WO1998049186A1 - Dipeptide surrogates containing asparagine-derived tetrahydropyrimidinones - Google Patents

Abstract

Dipeptide surrogates Fmoc-Xxx-(cyclo)Asn-OBu are prepared from asparagine, aromatic aldehydes, and Fmoc-protected amino acid chlorides. The tetrahydropyrimidinone structure exist in a preferred ring conformation, as observed in a single crystal X-ray analysis of one of the derived carboxylic acids. As with other molecules with similar substitution patterns, the six-membered ring is held in a boat conformation. These dipeptide building blocks are deprotected at the C-terminal and/or N-terminal positions by known protocols. In addition, they function as ready participants for solid phase peptide synthesis. Finally, the constrained asparagine residue can be transformed to the native amino acid within a polypeptide framework, allowing for the direct interchange of a restricted system of known conformation and configuration to the native state under mild conditions. These peptide surrogates containing (cyclo)Asn are useful therapy of mammalian disease conditions, particularly in therapy of brain disorders.

Description

DIPEPTIDE SURROGATES CONTAINING ASPARAGINE-DERIVED TETRAHYDROPYRIMIDINONES

BACKGROUND OF THE INVENTION

Related Applications

This application is a continuation-in-part application of U.S. provisional application serial number 60/044,828, filed April 25, 1997 which is incorporated herein by reference in its entirety.

Field of the Invention Dipeptide surrogates such as Fmoc-Xxx(cyclo) Asn-OBu*¹ (where Xxx is an amino acid) are prepared from asparagine, aromatic aldehydes and Fmoc-protected amino acid chlorides. These dipeptide units are deprotected at the C-terminal and/or the N-terminal positions by known protocols. These dipeptide surrogates function as useful participants for solid phase peptide synthesis to produce peptide analogues and pharmaceuticals useful in the treatment of mammalian diseases and conditions particularly of the brain.

Description of Related Art The amino acid asparagine is the focus for varied studies of polypeptide and protein structure and function. Asparagine acts as an efficient C-terminal α-helix cap

(H.X. Zhou, et al, J. Am. Che . Soc. 1994, Vol. 116, pp.

1139-40) , and serves as the linkage point for the oligosaccharide unit of N-linked glycopeptides (R.A. Dwek,

Chem. Rev. 1996, Vol. 96, pp. 683-720) . These ubiquitous cell-surface biomolecules function as recognition elements in cell-cell interactions, and are implicated in many biological functions and disease states. However, asparagine-containing polypeptides and proteins require special considerations upon attempted laboratory synthesis. Use of the amino acid with an unprotected side-chain amide can lead, by dehydration, to nitrile and/or succinimide derivatives and, even if the synthesis is successful, to general solubility difficulties (P. Sieber, et al, Tetrahedron Lett. 1991, Vol. 32, pp. 739-42) .

An array of protective groups for the primary amide, including trityl and others (P. Sieber et al (1991)) and, more recently, dimethylcyclopropylmethyl (L.A. Carpino, et al, J. Orσ. Chem. 1995, Vol. 60, pp. 7718-9) have been promoted to alleviate, or at least reduce, these difficulties. Figure 1 shows a retrosynthetic analysis of the structure of jasplakinolide. The total synthesis is reported by K.S. Chu et al, J. Orσ. Che _r (1991), Vol. 51, pp. 5196-5202.

Figure 2A is a reaction sequence representation of dihydropyrimidinone reagent in β-amino acid synthesis as reported by F. J. Lakner et al. Organic Synthesis, (1996) , Vol. 73, p. 201. Figure 2B is a similar reaction sequence using dihydropyrimidinone reagent in β-amino acid synthesis as reported by K.C. Chu et al. _, Amer. Chem. Soc. , (1992), Vol. 114, p. 1800.

The polypeptides of the present invention are prepared by adaptation of the solid phase synthesis techniques of U.S. Patents 4,318,905 and 3,531,258. Also, see the solid phase synthesis teachings of J.M. Stewart and J.D. Young "Solid Phase Peptide Synthesis" 2nd ed. , Pierre Chem. Co., Rockford, Illinois (1969) , and J. Meinenhofer "Hormonal Protein and Peptides", Academic Press (New York) Vol. 2, p. 46 ff (1979) .

All articles, patent applications, patents, etc., cited in this application are incorporated herein by reference in their entirety. A need exists to efficiently produce dipeptide surrogates which contain asparagine-derived tetrahydropyridimidinones to produce peptides and compositions useful in therapy for disease conditions, e.g. brain chemistry disorders. The present invention provides such a method and surrogates.

SUMMARY OF THE INVENTION In one aspect, the present invention concerns a process to produce (cyclo)Asn and its protected derivatives useful in solid phase peptide synthesis and peptide surrogates useful in therapy, which process comprises:

(a) combining asparagine ester, e.g. -t-butyl ester, with an aldehyde of the structure Q-(C=0)H wherein Q is selected from the group consisting of alkyl having 1 to 10 carbon atoms and aryl of the structure

wherein Y and Z are each independently selected from the group consisting of H-, F-, Cl, -Br-, CH₃-, CH₃CH₂-, CH₃CH₂CH₂-, CH₃(CH₃) CH-,(CH₃)₃ C-, and -N0₂, at ambient temperature for between about 2 to 10 hr with stirring to produce an imine;

(b) reacting the imine of step (a) with a protected amino acid chloride in anhydrous aprotic organic liquid and base, at a temperature of between about 40 and 90-C for between about 1 and 10 hr.; and

(c) recovering the (cyclo)Asn peptide. Preferably, Q is an aryl or substituted aryl group. In another aspect, the present invention concerns: a process to produce (cyclo)Asn and the protected derivatives optionally useful in solid phase synthesis of polypeptides thereof, which process comprises:

(a) combining available asparagine ester e.g. - t-butyl ester, with an aldehyde to produce the imine;

wherein Q is selected from the group consisting of alkyl having 1 to 10 carbon atoms and aryl of the structure:

wherein Y and z are each independently selected from the group consisting of H-, F-, C1-, Br-, CH₃-, CH₃CH₂-, CH₃(CH₃)CH-, (CH₃)₃ C-, and N0₂; and

R¹ is selected from alkyl having 1 to 10 carbon atoms or benzyl;

(b) reacting imine with protected amino acid chloride in anhydrous aprotic organic compound, such as benzene and base such as pyridine which reaction produces the heterocycle

R is as described herein in Table 1 and elsewhere and maybe selected from H-, (S)-Me-, (R)-Me-, (S)-CHMe₂, (S)-CH₂0Bn,

(S)-CH₂C₆H₄OBn, 7 ^ or any of the fv^Fmoc R groups of a conventional or synthetic amino acid; and

(c) obtaining the (cyclo)Asn peptide. In another embodiment, the present invention relates to a process wherein the (cyclo)Asn is further protected for use in solid phase peptide synthesis.

In another embodiment, the present invention relates to a process wherein an amino acid sequence which is obtained by the replacement of one proline or optionally more than one proline amino acid with at least one

(cyclo) Asn.

In another embodiment, the present invention relates to a process wherein an amino acid sequence which is obtained by the replacement of a proline with (cyclo) Asn.

In another embodiment, the present invention relates to a process wherein a pharmaceutical composition is obtained by the replacement of one proline or optionally more than one proline amino acid with at least one

(cyclo) Asn and a pharmaceutically acceptable excipient.

In another embodiment, the present invention relates to a process wherein a pharmaceutical composition comprises an amino acid sequence wherein at least one proline has been replaced by (cyclo) Asn and optionally includes a pharmaceutically acceptable excipient.

Another embodiment is an amino acid sequence in which includes the following sequence:

-Tyr- (cyclo) Asn-Trp-Phe-NH₂; -Tyr- (cyclo)Asn-Phe-Phe-NH₂ ;

-Tyr- (cyclo) Asn-Trp-Gly-NH₂ ; or combinations thereof. Preferably, the amino acid sequence has a total of 100 amino acids or less, more preferably 50 or less, and most preferably 20 or less. Another embodiment includes an amino acid having the following sequence:

Tyr- (cyclo) Asn-Trp-Phe-NH₂ ; Tyr- (cyclo) Asn-Phe-Phe-NH₂ ; Tyr- (cyclo) Asn-Trp-Gly-NH₂ ; or combinations thereof.

Another embodiment includes a pharmaceutical composition which comprises an effective amount of an amino acid having a sequence comprising of the structure: -Tyr- (cyclo) Asn-Trp-Phe-NH₂ -Tyr- (cyclo) Asn-Phe-Phe-NH , or -Tyr- (cyclo) Asn-Trp-Gly-NH₂; and a pharmaceutically acceptable excipient.

Preferably, this peptide has a total of 100 amino acids or less, more preferably 50 or less, and most preferably 20 or less.

Another embodiment includes a pharmaceutical composition which includes an effective amount of the amino acid sequence:

Tyr- (cyclo) Asn-Trp-Phe-NH₂ ; Tyr- (cyclo) Asn-Phe-Phe-NH₂ ; Tyr- (cyclo) Asn-Trp-Gly-NH₂ ;or combinations thereof.

