired that one of the amine-containing sidechains should be allowed to react with the S-substituted thiouronium compound (IV) the other amine-containing sidechain should be suitably protected prior to carrying out the method of the invention. For example, a Boc or Cbz group can be used. The selection of blocking groups when needed to protect sidechain functionalities can readily be made by a person of ordinary skill without undue experimentation based on well-known blocking groups for the kinds of sidechain functionalities that may be encountered. The compound of formula (IV) is an S-substituted thiourea or a thiouronium salt thereof. Preferably the compound of formula (FV) is a thiouronium salt, for example a hydriodide (HI) salt. The sulfur atom of the reagent bears a substituent R⁶ which can be substituted or unsubstituted alkyl or aryl. For example, R⁶ can be a methyl group. It is within the knowledge of a person of ordinary skill to modify R⁶ to confer a greater or a lesser degree of reactivity upon the thiouronium compound of formula (IV). For example, to increase the reactivity of the compound of formula (IV), for example when the compound of formula (V) bears a relatively unreactive NHR¹ group, such as where R¹ is electon-withdrawing, with the compound of formula (IV), a trifluoromethyl group could replace the methyl group as R⁶. This kind of substitution is well-known in the art to increase the reactivity of a thiouronium by increasing the electrophilicity of the central carbon atom. Alternatively R⁶ can be an substituted or unsubstituted aryl group, for example a phenyl group. To increase the reactivity of the thiouronium reagent of formula (IV), it would be within the knowledge of a person of ordinary skill to substitute the phenyl group with an electron-withdrawing group such as a nitro group in order to increase the reactivity of the thiouronium compound.

The compound of formula (FV) can also be substituted on its nitrogen atoms with substituents R⁴ and R⁵ which are at each occurrence independently H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein any alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl residue may be unsubstituted or substituted. It is understood that when one R⁵ group is a hydrogen atom, the tautomeric nature of guanido groups makes R⁴ and R⁵ interchangeable through a proton shift process. X can be OH, 0(Ci-C₆ alkyl), 0(C₆-Ci₀ aryl), NR₂, an aminoacid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; and Z can be H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an aminoacid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that when Z is NHR, X is an amino acid residue or a peptide residue, or is an aminoacid residue or a peptide residue respectively covalently bonded to a polymer. For example, Z can be H or alkyl, more specifically methyl. When Z is a nitrogen, X comprises at least a single aminoacid residue, which may or may not be covalently bonded to a polymer.

The compound of formula (IV) offers a major advantage over art reagents in that a greater number and variety of substituents can be disposed on the final arginine analog of formula (HI) than was previously possible in a single step. Using a method of the invention, an arginine analog bearing a substituted guanido group can be synthesized with as many as four substituents included, one substituent each on two of the three guanido nitrogen atoms and two substituents on the third guanido nitrogen atom.

The reaction can have molar ratios of reagents between about 1:1 compound of formula (V) and thiouronium compound of formula (IV) ranging up to about a 1 :10 ratio. Use of some excess of the thiouronium compound can be used to drive the reaction to completion and to increase the rate of the reaction, as well as in reducing unwanted side reactions.

The reaction between a compound of formula (V) and a compound of formula (IV) can be carried out in any of a variety of solvents, including water or organic solvents. For example, the reaction can be carried out in water, acetonitrile, methanol, ethaol, tetrahydrofuran, N,N-dimethylformamide, or a mixture thereof. Other suitable solvents will be known to a person of ordinary skill. The reaction can be carried out at any suitable temperature; for example, the reaction can be carried out within a time period of hours to days at a reaction temperature of about 20-80⁰C. The time period of the reaction can be as short as a few hours, for example about four hours, or can be in the order of days, for example up to about seven days. If necessary, reaction temperatures can be as high as about 100⁰C or 120⁰C using a solvent with a reflux temperature that exceeds the given temperature.

