Method of making an omega-functionalized amino acid derivative

FIELD OF THE INVENTION

0001The present invention relates to processes for the preparation of novel N^α,ω-functionalized amino acid building units.

BACKGROUND OF THE INVENTION

Peptidomimetics

0002As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical utilities. Thus in the last few years new methods have been established for the treatment and therapy of illnesses in which peptides have been implicated. However, the use of peptides as drugs is limited by the following factors: a) their low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; b) their poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; c) their rapid excretion through the liver and kidneys; and d) their undesired side effects in non-target organ systems, since.peptide receptors can be widely distributed in an organism.

0003Moreover, with few exceptions, native peptides of small to medium size (less than 30-50 amino acids) exist unordered in dilute aqueous solution in a multitude of conformations in dynamic equilibrium which may lead to lack of receptor selectivity, metabolic susceptibilities and hamper attempts to determine the biologically active conformation. If a peptide has the biologically active conformation per se, i.e., receptor-bound conformation, then an increased affinity toward the receptor is expected, since the decrease in entropy on binding is less than that on the binding of a flexible peptide. It is therefore important to strive for and develop ordered, uniform and biologically active peptides.

0004In recent years, intensive efforts have been made to develop peptidomimetics or peptide analogs that display more favorable pharmacological properties than their prototype native peptides. The native peptide itself, the pharmacological properties of which have been optimized, generally serves as a lead for the development of these peptidomimetics. However, a major problem in the development of such agents is the discovery of the active region of a biologically active peptide. For instance, frequently only a small number of amino acids (usually four to eight) are responsible for the recognition of a peptide ligand by a receptor. Once this biologically active site is determined a lead structure for development of peptidomimetic can be optimized, for example by molecular modeling programs.

0005As used herein, a "peptidomimetic" is a compound that, as a ligand of a receptor, can imitate (agonist) or block (antagonist) the biological effect of a peptide at the receptor level. The following factors should be considered to achieve the best possible agonist peptidomimetic a) metabolic stability, b) good bioavailability, c) high receptor affinity and receptor selectivity, and d) minimal side effects.

0006From the pharmacological and medical viewpoint it is frequently desirable to not only imitate the effect of the peptide at the receptor level (agonism) but also to block the receptor when required (antagonism). The same pharmacological considerations for designing an agonist peptidomimetic mentioned above hold for designing peptide antagonists, but, in addition, their development in the absence of lead structures is more difficult. Even today it is not unequivocally clear which factors are decisive for the agonistic effect and which are for the antagonistic effect.

0007A generally applicable and successful method recently has been the development of conformationally restricted peptidomimetics that imitate the receptor-bound conformation of the endogenous peptide ligands as closely as possible (Rizo and Gierasch, <u style="single">Ann. Rev. Biochem.,</u> 61:387, 1992). Investigations of these types of analogs show them to have increased resistance toward proteases, that is, an increase in metabolic stability, as well as increased selectivity and thereby fewer side effects (Veber and Friedinger, <u style="single">Trends Neurosci.,</u> p. 392, 1985).

0008Once these peptidomimetic compounds with rigid conformations are, produced, the most active structures are selected by studying the conformation-activity relationships. Such conformational constraints can involve short range (local) modifications of structure or long range (global) conformational restraints (for review see Giannis and Kolter, <u style="single">Angew. Chem. Int. Ed. Engl.</u> 32:1244, 1993).

Conformationally constrained peptides

0009Bridging between two neighboring amino acids in a peptide leads to a local conformational modification, the flexibility of which is limited in comparison with that of regular dipeptides. Some possibilities for forming such bridges include incorporation of lactams and piperazinones. γ-Lactams and 6-lactams have been designed to some extent as "turn mimetics"; in several cases the incorporation of such structures into peptides leads to biologically active compounds.

0010Global restrictions in the conformation of a peptide are possible by limiting the flexibility of the peptide strand through cyclization (Hruby <i>et al</i>., <u style="single">Biochem. J.,</u> 268:249, 1990). Not only does cyclization of bioactive peptides improve their metabolic stability and receptor selectivity, cyclization also imposes constraints that enhance conformational homogeneity and facilitates conformational analysis. The common modes of cyclization are the same found in naturally occurring cyclic peptides. These include side chain to side chain cyclization or side chain to end-group cyclization. For this purpose, amino acid side chains that are not involved in receptor recognition are connected together or to the peptide backbone. Another common cyclization is the end-to-end cyclization.

0011Three representative examples are compounds wherein partial structures of each peptide are made into rings by linking two penicillamine residues with a disulfide bridge (Mosberg <i>et al.</i>, <u style="single">P.N.A.S. US,</u> 80:5871, 1983), by formation of an amide bond between a lysine and an aspartate group (Charpentier <i>et al.</i>, <u style="single">J. Med. Chem.</u> 32:1184, 1989), or by connecting two lysine groups with a succinate unit (Rodriguez <i>et al.</i>, <u style="single">Int. J. Pept. Protein Res.</u> 35:441, 1990). These structures have been disclosed in the literature in the case of a cyclic enkephalin analog with selectivity for the δ-opiate receptor (Mosberg <i>et al</i>., ibid.); or as agonists to the cholecystokinin B receptor, found largely in the brain (Charpentier et al., ibid., Rodriguez <i>et al</i>., ibid.).

0012The main limitations to these classical modes of cycli.zation are that they require substitution of amino acid side chains in order to achieve cyclization.

0013Another conceptual approach to the conformational constraint of peptides was introduced by Gilon, <i>et al.,</i><u style="single">(Biopolymers,</u> 31:745, 1991) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. While the concept was envisaged as being applicable to any linear peptide of interest, in point of fact the limiting factor in the proposed scheme was the availability of suitable building units that must be used to replace the amino acids that are to be linked via bridging groups. The actual reduction to practice of this concept of backbone cyclization was prevented by the inability to devise any practical method of preparing building units of amino acids other than glycine (Gilon <i>et al.</i>, <u style="single">J. Org. Chem.,</u> 587:5687, 1992). While analogs of other amino acids were attempted the synthetic method used was unsuccessful or of such low yield as to preclude any general applicability.

0014In Gilon, EPO Application No. 564,739 A2; and <u style="single">J. Org</u>. <u style="single">Chem.,</u> 57:5687, 1992, two basic approaches to the synthesis of building units are described. The first starts with the reaction of a diamine with a general α bromo acid. Selective protection of the ω amine and further elaborations of protecting groups provides a building unit, suitable for Boc chemistry peptide synthesis. The second approach starts with selective protection of a diamine and reaction of the product with chloroacetic acid to provide the protected glycine derivative, suitable for Fmoc peptide synthesis.

0015Both examples deal with the reaction of a molecule of the general type X-CH(R)-CO-OR' (wherein X represents a leaving group which, in the examples given, is either Br or Cl) with an amine which replaces the X. The amine bears an alkylidene chain which is terminated by another functional group, amine in the examples described, which may or may not be blocked by a protecting group.

0016In all cases the α nitrogen of the end product originates in the molecule which becomes the bridging chain for subsequent cyclization. This approach was chosen in order to take advantage of the higher susceptibility to nucleophilic displacement of a leaving group next to a carboxylic group.

0017In a molecule where R is different than hydrogen there is a high tendency to eliminate HX under basic conditions. This side reaction reduces the yield of Gilon's method to the point where it is impractical for production of building units based on amino acids other than glycine. The diamine nitrogen is primary while the product contains a secondary nitrogen, which is a better nucleophile. So while the desired reaction may be sluggish, and require the addition of catalysts, the product may be contaminated with double alkylation products. There is no mention of building units with end group chemistries other than nitrogen, so the only cyclization schemes possible are backbone to side chain and backbone to C terminus.

Applications of conformationally constrained peptides

0018Conformationally constrained peptides find many pharmacological uses. Somatostatin is a cyclic tetradecapeptide found both in the central nervous system and in peripheral tissues. It was originally isolated from mammalian hypothalamus and identified as an important inhibitor of growth hormone secretion from the anterior pituitary. Its multiple biological activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut hormones and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central nervous system (for review see Lamberts, <u style="single">Endocrine Rev.,</u> 9:427, 1988).

0019Natural somatostatin (also known as Somatotropin Release Inhibiting Factor, SRIF) of the following structure: <ul id="ul0001" list-style="none" compact="compact"><li>H-Ala¹-Gly²-Cys³-Lys⁴-Asn⁵-Phe⁶-Phe⁷-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹-Thr¹²-Ser¹³-Cys¹⁴-OH</li></ul> was first isolated by Guillemin and colleagues (Bruzeau et al. <u style="single">Science,</u> 179:78, 1973). In its natural form, it has limited use as a therapeutic agent since it exhibits two undesirable properties: poor bioavailability and short duration of action. For this reason, great efforts have been made during the last two decades to find somatostatin analogs that will have superiority in either potency, biostability, duration of action or selectivity with regard to inhibition of the release of growth hormone, insulin or glucagon.

0020Structure-activity relation studies, spectroscopic techniques such as circular dichroism and nuclear magnetic resonance, and molecular modeling approaches reveal the following: the conformation of the cyclic part of natural somatostatin is most likely to be an antiparallel β-sheet; Phe⁶ and Phe¹¹ play an important role in stabilizing the pharmacophore conformation through hydrophobic interactions between the two aromatic rings; the four amino acids Phe⁷-Trp⁸-Lys⁹-Thr¹⁰ which are spread around the β-turn in the antiparallel β-sheet are essential for the pharmacophore; and (D)Trp⁸ is almost always preferable to (L)Trp⁸.

0021Nevertheless, a hexapeptide somatostatin analog containing these four amino acids anchored by a disulfide bridge: <chemistry id="chem0001" num="0001"><img file="EP0804468B1_D0001.tif" /></chemistry> is almost inactive both <i>in vitro</i> and <i>in vivo</i>, although it has the advantage of the covalent disulfide bridge which replaces the Phe⁶-Phe¹¹ hydrophobic interactions in natural somatostatin.

0022Four main approaches have been attempted in order to increase the activity of this hexapeptide somatostatin analog. (1) Replacing the disulfide bridge by a cyclization which encourages a cis-amide bond, or by performing a second cyclization to the molecule yielding a bicyclic analog. In both cases the resultant analog has a reduced number of conformational degrees of freedom. (2) Replacing the original amino acids in the sequence Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with more potent amino acid analogs, such as replacing Phe⁷ with Tyr⁷ and Thr¹⁰ with Val¹⁰. (3) Incorporating additional structural elements from natural somatostatin with the intention that these new elements will contribute to the interaction with the receptor. (4) Eliminating one of the four amino acids Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with the assumption that such analogs would be more selective.

0023The somatostatin analog, MK-678: cyclo(N-Me-Ala⁷-Tyr⁷- (D) Trp⁸-Lys⁹-Val¹⁰-Phe) is an example of a highly potent somatostatin analog designed using the first three approaches above (Veber, <i>et al.,</i><u style="single">Life Science,</u> 34:371, 1984). In this hexapeptide analog, a cis-amide bond is located between N-Me-Ala and Phe¹¹, Tyr⁷ and Val¹⁰ replace Phe⁷ and Thr¹⁰ respectively, and Phe¹¹ is incorporated from natural somatostatin.

0024Another group of somatostatin analogs (U.S. patents 4,310,518 and 4,235,886) includes octreotide: <chemistry id="chem0002" num="0002"><img file="EP0804468B1_D0002.tif" /></chemistry> the only somatostatin analog currently available. It was developed using the third approach described above. Here, (D)Phe⁵ and the reduced C-terminal Thr¹²-CH₂OH are assumed to occupy some of the conformational space available to the natural Phe⁶ and Thr¹², respectively.

0025The compound TT2-32: <chemistry id="chem0003" num="0003"><img file="EP0804468B1_D0003.tif" /></chemistry> is closely related to octreotide and is an example of implementing the fourth approach described above. The lack of Thr¹⁰ is probably responsible for its high selectivity in terms of antitumor activity.

0026These examples of highly potent somatostatin analogs indicate that the phenylalanines in positions 6 and 11 not only play an important role in stabilizing the pharmacophore conformation but also have a functional role in the interaction with the receptor. It is still an open question whether one phenylalanine (either Phe⁶ or Phe¹¹) is sufficient for the interaction with the receptor or whether both are needed.

0027It is now known that the somatostatin receptors constitute a family of five different receptor subtypes (Bell and Reisine, <u style="single">Trends Neurosci.</u>, <u style="single">16,</u> 34-38, 1993), which may be distinguished on the basis of their tissue specificity and/or biological activity. Somatostatin analogs known in the art may not provide sufficient selectivity or receptor subtype selectivity, particularly as anti-neoplastic agents (Reubi and Laissue, <u style="single">TIPS, 16,</u> 110-115, 1995).

0028Symptoms associated with metastatic carcinoid tumors (flushing and diarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas (watery diarrhea) are treated with somatostatin analogs. Somatostatin has been also approved for the treatment of severe gastrointestinal hemorrhages. Somatostatin may also be useful in the palliative treatment of other hormone-secreting tumors (e.g., pancreatic islet-cell tumors and acromegaly) and hormone dependent tumors (e.g., chondrosarcoma and osteosarcoma) due to its anti-secretory activity.

0029Another important peptide, Bradykinin, is a naturally occurring nonapeptide, Arg¹ -Pro² -Pro³ -Gly⁴ -Phe⁵ -Ser⁶ -Pro⁷- Phe⁸ -Arg⁹, formed and released from precursors in the blood in response to inflammatory stimuli. Elevated levels of bradykinin also appear in other body fluids and tissues in pathological states such as asthma, septic shock and common cold. No clinical abnormalities have been associated so far with bradykinin deficiency which indicates that bradykinin may not play a critical role in normal physiology.

0030However, bradykinin mediates its physiological activities by binding to a specific receptive molecule called the bradykinin receptor. Two such bradykinin receptors have been identified so far (these are called B1 and B2 receptors). Subsequent to binding, the bradykinin signal transduction pathway includes production of prostaglandins and leukotrienes as well as calcium activation. Through these mediators, bradykinin is involved in pain, inflammation, allergic reactions and hypotension. Therefore, a substance that can block the ability of bradykinin to bind to its receptor, namely a bradykinin antagonist, should have a significant therapeutic value for one of the following indications: asthma, inflammation, septic shock, pain, hypotension and allergy.

0031The analog used herein to exemplify backbone cyclization is: D-Arg⁰-Arg-R¹-Hyp³-Gly-Phe-R²-D-Phe-Phe⁷-Arg (wherein, R¹ is Pro, R² is Ser in native bradykinin). The change of proline at position 7 of native bradykinin to D-Phe confers antagonist activity. This compound was described in Steranka, <i>et al.,</i><u style="single">P.N.A.S. U.S.,</u> 85:3245-3249, 1988 and is one of a plethora of candidate sequences for modification by the current technology, i.e. backbone cyclization. In this regard, it is worth noting the applications: WO 89/01781, EP-A-0370453 and EP-A-0334244 which disclose a wide range of candidate structures. Antagonist peptides on which stability and/or tissue selectivity can be conferred by appropriate cyclization will be selected from the many such known sequences.

0032Kaljuste and Undén, Int. J. Peptide Protein Res. 43, 1994, 505-511 describes a general solid-phase method for the synthesis of backbone-to-backbone cyclized peptides.

0033EP 0 564 739 discloses backbone cyclic peptides, process for their preparation and pharmaceutical compositions containing them.

0034Byk and Chilon, J. Org. Chem. 1992, 57, 5687-5692 refers to building units for N-backbone cyclic peptides, in particular to the synthesis of protected N-(ω-amino-alkylene)amino acids and their incorporation into dipeptide units.

0035According to the present invention a novel synthetic approach is disclosed providing N^α(ω(functionalized)alkylene) amino acid building units that can be used to synthesize novel N^α-backbone cyclized peptide analogs such as, but not limited to, novel somatostatin and bradykinin analogs. None of the above-mentioned references teaches or suggests N^α-(ω (functionalized) alkylene) amino acids or the novel N^α-backbone cyclized peptide analogs.

SUMMARY OF THE INVENTION

0036The object of the present invention is directed to a method of making building units known as a N^α-ω-functionalized derivatives of the general Formula (VI) of amino acids which are prerequisites for the cyclization process: <chemistry id="chem0004" num="0004"><img file="EP0804468B1_D0004.tif" /></chemistry> wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R is the side chain of an amino acid, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes and alkyl halides; and A is a protecting group thereof.

0037It is preferred that X is alkylene; G is a thiol group, an amine group or a carboxyl group; R is phenyl, methyl or isobutyl; with the proviso that when G is an amine group, R is other than H.

0038Further it is preferred that R is protected with a specific protecting group.

0039More preferred are methods for making ω-functionalized amino acid derivatives of the Formulae: <chemistry id="chem0005" num="0005"><img file="EP0804468B1_D0005.tif" /></chemistry> wherein X, R, A and B are as defined above.

0040Specifically preferred ω-functionalized amino acid derivatives include the following: <ol id="ol0001" compact="compact" ol-style=""><li>1) N^α-(Fmoc) (3-Boc-amino propylene)-(S)Phenylalanine;</li><li>2) N^α-(Fmoc) (3-Boc-amino propylene)-(R)Phenylalanine;</li><li>3) N^α-(Fmoc)(4-Boc-amino butylene)-(S) Phenylalanine;</li><li>4) N^α-(Fmoc) (3-Boc-amino propylene)-(S)Alanine;</li><li>5) N^α-(Fmoc) (6-Boc-amino hexylene)-(S)Alanine;</li><li>6) N^α-(Fmoc) (3-Boc-amino propylene)-(R)Alanine;</li><li>7) N^α-(2-(benzylthio)ethylene)glycine ethyl ester;</li><li>8) N^α-(2- (benzylthio) ethylene) (S) leucine methyl ester;</li><li>9) N^α-(3-(benzylthio)propylene) (S) leucine methyl ester:</li><li>10) Boc-N^α- (2-(benzylthio) ethylene) glycine;</li><li>11) Boc-N^α-(2-(benzylthio)ethylene) (S) phenylalanine;</li><li>12) Boc-N^α-(3-(benzylthio)propylene) (S)phenylalanine;</li><li>13) Boc-L-phenylalanyl-N^α-(2-(benzylthio)ethylene)glycine-ethyl ester;</li><li>14) Boc-L-phenylalanyl-N^α-(2-(benzylthio)ethylene)-(S)phenylalanine methyl ester;</li><li>15) N^α(Fmoc)-(2-t-butyl carboxy ethylene) glycine;</li><li>16) N^α(Fmoc)-(3-t-butyl carboxy propylene) glycine;</li><li>17) N^α(Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;</li><li>18) N^α(Fmoc)(2-Boc amino ethylene)glycine;</li><li>19) N^α(Fmoc) (3-Boc amino propylene)glycine;</li><li>20) N^α(Fmoc) (4-Boc amino butylene)glycine; and</li><li>21) N^α(Fmoc) (6-Boc amino hexylene)glycine.</li></ol>

0041Thus the present invention is directed to novel, practical, generally applicable processes for the preparation of these N^α-ω-functionalized derivatives of amino acids.