Another embodiment includes the use of the pharmaceutical composition of the above sequences in an amount effective in therapy to alleviate conditions of brain disease or disorder. Another embodiment includes the use of any of pharmaceutical compositions of the above sequence wherein the brain disease or disorder includes, depression, anxiety, compulsive-obsessive disorder, drug addiction, alcohol addiction, nicotine addiction and the like. Another embodiment includes the use of the pharmaceutical composition of the above sequence in an amount effective as an analgesic or an anesthetic.

Another embodiment includes the amino acid sequence of selected from which is an agonist or an antagonist to the opioid receptor subtype, K, μ or δ.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic representation of the prior art retrosynthetic analysis of jasplakinolide.

Figures 2A and 2B are schematic representations of the prior art use of dihydropyrimidinone reagent in β-amino acid synthesis.

Figures 3A, 3B, and 3C are schematic representations of three prior art reaction sequences which show the limitations of direct cyclization. Figure 4A is a reaction sequence. Figure 4A is a schematic representation of the use of aromatic aldehydes as cyclization components in a reaction sequence. Figures 4B and 4C are two and three dimensional representations of a cyclic tetrahydropyrimidinone. Figures 4D and 4E are two and three dimensional representations of a cyclic tetra hydropyrimidinone.

Figure 5A and 5B are schematic representation of attempted direct cyclization and indirect acylation.

Figure 6 is a schematic representation of the limits of the cyclization reaction starting with an aromatic imine-amide.

Figures 7A and 7B are schematic representations of the synthesis of surrogate dipeptides according to the present invention. Figure 8 is a schematic representation of the deprotection of the amino and carboxyl groups of surrogate dipeptides.

Figure 9A is a two dimensional representation of a tetrahydropyrimidinone . Figure 9B is a schematic solid state representation of the cyclo tetrahydropyrimidinone.

Figure 10 is a three dimensional representation of the cyclotetrahydropyrimidone of Figure 9 but contains more molecules and indicates the unit cell. Figure 11 is a schematic representation showing the use of surrogate dipeptides in solid phase peptide synthesis. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Definitions As used herein:

"Alkyl" refers to the conventional saturated groups having 1 to 10 carbons such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. These groups may be linear or branched. "Amino acid" refers to all natural amino acids RCH(NH₂) (C=0)OH, in the L-configuration and in the D- configuration. Amino acid also refers to synthetic amino acids RCH(NH₂) (C=0)0H, wherein R is any alkyl, aryl, heteroalkyl, or heteroaryl group which is not conventional or found in nature. The R group for natural amino acids therefore includes for example -H, CH₃-, CH₃(CH₃)CH-, -CH₃CH(CH₃)CH₂-, CH₃CH₂CH(CH₃)-, phenyl-CH₂-, -CH₂OH, -CH(OH)CH₃, -CH₂SH, -CH₂CHSCH₃, HO -{O^CH, , -0^(0=0) OH, -&_ - (C=0)NH₂, -CH₂CH₂(C=0)NH₂, -CH₂CH₂CH₂CH₂NH₂ , -CH₂CH₂-CH₂NH (C=NH)NH₂ and -CH₂-ΓJ— N. It is understood that if multiple reactive groups NH are present in R they will be protected using protecting groups which are well known in this art.

"Aryl" refers to those conventional unsaturated structure having aromatic character. These include but are not limited to benzene, naphthalene, fluorene, biphenyl, terphenyl and the like. Heteroaromatic groups such as pyridine, pyrimidine, triazine and the like are included but are not limited to those recited. Benzene and substituted benzene (as described herein) are preferred.

"Base" refers to the base in steps (b) and includes strong organic base such as pyridine, trimethylamine, triethylamine, and the like.

"DIEA" refers to disopropyethylamine. "DMF" referes to di ithyformamide.

"Fmoc" refers to the protecting group florenemethyloxycarbonyl, and other standard protecting groups for amino acids may be used.

"HATU" refers to N- [ (dimethylamino) -1H-1, 2 , 3- triazolo[4 , 5-b]pyridin-l-ylmethylene] -N-methylmethanaminium hexafluorophosphate N-oxide.

"Substituted aryl" refers to the aryl groups recited hereinabove wherein one or more protons are replaced with another group e.g. Y and/or Z as are defined herein. Examples of such structures include but are not limited to:

"THF" refers to tetrohydrofuran.

A novel class of protected asparagine building blocks are disclosed herein. On one level, these cyclic surrogates function as protected, organic-soluble asparagine residues for peptide synthesis. However, as opposed to conventional protective groups, the tetrahydropyrimidines employed herein are of known absolute configuration and conformation. Such topological certainty allows for their incorporation into rationally designed polypeptides as stereochemically defined peptidomimetics (J. Gante, Angew Chem. Int Ed. Enαl. 1994, Vol. 33, pp. 1699-1720) . Finally, the heterocycle is easily transformed to the natural amino acid within the polypeptide framework (that is, Pseudo-prolines of serine, threonine (oxazolidine) , and cysteine thiazolidine have been described as reversible protecting groups. For example see ohr, et al, J. Am. Chem. Soc. 1996, Vol 118, pp. 9218- 27)). This situation allows for both the direct comparison between the conformationally restricted and native polypeptide and the observation of the changes that occur in the process.

Figure 3A is a reaction sequence showing the limits of direct cyclization. Figure 3B shows the research results of Seebach. Figure 3C shows the research results of J.P. Konopelski.

Figure 4A is the reaction sequence which shows the use of aromatic aldehyde or a cyclization reagent. Figure 4B is a representation of the 1993 x-ray research of Seebach. Figure 4C is a three dimensional representation of the structure of 4B. Figure 4D is a representation of the cyclic structure reported by J.P. Konopelski reported in 1991 using molecular mechanics and NMR. Figure 4E is three dimensional schematic representation of the structure of 4D.

Figure 5A is a representation of a number of unsuccessful reactions using asparagine and aromatic aldehyde. Figure 5B is a reaction sequence which shows one route which was not successful and produced asparagine. Figure 6 is a schematic representation of six possible approaches to cyclize the aromatic imine-amide, and one successful cyclization. In Reaction Sequence I, imine 3 is obtained in nearly quantitative yield from 4-chloro-3-nitrobenzaldehyde and commercially available asparagine t-butyl ester, in accord with the results of Seebach and coworkers (E. Juaristi, et al, J. Org. Chem. 1991, Vol. 56, pp. 2553-7) . Although no desired product came from the use of more conventional amino acid activated carboxyl residues (including p- nitrophenyl ester, anhydride, acyl fluoride, acyl imidazolide) , treatment of 3 with Fmoc-protected amino acid chlorides (L.A. Carpino, Ace. Chem. Res. 1996, Vol. 29, pp. 268-74) in anhydrous benzene with pyridine as base led to desired heterocycle 5. Yields of 5 vary from 58-66% (and in some cases up to about 85%) for a wide range of amino acid side chains (see Table 1) , including D-amino acids. Proline can also be employed in this reaction, although in more modest yield (The Pro-Asn dipeptide residue is found in a number of important biomolecules, including the circumsporozoite surface protein of the malaria parasite Plasmidium falciparum. (Also, see C. Bisang, et al, J. Am. Chem. Soc. 1995, Vol. 117, pp. 7904-15)

5 a-g

a) 4, (MeO)₃CH, 98%; b) Fmoc-Xxx-Cl, C₅H₅N, 58-66% wherein R is also partially defined in Table 1, and Xxx is a generic amino acid.

Figure 7 is a schematic representation of the formation of a tetrahydropyrimidinone having an aromatic group as a substituent as reported by J.P. Konopelski et al, ___ Am. Chem. Soc. (1997), Vol. 119, pp. 4305-6. Compounds 5a-g are sufficiently (and orthogonally) protected to function in peptide synthesis schemes. Standard treatment with trifluoroacetic acid (TFA) affords corresponding free acids 6 in excellent yields (See Table 1 below) . Isolation of free amines 7 after selective removal of the Fmoc group proved somewhat more difficult. Standard treatment with piperidine in DMF results in complete deprotection. Unfortunately, the product amines exhibit enough water solubility to make isolation of pure compounds difficult. Success was achieved with certain of the derivatives through use of Me₂NH as deprotection agent.

Under these conditions, excess base is removed by evaporation to leave the desired material contaminated with fluorene residue, which is easily removed by filtration. However, both 5b and 5d afforded variable amounts of a largely insoluble material, which has yet to be identified, upon attempted isolation of the desired free amine. While direct acetamide formation proved advantageous in most cases, the yield of 7b could not be brought to an acceptable level. Fortunately, these problems were not evident in a solid phase peptide synthesis (SPPS) deprotection sequence. For example, compound 6b was attached to a solid support and deprotected with piperidine/DMF under standard conditions. Acetamide formation and isolation of the product after cleavage from the resin occurs in 77% yield, as opposed to 40% for the solution phase synthesis. Finally, the aminal functionality can be opened with dilute aqueous acid, which also liberates the free asparagine carboxylic acid functionality to afford compound 8. Each of the compounds 8a-g is identical with the dipeptide formed by conventional methods .

