CA2192305A1 - Conformationally constrained backbone cyclized peptide analogs - Google Patents

Abstract

Novel backbone cyclized peptide analogs are formed by means of bridging groups attached via the alpha nitrogens of amino acid derivatives to provide novel non-peptidic linkages. Novel building units disclosed are N.alpha./(.omega.-functionalized) amino acids constructed to include a spacer and a terminal functional group. One or more of these N.alpha.(.omega.-functionalized) amino acids are incorporated into a peptide sequence, preferably during solid phase peptide synthesis. The reactive terminal functional groups are protected by specific protecting groups that can be selectively removed to effect either backbone-to-backbone or backbone-to-side chain cyclizations. The invention is exemplified by backbone cyclized bradykinin antagonists having biological activity. Further embodiments of the invention are somatostatin analogs having one or two ring structures involving backbone cyclization.

Description

21 923~
WO95/33765 rcl~.',r ''-CONFORNATI~T~r~Y CONSTRAINED P~rRRO~ cy~T,T7.Rn PE~TIDE ANALOGs FIELD OF T~E INVENTION

The present invention relates to conformationally constrained N~ backbone-cyclized peptide analogs cyclized via novel non-peptidic linkages, to novel N~,~-functionalized 10 amino acid building units, to processes for the preparation of these backbone cyclized peptides and building units, to ~ -methods for using these peptide analogs and to pharmaceutical compositions containing same.

BACRGRO~ND OF T~E lNV~Ll~N

Pe~tidomimetics As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared 20 in ~uantities sufficient for pharmacological and clinical utilities. Thus in the last few years new methods have been est~hl;qh~d for the treatment and therapy of illnesses in which peptides have been implicated. ~owever, the use of peptides as drugs i8 limited by the following factors: a) 25 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 30 kidneys; and d) their undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism.
Moreover, with few exceptions, native peptides of small to medium size (less than 30-50 amino acids) exist unordered 35 in dilute a~ueous solution in a multitude of conformations in dynamic e~uilibrium which may lead to lack of receptor selectivity, metabolic susceptibilities and hamper attempts to ... . .. . . .. .. _ . _ .. . . .. . . . . _ _ _ _ _ _ _ _ _ . .

21 923()$ ' ' WO 95J3376S F_lll.,,S.'C

determine the biologically active conformation. I~ 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 ~s 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 p~pt;~r.
In recent years, intensive efforts have been made to develop pepti~r-; tics or peptide analog6 that display more 10 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 dev~l~ L ~nt of these pept;~ ~ tics. However, a major problem in the development 15 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. Cnce this biologically active site is determined a 20 lead structure for development of peptidomimetic can be optimized, for example by molecular r '~l ;ng ~LUyL~",3.
As 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 25 receptor level. The following factors should be ~onrid~red to achieve the best possible agonist pepti,~ --t;c a) metabolic stability, b) good bicavA;lAhil;tyl c) high receptor affinity and receptor selectivity, and d) minimal side effects.
From the pharmacological and medical viewpoint it i5 30 fre~uently desirable to not only imitate the effect of the peptide at the receptor level (agonism) but also to block the receptor when re~uired (Antng~n;rm) The aame phArr--olog;
considerations for designing an agonist pept;~ tic mentioned above hold for designing peptide AntAg~n;rts, but, 35 in addition, their development in the absence of lead structures is more ~;ff;C-llt. Even today it is not SUBSTITUTE SHEET (RULE 26) 2 1 9230~
095/33765 P~~ 5'~C''~

une~uivocally clear which factors are decisive for the agonistic effect and which are for the antagonistic effect.
A generally applicable and successful method recently has been the development of conformationally restricted 5 pepti~; t;cs that imitate the receptor-bound conformation of the endogenous peptide ligands as closely as possible (Rizo and Gierasch, Ann. Rev. ~iochem., 61:387, 1992).
Investigations of these types of analogs show them to have increased resistance toward proteases, that is, an increase in lO metabolic stability, as well as increased selectivity and thereby fewer side effects (Veber and Friedinger, Trends Neurosci.. p. 392, 1985).
Once these pept~ ; 'ic compounds with rigid conformations are produced, the most active structures are 15 selected by studying the conformation-activity relAti~nch;ps.
Such conformational constraints can involve short range (local) modifications of structure or long range (global) conformational restraints (for review see Giannis and Kolter, ~n~eW~ Chem. Int. Ed. Enal, 32:1244, 1993).
20 Conformati~n~llv cgnstr~;n~ ~e~tides ~ ridging between two n~;shhnring amino acids in a peptide leads to a local confor~-t;~n~l modification, the flexibility of which is limited in comparison with that of regular dipeptides. Some possibilities for forming such bridges 25 include incorporation of lactams and piper~7;n~n~R. y-~actams and ~-lactams have been designed to some extent as "turn tirq~; in several ca3es the incorporation of such 3tructures into peptides leads to biologically active compounds.
Global restrictions in the conf~r~-t;~n of a peptide are possible by limiting the f 1~; h; 1; ty of the peptide strand through cy~1;7~ti~n (Hruby e~ al., Biochem. J.. 268:249, 1990). Not only does cyclization of bioactive peptides improve their --t~hol;c stability and receptor selectivity, 35 cy~l;zatior algo impoges constraints that enhance confor~-t;on~l homogeneity and f~ ;t~t~ confor7-t;~n~l analysis. The common modes of cy~l;7at;~n are the same found SU3STITLITE SHEET (RULE 26) W095l33765 2 1 9 2 3 o 5 r~

in naturally occurring cyclic peptides. These include side chain to Gide chain cyclization or side chain to end-group cyrl;7~t;nn- Eor this purpose, amino acid side chains that are not involved in receptor recognition are connected 5 together or to the peptide barkhnnP. Another common cyclization is the end-to-end cyclization.
Three representative examples are compounds wherein partial structures of each peptide are made into rings by linking two penir;llAm;nP residues with a ~;CI1lfi~P bridge 10 (Mosberg et al., P.N.A.S. U5, 80-5871, 19831, by formation of an amide bond between a lysine and an aspartate group (Charpentier et al., J. Med. Chem. 32:1184, 1989), or by connecting two lysine groups with a succinate unit (Rodriguez et al., Int. J. Pe~t. Protein Res. 35:441, 1990). These 15 structures have been disclosed in the literature in the case of a cyclic Pnk~r~l ;n analog with selectivity for the ~-opiate receptor (Mosberg et al., ibid.); or as agonists to the cholecystokinin B receptor, found largely in the brain (Charpentier et al., ibid., Rodriguez et al., ibid ).
The main limitations to these rlAccir~l modes of cyclization are that they rer~uire substitution of amino acid side chains in order to achieve cyclization.
Another:conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., 25 (Bio~olvmers, 31:745, 1991) who propo8ed h~rkho~P to b~rkh~nP
cyrl;7stinn of peptides. The theoretical advantages of this strategy include the ability to effect cyrl;7at;rln via the carbons or nitrogens of the peptide bsrkh~np without interfering with side chains that may be crucial for 30 interaction with the specific receptor of a given peptide.
While the concept was envisaged as being ~rplic~hl~ to any linear peptide of interest, in point of fact the limiting factor in the proposed scheme was the av~ h;l;ty of suitable building units that must be used to replace the amino acids 35 that are to be linked via bridging groups. The actual reduction to practice of this concept of barkhrn~ cyclization was prevented by the inability to devise any practical method SUBSTITUTE SHEET ~RULE 26) 2 1 92~Q~5 95/33765 r~.,~ ~/~ --of preparing building units of amino acids other than glycine (Gilon et al., J. Orq. Chem., 587:5687, 1992). While analogs of other amino acids were attempted the synthetic method used was unsucces~ful or of guch low yield as to preclude any 5 general applicability.
In Gilon, EP3 Application No. 564,739 A2; and J. Orq.
Chem,, 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 ~ bromo acid. Selective 10 protection of the ~ amine and further elaborations of protecting groups provides a building unit, suitable for Boc chemistry peptide synthesis. The ~econd approach starts with selective protection of a diamine and reaction of the product with chloroacetic acid to provide the protected glycine 15 derivative, suitable for Fmoc peptide synthesis.
Both 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 20 chain which is terminated by another functional group, amine in the examples described, which may or may not be blocked by a protecting group.
In all cases the ~ nitrogen of the end product originates in the molecule which becomes the bridging chain for 25 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.
In a molecule where R is different than hydrogen there is a high tendency to eliminate HX under basic conditions. This 30 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 35 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 .... . . . .. .. , .. . . _ _ _ _ _ _ .

21923~5 W095/3376s rcl~,5~

than nitrogen, 60 the only cyclization schemes pos~ible are hArkhnn~ to side chain and b~rkhrnp to C terminus.
A~lications of conformationallv constrained ~e~tides Conformationally constrained peptides find many 5 pharmacological uses. Somatostatin is a cyclic tetradecapeptide found both in the central nervous system and in peripheral tissues. It was originally ;cr,lAt~d from , - l;An hypothAlAmllR and ;~nt;fied as an important inhibitor of growth hormone secretion from the anterior 10 pituitary. Its multiple b;o10g;rAl activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut l l-c and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central 15 nervous system (for review see Lamberts, Fndocrine Rev., 9:427, 1988).
Natural somatostatin (also known as Somatotropin Release Inhibiting Factor, SRIF) of the following structure:

20 H-Alal-Gly2-Cys3-Lysi-Asns-Phe6-Phe'-TrpC-Lys9-Thrl0-Phell-ThrI2-Serl'-cysl4-oH

was first isolated by Gn;llPmin and colleagues (Bruzeau et al.
Scie~ce, 179:78, 1973). In its natural form, it has limited 25 use as a therapeutic agent since it exhibits two undesirable properties: poor bioavA;lAh;l;ty 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, ~nrAt~rn of 30 action or selectivity with regard to inhibition of the release of growth hormone, insulin or glucagon.
Structure-activity relation studies, spectroscopic techniques guch as circular dichroism and nuclear ~~gn~t; r r~crnAnre, and molecular ~~l;ng approaches reveal the 35 following: the conformation of the cyclic part of natural somatostatin is most likely to be an antiparallel ~'-sheet;
Phe6 and Phell play an important role in stabilizing the SU5STITUTE SHEET (RULE 26) 21~Z305 095/3376~ ~"~,5,~

pharmacophore conformation through hydrophobic interactions between the two aromatic rings; the four amino acids Phe7-TrpP-~ys9-Thr10 which are spread around the ~-turn in the antiparallel ~-sheet are PA~Pnt;Al for the pharmacophore; and 5 (D)Trp8 is almost always preferable to (D)Trp8.
Nevertheless, a he~dp~Lide somatostatin analog rnntA;ninS these four amino acids anchored by a disulfide bridge:

10 Cys-phe7-(D)Trp3-Lys9-Thrlo-cy8 is almost inactive both in vitro and in vlvo, although it has the advantage of the covalent disulfide bridge which replace6 15 the Phe6-Phell hydrophobic ;ntPrArtinnr in natural somatostatin.
Four main approaches have been attempted in order to increase the activity of this hexapeptide somatostatin analog.
(l) RPplAr;ng the disulfide bridge by a cyclization which 20 encourages a cis-amide bond, or by performing a second cyr7; 7~t;nn 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 se~uence Phe'-(D)Trp2-Lysg-Thrl0 with more 25 potent amino acid analogs, such as rpplAr;ng Phe7 with Tyr7 and Thrl~ with Val10. (3) Incorporating additional structural elements from natural somatostatin with the ;rtPnt;nn that these new elements will contribute to the interaction with the receptor. (4) ~l;m;nAt;ng one of the four amino acids 30 Phe7-(D)Trp8-Lys9-Thrl0 with the assumption that such analogs would be more selective.
The somatostatin analog, MK-678:

cyclo(N-Me-Ala7-Tyr7-(D)Trp8-Lys9-Vall0-Phe) is an example of a highly potent somatostatin analog designed using the first three approaches above (Veber, et al., Life SUBSTITUTE SHEET (RULE 26) _ _ _ _ 21 92~05 w09~765 ..l/~.''~ -' Science, 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 Phel1 is incorporated from natural somatostatin.
Another group of somatostatin analogs (U.S. patents 4,310,518 and 4,235,886) includes octreotide:
H-(D)Phe-Cys-Phe -(D)Trp8-Lys9-Thr10-Cys-Thr-CH2OH

10 the only somatostatin analog currently available. It was developed using the third approach described above. Here, (D)Phes and the reduced C-terminal Thr1Z-CH2OH are assumed to occupy some of the conformational space available to the natural Phe6 and Thr17, respectively.

The compound TT2-32:
H-(D)Phe-Cys-Phe -(D)Trp8-Lys -Cys-Thr-NH2 I

is closely related to octreotide and is an example of impl~ -- ; ng the fourth approach described above The lack of Thr10 is probably r~crnncihle for its high selectivity in terms of antitumor activity.
These ~ ~ PR of highly potent somatostatin analogs indicate that the phenyl~l An; n~c in positions 6 and 11 not only play an important role in 5t~h;1 i7;ng the phar~-cnphnre conformation but also have a functional role in the interaction with the receptor. It is still an open question 30 whether one phenyl~lcn;n~ (either Phe6 or Phe1') is snff;~i~nt for the interaction with the receptor or whether both are needed.
It is now known that the somatostatin receptors constitute a family of five different receptor subtypes (Bell 35 and Reisine, Trends Neurosci., 1~ 34-38, 1993), which may be distinguished on the basis of their tissue specificity and/or biological activity. Somatostatin analogs known in the art SUBSTITUTE SHEET(RULE 26) 2 1 9230~
09sl3376s r~l,~,ic-~ss may not provide sufficient selectivity or receptor subtype 6electivity, particularly as anti-neoplastic ayents (Reubi and Laissue, IL~, 16, 110-115, 1995).
Symptoms associated with metastatic carcinoid tumors 5 (flushing and diarrhea) and vasoactive intestinal peptide (VIP) secreting ~n~ ~ (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 10 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.
Another important peptide, sradykinin, is a naturally 15 occurring nonapeptide, Argl-Pro7-Pro3-Gly~-Phes-Ser6-Pro7-Phe8-Arg9, formed and released from precursors in the blood in response to ;nf1: tory stimuli. Elevated levels of bradykinin also appear in other body fluids and tissues in pathological states 20 such as asthma, septic shock and common cold. ~o clinical abnormalities have been associated 80 far with bradykinin deficiency which indicates that bradykinin may not play a critical role in normal physiology.
~owever, bradykinin mediates its physiological activities 25 by binding to a specific receptive molecule called the bradykinin receptor. Two such bradykinin receptors have been i~n~;f;ed 80 far (these are called B1 and B2 receptors).
Subsequent to binding, the bradykinin signal transduction pathway includes production of prcs~gl~n~;n~ and leukotrienes 30 as well as calcium activation. ,Through these ~;a~ors, bradykinin is involved in pain, ;nf1; t;on, 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 35 therapeutic value for one of the following indications:
asthma, ;nf1: tion, septic shock, pain, hypotension and allergy.
_ g _ SUBSTITUTE SHEET (RUEE 26~

W09s/~76s P~l/~,5:C[I55 The analog used herein t~o exemplify backbone cyclization i5:
D-Arg~-Arg-R3-Hyp3-Gly-Phe-R2-D-Phe-Phe'-Arg (wherein, R1 is Pro, R2 is Ser in native bradykinin). The 5 change of proline at position 7 of native bradykinin to D-Phe confers antagonist activity. This , ~ ~ was described in Steranka, et al., P.N.A.S, U.S., 85:3245-3249, 1988 and is one of a plethora of r~n~ te sequences for r-~; f i cAtion by the current technology, i.e. h~rkhnnP cyclization. In this 10 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 15 sequences.
According to the present invention a novel synthetic approach is disclosed providing N3(~(functinn~1;7ed)alkylene) amino acid building units that can be used to synthesize novel N~-barkhnnP cyclized peptide analogs such as, but not limited 20 to, novel somatostatin and bradykinin analogs. None of the above- -; nnPi references teaches or suggests N3-(~(functinn~1;7P~)alkylene) amino acids or the novel N3-bPrkhnnP cyclized peptide analogs of the present invention.