0042As such, an object of this invention is a method of making an ω-functionalized amino acid derivative of the general Formula: <chemistry id="chem0006" num="0006"><img file="EP0804468B1_D0006.tif" /></chemistry> wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R is the side chain of an amino acid, such as H, CH₃, etc.; A and B are protecting groups selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; comprising the steps of : <ol id="ol0002" compact="compact" ol-style=""><li>i) reacting a diamine compound of the general Formula: <chemistry id="chem0007" num="0007"><img file="EP0804468B1_D0007.tif" /></chemistry> wherein A, B and X are as defined above, with a triflate of Formula CF₃SO₂-O-CH(R)-CO-E wherein E is a carboxyl protecting group and R is as defined above; to yield a compound of Formula: <chemistry id="chem0008" num="0008"><img file="EP0804468B1_D0008.tif" /></chemistry> wherein A, B, E, R and X are as defined above</li><li>ii) and deprotecting the carboxyl to yield an N^α ω-functionalized amino acid derivative, wherein the ω-functional group is an amine.</li></ol>

0043A further object of this invention is a method of making an ω-functionalized amino acid derivative of the general Formula: <chemistry id="chem0009" num="0009"><img file="EP0804468B1_D0009.tif" /></chemistry> where B is a protecting group selected from the group of substituted alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the side chain of an amino acid, such as H, CH₃, etc.; X is a spacer group selected from the group of alkylene, substituted alkylene, arylene, cyclcalkylene or substituted cycloalkylene; and A is a protecting group selected from the group of alkyl or substituted alkyl, thio ethers or aryl or substituted aryl thio ethers; comprising the steps of: <ol id="ol0003" compact="compact" ol-style=""><li>i) reacting a compound of the general Formula B-NH-X-S-A with a triflate of the general Formula CF₃SO₂-O-CH(R)-CO-E wherein E is a carboxyl protecting group and A, X and R are as defined above, to give a compound of the Formula: <chemistry id="chem0010" num="0010"><img file="EP0804468B1_D0010.tif" /></chemistry></li><li>ii) selectively removing the protecting group E, and</li><li>iii) protecting the free amino group to yield an N^α-(ω-functicnalized) amino acid derivative, wherein the ω-functional group is a thiol.</li></ol>

0044A further object of this invention is a method of making an ω-functionalized amino acid derivative of the general Formula: <chemistry id="chem0011" num="0011"><img file="EP0804468B1_D0011.tif" /></chemistry> where B is a protecting group selected from the group of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the side chain of an amino acid, such as H, CH₃, etc.; X is a spacer group selected from the group of alkylene, substituted alkylene, arylene, cycloalkylene or substituted cycloalkylene; and A is a protecting group selected from the group of alkyl or substituted alkyl, esters, or thio esters or substituted aryl esters or thio esters; comprising the steps of: <ol id="ol0004" compact="compact" ol-style=""><li>i) reacting a compound of the general Formula B-NH-X-CO-A with a triflate of the general Formula CF₃SO₂-O-CH(R)-CO-E wherein E is a carboxyl protecting group and A, B, X and R are as defined above, to give a compound of Formula: <chemistry id="chem0012" num="0012"><img file="EP0804468B1_D0012.tif" /></chemistry></li><li>ii) and selectively removing protecting group E, to yield an N^α(ω-functionalized) amino acid derivative, wherein the ω-functional group is a carboxyl.</li></ol>

BRIEF DESCRIPTION OF THE DRAWINGS

0045<ul id="ul0002" list-style="none" compact="compact"><li>Figure 1 is a graph showing in vitro biostability of somatostatin and three analogs thereof in human serum. The graph depicts the percentage of undegraded molecules for each of the compounds initially and after various periods of time.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

Definitions

0046All abbreviations used are in accordance with the IUPAC-IUB recommendations on Biochemical Nomenclature (<u style="single">J. Biol. Chem.,</u> 247:977-983, 1972) and later supplements.

0047As used herein and in the claims, the phrase "an amino acid side chain" refers to the distinguishing substituent attached to the α-carbon of an amino acid; such distinguishing groups are well known to those skilled in the art. For instance, for the amino acid glycine, the R group is H; for the amino acid alanine, R is CH₃, and so on. Other typical side chains of amino acids include the groups: (CH₃)₂CH-, (CH₃) ₂CHCH₂-, CH₃CH₂CH(CH₃) -, CH₃S(CH₂)₂-, HOCH₂-, CH₃CH (OH)-, HSCH₂-, NH₂C (=O) CH₂-, NH₂C(=O) (CH₂)₂-, NH₂(CH₂)₃-, HOC (=O) CH₂-, HOC (=O) (CH₂)₂-, NH₂(CH₂)₄-, C(NH₂)₂ NH(CH₂)₃-, HO-phenyl-CH₂-, benzyl, methylindole, and methylimidazole.

0048As used herein and in the claims, the letters "(AA)" and the term "amino acid" are intended to include common natural or synthetic amino acids, and common derivatives thereof, known to those skilled in the art, including but not limited to the following. Typical amino-acid symbols denote the L configuration unless otherwise indicated by D appearing before the symbol. <tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="41mm" /><thead><row><entry valign="top">Abbreviated Designation</entry><entry valign="top">Amino Acids</entry></row></thead><tbody><row rowsep="0"><entry>Abu</entry><entry>α-Amino butyric acid</entry></row><row rowsep="0"><entry>Ala</entry><entry>L-Alanine</entry></row><row rowsep="0"><entry>Arg</entry><entry>L-Arginine</entry></row><row rowsep="0"><entry>Asn</entry><entry>L-Asparagine</entry></row><row rowsep="0"><entry>Asp</entry><entry>L-Aspartic acid</entry></row><row rowsep="0"><entry>βAsp (Ind)</entry><entry>β-Indolinyl aspartic acid</entry></row><row rowsep="0"><entry>Cys</entry><entry>L-Cysteine</entry></row><row rowsep="0"><entry>Glu</entry><entry>L-Glutamic acid</entry></row><row rowsep="0"><entry>Gln</entry><entry>L-Glutamine</entry></row><row rowsep="0"><entry>Gly</entry><entry>Glycine</entry></row><row rowsep="0"><entry>His</entry><entry>L-Histidine</entry></row><row rowsep="0"><entry>Hyp</entry><entry>trans-4-L-Hydroxy Proline</entry></row><row rowsep="0"><entry>Ile</entry><entry>L-Isoleucine</entry></row><row rowsep="0"><entry>Leu</entry><entry>L-Leucine</entry></row><row rowsep="0"><entry>Lys</entry><entry>L-Lysine</entry></row><row rowsep="0"><entry>Met</entry><entry>L-Methionine</entry></row><row rowsep="0"><entry>Nal</entry><entry>β-Naphthyl alanine</entry></row><row rowsep="0"><entry>Orn</entry><entry>Ornithine</entry></row><row rowsep="0"><entry>Phe</entry><entry>L-Phenylalanine</entry></row><row rowsep="0"><entry>Pro</entry><entry>L-Proline</entry></row><row rowsep="0"><entry>Ser</entry><entry>L-Serine</entry></row><row rowsep="0"><entry>Thr</entry><entry>L-Threonine</entry></row><row rowsep="0"><entry>Trp</entry><entry>L-Tryptophane</entry></row><row rowsep="0"><entry>Tyr</entry><entry>L-Tyrosine</entry></row><row rowsep="0"><entry>Val</entry><entry>L-Valine</entry></row></tbody></tgroup></table></tables>

0049Typical protecting groups, coupling agents, reagents and solvents such as but not limited to those listed below have the following abbreviations as used herein and in the claims. One skill in the art would understand that the compounds listed within each group may be used interchangeably; for instance, a compound listed under "reagents and solvents" may be used as a protecting group, and so on. Further, one skill in the art would know other possible protecting groups, coupling agents and reagents/solvents; these are intended to be within the scope of this invention. <tables id="tabl0002" num="0002"><table frame="none"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="45mm" /><thead><row><entry valign="top">Abbreviated Designation</entry><entry valign="top">Protecting Groups</entry></row></thead><tbody><row rowsep="0"><entry>Ada</entry><entry>Adamantane acetyl</entry></row><row rowsep="0"><entry>Alloc</entry><entry>Allyloxycarbonyl</entry></row><row rowsep="0"><entry>Allyl</entry><entry>Allyl ester</entry></row><row rowsep="0"><entry>Boc</entry><entry>tert-butyloxycarbonyl</entry></row><row rowsep="0"><entry>Bzl</entry><entry>Benzyl</entry></row><row rowsep="0"><entry>Fmoc</entry><entry>Fluorenylmethyloxycarbonyl</entry></row><row rowsep="0"><entry>OBzl</entry><entry>Benzyl ester</entry></row><row rowsep="0"><entry>OEt</entry><entry>Ethyl ester</entry></row><row rowsep="0"><entry>OMe</entry><entry>Methyl ester</entry></row><row rowsep="0"><entry>Tos (Tosyl)</entry><entry>p-Toluenesulfonyl</entry></row><row rowsep="0"><entry>Trt</entry><entry>Triphenylmethyl</entry></row><row rowsep="0"><entry>Z</entry><entry>Benzyloxycarbonyl</entry></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="none"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="115mm" /><thead><row><entry valign="top">Abbreviated Designation</entry><entry valign="top">Coupling Agents</entry></row></thead><tbody><row rowsep="0"><entry>BOP</entry><entry>Benzotriazol-1-yloxytris-(dimethyl-amino)phosphonium hexafluorophosphate</entry></row><row rowsep="0"><entry>DIC</entry><entry>Diisopropylcarbodiimide</entry></row><row rowsep="0"><entry>HBTU</entry><entry>2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate</entry></row><row rowsep="0"><entry>PyBrOP</entry><entry>Bromotripyrrolidinophosphonium hexafluorophosphate</entry></row><row rowsep="0"><entry>PyBOP</entry><entry>Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate</entry></row><row rowsep="0"><entry>TBTU</entry><entry>O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate</entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="none"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="86mm" /><thead><row><entry valign="top">Abbreviated Designation</entry><entry valign="top">Reagents and Solvents</entry></row></thead><tbody><row rowsep="0"><entry>ACN</entry><entry>Acetonitrile</entry></row><row rowsep="0"><entry>AcOH</entry><entry>Acetic acid</entry></row><row rowsep="0"><entry>Ac₂O</entry><entry>Acetic acid anhydride</entry></row><row rowsep="0"><entry>AdacOH</entry><entry>Adamantane acetic acid</entry></row><row rowsep="0"><entry>Alloc-Cl</entry><entry>Allyloxycarbonyl chloride</entry></row><row rowsep="0"><entry>Boc₂O</entry><entry>Di-tert butyl dicarbonate</entry></row><row rowsep="0"><entry>DMA</entry><entry>Dimethylacetamide</entry></row><row rowsep="0"><entry>DMF</entry><entry>N,N-dimethylformamide</entry></row><row rowsep="0"><entry>DIEA</entry><entry>Diisopropylethylamine</entry></row><row rowsep="0"><entry>Et₃N</entry><entry>Triethylamine</entry></row><row rowsep="0"><entry>EtOAc</entry><entry>Ethyl acetate</entry></row><row rowsep="0"><entry>FmocOSu</entry><entry>9-fluorenylmethyloxy carbonyl N-hydroxysuccinimide ester</entry></row><row rowsep="0"><entry>HOBT</entry><entry>1-Hydroxybenzotriazole</entry></row><row rowsep="0"><entry>HF</entry><entry>Hydrofluoric acid</entry></row><row rowsep="0"><entry>MeOH</entry><entry>Methanol</entry></row><row rowsep="0"><entry>Mes (Mesyl)</entry><entry>Methanesulfonyl</entry></row><row rowsep="0"><entry>NMP</entry><entry>1-methyl-2-pyrrolidinone</entry></row><row rowsep="0"><entry>nin.</entry><entry>Ninhydrin</entry></row><row rowsep="0"><entry>i-PrOH</entry><entry>Iso-propanol</entry></row><row rowsep="0"><entry>Pip</entry><entry>Piperidine</entry></row><row rowsep="0"><entry>PP</entry><entry>4-pyrrolidinopyridine</entry></row><row rowsep="0"><entry>Pyr</entry><entry>Pyridine</entry></row><row rowsep="0"><entry>SRIF</entry><entry>Somatotropin release inhibiting factor</entry></row><row rowsep="0"><entry>SST</entry><entry>Somatostatin</entry></row><row rowsep="0"><entry>SSTR</entry><entry>Somatostatin receptor</entry></row><row rowsep="0"><entry>TEA</entry><entry>Triethylamine</entry></row><row rowsep="0"><entry>TFA</entry><entry>Trifluoroacetic acid</entry></row><row rowsep="0"><entry>THF</entry><entry>Tetrahydrofuran</entry></row><row rowsep="0"><entry>Triflate (Trf)</entry><entry>Trifluoromethanesulfonyl</entry></row><row rowsep="0"><entry>Trf₂O</entry><entry>Trifluoromethanesulfonic acid anhydride</entry></row></tbody></tgroup></table></tables>

0050The compounds herein described may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention.

0051By "stable compound" or "stable structure" is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and Formulation into an efficacious therapeutic agent.

0052As used herein and in the claims, "alkyl" or "alkylenyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having one to ten carbon atoms; "alkenyl" is intended to include hydrocarbon chains of either a straight or branched configuration and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl, propenyl, and the like; and "alkynyl" is intended to include hydrocarbon chains of either a straight or branched configuration and one or more triple carbon-carbon bonds which may occur in any stable point along the chain, such as ethynyl, propynyl, and the like.

0053As used herein and in the claims, "aryl" is intended to mean any stable 5- to 7-membered monocyclic or bicyclic or 7-to 14-membered bicyclic or tricyclic carbon ring, any of which may be saturated, partially unsaturated or aromatic, for example, phenyl, naphthyl, indanyl, or tetrahydronaphthyl tetralin, etc.

0054As used herein and in the claims, "alkyl halide" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having one to ten carbon atoms, wherein 1 to 3 hydrogen atoms have been replaced by a halogen atom such as Cl, F, Br, and I.

0055As used herein and in the claims, the term "heterocyclic" is intended to mean any stable 5- to 7- membered monocyclic or bicyclic or 7- to 10- membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of N, O and S and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure. The heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. Examples of such heterocycles include, but are not limited to pyridyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl, piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, or octahydroisoquinolinyl and the like.

0056As used herein and in the claims, the phrase "therapeutically effective amount" means that amount of novel backbone cyclized peptide analog or composition comprising same to administer to a host to achieve the desired results for the indications described herein, such as but not limited of inflammation, septic shock, cancer, endocrine disorders and gastrointestinal disorders.

0057The term, "substituted" as used herein and in the claims, means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.

0058When any variable (for example R, x, z, etc.) occurs more than one time in any constituent or any other Formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

Synthetic Approach

0059Peptide analogs are cyclized via bridging groups attached to the alpha nitrogens of amino acids that permit novel non-peptidic linkages. In general, the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. The innovation requires replacement of one or more of the amino acids in a peptide sequence by novel building units of the general Formula: <chemistry id="chem0013" num="0013"><img file="EP0804468B1_D0013.tif" /></chemistry> wherein R is the side chain of an amino acid, X is a spacer group and G is the functional end group by means of which cyclization will be effected. The side chain R is the side chain of any natural or synthetic amino acid that is selected to be incorporated into the peptide sequence of choice. X is a spacer group that is selected to provide a greater or lesser degree of flexibility in order to achieve the appropriate conformational constraints of the peptide analog. Such spacer groups include alkylene chains, substituted, branched and unsaturated alkylenes, arylenes, cycloalkylenes, unsaturated and substituted cycloakylenes. Furthermore, X and R can be combined to form a heterocyclic structure.

0060This synthetic approach utilizes alkylene chains containing from two to ten carbon atoms.

0061The terminal (ω) functional groups to be used for cyclization of the peptide analog include but are not limited to: <ol id="ol0005" compact="compact" ol-style=""><li>a. Amines, for reaction with electrophiles such as activated carboxyl groups, aldehydes and ketones (with or without subsequent reduction), and alkyl or substituted alkyl halides.</li><li>b. Alcohols, for reaction with electrophiles such as activated carboxyl groups.</li><li>c. Thiols, for the formation of disulfide bonds and reaction with electrophiles such as activated carboxyl groups, and alkyl or substituted alkyl halides.</li><li>d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.</li><li>e. Alkynes or Substituted Alkynes, for reaction with nucleophiles such as amines, thiols or carbanions; free radicals; electrophiles such as aldehydes and ketones, and alkyl or substituted alkyl halides; or organometallic complexes.</li><li>f. Carboxylic Acids and Esters, for reaction with nucleophiles (with or without prior activation), such as amines, alcohols, and thiols.</li><li>g. Alkyl or Substituted Alkyl Halides or Esters, for reaction with nucleophiles such as amines, alcohols, thiols, and carbanions (from active methylene groups such as acetoacetates or malonates); and formation of free radicals for subsequent reaction with alkenes or substituted alkenes, and alkynes or substituted alkynes.</li><li>h. Alkyl or Aryl Aldehydes and Ketones for reaction with nucleophiles such as amines (with or without subsequent reduction), carbanions (from active methylene groups such as acetoacetates or malonates), diols (for the formation of acetals and ketals).</li><li>i. Alkenes or Substituted Alkenes, for reaction with nucleophiles such as amines, thiols, carbanions, free radicals, or organometallic complexes.</li><li>j. Active Methylene Groups, such as malonate esters, acetoacetate esters, and others for reaction with electrophiles such as aldehydes and ketones, alkyl or substituted alkyl halides.</li></ol>

0062It will be appreciated that during synthesis of the peptide these reactive end groups, as well as any reactive side chains, must be protected by suitable protecting groups. Suitable protecting groups for amines are alkyloxy, substituted alkyloxy, and aryloxy carbonyls including, but not limited to, tert butyloxycarbonyl (Boc), Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) and Benzyloxycarbonyl (Z).

0063Carboxylic end groups for cyclizations may be protected as their alkyl or substituted alkyl esters or thio esters or aryl or substituted aryl esters or thio esters. Examples include but are not limited to tertiary butyl ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethyl ester and 9-methyl fluorenyl.

0064Thiol groups for cyclizations may be protected as their alkyl or substituted alkyl thio ethers or disulfides or aryl or substituted aryl thio ethers or disulfides. Examples of such groups include but are not limited to tertiary butyl, trityl(triphenylmethyl), benzyl, 2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl, carboxy-methyl, 2-thio-4-nitropyridyl.

0065It will further be appreciated by the artisan that the various reactive moieties will be protected by different protecting groups to allow their selective removal. Thus, a particular amino acid will be coupled to its neighbor in the peptide sequence when the N^α is protected by, for instance, protecting group A. If an amine is to be used as an end group for cyclization in the reaction scheme the N^ω will be protected by protecting group B, or an ε amino group of any lysine in the sequence will be protected by protecting group C, and so on.