In a method of the invention for preparation of a compound of formula (III) from a compound of formula (II), the two reactions described herein can be combined to provide a single process for carrying out the transformation of an aminoacid residue with a sidechain -(CH₂)_n+i-Y to an aminoacid residue bearing a substituted guanido group, with the provision that the intermediate aminoacid residue bearing an amine sidechain must bear at least a single hydrogen atom on the sidechain amino group to allow for condensation with the thiouronium reagent. It may be advantageous under certain circumstances to carry out the two synthetic conversions sequentially, for example, to obtain a product with a modified arginine analog residue in a molecule that also contains a lysine or lysine analog residue. If one were to synthesize the precursor peptide with the residues that were to be modified arginine residues present with the -(CH2)_n+i-Y sidechain, and the lysine residues present as protected lysine, a high degree of selectively could be obtained wherein no inadvertent conversion of lysine residues to substituted arginine residues took place.

There are numerous peptides for which use of a method of the invention could provide a target compound, including an analog, of interest. For example, the peptide neurotensin is a highly cationic peptide of medicinal interest, as is described in PCT Application Publication No. WO2006/009902, which is incorporated herein by reference. This same PCT Published Application also discusses the C-terminal region of neurotensin, aminoacids number 8-13 as a structural lead for the preparation of peptide derivatives with analgesic activity. . For example, the molecule wherein the N-terminal arginine of the NT(8-13) fragment is converted to a desamino alkyl derivative, i.e., wherein the N- terminal amino group is replaced by a methyl group, possesses desirable bioactivity and the preparation and biological testing of a series of analogs, such as can be prepared by a method of the current invention, is of interest in the field.

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims. All documents referred to herein are incorporated by reference in their entireties.

Examples Example 1

Synthesis of 5-oxo-Pro-His-Tφ-Ser-Tyr-DLeu-Leu-(N^ω-Me-Arg)-Pro-NHEt • 3 TFA (2)

A sample of 5-oxo-Pro-His-Tφ-Ser-Tyr-DLeu-Leu-Orn-Pro-NHEt • 3 TFA (1) (100 mg, 0.066 mmole) was dissolved in 10 mL of acetonitrile / methanol (2:1 v/v), to which was added 1 ,2-dimethylisothiourea hydriodide (20 mg, 0.08 mmole, 1.2 eq) and triethylamine (33 mg, 0.33 mmole, 5 eq). The solution was stirred for 14 days at 40⁰C. The solution was then concentrated, purified by HPLC, and lyophilized to yield 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu- LeU-(N⁰^Me-ATg)-PrO-NHEt • 3 TFA (2) as a fluffy, colorless solid. MS (m/e)+ = 1222.29, calc. = 1222.66.

Example 2

Synthesis of butyryl-His-DPhe-πsr-ErtArg-Trp-Sar-NH_?- 3 TFA (4)

A sample of butyryl-His-DPhe-Orn-Trp-Sar-NH₂» 3 TFA (3) (100 mg, 0.092 mmole) was dissolved in 10 mL of acetonitrile / methanol (2: 1 v/v), to which was added l-ethyl-2-methylisothiourea hydriodide (113 mg, 0.46 mmole, 5 eq) and triethylamine (92 mg, 0.92 mmole, 10 eq). The solution was stirred for 2 days as 40⁰C. The solution was then concentrated, purified by HPLC, and lyophilized to yield butyryl-His-DPhe-(N^ω-Et)Arg-Trp-Sar-NH₂ ^« 3 TFA (4) as a fluffy, colorless solid. MS (m/e)+ = 812.63, calc. = 812.44.

Claims

ClaimsWhat is claimed is:

1. A method of preparation of a compound of Formula (I): R³

(D, or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(Ci-C₆ alkyl), 0(C₆-Ci₀ aryl), NR₂, an aminoacid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR2, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an aminoacid residue, or a peptide residue; or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer;

2. A method for the preparation of a compound of Formula (III):

(HI), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(C_I-C_O alkyl), 0(C₆-C₁₀ aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an amino acid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer; R is independently at each occurrence H, alkyl, aryl, heterocyclyl, heteroaryl, a nitrogen protecting group, or a nitrogen capping group, or two R groups together with the nitrogen atom to which they are bound form a 5-7 membered heterocyclic ring that can further comprise 1-3 additional heteroatoms N, O or S;

R⁴ and R⁵ are independently at each occurrence H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein any alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl residue may be unsubstituted or substituted; provided that R or R is not a Boc group; the method comprising contacting a compound of Formula (V):