SUMMARY OF ~EE lNV~~

It is an object of the present invention to provide harkhnnP cyclized peptide analogs that comprise peptide sequences which in~ Ldte at least two bn;l~;ng units, each 30 of which cnnt~;n~ one nitrogen atom of the peptide b~ckhnnp rnnnPctpd to a bridging group as described below. In the present invention, one or more pairs of the bn;l~;nr~ units is joined together to form a cyclic structure. Thus, according to one aspect of the present invention, backbone cyclized 35 peptide analogs are provided that have the general Formula (I):

SUBSTITUTE SHEET (RULE 26) 2 t ~23~
095/3376S ~ S~-l55 ~ I I
lI2N-(AA)d-CO-N-C~tR)-(AA)a-CO-N-(AA.) -CO-N-CE~R')-(AA)b-CO-N-(AA)f-CO-E

Formula (I) wherein: a and b each independently designates an integer from 1 to 8 or zero; d, e, and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same 10 or different; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or CO-E can be reduced to CH,-OH; R and R' each designates an amino acid side-chain such as H, CH3, etc., optionally bound with a specific protecting group; and the lines independently designate a bridging group 15 of the Formula: (i) -X-M-Y-W-Z- ; or (ii) -X-M-Z-wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, amide, thioetherr thioesters, imines, ethers and alkenes; and X, Y
and Z are each independently selected from the group 20 consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene.
In certain preferred embodiments, the CO-E group of Formula (I) is reduced to a CH2OH group.
Another embodiment of the present invention involves N-25 hSrkh~n~ to side chain cyclized peptides of the generalformula (II):

H2N-(AA)d-CO-N-CH(R)-CO-NH-(AA)e-CO-NH-CH-C3-NH(AA)f-CO-E

Formula (II) wherein the substituents are as defined above.
A preferred embodiment of the present invention involves the harkh~n~ cyclized peptide analog of Formulae I or II
35 wherein the line designates a bridging group of the Formula:
-(CH2)x-M-(cH2)y-w-(cH2)z- wherein M and W are independently selected from the group consisting of disulfide, amide, SUBSTITUTE SHEET (RULE 26) ~1 92305 W0 95/33765 r~

thioether, thioesters, imines, ethers and alkenes; x and z each independently designates an integer from l to=10, and y is zero or an integer of from 1 to 8, with the proviso that if y is zero, W is absent.
Further preferred are hA~khnnP cyclized peptide analogs of the Formula I or II wherein R and R' are other than H, such as CH3, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, CH3S(CH2) 2- ' HOCH,-, CH3CH(OH)-, HSCH2-, NH2C(=O)CH2-, NH2C(=O)(CH2) 2- ~
NH2(CH2)3-, HOC(=O)CH2-, HOC(=O)(CH2) 2-, NH2(CH,) 4-, C (NH2) 2 10 NH(CH2)3-, HO-phenyl-CH2-, benzyl, methylindole, and methylimidazole.
A more preferred ~ of the pre~ent invention is directed to bA~khnnp cyclization to stabilize the ~-turn conformation~of bradykinin analogs of the general Formula 15 (III):

(CH2)x ~ (CH2) Z
K-~D)Arg-Arg-N-Gly-Hyp-Gly-N-Phe-Ser-(D)Phe-Phe-Arg ao Formula III
wherein M is an amide bond, x and z are each independently an integer of 1 to 10, and K is H or an acyl group.
Also more preferred are backbone cyclized peptide analogs 25 of the present invention comprising bradykinin analogs of the general Formula (IVa):

(CH2)X M (CH2)z K-(D)Arg-Arg-N-Gly-Hyp-Gly-Phe-N-R6-(D)Phe-Phe-Arg Formula (IVa) wherein M is an amide bond, x and z are each ;n~p.~ ly an integer of 1 to 10, K is H or an acyl group, and R6 is Gly or 8er; or the general Formula (IVb):

SUBSTITUTE SHEET(RULE 2 2 1 ~2~ ~
W095/33765 P~ 5 HN CO
(CH2)x ( 2~z K-(D)Arg-Arg-N-Gly-Hyp-Gly-Phe-(R )-(D)Phe-Phe-Arg Formula (IVb) wherein x is an integer of 1 to 10; K is H or an acyl group;
(R6) is selected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z is according to the amino acid specified: 1 in 10 case of D and L-~sp, and 2 in the case of D and L Glu.
Further more pre~erred b~nkhnnr cyclized peptide analogs accordiny to the present invention having bradykinin antagonist activity have the Formula (V):

(CH2)X M (CH2)z ~-(D)Arg-Arg-Pro-Hyp-N-Gly-Phe-Ser-N-(D)Phe-Phe-Arg Form. ula (V) wherein M i5 an amide bond, x and z are each independently an 20 integer of 1 to 10, and K is H or an acyl group.
Specifically preferred h~c~hnn~ cyclized peptide analogs of the present invention are:
1) Ada-(D)Arg-Arg-cyclo(N~(1-(6-Am;nohPYylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;
25 2) H-D-Arg-Arg-cyclo(N~(1-(4-propanoyl))Gly-Hyp-Phe-N~(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH; and 3) H-D-Arg-Arg-cyclo(N~(4-propanoyl)Gly-Hyp-Phe-N~(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.
Another preferred aspect of the p~esent invention i8 30 directed to backbone cy~l;7atio~n to generate novel somatostatin analogs linked between positions 6 and ll, leaving the pheny~cl~n;n~ side chains lln~ollrh~d This confor~-~;nn~l 5~h;1;7ation is much more rigid than the Phe6, Phe1~ hydrophobic interaction in natural somatostatin and is 35 more stable to reduction/oxidation r~c~;n~c than the Cys-Cys disulfide bridge. In other words, for the first time a stable SUBSTITUTE SHEET (RUEE 26) W095/33765 21 ~ 2 3 0 5 r l,~ s ~ - ~

covalent bridge can be achieved vhile either one or both of the original Phe6 and Phell are r~t~;n~d.
Moreover, bA~kh~n~ cyclizations can also be used to anchor the ~7-turn, not only in positions 6 and 11 but also 5 inside the active reaction of Phe7-(D)Trp6-Lys9-Thrl0, yielding either a monocyclic analog with a preferable conformation or a very rigid bicyclic analog. Here again, the side chains of the pharmacologically active amino acids remain untouched and the only change is in limiting the conformational space.
As used herein and in the claims in the following more preferred hackhnn~ cyclized peptide analogs, the superscript numbers following the amino acids refer to their position numbers in the native Somatostatin.
A more preferred hA~kh~n~ cyclized peptide novel analog 15 is the Formula (XIVa):
R5-NR6-R7-~D)Trp-LyS-RlO-NRll-R -X

L (CH2)m~Y ~(C~2)n Formula (XIVa) with a most preferred analog being the Formula (XIVb):
25 H- (D)Phe-NR6-R7- (D)TrP-~YS-R1O-NR11-Thr-X
L(CH2) m~Y ~(CH2)n Figure (XIVb) wherein m and ~ are l, 2 or 3; X is CH20H or CONH2; Rs is absent or is Gly, ~D)- or ~L)-Ala, Phe, Nal and ~-Asp~Ind); R6 and Rll _re ;n~p~n~n~ly Gly or ~D)- or ~L)-Phe; R7 is Phe or Tyr; Rl~ is absent or is Gly, Abu, Thr or Val; Rl7 is absent or is Thr or Nal, and y2 is selected from the group consisting of 35 amide, disulfide, th;~eth~r, imines, ethers and alkenes. In these monocyclic somatostatin analogs, a bA~kh~n~ cyclization replaces the Cys6-Cysll ~ lf;~ bridge, leaving the SUBSTITUTE SHEET (RULE 26) ~1 923~5 095l3376s F~~ t'' phenylAlAn;n~ side chains as in the natural somatostatin.
Still more preferred is the analog wherein Phe7 is replaced with Tyr7 and Thrl~ is replaced with Vall~.
Other more preferred monocyclic analogs that anchor the 5 molecule in positions inside the active region rather than in positions 6 and 11 are formulae XV (a and b) and XVI (a-c):

~ (CH2 ) i-Y - (CH2 ) jl R5-R6-NPhe - (D)Trp - NLys-R10-Rll-Rl2-X
Formula (XVa) ~(CH2)i-Y -(CH2)jl 15 H-(D)Phe-R -NPhe - (D)Trp - NLys-Thr-R11-Thr-X
Formula (XVb) ~(CH2)i-Y -(CH2)jl R5-R6-NPhe-(D)Trp- Lys-NR10-Rll-Rl2-X
Formula (XVIa) ~(CH2)i-Y -(CH2)jl H-(D)Phe-R6-NPhe- (D)Trp-Lys-NR10-Rll-Thr-X
Formula (XVIb) ~ ( CH2 ) i--Y ( CH2 ) j 1 R5-NR6 -NPhe-(D)Trp-Lys-NR10-Phe-Rl2-X
- Formula (XVIc) wherein i and j are ;n~er~n~ntly 1, 2 or 3; X is CH20H or 35 CON~2; Rs is aksent or is (D)- or (L)-Phe, Nal, or ~7-Asp(Ind);
R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr; and SUBSTITUTE SHEET (RULE 26) ~ ~23Q~
W095/33765 I~~

is (D)- or (L)-Phe; ~12 i8 absent or is Thr or Nal, and Yl i8 selected from the group consisting of amide, disulfide, thioether, imine~, ethers and alkenes.
Still other more preferred analogs incorporate barkhr~nr 5 cyclization=in positions 6 and 11 as in Formula XIV, t.ogether with the hackhrnr cyclizations as in Formula XV and XVI, yielding rigid bicyclic analogs of the Formulae XVII (a and b) and XVIII (a and b):

~(CH2) i-Y - (CH2) jl R5~NR6-NPhe - (D)Trp - NLys_R10_~Rl1_R12_x (CH2)m - Y - (CH2)n Formula (XVIIa) ~(CH2) i-Y - (CX2) jl H-(D)Phe-NR -NPhe - (D)Trp - NLys-Thr-NR -Thr-X
( CH2 ) m - Y - ( CH2 ) n Formula (XVIIb) ~ (CH2) i-Y - (CH2) il R5-NR6-NPhe-(D)Trp-~ys- NR10-NR l-Rl -X
~ (CH2)m~Y ~(CH2)n Formula (XVIIIa) (CH ) yl (CH ) 30 H-(D)Phe-NR6-NPhe- (D)Trp -Lys-NR10-NRll-Thr-X
(CH2)m~Y ~(CH2)n Formula (XVIIIb) wherein i, j, m and n are independently 1, 2 or 3; X i8 35 CHlOH or NH,; Rs is absent or is (D)- or (k)-Phe, Nal, or ~-Asp(Ind); R~ and Rll are independently Gly or (D)- or (L)-Phe;

2 1 9230~
W095/3~765 P~1~,5'G -' Rl~ i~ dbsent or is Gly, Abu, Val or Thr; Rl2 is absent or is Thr or Nal; and yl and y2 are independently selected from the group consisting of amide, disulfide, thioether, imines, ethers and alkenes.
Other mor~ preferred bicyclic analogs differ from Formulae XVII and XVIII by the replacement of the amino acids at positions 6 and ll by cysteines which form a disulfide bond, leaving only one h~rkhn"~ cyclization in the XIX (a and b) and XX (a and b):

(CH ) yl (CH ) R -Cys- NPhe - (D)Trp - NLys-RlO-Cys-Rl2-X

Formula (XIXa), (CH ) yl (CH ) H-(D)Phe-Cys- NPhe - (D)Trp - NLys-Thr-Cys-Thr-X

Formula (XIXb), (CH ) yl (CH ) R -Cys-NPhe- (D)Trp-Lys- NRlO-Cys-Rl2-X

Formula (XXa), and (CH ) yl (CH ) H-(D)Phe-Cys-NPhe -(D)Trp- Lys- -Cys-Thr-X

Formula (XXb~

wherein i and j are independently l, 2 or 3; X is CH20H
or NHI; Rs is absent or is (D)- or (L)-Phe, Nal, or ~-- 17 _ Sl ia5TiTUTE S~iEE~ F-u WO95/33765 r~l~,5:~[~55 Asp(Ind); R6 and Rll are ;n~Pp~n~n~ly Gly or Phe; R~~ i9 absent or is Gly, Abu or Thr; R~2 is absent or is Thr or Nal; and i9 selected from the group consîsting of amide, disulfide, thioether, imines, ethers and alkenes.
Another aspect of the present invention is a method for the preparation of cyclic peptides of the general Formula (I):