0066The coupling of the amino acids to one another is performed as a series of reactions as is known in the art of peptide synthesis. Building units, namely the N^α-ω functionalized amino acid derivatives are incorporated into the peptide sequence to replace one or more of the amino acids. If only one such N^α-ω functionalized amino acid derivative is selected, it will be cyclized to a side chain of another amino acid in the sequence. For instance: (a) an N^α-(ω-amino alkylene) amino acid can be linked to the carboxyl group of an aspartic or glutamic acid residue; (b) an N^α-(ω-carboxylic alkylene) amino acid can be linked to the ε- amino group of a lysine residue; (c) an N^α-(ω-thio alkylene) amino acid can be linked-to the thiol group of a cysteine residue; and so on. A more preferred embodiment of the invention incorporates two such N^α-ω-functionalized amino acid derivatives which may be linked to one another to form N-backbone to N-backbone cyclic peptide analogs. Three or more such building units can be incorporated into a peptide sequence to create bi-cyclic peptide analogs as will be elaborated below. Thus, peptide analogs can be constructed with two or more cyclizations, including N-backbone to N-backbone, as well as backbone to side-chain or any other peptide cyclization.

0067As stated above, the procedures utilized to construct above peptide analogs from novel building units generally rely on the known principles of peptide synthesis. However, it will be appreciated that accommodation of the procedures to the bulkier building units of the present invention may be required. Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl), benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate (BOP), 1-oxo-1-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.

0068It has now been found that coupling of the bulky building units of the present invention may require the use of additional coupling reagents including, but not limited to: coupling reagents such as PyBOP^® (Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP^® (Bromo-tris-pyrrolidino-phosphonium hexafluoro-phosphate), HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate).

0069Novel coupling chemistries may be used, such as pre-formed urethane-protected N-carboxy anhydrides (UNCA's) and pre-formed acyl fluorides. Said coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone) or mixtures of the above.

0070Preparation of backbone to side chain cyclized peptide analogs is exemplified in Scheme I below. In this schematic example, the bridging group consists of alkylene spacers and an amide bond formed between an acidic amino acid side chain (e.g. aspartic or glutamic acid) and an ω-functionalized amino acid having a terminal amine.

(Scheme I)

Preparation of peptides with Backbone to Side Chain cyclization.

0071One procedure for preparing the backbone cyclic peptides involves the stepwise synthesis of the linear peptides on a solid support and the backbone cyclization of the peptide either on the solid support or after removal from the support. The C-terminal amino acid is bound covalently to an insoluble polymeric support by a carboxylic acid ester or other linkages such as amides. An example of such support is a polystyrene-co-divinyl benzene resin. The polymeric supports used are those compatible with such chemistries as Fmoc and Boc and include for example PAM resin, HMP resin and chloromethylated resin. The resin bound amino acid is deprotected for example with TFA to give (1) below and to it is coupled the second amino acid, protected on the N^α for example by Fmoc, using a coupling reagent like BOP. The second amino acid is deprotected to give (3) using for example piperidine 20% in DMF. The subsequent protected amino acids can then be coupled and deprotected at ambient temperature. After several cycles of coupling and deprotection that gives peptide (4), an amino acid having for example carboxy side chain is coupled to the desired peptide. One such amino acid is Fmoc-aspartic acid t-butyl ester. After deprotection of the N^α Fmoc protecting group that gives peptide (5), the peptide is again elongated by methods well known in the art to give (6). After deprotection a building unit for backbone cyclization (the preparation of which is described in Schemes III-VIII) is coupled to the peptide resin using for example the coupling reagent BOP to give (7). One such building unit is for example Fmoc-N^α(ω-Boc-amino alkylene)amino acid. After deprotection the peptide can then be elongated, to the desired length using methods well known in the art to give (8). The coupling of the protected amino acid subsequent to the building unit is performed by such coupling agents exemplified by PyBrOP^® to ensure high yield.

0072After the linear, resin bound peptide, e.g. (8), has been prepared the ω-alkylene protecting groups for example Boc and t-Bu are removed by mild acid such as TFA to give (9). The resin bound peptide is then divided into several parts. One part is subjected to on-resin cyclization using for example TBTU as cyclization agent in DMF to ensure high yield of cyclization, to give the N-backbone to side chain cyclic peptide resin (10). After cyclization on the resin the terminal amino protecting group is removed by agents such as piperidine and the backbone to side chain cyclic peptide (11) is obtained after treatment with strong acid such as HF. Alternatively, prior to the removal of the backbone cyclic peptide from the resin, the terminal amino group is blocked by acylation with agents such as acetic anhydride, benzoic anhydride or any other acid such as adamantyl carboxylic acid activated by coupling agents such as BOP.

0073The other part of the peptide-resin (9) undergoes protecting of the side chains used for cyclization, for example the ω-amino and carboxy groups. This is done by reacting the ω-amino group with for example Ac₂O and DMAP in DMF and activating the free ω-carboxy group by for example DIC and HOBT to give the active ester which is then reacted with for example Dh₃NH₂ to give the linear analog (13) of the cyclic peptide (10). Removal of the peptide from the-resin and subsequent removal of the side chains protecting groups by strong acid such as HF to gives (14) which is the linear analog of the backbone to side chain cyclic peptide (11).

0074The linear analogs are used as reference compounds for the biological activity of their corresponding cyclic compounds.

[Reaction Scheme I follows at this point]

0075<chemistry id="chem0014" num="0014"><img file="EP0804468B1_D0014.tif" /></chemistry><chemistry id="chem0015" num="0015"><img file="EP0804468B1_D0015.tif" /></chemistry><chemistry id="chem0016" num="0016"><img file="EP0804468B1_D0016.tif" /></chemistry>

0076The selection of N^α and side chain protecting groups is, in part, dictated by the cyclization reaction which is done on the peptide-resin and by the procedure of removal of the peptide from the resin. The N^α protecting groups are chosen in such a manner that their removal will not effect the removal of the protecting groups of the N^α(ω-aminoalkylene) protecting groups. In addition, the removal of the N^α(ω-aminoalkylene)protecting groups or any other protecting groups on ω-functional groups prior to the cyclization, will not effect the other side chain protection and/or the removal of the peptide from the resin. The selection of the side chain protecting groups other than those used for cyclization is chosen in such a manner that they can be removed subsequently with the removal of the peptide from the resin. Protecting groups ordinarily employed include those which are well known in the art, for example, urethane protecting substituents such as Fmoc, Boc, Alloc, Z and the like.

0077It is preferred to utilize Fmoc for protecting the α-amino group of the amino acid undergoing the coupling reaction at the carboxyl end of said amino acid. The Fmoc protecting group is readily removed following such coupling reaction and prior to the subsequent step by the mild action of base such as piperidine in DMF. It is preferred to utilize Boc for protecting ω-amino group of the N^α(ω-aminoalkylene) group and t-Bu for protecting the carboxy group of the amino acids undergoing the reaction of backbone cyclization. The Boc and t-Bu protecting groups are readily removed simultaneously prior to the cyclization.

(Scheme II)

Preparation of peptides with Backbone to Backbone cyclization.

0078Preparation of N-backbone to N-backbone cyclized peptide analogs is exemplified in scheme II. In this schematic example, the building group consists of alkylene spacers and two amide bonds.

0079A building unit for backbone cyclization (the preparation of which described in Schemes III-VIII) is coupled to a peptide resin, for example peptide-resin (4), using for example the coupling reagent BOP to give (16). One such building unit is for example Fmoc-Nα(ω-Boc-amino alkylene)amino acid. The side chain Boc protecting group is removed by mild acid such as TFA in DCM and an N-Boc protected ω-amino acid, or any other Boc protected amino acid, is coupled to the side chain amino group using coupling agent such as BOP to give peptide-resin (17).

0080After deprotection of the N^α Fmoc protecting group by mild base such as piperidine in DMF, the peptide can then be elongated, if required, to the desired length using methods well known in the art to give (18). Alternatively, the deprotection of the N^α Fmoc and subsequent elongation of the peptide can be done before deprotection of the' side chain Boc protecting group. The elongation of the N-alkylene side chain allow control of the ring size. The coupling of the protected amino acid subsequent to the building unit is performed by such coupling agents exemplified by PyBrOP^® to ensure high yield.

0081After deprotection pf the terminal N^α Fmoc group, a second building unit, for example Fmoc-N^α(ω-t-Bu-carboxyalkylene)amino acid is coupled to the peptide-resin to give (19). After deprotection of the N^α Fmoc protecting group, the peptide can then be elongated, if required, to the desired length using methods well known in the art to give (20). The coupling of the protected amino acid subsequent to the building unit is performed by such coupling agents exemplified by PyBrOP^® to ensure high yield. After the linear, resin bound peptide, e.g. (20), has been prepared the ω-alkylene-protecting groups for example Boc and t-Bu are removed by mild acid such as TFA to give (21). The resin peptide is then divided into several parts. One part is subjected to on-resin cyclization using for example TBTU as cyclization agent in DMF to ensure high yield of cyclization, to give the N-backbone to N-backbone cyclic peptide resin (22). After cyclization on the resin the terminal amino protecting group is removed by agents such as piperidine and the backbone to backbone cyclic peptide (23) is obtained after treatment with strong acid such as HF. Alternatively, prior to the removal of the backbone cyclic peptide from the resin, the terminal amino group of (22) is blocked, after deprotection, by acylation with agents such as acetic anhydride, benzoic anhydride or any other acid such as adamantyl carboxylic acid activated by coupling agents such as BOP to give the N-terminal blocked backbone to backbone cyclic peptide (24).

0082The other part of the peptide-resin (21) undergoes protecting of the side chains used for cyclization, for example the ω-amino and carboxy groups. This is done by reacting the ω- amino group with for example Ac₂O and DMAP in DMF and activating the free ω-carboxy group by for example DIC and HOBT to give the active ester which is then reacted with for example MeNH₂. Removal of the peptide from the resin and subsequent removal of the side chains protecting groups by strong acid such as HF to gives (26) which is the linear analog of the backbone to backbone cyclic peptide (23). The linear analogs are used as reference compounds for the biological activity of their corresponding cyclic compounds.

[Reaction Scheme II follows at this point]

0083<chemistry id="chem0017" num="0017"><img file="EP0804468B1_D0017.tif" /></chemistry><chemistry id="chem0018" num="0018"><img file="EP0804468B1_D0018.tif" /></chemistry><chemistry id="chem0019" num="0019"><img file="EP0804468B1_D0019.tif" /></chemistry><chemistry id="chem0020" num="0020"><img file="EP0804468B1_D0020.tif" /></chemistry>

Novel Synthesis of Building Units

0084The novel synthesis according to the present invention providing N(ω-(functionalized) alkylene) amino acids used to generate backbone cyclic peptides is depicted in schemes III - VIII. In this approach we have implemented the following changes in order to devise a practical, general synthesis: <ol id="ol0006" compact="compact" ol-style=""><li>1. The nucleophile is a secondary nitrogen, which is a better nucleophile than the primary nitrogen previously used. This also prevents the possibility of double alkylation.</li><li>2. The leaving group was changed to trifluoromethanesulfonyl (triflate), which has a much lower tendency to eliminate than a halogen, thus making it possible to implement the synthesis with amino acids other than glycine. Furthermore, the triflate leaving group prevents racemization during the alkylation reaction.</li><li>3. The carboxylate is esterified prior to the substitution reaction, to facilitate the substitution by removing the negative charge next to the electrophilic carbon.</li></ol>

(Scheme III)

Preparation of N^α, N^ωprotected ω-amino alkylene amino acids building units.

0085One preferred procedure for the preparation of protected N^α(ω- amino alkylene) amino acids involves the N^α alkylation of suitably protected diamino alkanes. One preferred N^α, N^ω di- protected diamino alkane is for example N^α-Benzyl, N^ω-Boc diamino alkane (27). This starting material contains one protecting group such as Boc which is necessary for the final product, and a temporary protecting group such as Bzl to minimize unwanted side reactions during the preparation of the titled compound. One preferred procedure for the preparation of the starting material (27) involves reductive alkylation of N-Boc diamino alkane with aldehydes such as benzaldehyde. The temporary protection of the N^α amino group, which is alkylated in the reaction by such protecting groups as Bzl, minimizes the dialkylation side reaction and allows removal by such conditions that do not remove the N^ω-protecting group.

0086The N^α,N^ω di-protected diamino alkane is reacted with for example chiral α-hydroxy α-substituted acid esters where the hydroxyl moiety is converted to a leaving group for example Triflate.

0087The use of Triflate as the leaving group was found to be superior to other leaving groups such as halogens, Tosyl, Mesyl, etc., because it prevents the β-elimination reaction encountered with the other leaving groups. The use of Triflate as the leaving group also ensures high optical purity of the product (28). The temporary N^α protecting group, such as Bzl, and the carboxyl protecting group, such as methyl ester, are removed by mild conditions, such as catalytic hydrogenation and hydrolysis, that do not remove the N^ω protecting group such as Boc to give the N^ω protected amino acid (29). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N^α(N^ω protected amino alkylene) amino acid (30).

0088The choice of the N^α and the N^ω protecting groups is dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. Combinations of N^α and N^ω protecting groups are for example: N^α-Fmoc, N^ω-Boc ; N^α-Fmoc , N^ω-Alloc; N^α-Boc, N^ω-Alloc. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution.

(Scheme IV)

Preparation of N^α, N^ωprotected ω-amino alkylene glycine building units.

0089One preferred procedure for the preparation of protected N^α(ω- amino alkylene) glycines involves the reaction of the N^α, N^ω di-protected di amino alkane (27) with commercially available α-activated carboxylic acid esters, for example benzylbromo acetate. Since the titled compound is achiral, the use of leaving groups such as Trf, Tos or Mes is not necessary. The use of the same temporary protecting groups for the N^α and the carboxy groups, for example the Bzl protecting group, ensures the prevention of the undesired dialkylation side reaction and allows concomitant removal of the temporary protecting groups thus giving high yield of the N^ω protected amino acid (32). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N^α (N^ω protected amino alkylene) glycines (33).

0090The choice of the N^α and the N^ω protecting groups is dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. Combinations of N^α and N^ω protecting groups are for example : N^α Fmoc, N^ω Boc ; N^α Fmoc, N^ω Alloc; N^α Boc, N^ω Alloc. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution. <chemistry id="chem0021" num="0021"><img file="EP0804468B1_D0021.tif" /></chemistry><chemistry id="chem0022" num="0022"><img file="EP0804468B1_D0022.tif" /></chemistry>

(Scheme V)

Preparation of N^α, ω-carboxy protected ω-carboxy alkylene amino acids.

0091One preferred procedure of the present invention for the preparation of protected N^α(ω- carboxy alkylene) amino acids involves the N^α-alkylation of suitably N^α, ω-carboxy deprotected amino acids. One preferred deprotected amino acid is N^α-Benzyl ω-amino acids t-butyl esters (34). This starting material contains one protecting group such as t-Bu ester which is necessary for the final product, and a temporary protecting group such as N^α Bzl to minimize side reactions during the preparation of the titled compound. One preferred procedure for the preparation of the starting material (34) involves reductive alkylation of ω-amino acids t-butyl esters with aldehydes such as benzaldehyde. The temporary protection of the amino group which is used as nucleophile in the proceeding alkylation reaction by such protecting groups as Bzl minimizes the dialkylation side reaction.

0092The N^α, ω-carboxy deprotected amino acids (34) are reacted with, for example, chiral α-hydroxy α-substituted acid esters where the hydroxyl moiety is converted to a leaving group, for example, Triflate. The use of Triflate as the leaving group was found to be superior to other leaving groups such as halogens, Tosyl, Mesyl ; etc., because it prevents the β-elimination reaction encountered with the other leaving groups. The use of Triflate as the leaving group also ensures high optical purity of the product, for example (36). The temporary N^α protecting group, such as Bzl, and the α-carboxyl protecting group, such as benzyl ester, are concomitantly removed by mild condition, such as catalytic hydrogenation, that to not remove the ω-carboxy protecting group such as t-Bu to give the N^α (protected ω-carboxy alkylene) amino acid (36). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N^α(ω protected carboxy alkylene) amino acid (37).

0093The choice of the N^α and the ω-carboxy protecting groups is dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. A combination of N^α and ω-carboxy protecting groups are for example: N^α-Fmoc, ω-carboxy t-Bu; N^α-Fmoc, ω-carboxy Alloc; N^α-Boc, ω-carboxy Alloc. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution.

(Scheme VI)

Preparation of N^α, ω-carboxy protected ω-carboxy alkylene glycine building units.

0094One preferred procedure of the present invention for the preparation of protected N^α(ω-carboxy alkylene)glycines involves the N^α-alkylation of suitably N^α, ω-carboxy deprotected amino acids(34) with commercially available α-activated carboxylic acid esters for example, benzyl bromo acetate. Since the titled compound is achiral, the use of leaving groups such as Trf, Tos or Mes is not necessary.

0095The use of the same temporary protecting groups for the N^α and the α-carboxy groups, for example the Bzl protecting group, ensures the prevention of the undesired dialkylation side reaction and allows concomitant removal of the temporary protecting groups thus giving high yield of the N^α(protected ω-carboxy alkylene) glycines (39). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N^α(ω protected carboxy alkylene) glycines (40).

0096The choice of the N^α and the ω-carboxy protecting groups is dictated by the use of the building units in peptide synthesis.. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. A combination of N^α and ω-carboxy protecting groups are, for example: N^α Fmoc, ω-carboxy t-Bu; N^α Fmoc, ω-carboxy Alloc; N^α Boc, ω-carboxy Alloc. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution. <chemistry id="chem0023" num="0023"><img file="EP0804468B1_D0023.tif" /></chemistry>

(Scheme VII)

Preparation of N^α S^ω protected ω-thio alkylene amino acid building units.

0097One preferred procedure of the present invention for the preparation of N^α, S^ω-deprotected N^α(ω-thio alkylene) amino acids involves the N^α-alkylation of suitably S^ω protected ω-thio amino alkanes. Suitable S^ω protecting groups are, for example, Bzl, t-Bu, Trt. One preferred S^ω-protected ω-thio amino alkanes is for example ω-(S-Benzyl) amino alkanes (41). One preferred procedure for the preparation of the starting material (41) involves the use of salts of S-protected thiols as nucleophiles for a nucleophilic substitution reaction on suitably N^α-protected ω-activated amino alkanes. Removal of the amino protection gives the starting material (41).

0098The S-protected ω-thio amino alkanes (41) are reacted with for example chiral α-hydroxy α-substituted acid esters where the hydroxyl moiety is converted to a leaving group for example Triflate. The use of Triflate as the leaving group was found to be superior to other leaving groups such as halogens, Tosyl, Mesyl etc. because it prevents the β-elimination reaction encountered with the other leaving groups. The use of Triflate as the leaving group also ensures high optical purity of the product for example (42). The temporary α-carboxyl protecting group, such as methyl ester, is removed by mild condition, such as hydrolysis with base, that to not remove the ω-thio protecting group such as S-Bzl to give the N^α (S-protected ω-thio alkylene) amino acid (43). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N,S protected N^α (ω-thio alkylene) amino acid (44).