In another aspect, the 4-chloro-3-nitrobenzaldehyde recited in Reaction Sequence I is represented as Q (C=0)H wherein Q is an alkyl or aryl group such as phenyl, biphenyl, terphenyl, napthyl and the like. Q may also be heteromatic containing one or more heteroatoms such as N, S, or O. Further Q may be substituted by Y and Z which are each independently selected from the group consisting of H- , F-, C1-, Br-, CH₃-, CH₃CH₂-, CH₃CH₂CH₂-, CH₃(CH₃)CH-, CH₃C- and -N0₂.

TABLE 1

Yields of free acid 6 . amine 7 . and native dineDtide 8 from 5_.

Entry R % yield % yie Id % yield % yield

5 5-6 5-7 5-8

a H 61 88 82¹ 91 b ( S ) -Me 65 95 40¹ ( 77² ) 90 c (R) -Me 58 90 90¹ 78 d ( S ) -CHMe₂ 66 95 75¹ ( 80² ) 85 e ( S ) -CH₂OBn 63 87 87¹ 78 ( S ) -CH₂C₆H₄OBn 62 89 90 75

1) Prepared by treatment with Me,NH: isolated as the corresponding acetamide by direct treatment with Ac₂0. 2) Prepared by binding corresponding acid 6 to NovaSyn® PR-500 resin and treating with piperidine/DMF. Cleavage from resin results in carboxa ide, isolated as the corresponding acetamide derivative by direct treatment with Ac₂0.

The structure obtained from a single crystal X-ray analysis of compound 6b is depicted in Figure 9, (The crystals of 6b are orthorhombic, α=7.654(2), b=12.190(3), c=32.529(6) Λ, space group P2₁2₁2₁, Z=4 , r=1.337 g/cm³ for C₂₉H₂₇C1N₄0₉. A total of 2934 independent reflections were measured with nickel-monochromated Cu Ka radiation at 130(2) K on a Siemens P4 diffractometer in the -θ range of 2.72 to 56.06°. The structure was solved by using direct methods and refined to a final R value of 4.01%. The primary program used was SHELXTL, version 5.03, 1994, by G.M. Sheldrick) and has similarity to related compounds studied both in this laboratory (see J.P. Konopelski, et al, J. Orα. Chem. _r (1991), Vol. 56, pp. 1355-6 and K.S. Chu, et al, J. Am. Chem. Soc. (1992), Vol. 114, pp. 1800- 1812) , and others (D. Seebach, et al, Helv. Chi . Acta 1992, Vol. 75, pp. 913-34 and F. Beaulieu, et al, J. Am. Chem. Soc. 1996, Vol. 118, pp. 8727-8). In particular, the peptide linkage is maintained in the equatorial plane to allow maximum amide resonance. This comes at the expense of the C2 aryl group, which is stationed as a flgpole substitutent on the six-me bered ring in a boat conformation. The two flagpole bonds are nearly parallel, with a dihedral angle of only 1.7°, and these substituents are held in close proximity (2.71 A). The key torsion angle around the amino acid residue (C (Ala) -N1-C6-C0₂H) is 73.2°, somewhat larger than that found in proline (approximately 65°) . Intermolecular hydrogen bonds between the free asparagine acid functionality and the adjacent lactam system afford linear arrays of molecules in the solid state. Adjacent linear sequences are held together by edge-to-face IT-stacking forces between the fluorene fragments of the Fmoc protection group (See Figures 9 and 10) .

Compounds 5a-g display the full range of cis-trans amide iso er populations, with 5c and 5g appearing as single isomers, while 5f, derived from L-tyrosine benzyl ether, affords two sets of ^XH and ¹³C NMR (CDC1₃) resonances in almost equal amounts. Experiments with 5f in DMSO-d₆ over the range from room temperature to 100°C suggest that this cis-trans isomerization around the dipeptide bond has a relatively low barrier, with a coalescence temperature of 70°C (F. Beaulieu, et al, J. Am. Chem. Soc. 1996, Vol. 118, pp. 8727-8) .

It is of interest to determine if these dipeptides could be employed in the SPPS of polypeptides, particularly with the heterocycle at the C-terminal position (Asparagine has been a troublesome amino acid when directly linked to the resin. See a) F. Albericio, et al, Int. J. Peptide Protein Res. 1990, Vol. 35, pp. 284-6. b) R.T. Carey, et al, Ibid (1996), Vol. 47, pp. 209-13). To this end, the previously described 6b-resin adduct was deprotected with piperidine/DMF. Resulting free amine 9 was coupled with Fmoc-Val-OH to give the desired tripeptide, followed by deprotection and coupling wtih Fmoc-Gly-OH in similar manner to afford 10. Cleavage from the resin with TFA gave expected tetrapeptide 11 as the Fmoc-protected primary amide in 82% yield from 6b. Compound 11, which is readily soluble in common organic solvents, was treated with mild aqueous acid to afford tetrapeptide 12 , identical with material made in the conventional manner in all respects. The solubility of 12 in organic solvents is poor.

Figure 11 is a schematic representation of a series of reactions of surrogate dipeptides in solid phase peptide synthesis as is described herein.

a) diisopropyocarbodiimide, HOBt; b) 30% piperidine, DMF; c) Fmoc-Xxx-OH, HATU, DIEA, DMF; d) 95% TFA; e) 0.25 M HCI , THF

These dipeptide surrogates offer attractive scaffolds from which to explore issues of polypeptide synthesis and structure. Incorporation of these building blocks into model polypeptides and the elaboration of their influence on local conformations are the subject of ongoing studies in these laboratories and will be reported in due course. Figure 8 is a reaction scheme showing the deprotection of surrogate dipeptides as described by J.P. Konopelski, et al. J. Amer. Chem. Soc. _r (1997) Vol. 119, pp. 4305-4306. Figure 9 is a schematic solid two dimensional state representation of two molecules of the cyclic tetrahydropyrimidone. Figure 10 is a three dimensional schematic representation of many molecules of cyclic tetrahydropyrimidinone and shows the unit cell 21.

Detailed experimental procedures and H NMR spectra for key compounds, and X-ray data for 6b (62 pages) (see J.P.

Konopelski, et al. J. A er . Chem, Soc. _r (1997) Vol. 119

(#18), pp. 4305-4306. See any current masthead page for _l__ Amer. Chem. Soc, for ordering and Internet access instructions .

The following examples are presented for the purposes of explanation and illustration. They are not to be construed to be limiting in any way. General

Melting point determinations are uncorrected. Infrared spectra were recorded as thin films on salt plates. Proton (^XH NMR) and carbon (¹³C NMR) magnetic resonance spectra were obtained in CDCL₃ (unless otherwise noted) at 500 and 62 MHz, respectively. Flash chromatography was performed on Merck Grade 60, 40-63 mesh silica gel using chloroform-methanol as solvent, unless otherwise indicated. Ultraviolet (UV) absorption maxima were determine dusing a diode array detector for HPLC analysis.

Example 1 Imine Derivative of H-Asn-Ot-Bu with 4-chloro-3-nitrobenzaldehyde ( 3 ) To 0.188 g (1.0 mmol) H-Asn-0Bu^ϋ in 5 L of trimethylorthoformate was added 0.185 g (1.0 mmol) of 4- chloro-3-nitrobenzaldehyde (4) . The resulting solution was stirred at room temperature for 8 h. and solvent was removed in vacuo . Analysis indicated complete transformation and the material was employed in the next step without further treatment. The imine is very stable and could be stored at room temperature under N₂ without noticeable decomposition. Yield 0.35g (98%); .p. 140-141 °C; [α]_D = +16.8 (c 3.52, CHCl₃) ; ^XH NMR δ 1.47 (s, 9H) , 2.78 (dd, J = 8.5, 15.5 Hz, 1H) , 2.95 (dd, J = 5.0, 15.0 Hz, 1H) , 4.45 (dd, J = 5.0, 8.0 Hz, 1H) , 5.43 (br s, 1H) , 5.9 (br s, 1 H) , 7.61 (d, J = 8.5 Hz, 1 H) , 7.88 (dd, J = 2.0, 5.0 Hz, 1H) , 8.29 (d, J = 2.0 Hz, 1H) , 8.36 (s, 1H) ; ¹³C NMR δ 27.8, 38.8, 69.3, 82.4, 124.8, 129.2, 132.1, 132.4, 135.4, 148.1, 161.4, 169.4, 172.1; IR (thin film) 1671, 1536, 1367, 1155, 1049, 752 cm^"1.