~12N~ d-CO-N-CH ~R) - (AA) -CO-N- (A~) e-CO-N-C~I (R' ) - (A~) b-CO-N- (AA) f -CO-~

Formula ~I) wherein: a and b each independently designates an integer from l to 8 or zero; d, e, and f each independently designates 15 an integer from 1 to 10; (AA) designates an amino~acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carbcxyl protecting group or an amino group, or CO-E can be reduced to CH2-OH; R and R' each designates an amino acid side-chain 20 optionally bound with a specific protecting group; and the lines designate a bridging group of the Formula:
(i) -X-M-Y-W-Z- ; or~ (ii) -X-M-Z-wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, 25 amide, thioether, thioesters, imines, ethers and alkenes; and X, Y and Z are each ;n~p~n~n~ly selected from the group consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene. This method comprises the steps of incorporating at least one N~
30 functinn~1; 7~ derivative of amino acids of Formula (VI):

s-N-CH(R~)-CO-OH
X
A G
Formula (VI) ~ 219~05 wosst~76s P~~ r~

wherein X i8 a spacer group selected from the group con6isting of alkylene, substituted alkylene, arylene, cycloalkylene and sub6tituted cycloalkylene; R' is an amino acid side chain, optionally bound with a specific protecting group; B is a 5 protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is a functional group selected from the group con6isting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting 10 group of G; into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence or with another ~-functionalized amino acid derivative.
A further object of the present invention is directed to 15 building units known as a N~-~-functi~n~liz~ derivatives of the general Formula (VI) of amino acids which are prerequisites for the cyclization process:

_ B-N-C~R)-CO-0 X

A - G

Formula (VI) 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 30 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 35 group thereof.
Preferred building units are the ~-functionalized amino acid derivatives wherein X is alkylene; G is a thiol group, an W095/33765 ~1 923~5 P~

amine group or a carboxyl group; R is phenyl, methyl or isobutyl; with the proviso that when G i8 an amine group, R is other than X.
Further preferred are ~-functionalized:amino acid 5 derivatives wherein R is protected with a specific protecting group.
More preferred are ~-fnn~t;~n~l; 7~d amino acid derivatives of the F~ 1 A~:

B-N-CH(R)-CO-OH B-N-CH(R)-CO-OH B-N-C~(R)-CO-OH
X X -X
A - NH , A - CO and A S

15 wherein X, R, A and B are as de_ined above.
Sp~;fi~lly preferred ~-functinn~l;7ed amino acid derivatives include the following:
1) N~-(Fmoc)(3-Boc-amino propylene)-(s)phenyl~l~n;n~;
2) N~-(Fmoc)(3-Boc-amino propylene)-(R)Phenyl~l~n;n~;
20 3) N~-(Fmoc)(4-Boc-amino butylene)-(S)Phenyl~lAn;n~
~) N~-(Fmoc)(3-Boc-amino propylene)-(S)Alanine 5) N~-(Fmoc)(6-Boc-amino hexylene)-(S)Alanine;
6) N~-(Fmoc)(3-Boc-amino propylene)-(R)Alanine;
7) N~-(2-(benzylthio)ethylene)glycine ethyl ester;
25 8) Nh-(2-(benzylthio)ethylene)(S)leucine methyl ester;
9) N~-(3-(benzylthio)propylene)(S)leucine methyl ester:
10) Boc-N~-(2-(benzylthio)ethylene)glycine;
11) Boc-N~-(2-(benzylthio)ethylene)(s)phenyl:llAn;n~;
12) Boc-N~-(3-(benzylthio)propylene)(S)phenyl~l~n;n~;
30 13) Boc-L-phenylalanyl-N3-(2-(b~nzylthio)ethylene)glycine-ethyl ester;14) Boc-L-phenylalanyl-D~-(2-(benzylthio)ethylene)-(S)phenyl~l~n;n~ methyl ester;15) N~(Fmoc)-(2-t-butyl carboxy ethylene)glycine;
35 16) N~(Fmoc)-(3-t-butyl carboxy propylene)glycine;
17) N~(Fmoc)(2-t-butyl carboxy ethylene)(S)phenyl~l~n;n~;

SUBSTITUTE SHEET (RULE 2~) ~ WOgs/3376s ~1923 05 ~ c~l .

18) N~(Fmoc)(2-Boc amino ethylene)glycine;
19) NP(Fmoc)t3-Boc amino propylene)glycine;
20) N~(Fmoc)(4-Boc amino butylene)glycine; and 21) N~(Fmoc)(6-Boc amino hexylene)glycine.
Novel, practical, generally applicable processes for the preparation of these N~-~-functionalized derivatives of amino acids are a further aspect of this invention.
As such, an object of this invention is a method of 10 making an ~-functionalized amino acid derivative of the general Formula:
B-N-CH(R)-CO-OH
X

wherein X is a spacer group gelected 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, Cx3, etc.; A and B are 20 protecting groups selec~ed from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls;
comprising the steps of:
i) reacting a diamine compound of the general Formula:

B-NH

X
A NH

wherein A, B and X are as defined above, 3~ 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:

B-N-CH(R)-CO-E
X
A NH

~ 1 9~05 WO95/33?65 r~ r '~

wherein A, s, ~, R and X are as defined above ii) and deprotecting the carboxyl to yield an N~-funct;~n~l;7ed amino acid derivative, wherein the ~-functional group is an amine.
A further object of this invention is a method of making an ~-funct;rn~ ed amino acid derivative of the general Formula:
B-N-CH(R)-CO-0~ 1 X
A - S

where B i8 a protecting group selected from the group of substituted alkyloxy, substituted alkyloxy, or aryloxy 15 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, thio 20 ethers or aryl or substituted aryl thio ethers;
comprising the steps of:
i) reacting a , _o--n~ of the general Formula B-NH-X-S-A
with a triflate of the general Formula CF3SO~-0-CH(R)-CO-E
wherein B is a carboxyl protecting group and A, X and R are as 25 defined above, to give a compound of the Formula:
B-N-CH(R)-CO-E
X
A S

ii) selectively removing the protecting group E, and iii) protecting the free amino group to yield an N~
functionalized) amino acid derivative, wherein the ~-35 fllnrt;rn~l group is a thiol.

SUBSTITUTE SHEET (RULE 26) 21 ~23Q~
095/3376s ~ ~,5'~ ''' A further object of this invention is a method of making an ~-funct;nn~l;zed amino acid derivative of the general Formula:
B-N-CH(R)-C0-OX
. I
X
--CO
where B i9 a protecting group selected from the group of alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the 10 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 15 aryl esters or thio esters;
comprising the steps of:
i) reacting a compound of the general Formula B-NH-X-C0-A
with a triflate of the general Formula CF3S0l-0-CH(R)-C0-E
wherein E is a carboxyl protecting group and A, B, X and R are 20 as defined above, to give a compound of Formula:

B-N-CH(R)-C0-E
X
25 A _ C0 ii~ and selectively removing protecting group E, to yield an N~(~-funct;nn~ ed) amino acid derivative~ wherein the ~-functional group is a carboxyl.
A further aspect of this invention is to provide methods . for the preparation of novel backbone cyclic peptides, - comprising the steps of incorporating at least one N~-~-functionalized derivatives of amino acids into a peptide sequence and subsequently selectively cyclizing the functional 35 group with one of the side chains of the amino acids in said peptide sequence, or with another ~-functionalized amino acid derivative.

21 ~23~5 W095/3376s r~l~,3~

sackbone cyclized analogs of the present invention=may be used as pharmaceutical compositions and for methods for the treatment of disorders including: acute asthma, septic shock, brain trauma and other traumatic injury, post-surgical pain, 5 all types of ;n~ tion, cancers, endocrine disorders and gastrointestinal disorders.
Therefore, iurther objects of the present invention are directed to pharmaceutical compositions comprising pharmacologically active backbone cyclized peptide agonists 10 and antagonists prepared according to the methods disclosed herein and a pharmaceutically acceptable carrier or diluent;
and methods for the treatment of ;nfl i tion, septic shock, cancer or endocrine disorders and gastrointestinal disorders therewith.
BRIEF DESCRIPTION OF T~E DRAWINGS

Figure 1 is a graph bhowing in vitro biostability of somatostatin and three analogs thereof in human serum. The 20 graph depicts the perce~tage of undegraded molecules for each of the compounds initially and after various periods of time.

nET~TT.Fn DES~LlUN OF T~E INVENTION

2S Defin;tions All abbreviations used are in accordance with the IUPAC-IU3 r~ 2tions on Biochemical N ~nCli~ture (J~ Bic~.
Chem., 247:977-983, 1972) and later~supplements.
As used herein and in the claims, the phrase "an amino 30 acid side chain" refers to the distinguishing substituent attached to the ~-carbon of an amino acid; such distinguishing groups are well known to those skilled in the art. For instance, ~or the amino acid glycine, the R group is H; for the amino acid alanine, R is CH" and so on. Other typical 35 side chains of amino acids include the groups: (CH3),CH-, (CH~)2CHCH,-, CH3CH2CH(CX3)-, CH3S(CH2) 2-, HOCX,-, CH,CH(OH)-, HSCH,-, NH2C(=O)CH2-, NH2C(=O)(CH2) 2-, NH2(CX2)3-, HOC(=O)CH2-, _ _ . _ .. . _ .. . .. . . .

2 } 923a5 W095/3376s HOC~=O)(CH2)2-, NH2(CH2)~-, C(NH2)2 NH~CH2)3-, HO-phenyl-CH2-, benzyl, methylindole, and methylimidazole.
As used herein and in the claims, the letters "(AA)" and the term "amino acid" are intended to include common natural 5 or synthetic amino acids, and common derivatives thereof, known to those ~killed 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.

A'obreviated Desiqnation ~m;nn Acids Abu ~-Amino butyric acid Ala L-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic acid ~Asp(Ind) ~-Indolinyl aspartic acid Cys L-Cysteine Glu L-Glutamic acid Gln L-Glutamine Gly Glycine His L-Histidine Hyp trans-4-L-Hydroxy Proline Ile L-Isoleucine Leu L-Leucine Lys L-Lysine Met L-M~th;~n;n~
Nal ~-Naphthyl alanine Orn Ornithine Phe L-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine Trp L-Tryptophane Tyr L-Tyrosine Val L-Valine Typical 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 35 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, W095l3376s ~ 5~

coupling agents and reagents/solvent5; these are intended to be within the scope of this invention.
~hhreviated Desicnation . Protectinq Groups Ada Adamantane acetyl . Alloc Allyloxycarbonyl Allyl Allyl ester Boc tert-butyloxycarbonyl Bzl Benzyl Fmoc Fluorenylmethyloxycarbonyl OBzl Benzyl ester OEt Ethyl ester OMe Methyl ester Tos (Tosyl) p-Toluenesulfo~yl Trt Triphenylmethyl Z Benzyloxycarbonyl Abbreviated Desiqnation Cou~linq Aqents BOP Bènzotriazol-1-yloxytris-~' (dimethyl-amino)phosphonium hexafluorophosphate DIC Diisopropyl~rho~;;m;de XBTU ~ 2-(lX-Benzotriazol-l-yl)-1,1~3,3-tetramethyluroniu,m,.
_ 1~ n:~f1~ rcpho5phat e PyBrOP Bromotripyrrolidinophosphonium l~P~r~ f 1 1lr~ropho5phate PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate TBTU 0-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate Abbreviated Reagents DesiGnation and Solvents ACN Acetonitrile AcOX Acetic acid Ac~O Acetic acid anhydride AdacOX Adamantane acetic acid Alloc-Cl ,Allyloxycarbonyl chloride Boc20 Di-tert butyi dicarbonate DMA Dimethylacetamide DMF N,N-dimethylformamide DIEA Diisopropylethylamine Et3N Triethylamine . _ ...... _ .... _ 2 1 ~23~5 wosst3376s P~,~,~C~--EtCAc Ethyl acetate FmocOSu 9-fluorenylmethyloxy carbonyl N-hydroxysuccinimide ester HOBT l-Hydroxybenzotriazole Hydrofluoric acid MeOH Methanol Mes (Mesyl) Methanesulfonyl MP l-methyl-2-pyrrolidinone nin. Ninhydrin i-PrOH Iso-propanol Pip Piperidine PP 4-pyrrolidinopyridine Pyr Pyridine SRIF Somatotropin release inhibiting SST Somatostatin SSTR Somatostatin receptor TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran Triflate (Trf) Trifluoromethanesulfonyl Trf20 Trifluoromethanesulfonic acid anhydride The compounds herein described may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of 20 olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention.
By "stable compound" or "stable structure~ is meant herein a compound that is sufficiently robust to survive 25 i601ation to a useful degree of purity from a reaction mixture, and Formulation into an efficacious therapeutic agent.
As used herein and in the claims, "alkyl~' or "alkylenyl"
is ;nt~n~d to include both branched and straight-chain 30 saturated aliphatic hydrocarbon groups having the Cl to C10 carbon atoms; "alkenyl" is ;n~n~d 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 35 the like; and "alkynyl" is intended to include hydrocarbon chains of either a straight or branched configuration and one W095l33765 2 1 9 2 3 0 5 PCTAsgs/004ss or more triple carbon-carbon bonds which may occur in any stable point along the chain, such as ethynyl, propynyl, and the like.
As used herein and in the claims, '~aryl" is intended to 5 mean any stable 5- to 7-membered monocyclic or bicyclic or 7-to 14-~ d bicyclic or tricyclic carbon ring, any of which may be saturated, partially unsaturated or aromatic, for example, phenyl, naphthyl, indanyl, or tetrahydronaphthyl tetralin, etc.
As used herein and in the claims, ~'alkyl halide" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the C1 to C10 carbon atoms, wherein 1 to 3 hydrogen atoms have been replaced by a halogen atom such as Cl, E, Br, and I
As 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 lO- 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 20 group consisting of N, O and S and wherein the nitrogen and sulfur atoms may optionally be ~ ; 7~ 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 25 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, 30 pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl, piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, or 35 octahydroisoquinolinyl and the like.
As used herein and in the claims, the phrase ~therapeutically effective amount~ means that amount of novel ________ _ , g~/33765 F~~

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 5 gastrointestinal disorders.
The 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 10 PxreP~Pd, and that the substitution results in a stable compound.
When any variable (for example R, x, z, etc.) occurs more than one time in any constituent or in Formulae (I to XX) or any other Formula herein, its definition on each occurrence is 15 ;ndPrPn~Pnt of its definition at every other occurrence.
Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

20 Synthetic Approach According to the present invention peptide 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 25 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 reguires replA~ ~ t of one or more of the amino acids in a peptide se~uence by novel 30 building units of the general Formula:
HN-C~(R)-COOH
X
b 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 ~1 9~30~
WO 95/33765 r~l/lb7~

cyclization will be effected. The side chain R is t~e 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 5 degree of fl~;h;l;ty in order to achieve the a~ Liate conformational constraints of the peptide analog. Such spacer groups include alkylene chains, substituted, branched and unsaturated alkylenes, arylenes, cycloalkylenes, unsaturated and sub8tituted cycloakylenes. Furthermore, X and R can be 10 ~C. ';n~d to form a heterocyclic structure.
A preferred ~hn~;~Ant of the present invention utilizes alkylene chains cnn~A;n;ng from two to ten carbon atoms.
The terminal ~) functional groups to be used for cyclization of the peptide analog include but are not limited 15 to:
a. Amines, for reaction with electrophiles such as activated carboxyl groups, aldehydes and ketones (with or without subse~uent reduction), and alkyl or substituted alkyl halides.
b. AlAnhn~A, for reaction with electrophiles such as 20 activated carboxyl groups.
c. Thiols, for the formation of disulfide bonds and reaction with electrophiles such as activated carboxyl groups, and alkyl or substituted alkyl halides.
d. 1,2 and 1,3 Diols, for the formation of acetals and 25 ketals.
e. Alkynes or Substituted Alkynes, for reaction with nucleophiles such as amines, thiols or ~AArhAn;nnc; free radicals; electrophiles such as aldehydes and ketones, and alkyl or substituted alkyl halides; or organometallic 30 complexes.
f. Carboxylic Acids and Esters, for reaction with nucleophiles (with or without prior activation), such as amines, alcohols, and thiols.
g. Alkyl or Substituted Alkyl Halides or Esters, for 35 reaction with nucleophiles such as amines, alcohols, thiols, and carbanions (from active methylene groups such as acetoacetates or malonates); and formation of free radicals _ _ _ _ _ _ .