0099The choice of the N^α and the ω-thio protecting groups is dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. A combination of N^α and ω-thio protecting groups are for example: N^α Fmoc, S^ω t-Bu; N^α Fmoc, S^ω Bzl; N^α Fmoc, S^ω Trt; N^α Boc, S^ω Bzl. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution.

(Scheme VIII)

Preparation of N^α, S^ω protected ω-thio alkylene glycine building units.

0100One preferred procedure of the present invention for the preparation of N^α, S^ω-deprotected N^α(ω-thio alkylene) amino acids involves the N^α-alkylation of suitably S^ω protected ω-thio amino alkanes (41) with commercially available α-activated carboxylic acid esters for example ethyl bromo acetate. Since the titled compound is achiral, the use of leaving groups such as Trf, Tos or Mes is not necessary.

0101Suitable protecting groups for the ω-thio groups are for example Bzl, t-Bu, Trt. One preferred S-protected ω-thio amino alkanes is for example ω-(S-Benzyl) amino alkanes (41). The N-alkylation reaction gives the ester (45). The temporary α-carboxyl protecting group, such as ethyl ester, is removed by mild conditions, such as hydrolysis with base, that to not remove the ω-thio protecting group such as S-Bzl to give the N^α (S-protected ω-thio alkylene) glycines (46). Introduction of the N^α protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N^α, S^ω-deprotected N^α (ω-thio alkylene) glycines (47).

0102The choice of the N^α and the ω-thio protecting groups is dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthogonal to the other side chains protecting groups in the peptide. A combination of N^α and ω-thio protecting groups are for example: N^α Fmoc, S^ω t-Bu; N^α Fmoc, S^ω Bzl; N^α Fmoc, S^ω Trt; N^α Boc, S^ω Bzl. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution. <chemistry id="chem0024" num="0024"><img file="EP0804468B1_D0024.tif" /></chemistry>

REFERENCE EXAMPLES OF PEPTIDES

0103Preparation of the novel backbone cyclized peptide analogs using the schematics outlined above will be illustrated by the following reference examples:

EXAMPLE 1

Ada-(D) Arg-Arg-cyclo (N

α

(1-(6-aminohexylene) Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH

STAGE 1

Boc-Arg(Tos)-O-resin ---&gt; Fmoc-Phe-Arg (Tos)-O-resin

0104Boc-L-Arg(Tos)-O-resin (0.256 g, 0.1 mmole, 0.39 meq of nitrogen/g) was placed in a shaker flask and swelled for two hours by the addition of DCM. The resin was then carried out through the procedure in Table 1 which includes two deprotections of the Boc protecting group with 55% TFA in DCM for a total of 22 minutes, washing, neutralization with 10% DIEA in NMP and washing (Table 1 steps 1-8). After positive ninhydrin test, as described in Kaiser <i>et al.</i>, <u style="single">Anal Biochem.,</u> 34:595, 1970 and is incorporated herein by reference in its entirety, coupling (Table 1 steps 9-10) was achieved in NMP by the addition of Fmoc-L-Phe (0.232 g, 0.6 mmole) and after 5 minutes of shaking, solid BOP reagent (0.265 g, 0.6 mmole) was added to the flask. <tables id="tabl0005" num="0005"><table frame="none"><title><u style="single">TABLE 1 PROCEDURE FOR 0.1 mMOLE SCALE</u></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="37mm" /><thead><row><entry valign="top">STEP NO.</entry><entry valign="top">SOLVENT/ REAGENT</entry><entry valign="top">VOLUME (ML)</entry><entry valign="top">TIME (MIN)</entry><entry valign="top">REPEAT (XS)</entry><entry valign="top">COMMENT</entry></row></thead><tbody><row rowsep="0"><entry>1</entry><entry>DCM</entry><entry>5</entry><entry>120</entry><entry>1</entry><entry>Swells resin</entry></row><row rowsep="0"><entry>2</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>3</entry><entry>TFA/DCM 55%</entry><entry>5</entry><entry>2</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>4</entry><entry>TFA/DCM 55%</entry><entry>5</entry><entry>20</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>5</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>6</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry>check for positive nin.</entry></row><row rowsep="0"><entry>7</entry><entry>DIEA/NMP</entry><entry>5</entry><entry>5</entry><entry>2</entry><entry>Neutralization</entry></row><row rowsep="0"><entry>8</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>5</entry><entry /></row><row rowsep="0"><entry>9</entry><entry>Fmoc-AA in NMP</entry><entry /><entry>5</entry><entry>5</entry><entry>Coupling add BOP 6 eq. add DIEA 120 600 1 12 eq. Check pH, adjust to pH 8 with DIEA</entry></row><row rowsep="0"><entry>10</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>5</entry><entry>check for negative nin.</entry></row><row rowsep="0"><entry>11</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>12</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>13</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>check for positive nin.</entry></row></tbody></tgroup></table></tables>

0105After shaking for 10 minutes, the mixture was adjusted to pH 8 (measured with wetted pH stick) by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 10 hours at ambient temperature. The resin was then washed and subjected to ninhydrin test. After negative ninhydrin test the resin was used for the next coupling.

STAGE 2

Fmoc-Phe-Ara(Tos)-O-resin ----&gt; Fmoc-N^α(6-Boc amino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-o-resin

0106The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to two deprotections of the Fmoc protecting group by 20% Pip in NMP (Table 1 steps 11-13). After washing and ninhydrin test (Method J, below), coupling of Fmoc-D-Phe was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1 steps 11-13). After washing and ninhydrin test, coupling of Fmoc-D-Asp(t-Bu) was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Asp(t-Bu) (0.247 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1 steps 11-13). After washing and ninhydrin test, coupling of Fmoc-L-Phe was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-L-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1 steps 11-13). After washing and ninhydrin test, coupling of Fmoc-L-Hyp(OBzl) was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-L-Hyp(OBzl) (0.266 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1 steps 11-13). The resin was washed and subjected to picric acid test (Method K). Coupling of Fmoc-N°(6-Boc amino hexylene) glycine was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-N^α (6-Boc amino hexylene)glycine (0.3 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was then washed and subjected to the picric acid test (Method K below). After negative test the resin was used for the next coupling.

STAGE 3

Fmoc-N

α

(6-Boc amino hexylene) Gly-Hyp (OBzl)-Phe-D-Asp (t-Bu)-D-Phe-Phe-Arg (Tos)-O-resin ----&gt; Fmoc-D-Arg (Tos)-Arg (Tos)-N

α

(6-Boc amino hexylene) Gly-Hyp (OBzl)-Phe-D-Asp (t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

0107The Fmoc-N^α(6-Boc amino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg (Tos) -O-resin (Stage 2) was subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2 steps 1-2). After washing, the picric acid test (Method K) was performed. If the test did not show 98±2%, deprotection of the peptide resin was subjected again to 3 deprotection steps (Table 2 steps 1-2), washing and picric acid test (Method K). Coupling of Fmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyB0P reagent (0.28 g, 0.6 mmole) was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 (measured with wetted pH stick) by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature. The resin was then washed and subjected to a second coupling by the same procedure for 20 hours. After washing the resin was subjected to picric acid test (Method K) (Table 2 steps 3-6). If the test did not show 98±2% coupling the peptide resin was subjected again to a third coupling for 2 hours at 50 °C (Table 2 step 7). The resin was washed subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2 steps 1-2). After washing picric acid test (Method K) was performed. <tables id="tabl0006" num="0006"><table frame="none"><title><u style="single">TABLE 2 PROCEDURE FOR 0.1 mMOLE SCALE</u></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="38mm" /><thead><row><entry valign="top">STEP NO.</entry><entry valign="top">SOLVENT/ REAGENT</entry><entry valign="top">VOLUME (ML)</entry><entry valign="top">TIME (MIN)</entry><entry valign="top">REPEAT (XS)</entry><entry valign="top">COMMENT</entry></row></thead><tbody><row rowsep="0"><entry>1</entry><entry>Piperidine/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>3</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>2</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Picric acid test.</entry></row><row rowsep="0"><entry>3</entry><entry>Fmoc-AA in NMP add PyBroP add DIEA</entry><entry>5</entry><entry>5 150</entry><entry>1</entry><entry>Coupling 6 eq. 12 eq. Check pH, adjust to pH 8 with DIEA.</entry></row><row rowsep="0"><entry>4</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>3</entry><entry>check for negative nin.</entry></row><row rowsep="0"><entry>5</entry><entry>Fmoc-AA in NMP add PyBroP add DIEA</entry><entry>5</entry><entry>5 20 hr.</entry><entry>1</entry><entry>Coupling 6 eq. 12 eq. Check pH, adjust to pH 8 with DIEA.</entry></row><row rowsep="0"><entry>6</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry>Picric acid test. If less than 98±2% coupling repeat Steps 4-5</entry></row><row rowsep="0"><entry>7</entry><entry>Fmoc-AA in NMP add PyB0P add DIEA</entry><entry>5</entry><entry>5 120</entry><entry>1</entry><entry>Coupling at 50°C 6 eq. 12 eq. Check pH, adjust to pH 8 with DIEA.</entry></row><row rowsep="0"><entry>8</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry /></row></tbody></tgroup></table></tables>

0108If the test did not show 98±2% deprotection, the peptide resin was subjected again to 3 deprotection steps (Table 2 steps 1-2), washing and the picric acid test (Method K). Coupling of Fmoc-D-Arg(Tos) was achieved in NMP as described in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Arg(Tos) (0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed 6 times with NMP (Table 1 step 15) and used in the next stages.

STAGE 4

Fmoc-D-Arg(Tos)-Arg(Tos)-N^α(6-Boc amino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Ara(Tos)-O-resin ----&gt; Ada-D-Arg-Arg-cyclo(N^α(1-(6-amidohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH

0109The Fmoc-D-Arg(Tos)-Arg(Tos)-N^α(6-Boc amino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 3) was subjected to deprotection of the Boc and t-Bu protecting groups and on resin cyclization according to Table 3. The peptide resin was washed with DCM and deprotected as described in Stage 1 by 55% TFA in DCM. After washing and neutralization by 10% DIEA in NMP and washing 6 times with DCM the peptide resin was dried <i>in vacuo</i> for 24 hours. The dry peptide resin weight, 0.4 g, it was divided into two parts. 0.2 g of the peptide resin was swollen 2 hours in 5 mL NMP and cyclized as follows: Solid TBTU reagent (0.19 g, 6 mmole) was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature. The resin was then washed and subjected to a second coupling by the same procedure for 20 hours. After washing the resin was subjected to picric acid test (Method K) (Table 3 steps 8-11). If the test did not show 98±2% cyclization the peptide resin was subjected again to a third cyclization for 2 hours at 50°C (Table 2 step 12). The resin was washed, subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2 steps 1-2). After washing and ninhydrin test, the N-terminal amino group was blocked by Ada. Adamantane acetic acid (0.108 g, 6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole) were added and the flask shaken for 2 hours. After washing 6 times with NMP (Table 2 step 13), ninhydrin test (Method J) was performed. If the test was positive or slightly positive the protecting with adamantane acetic acid was repeated. If the ninhydrin test was negative, the peptide resin was washed 6 times with NMP and 6 times with DCM. The resin was dried under vacuum for 24 hours. The dried resin was subjected to HF as follows: to the dry peptide resin (0.2 g) in the HF reaction flask, anisole (2 mL) was added and the peptide treated with 20 mL liquid HF at -20 °C for 2 hours. After the evaporation of the HF under vacuum, the anisole was washed with ether (20 mL, 5 times) and the solid residue dried in vacuum. The peptide was extracted from the resin with TFA (10 mL, 3 times) and the TFA evaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH and lyophilized. This process was repeated 3 times. The crude peptide was purified by semiprep HPLC (Method H). The final product was obtained as white powder by lyophilization from dioxane, which gave 42 mg (56%) of the title compound. HPLC (Method G) RT 32.15 minutes, 95% TOF MS: 1351.4 (M⁺) AAA in agreement with the title compound

[Table 3 follows at this point.]

0110<tables id="tabl0007" num="0007"><table frame="none"><title><u style="single">TABLE 3 PROCEDURE FOR 0.05 mMOLE SCALE</u></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="37mm" /><thead><row><entry valign="top">STEP NO.</entry><entry valign="top">SOLVENT/ REAGENT</entry><entry valign="top">VOLUME (ML)</entry><entry valign="top">TIME (MIN)</entry><entry valign="top">REPEAT (XS)</entry><entry valign="top">COMMENT</entry></row></thead><tbody><row rowsep="0"><entry>1</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>2</entry><entry>TFA/DCM 55%</entry><entry>5</entry><entry>2</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>3</entry><entry>TFA/DCM 55%</entry><entry>5</entry><entry>20</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>4</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>5</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry /></row><row rowsep="0"><entry>6</entry><entry>DIEA/NMP 10%</entry><entry>5</entry><entry>5</entry><entry>2</entry><entry>Neutralization</entry></row><row rowsep="0"><entry>7</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>5</entry><entry /></row><row rowsep="0"><entry>8</entry><entry>TBTU/NMP/DIEA</entry><entry>5</entry><entry>150</entry><entry>3</entry><entry>Cyclization</entry></row><row rowsep="0"><entry>9</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry>Picric acid test. If less than 98±2% coupling perform Steps 10-12. If above 98±2%, go to step 13.</entry></row><row rowsep="0"><entry>10</entry><entry>TBTU/NMP/DIEA</entry><entry>5</entry><entry>20hr</entry><entry>3</entry><entry>Cyclization. Check pH, adjust to pH 8 with DIEA.</entry></row><row rowsep="0"><entry>11</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry>Picric acid test. If less than 98±2% coupling perform Steps 12. If above 98±2%, go to step 13</entry></row><row rowsep="0"><entry>12</entry><entry>TBTU/NMP/DIEA</entry><entry>5</entry><entry>120</entry><entry>3</entry><entry>Cyclization, 50 C Check pH, adjust to pH 8 with DIEA.</entry></row><row rowsep="0"><entry>13</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry /></row><row rowsep="0"><entry>14</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>15</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>16</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Check for positive nin.</entry></row><row rowsep="0"><entry>17</entry><entry>AdacOH/BOP /NMP</entry><entry>5</entry><entry>2</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>18</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Check for negative nin.</entry></row><row rowsep="0"><entry>19</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry /></row></tbody></tgroup></table></tables>

EXAMPLE 2

NON-CYCLIZED PEPTIDE (Control for biological assays)

Ada-D-Arg-Ara-N^α(6-acetamidohexylene)Gly-Hyp-Phe-D-Asp(NH-Me)-D-Phe-Phe-Arg-OH

0111The Fmoc-D-Arg(Tos)-Arg(Tos)-N^α(6-amino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp-D-Phe-Phe-Arg(Tos)-O-resin (0.2 g) which was prepared in Example 1 Stage 4 was subjected to acetylation of the 6-amino side chain of N^α (6-acetamidohexylene)Gly and to methyl amidation of the carboxylic group of D-Asp as described in Table 4. The peptide resin was swollen in 5 mL NMP for 2 hours and AcO (0.113 mL, 12 mmole) and PP (17 mg) were added. After 30 minutes, the resin was washed with NMP 6 times and subjected to ninhydrin test. If the test was positive or slightly positive the acetylation reaction was repeated. If the ninhydrin test was negative, the carboxy group of D-Asp was activated by the addition of HOBT (0.040 g, 0.3 mmole) and DIC (0.047 mL, 0.3 mmole) to the peptide resin in NMP. The mixture was shaken for half an hour and a solution of 30 % methylamine in EtOH (0.2mL) was added. After one hour, the resin was washed 6 times with NMP and the terminal Fmoc group removed by 20% Pip in NMP (Table 4 steps 7-9). After washing with NMP the N-terminal amino group was blocked by Ada as described in Example 1 Stage 4 and the resin was washed with NMP and DCM (Table 4 steps 10-12) and the resin dried in <i>vacuo.</i> The peptide was deprotected and cleaved from the resin by HF. To the dry peptide resin (0.2 g) in the HF reaction flask, anisole (2 mL) was added and the peptide treated with 20 mL liquid HF at -20 °C for 2 hours. After the evaporation of the HF under vacuum, the anisole was washed with ether (20 mL 5 times) and the solid residue dried in <i>vacuo.</i> The peptide was extracted from the resin with TFA (10 mL, 3 times) and the TFA evaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH and lyophilized. This process was repeated 3 times. The crude peptide was purified by semiprep HPLC (Method H). The final product was obtained as white powder by lyophilization from dioxane, which gave 48 mg (64%) of the title compound. HPLC (Method G) RT 27.70 minutes, 93% TOF MS: 1424.6 (M⁺) AAA in agreement with the title compound <tables id="tabl0008" num="0008"><table frame="none"><title><u style="single">TABLE 4 PROCEDURE FOR 0.05 mMOLE SCALE</u></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="37mm" /><thead><row><entry valign="top">STEP NO.</entry><entry valign="top">SOLVENT/ REAGENT</entry><entry valign="top">VOLUME (ML)</entry><entry valign="top">TIME (MIN)</entry><entry valign="top">REPEAT (XS)</entry><entry valign="top">COMMENT</entry></row></thead><tbody><row rowsep="0"><entry>1</entry><entry>NMP</entry><entry>5</entry><entry>120</entry><entry>1</entry><entry>Swells resin</entry></row><row rowsep="0"><entry>2</entry><entry>AC₂O/PP/NMP</entry><entry>5</entry><entry>30</entry><entry>1</entry><entry>Protecting of side chain</entry></row><row rowsep="0"><entry>3</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Check for negative nin.</entry></row><row rowsep="0"><entry>4</entry><entry>DIC/HOBT/NMP</entry><entry>5</entry><entry>30</entry><entry>1</entry><entry>Activation of COOH side chain</entry></row><row rowsep="0"><entry>5</entry><entry>MeNH₂/EtOH/</entry><entry>5</entry><entry>60</entry><entry>1</entry><entry>Protecting of side chain</entry></row><row rowsep="0"><entry>6</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry /></row><row rowsep="0"><entry>7</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry>Deprotection</entry></row><row rowsep="0"><entry>8</entry><entry>Pip/NMP 20%</entry><entry>5</entry><entry>10</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>9</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Check for positive nin.</entry></row><row rowsep="0"><entry>10</entry><entry>AdacOH/BOP/ NMP</entry><entry>5</entry><entry>2</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>11</entry><entry>NMP</entry><entry>5</entry><entry>2</entry><entry>6</entry><entry>Check for negative nin.</entry></row><row rowsep="0"><entry>12</entry><entry>DCM</entry><entry>5</entry><entry>2</entry><entry>4</entry><entry /></row></tbody></tgroup></table></tables>

Reference EXAMPLE 3

H-D-Arg-Arg-cyclo (N^α (1- (4 -bropanoyl)) Gly-Hyp-Phe-N

α

(3 - amido-propylene) Gly)-Ser-D-Phe-Phe-Arg-OH

STAGE 1

Fmoc-Phe-Arg(Tos)-O-resin ----&gt; Fmoc-N

α

(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^α(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

0112Fmoc-Phe-Arg(Tos)-O-resin prepared from Boc-Arg(Tos)-O-Resin (0.3 g, 0.1 mmole) (Example 1, Stage 1) was subjected to two deprotection of the Fmoc protecting group by 20% piperidine in NMP (Table 1, steps 11-13). After washing and ninhydrin test (Method J), coupling of Fmoc-D-Phe was achieved as described in Stage 1 (Example 1) (Table 1 steps 9-10) using Fmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1, steps 11-13). After washing and ninhydrin test (Method J), coupling of Fmoc-Ser (BzL) was achieved as described in Stage 1 (Example 1) (Table 1 steps 9-10) using Fmoc-Ser (Bzl) (0.25 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc group deprotected as described above (Table 1, steps 11-13). After washing and picric acid test (Method K), coupling of Fmoc-N^α(3-Boc amino propylene)glycine was achieved as described in Table 1, steps 9-10 using Fmoc-N^α(3-Boc amino propylene) Gly (0.272 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test (Method K) was performed. If the test did not show 98±2% deprotection the peptide resin was subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and picric acid test (Method K). Coupling of Fmoc-L-Hyp(OBzl) was achieved in NMP by the addition of Fmoc-L-Hyp(OBzl) (0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyBr0P reagent (0.28 g, 0.6 mmole) was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature. The resin was then washed and subjected to a second coupling by the same procedure for 20 hours. After washing the resin was subjected to picric acid test (Method K) (Table 2, steps 3-5). If the test did not show 98±2% coupling the peptide resin was subjected again to a third coupling for 2 hours at 50 °C. (Table 2, step 7). The resin was washed subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test (Method K) was performed. If the picric acid test did not show 98±2% deprotection, the resin was subjected again to deprotections steps (Table 2, steps 1-2). Coupling of Fmoc-Phe was achieved in NMP by the addition of Fmoc-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and after picric acid test (Method K) the Fmoc group deprotected as described above (Table 2, steps 1-2). After washing picric acid test (Method K) was performed. If the test did not show 98±2t deprotection the peptide resin was subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and picric acid test (Method K). Coupling of N^α(3-t-Bu carboxy propylene)Gly was achieved as described in Table 1 steps 9-10 using N^α(3-t-Bu carboxy propylene)Gly (0.264 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was then washed and subjected to the picric acid test (Method K). After negative test the resin was used for the next coupling.