Example 2

General Procedure For The Synthesis of Dipeptide Surrogates

Imine 3 (1.0 mmol) was dissolved in 10 mL of benzene and 120 μL (1.5 mmol) of pyridine was added. The resulting mixture was heated to 50-65°C for 30 min, during which time the majority of the solid imine dissolved. The solution was allowed to cool to room temperature (solid appeared) and 1.35 mmol of the desired Fmoc-protected amino acid chloride was added. This solution was stirred at 65°C for 5 h. After allowing to cool, the benzene layer was separated, solvent evaporated, and residue purified by column chromatography on silica using chloroform - methanol or ethyl acetate - hexanes as eluent.

Example 3

Fmoc-Gl y- tcyclo^Asn-OBu (5a^ The procedure described in Example 2 was followed using the corresponding Fmoc-protected amino acid chloride. After the described purification, the product was recovered in a yield of 61%; m.p. = 124-127 °C; [ ]_D = 12.6 (c 1.13, EtOH) ; ^XH-NMR δ 1.38 and 1.46 (s, 9 H) , 2.62 (dd, J = 8.5, 16.2 Hz, 1 H) , 2.83 (dd, J = 6.5, 16.2 Hz, 1 H) , 3.98. (m, 1 H) , 3.98 (m, 1 H) , 4.27 (m, 2 H) , 4.41 (d, J = 7.1 Hz, 2 H) , 4.66 (m, 1 H) , 5.70 (br s, 1 H) 6.99 and 6.37 (br s 1 H) , 7.32 ( t, J = 7.5 Hz, 2 H) , 7.41 (t, J = 7.5 Hz, 2H) , 7.54-7.68 (m, 5 H) , 7.77 (d, J = 7.0 Hz, 2 H) , 8.00 and 8.21 (br s, 1 H) ; ¹³C-NMR δ 27.6, 42.8, 46.9, 53.4, 63.0, 67.3, 84.6, 119.9, 123.7, 125.0, 127.0, 127.7, 131.4, 132.1, 138.6, 141.2, 143.6, 156.5, 168.2, 169.2, 171.0; IR (thin film); 1675, 1537, 1151, 759 cm^"1; UV λ_max = 208.4, 263.0, 298.8 nm.

Example 4

F oc- (L) -Ala- tcyclo Asn-OBu* (5b^ The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 65% yield; m.p. = 129-134 °C; [α]_D = 26.5 (c 1.75, EtOH) ; ^-NMR δ 1.31 and 1.47 (s, 9 H) , 1.40 (d, J = 7.0 Hz, 3H) 2.56 and 2.78 (m, 2 H) , 4.17-4.23 (m, 1 H) , 4.35-4.43 (m, 3 H) , 4.63 - 4.67 (m, 1 H) , 5.46 and 5.78 (d, J = 7.5 Hz, 1 H) , 6.95 (br s, 1 H) , 7.30 - 7.43 (m, 4 H) , 7.53-7.96 ( , 7 H) , 8.00 and 8.26 (br s, 1 H) ; ¹³C-NMR δ 18.8, 19.4, 27.4, 27.8, 33.3, 46.8, 53.7, 54.9, 63.5, 67.3, 82.9, 120.0, 123.8, 124.9, 127.0, 127.4, 127.8, 130.9, 132.1, 132.4, 140.1, 141.2, 143.3, 148.1, 156.2, 168.3, 169.6, 173.6; (IR (thin film) 1701, 1538, 1251, 771 cm^"1; UV λ,,. = 216.2, 264.4, 299.2 nm. In this case, 5% of D-Ala-isomer 5c was isolated.

Example 5 Fmoc- tD^ -Ala- tcyclo) Asn-OBu^t t5c^ The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 58% yield; m.p. = 130-134 °C; [α]_D = +18.1 (c 1.26; EtOH); ^-NMR δ 1.41 (s, 9 H) , 1.43 (br s, 3 H) , 2.70 (dd, J = 7.5, 14.0 Hz, 1H) , 2.82 (dd, J = 6.5, 17.2 Hz, 1 H) , 4.20 (m, 1 H) 4.35-4.49 (m, 3H) 5.30-5.40 (m, 2H) , 6.81 (d, J = 4.5 Hz, 1 H) 7.32 (t, J = 6.5 Hz, 2 H) , 7.42 (t, J = 7.0 Hz, 1 H) , 7.55-7.68 (m, 5 H) , 7.77 (d, J = 7.0 Hz, 2 H) , 8.02 and 8.24 (br s, 1 H) ; ¹³C-NMR δ 18.0, 18.5, 27.6, 27.7, 33.3, 46.9, 47.1, 54.3, 55.0, 63.3, 67.1, 82.9, 84.0, 119.9, 123.8, 125.0, 127.0, 127.7, 131.4, 132.0, 139.1,

141.2, 143.5, 147.7, 156.2, 168.9, 169.6, 172.1, 174.9; IR (thin film) 1699, 1538, 1219, 722 cm^"1; UN λ_^ = 209.2, 263.0, 298.8 nm.

Example 6

The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 66% yield; m.p. = 145-150 °C; [ ]_D = -65.7 (c 1.44, EtOH); ^-NMR δ 0.96 (d, J = 7.0 Hz, 3 H) , 1.00 (d, J = 7.0 Hz, 3 H) , 1.28 and 1.46 (s, 9 H) , 2.12 (m, 1 H) , 2.51 and 2.73 (m, 2 H) , 3.96-4.48 (m, 4 H) 4.64 (dd, J = 5.0, 14.0 Hz, 1 H) , 5.32 and 5.64 (d, J = 10.0 Hz, 1 H) , 6.93 and 7.02 (d, J = 6.0 HZ, 1 H) , 7.30 - 7.43 (m, 4 H) , 7.52 - 7.63 ( , 5 H) , 7.77 (d, J = 6.5 Hz , 2 H) , 7.99 (d, J = 6.5 Hz, 1 H) , 8.31 (d, J = 2.0 Hz, 1 H) ; ¹³C-NMR δ 17.1, 17.9, 19.5, 27.4, 27.8, 31.3, 33.3, 46.9, 55.0, 55.9, 65.2, 67.3, 82.5, 84.6, 120.0, 124.9, 127.3, 127.8, 131.1, 132.2,

140.1, 141.2, 143.4, 148.0, 156.5, 168.4, 169.6, 172.8; IR (thin film) 1700, 1538, 1151, 771 cm^"1; UV λ,._ax = 207.2, 263.0, 298.9 nm.

Example 7

Fmoc- fL) -OBn-Ser- t cyclo^ Asn-OBu* f5e)

The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 63% yield; m.p. = 103-105 °C; [ ]_D = -22.1 (c 1.16, EtOH) ; ^-NMR δ 1.29 and 1.45 (s, 9 H) , 2.23-2.72 (m, 2 H) , 3.53- 3.80 (m, 2 H) , 4.21 (m, 1 H) , 4.30-4.64 (m, 5 H) , 4.92 and 5.10 (m, 1 H) , 5.43 and 5.66 (d, J = 7.0 Hz, 1 H) , 6.87- 6.95 (m, 2 H) , 7.25-7.43 ( , 10 H) , 7.52-8.00 ( , 5 H) , 8.26 (br s, 1 H) ; ¹³C-NMR δ 27.4, 27.7, 31.9, 51.2, 53.2, 63.4, 65.4, 67.1, 69.7, 71.5, 73.8, 84.3, 119.9, 123.9, 125.0, 127.0, 127.7, 127.9, 128.3, 128.4, 128.6, 131.5, 132.1, 136.7, 138.7, 141.2, 143.3, 143.5, 143.7, 148.0, 155.0, 168.1, 169.1, 172.8; IR (thin film) 1697, 1537, 1151, 771 cm^"1; UV λ_max = 207.6, 263.2, 298.8 nm. Example 8

Fmoc- tT -OBn-Tyr- (cyclop Asn-OBu'J 5f )

The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 62% yield; m.p. = 117-121°C; [α]_D = -32.8 (c 1.7,

EtOH); ²H-NMR δ 1.28 and 1.47 (s, 9 H) , 2.14-2.67 (m, 2 H) ,

2.84-3.27 (m, 2 H) , 3.87 and 4.17 (t, J = 7.5 Hz, 1 H) ,

4.23-4.70 (m, 4 H) , 5.08 and 5.10 (s, 1 H) , 5.38 and 5.74 (d, J = 8.0 Hz, 1 H) , 6.58 and 6.78 (d, J = 5.5 Hz, 1 H) (,

6.90-7.80 (m, 20 H) , 7.91 and 7.95 (br s, 1 H) ; ¹³C-NMR δ

27.4, 27.8, 38.0, 40.4, 46.8, 47.0, 52.7, 53.4, 55.4, 67.0,

67.3, 69.9, 82.5, 114.9, 115.3, 120.0, 123.3, 123.7, 124.9,

127.0, 127.1, 127.4, 127.6, 127.7, 128.0, 128.5, 130.2, 130.7, 131.5, 132.0, 136.7, 138.7, 139.7, 141.2, 143.3,

143.6, 147.9, 155.1, 156.1, 158.1, 158.3, 167.8, 168.3, 168.6, 169.7, 172.3, 173.0; IR (thin film) 1696, 1537, 1219, 771 cm^"1; UV λ_max = 207.0, 265.0, 299.2 nm.