2t~23~5 ~ WO95133765 P~l/~3~['' for subsequent reaction with alkeneg or substituted alkenes, and alkynes or substituted alkynes h. Alkyl or Aryl Aldehydes and Ketones for reaction with nl~rlPorh;les such as amines ~with or without subsequent 5 reduction), carbanions (from active methylene groups such as acetoacetates or malonates), diols (for the formation of acetals and ketals).
i. Alkenes or Substituted Alkenes, for reaction with nucleophiles such as amines, thiols, carbanions, free 10 radicals, or organometallic complexes.
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.
It 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, 20 substituted alkyloxy, and aryloxy carbonyls including, but not limited to, tert butyloxycarbonyl (Boc), Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) and Benzyloxycarbonyl (Z).
Carboxylic end groups for cyclizations may be protected 25 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.
Thiol 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, 35 pixyl(9-phenylxanthen-9-yl), acetamidomethyl, carboxy-methyl, 2-thio-4-nitropyridyl.

W09s/33765 21 g23Q5 It will further be appreciated by the artisan that the various reactive moieties will be protected by different protecting groups to allow their selective ~moval. Thus, a particular amino acid will be coupled to its n~;ghhor in the 5 peptide sequence when the N~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~ will be protected by protecting group B, or an ~ amino group of any lysine in the sequence will be protected by protecting group 10 C, and so on.
The coupling of the amino acids to one another is performed as a series of reactions as is known in the art of peptide synthesis. Novel building units of the invention, namely the N~-~ functionalized amino acid derivatives are lS incorporated into the peptide sequence to replace one or more of the amino acids If only one such N~-~ 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~ -amino alkylene) amino acid can be 20 linked to the carboxyl group of an aspartic or glutamic acid residue; (b) an N~ -carboxylic alkylene) amino acid can be linked to the ~- amino group of a lysine residue; (c) an N~-(~-thio alXylene) amino acid can be linked to the thiol group of a cysteine residue; and so on. A more preferred embodiment a5 of the invention incorporates two such N~-~-functi~n~1;7ed 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 30 elaborated below. Thus, peptide analogs can be constructed with two or more cyclizations, including N-h~kh~ne to N-backbone, as well as backbone to side-chain or any other peptide cyclization.
As stated above, the procedures utilized to construct 35 peptide analogs of the present invention from novel building units generally rely on the known principles of~peptide synthesis. However, it will be appreciated that accommodation 2 1 ~2~
~ WO 95/33765 P~ ,,S.'~

of the procedures to the bulkier building units of the present invention may be re~uired. Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a ~ourl ;ng agent such as but not limited to 5 dicyclohexycarbodiimide (DCC), bis(2-oxo-3-~7ol;~; nyl) rh~6rh;n;c chloride ~BOP-Cl), benzotriazolyl-N-oxytrisdimethyl-am;n~rhn6rh~n;um hexafluoro phosphate (BOP), l-oxo-l-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.
It has now been found that coupling of the bulky building units of the present invention may re~uire the use of additional coupling reagents including, but not limited to:
coupling reagents such as PyBOP~ (Benzotriazole-l-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP~ (Bromo-15 tris-pyrrolidino-phosphonium hexafluoro-phosphate), HBTU (2-(lH-Benzotria7cle-l-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate), TBTU (2-(lH-Benzotriazole-l-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate).
Novel coupling chemistries may be used, such as pre-20 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 25 pyrrolidinone) or mixtures of the above.

One object of the present invention is a method for the preparation of h5~kh~n~ cyclized peptide analogs of Formula (I):

iI2N-(AA)d-CO-N-CiI(R)-(~R)a-CO-N-(A~)e-CO-N-C~l(R'~-(AA~b-CO-N-(AA~-CO-E:

Formula (I) wherein the substituents are as defined above;

~t 92~G5 w095/33765 comprising the steps of i~corporating at least one N~
functionalized derivatives of amino acids of Formula (VI):
B-N-CH(R')-CO-OH
X
A G
Formula (VI) wherein X i9 a spacer group selected from the group 10 consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R' is an amino acid side chain such as H, CH~, etc., optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, 15 or aryloxy carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting group of G;
with a compound of the Formula (VII):
H2N-(A~)f-CO-B
Formula (VII) wherein f is an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues may be the same or different, and E is a hydroxyl, a carboxyl protecting 25 group or an amide to give a com~ound of the_general Formula:

B-N-CH(R')-CO-NH-(AA)f-CO-E
X

A G
Formula (VIII) (ii) selectively removing protecting group B and reacting the unprotected compound with a c ~ ~ of Formula:
B-NH-(AA)e-COOH
Formula (IX) ~ WO95/~765 rcl,~.'~( ''' wherein B and (AA) are as described above and e is an integer from 1 to 10, to give a compound of Formula:
5B-NH-(AA)e-CO-N-CH(R')-CONH-(AA)f-CO-E

A G
Eormula (X) wherein B, (AA), e, R3, and f are as described above;
(iii) removing the protecting group B from the compound of the Formula (X) and reacting the unprotected compound with a compound of Formula:

15B-N-CH(R)-COOH
X' A' l' Formula (VIa) 20wherein X' is a spacer group selected from the group consisting of alkylenes, substituted alkylenes, arylenes, cycloalkylenes and substituted alkylenes; G' is a functional group selected from amines, thiols, carboxyls, aldehydes or alcohols; A' is a specific-protecting group thereof; R3 is an 25 amino acid side chain such as H, CH3, etc., optionally bound with a specific protecting group; and B is a protecting group;
to yield a compound of Formula:

B-N-CH(R)-CO-NH-(AA)e-CO-N-CH(R')-CO-NH-(AA)f-CO-E
X ' X
- A' G' A - G
Formula (XI) 35(iv) removing the protecting group 3 and reacting the unprotected ~ onn~ with a compound of Formula:
B-NH-(AA)d-COOH

WO9S/33765 2 1 ~ 2 3 ~ 5 Formula (IXa) to yield a compound of Pormula:

5 B-NH-[AA)d-CO-N-CH(R)-CO-NH-(AA)e-CO-N-CH(R~)-CO-NH-(~A)f-CO-E

X ~ X
A' G' A - G
Formula (XII) (v) selectively removing protecti~g groups A and A' and reacting the terminal groups G and G' to form a compound of the Formula:

B-NH-(AA)d-CO-N-CH(R)-CO-NH-(AA)e-CO-N-CH(R')-CO-~H-(AA)f-CO-E
Formula (XIII) wherein d, e and f are independently an integer from 1 to 10; (AA) is an amino acid residue wherein the amino acid 20 residues in each chain may be the same or different; E is an hydroxyl group, a carboxyl protecting group or an amino group;
R and R' are independently an amino acid side-chain such as H, CH~, etc.; and the line designates a bridging group of the Eormula: -X-M-Y-W-Z-wherein M and W are in~PpPn~Pntly selected from the group consisting of disulfide, amide, thioether, imine, ether, and alkene; X, Y and Z are independently selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene, and substituted cycloalkylene;
(vi) removing all re--;n;ng ~rotecting groups to yiel~d a compound of Formula (I).
sicyclic analogs are prepared in the same manner, that is, by repetition of steps (v) and (vi). The determination of which residues are cyclized with which other residues is made 35 through the choice of blocking groups. The various blocking groups may be removed selectively, thereby exposing the selected reactive groups for cyclization.

~ 92305 WO9S/33765 r~.~,s,~c ~

Preferred are methods for the preparation of backbone cyclized peptide analogs of Formula ~I) wherein G i8 an amine, thiol or carboxyl group; R and R3 are each other than H, such as CH3, ( CH3 ) 2CH-, ( CH3 ) 2CHCH2-, CH3CH2CH ( CH3 ) -, CH3S ( CH2) 2 -5 HOCH2-, CH3CH~OH~-, HSCH2-, NH2C(=O)CH2-, NH2C(=O) (CH2) 2--, HOC (=O) CH2-, HOC (=O) (CH2) 2- ~ NH2 (CH2) ~-, C (NH2) 2 NH (CH2) 3-, HO-phenyl-CH2-, benzyl, methylindole, and methylimidazole, and wherein E is covalently bound to an insoluble polymeric support.
Another object of the present invention is a method for the preparation of ksrkh~nr cyclized peptide analogs of Formula (II):

15 H2N- (AA)d-CO-N-CH(R) -CO-NH- (AA)e-CO-NH-CH-CO-~X(AA)f-CO-E
Formula (II) wherein the substituents are as defined above;
comprising the ~teps of: incorporating at least one ~-20 functionalized amino acid derivative of the general Eormula(VI):
B-N-CH (R) -CO-OH

X
A - G
Formula (VI) wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R is the side 30 chain of an amino acid, such as H, CH3, etc.; 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 s amines, thiols, alcohols, carboxylic acids and esters or alkyl 35 halides and A is a protecting group thereof;

Wo9sl3376s 71 ~2~0S r~ s~LL~ss into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence.
Preferred is the method for the preparation of backbone 5 cyclized peptide analogs of Formula (II) wherein G is a carboxyl group or a thiol group; R is CH3, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, CH3S(CH2) 2-, HOCH2-, CH3CH(OH)-, HSCH2-, NH2C(=O)CH2-, NH2C(=O) (CH2)2-~ HOC(=O)CH2-, HOC(=O)(CH2) 2- ~ NH2 (CH2) ~-, C (NH2) 2 NH(CH2)3-, HO-phenyl-CH2-, 10 benzyl, methylindole, and methyl;m'~7~1e, and wherein E is covalently bound to an insoluble polymeric support.

Preparation of backbone to side chain cyclized peptide analogs is exemplified i~ Scheme I below. In this schematic 15 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 ~-functionalized amino acid having a terminal amine.

20 (Scheme I) Preparation of peptides with p~kh~n~ to Side Chain cyclizatio One preferred procedure for preparing the desired backbone cyclic peptides involves the stepwise synthesis of 25 the linear peptides on a solid support and the h~kh~n~
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 30 example of such support is a polystyrene-co-divinyl benzene resin. The polymeric supports used are those compatible with such chemistries as Pmoc and Boc and include for example PAM
resin, HMP resin and chloromethylated resin. The resin bound amino acid is deprotected for example with TPA to give (1) 35 below and to it is coupled the second amino acid, protected on the N~ for example by Fmoc, using a coupling reagent like BOP
The second amino acid is deprotected to give (3) using for 2 1 92~0~
w095~3376s ~ 55 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 5 example carboxy side chain is coupled to the desired peptide.
One such amino acid i9 Fmoc-aspartic acid t-butyl ester.
After deprotection of the N~ 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 10 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~(~-Boc-amino alkylene~amino acid. After deprotection the peptide can then 15 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 PysrOPr to ensure high yield.
After the linear, resin bound peptide, e.g. (8), has been 20 prepared the ~-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 cy~l; 7~tinn using for example TBTU as cyclization agent in DMF to ensure high yield of 25 cyclization, to give the ~-barkhnn~ to side chain cyclic peptide resin (10). After cyclization on the resin the terminal amino protecting group i8 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.
30 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.
The other part of the peptide-resin (9) undergoes protecting of the side chains used for cyclization, for example the ~-amino and carboxy groups. This is done by wogs/33~65 Z 1 92305 rc~ 5~ ~

reacting the ~-amino group with for e~ample AC20 and DMAP in DMF and activating the free ~-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 5 cyclic peptide (10). Removal of the peptide from the-resin and subse~uent removal of the side chains protecting groups by strong acid such as HF to gives (14) which is the linear analog of the b~nk~nn~ to side chain cyclic peptide (11).
The linear analogs are used as reference compounds for 10 the biological activity of their corresponding cyclic compounds.