STAGE 2

Fmoc-N

α

(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^α(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin ----- &gt; Fmoc-D-Arg(Tos)-Arg(Tos)-N

α

(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^α(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg (Tos)-O-resin

0113Fmoc-N^α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^α(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to three deprotection of the Fmoc protecting group by 20% Piperidine in NMP (Table 2, steps 1-2). After washing picric acid (Method K) test was performed. If the test did not show 98±2% deprotection the peptide resin was subjected again to 3 deprotection steps (Table 6, steps 1-2), washing and picric acid test. Coupling of Fmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyBroP reagent (0.28 g, 0.6 mmole) was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature. The resin was then washed and subjected to a second coupling by the same procedure for 20 hours. After washing the resin was subjected to picric acid test (Method K) (Table 2, steps 3-6). If the test did not show 98±2% coupling the peptide resin was subjected again to a third coupling for 2 hours at 50°C. (Table 2, step 7). The resin was washed subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test (Method K) was performed. If the test did not show 98±2% deprotection the peptide resin was subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and picric acid test (Method K). Coupling of Fmoc-D-Arg(Tos) was achieved in NMP as described in Stage 1 (Table 1, steps 9-10) using Fmoc-D-Arg(Tos) (0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed 6 times with NMP (Table 1, step 15) and used in the next stages.

STAGE 3

Fmoc-D-Ara(Tos)-Arg(Tos) -N^α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^α(3-Boc amino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin ----- &gt; H-D-Arg-Arg-cyclo(N^α(4- propanoyl))Gly-Hyp-Phe-N<i>^α</i>(3 -amido-propyl)Gly)-Ser-D-Phe-Phe-Arg-OH

0114Fmoc-D-Arg(Tos)-Arg(Tos)-N^α(4-t-Bu-propanoyl)Gly-Hyp(OBzl)- Phe-N^α(3-Boc amino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 2) was subjected to deprotection of the Boc and t-Bu protecting groups and on resin cyclization according to Table 5. The peptide resin was washed with DCM and deprotected as described in Stage 1 by 55% TFA in DCM. After washing and neutralization by 10% DIEA in NMP and washing 6 times with and NMP (Table 5, steps 1-5) the peptide was cyclized as follow: solid TBTU reagent (0.19 g, 6 mmole) was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature. The resin was then washed and subjected to a second coupling by the same procedure for 20 hours. After washing the resin was subjected to picric acid test (Method K) (Table 3, steps 8-11). If the test did not show 98±2% cyclization the peptide resin was subjected again to a third cyclization for 2 hours at 50°C. (Table 2, step 12). The resin was washed, subjected to three deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 5, steps 14-15). After washing 6 times with NMP and 4 times with DCM, the resin was dried <i>in vacuo</i> for 24 hours. The dried resin was subjected to HF as follows: to the dry peptide resin (0.4 g) in the HF reaction flask, anisole (2 mL) was added and the peptide treated with 20 mL liquid HF at -20°C for 2 hours. After the evaporation of the HF under vacuum, the anisole was washed with ether (20 mL, 5 times) and the solid residue dried in <i>vacuo.</i> The peptide was extracted from the resin with TFA (10 mL 3 times) and the TFA evaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH and lyophilized. This process was repeated 3 times. The crude peptide was purified by semipreparative HPLC (Method H). The final product was obtained as white powder by lyophilization from dioxane, which gave 59 mg (34%) of the title compound.

[Table 5 follows at this point.]

0115<tables id="tabl0009" num="0009"><table frame="none"><title><u style="single">TABLE 5 PROCEDURE FOR 0.1 mMOLE SCALE</u></title><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="37mm" /><thead><row><entry rowsep="1" valign="top">STEP NO.</entry><entry rowsep="1" valign="top">SOLVENT/ REAGENT</entry><entry rowsep="1" valign="top">VOLUME (ML)</entry><entry rowsep="1" valign="top">TIME (MIN)</entry><entry rowsep="1" valign="top">REPEAT (XS)</entry><entry rowsep="1" valign="top">COMMENT</entry></row></thead><tbody><row><entry>1</entry><entry>DCM</entry><entry>10</entry><entry>2</entry><entry>3</entry><entry /></row><row><entry>2</entry><entry>TFA/DCM 55%</entry><entry>10</entry><entry>2</entry><entry>1</entry><entry>Deprotection</entry></row><row><entry>3</entry><entry>TFA/DCM 55%</entry><entry>10</entry><entry>20</entry><entry>1</entry><entry>Deprotection</entry></row><row><entry>4</entry><entry>DCM</entry><entry>10</entry><entry>2</entry><entry>3</entry><entry /></row><row><entry>5</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>4</entry><entry /></row><row><entry>6</entry><entry>DIEA/NMP 10%</entry><entry>10</entry><entry>5</entry><entry>2</entry><entry>Neutralization</entry></row><row><entry>7</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>5</entry><entry /></row><row><entry>8</entry><entry>TBTU/NMP / DIEA</entry><entry>10</entry><entry>150</entry><entry>3</entry><entry>Cyclization</entry></row><row><entry>9</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>4</entry><entry>Picric acid test.</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry>If less than 98±2% Coupling perform Steps 10.</entry></row><row><entry>10</entry><entry>TBTU/NMP /DIEA</entry><entry>10</entry><entry>20h</entry><entry>3</entry><entry>Cyclization, Check pH, adjust to pH 8 with DIEA.</entry></row><row><entry>11</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>4</entry><entry>Picric acid test. If less than 98±2% coupling perform Step 12.</entry></row><row><entry>12</entry><entry>TBTU/NMP /DIEA</entry><entry>10</entry><entry>120</entry><entry>3</entry><entry>Cyclization, 50 °C Check pH, adjust to pH 8 with DIEA.</entry></row><row><entry>13</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>6</entry><entry /></row><row><entry>14</entry><entry>Piperidine/ NMP 20%</entry><entry>10</entry><entry>10</entry><entry>1</entry><entry>Deprotection</entry></row><row><entry>15</entry><entry>Piperidine/ NMP 20%</entry><entry>10</entry><entry>1</entry><entry /><entry /></row><row><entry>16</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>6</entry><entry>Check for positive nin.</entry></row><row><entry>17</entry><entry>DCM</entry><entry>10</entry><entry>2</entry><entry>4</entry><entry /></row></tbody></tgroup></table></tables> HPLC (Method G)RT 33.62 minutes (91%) TOF MS: 1278 (M⁺) AAA in agreement with the title compound

Reference EXAMPLE 4

H-D-Arg-Arg-cyclo(N

α

(4-propanoyl)Gly-Hyp-Phe-N

α

(3-amidopropyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH

0116Title compound was synthesized according to Example 3 except that in stage 1, Fmoc-N^α(3-Boc-amino-propylene)-S-Phe (0.326) was substituted for Fmoc-N°(3-Boc amino propylene)Gly. A total of 0.643 g Boc-L-Arg(Tos)-O-resin (0.39 meq/g, 0.250 mmole) was used and reagent quantities were adjusted accordingly. Cyclic peptide yield (from half the total resin used) was 74 mg (42%) of the title compound.

SPECIFIC EXAMPLES OF BUILDING UNITS

0117The following specific examples of novel building units are provided for illustrative purposes not meant to be limiting. The following is described in sections, including "Procedures", "Methods",- "Compounds" and "EXAMPLES". "Procedures" are detailed stepwise descriptions of synthetic procedures according to the more general schemes. "Methods" II are general descriptions of analyses used to determine the progress of the synthetic process. Numbered "Compounds" are either the starting material or intermediates for further numbered "Compounds" the synthesis of which progresses according to the specified "Procedure." Several "Compounds" used in series produce "EXAMPLES" of novel building units of the present invention. For instance, "Compounds" 27-29, 41-44, 46-47, 51-55, 62, 63, 67 and 76-79 are actually "EXAMPLES" 5-25, respectively, of novel building units of the present invention. "Compounds" 1-26, 30-40, 45, 48-50, 56-61, 64-66, 68-75 are starting materials or intermediates (only) for the synthesis of "EXAMPLES".

Procedure 1

Synthesis of N-Boc alkylene diamines (BocNH (CH

<u style="single">2</u>

)

<u style="single">n</u>

NH

<u style="single">2</u>

) (known compounds).

0118To a solution of 0.5 mole alkylene diamine in 0.5 L CHCl₃ cooled in an ice-water bath, was added dropwise, with stirring, a solution of 10.91 g (0.05 mole) Boc₂O in 0.25 L CHCl₃ for 3 h. The reaction mixture was stirred for 16 h. at room temperature and then washed with water (8 X 250 mL). The organic phase was dried over Na₂SO₄ and evaporated to dryness <i>in vacuo</i>.

Procedure 2

Synthesis of N-Boc, N-Bzl alkylene diamines (BocNH(CH

2

)

n

NH-Bzl).

0119To a solution of 0.05 mole of mono Boc alkylene diamine in 60 mL MeOH was added 2.77 mL (0.02 mole) Et₃N, 9.02 g (0.075 mole) MgSO₄, and 5.56 mL (0.055 mole) of freshly distilled benzaldehyde. The reaction mixture was stirred under room temperature for 1.5 h. Then 11.34 g (0.3 mole) of NaBH₄ were added in small portions during 0.5 h with cooling to -5°C. The reaction mixture was then stirred for 1 h at - 5°C and for another 1 h at 0°C. The reaction was stopped by addition of 200 mL water and the product was extracted with EtOAc (3 X 200 mL). The combined EtOAc extracts were washed with water (4 X 100 mL). The organic phase was extract with 0.5 N HCl (4 X 100 mL) and the aqueous solution was neutralized under cooling with 25 mL 25% NH₄OH, extracted with CHCl₃ (3 X 100 ml) and the combined extracts were washed with water (3 X 80 mL), dried over Na₂SO₄ and evaporated to dryness <i>in vacuo.</i>

Procedure 3

Synthesis of (R) or (S) α-hydroxy acids (known compounds).

0120To a solution of 16.52 g (0.1 mole) (R) or (S) amino acid in 150 ml 1N H₂SO₄ was added dropwise a solution of 10.35 g (0.15 mole) NaNO₂ in 100 mL H₂O during 0.5 h with stirring and cooling in an ice bath. The reaction mixture was stirred 3 h. at 0°C and additional 18 h at room temperature, then the (R)- or (S)- hydroxy acid was extracted with ether in a continuous ether extractor. The etheral solution was washed with 1N HCl (2x50 mL), H₂O (3x80 mL), dried over Na₂SO₄ and evaporated to dryness. The product was triturated twice from ether:petrol-ether (40-60°C) (1:10). The precipitate was filtered, washed with 50 mL petrol-ether and dried.

Procedure 4

Synthesis of (R) or (S) α-hydroxy acid methyl esters (known compounds)

.

0121To a suspension of 0.065 mole (R)- or (S)- hydroxy acid in 100 mL ether was added under cooling in an ice bath 300 mL of an etheral solution of CH₂N₂ until stable yellow color of reaction mixture was obtained. Then the ether solution was washed with 5% KHCO₃ (3x100 mL) and H₂O (2x80 mL), dried over Na₂SO₄ and evaporated to dryness. The product was dried in <i>vacuo.</i>

Procedure 5

Synthesis of triflate of (R) or (S) α-hydroxy acid methyl esters.

0122To a cooled solution of 2.67 ml (0.033 mole) pyridine in 20 mL dry DCM was added 5.55 mL (0.033 mole) Trf₂O at -20°C (dry ice in EtOH bath), then after 5 min a solution of 0.03 mole (R) or (S) α-hydroxy acid methyl ester in 20 mL dry DCM was added dropwise. The reaction mixture was stirred at room temperature for 45 min, then was passed through a short silica gel column (2 cm). The product was eluted with 400 mL of petrol-ether:methylene-chloride (1:1). The solvent was evaporated in <i>vacuo.</i>

Procedure 6

Synthesis of (R) or (S) N

α

(Bzl) (N^ω-Boc-amino alkylene) amino acid methyl esters ((R) or (S) BocNH(CH

2

)

n

N(Bzl)CH(R)COOMe).

0123To a solution of 0.022 mole of N^α-Boc, N^ω-Bzl alkylene diamine in 20 mL of dry DCM was added 3.04 mL (0.022 mole) Et₃N. Then a solution of 0.02 mole of (R) or (S) α-hydroxy acid methyl ester triflate in 25 mL dry DCM was added dropwise (0.5 h.) under cooling in an ice-water bath. The reaction mixture was stirred at room temperature for 18 h. Then 150 mL of CHCl₃ was added and the yellow solution was washed with water (3 X 80 mL). The organic phase was dried over Na₂SO₄ and adsorbed on silica-gel and dried <i>in vacuo.</i> The silica-gel was washed on filter with 0.5 L of petrol-ether and with 0.5 L of 2% EA in PE. Then the product was eluted from silica with 0.5 L of mixture petrol-ether:ethyl-acetate (4:1). The solvent was evaporated <i>in vacuo.</i> If the product was not clean it was further purified on a small column of silica-gel (250 mL). The first impurities were eluted with 0.8 L of hexane then the product was eluted with 1.5 L of mixture of petrol-ether: ethyl-acetate (4:1).

Procedure 7

Hydrolysis of methyl esters

0124To a solution of 0.015 mole of methyl ester in 40 mL MeOH was added 10 mL 7.5 N NaOH cooled in an ice-water bath. The reaction mixture was stirred at room temperature for approximately 24 h (until the methyl ester spot disappears on TLC). Then 100 mL of water were added and the reaction mixture was washed with petrol-ether (3 X 80 mL). The aqueous solution was acidified under cooling by addition of 40 mL 2N HCL. The product was extracted with a mixture of CHC₃:i-PrOH (3:1) (3 X 80 mL), dried over Na₂SO₄, evaporated to dryness and dried <i>in vacuo</i> to obtain a white foam in quantitative yield.

Procedure 8

Removal of Bzl by Hydrogenation with Pd/C

0125To a solution of 0.012 mole (R) or (S) N^α (Bzl) (N^ω-Boc-amino alkylene) amino acid in 60 mL MeOH-DMF (11-1) was added 0.5 g 10% Pd/C. The solution was hydrogenated for 4 h under a pressure of 45-50 Psi at room temperature. Then 200 mL of a mixture of DMF:MeOH:H:₂O:glacial AcOH (1:3:5:1) was added. The catalyst was filtrated off and washed (is the acetic acid?) with H₂O or MeOH (2X15 mL). The combined filtrate was evaporated to dryness and recrystallized from methanol: ether (15 mL:250). The precipitate was filtered and dried <i>in vacuo</i>.

Procedure 9

Synthesis of (R) or (S) N^α (Fmoc)(N^ω-Boc-amino alkylene) amino acid.

0126To 50 mL water was added 0.07 mole of (R) or (S) N^α (N^ω-Boc-amino alkylene) amino acid and 1.95 mL (0.014 mole) Et₃N. The suspension was stirred 2-3 h until a clear solution was obtained. Then a solution of 2.25 g (0.07 mole) of FmocOSu in 100 mL ACN was added. The reaction mixture was stirred 18 h at room temperature, then 150 mL water was added and the solution was washed with petrol-ether (3 X 100 mL) and with ether:petrol-ether (1:4). The aqueous solution was acidified by addition of 14 mL 1N HCL. The product was extracted with EtOAc (4 X 100 mL) and the organic phase was washed with 0.5 N HCl (2 X 50 mL), H₂O (3 X 80 mL), dried over Na₂SO₄, evaporated to dryness and recrystallized from ether: petrol-ether (80 mL: 200 mL)

Procedure 10

Synthesis of S-benzylcysteamine (Bzl-S-(CH

<u style="single">2</u>

)

<u style="single">2</u>

-NH

<u style="single">2</u>

) (known compound).

0127To a suspension of 0.1 mole cysteamine hydrochloride in 20 mL methanol were added 13.6 mL of 25% ammonia solution, followed by dropwise addition of 0.12 mole benzyl bromide at room temperature. The mixture was stirred for 0.5 h, and the formed precipitate of S-dibenzylcysteamine was collected by filtration. The product was extracted with ether (3 X 100 mL) and the organic phase was successively washed with brine (2 X 100 mL), dried over MgSO₄ and the solvent evaporated <i>in vacuo.</i> The crude product was essentially pure enough for the next step. It could, however be recrystallized from ethyl acetate. Yield 86%, of white solid. m.p. 85-6°C NMR (CDCl₃) in agreement with the title compound.

Procedure 11

synthesis of N

α

-(ω-(benzylthio)alkylene)phthalimides ((Bzl-S-(CH

<u style="single">2</u>

)

<u style="single">n</u>

-N=Pht))

(known compounds)

.