Example 9 Fmoc-(L)-Pro-fcvclo)Asn-Obu^t(5g)

The procedure described in Example 2 was followed by using the corresponding Fmoc-proteced amino acid chloride. After the described purification, the product was recovered in a 30% yield; m.p. = 119-122°C; [ ]_D = -10.1 (c 1.32, EtOH): ^αH-NMR δ 1.46 (s, 9 H) , 1.90-2.25 (m, 4 H) , 2.36 (m, 1 H) , 2.71 (dd, J = 4.0, 15.7 Hz, 1 H) , 3.50-3.71 ( , 2 H) , 4.19-4.34 (m, 3 H) , 4.68 (dd, J = 4.5, 13.5 Hz, 1 H) , 7.05 (br s, 1 H) , 7.28-7.93 (m, 11 H) , 8.26 (br s, 1 H) ; ¹³C-NMR δ 24.8, 27.8, 30.6, 33.3, 46.8, 46.9, 54.9, 54.3, 65.4, 67.8, 82.7, 119.7, 119.9, 120.9, 123.8, 124.8, 125.0, 126.9, 127.0, 127.1, 127.7, 128.7, 130.9, 132.4, 140.8,

141.1, 143.3, 143.7, 148.1, 168.7, 169.7, 173.8; IR (thin film) 1673, 1537, 740 cm^"1; UV λ,^ = 207.0, 265.0, 299.2 nm.

Example 10

General Method For Removal of t-Bu Group The requisite dipeptide surrogate 5 (0.15 mmol) was dissolved in 1 L of neat TFA and the solution was stirred at room temperature for 1 h. TFA was removed in vacuo and the residue was recrystallized or purified on silica gel using chloroform - methanol mixtures as eluent.

Example 11

F oc-Gly- (cyclo) Asn-OH (6a) The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in an 88% yield; m.p. 165- 167°C; [ ]_D = 23.7° 1.01, acetone); ^-NMR (acetone-d_e) , δ 39.1, 41.9, 42.5, 52.1, 56.5, 63.1, 72.5, 76.1, 129.4, 133.7, 134.8, 136.6, 137.2, 137.7, 140.6, 141.1, 141.7,

150.1, 153.6, 166.1, 172.9, 177.5, 179.4, 180.5; IR (thin film) 1670, 1536, 1049, 741 cm^"1; UV 215.4, 264.8, 292.0 nm.

Example 12

Fmoc- HΛ -Ala- (cyclop Asn-OH t6b^

The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in a 95% yield; m.p. 249-

250°C; [ ]_D = 10.3° (c 1.17, MeOH) ; ^-NMR (acetone-d₆) δ

1.38 (dd, J=5.5, 25.0 Hz, 3 H) , 2.38-2.83 (m, 2 H) , 4.22-

4.34 (m, 3 H) , 4.54-4.77 (m, 2 H) , 5.25 (br s, 1 H) , 6.95 and 7.09 (br s, 1 H) , 7.33 (t, J=7.0 Hz, 2 H) , 7.42 (t, J=7.5 Hz, 2 H) , 7.66-7.87 (m, 6 H) , 8.14 and 8.53 (m, 2 H) ;

¹³C-NMR (acetone-d₆) δ 17.4, 42.5, 56.1, 70.5, 74.0, 75.3,

129.6, 133.6, 134.7, 136.6, 137.1, 141.5, 145.4, 150.2,

153.2, 158.1, 159.7, 174.5, 179.6, 180.7; IR (thin film); and 1666, 1537, 1199, 740 cm^"1; UV 219.8; 264.2, 299.2 nm.

Example 13

Fmoc- (Ω) -Ala- (cyclop Asn-OH ( 6c)

The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in a 90% yield; m.p. 234-

236°C; [ ]_D = 23.9° (c 2.81, MeOH); ^XH-NMR (acetone-d₆) δ

1.40 (d, J^"=6.5 Hz, 3 H) , 2.38-2.83 (m, 2 H) , 4.03-4.88 (m,

5 H) , 5.57 (t, J=6.5 Hz, 1 H) , 6.68 and 6.96 (br s, 1 H) , 7.33 (t, J=7.5 Hz, 2 H) , 7.42 (t, J=l .5 Hz, 2 H) , 7.64-7.87 ( , 6 H) , 8.13 and 8.49 (m, 1 H) ; ¹³C-NMR (acetone-d₆) δ 17.4, 34.1, 55.3, 60.0, 60.4, 62.6, 74.5, 119.5, 120.1, 122.8, 124.4, 125.0, 125.6, 126.0, 127.1, 127.2, 132.5, 130.2, 132.0, 133.7, 142.2, 142.5, 147.3, 150.0, 150.2, 159.6, 177.8, 180.1, 181.0; IR (thin film) 1682, 1536, 1348, 739 cm^"1; and UN 209.8, 263.0, 298.8 nm.

Example 14 Fmoc- t L> -Val- t cyclo^ Asn-OH t 6d ^

The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in a 95% yield; m.p. 241- 242°C; [ ]_D = 24.4° (c 1.22, DMSO) ; ^XH-ΝMR (acetone-d₆) δ 0.95-0.98(m, 6 H) , 1.70-2.28 (m, 1 H) , 2.37-2.18 (m, 2 H) , 4.16-4.42 ( , 4 H) , 4.74 (dd, J=5.0 , 13.7 Hz, 1 H) , 4.66 and 5.32 (br s, IH) , 7.00 and 7.16 (br s, IH) , 7.33 (t, J = 6.5 Hz, 2 H) , 7.42 (t, J = 7.5 Hz, 2 H) , 7.65-7.87 (m, 6 H) , 8.14 (d, J = 10.0 Hz, 1 H) , 8.59 (s, 1 H) ; ¹³C-NMR (acetone-d₆) δ 27.7, 28.6, 39.6, 42.6, 56.1, 63.6, 66.6, 73.8, 75.5, 129.6, 133.7, 134.8, 136.6, 137.2, 141.4, 141.6, 150.2, 151.4, 153.1, 153.3, 156.6, 165.9, 177.9,

180.8, 181.6; IR (thin film) 1659, 1028, 824, 762 cm^"1; and UV 207.8, 265.4, 294.8 nm.

Example 15

Fmoc- tL^ -OBn-Ser- (cyclop Asn-OH t 6e^

The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in an 85% yield; m.p. 182-

183°C; [α]_D = -13.9 (c 4.42, MeOH), ^XH-NMR (acetone-d₆) δ

2.36-2.78(m, 2 H) , 3.17 - 3.82 (m, 2 H) , 4.22 - 4.76 (m, 7

H) , 5.04 and 5.40 (br s, 1 H) , 6.96 and 7.11 (br s, 1 H) ,

7.31 (m, 7 H) , 7.41 (t, J = 7.0 Hz, 2 H) , 7.48-7.87 (m, 6 H) , 8.16 and 8.50 (m, 1 H) ; ¹³C-NMR (acetone-d₆) δ 34.1,

60.1, 61.5, 62.2, 65.5, 70.0, 75.1, 76.5, 119.9, 120.1,

123.9, 126.4, 126.9, 127.0, 127.2, 127.5, 128.3, 132.0, 132.5, 136.3, 136.5, 142.9, 146.4, 150.0, 157.9, 175.8, 178.9, 180.0; IR (thin film) 1684, 1536, 1048, 740 cm^"1; and UV 206.4, 264.6, 299.2 nm.

Example 16 Fmoc- (L) -OBn-Tyr- fcvclol Asn-OH (6 )

The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in an 89% yield; m.p. 158- 160°C; [ ]_D = -22.6 (c 1.07, MeOH), 'Ή-NMR (acetone-d₆) δ 2.35-2.64(m, 2 H) , 2.91-3.16 (m, 2 H) , 4.17-4.32 (m, 3 H) , 4.56-4.90 (m, 2 H) , 5.05 (br s, 1 H) , 6.85-6.92 ( , 3 H) , 7.07-7.45 (m, 12 H) , 7.63-7.86 (m, 6 H) , 8.12 and 8.37 (m, 1 H) ; ¹³C-NMR (acetone-d₆) δ 34.1, 52.1, 56.7, 60.4, 62.6, 69.4, 69.8, 117.6, 117.8, 119.6, 122.8, 124.5, 125.6, 125.7, 127.0, 127.3, 128.2, 128.7, 130.3, 130.4, 133.9,

137.0, 141.8, 141.9, 145.6, 147.9, 148.0, 156.9, 158.4,

176.1, 177.0, 178.9; IR (thin film) 1696, 1510, 1032, 739 cm^"1; and UV 211.8, 265.4, 298.2 nm.