[Reaction Scheme I follows at thi~3 point~

-~ 1 9~3Q5 WO9S133765 r~ 155 SCHEM~ I

BOP
8ec-NH-[M~-COOH + E-Resln ~ Boc-NH-IM]-CO-E-Resin TFA
(1) r Nll IM']-COOH ~ Fmoc-NH-lAAl-CO-NH-lM] -CO-E-Resln (2) plp coupling deprotection NH2-lM']-CO-NH lM] -CO-E-Rcsin (3) Fmo~NH-CH-C~NH{M] ~E-Resin NH2-lMIf-CO-E-Resin ~ (CH ) Fmo~NH CH-COOH I 2 z (CH2 ) z COO-t-8u COO-t-Bu NH2-CH-CO-NH-[M]~-CO-E-Resin 1. BOP I Fmoc 1111 IM]e-COOH
PIP
NMP (CH2)z 2.PIP
OO-t-Bu BOP
Fmoc-N-CH(R)-COOH
NH2-1M]e-CO-NH-CH CO-NH-lM] ~-CO-E-Resin (CH2)Z (I H2)X
3 0 1 (6) NH-Boc COO*Bu Fmoc-N-CH(R)-CO-NH-lM]e-CO-NHCH-CO-NH-lM]fCO-E-Resln (CH2 ) X CO
3 5 H-Boc O-t-Bu WO 95/33765 r~5.'~

. 3_ ~f. . , /~/
~' i ~1 ~Z305 ~ W0 95/33765 r~ ~ 5'1~ ''' SCEIEME I (continuec) . 1. PIP
2. FyBrOP I r N:l [M]d COOH
F I J: I IMI d-CO N-CH(R)-CO-NH-lM] e-CO-NH-CH-CO-NH{MI rCO-E-Resln (CH2)x (CH2~ Z
1 1 (8) TFA H-Poc COO-t-Bu DCM
r. ~ 1: IMI d CO N-CH(R)-CO-NH-[M] e-CO-NH-CH-CO-NH-l~AI rCO-E-Resin (1ChH2)X (CH2) Z

TBTU
NMP
r~ I1 H IMl d CO N-CH(R)-CO-NH-IM] e-CO-NH-CH-CO-NH~MI rCO-E-Resin I. l (CH2)x (CH2) Z
(10) hH CO
1. PIP
2. HFlAnisole 3. AcOH-H 2~

AcOH.NH2-lMI d-CO-N-CH(R)-CO~NH-[MI e-CO-NH-CH-CO-NH-IMI f-CO-E

(CH2)x (CH2) Z
H

1. PIP 2. i30Pf D-COOH
3. HFlAnlsole 4. AcOH-H 2~
D-CO-NH-lMld-CO-N-CH(R)-CO-NH-lMle-CO-NH-CH-CO-NH-lMlf-CO-E

(CH2)x (CH2)z (12) 3 5 hH CO

2~1 92~05 WO9~/33~65 J~l~ .,5.'1 ''' SCE~EM~ I (con~nued) 1. Ac20/DMAP 2. DIC/HOBT
3. CH3NH2/EtOH
Fmoc-NH-IMld-CO-N-CH(R)-CO-NH-lMle -CO-NH-CH-CO-NH-lMIf -CO-E-Pesin (CH2)x (CH2)Z

1. PIP H-Ac CO'NH2-CH3 2. HFlAnisole 3. AcOH-H20 AcOH.NH2~MId-CO-N-CH(R) CO-NH-IM~e-CO-NH-CH CO-NH-lAAlf -CO-E
(CH2)X (CH2)z (14) NH-Ac CO NH2 CH3 1. PIP 2. BOP I D-COOH
(1 3) 3. HF/Anisole 4. AcOH-H20 D-CO-NH-lM]d -CO-N-CH(R)-CO-NH-IMle -CO-NH-CH-CO-NH-lM]f -CO-E

(CH2)x (CH2)Z
H-Ac CO-NH 2 -CH 3 21 923~
095/33765 r~l,~,s~ --The selection of N~ and side chain protecting groups i8, 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~ protecting groups are chosen 5 in such a manner that their removal will not effect the removal of the protecting groups of the N~ m;n~lkylene) protecting groups. In addition, the removal of the N~
Am;n~Alkylene)protecting groups or any other protecting groups on ~-functional groups prior to the cyclization, will not 10 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 subse~uently with the removal of the peptide from the resin Protecting 15 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.
It is preferred to utilize Fmoc for protecting the ~-amino group of the amino acid undergoing the coupling reaction 20 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 soc for protecting ~-amino group of the N~(~-aminoalkylene) group and 25 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.

30 (Scheme II) Preparation of peptides with P~kh~n~ to Pa~h~ne cyclization.
Preparation of N-backbone to N-backbone cyclized peptide analogs is exemplified in scheme II~ In this schematic example, the ~uilding group consists of alkylene spacers and 35 two amide bonds A building unit for backbone cyclization ~the preparation of which described in Schemes III-VIII) is coupled to a 2t9230~
W095/33765 I~~

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-N~ Boc-amino alkylene)amino acid. The side chain Boc protecting group is 5 removed by mild acid such as TFA in DCM and an N-Boc protected ~-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).
After deprotection of the N~ Emoc protecting group by 10 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~ Fmoc and subsequent elongation of the peptide can be done before deprotection of the side chain Boc 15 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 r~r~pl; ~ied by PyBrOP- to ensure high yield.
After deprotection ~f the terminal N' Fmoc group, a second bn;l~;ng unit, for example Fmoc-N~(~-t-Bu-carboxy-alkylene)amino acid is coupled to the peptide-resin to give (19~. After deprotection of the N~ Fmoc protecting group, the peptide can then be elongated, if required, to the desire-d 25 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 ~-alkylene-30 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 cycllzation agent in DMF
to ensure high yield of cyclization, to give the N-backbone to 35 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 ~ i 923 QS
095l3376s r~l,~ sc --peptide (23) is obtained after treatment with strong acid such as XF. Alternatively, prior to the removal of the backbone cyclic peptide from the resin, the t~r~; n~l amino group of (22) is blocked, after deprotection, by acylation with agents 5 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-~r~;n-~l blocked backbone to backbone cyclic peptide (24).
The other part of the peptide-resin (21) undergoes 10 protecting of the side chains used for cyclization, for example the ~-amino and carboxy groups. This is done by reacting the ~- amino group with for example Ac20 and DMAP in DMF and activating the free ~-carboxy group by for example DIC
and HOBT to give the active ester which is then reacted with 15 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 ha~khnn~ cyclic peptide (23). The linear analogs are used as reference ~c~pollnAq for the 20 biological activity of ~heir corresponding cyclic compounds.

[Reaction Scheme II follows at thi~ point]

Z 1 9~3 ~
WO 95/33765 ~ IB95/00455 ' BOP
Boc NH-[M]-COOH ~ E-Resin ~ Boc-NH-IMl -CO E-Resin > TFA NH-[Ml-CO-E-Resin DCM (1) Fmoc NH-lM' ] -COOH ~ Fmoc-NH [M'I -CO-NH-[M] CO-E-Resin NMP
(2) plp coupling deprotection NH2-lM']-CO-NH-[M] -CO-E-Resin s NMP
(3) Fmoc-NH CH-CO-NH [MI t-CO-E-Resin BOP ¦
NH2-[M]f-CO-E-Resin ~ (CH2) Fmoc-NH CH-COOH I z (CH2) z COO-t-Bu ~ I
COO-t-Bu NH2-CH-CO-NH-[M] t-CO-E-Resin 1. BOP t Fmoc-NH-[M~e-COOH
~ (CH2) 2. PIP

O-t-Bu Fmoc-N-CH(R)-COOH
NH 2-[M]e-CO-NH~CH-CO-NH-[M] ~-CO-E-Resin (CH2 ) X

(ICcH2)z (6) NH-Boc 3 ~ O-t-Bu Fmoc-N-CH(R)-CO-NH [M] e-CO-NH CH-CO-NH [M] t-CO-E-Resln (IH2)x (CH2) z ~7 3 5 H-BocCOO-t-Bu ~92~5 WO 95/33765 r~ lL~5 . SCHEME ~

BOP Fmoc N-CH(R~ )-CO-NH-[M]~ -CO-E-Resln NHz-[M~t-CO-E-Resin Fmoc-N-CH(R)~OOH (CH2)X
(CH2)X NH-Boc (16) NH-Boc Fmoc-N-CH(Rl )-CO-NH-[MI~ -CO-E-Resin 1. TFA/DCM
152~ BOC-NH~CHz)r-COOHf BOP (CH2)X (17) NH-CO-(CH2)y -NH-Boc 1. PIP
2~ PyBrOP I Fmoo-NH-[M],-COOH

r. NH IM], CO-N-CH(Rl )-CO-NH-[MI~ -CO-E-Rosin (CHt)x (18) NH-CO-(CH2)y -NH-Boo 1. PIP
~ _ Fmoc-N-CH(RZ )-COOH
2~ BOP I (CH2~2 COO-t-Bu Fmoc-N-CH(RZ ~-CO-NH IM], -CO-N-CH(R1 ~-CO-NH-lM]t -CO-E-Resin (CHz)2 (CH2)y 00-t-Bu NH-CO-(CH2)y~NH~BOO (19) 21 ~3Q5 Wo 9513376S F~ llJ.,,' 'C ~ Iss SCE~ME n (condm~f~d) 1. PIP
2. PyBrOP I Fmoc-NH-IMjd-COOH

FmocNH-[AAld CO-N-CH(R2)CO-NH-[M]~CO-r-CH(R~)CO-NH-[M]t CO-E-Resh (CH2)z (CH2)x COO-t-Bu NH-CO-(CH2)y-NH-Bcc t20) TFA
DCM
Fmoc-N H-lMld -CO-N-CH(R2 )CO-N H-lMI .CO-N-CH(R1 ) CO-NH-[M] f CO-E-Resln (1H2)2 (CH2)r 100H NH-CO-(CH2)yNH2.TFA
TBTU
Nlhl~ ~

r, ~ M]d CO-N-CH(RZ)-Co-NH-[MI,CO-N-CH(R1)CO-NH-[MltCO-E-Resln (CH2)2 (CH2)s t22) CO ll: l (CH2)y--CO-NH
l. PIP
2. HF/Anisole 3.AcOH-H20 AcOH.NH2-[M]dCO-N-CH(R2)CO-NH-[MI.CO-N CH(R1)CO-NH-[M]fCO-E
(CH2)2 (CH2)x CO-NH (CH2)~CO-NH (23) 1. PIP 2. BOP t D-COOH
( ) 3. HF/Anisole 4.AcOH-H20 ~ ~ ~ ~~
D-CO-NH-lM]dCO-N-CH(R2)-CO-NH-lM]~CO-N-CH(R1)-CO NH-[MIf CO-E
(lH2)2 (CH2)X
t24) CO-NH (CH2)~CO-NH

2~ ~2~05 WO 95/33765 p SCBMli ~ (co~tinued) 10 ~l) 1. Ac20/DMAP ~ DIC/HOBT
3. CH3NH2/EtOH

Fmoc-NH-IM]d-CON-CH(R2)-CO-NH-[M]OCO-r-CH(Rl )-CO-NH-[MI, -CO-E-Resin (CH2)z (CH2)x 1 1 (25) CO-NH-CH3 NH-CO-(CH2)y-NH~Ac 1. PIP
2. HF/Anisole 3.AcOH-H20 2 0 AcOH.NH2 -IMld-CO-N-CH(R 2-~CO-NH-IM] ~-CO-N-CH (R1 )-CO-NH-lM] ~ -CO-E

(CH2)z (CH2)x CO-NH-CH3 NH-CO-(CH2 )y -NH-Ac (2 1. PIP 2. BOP + D-COOH
(~1) 3.HF/Anisole 4.AcOH-H20 D-CO-NH-[Mld CO-N-CH(R2)-CO-NH-[Mle~CO-N-CH(R1 )-CO-NH-IM]~ -CO-E
(CH2)~ (CH2)X
CO-NH-CH3 NH-CO-(CHz)y~NH-Ac w09s/33765~i g ~ ~ 5 r~ I /~ e ~ . rr ~

Novel Synthesis o~ Bu;l~;ng Units The novel synthesis providing N(~-(functinn~li7ed) alkylene) amino acids used to generate backbone cyclic peptides i9 depicted in schemes III - VIII. In this approach 5 we have impll -nte~ the following changes in order to devise a practical, general synthesis:
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.
10 2. The leaving group was changed to triflucromethanesulfonyl (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 15 alkylation reaction.
3. The carboxylate is esterified prior to the substitution reaction, to facilitate the substitution by removing the negative charge next to the electrophilic carbon.

(Scheme III) Preparation of N~, N~protected ~-amino alkylene amino acids building units.
One preferred procedure for the preparation of protected 25 N~(~- amino alkylene) amino acids involves the N~ alkylation of suitably protected diamino alkanes. One preferred N~,N~
di- protected diamino alkane is for example N~-Benzyl, N~-Boc diamino alkane (27). This starting material r~nt~;nq one protecting group such as Boc which is necessary for the final 30 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 35 temporary protection of the N~ amino group, which is alkylated in the reaction by such protecting groups as Bzll minimizes 2 t 92305 ~ W09~33765 I~l/~95~r~--the dialkylation side reaction and allows removal by suchconditions that do not remove the NJ-protecting group.
he N~,NJ di-protected diamino alkane is reacted with for example chiral ~-hydroxy r~-substituted acid esters where the 5 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 ~-elimination reaction 10 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~ protecting group, such as Bzl, and the carboxyl protecting group, such as methyl ester, are removed by mild conditions, such as catalytic 15 hydrogenation and hydrolysis, that do not remove the N~
protecting group such as Boc to give the N~protected amino acid (29). Introduction of the N~ protecting group sui~able for peptide synthesis is accomplished by methods well known in the art, to give the protected N~(N~ protected amino alkylene) 20 amino acid (30).
The choice of the N~ and the N~ 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 orth~gon~l to the other side chains protecting 25 groups in the peptide. Combinations of N~ and N= protecting groups are for example: N~-Fmoc, N~-Boc; N~-Fmoc, N~-Alloc; N~-Boc, N~-Alloc. These combinations are suitable for peptide synthesis and hac~h~n~ cycl;7ati~n, either on solid support or in solution.

~ (Schene IV) Preparation of N~, Nrprotected ~-amino alkylene glycine ~ building units.
One preferred procedure for the preparation of protected NY~- amino alkylene) glycines involves the reaction of the N~, N~ di-protected di amino alkane (27) with commercially 21 q~305 w09s~3376s r~ c --available ~-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 i3 not nec~RR~ry. The use of the same temporary protecting groups 5 for the N~ 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~ protected amino acid (32~. Introduction of the N~
10 protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N~ (N= protected amino alkylene) glycines (33).
The choice of the N~ and the N~ protecting groups is dictated by the use of the building units in peptide 15 synthesis. The protecting groups have to be orthogonal to each other and orth~gnn~l to the other side chains protecting groups in the peptide. Combinations of N~ and N~ protecting groups are for example: N~ Fmoc, N~ Boc ; N~ Fmoc, N~ Alloc; N~
soc, N~ Alloc. These combinations are suitable for peptide 20 synthesis and h~hnn~ cyclization, either on solid support or in solution.