0128N-(ω-bromoalkylene)phthalimide (0.1 mole) and benzyl mercaptane (0.11 mole) were stirred with 0.1 mole potassium carbonate in 100 mL DMSO at 50°C for 24 hours. The mixture was poured into ice-water and the product was allowed to crystallize for 0.5 hour, collected by filtration and recrystallized from i-PrOH.

Procedure 12

Synthesis of N^α-(ω-(benzylthio)alkyl)amines (Bzl-S-(CH

<u style="single">2</u>

)-NH

<u style="single">2</u>

) (known compounds).

0129Hydrazinolysis of the phthalimide group was performed by refluxing 0.09 mole of N^α-(ω-(benzylthio) alkylene) - phthalimide (Procedure 11) with 120 mL 1M solution of hydrazine hydrate in ethanol (diluted with additional 220 mL ethanol) for 2 hours. The formed precipitate was collected by filtration and hydrolyzed with 180 mL 2N HCl at 50°C for 0.5 hour. The water was evaporated <i>in vacuo</i> and the crude hydrochloride dissolved in 50 mL 25% ammonia solution. The free amine was extracted with DCM (4 X 100 mL) and the organic phase was washed with brine (2 X 100 mL), dried over MgSO₄ and the solvent evaporated <i>in vacuo</i>. The crude N-(ω-(benzyl-thio)alkyl)amine was distilled under reduced pressure, and appeared as colorless oil, which could be kept refrigerated under nitrogen for prolonged periods.

Procedure 13

Synthesis of (R) and (S) N^α- (ω-(benzylthio)alkylene) amino acids methyl esters ((R) or (S) (Bzl-S-(CH

<u style="single">2</u>

)-NH-CH(R)-COOMe)).

0130To a solution of 15 mmol N-(ω-(benzylthio)alkyl)amine and 15 mmol DIEA in 55 mL DCM was added dropwise a solution of 15 mmol (R) or (S) α-hydroxy acid methyl ester triflate (Procedure 5) in 55 mL DCM. at 0°C. The reaction mixture was then stirred at room temperature for 18 h. The mixture was then diluted with 100 mL DCM and washed with water (3 X 100 mL). The crude product was cleaned on a silica-gel column with DCM:MeOH(99:1), and was further crystallized from DIE:hexane.

0131In the case of Glycine, bromoacetic acid esters were suitable starting materials. Identical results were obtained when both these substrates were reacted with N-(ω-(benzylthio) alkyl)amines.

Procedure 14

Synthesis of (R) or (S) Boc-N

α

-(ω-(benzylthio)alkylene) amino acids ((R) or (S) (Bzl-S-(CH

<u style="single">2</u>

)

<u style="single">n</u>

-N(Boc)-CH(R)-COOH))

.

013210 mmol (R) or (S) N-(ω-(benzylthio)alkylene) amino acid methyl ester was dissolved in 50 mL 1,4-dioxane and 50 mL 1N NaOH were added. The mixture was stirred at room temperature overnight. The disappearance of the starting material was followed by TLC (Silica gel+ F₂₅₄, CHCl₃:MeOH-1:4). When all the ester was hydrolyzed, 50 mL water were added, followed by 30 mmol Bac₂O. The mixture was stirred overnight, then the dioxane was evaporated <i>in vacuo,</i> the mixture was cooled in an ice-water bath, covered with 100 mL EtAc and acidified with saturated KHSO₄ to pH 2-3. The layers were separated, and the aqueous layer was extracted with additional 2X 100 mL EtOAc. The organic layer was washed with water (2 X 100 mL), dried over MgSO₄ and the solvent evaporated <i>in vacuo</i>. The crude product was cleaned on a silica-gel column with DCM:MeOH-99:1 or crystallized from DIE:hexane.

Procedure 15

Synthesis of dipeptides (S, S) -Boc-amino acid-N^α-(ω-(benzylthio)alkylene) amino acids esters.

0133A solution of 1.1 mmol Boc-amino acid, 1 mmol N^α-(ω-(benzylthio)alkylene) amino acid ester, 1.1 mmol BOP and 3 mmol DIEA in 10 mL DCM was stirred at room temperature for 2 hours. Then the mixture was diluted with 40 mL of DCM and washed successively with saturated KHSO₄ (3 X 100 mL), saturated KHCO₃ (3 X 100 mL) and brine (2 X 100 mL), dried over MgSO₄ and the solvent evaporated <i>in vacuo</i>. The crude product was cleaned on a silica-gel column with DCM:MeOH-99:1 or crystallized from DIE:hexane.

Procedure 16

Synthesis of N

α

-Bzl ω-amino acids t-butyl esters (Bzl-NH-(CH

<u style="single">2</u>

)

<u style="single">n</u>

-COO-t-Bu).

0134A solution of 0.05 mole of amino acid t-butyl ester acetate in 200 mL H₂O was acidified to pH 2 with AcOH, washed with PE (70 mL X5) cooled and the pH adjusted to 9 by NH₄OH 25%. The free amino acid t-butyl ester was extracted with i-Pr:CHCl (3:1, 3X100 mL). The combined extracts were dried on Na₂SO₄ and evaporated to dryness under vacuum. The benzylation reaction was performed according to Procedure 2.

Procedure 17

Synthesis of N^α-(Bzl) (wt-Bu carboxy alkylene) glycine Bzl ester (N- (Bzl) (CH

<u style="single">2</u>

)

<u style="single">n</u>

-COO-t-Bu) CH

<u style="single">2</u>

-COO-Bzl)

0135To a stirred solution of 0.015 mole of N-Bzl ω-amino acid t-butyl ester in 10 mL DMF at 0°C were added 2.61 mL of DIEA and 2.38 mL of benzyl bromo acetate. The reaction mixture was stirred 30 min at 0°C and 3 h at room temperature. After the addition of 200 mL of ether, the precipitate was removed by filtration and the organic phase washed with H₂O (3X80 mL), 1N HCl (3X80 mL), H₂O (3X80 mL), dryed over Na₂SO₄ and evaporated to dryness under vacuum. The resulting oil was dried under vacuum.

METHODS

0136<u style="single">ANALYTICAL TLC</u> was performed on TLC plates of silica gel E (Merck F₂₅₄), using the following solvent systems: <u style="single">METHOD A</u> DCM:MeOH:AcOH 16:4:0.5 <u style="single">METHOD B</u> PE : EtOAc 1:1 <u style="single">METHOD C</u> PE: EtOAc 4:1 <u style="single">METHOD D</u> CHCl₃: EtOAc 4:1 <u style="single">METHOD E</u> PE: EtOAc 9:1 <u style="single">METHOD F</u> CHC1₃: EtOAc 19:1 <u style="single">METHOD G ANALYTICAL REVERSE PHASE HPLC</u><tables id="tabl0010" num="0010"><table frame="none"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="32mm" /><colspec colnum="2" colname="col2" colwidth="68mm" /><tbody><row><entry colsep="0" rowsep="0">Column</entry><entry colsep="0" rowsep="0">Merck LICHROCART RP-18 5 µm, 250X4 mm.</entry></row><row><entry colsep="0" rowsep="0">Mobile Phases</entry><entry colsep="0" rowsep="0">A = 0.1% TFA in H₂O</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">B = 0.1% TFA in ACN</entry></row><row><entry colsep="0" rowsep="0">Gradient</entry><entry colsep="0" rowsep="0">T = 0-5 min A(75%), B(25%)</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">T = 30 min A(50%), B(50%)</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">T = 40 min A(100%), B(0%)</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">T = 50 min A(100%), B(0%)</entry></row><row><entry colsep="0" rowsep="0">Flow = 1 mL/minute</entry><entry colsep="0" rowsep="0">Temperature = 23 °C</entry></row></tbody></tgroup></table></tables><u style="single">METHOD H - SEMIPREPARATIVE REVERSE PHASE HPLC</u> The crude peptide was dissolved in MeOH (1mL) and chromatographed using reverse phase semipreparative HPLC with the following conditions: <tables id="tabl0011" num="0011"><table frame="none"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="32mm" /><colspec colnum="2" colname="col2" colwidth="81mm" /><tbody><row><entry colsep="0" rowsep="0">Column</entry><entry colsep="0" rowsep="0">Merck Hibar LICHROSORB RP-18 7 µm, 250 X 410 mm</entry></row><row><entry colsep="0" rowsep="0">Mobile Phases</entry><entry colsep="0" rowsep="0">A = 0.1% TFA in H₂O</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">B = 0.1% TFA in ACN</entry></row><row><entry colsep="0" rowsep="0">Gradient</entry><entry colsep="0" rowsep="0">T = 0-10 min A(80%), B(20%)</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">T = 60 min A(100%), B (0%)</entry></row><row><entry colsep="0" rowsep="0" /><entry colsep="0" rowsep="0">T = 70 min A(100%), B(0%)</entry></row><row><entry colsep="0" rowsep="0">Flow = 4mL/minute</entry><entry colsep="0" rowsep="0">Temperature = 23 °C</entry></row></tbody></tgroup></table></tables><u style="single">METHOD J</u> NINHYDRIN TEST (NIN. TEST) The test was performed according to Kaiser <i>et al.</i><u style="single">Anal. Biochem.,</u> 34:595 (1970) which is incorporated herein by reference in its entirety. Test was considered negative when the resin did not change color after heating to 11°C for 2 minutes with the test mixture. Test was considered positive or slightly positive when the resin was dark or faint purple after heating to 110°C for 2 minutes with the test mixture. <u style="single">METHOD K -</u> QUANTITATIVE PICRIC ACID TEST Picric acid test was performed after removal of the Fmoc protecting group from the amino acid preceding the coupling of protected Nα(ω-alkylene) building unit. This absorbance was taken as 100% free amines. After coupling the test was used to check yield of % coupling by comparison.

0137The resin 0.1 mmole was treated according to steps 1-8, Table 1. After step 8 the resin was introduced into a centrifuge tube and shaken with 40 mL of 5% DIEA:95% DCM for ten minutes. The resin was centrifuged 5 minutes and 4 mL of the solution were pipette into 40 mL. EtOH and the absorbance at 358 nm measured. This procedure was repeated 3 times and the average absorbance calculated. <tables id="tabl0012" num="0012"><table frame="none"><title><u style="single">TABLE 6 PROCEDURE FOR 0.1 mMOLE SCALE</u></title><tgroup cols="6" colsep="0"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="22mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="22mm" /><thead><row><entry valign="top">STEP NO.</entry><entry valign="top">SOLVENT/ REAGENT</entry><entry valign="top">VOLUME (ML)</entry><entry valign="top">TIME (MIN)</entry><entry valign="top">REPEAT (XS)</entry><entry valign="top">COMMENT</entry></row></thead><tbody><row rowsep="0"><entry>1</entry><entry>NMP</entry><entry>10</entry><entry>1</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>2</entry><entry>DCM</entry><entry>10</entry><entry>1</entry><entry>2</entry><entry /></row><row rowsep="0"><entry>3</entry><entry>Picric acid 0.05M/DCM</entry><entry>10</entry><entry>1</entry><entry>2</entry><entry /></row><row rowsep="0"><entry>4</entry><entry>DMF</entry><entry>10</entry><entry>0.5</entry><entry>10</entry><entry /></row><row rowsep="0"><entry>5</entry><entry>NMP</entry><entry>10</entry><entry>1</entry><entry>2</entry><entry /></row><row rowsep="0"><entry>6</entry><entry>NMP</entry><entry>10</entry><entry>2</entry><entry>3</entry><entry /></row><row rowsep="0"><entry>7</entry><entry>10% EtOH/DCM</entry><entry>10</entry><entry>1</entry><entry>1</entry><entry /></row><row rowsep="0"><entry>8</entry><entry>DCM</entry><entry>10</entry><entry>1</entry><entry>2</entry><entry /></row></tbody></tgroup></table></tables>

COMPOUND 1

N-Boc diamino ethane (known compound)

0138A solution of 33.4 mL of ethylene diamine in CHCl₃ and 10.91 g of Boc₂O were used (Procedure 1). Yield 97% of colorless oil. TLC (Method A) Rf 0.2-0.24 (one spot) NMR (CDCl₃) in agreement with the title compound

COMPOUND 2

N-Boc 1,3 diamino propane (known compound).

0139A solution of 41.7 mL of 1,3 diamino propane in CHCl₃ and 10.91 g of Boc₂O were used (Procedure 1). Yield 96% of colorless oil. TLC (Method A) Rf 0.27-0.3 (one spot) NMR CDCl₃) in agreement with the title compound

COMPOUND 3

N-Boc 1,4 diamino butane (known compound).

0140A solution of 44.08 g of 1,4 diamino butane and 10.91 g of Boc₂O were used (Procedure 1). Yield 98% of white oil. TLC (Method A) Rf 0.32-0.35 (one spot). NMR (CDCl₃) in agreement with the title compound.

COMPOUND 4

N-Boc 1,6 diamino hexane (known compound).

0141A solution of 58.10 g of 1,6 diamino hexane in CHCl₃ and 10.91 g of Boc₂O were used (Procedure 1). Yield 70% of colorless oil after purification on a silica gel column and elution with CHCl₃-MeOF (4:1) TLC (Method A) Rf 0.50-0.54 (one spot) NMR (CDCl₃) in agreement with the title compound

COMPOUND 5

N-Boc. N-Bzl 1.2 diamino ethane

0142A solution of 8.01 g of Boc ethylene diamine (COMPOUND 1) was used (Procedure 2). Yield 65% of colorless oil. TLC (Method A) Rf 0.62-0.65 (one spot) NMR (CDCl₃) in agreement with the title compound

COMPOUND 6

N-Boc. N-Bzl. 1.3 diamino propane.

0143A solution of 8.71 g of N-Boc 1,3 diamino propane (COMPOUND 2) was used (Procedure 1). Yield 75% of colorless. TLC (Method A) Rf 0.63-0.68 (one spot) NMR (CDCl₃) in agreement with the title compound

COMPOUND 7

N-Boc. N-Bzl 1.4 diamino butane

0144A solution of 9.41 g of N-Boc 1,4 diamino butane (Compound 3) was used (Procedure 1). Yield 63% of white oil. TLC (Method A) Rf 0.65-0.72 (one spot) NMR (CDCl₃) in agreement with the title compound

COMPOUND 8

N-Boc. N-Bzl 1.6 diamino hexane

0145A solution of 10.82 g of N-Boc 1,6 diamino hexane (Compound 4) was used (Procedure 1). The ethyl acetate solution after extraction was dried over Na₃SO₄ and evaporated under vacuum to dryness. The remaining crude product was dissolved in 400 mL chloroform and washed with 0.5 N HCl (3 X 80 mL, 0.12 mole), water (2 X 100 mL) dried over Na₂SO₄ and evaporated to dryness. Then 200 mL of ether was added. The precipitate was filtered, washed with ether (3 X 50 mL) and dried under vacuum. Yield 70% of white solid mp 150-152°C. TLC (Method A) Rf 0.8 (one spot)

0146To remove HCl from product with n=6, the HCl salt was dissolved in CHCl₃ , washed with an alkali solution (0.5% NH₄OH), dried over Na₂SO₄ and evaporated to dryness. NMR (CDCl₃) in agreement with the title compound.

COMPOUND 9

(S)-3-Phenylacetic acid methyl ester (known compound)

0147A suspension of 10.8 g of (S)-3-Phenylacetic acid in 100 mL ether was treated with diazomethane (Procedure 4). Yield 85%. TLC (Method B) Rf 0.6-0.65 (one spot) (α)_D= +3,3 (c=1, MeOH) NMR (CDCl₃) in agreement with the title compound

COMPOUND 10

(R)-3-Phenylacetic acid methyl ester (known compound).

0148A suspension of 10.8 g (R)-3-Phenylacetic acid in 100 mL ether was treated with diazomethane (Procedure 4). Yield 84%. TLC (Method B) Rf 0.6-0.65 (one spot) (α)_D= -3,3 (c=1, MeOH) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 11

(S)-O-Trf-3-Phenylacetic acid methyl ester

0149To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), a solution of 5.4 g of (S)-3-Phenylacetic acid methyl ester was added. After the workup (Procedure 5), the yield was 74%. The product was used immediately or kept in a cold desiccator under Ar.

COMPOUND 12

(R)-O-Trf-3-Phenylacetic acid-methyl ester

0150To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), a solution of 5.4 g of (R)-3-Phenylacetic acid methyl ester was added. After the workup (Procedure 5), the yield was 74%. The product was used immediately or kept in a cold desiccator under Ar.

COMPOUND 13

N

α

(Bzl)(2-Boc-amino ethylene) (R)Phenylalanine methyl ester

0151A solution of 6.24 g of (S)-O-Trf-3-Phenyllactic acid methyl ester in dry DCM (Compound 11) was added to a solution of 5.51 g of N-Boc, N-Bzl-diamino ethane (Compound 5) in dry DCM (Procedure 6). Yield 69.2% (α)_D= +64.0 (c=1, MeOH) . TLC (Method C) Rf =0.41 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 14

N^α (Bzl) (3-Boc-amino propylene) (S) Phenylalanine methyl ester

0152A solution of 6.24 g of (R)-O-Trf-3-Phenyllactic acid methyl ester in dry DCM (Compound 12) was added to a solution of 5.82 g of N-Boc, N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 67.7% (α)_D= -55.8 (c=1, MeOH) TLC (Method C) Rf-0.38 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 15

N

α

(Bzl) (4-Boc-amino butylene) (S) Phenylalanine methyl ester

0153A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester in dry DCM (Compound 12) was add to a solution of 6.12 g of N-Boc, N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 58.6% (α)_D = -62.6 (e=1, MeOH) Rf (Method C) 0.42 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 16

N^α (Bzl) (6-Bac-amino hexylene) (S) Phenylalanine methyl ester

0154A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester in dry DCM (Compound 12) was add to a solution of 6.74 g of N-Boc, N-Bzl-diamino hexane (Compound 8) in dry DCM (Procedure 6). Yield 78.9% (α)_D= -60.0 (c=1, MeOH) TLC (Method C) Rf=0.47 (one spot) NMR (CDCl₃) in agreement with the title compound.