(V) wherein R is hydrogen, alkyl, aryl, aralkyl, cycloalkyl, heterocycles, or heteroaryl; wherein any alkyl can further contain 0-3 heteroatoms comprising N, O, or S, wherein any alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl may be unsubstituted or substituted; with a compound of Formula (IV):

3. A method of preparation of a compound of formula (in) :

N-^R5

R⁵

(πi), or a salt, hydrate, solvate, stereoisomer, or tautomer thereof, wherein X is OH, 0(C₁-C₆ alkyl), 0(C₆-Ci₀ aryl), NR₂, an amino acid residue or a peptide residue, or wherein X is O, NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer;

Z is H, NR.₂, alkyl, aryl, aralkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, an amino acid residue, or a peptide residue, or is NR, an aminoacid residue, or a peptide residue, respectively covalently bonded to a polymer; provided that X and Z are not both bonded to polymers; and provided that at least one of X or Z comprises an amino acid residue or a peptide residue, or an aminoacid residue or a peptide residue respectively covalently bonded to a polymer;

R⁴ and R⁵ are at each occurrence independently H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein any alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl residue may be unsubstituted or substituted; provided that R or R is not a Boc group; from a compound of formula (II):

(II), wherein Y is a leaving group; the method comprising contacting the compound of formula (H) with an amine of the formula R¹Nt^, in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (V):

(V), then, contacting the compounc 1 of formula (V) with a compound of

Formula (IV):

(IV), wherein R⁶ is substituted or unsubstituted alkyl or aryl, or a salt thereof; in a solvent for a time period and at a temperature sufficient for the formation of the compound of Formula (IH).

4. The method of claim 1 wherein Z is not methyl.

5. The method of any one of claims 1, 3, or 4 wherein Y is halo, alkylsulfonoxy, arylsulfonoxy or alkarylsulfonoxy, wherein the alkyl or aryl is unsubstituted or is substituted with electron-withdrawing groups.

6. The method of any of claims 1 -4 wherein Z is H.

7. The method of any one of claims 1-3 wherein Z is methyl.

8. The method of any one of claims 1, 3, or 4 wherein the compound of Formula (I) comprises a lysine residue, a N-methyllysine residue, a N,N- dimethyllysine residue, or an N,N- dimethyllysine N-metho salt residue.

9. The method of any of claims 1 through 4 wherein the compound of Formula (I) or the compound of Formula (III), respectively, is a neurotensin analog or an analog of a neurotensin fragment.

10. The method of any of claims 1 through 4 wherein X comprises a Pro- Tyr-Ile residue.

11. The method of any of claims 1 through 4 wherein Z comprises a PGIu- Leu-Tyr residue.

12. The method of claim 2 or 3 wherein R⁶ is methyl or ethyl.

13. A compound of Formula (I) prepared by the method of claim 1.

14. A compound of Formula (IH) prepared by the method of claim 2 or claim 3.

15. A compound of Formula (FV) comprising a salt thereof.

16. The compound of claim 15 wherein the salt is a hydriodide salt.

17. The method of any one of claims 1 -4 wherein the solvent is water, methanol, or ethanol, or a mixture thereof.

18. The method of any one of claims 1-4 wherein the solvent is DMSO, DMF, DMAc, or NMP, or a mixture thereof.

19. The method of claim 2 wherein the solvent is water, acetonitrile, methanol, ethanol, tetrahydrofuran, or N,N-dimethylformamide, or a mixture thereof.

20. The method of any one of claims 1 to 3 wherein L is a linear or cycloalkylalkyl, saturated hydrocarbyl of 1 to about 10 carbon atoms.

21. A method of use of a peptide analog prepared by any of the methods of claims 1 -3 for treatment of a malcondition in a patient in need thererof, the method comprising administering the analog in a dosage, at a frequency, and over a period of time sufficient to provide a beneficial effect to the patient.

22. The method of claim 21 wherein the peptide analog is a neurotensin analog.

23. The method of claim 21 wherein the malcondition comprises pain.

24. The method of claim 21 wherein the malcondition comprises psychosis.

25. The method of claim 21 wherein the malcondition comprises obesity.