2 I q2~5 SC~EME I

BOP
i30cNH-[M]-COOH I E-Rcsin ~ Boc-NH-IM] -CO-E-Resin NMP
~ TFA NH-[MI-CO-E-Resin OCM (1) Fmoo NH-[M''i -COOH s Fmoc NH [Ml CO-NH-IAAl -CO-E-Resin (2) PIP coupling deprotecton s NH2-[M'l-CO-NH-[M]-CO-E-Resin (3) Fmo~NH-CH-CO-NH-IMI f-CO-E-Resin BOP
NH2-1M]~-CO-E-Resin ~ (CH2) Fmo~NH-CH-COOH I z (CH2)z COO-t-Bu COO t-Bu NH2-CH-CO-NH-[M] t-CO-E-Resin 1. BOP IFmoc-NH-IMIe-COOH
~ (CH2) 2.PIP
NMP CO ROP
O-t-Bu Fmoc-N CH~)-COOH
NH2-lAAle-CO-NH-CH-CO-NH-IAA] I-CO-E-Resin (CH2 ) X

(CH2)z (6) NH-i30c 3 ~ COO-t-Bu Fmw-N-CH(R)-CO-NH-[MI~-CO-NHCH-CO-NH-~M]fCO-E-Resin (CH2)X (CH2)Z

3 5 H-Boc O-t-Bu ~1 92~05 W0 95/33765 P~ IJ~ ~

scH~ME m CH Ph-CHOlM~OH~ ~CH T~-O-CH(R)-CO-E
(I 2)X NaBH4lEt3N I 2 PylDCM
NH-A NH-A
(27) Bzl-N-CH(R)-CO-E HCI.H2N-CH(R)-COOH
(CH2)x 1.NaOHlH20 (CH2)x B-L
~H-A 2. H21Pd/C(HCI) NH-A
(28) t29) B-N-CH(R)-COOH A- for e~mple Boc, Fmoc, A~oc.
¦ B- for e~ample Fmoc, Boc, Alloc.
tCH2) x B-L- for e~cample Fmoc-Osu, Boc2 O, Alloc-a NH-A E- for e;ample OMe, OEt, OBzl.
(30) R ~mino acid side chains 2 0 - SC~lE IV
Bzl-N-CH2-CO-E
2~ X-CH2-CO-E , (~H2)x H2/PdlC(HCI) NH-A
HCI.H2N-CH2-COOH (31) B-N-CHz-COOH

(IH2)x B-L ~ (IH2)x NH-A NH-A
(32) (33) A- for e~ample Boc~ Fmoc, AUoc.
B- for e~2mple Fmoc, Boc, Alloc.
B-L for e~ample Fmoc Osu, Boc2 O, Alloc{:l E- for e~ample OMe, OEt, OBzl.
~- 1-10 X- ~, Br, r 2 1 9~05 WO95/33765 r~ IJS

~Scheme V) ~reparation of N~ carboxy protected ~-carboxy alkylene ~i o acida.
One preferred procedure for the preparation of protected 5 N~(~- carboxy alkylene) amino acids involves the N~-alkylation of suitably N~, ~-carboxy deprotected amino acids. One preferred deprotected amino acid is N~-Benzyl ~-amino acids t-butyl esters (34). This starting material rnnt~;nC one protecting group such as t-Bu ester which is necessary for the 10 final product, and a temporary protecting group such as N~ 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 ~-amino acids t-butyl esters with aldehydes such as 15 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.
The N3, ~-carboxy deprotected amino acids (34) are 20 reacted with, for examp~e, chiral ~-hydroxy ~-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 25 ~-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~ protecting group, such as Bzl, and the ~-carboxyl protecting group, such as benzyl ester, are concomitantly 30 removed by mild condition, such as catalytic hydrogenation, that to not remove the ~-carboxy protecting group such as t-Bu - to give the N~(protected ~-carboxy alkylene) amino acid (36).
ntroduction of the N~ protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to 35 give the protected N~(~ protected carboxy alkylene) amino acid (37).

~192~5 The choice of the N~ and the ~-carboxy protecting groups i9 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 5 groups in the peptide. A combination of N~ and ~-carboxy protecting groups are for example. N~-Fmoc, ~-carboxy t-Bu;
N~-Fmoc, ~-carboxy Alloc; N~-Boc, ~-carboxy Alloc. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution.
10 (Scheme VI) Preparation of Na, ~-carboxy protected ~-carboxy alkylene glycine building units.
One preferred procedure for the preparation of protected N~(~-carboxy alkylene)glycines involves the N~-alkylation of 15 suitably N~, ~-carboxy deprotected amino acids(34) with commercially available ~-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 n~rGcS~ry.
The use of the same temporary protecting groups for the N~ and the ~-carboxy groups, for example the 8zl 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~protected 25 ~-carboxy alkylene) glycines (39). Introduction of the N~
protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N~(~ protected carboxy alkylene) glycines (40).
The choice of the N~ and the ~-carboxy protecting groups 30 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~ and ~-carboxy protecting groups are, for example: N~ Fmoc, ~-carboxy t-Bu;
35 N~ Fmoc, ~-carboxy Alloc; N~ Boc, ~-carboxy Alloc. These combinations are suitable for peptide synthesis and backbone =~
cyclization, either on solid support or in solution.

~ 1 9Z3~5 95l33765 r~~ c 'r SCHEME V
NH2 NH-Bzl Ph-CHOlMeOH (1H ) Tr~-O-CH(R)-COO-E
( I H2)z NaBH41Et3N ~ PylDCM
CO-A CO-A
~4) Bzl-N-CH(R)-COO-E HCI . H 2 N-CH(R)-COOH
(CH2)Z H21PdlC(HCI) ( ICH2)z B-L
OO-A COO-A
(35) (36) B-N-CH(R)-COOH A- for e~ample O-t-Bu, O-AII
B- for ex~lmple Fmoc, Boc (CH2)z B-L~ for e~ample Fmoc-Osu, Boc2 0 E~ for example OMe, OEt, OBzl COO-A z- 1-10 (37) R- amino acid side chains ~ SCE~D3 Vl Bzl-N-CH2-CO-Bzl X-CH2-CO-Bzl ~ H H2/PdlC (HCL) PylDCM (I 2)2 (38) HCI.H2N-CH2-COOH Fmo~N-CH2COOH
(I~H2)Z FmocOSu ((!'H.)z O-A A
(39) (40) A~ for e~ample O-t-Bu, O-AII
B~ for e~ample Fmoc, Boc 8-L- for e~amplc Fmoc-Osu, Boc2 0 E- for e2ample O~, OEt, OBzl ze 1-10 2 ~ ~23~5 W09sl33765 r ~ ~s (Scheme VII) Preparation of N3 SWprotected ~-thio alkylene amino acid b~ ;n~ unita.
Qne preferred procedure for the preparation of N3, SW-deprotected N3(~-thio alkylene) amino acids involves the N3-alkylation of suitably sw protected ~-thio amino alkanes.
Suitable sw protecting groups are, for example, Bzl, t-Bu, Trt. One preferred SW-protected ~-thio amino alkanes is for 10 example ~-(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 subgtitution reaction on suitably N3-protected ~-activated amino alkanes. Removal of 15 the amino protection gives the starting material (41).
The S-protected ~-thio amino alkanes (41) are reacted with for example chiral ~-hydroxy ~-substituted acid esters where the hydroxyl moiety is converted to a leaving group for example Triflate. The use of Triflate as the leaving group ' 20 was found to be superior to other leaving groups such as halogens, Tosyl, Mesyl etc. because it prevents the ~-elimination reaction encountered with the other leaving groups. The use of Triflate as the beaving group also ensures high optical purity of the product for example (42). The 25 temporary ~-carboxyl protecting group, such as methyl ester, is removed by mild condition, such as hydrolysis with base, that to not remove the ~-thio protecting group such as S-Bzl to give the N3 (S-protected ~-thio alkylene) amino acid (43).
Introduction of the ~3 protecting group suitable for peptide 30 synthesis is ~c~ h~ by methods well known in the art, to give the protected N,S protected N3 (~-thio alkylene) amino acid (44).
The choice of the N3 and the ~-thio protecting groups is dictated by the use of the building units in peptide 35 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 N3 and ~-thio 2 ~ 923Q~
W095/33765 r~

protecting groups are for example: N3 Fmoc, S~ t-Bu; N3 Fmoc, S~ Bzl; N3 Fmoc, S~ Trt; N3 Boc, S~ Bzl. These combinations are suitable for peptide synthesis and backbone cyclization, either on solid support or in solution.

(Scheme VIII) Preparation of N3, S~ protected ~-thio alkylene glycine bu~l~;n~ units.
One preferred procedure for the preparation of N~, S~-deprotected NY(~-thio alkylene) amino acids involves the N'-alkylation of suitably S~ protected ~-thio amino alkanes (41) with commercially available ~-activated carboxylic acid esters for example ethyl bromo acetate. Since the titled compound is 15 achiral, the use of leaving groups such as Trf, Tos or Mes i8 not necessary.
Suitable protecting groups for the ~-thio groups are for example Bzl, t-Bu, Trt. One preferred S-protected ~-thio amino alkanes is for example ~-(S-Benzyl) amino alkanes (41).
20 The N-alkylation reaction gives the ester ~45). The temporary ~-carboxyl protecting group, such as ethyl ester, is removed by mild conditions, such as hydrolysis with base, that to not remove the ~-thio protecting group such as S-Bzl to give the N3 (S-protected ~-thio alkylene) glycines (46). Introduction 25 of the N' protecting group suitable for peptide synthesis is accomplished by methods well known in the art, to give the protected N3, S~-deprotected N3 ( ~-thio alkylene) glycines (47)-The choice of the N~ and the ~-thio protecting groups is 30 dictated by the use of the building units in peptide synthesis. The protecting groups have to be orthogonal to each other and orthrgrn~1 to the other ~ide chains protecting groups in the peptide. A combination of N3 and ~-thio - protecting groups are for example: N3 Fmoc, S~ t-Bu; N3 35 Fmoc, S~ Bzl; N3 Fmoc, S~ Trt; N3 Boc, S~ Bzl. These combinations are suitable for peptide synthesis and b~rk~rnr cyclization, either on solid support or in solution.

.. ,,, . , , , , . , _ _ _ _ _, _ _ _ _ _ _ , wo 95~3376s 2 1 9 2 3 ~ 5 1 1 ,~ 5 ~c SCHE;~ VII
NH2 HN-CH(R)-CO-E
(CH ) T~ O CH(R~ C~~ E (CH ) NaOH/H2O, Py/DCM I x S-A S-A
(41) (42) HCI.H2N-CH(R)-COOH B-N-CH(R)-COOH
(1H2)X B-L (I H2)X
S-A S-A
(43) (44) A~ for a~nmple Bil, t-13u, Trt B- for examplc Fmoc, Boc, AUoc B-L for e~ample Fmoc-Osu, Boc2 O, Alloc-CI
E- for e~amplc OMe, OEt ~ 1 0 R- amino cid ride chahls SCHE~E vm HN-CHz -CO-E
X-CH2~CO-E I NaOH/H20 (41)PyIDCM (CH2)x ~ _ g-A
(45) HCI.H2N-CH2-COOH B-N-CH2-COOH
(CH2)x B-L (CH21x ~;-A S-A
(46) (47) A- for e~ample B71, t-Bu, Trt B- for ex2mple Fmoc, Boc, Alloc B-L- for e~ample Fmoc-Osu, Boc2 O, AUoc-CI
E- for example OMe, OEt ~- 1-10 X~ Cl, Br, 1.

~ WO 95J33765 P ~ 5 SPECIFIC EXA~PLES OF ~h~ S
Preparation of the novel backbone cyclized peptide analogs using the schematics outlined above will be 5 illustrated by the following non-limiting specific examples:

Ada-(D~Arq-Arq-cvclo(N~(1-(6-~m;~h~vlene)Glv-HYo-Phe-D-As~)-D-Phe-Phe-Arq-OH

Boc-Arq(Tos)-O-resin ---> Fmoc-Phe-Arq(Tos)-O-resin Boc-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 15 hours by the addition of DCM. The resin was then carried out through the procedure in Table l 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 l steps 1-8). After positive 20 ninhydrin test, as described in Kaiser et al., Anal Biochem., 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 25 added to the flas~.

~192~05 WO95/33765 r~l~,s.'t~

TAB~E 1 PROCED~RE FOR 0.1 mMCLE SCAL~
STEP SOLVENT/VOLVME TIME REPEAT
~o- B5~ (ML) ~MIN) ~XS) COMMENT
1 DCM 5 120 1 Swells resin 3 TF~/DCM 55% 5 a 1 Deprotection 4 TFA/DCM 55% 5 20 1 Deprotection 6 NMP 5 2 4 check for positive nin.
7 DIEA/NMP 5 5 2 ~entr~ t;~n 9 Fmoc-AA in NMP S S Coupling add BOP 6 eq. add 12 eq. Check p~, adjust to p~ 8 with DIEA
NMP 5 2 5 check ~or negative nin.
11 Pip/NMP 20~ 5 10 1 Deprotection 12 Pip/NMP 20% 5 10 ao 13 NMP 5 2 6 check ior - positive nin.

After shaking for 10 minutes, the mixture was adjusted to pH 8 (measured with wetted p~ stick) by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 10 hours 25 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.

Fmoc-Phe-Arq(Tos)-O-resin ----~ Fmoc-~(6-Boc amino hexvlene)Glv-Hvo(o8zl)-phe-D-As~ (t-Bu) -D-Phe-Phe-Arq(Tos)-o-~in The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to two deprotections of the Fmoc protecting group by 20~ Pip 35 in NMP (Table 1 steps 11-13). After washing and ninhydrin test (Method J, below), coupling of Fmoc-D-Phe was achieved as ~ 1 ~23Q5 W095/33765 r~ r4ss 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 re6in was washed and the Fmoc group deprotected as described above (Table 1 steps 11-13).

5 After washing and ninhydrin test, coupling of Fmoc-D-Asp(t-Bu) was achieved a~ 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 10 above (Table 1 steps 11-13). After washing and ninhydrin test, coupling of Fmoc-L-Phe wag achieved as described in Stage 1 (Table 1 steps 9-10) uging 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 15 deprotected as described above (Table 1 steps 11-13). After washing and ninhydrin tegt, 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 20 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~(6-Boc amino hexylene)glycine (0.3 25 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.

Fmoc-N~(6-Boc ~m;no hexvlene)Gly-H~,rp(oBzl)-phe-D-Aso(t-Bu)-D
Phe-Phe-Arq ITos)-O-resin ----3 Fmgc-D-Ara(Tos)-Arq(Tos)-~(6-Boc ~m;n~ hexvlene)Glv-Hv~(OBzl)-Phe-D-As~(t-Bu)-D-Phe-he-Arq(TDs)-3-resin The Fmoc-N~(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 . , . . . ... .. _ . .. .. . , ., _,, .,, _ _ _ _ _ _ _ _ _ _ _ _ . .... ..

~1 92305 w095/3376s P~l~ C

in NMP ~Table 2 steps 1-2). After washing, the picric acid test (Method K) was performed. If the test did not show 98i2~, deprotection of the peptide resin was subjected again to 3 deprotection steps (Table 2 steps 1-2), washing and 5 picric;acid test (Method K). Coupling o~ 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 PyBOP 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
10 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 15 test did not show 98i2~ coupli~g the peptide resin was subjected again to a third ~nrl lng for 2 hours at 50 ~~
(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 20 K) was performed.

2t 9230S
W 095/33765 P~l/1L.

TART.~ 2 PRo~n~TRT~ FOR 0.1 mMOT~R SCALE~
STLP SOLVENT/VOLUME TIME REPEAT
~ E~2~ (ML) (MIN)~Xs) ~ E~
1 Piperidine/NMP s 10 3 Deprotection 20~
_ 5 2 NMP 5 2 6 Picric acid test.
3 ~moc-A~ in NMP S S Coupling add PyBroP 6 eq.
add DIEA 150 1 12 eg. Check p~, adjust to pH 8 with DIEA.
10 4 NMP 5 2 3 check for negative s Fmoc-~ in NMP S S Coupling add PyBroP 6 eq.
add DIEA 20 hr. 1 12 eq.
Check p~, adjuGt to p~ 8 with DIEA.
15 6 NMP 5 2 4 Picric acid test.
If less than 98~2 coupling repeat Steps 4-s 7 Fmoc-~A in NMP S S Coupling at 50 ~C
add PyBOP 6 eq.
add DIEA 120 1 12 eq.
Check p~, adjust to p}~ 8 with DIEA.