Example 17

Fmoc-(L) -Pro- (cyclo) Asn-OH ( 6a) The method of Example 10 was followed starting with the corresponding dipeptide surrogate. After purification, the acid compound was obtained in an 85% yield; m.p. 233- 235°C; [ ]_D = -18.1° (c 1.18, DMSO) ; ^-NMR (DMSO-d₆) δ 1.88-2.22 (m, 4 H) , 2.25-2.64 (m, 2 H) , 3.55-3.70 (m, 2 H) , 4.16-5.03 ( , 6 H) , 6.65-6.77 (m, 1 H) , 7.30-8.08 (m, 11 H) , 8.48 and 8.64 (br s, 1 H) ; ¹³C-NMR (DMSO-d₆) δ 24.6, 33.8, 39.5, 47.9, 56.1, 63.4, 65.6, 74.2, 76.3, 129.6, 133.6, 134.6, 136.6, 137.2, 141.6, 150.2, 151.3, 152.8, 156.6, 163.2, 177.8, 181.2, 182.9; IR (thin film) (DMSO) 1674, 1426, 1032 cm^"1; and UV 208.6, 262.4, 298.6 nm.

Example 18 General procedure for removal of Fmoc group

Method 1 (solution phased The requisite dipeptide surrogate (5) (0.08 mmol) was dissolved in 1 mL of Me₂NH (2 M solution in THF) and stirred at room temperature for 10-15 minutes. Solvent was removed and the residue was treated with petroleum ether. Filtration removed most of the soluble dibenzofulvene-Me₂NH adduct. The remaining solid was dissolved in a small amount of THF or diethyl ether, and desired amine was precipitated by addition of petroleum ether. In most cases these amines were isolated as the corresponding acetamide derivative through treatment with excess acetic anhydride.

Example 19

Ac-Gly-rcyclop Asn-OBu' t7a) The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 82% yield; m.p. 113-115°C; [ ]_D = 12.1 (c 1.16, MeOH); ²H-NMR δ 1.37 and 1.45 (s, 9 H) , 2.01-2.06 (s, 3 H) , 2.23-2.91 (m, 2 H) , 3.91-4.00 (m, 1 H) , 4.33-4.77 ( , 2 H) , 6.48 and 6.97 (m, 1 H) , 6.64 and 6.72 (br s, 1 H) , 7.54-8.53 (m, 4 H) ; ¹³C-NMR δ 22.7, 27.6, 27.7, 41.5, 53.5, 62.8, 84.5, 123.8, 128.0, 131.4, 132.0, 138.6, 168.2, 170.0, 170.7; IR (thin film) 1679, 1539, 1369, 1154, 1048 cm^"1; and UV 205.2 nm.

Example 20

The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 40% yield; m.p. 117-120°C; [ ]_D

= -23.3 (c 1.26, MeOH); ^-NMR δ 1.29 and 1.38 (d, J = 7.5 Hz, 3 H) , 1.93 and 1.95 (s, 3 H) , 2.31-3.01 (m, 2 H) , 4.45 and 4.61 (m, 1 H) , 4.78 and 5.18 (m, 1 H) , 6.92 and 6.99

(br s, 1 H) , 7.59 and 8.05 (d, J = 8.5 Hz, 1 H) , 7.70 (d,

J = 8.0 Hz, 1 H) , 8.00 and 8.55 (s, 1 H) ; ¹³C-NMR δ 15.2,

22.0, 26.5, 34.1, 50.3, 52.3, 61.0, 122.5, 127.6, 129.6, 132.9, 138.5, 148.1, 166.4, 168.1, 168.9, 171.3, 175.5; IR

(thin film) 1681, 1537, 1203, 1050 cm^"1; and UV λ^ 214.2 nm. Example 21

Ac- (D) -Ala- ( cyclo) Asn-OBu* (7c)

The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 90% yield; m.p. 112-113°C; [α]_D

= 22.5 (c 1.34, MeOH); ^-NMR δ 1.28 and 1.39 (s, 9 H) , 1.43

(d, J = 7.0 Hz, 3 H) , 1.98 (s, 3 H) , 2.62 (dd, J = 8.0,

16.5 Hz, 1 H) , 2.83 (dd, J = 7.0, 16.5 Hz, 1 H) , 3.95 and

4.61 (m, 1 H) , 5.01 and 5.50 (t, J = 7.5 Hz, 1 H) , 6.39 and 6.85 (d, J = 4.0 Hz, 1 H) , 6.64 and 6.99 (d, J = 7.5 Hz, 1

H) , 7.51 (d, J = 8.0 Hz, 1 H) , 7.59 and 7.63 (d, J = 8.5

Hz, 1 H) , 7.88 (d, J - 3.0 Hz, 1 H) , 7.95 and 8.29 (d, J =

1.5 Hz, 1 H) ; ¹³C-NMR δ 17.6, 22.5, 27.6, 27.7, 29.6, 33.3,

45.7, 54.4, 63.1, 83.9, 123.8, 127.0, 131.3, 132.1, 139.3, 147.7, 169.5, 170.8, 175.3; and IR (thin film) 1651, 1539,

1261, 1154 and 1050 cm^"1.

Example 22 Ac- (L)-Val- (cyclo') Asn-OBu' (7d^ The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in a 75% yield; m.p. 158-160°C; [ ]_D = -58.4 (c 1.78, MeOH); ^-NMR δ 0.95 (d, J = 6.5 Hz, 3 H) , 1.02 (d, J = 6.0 Hz, 3 H) , 1.32 and 1.46 (s, 9 H) , 2.06 and 2.08 (s, 3 H) , 2.12-2.19 (m, 1 H) , 2.21-2.28 ( , 1 H) , 2 . 69-2 . 14 ( , 1 H) , 4.27 and 4.80 (t, J = 8.5 Hz, 1 H) , 4.59 and 4.84 (dd, J = 5.0, 14.0 Hz, 1 H) , 6.32 (d, J = 9.0 Hz, 1 H) , 7.26 (d, J = 7.0 Hz, 1 H) , 7.62 (d, J = 8.5 Hz, 1 H) , 8.08 (dd, J = 2.0, 8.5 Hz, 1 H) , 8.00 and 8.17 (d, J = 6.0 Hz, 1 H) , 8.31 (d, J = 2.5 Hz, 1 H) ; ¹³C-NMR δ 18.2, 19.4, 19.5, 22.7, 27.4, 27.8, 29.6, 31.3, 33.4, 55.3, 55.5, 65.3, 82.8, 124.1, 127.2, 131.1, 132.1, 140.4, 147.8, 168.5, 169.4, 173.0; IR (thin film) 1646, 1538, 1205, 1150 cm^"1; and UV 217.6 nm.

Example 23 Ac- (I -OBn-Ser- (cyclop Asn-OBu' f7e The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 87% yield; m.p. 88-90°C; [α]_D = -23.2 (c, 1.55, MeOH); ^-NMR δ 1.31 and 1.43 (s, 9 H) , 1.98 and 2.01 (s, 3 H) , 2.17-2.71 (m, 2 H) , 3.45-3.81 (m, 2 H) , 4.44-4.52 (m, 2 H) , 4.59 and 4.84 (d, J = 8.5 Hz, 1 H) , 5.10-5.16 (m, 1 H) , 6.32 (overlapping doublets, apparent J = 7.5 Hz, 1 H) , 6.81 and 7.09 (m, 2 H) , 7.24-7.37 ( , 5 H) , 7.54 and 7.66 (d, J = 8.0 Hz, 1 H) , 7.73 and 7.92 (d, J = 8.0 Hz, 1 H) , 7.98 and 8.28 (s, 1 H) ; ¹³C-NMR δ 22.7, 27.8, 32.4, 49.9, 50.9, 53.5, 55.5, 63.6, 65.4, 69.6, 65.7, 69.6, 73.9, 82.8, 84.5, 124.1, 128.1, 128.3, 128.5, 128.8, 129.1, 131.6, 131.8, 137.0, 139.0, 140.0, 148.1, 168.3, 169.3, 171.5, 172.6; IR (thin film) 2300, 1650, 1539, 1154, 1050 cm^"1; and UV 213.6 nm.

Example 24 H- L^ -OBn-Tyr (cyclop Asn-OBu*¹ (7f ) The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 90% yield; m.p. 130-132 °C; [ ]_D = -59.3 (c 0.62, MeOH); ^XH-NMR (MeOH-d₄) δ 1.31 and 1.43 (s, 9 H) , 2.04-2.79 (m, 2 H) , 2.96-3.21 (m, 2 H) , 4.81 and 4.05 (m, 1 H) , 4.50 and 4.60 (m, 1 H) , 5.04-5.11 (m, 2 H) , 6.42 and 6.76 (s, 1 H) , 6.82 and 6.92 (d, J = 8.0 Hz, 2 H) , 7.01-7.90 (m, 10 H) ; ¹³C-NMR (MeOH-d₄) δ 26.7, 34.5, 35.1, 52.5, 55.3, 61.2, 70.2, 82.0, 115.8, 122.7, 126.8, 127.6, 127.9, 128.2, 128.8, 129.6, 130.5, 133.0, 136.8, 138.6, 148.4, 158.6, 166.7, 167.3, 169.1, 173.4; IR (thin film) 1679, 1539, 1050 cm^"1; and UV 204.6 n .