<u style="single">COMPOUND 17</u>

N^α (Bzl) (3-Boc-amino propylene) (R)Phenylalanine methyl ester

0155A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester in dry DCM (Compound 11) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 51.5% (α)_b= +58.8 (c=1, MeOH) TLC (Method C) Rf=0.35 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 18

N^α (Bzl)(4-Boc-amino butylene

)

(R) Phenylalanine methyl ester

0156A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester in dry DCM (Compound 11) was add to a solution of 6.12 g of N-Soc, N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 66.8% (α)_D= +59.0 (c=1, MeOH) TLC (Method C) Rf=0.33 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 19

N

^α

(Bzl)(3-Boc-amino propylene) (S) Phenylalanine

0157A solution of 6.61 g of N^α(Bzl)(3-Boc-amino propylene) (S)Phenylalanine methyl ester in MeOH (Compound 14) was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 89.5% (α)_b= -24.0 (c=1, MeOH) TLC (Method B) Rf=0.16 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 20

N^α (Bzl) (4-Boc-amino butylene)

(S)Phenylalanine

0158A solution of 6.61 g of N^α (Bzl)(4-Boc-amino butylene) (S)Phenylalanine methyl ester in MeOH (Compound 15) was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 73.5% (α)_b= -12.0 (c=1, MeOH) TLC (Method D) Rf=0.6 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 21

N° (Bzl) (3 -Doc-amino propylene) (R) Phenylalanine

0159A solution of 6.40 g of N^α (Bzl) (3-Boc-amino propylene) (R)Phenylalanine methyl ester in MeOH (Compound 17) was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 100% (α) _D= +15.33 (c=1, MeOH) TLC (Method B) Rf=0.38 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 22

N^α (Bzl) (4-Boc-amino butylene) (R)Phenylalanine

0160A solution of 6.61 g of N^α (Bzl)(4-Boc-amino butylene) (R)Phenylalanine methyl ester in MeOH (Compound 18) was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 100% (α)_D= +12.0 (c=1, MeOH) TLC (Method D) Rf=0.54 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 23

N<i>^α</i>(3-Boc-amino propylene) (S) Phenylalanine HCl

0161A solution of 5.39 g of N^α (Bzl) (3-Boc-amino propylene) (S)Phenylalanine (Compound 19) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 86.1% TLC (Method A)Rf = 0.51 (one spot) NMR (D₂O+Na₂CO₃) in agreement with the title compound.

COMPOUND 24

N^α (4-Boc-amino butylene) (S)Phenylalanine HCl

0162A solution of 5.56 g of N^α (Bzl) (4-Boc-amino butylene) (5)Phenylalanine (Compound 20) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 79.25% TLC (Method A) Rf = 0.50 (one spot) NMR (D₂O+Na₂CO₃) in agreement with the title compound.

COMPOUND 25

N^α (3-Boc-amino propylene) (R)Phenylalanine HCl

0163A solution of 5.39 g of N^α (Bzl) (3-Boc-amino propylene) (R)Phenylalanine (Compound 21) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 75.5% TLC (Method A)Rf = 0.51 (one spot) NMR (D₂O+Na₂CO₃) in agreement with the title compound.

COMPOUND 26

N^α (4-Boc-amino butylene) (R)Phenylalanine HCl

0164A solution of 5.56 g of N^α (Bzl) (4-Boc-amino butylene) (R)Phenylalanine (Compound 22) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 73.85% TLC (Method A) Rf - 0.50 (one spot) NMR (D₂O+Na₂CO₃) in agreement with the title compound.

<u style="single">EXAMPLE 5:</u>

COMPOUND 27: N° (Fmoc) (3-Boc-amino propylene) (S) Phenylalanine

0165A solution of 2.51 g of N^α (3-Boc-amino propylene) (S)Phenylalanine.HCl (Compound 23) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 64.84% TLC (Method D) Rf = 0.74 (one spot) (α)_D= -87 (c=1, MeOH) HPLC (Method G) 92 % NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 6:</u>

COMPOUND

28:

N^α(Fmoc) (3-Boc-amino propylene) (R)Phenylalanine

0166A solution of 2.51 g of N^α (3-Boc-amino propylene) (R)Phenylalanine.HCl (Compound 25) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 61.56% TLC (Method D) Rf = 0.62 (one spot) (α)_D= +79.6 (c=1, MeOH) HPLC (Method G) 94 % NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 7:</u>

COMPOUND 29: N^α (Fmoc) (4-Boc-amino butylene) (S) Phenylalanine

0167A solution of 2.61 g of N^α (4-Boc-amino butylene) (S) Phenylalanine.HCl (Compound 24) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 56% TLC (Method D) Rf = 0.64 (one spot) HPLC (Method G) 89% NMR (CDCl₃) in agreement with the title compound.

COMPOUND 30

O-Trf-(S)-Lactic acid methyl ester

0168To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5), a solution of 2.9 mL of (S)lactic acid methyl ester was added. After the workup (Procedure 5), the yield was 70%. The product was used immediately or kept in a cold desiccator under Ar.

COMPOUND 31

O-Trf-(R)-Lactic acid methyl ester

0169To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5), a solution of 2.9 mL of (R) lactic acid methyl ester was added. After the workup (Procedure 5), the yield was 70%. The product was used immediately or kept in a cold desiccator under Ar.

COMPOUND 32

N

α

(Bzl)(3-Boc-amino propylene) (S) Alanine methyl ester

0170A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM (Compound 31) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 69.5% (α)_D = -6.6 (C=1, MeOH) TLC (Method C) Rf =0.42 (one spot) TLC (Method D) Rf = 0.92 (one spot) TLC (Method E) Rf= 0.13 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 33

N^α(Bzl) (3-Boc-amino propylene) (R)Alanine methyl ester

0171A solution of 4.72 g of O-Trf-(S-lactic acid methyl ester in dry DCM (Compound 30) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 71% (α)_D = +6.53 (C=1, MeOH) TLC (Method C) Rf = 0.42 (one spot) TLC (Method D) Rf = 0.93 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 34

N^α(Bzl)(6-Boc-amino hexylene) (S)Alanine methyl ester

0172A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM (Compound 31) was add to a solution of 6.74 g of N-Boc, N-Bzl-diamino hexane (Compound 8) in dry DCM (Procedure 6). Yield 81.42 % (α)_D = -6.76. (C=1, MeOH) TLC (Method D) Rf = 0.95 (one spot) TLC (Method E) Rf = 0.26 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 35

N

α

(Bzl)(2-Boc-amino propylene) (S)Alanine

0173A solution of 5.25 g of N^α (Bzl)(2-Boc-amino ethylene) (S)Alanine methyl ester in MeOH (Compound 32) was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 100 % of white solid, mp 64°C (α)_D = +0.5 (C=l, MeOH) TLC (Method A) Rf = 0.64 (one spot) TLC (Method D) Rf = 0.47 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 36

N^α (Bzl) (6-Boc-amino hexylene) (S) Alanine

0174A solution of 5.88 g of N^α (Bzl) (6-Boc-amino hexylene) (S)Alanine methyl ester (Compound 34) in MeOH was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 100 % (α)_D= +0.7 (C=1, MeOH) TLC (Method D) Rf = 0.51 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 37

N

α

(Bzl) (3-Boc-amino propylene) (R)Alanine

0175A solution of 5.25 g of N^α (Bzl)(2-Boc-amino ethylene) (R)Alanine methyl ester (Compound 35) in MeOH was hydrolyzed by NaOH 7.5N (Procedure 7). Yield 100 % (α)_D = -0.5 (C-1, MeOH) TLC (Method D) Rf = 0.51 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 38

N^α (3-Boc-amino propylene) (S)Alanine.HCl

0176A solution of 4.47 g of N^α (Bzl)(3-8oc-amino propylene) (S)Alanine (Compound 35) in MeOH was hydrogenated on Pd/C (Procedure 8). Yield 75 % TLC (Method A) Rf = 0.42 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 39

N

α

(6-Boc-amino hexylene) (S)Alanine HCl

0177A solution of 5 g of N^α(Bzl)(4-Boc-amino hexylene) (S)Alanine (Compound 36) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 64.5 % of white solid, mp 134-136 °C TLC (Method A) Rf = 0.39 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 40

N° (3-Boc-amino propylene) (R)Alanine .HCl

0178A solution of 5 g of N^α (Bzl)(4-Boc-amino propylene) (R) Alanine (Compound 37) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield 79.1 %. TLC (Method A) Rf = 0.39 (one spot) NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 8:</u>

COMPOUND 41 : N^α (Fmoc) (3-Boc-amino propylene) (S) Alanine

0179A solution of 2.82 g of N^α(Bzl) (4-Boc-amino propylene) (S)Alanine .HCl (Compound 38) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 75 % of white solid, mp 70-72°C TLC (Method D) Rf = 0.65 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0013" num="0013"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">66.40</entry><entry align="char" char="." charoff="20">6.78</entry><entry align="char" char="." charoff="20">5.63</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">66.65</entry><entry align="char" char="." charoff="20">6.88</entry><entry align="char" char="." charoff="20">5.93</entry></row></tbody></tgroup></table></tables>

EXAMPLE 9:

COMPOUND 42: N

α

(Fmoc)(6-Boc-amino hexylene) (S)Alanine

0180A solution of 3.25 g of N^α (Bzl)(4-Boc-amino hexylene) (S)Alanine .HCl (Compound 39) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 72.8 % of white solid, mp 70-72°C TLC (Method D) Rf = 0.7 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0014" num="0014"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">68.37</entry><entry align="char" char="." charoff="20">7.40</entry><entry align="char" char="." charoff="20">5.23</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">68.21</entry><entry align="char" char="." charoff="20">7.50</entry><entry align="char" char="." charoff="20">5.49</entry></row></tbody></tgroup></table></tables> HPLC (Method G) 90%

<u style="single">EXAMPLE 10:</u>

COMPOUND 43: N^α(Fmoc) (3-Boc-amino propylene) (R) Alanine

0181A solution of 2.82 g of N^α (Bzl)(4-Boc-amino propylene) (S)Alanine .HCl (Compound 40) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield 75.9 % of white solid, mp 70-72°C TLC (Method D) Rf = 0.5 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0015" num="0015"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry>Found:</entry><entry align="char" char="." charoff="31">66.4</entry><entry align="char" char="." charoff="20">6.78</entry><entry align="char" char="." charoff="20">5.63</entry></row><row rowsep="0"><entry>Calc:</entry><entry align="char" char="." charoff="31">66.65</entry><entry align="char" char="." charoff="20">6.88</entry><entry align="char" char="." charoff="20">5.93</entry></row></tbody></tgroup></table></tables>

EXAMPLE 11

:

COMPOUND 44: N-(2-(benzylthio)ethylene)glycine ethyl ester

0182The title compound was prepared according to procedure 13 from ethyl bromo acetate. Yield 75% of colorless oil.

0183NMR (CDCl₃) in agreement with the title compound. Elemental analysis-calculated: C-61.16, H-7.70, N-3.96 ; found: C-61.45, H-8.03, N-3.49.

COMPOUND 45

N-(3-(benzylthio)propylene)glycine methyl ester

0184The title compound was prepared according to procedure 13 from methyl bromo acetate. Yield 74% of colorless oil. NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 12:</u>

COMPOUND 46: N<i>-</i> (2- (benzylthio) ethylene) (S) leucine methyl ester

0185The title compound was prepared according to procedure 13 from the Triflate of (R) leucine methyl ester (Procedure 5). Yield 70% of colorless oil. NMR (CDCl₃) in agreement with the title compound. Elemental analysis-calculated: C-65.05, H-8.53, N-4.74 ; found: C-66.29, H-9.03, N-4.49. (a)_D14=-51.2° (C 0.94,DCM).

<u style="single">EXAMPLE 13:</u>

COMPOUND 47: N-(3-(benzylthio)propylene) (S) leucine methyl ester

0186The title compound was prepared according to procedure 1.3 from the Triflate of (R) leucine methyl ester (Procedure 5). Yield 60% of colorless oil. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-65.98, H-8.79, N-4.53 ; found: C-67.09, H-9.20, N-4.54. (a)_D23=-17.4° (C 1.44, DCM) .

COMPOUND 48:

N-(2-(benzylthio)ethylene) (S) phenylalanine methyl ester

0187The title compound was prepared according to procedure 13 from the Triflate of (R)phenyl lactic acid methyl ester (Procedure 5). Yield 82% of white crystals. m.p.=48-49°C. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-69.27, H-7.04, N-4.25 ; found: C-69.55, H-7.21, N-4.08. (a)_D14=-23 .3° (C =1.01, DCM).

COMPOUND 49

N-(3-(benzylthio)propylene) (S) phenylalanine methyl ester

0188The title compound was prepared according to procedure 13 from the Triflate of (R)phenyl lactic acid methyl ester (Procedure 5). Yield 71% of white crystals. m.p.=38-39°C. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-69.94, H-7.34, N-4.08 ; found: C-69.66, H-7.39, N-4.37. (a) _D26=+2.0°(C 1.00, DCM) .

COMPOUND 50

N-(4-(benzylthio)butylene)(S)phenylalanine methyl ester

0189The title compound was prepared according to procedure 13 from the Triflate of (S)phenyl lactic acid methyl ester (Procedure 5). Yield 81% of colorless oil. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-70.55, H-7.61, N-3.92 ; found: C-70.51, H-7.69, N-4.22. (a)_D26=+4.9° (C 1.00, DCM).

<u style="single">EXAMPLE 14:</u>

COMPOUND 51: Boc-N-(2-(benzylthio)ethylene)glycine

0190The title compound was prepared from Compound 44 by hydrolysis according to Procedure 11. Yield 88% of white crystals. m.p.=71-72°C. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-59.05, H-7.12, N-4.30 ; found: C-59.39, H-7.26, N-4.18.

<u style="single">EXAMPLE 15:</u>

COMPOUND 52: Boc-N- (2 - (benzylthio) ethylene) (S) phenylalanine

0191The title compound was prepared from Compound 48 by hydrolysis according to Procedure 11. Yield 78% of white crystals. m.p.=82-83°C. NMR (CDCl₃) in agreement with the title compound. (a)_D25=-105.9° (C 1.01, DCM) .

<u style="single">EXAMPLE 16:</u>

COMPOUND 53: Boc-N-(3-(benzylthio)propylene)(S) phenylalanine

0192The title compound was prepared from Compound 49 by hydrolysis according to Procedure 11. Yield 99% of white crystals. m.p.=63-64°C. NMR (CDCl₃) in agreement with the title compound. (a)_D25=-87.4° (C 1.01, DCM).

<u style="single">EXAMPLE 17:</u>

COMPOUND 54: Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

0193Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester (Compound 44) according to Procedure 12. Yield 32% of colorless oil. NMR (CDCl₃) in agreement with the title compound. Elemental analysis- calculated: C-64.77, H-7.25, N-5.60 ; found: C-64.39, H-7.02, N-5.53. (a)_D16 =+4.5 ° (C 0.88, DCM).

<u style="single">EXAMPLE 18:</u>

COMPOUND 55: Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methyl ester

0194Boc-L-Phe was coupled to N-(2-(benzylthio)ethylene) (S)phenylalanine methyl ester (Compound 48) according to Procedure 12. Yield 46% of colorless oil. NMR (CDCl₃) in agreement with the title compound. (a)_D26=-115. 9° (C 1.0, CHCl₃).

COMPOUND 56

N-Bzl-β- alanine t-butyl ester

0195A solution of 6.16 g of β- alanine t-butyl ester acetate in 150 mL water was reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5% yield TLC (Method A) Rf=0.78 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 57

N-Bzl-γ- amino butyric acid t-butyl ester

0196A solution of 6.58 g of γ- aminobutyric acid t-butyl ester acetate in 150 mL water was reacted with benzaldhyde (Procedure 2) to give 4.24 g, 57.9% yield TLC (Method A) Rf=0.74 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 58

N

α

(Bzl)(2-t-butyl carboxy ethylene) glycine benzyl ester

0197A solution of 3.53 g of N-Bzl-β- alanine t-butyl ester (Compound 56) in DMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17). Yield 86.9% TLC (Method F) Rf=0.95 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 59

N^α (Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester

0198A solution of 3.53 g of N-Bzl-γ- aminobutyric acid t-butyl ester (Compound 57) in DMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17). Yield 83% TLC (Method F) Rf=0.92 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 60

N

α

(2-t-butyl carboxy ethylene) glycine

0199A solution of N^α (Bzl) (2-t-butyl carboxy ethylene)glycine benzyl ester (Compound 58) in MeOH was hydrogenated (Procedure 8). Yield 87.8% TLC (Method A) Rf=0.56 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 61

N^α(3-t-butyl carboxy propylene)glycine

0200A solution of N^α (Bzl) (3-t-butyl carboxy propylene)glycine benzyl ester (Compound 59) in MeOH was hydrogenated (Procedure 8). Yield 94% TLC (Method A) Rf=0.3 (one spot) NMR (CDCl₃) in agreement with the title compound.

<b>EXAMPLE 19:</b> COMPOUND 62

N^α(Fmoc) (2-t-butyl carboxy ethylene) glycine

0201A solution of N^α (2-t-butyl carboxy ethylene)glycine (Compound 60) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 90% TLC (Method D) Rf=0.5 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0016" num="0016"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">67.38</entry><entry align="char" char="." charoff="20">6.34</entry><entry align="char" char="." charoff="20">3.11</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">67.75</entry><entry align="char" char="." charoff="20">6.40</entry><entry align="char" char="." charoff="20">3.29</entry></row></tbody></tgroup></table></tables>

<u style="single">EXAMPLE 20:</u>

COMPOUND 63: N^α(Fmoc)(3-t-butyl carboxy propylene)glycine

0202A solution of N^α(3-t-butyl carboxy propylene)glycine (Compound 61) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 82% TLC (Method D) Rf=0.58 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0017" num="0017"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">68.29</entry><entry align="char" char="." charoff="20">6.83</entry><entry align="char" char="." charoff="20">3.88</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">68.32</entry><entry align="char" char="." charoff="20">6.65</entry><entry align="char" char="." charoff="20">3.19</entry></row></tbody></tgroup></table></tables>

COMPOUND 64

(R)-O-Trf-3-Phenyllactic acid benzyl ester

0203To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), a solution of 5.3 g of (R)-3-Phenyllactic acid benzyl ester was added. After the workup (Procedure 5), the yield was 91.43%. The product was used immediately or kept in a cold desiccator under Ar.