If the test did not show 98i2~ deprotection, the peptide resin was subjected again to 3 deprotection steps 25 (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 l (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 DIBA (0.209 mD, 1.2 mmole). The resin was washed 6 times 30 with NMP (Table 1 step 15) and used in the next stages.

Fmoc-P-Arq~Tos)-Arq(Tos)-N"(6-Boc amino hexYlene)GlY-Hv~(OBzl)-Phe-D-AsT~(t-Bu)-D-Phe-Phe-Arc(Tos)-O-resin ----~
35 Ada-D-Arq-Arq-cYclo(N"(1-(6-amidohexYlene)GlY-HYo-Phe-D-As~)-D-Phe-Phe-Arq-OH

Wogs/33765 Z ~ 9 2 3 0 5 r 1/~ s ~ - 5 The Fmoc-D-Arg~Tos)-Arg(Tos)-N~(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 accordirg to 5 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 ~n vacuo for 24 hours. The dry peptide resin weight, 0.4 g, it was divided 10 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 15 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 20 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 25 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 ~) was performed. If the test was positive or slightly positive the protecting with a~m~nt~np acetic acid 30 was repeated. If the ninhydrin test was negative, the peptide resin was washed 6 times with NMP and 6 ti~es 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 m~) was added and the 35 peptide treated with 20 mL liquid HF at -20 ~C for 2 hours.
After the evaporation cf the HF=under vacuum, the anisole was washed with ether (20 mL, 5 times) and the solid residue dried 2~q~3Q5 WO 95133765 P~ SS

in vacuum. The peptide was extracted from the resin with TFA
(10 m~, 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 5 by semiprep HPLC (Method H). The final product was nh~;n~d 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') A~A in agreement with the title compound [Table 3 follows at this point.]

i~ 1 9 wog5n376s r ~ ,s~ ~s~

TABLE 3 PROr~r)UEU3 FOR 0 . 05 ' ')I,E SCALE
STEPSOLVENT/ VO~UME TIME REPEAT
N0. ~EAGENT (ML~ (MIN)~XS) COMMENT

2 TFA/DCM 55~ 5 2 l Deprotection 3 'TFA/DCM sSt s 20 1 Deprotection 6 DIEA/NMP 10t 5 s 2 ~ tY~l;7At;nn 8 T8TU/NMP/DIEA 5 150 3 Cynl;7~t;nn 9 NMP 5 2 4 Picric acid te~t.
I~ less than 98~2t coupling per~orm Steps 10-12. If above 98~2t, go to 3tep 13.
TBTU/NMP/DIEA s 20hr 3 Cy~ t;nn. Check p~, adjust to p~ 8 with DIEA.
11 NMP 5 2 4 Picric acid test.
I~ less than 98~2t coupling per~or~
Steps 12. I~ above 98+2t, go to step 13 12 T87U/NMP/DIEA s 120 3 Cyclization, 50 C
Check p~, ad~ust to r pX 8 with DIEA.

14 Pip/NMP 20t s 10 l Deprotection Pip/NMP 20t 5 10 25 16 NMP 2 6 check ~or positive nin.
17 Ad~cO~/BOP 5 2 /NMP
18 NMP s 2 6 Check ~or negative nin .
19 DCM s 2 4 2~ ~2305 WOgs/3376s P~~ CCl55 EXAMPLE 2 NON-CYCLIZED PEPTIDE (Control for biological assays) Ada-D-Arq-Arq-N~(6-acet~m;dohexvlçne~Glv-Hv~-Phe-D-As~(NH-~e)-D-Phe-Phe-Arq-OH
The Fmoc-D-Arg(Tos)-Arg(Tos)-N~(6-amino hexylene)Gly-5 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~(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

10 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 ~MP 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 15 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 20 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 vacuo. The peptide was deprotected and cleaved 25 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 30 vacuo. 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 35 white powder by lyophilization from dioxane, which gave 48 mg (64~) of the title compound.

Z192~G5 WO95/33765 Y~

~PLC (Method G) RT 27.70 minutes, 93 TOF MS: 1424.6 (Mt) AAA in agreement with the title compound Tl~RT,T~ 4 PROCEDURT~ FOR 0.05 mMOLE SCAI.E - _ 6TEP SOLVENT/ VOLUME T}ME REPEAT
NO. B~ 3~ (MD) (MI~(XS) 5~
l NMP s 120 1 Swells resin 2 Ac,O/PP/NMP S 30 1 Protecting o~ side chain 10 3 NMP s 2 6 Check for negative nin.
4 DIC/HOBT/NMP S 30 1 Activation of COO~
aide chain s MeN~,/EtO~/ 5 60 1 Protecting o~ side chain ' 7 Pip/NMP 20~ 5 10 1 Deprotection 8 Pip/NMP 20i 5 10 9 NMP 5 2 6 Check for positive nin.
AdacO~/BOP/ 5 2 NMP -11 NMP s 2 6 Check for negative nin.

2 1 ~2~Q5 W095/33765 1~l~,5.~-!5' H-D-Arq-Arc-cYclo(N~(l-(4-Pro~anovl~)Gly-HvP-phe-N~(3-amid 5 ProPvlene)Glv)-Ser-D-Phe-Phe-~rc-OH

STAGE l Fmoc-Phe-Arq~To8)-O-resi~ ----> Fmoc-N~(4-t-Bu-propanovl)G
10 HvP(OBzl)-Phe-Na(3-Boc amino ProPvlene)-Glv-Ser(Bzl)-D-Phe-Phe-Arq(Tos)-O-resin Fmoc-Phe-Arg(Tos)-o-regin prepared from Boc-Arg(Tos)-O-Resin (0.3 g, 0.1 mmole) (Example 1, Stage l) was subjected to 15 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 20 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, 25 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~(3-Boc amino propylene)glycine was achieved as described in 30 Table 1, steps 9-10 using Fmoc-D~(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 re8in 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 35 acid test (Method K) was performed. If the test did not show 98I2~ deprotection the peptide resin was subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and picric W095/33765 2 1 9 ~ 3 0 5 r~ "~ 5~

acid test (Method K). Coupling of Emoc-L-Hyp(OBzl) wa6 achieved in NMP by the addition of Fmoc-L-Hyp(OBzl) (0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid Py3r0P
reagent (0.28 g, 0.6 mmole) was added to the flask. After 5 ~haking 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 wa8hed and subjected to a second rollrl;~3 by the same procedure for 20 hours. After washing the resin was subjected 10 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
15 (Table 2, steps 1-2). After washing picric acid test (Method X) 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
20 reagent (0.26S g, 0.6 mm~ole) 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+2~ deprotection the peptide resin 25 was subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and picric acid test (Method R). Coupling of N~(3-t-Bu carboxy propylene)Gly was achieved as described in Table 1 steps 9-10 using N~(3-t-Bu carboxy propylene)Gly (0.264 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and 30 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.

~1 92~05 W095~33765 P ~ rr FmOc-N~(4-t-Bu-lJ~Luuall~Jvl)Gly-Hvl~(cBzl)-phe-N~(3-Boc ;:m;nn~
~ro~ylene) -Gly-ser(Bzl) -D-phe-phe-Arq(Tos) -o-res;n -----~
Fmoc-D-Arq(Tos)-Arq(Tos)-NQ(4-t-Bu- ~ro~nnYl)Gl~-Hv~(OBzl)-5 Phe-N~3-Boc ~m;nn ~ro~:)vlene)-Glv-Ser~Bzl)-D-Phe-Phe-Arq~Tos)-O-resin - -Fmoc-N~4-t-Bu-propanoyl)Gly-Hyp~OBzl)-Phe-N~(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to three deprotection of the Fmoc 10 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 98i2~ 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) 15 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 20 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 R) (Table 2, steps 3-6). If the test did not show 98+2~ coupling the peptide resin was subjected again to a 25 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 98i2~ deprotection the peptide resin was 30 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).
35 The resin was washed 6 times with NMP (Table 1, step 15) and used in the r,ext stages.

21 ~305 WO 95/3376S P~

Fmoc-D-Arq(Tos)-Arq(Tos)-Na(4-t-Bu-pro~anovl)Glv-Hv~(oB
~S:~(3-BoC amino-~ro~ylene)Glv-ser(Bzl)-D-phe-phe-Arq(Tos)-O-resin -~ H-D-Arq-Arc-cvclQ(N~(4- Pro~anovl))Glv-Hvo-Phe-5 _~(3-amido-~ro~vl)GlY)-Ser-D-Phe-Phe-Arq-OH
Fmoc-D-Arg(Tos)-Arg~Tos)-N~(4-t-Bu-propanoyl)Gly-Hyp(OBzl)- Phe-N~(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 ~rotecting groups and on resin cyclization 10 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 reagen~ (0.19 g, 6 mmole) 15 was added to the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209 ~L, 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 20 washing the resin was subjected to picric acid test (Method R) (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 25 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 in vacuo 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 30 peptide treated with 20 mL liquid XF 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 vacuo. The peptide was extracted from the resin with TFA
(10 mL 3 times) and the TFA evaporated under vacuum. The 35 residue was dissolved in 20 mL 30~ AcOH and lyophilized.
This process was repeated 3 times. The crude peptide was purified by semipreparative HPDC (Method X). The final .. .... ..... . . .

~1 92305 ~ woss/3376s r~l~,s~ ''' product was obtained as white powder by lyophilization from dioxane, which gave 59 mg ( 34~) of the title co~ronn~.

. ~Table 5 f ollows at this point.]

2t 92~
W095/33765 r~

TABLE S BRO~u~X FOR 0.1 mMOLE SCAiE
STEP SOLVENT/ VOLUME TIME REPEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT

5 2 TFA/DCM 10 2 1 Deprotection 3 TFA/DCM 10 20 1 Deprotection 55~

10 5 NMP lû 2 4 6 DIEA/NMP 10 5 2 Neutralization 10~

8 TBTU/NMP 10 150 3 Cy~l;z~t;~n / DIEA
9 NMP 10 2 4 Picric acid test If less than 98+2~ Coupling perform Steps 10.

TBTU/NMP 10 2Oh 3 Cyclization, / DIEA Check pH, adjust to pH 8 with DIEA.
11 ~MP 10 2 4 Picric acid test.
If less than 98~2~ coupling perform Step 12.
12 TBTU/NMP 10 120 3 C~v~l;z~tion, 50 / DIEA ~C Check pH, adjust to pH 8 with DIEA

30 14 Piper- 10 10 l Deprotection idine/
NMP 20~
15 Piper- 10 idine/
NMP 20~
35 16 NMP 10 2 6 Check for positive nin.

2192~05 ~ WOgs/3376 HPLC ~Method G)RT 33.62 minutes (91~) TOF MS: 1278 (M~) A~A in agreement with the title compound .

E~XAMPLE 4 H-D-Arq-Arc-cvclo(N~(4-~ro~anoYl)Glv-Hv~-phe-N~(3-amid ~ro~vl)-S-Phe)-Ser-D-Phe-Phe-Arq-OH
Title compound was synthesized according to Example 3 except that in stage 1, Fmoc-N~(3-Boc-amino-propylene)-S-Phe (0.326) was substituted for Fmoc-D~(3-Boc amino propylene)Gly.
10 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 ~MP~ES OF BUILDING UNITS
The following speci~ic exam~les of novel building units are provided for illustrative purposes not meant to be limiting. The following is described in sections, including 20 "Procedures", "Methods",- "Compounds" and "EXAMPLES".
"Procedures" are detailed stepwise descriptions of synthetic procedures according to the more general schemes. "Methods"
are general descriptions of analyses used to determine the progress of the synthetic process. Numbered "Compounds" are 25 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, 41-30 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".

~1 9~305 W095l33765 I~~ .'C ''' Procedure 1 Svnthesis of N-Boc alkvlene diamines (BocNH(CH.) NH,) (known com~ounds ) .
To a solution of 0.5 mole alkylene diamine in 0.5 L
5 CHCl~ 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 Na2SO~ and evaporated to dryness 10 in vacuo.

Procedure 2 _ _ Svnthesis of N-Boc, N-Bzl alkylene ~;~m;n~ (BocNH(CH.) NH-~L
To 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) MgSO~, and 5.56 mL (0.055 mole) of f~shly distilled b~n7~ hyde. The reaction mixture was stirre under room temperature for 1.5 h. Then 11.34 g (0.3 mole) of 20 NaBH~ 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 25 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~ NH~OH, extracted with CHCl3 (3 X 100 ml) and the co~h;nGd extracts were~washed with water (3 X 80 mL), dried over Na2SO~and evaporated to dryness 30 in vacuo.

Procedure 3 SYnthesis of tR) or (S) ~-hvdroxv acids (known com~ounds).
To a solution of 16.52 g (0.1 mole) (R) or (S) amino 35 acid in 150 ml lN 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 2 1 ~3a5 W095/33765 ~ C-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 lN XCl (2x50 mL), H2O (3x80 mL), dried over Na2SO and _ 5 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 10 Svnthesis of (R~ or (S~ ~-hvdroxv acid methvl esters (known C~-~n~o~
To 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 CH,N2 until stable yellow color of 15 reaction mixture was obtained. Then the ether solution was washed with 5~ KHCO3 (3x100 mL) and X2O (2x80 mL), dried over Na25O4 and evaporated to dryness. The product was dried in vacuo.

20 Procedure 5 Svnthesis of triflate of (R~ or (S~ ~-hvdroxv acid methvl esters.
To 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
25 (dry ice in EtOH bath), then after 5 min a solution of 0.03 mole (R) or (S) ~-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 cmj. The product was eluted with 400 mL of 30 petrol-ether:methylene-chloride ~1:1). The solvent was evaporated in vacuo.

PrDcedure 6 - Svnthesis of (R~ or (S) ~ (Bzl)(N~-Boc-amino alkvlene ~ amino 35 acid methvl esters ((R) or (S) BocNH(CH,)~N(Bzl~CH(R)COOMe).
To a solution of 0.022 mole of N~-Boc, N~-Bzl alkylene diamine in 20 mL of dry DCM was added 3.04 mL (0.022 mole) Wogs/33765 2192305 r~ ss Et,N. Then a solution of 0.02 mole o~=(R) or (S) ~-hydroxy acid methyl ester triflate in 25 m~ 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
5 of CHCl3 was added and the yellow solution was washed with water (3 X 80 mL). The organic phase was dried over Na2SO~
and adsorbed on silica-gel and dried i~ vacuo. The silica-gel was washed on filter with 0.5 ~ of petrol-ether and with 0.5 L
of 2~ EA in PE. Then the product was eluted from silica with 10 0.5 L of mixture petrol-ether:ethyl-acetate (4:1). The solvent was evaporated in vacuo. 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 Q.8 ~ of hexane then the product was eluted with 1.5 ~ of mixture of pe~rol-15 ether: ethyl-acetate (4:1).