Example 25

H-(L) -Pro- (cycloϊ Asn-OBu" (la)

The method of Example 18 was followed using the corresponding dipeptide surrogate. Upon purification, a product was obtained in an 85% yield; m.p. 230°C (dec);

[α]_D = -42.2 (c 0.46, MeOH); ^αH-NMR (MeOH-d δ 1.31 and 1.43

(s, 9 H) , 2.04-2.79 (m, 2 H) , 2.96-3.21 (m, 2 H) , 4.81 and

4.05 ( , 1 H) , 4.50 and 4.60 (m, 1 H) , 5.04-5.11 ( , 2 H) , 6.42 and 6.76 (s, 1 H) , 6.82 and 6.92 (d, J= 8.0 Hz, 2 H) , 7.01-7.90 (m, 10 H) ; ¹³C-NMR (MeOH-d₄) δ 25.2, 27.4, 27.8, 30.6, 33.4, 47.2, 60.1, 65.3, 66.9, 81.8, 124.1, 127.2, 131.1, 132.1, 136.1, 147.8, 168.5, 173.0, 180.2; IR (thin film) 1680, 1538, 1051 cm^"1; and UV 205.6 nm.

Example 26

Method A (solid state)

NOVASYN® PR-500 resin (0.34 g) was deprotected by treatment with 30% piperidine in DMF (15 min), and 0.1 mmol of compound 6 was introduced in the presence of 15 mg (0.11 mmol) of HOBt and 0.017 mL of diisopropylcarbodiimide in 1 mL of DMF. The reaction was continued for 12-14 h, at which time the Fmoc-group was removed by treatment with 30% piperidine in DMF (15 min) , and the product directly reacted with acetic anhydride (3 mL of 0.5 M solution in

DMF) . The resin was washed with DMF, isopropanol, methanol, and ether, dried in vacuo , and cleaved from the resin by treatment with 95% TFA/5% H₂0 for 1 h, which resulted in the isolation of the corresponding carboxamide.

Example 27 Ac- (L -Ala (cyclo) Asn-NH, The method of Example 26 was repeated using the appropriate compound for compound (6) . After purification, a product was obtained in a 77% yield; m.p. 125-126°C; [ ]_D = -6.5 (c 1.85, MeOH); ^XH-NMR (MeOH-d₄) δ 1.29 and 1.38 (d, J = 7.5 Hz, 3 H), 1.33 (overlapping doublets, apparent J = 3.0 Hz), 1.93 and 1.95 (s, 3 H) , 2.31 - 3.01 ( , 2 H) , 4.45 and 4.61 (m, 1 H) , 4.78 and 5.18 (m, 1 H) , 6.92 and 6.99 (br s, 1 H) , 7.59 and 8.05 (d, J = 8.5 Hz, 1 H) , 7.70 (overlapping doublets, apparent J = 8.0 Hz , 1 H) , 8.00 and 8.55 (s, 1 H) ; IR (thin film) 3277, 1681, 1537, 1203 cm^"1; and UV 227.2, 257.8 nm.

Example 28 Ac- (L)-Val (cyclo') Asn-NH-, The method of Example 26 was repeated using the appropriate compound for compound (6) . After purification, a product was obtained in a 80% yield; m.p. 137-139^CC; [ ]_D = -55.6 (c 1.31, MeOH); ^-NMR (MeOH-d₄) δ 0.90 and 1.06 (dd, J = 6.5, 23.5 Hz, 6 H) , 1.92 and 1.98 (s, 3 H) , 2.11 ( , IH) , 2.31-2.96 (m, 2 H) , 4.13 and 4.55 (d, J = 9.0 Hz, 1 H) , 4.58 and 4.80 (m, 1 H) , 5.31 (m, 1 H) , 7.00 (s, 1 H) , 7.58 and 7.71 (d, J = 8.0 Hz , 1 H) , 7.66 and 8.08 (d, J = 8.0 Hz, 1 H) , 7.98 and 8.64 (s, 1 H) ; IR (thin film) 1638,

1537, 1204, 1150, 1050 cm^"

Example 29 Solid state synthesis of Fmoc-Gly-Val-Ala- (cyclo) Asn-NH_; mx

NOVASYN® PR-500 resin (0.45 g) was deprotected by treatment with 30% piperidine in DMF (15 min) , and (6b) (0.13 mmol, 80 mg) was introduced in the presence of 21 mg (0.15 mmol) of HOBt and 0.022 mL of diisopropylcarbodiimide in 1 mL of DMF. The reaction was continued for 12-14 h, at which time the Fmoc-group was removed by treatment with 30% piperidine in DMF (15 min) . The peptide was elongated consecutively with Fmoc-Val-OH and Fmoc-Gly-OH used in three-fold excess with the following equivalents: Fmoc- a ino acid:HATU:DIEA (1:1:3) dissolved in 1.5 mL of DMF. Coupling time for each amino acid was 15 min. The resulting resin was washed with DMF, isopropanol, methanol, and ether, dried in vacuo , and cleaved from the resin by treatment with 95% TFA/5% H₂0 for 1 h, which resulted in the isolation of the corresponding carboxamide. Final purification was achieved by recrystallization from ethyl acetate - methanol. After purification, a product was obtained 0.80 g (82% yield); m.p. 170-172°C; [ ]_D = -33.6 (c 1.0, MeOH); ^-NMR (DMSO-d₆) δ 0.77-0.87 (m, 6 H) , 1.13 and 1.27 (m, 3 H) , 1.84-1.94 (m, 1 H) , 2.09-2.70 (m, 2 H) , 3.64 (m, 2 H) , 3.93 (d, J = 5.5 Hz, 1 H) , 4.14-4.27 (m, 4 H) , 4.40-5.11 (m, 1 H) 6.70 and 6.88 (m, 1 H) , 7.05 and 7.20 (br s, 1 H) , 7.32 (t, J = 7.0 Hz, 3 H) , 7.42 (t, J = 8.0 Hz, 2 H) , 7.46-8.74 (m, 15 H) , 8.88 and 9.35 (d, J = 4.5 Hz, 1 H) ; UV 227.0, 250.8, 298.6 nm.

Example 30

F oc-Gly-Val-Ala-Asn-NH (12) (Method 1) NOVASYN® PR-500 resin (0.4 g) was deprotected with 30% piperidine in DMF, and 0.17 g (0.28 mmol, two fold excess compared to the capacity of resin) of Fmoc-(Trt)- Asn-OH was introduced in the presence of 0.1 g (0.28 mmol) of HATU and 0.15 mL (0.84 mmol) of DIEA in 1 mL of DMF within 15 min. The remaining transformations proceeded as described above. Yield 0.056 g (70%).

Example 1

Fmoc-Gly-Val-Ala-Asn-NHo (12) (Method 2) Tetrapeptide surrogate (11) (0.6 g, 0.08 mmol) in 2 mL of 0.25 M HCl in THF was refluxed for 2 h, solvent was removed, residue dried in vacuo and refluxed with ethyl acetate - hexane mixture to remove 4-chloro-3- nitrobenzaldehyde. The residue was recrystallized from methanol. Yield 0.04 g (88%); ^:H-NMR (DMSO-d₆) δ 0.84 (dd, J^" = 7.0, 17.7 Hz, 6 H) , 1.21 (d, J = 6.5 Hz, 3 H) , 1.97 (m,

1 H) , 2.46 (d, J = 6.5 Hz, 2 H) , 3.68 (dd, J = 3.5, 6.0 Hz, 2 H) , 4.18-4.29 (m, 5 H) , 4.40 (overlapping doublets, apparent J = 8.0 Hz, 1, H) , 6.87 (br s, 1 H) , 7.05 (d, J = 3.5 Hz, 2 H) , 7.33 (t, J = 7.0 Hz, 3 H) , 7.42 (t, J = 7.0 Hz, 2 H) , 7.55 (t, J = 5.0 Hz, 1 H) , 7.71 (d, J = 7.5 Hz,

2 H) ; 7.81 (d, J = 8.5 Hz, 1 H) ; 7.90 (t, J = 7.5 Hz, 3 H) ; 8.21 (d, J = 6.5 Hz, 1 H) ; UN 216.2, 263.2, 298.6 nm.

Example 32

GENERAL PROCEDURE FOR RING OPENING

The specific surrogate (0.08 mmol) was dissolved in THF and treated with 2mL of a 0.25 M HCl solution. The resulting solution was refluxed for 2 h, solvent was removed, residue dried in vacuo , and refluxed with ethyl acetate - hexane to remove 4-chloro-3-nitrobenzaldehyde. The residue was generally recrystallized. Material obtained by this procedure was identical with material prepared by conventional peptide synthesis.