COMPOUND 65

N^α(Bzl) (2-t-butyl carboxy ethylene) (S) Phenylalanine benzyl ester

0204A solution of 5.48 g of N-Bzl-β- alanine t-butyl ester (Compound 56) in DCM was reacted with 7.35 g of (R)-O-Trf-3-Phenyllactic acid benzyl ester (COMPOUND 64) in dry DCM (Procedure 6). After workup the crude product was purified by flash chromatography. PE:EtOAc (4:1) 1.5 L. After solvent evaporation under vacuum, the product was dried under vacuum. Yield 71.5% TLC (Method C) Rf=0.77 (one spot) (α)_D = -62.7 (C=1, MeOH) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 66

N^α(2-t-butyl carboxy ethylene)(S) Phenylalanine

0205A solution of 6.3 g of N^α (Bzl) (2-t-butyl carboxy ethylene) (S) Phenylalanine benzyl ester (Compound 65) in MeOH was hydrogenated (Procedure 8). Yield 48.6% TLC (Method A) Rf=0.52-0.54 (one spot) NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 21:</u>

COMPOUND 67: N

α

(Fmoc)(2-t-butyl carboxy ethylene) (S) Phenylalanine

0206A solution of 2.13 g of N^α (2-t-butyl carboxy ethylene) (S) Phenylalanine (Compound 66) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 38% TLC (Method D) Rf=0.77 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0018" num="0018"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">71.92</entry><entry align="center">639</entry><entry align="char" char="." charoff="20">2.87</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">72.21</entry><entry align="center">6.45</entry><entry align="char" char="." charoff="20">2.72</entry></row></tbody></tgroup></table></tables> HPLC (Method G) 93%

COMPOUND 68

N

α

(Bzl)(2-Boc amino ethylene) glycine benzyl ester

0207A solution of 0.0325 mole of N-Boc, N-Bzl,1,2 diaminoethane (Compound 5) in DMF was reacted with 5.15 mL benzyl bromoacetate (Procedure 17). Yield 97.9% TLC (Method F) Rf=0.78 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 69

N^α (Bzl) (3-Boc amino propylene)glycine benzyl ester

0208A solution of 0.0325 mole of N-Boc, N-Bzl,1,3 diaminopropane (Compound 6) in DMF was reacted with 5.15 mL benzyl bromoacetate (Procedure 17). Yield 98.2% TLC (Method F) Rf=0.78 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 70

N

α

(Bzl)(4-Boc amino butylene)glycine benzyl ester

0209A solution of 0.0325 mole of N-Boc, N-Bzl,1,4 diaminobutane (Compound 7) in DMF was reacted with 5.15 mL benzyl bromoacetate (Procedure 17). Yield 98.8% TLC (Method F) Rf=0.82(one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 71

N

^α

(Bzl) (6-Boc amino hexylene)glycine benzyl ester

0210A solution of 0.0325 mole of N-Boc, N-Bzl,1,6 diaminohexane (Compound 8) in DMF was reacted with 5.15 mL benzyl bromoacetate (Procedure 17). Yield 98.8% TLC (Method F) Rf=0.79(one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 72

N^α(2-Boc amino ethylene)glycine

0211A solution of 0.025 mole of N^α(Bzl)(2-Boc amino ethylene)glycine benzyl ester (Compound 68) in 60 mL MeOH was hydrogenated (Procedure 8). Yield 85% of white solid. mp 200- 2°C. TLC (Method A) Rf=0.22 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 73

N^α(3-Boc amino propylene)glycine

0212A solution of 0.025 mole of N^α (Bzl) (3-Boc amino propylene)glycine benzyl ester (Compound 69) in 60 mL MeOH was hydrogenated (Procedure 8). Yield 74% of white solid. mp 214-6 °C. TLC (Method A) Rf=0.27 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 74

N^α(4-Boc amino butylene)glycine

0213A solution of 0.025 mole of N^α (Bzl) (4-Boc amino butylene)glycine benzyl ester (Compound 70) in 60 mL MeOH was hydrogenated(Procedure 8). Yield 89.5% of white solid. mp 176-8 °C. TLC (Method A) Rf=0.23 (one spot) NMR (CDCl₃) in agreement with the title compound.

COMPOUND 75

N^α(6-Boc amino hexylene)glycine

0214A solution of 0.025 mole of N^α (Bzl)(6-Boc amino hexylene)glycine benzyl ester (Compound 71) in 60 mL MeOH was hydrogenated (Procedure 8). Yield 80% of white solid. mp 172- 4°C. TLC (Method A) Rf=0.26 (one spot) NMR (CDCl₃) in agreement with the title compound.

<u style="single">EXAMPLE 22:</u>

COMPOUND 76:

N

α

(Fmoc) (2-Boc

amino

ethylene) glycine

0215A solution of 0.02 mole of N^α (2-Boc amino ethylene)glycine (Compound 72) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9), Yield 80% of white solid. mp 130-132 °C. TLC (Method D) Rf=0.5 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0019" num="0019"><table frame="none"><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry>Elemental Analysis:</entry><entry align="center">% C</entry><entry align="center">% H</entry><entry align="center">% N</entry></row><row><entry align="right">Found:</entry><entry align="char" char="." charoff="31">65.18</entry><entry align="char" char="." charoff="20">6.11</entry><entry align="char" char="." charoff="20">5.91</entry></row><row><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">65.43</entry><entry align="char" char="." charoff="20">6.40</entry><entry align="char" char="." charoff="20">6.63</entry></row></tbody></tgroup></table></tables>

<u style="single">EXAMPLE 23:</u>

COMPOUND 77: N^α(Fmoc)(3-Boc amino propylene)glycine

0216A solution of 0.02 mole of N^α(Fmoc) (3-Boc amino propylene)glycine (Compound 73) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 85% of white solid. mp 125°C. TLC (Method D) Rf=0.5-0.6 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0020" num="0020"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry>Found:</entry><entry align="char" char="." charoff="31">66.05</entry><entry align="char" char="." charoff="20">6.65</entry><entry align="char" char="." charoff="20">6.00</entry></row><row rowsep="0"><entry>Calc:</entry><entry align="char" char="." charoff="31">66.06</entry><entry align="char" char="." charoff="20">6.65</entry><entry align="char" char="." charoff="20">6.16</entry></row></tbody></tgroup></table></tables>

<u style="single">EXAMPLE 24:</u>

COMPOUND 78: N^α(Fmoc) (4-Boc amino butylene)glycine

0217A solution of 0.02 mole of N^α(Fmoc) (4-Boc amino butylene)glycine (Compound 74) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 79.4% of white solid. mp 150 - 152 °C. TLC (Method D) Rf=0.42-0.47 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0021" num="0021"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry>Found:</entry><entry align="char" char="." charoff="31">66.35</entry><entry align="char" char="." charoff="20">6.84</entry><entry align="char" char="." charoff="20">5.77</entry></row><row rowsep="0"><entry>Calc:</entry><entry align="char" char="." charoff="31">66.06</entry><entry align="char" char="." charoff="20">6.88</entry><entry align="char" char="." charoff="20">5.98</entry></row></tbody></tgroup></table></tables>

<u style="single">EXAMPLE 25:</u>

COMPOUND 79: N^α(Fmoc)(6-Boc amino hexylene)glycine

0218A solution of 0.02 mole of N^α(Fmoc) (6-Boc amino hexylene)glycine (Compound 75) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 81.5% of white solid. mp 78-80 °C. TLC (Method D) Rf=0.7 (one spot) NMR (CDCl₃) in agreement with the title compound. <tables id="tabl0022" num="0022"><table frame="none"><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="13mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><tbody><row><entry rowsep="0">Elemental Analysis:</entry><entry rowsep="0">% C</entry><entry rowsep="0">% H</entry><entry rowsep="0">% N</entry></row><row rowsep="0"><entry align="right">Found:</entry><entry align="char" char="." charoff="31">68.02</entry><entry align="char" char="." charoff="20">7.08</entry><entry align="char" char="." charoff="20">5.37</entry></row><row rowsep="0"><entry align="right">Calc:</entry><entry align="char" char="." charoff="31">67.72</entry><entry align="char" char="." charoff="20">7.31</entry><entry align="char" char="." charoff="20">5.64</entry></row></tbody></tgroup></table></tables>

<u style="single">SYNTHETIC EXAMPLES</u>

0219Two series of octapeptide somatostatin analogs of the present invention were synthesized, characterized, and tested for biological activity (not encompassed in the present invention) <ol id="ol0007" compact="compact" ol-style=""><li>1) The first series of compounds corresponds to the general Formula (XIVb); this series comprises compounds of the specific formula H- (D) Phe-R⁶-Phe- (D) Trp-Lys-Thr-R¹¹-Thr-NH₂ wherein R⁶ and R¹¹ are N^α ω-functionalized alkylene amino acid building units.</li><li>2) The second series of compounds corresponds to the general Formula (XVIc); this series comprises compounds of the specific formula H-(D)Phe-R⁶-Phe-(D)Trp-Lys-R¹⁰-Thr-NH₂</li></ol> wherein R⁶ and R¹⁰ are N^α ω-functionalized alkylene amino acid building units.

0220The structures of these novel synthetic peptide analogs into which N^α ω-functionalized amino acid building units were incorporated, are summarized in Tables 7 and 8. In both series, the building units used were glycine building units in which the bridging groups, attached via the alpha nitrogens to the peptide backbone, were varied.

0221For the sake of simplicity, these two series are referred to herein as the SST Gly⁶,Gly¹¹ and SST Gly⁶, Gly¹⁰ series, respectively.

0222In each series, the position of the cyclization points was constant, while the length and direction of the bridge was varied. Thus, C2,N2 refers to a bridge consisting of an amide bond in which the carbonyl group is closer to the amino end of the peptide and which contains two methylene groups between the bridge amide and each of the backbone nitrogens involved in the bridge.

0223Peptide assembly was carried out either manually or with an automatic peptide synthesizer (Applied Biosystems Model 433A). Following peptide assembly, de-protection of bridging groups that form the cyclization arms was carried out with Pd(PPh₃)₄ (palladium tetrakis triphenyl phosphine) in the case of Allyl/Alloc protecting groups or with TFA in the case of tBu/Boc protecting groups. For obtaining the linear (non-cyclized) analog, the peptides were cleaved from the resin at this stage. Cyclization of the peptides was carried out with PyBOP. Cleavage of the peptides from the polymeric support was carried out with suitable reagents depending on the type of resin used, e.g., with TFA for Rink amide type resins and with HF for mBHA (para-methyl benzhydryl amine) type resins. The crude products were characterized by analytical HPLC. The peptides were purified by preparative reversed phase HPLC. The purified products where characterized by analytical HPLC, mass spectroscopy, and amino acid analysis. <tables id="tabl0023" num="0023"><table frame="all"><title><b>Table 7</b></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="33mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="21mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><b>SST Gly⁶, Gly¹¹</b></entry></row><row><entry valign="top">Example No.</entry><entry valign="top">Bridging Groups</entry><entry valign="top">Compound Number</entry><entry valign="top">Method</entry><entry valign="top">Crude Yield</entry></row></thead><tbody><row><entry>26</entry><entry>C1,N2 Cyclic</entry><entry>DE-3-32-4</entry><entry>1</entry><entry>NA**</entry></row><row><entry>27</entry><entry>C1,N2 Linear</entry><entry>DE-3-32-2</entry><entry>1</entry><entry>NA</entry></row><row><entry>28</entry><entry>C1,N3 Cyclic</entry><entry>PTR 3004</entry><entry>2</entry><entry>79 mg</entry></row><row><entry>29</entry><entry>C1,N3 Linear</entry><entry>PTR 3005</entry><entry>2</entry><entry>34 mg</entry></row><row><entry>30</entry><entry>C2,N2 Cyclic</entry><entry>PTR 3002</entry><entry>1</entry><entry>NA</entry></row><row><entry>31</entry><entry>C2,N2 Linear</entry><entry>PTR 3001</entry><entry>1</entry><entry>NA</entry></row><row><entry>32</entry><entry>C2,N3 Cyclic</entry><entry>PTR 3007</entry><entry>2</entry><entry>40 mg</entry></row><row><entry>33</entry><entry>C2,N3 Linear</entry><entry>PTR 3008</entry><entry>2</entry><entry>40 mg</entry></row><row><entry>34</entry><entry>N2,C2 Cyclic</entry><entry>YD-9-166-1</entry><entry>2</entry><entry>NA</entry></row><row><entry>35</entry><entry>N2,C2 Linear</entry><entry>YD-9-168-1</entry><entry>2</entry><entry>NA</entry></row><row><entry>36</entry><entry>N3,C2 Cyclic</entry><entry>PTR 3010</entry><entry>2</entry><entry>100 mg</entry></row><row><entry>37</entry><entry>N3,C2 Linear</entry><entry>PTR 3011</entry><entry>2</entry><entry>NA</entry></row><row><entry>38</entry><entry>Linear*</entry><entry>PTR 3003</entry><entry>3</entry><entry>96 mg</entry></row></tbody></tgroup><tgroup cols="5" rowsep="0"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="33mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="21mm" /><tbody><row><entry namest="col1" nameend="col5" align="justify">* Linear refers to the identical sequence with Gly residues in place of R⁶ and R¹¹. ** NA denotes not available. Table 7 methods: 1) Manual synthesis on mBHA resin. HF cleavage. 2) Manual synthesis on Rapp tentagel resin. TFA cleavage. 3) Rink amide resin; assembly in automated peptide synthesizer, 0.1 mmol scale.</entry></row></tbody></tgroup></table></tables><tables id="tabl0024" num="0024"><table frame="all"><title><b>Table 8</b></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="41mm" /><colspec colnum="3" colname="col3" colwidth="33mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="21mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="top"><b>SST Gly⁶,Gly¹⁰</b></entry></row><row><entry valign="top">Example No.</entry><entry valign="top">Bridging Groups</entry><entry valign="top">Compound Number</entry><entry valign="top">Method</entry><entry valign="top">Crude Yield</entry></row></thead><tbody><row><entry>39</entry><entry>C1,N2 Cyclic</entry><entry>YD-9-171-3</entry><entry>1</entry><entry>20 mg</entry></row><row><entry>40</entry><entry>C1,N2 Linear</entry><entry>YD-9-171-2</entry><entry>1</entry><entry>10 mg</entry></row><row><entry>41</entry><entry>C1,N3 Cyclic</entry><entry>YD-9-175-3</entry><entry>1</entry><entry>44.9 mg</entry></row><row><entry>42</entry><entry>C1,N3 Linear</entry><entry>YD-9-175-2</entry><entry>1</entry><entry>25.4 mg</entry></row><row><entry>43</entry><entry>C2,N2 Cyclic</entry><entry>PTR 3019</entry><entry>1</entry><entry>40 mg</entry></row><row><entry>44</entry><entry>C2,N2 Linear</entry><entry>PTR 3020</entry><entry>1</entry><entry>26 mg</entry></row><row><entry>4.5</entry><entry>C2, N3 Cyclic</entry><entry>YD-5-28-3-3</entry><entry>3</entry><entry>101.5 mg</entry></row><row><entry>46</entry><entry>C2,N3 Linear</entry><entry>YD-5-28-2</entry><entry>3</entry><entry>48.3 mg</entry></row><row><entry>47</entry><entry>N2,C2 Cyclic</entry><entry>PTR 3016</entry><entry>2</entry><entry>60 mg</entry></row><row><entry>48</entry><entry>N2,C2 Linear</entry><entry>PTR 3017</entry><entry>2</entry><entry>40 mg</entry></row><row><entry>49</entry><entry>N3,C2 Cyclic</entry><entry>YS-8-153-1</entry><entry>'2</entry><entry>93 mg</entry></row><row><entry>50</entry><entry>N3,C2 Linear</entry><entry>YS-8-152-1</entry><entry>2</entry><entry>54 mg</entry></row><row><entry>51</entry><entry>* Linear</entry><entry>PTR 3021</entry><entry>1</entry><entry>100 mg</entry></row><row><entry /><entry /><entry /><entry /><entry /></row><row><entry /><entry>** Acetylated Des-D-Phe⁵</entry><entry /><entry /><entry /></row><row><entry>52</entry><entry>N3,C2 Cyclic</entry><entry>PTR 3013</entry><entry /><entry>67 mg</entry></row><row><entry>53</entry><entry>N3,C2 Linear</entry><entry>PTR 3014</entry><entry /><entry>48 mg</entry></row></tbody></tgroup><tgroup cols="5" rowsep="0"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="41mm" /><colspec colnum="3" colname="col3" colwidth="33mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="21mm" /><tbody><row><entry namest="col1" nameend="col5" align="justify">* Linear refers to the identical sequence with Gly residues in place of R⁶ and R¹⁰. ** Acetylated Des-D-Phe⁵ refers to the same sequence in which the N terminal D-Phe⁵ is absent and the N-terminus is acetylated. Table 8 methods: 1) Assembly in automated peptide synthesizer; 0.1 mmol scale. (HBTU). 2) Manual synthesis; PyBrop. 3) Assembly in automated peptide synthesizer, 0.25 mmol scale. (HBTU).</entry></row></tbody></tgroup></table></tables>

Synthesis of SST Gly⁶, Gly¹⁰ N3,C2:

0224Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelled in N-methylpyrrolidone (NMP) in a reaction vessel equipped with a sintered glass bottom and placed on a shaker. The Fmoc protecting group was removed from the resin by reaction with 20% piperidine in NMP (2 times 10 minutes, 25 ml each). Fmoc removal was monitored by ultraviolet absorption measurement at 290 nm. A coupling cycle was carried out with Fmoc-Thr(OtBu)-OH (3 equivalents) PyBrop (3 equivalents) DIEA (6 equivalents) in NMP (20 ml) for 2 hours at room temperature. Reaction completion was monitored by the qualitative ninhydrin test (Kaiser test). Following coupling, the peptide-resin was washed with NMP (7 times with 25 ml NMP, 2 minutes each). Capping was carried out by reaction of the peptide-resin with acetic anhydride (capping mixture: HOBt 400 mg, NMP 20 ml, acetic anhydride 10 ml, DIEA 4.4 ml) for 0.5 hours at room temperature. After capping, NMP washes were carried out as above (7 times, 2 minutes each). Fmoc removal was carried out as above. Fmoc-Phe-OH was coupled in the same manner, and the Fmoc group removed, as above. The peptide resin was reacted with Fmoc-Gly-C2 (Allyl) building unit: coupling conditions were as above. Fmoc removal was carried out as above. Fmoc-Lys(Boc)-OH was coupled to the peptide resin by reaction with HATU (3 equivalents) and DIEA (6 equivalents) at room temperature overnight and then at 50°C for one hour. Additional DIEA was added during reaction to maintain a basic medium (as determined by pH paper to be about 9). This coupling was repeated. Coupling completion was monitored by the Fmoc test (a sample of the peptide resin was taken and weighed, the Fmoc was removed as above, and the ultraviolet absorption was measured). Fmoc-D-Trp-OH was coupled to the peptide resin with PyBrop, as described above. Following Fmoc removal, Fmoc-Phe-OH was coupled in the same way. Synthesis was continued with one-fifth of the peptide resin.

0225Following Fmoc removal, the second building unit was introduced: Fmoc-Gly-N3 (Alloc)-OH by reaction with PYBrop, as described above. Capping was carried out as described above. Following Fmoc removal, the peptide-resin was divided into two equal portions. Synthesis was continued with one of these portions. Boc-D-Phe-OH was coupled by reaction with HATU, as described above for Fmoc-Lys(Boc)-OH. Capping was carried out as above.

0226The Allyl and Alloc protecting groups were removed by. reaction with Pd(PPh₃)₄ and acetic acid 5%, morpholine 2.5% in chloroform, under argon, for 2 hours at room temperature. The peptide resin was washed with NMP as above. Two-thirds of the resin were taken for cyclization. Cyclization was carried out with PyBOP 3 equivalents, DIEA 6 equivalents, in NMP, at room temperature overnight. The peptide resin was washed and dried. The peptide was cleaved from the resin by reaction with TFA 81.5%, phenol 5%, water 5%, EDT 2.5%, TIS (triisopropyl-silane) 1%, and 5% methylene chloride, at 0°C for 15 minutes and 2 hours at room temperature under argon. The mixture was filtered into cold ether (30 ml, 0°C) and the resin was washed with a small volume of TFA. The filtrate was placed in a rotary evaporator and all the volatile components were removed. An oily product was obtained. It was triturated with ether and the ether decanted, three times. A white powder was obtained. This crude product was dried. The weight of the crude product was 93 mg.