Example 33

PHARMACEUTICAL COMPOSITIONS The following are typical pharmaceutical compositions containing, as active ingredient, an (cyclo) Asn analogue of the present invention, for example, by itself or as a pharmaceutically acceptable salt, e.g. the acetic acid addition salt, the zinc salt, the zinc tannate salt, etc. A. Tablet formulations for buccal (e.g. sublingual) administration:

1. (Cyclo)Asn Analogue 50.0 μg

Compressible Sugar, USP 96.0 mg

Calcium Stearate 4.0 mg

2. (Cyclo) Asn Analogue 30.0 μg Compressible Sugar, USP 98.5 mg Magnesium Stearate 1.5 mg

3. (Cyclo) Asn Analogue 25.0 μg Mannitol, USP 88.5 mg Magnesium Stearate 1.5 mg Pregelatinized Starch, USP 10.0 mg 4. (Cyclo)Asn Analogue 200.0 μg

Lactose, USP 83.3 mg

Pregelatinized Starch, USP 15.0 mg

Magnesium Stearate 1.5 mg

Method of Manufacture a. The (cyclo) Asn analogue is dissolved in water, a sufficient quantity to form a wet granulation when mixed with the sugar portion of the excipients. After complete mixing, the granulation is dried in a tray or fluid-bed dryer. The dry granulation is then screened to break up any large aggregates and then mixed with the remaining components. The granulation is then compressed on a standard tabletting machine to the specific tablet weight. b. In this manufacturing method, all formulations would include 0.01% gelatin, USP. The gelatin would be first dissolved in the aqueous granulation solvent followed by the (cyclo) Asn analog. The remaining steps are as in (a) above.

Formulation 4 could also be used as a tablet for oral administration.

B. Long Acting intramuscular injectable formulation. ϊ^~. Long Acting l.M. Injectable - Sesame Oil Gel

(Cyclo)Asn Analogue 1.0 mg

Aluminum monostearate, USP 20.0 mg

Sesame oil q.s.ad 1.0 ml

The aluminum monostearate is combined with the sesame oil and heated to 125-C with stirring until a clear yellow solution forms. This mixture is then autoclaved for sterility and allowed to cool. The (cyclo)Asn analogue is then added aseptically with trituration. Particularly preferred (cyclo) Asn analogues are salts of low solubility, e.g. zinc salts, zinc tannate salts, pamoate salts, and the like. These exhibit exceptionally long duration of activity.

2. Long Acting l.M. Injectable - Biodegradable Polymer

Microcapsules

(Cyclo) Asn Analogue 1%

25/75 glycolide/lactide 99% copolymer (0.5 intrinsic viscosity)

Microcapsules (0-150 μ) of above formulation suspended in:

Dextrose 5.0%

CMC, sodium 0.5% Benzyl alcohol 0.9%

Tween 80 0.1% Water, purified q.s. 100.0%

25 mg of microcapsules would be suspended in 1.0 ml of vehicle. C. Aqueous Solution for Intramuscular Injection

Cyclo (Asn) Analogue 25 mg Gelatin, nonantigenic 5 mg

Water for injection, q.s.ad 100 ml

Dissolve gelatin and (cyclo) Asn analogue in water for injection, then sterile filter solution.

D. Aqueous Solution for Nasal Administration

(Cyclo) Asn Analogue 250 mg

Dextrose 5 gm Benzyl alcohol 0.9 gm

Water, purified q.s.ad 100 ml

Dissolve (cyclo) Asn analogue, dextrose, benzyl alcohol in purified water and q.s. to volume.

Suppository Vehicle for Rectal Administration

(Cyclo) Asn Analogue 500 μg

Witepsol H15 20.0 gm

The (cyclo) Asn analogue is combined with the molten Witepsol H15, mixed well and poured into 2 gm molds.

For those polypeptide agents useful in treatment of brain disorders, it is known that for these agents to be effective they must cross the blood brain barrier. Any formulation according to Remington which facilitates this transfer can be used. While only a few embodiments of the invention have been shown and described herein, it will become apparent to those skilled in the art that various modifications and changes can be made in the synthesis of dipeptide surrogates containing asparagine derived tetrahydropyrimidinones and the formation of peptides useful in brain therapy without departing from the spirit and scope of the present invention. All such modifications and changes coming within the scope of the appended claims are intended to be carried out thereby.

Claims

We Claim :

1. A process to produce (cyclo)Asn and protected derivatives which are useful in solid phase peptide synthesis reactions and as peptide surrogates useful in therapy, which process comprises:

(a) combining asparagine ester with an aldehyde of the structure Q-(C=0)H wherein Q is selected from the group consisting of alkyl having 1 to 10 carbon atoms and aryl of the structure

wherein Y and Z are each independently selected from the group consisting of H-, F-, C1-, Br-, CH₃-, CH₃CH₂-, CH₃CH₂CH₂-, CH₃(CH₃) CH-, (CH₃)₃C-, and -N0₂, at ambient temperature for between about 2 to 10 hr with stirring to produce an imine _n

wherein R¹ is an alkyl having from 1 to 10 carbon atoms;

(b) reacting the imine of step (a) with a protected amino acid chloride in anhydrous aprotic organic liquid and base, at a temperature of between about 40 and 90┬░C for between about 1 and 10 hr to produce

wherein R¹ and Q are defined above and wherever R is any alkyl or aryl group found in an amino acid; and (c) recovering the (cyclo)Asn peptide.

2. The process of Claim 1 wherein Q is alkyl.

3. The process of Claim 1 wherein Q is aryl.

4. The process of Claim 3 wherein X and Y are each hydrogen.

5. The process of Claim 3 wherein X is Cl and Y is N0₂.

6. A process to produce (cyclo) Asn and the protected derivatives optionally useful in solid phase synthesis of polypeptides thereof, which process comprises:

(a) combining commercially available asparagine- t-butyl ester with an aromatic aldehyde to produce the imine

wherein and R¹ is alkyl having from 1 to 10 carbon atoms, and Q is

wherein Y and Z are each independently selected from the group consisting of H-, F-, C1-, Br-, CH₃-, CH₃CH₂CH₂-, CH₃(CH₃)CH-, (CH₃)₃C-, and -N0₂ , at ambient temperature for between about 2 to 10 hr with stirring,

(b) reacting the imine with protected amino acid chloride in anhydrous aprotic organic liquid compound at a temperature of between about 40 and 90┬░C for between about 1 and 10 hr to produce and base which reaction produces the heterocycle

Wherein R¹ and Q are defined herein and

R is any alkyl or aryl group found in an amino acid and (c) recovering the (cyclo) Asn peptide.

7. The process of 6 wherein the (cyclo) Asn is protected for use in solid phase peptide synthesis.

8. An amino acid sequence which is obtained by the replacement of one or optionally more proline amino acids with at least one (cyclo) Asn.

9. An amino acid sequence which is obtained by the replacement of a proline with (cyclo) Asn.

10. Pharmaceutical composition obtained by the replacement of one or optionally more proline amino acids with at least one (cyclo) Asn and a pharmaceutically acceptable excipient.

11. A pharmaceutical composition which comprises an amino acid sequence of claim 8 or 9 above wherein at least one proline has been replaced by (cyclo) Asn and a pharmaceutically acceptable excipient.

12. An amino acid sequence in which includes the following sequence:

-Tyr- (cyclo) Asn-Trp-Phe-NH₂ ; -Tyr- (cyclo) Asn-Phe-Phe-NH₂ ;

-Tyr- (cyclo) Asn-Trp-Gly-NH₂ ; or combinations thereof

13. An amino acid sequence having the following sequence: Tyr- (cyclo)Asn-Trp-Phe-NH₂;

Tyr- (cyclo) Asn-Phe-Phe-NH₂; Tyr- (cyclo) Asn-Trp-Gly-NH₂ ; or combinations thereof.

14. A pharmaceutical composition which comprises an effective amount of an amino acid having a sequence comprising of the structure:

-Tyr- (cyclo) Asn-Trp-Phe-NH₂ -Tyr- (cyclo) Asn-Phe-Phe-NH₂, or -Tyr- (cyclo) Asn-Trp-Gly-NH₂ ; and a pharmaceutically acceptable excipient.

15. A pharmaceutical composition which includes an effective amount of the amino acid sequence: Tyr- (cyclo) Asn-Trp-Phe-NH₂ ;

Tyr- (cyclo) Asn-Phe-Phe-NH₂ ;

Tyr- (cyclo) Asn-Trp-Gly-NH₂; or combinations thereof.

16. The use of the pharmaceutical composition of claims 10, 11, 12 or 15 above in an amount effective in therapy to alleviate conditions of brain disease or disorder.

17. The use of any of pharmaceutical compositions of claims 14 or 15 wherein the brain disease or disorder includes, depression, anxiety, compulsive-obsessive disorder, drug addiction, alcohol addiction, nicotine addiction and the like.

18. The use of the pharmaceutical composition of claims 10, 11, 15 or 16 above in an amount effective as an analgesic or an anesthetic.

19. The amino acid sequence of claims 12 or 13 above which is an agonist or antagonist to the opioid receptor subtype, K, ╬╝ or ╬┤.