Prooedure 7 Hvdrolvsis of methvl esters _ __ _ To a solution of 0.015 mole of methyl ester in 40=mL
20 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 25 solution was acidified u~der cooling by addition of 40 mL 2N
HCL. The product was extracted with a mixture of CHCl~:i-PrOH
(3:1) (3 X 80 mL), dried over Na2SO~, evaporated to dryness and dried in vacuo to obtain a white foam in quantitative yield.
Procedure 8 Removal of Bzl bv Hvdroce~ation with Pd/C __ __ , To a solution of 0.012 mole (R) or (S) N3(Bzl)(N~-Boc-amino alkylene) amino acid in 60 mL MeOH-DMF (11-1) was added 35 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,O:glacial AcOH (1:3:5:1) was added.

~1 92305 WO 95/33765 1~ 1 /lL 5~C ~

The catalyst was filtrated off and washed (is the acetic acid~) with H20 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 in vacuo.
Proc~llre 9 Svnthesi6 of (R) or (S) N~Fmoc)(N=-Boc-amino alkvlene ) amino acid.
To 50 mL water was added 0.07 mole of (R) or (S) N~(NJ-10 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 15 solution was washed with petrol-ether (3 X 100 mL) and with ether:petrol-ether (1:4). The ac~ueous solution was acidified by addition of 14 mL lN 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), H20 (3 X 80 mL), dried over Na~SO~, evaporated 20 to dryness and recrystallized from ether: petrol-ether (80 mL:
200 mL) Procedure 10 Svnthesis of S-benzvlcvsteamine (Bzl-S-(CH~)~-NH,) (known 25 comPound).
To 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 30 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 MgSO~ and the solvent evaporated in vacuo. The crude product was essentially pure enough for the 35 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.

21 923(;~5 W095/33765 r~l,~,s~

Procedure 1}
Synthesis of N'-~-(benzYlthio)alkYlene)ohthallmides ~(Bzl-S-,-N~Pht)) ~known comDounds).
N-~-bromoalkylene)phthalimide (0.1 mole) and b-nzyl S mercaptane (0.11 mole~ were ~tirred with 0.1 mole potassium earbonate in 100 mL DMSO at 50~C for 24 hours. The mixture was poured into ice-water and the product was allowed to cryst~llize for 0.5 hour, collected by filtration and recrystallized from i-PrOH.
Pro~d~re ~2 S~r~thesis of N~ enzvlt~o)alkYl)~m;n~~ (9zl-S-(C~.) -NH~) ~ ~ rr---~pnl~n~lsl), Hydrazinolysi~ of the phthalimide group wab performed lS by refluxing 0.09 mole of N~-~w-(bcn2ylthio)alkylene)-phthalimide (P~Ce~LC 11) with 120 mL lM ~clution of hydrazine hydrate in ethanol (diluted with additional 220 mt ethanol) for 2 hours. The formed precipitate WaJ collected by filtration and hydrolyzed wlth 180 mL 2N HCl at 50~C for 0.5 20 hour. The water was evaporated in vacuo and the crude hydrochloride di~olved in 50 mL 25~ ammonia colution. Tho free amine was extracted with DCM (4 X 100 mL) and the organic pha~e was washed with brine ~2 X 100 mL), dried over MgSO~ and the solvent evaporated in vacuo. She crude N-(~-~5 (benzyl-thio)alkyl)amine wa~ distilled under reduced preYsure, and appeared as colorless oil, which could be kept refri~erated under nitrogen for prolonged periods.

Procedure 13 30 Svnthe~is of (R~ and (S) N~ benzylth~o~alkylen~ a~n~
acid~ meehvl est~rs ((R) or (S~ (3zl-S-(CX~r-NH-CH(Ri-COOMe~).
To a solution of 15 mmol N-~ benzylthio)alkyl)amine and 15 mmol D}EA in 55 mL DCM was added dropwi~e a solution of 35 15 ~mol (R) or (S) ~-hydroxy acid methyl ester triflate (Procedure 5) in 55 mL DCM at 0~C. The reaction mixture wau then stirred at room temperature for 18 h. The mixture was 2t 923~5 ~ W095/33765 r~

then dilueed with lOo mL DCM and washed with water (3 X lOo mL) The crude product wa~ cleaned on a silica-gel column with DCM MeOH(99 1), and was further crystallized from DIE hexan-In the case of Glycine, bromoacetic acld ester& were ~uita~le ~tarting material~ Identical re~ult~ were o~tained when both these substrates were reacted with ~ benzylthio) alkyl)amine~

10 Procedure 14 Svnthesi~ of ~R~ or ~s) Boc-N~ n~ylt~io~alkYl~nP) amino acids ((R~ or (S) (Bzl-S-(CH) -~(Boc)-CH(~)-COOH)) 10 mmol (R) or ~S) N-(~-lbenzylthio)alkylene) amino acid methyl eJter was di~solvcd in 50 m~ -dloxane and SD m~
15 lN NaO~ were added The mixture wa~ stirred at room temperature overnight The disappearance of thc ~tarting material was followed by TLC (Sillca gelL F,~, CHCl~ MeOH-~ 4) ~hen all the ester was hydrolyzed, so mL water were added, followed by 30 mmol Boc,O The mixture wa- ~tirred 20 overnight, then the dioxane was evaporated ln vacuo, the mixture was cooled in an ice-water bath, covered with loo m~
EtAc and acidified with saturated ~HSO~ to pH Z-3 The layers wer- ~eparated, and the aqueou~ layer wa~ extracted with additional 2X lOo m~ EtOAc The organic layer wa~ washed with 25 watcr (2 X loo mL), dried over MgSO~ and the solvent evaporated in VJC~0. The crude product was cleaned on a silica-gel column with DCM MeO~-99 1 or crystallized from DIE hexane 30 ~ L ~ 15 Svnthesis of ~;pe~tide~ (S S)-Boc-~m;n~ acid-N~-~-vlthio)alkYl~n~) amino acids e~ters A solution o~ 1 1 mmol Boc-amino acid, 1 mmol N~-(~-- (benzylthio~alkylene) amino acid eJtcr, 1 1 mmol ~OP and 3 35 mmol DIEA in 10 m~ DCM was otirred at room temperature for 2 hours Then the mixture wa~ diluted with 40 mL of DCM and wa~hed succes~ively with ~aturated RKSO~ ~3 X 100 m~), - 8~ -~192~Q5 wos~376s ~aturated XHCO~ (3 X 100 mL) and brine (2 X 100 mL), dried over MgSO~ and the solvent evaporated ~'~ vacuo. The crude product was cleaned on a sllica-gel column with DCM MeOH-9g 1 or cry~tallized ~rom DIE hexane Pror~ re 16 ~vnth~ls of Nr-T~ -2mi~ aclds t-butY1 ester~ (3zl-~H-(r~-~ -COO-t-Bu) A ~olution of 0 05 mole of amlno ac~d t-~utyl ester 10 acetate in 200 mL H2O was ~cidified to pH 2 with AcOH, washed with PE (70 mL X5) cooled and the pH adju-ted to 9 by NH~OH
25~ The free amino acid t-butyl ester wa6 extracted with i-Pr CHCl (3 1, 3X100 mL) The cv~'ir~d extract~ wcre dried on Na~SO, and e~aporated to dryness under vacuum The 15 benzylation reactlon was performed according to P~ocel~r~ 2 Proced~re 12 Svnthesi~ of N~-t~zl) (~t-~u carboxv alkYlene) qlyc;nP BZ1 ester (N-(9zl)~c~)r-coo-t-Bu~r~ -COO-Bzl) To a ~tirred ~olution of 0 015 mole of N-~zl ~-amlno acid t-butyl ehter in 10 mL DMF at 0~C were added 2 61 mL of DIEA and 2 38 mr of benzyl bromo acetate The reaction mixture was ~tirred 30 min at 0~C and 3 h at room temperature After the addition o~ 200 m~ of ether, the precipi~aoe was 25 removed by ~iltration and the organic pha~e wa~hed wlth ~O
l3XB0 mT~, lN ~Cl (3X80 mL), H~O l3X80 mL), dryed over NalSO~
and evaporated to dryness under vacuum The re-ulting oil was dried under vacuum ANAT,YT~C~T TLC wa~ performed on TLC plate~ of silica gel E
(Merck Fls",using the following solvent sy~tem~

MT'THOD A DCM MeOH AcOH 16 4 0,5 35 M~THOD B P~ EtOAc 1 1 M~THOD C PE EtOAc 4 1 ~THoD D CBCl~ EtOAc 4 1 .. . . . . _ . _ .

~ 1 92305 WO 95133765 r~ L.

M~TXOD E P~: ~tOAc 9:1 MRTXOD F CHCl~: EtOAc 19:1 .

~THOD G ANAT~YTI~Ar~ ~V~RC~ p~c~ ~PLC
Column Merck LICHROCART RP-l~ 5 ym, 250X4 mm.
Mobile Phases A ~ 0.1~ TFA in H~O
B - 0.1% TFA in ACN
Gradient T - 0-5 mln A(75~), B(25~) T ~ 30 min A~50t), B(50~
T - 40 min A(100~), B(O~) T ~ 50 min A~100%), P(0%) Flow . 1 mLtminute Tcmperature ~ 23 ~C
.

MFTHOD H - SEMIP~ARATIv~ REvE~S~ P~ UPLC
The crude peptide was di~solved in MeOH (lm~) and chromatographed using reverse pha-e ~emipreparative HPEC with the following conditlons:
Column Merck Hibar LIru~osop~ RP-18 7 ~m, 250 X 410 mm ~obile Pha-es A - 0.1~ TFA in H~O
B ~ 0.1% TFA in ACN
Gradient T - 0-10 min A(80~), 3(20%) T ~ 60 min A(100~), B(O~) T - 70 min A(100~), 3(0 Flow ~ 4 mL/minute Temperature - 23~C
M~THOD J NINHYDRIN TEST (NIN. TEST) The test was performed accordlng to Kal~er et al. ~31~
Bl~h~m., 34:5g5 ~1970) which is incorporated herein by refer-nce ln i~s entirety. Test was consldered negatlve when 30 the re~in did not change color after heating to 11~C for 2 minutes with the te~t mixture. Te~t was consldered posl~lve or ~lightly po~itive when the re~in was dark or fAlnt purple after heating to 110~C for 2 minute~ wlth the te~t mixture.

35 M~THOD X - QUANTITATIVE PICRIC ACID TEST
Picric acid te~t was performed af~er removal of the Fmoc protecting group from the amino acld precedlng the 21 q2305 wo9st3376s r~,~,s~

coupling of protected N~ alkylene) building unit. This absorbance was taken as lCOS free ~mines. After coupling the test was u~ed to check yielt of ~ coupling by comparison.
The resin 0.1 mmole wa~ tre~ted according to ~tep~ 1-8, 5 T~kle 1. After ctep 3 the re3in wa- introduced into ~
centrifuge tube and shaken wlth 40 mL Or 5~ DI~A:95~ DCM for ten minute~. ~he resin wa~ centrifuged 5 minute8 and 4 mL of the solution were pipette into 40 mL. EtOH and the ab~orbance at 358 nm measurea. This procedure wa~ repeated 3 time~ and lO the average ~orb~-~e calculatet.

T~T~n~ 6 PRG~ u Fo~ 0.1 -MO~ 6r~r 8~EP 58LVENT/ VOLI~ME ~IME REPEAT
NO. B~ ~ML~ (MlN1 tX51 ~ot~E!~r 15 2 ~M l0 1 2 3 ~lcric ~cl~lO 1 2 M nsM/D~M
DM~ _ 0 0 . S . 0 I;M ~ . o ~ . _ 6 NM~ .0 7 10 r Eto!~tDcM .0 S D~M .0 _ t r~ .,".~, 1 N-Boc diamlno ethane Iknown ') A ~olution o~ 33.4 mL of ethylene diamine in C~Cl~ and 25 lO.gl g of Boc~O were used (Procedure 1). Yield 97~ of colorless oil.
TLC (Method A) ~ 0.2-0.24 (one ~pot) NMR (CDCl~) in a~ r-nt with the title .v ,~.o~A

30 ~n~POUND Z
N-Boc 1.3 d;~miT~ pro~ane Iknown com~ound~.
A solution of 41.7 mL of 1,3 diamino propane ln CHCl~
and 10.91 g of Boc20 were used ~Procedure 1). Yield 9~% o~
colorle~ oil.
3~ TLC tMethod A) Rf 0.27-0.3 (one spot) NMR tCDCl~) in agreement with the title - ,_u 2 ~ 92305 N-Boc 1,4 ~;~m;r~ butane ~n~wn com~ound).
A solution of ~4.08 g of 1,4 diamino butane and 10.91 g of Boc~O were u~ed (Procedure 1). Yield sas of white oil.
- 5 TBC (Method A) Rf 0.32-0.35 (one spot).
NMR (CDCl~) ln agr-ement with the ticle compound.

SQ~Por~n 4 N-3OC 1.6 di~-~ n~ hexane Iknown co~noun~).
A solution of 58.10 g of 1,6 diamino hexane in CHCl, and 10.91 g of 3OC,O were used lProcedure 1). Yield 70~ of colorless oil after purification on a ~ilica gel column and elution with C~Cl~-MeOH (4:1).
TBC (Method A) Rf 0.50-0.54 (one apot) 15 NMR (CDCl~) in ayL~_ - t with ehe citle ro-rour~

r r.~po~Nn S
N-Boc. N-Bzl 1.2 diamlno ethane A solution of 8.01 g of Boc ethylene diamine (e .r~U~
20 1) wa~ u~ed (PLoccdu~e 2). Yield 65~ of colorless oil.
TBC (Method A) ~f 0.62-0.65 (one ~pot) NMR (CDCl) in ugreemeent wi~h the title compound CC..~O~NJ 6 25 ~-Boc. N-Bzl. 1.3 diamino ~r~nP.
A ~olution of 8.71 g of N-Boc 1,3 diamlno propano lCC.l~Ou..u 2) was uced (PLUCe1ULe 1). Yield 75~ of colorless.
TTC lMethod A) Rf 0.63-0.63 lone ~pot) NMR ~CDCl,) in a~L~ with the title cv~ u~
30 ~ uu~ 7 N-Boc. N-Bzl 1,4 dlamino butane A ~olutlon of 9.41 g of N-3OC 1,4 diamino butane lCompound 3) w~8 u~ed lProcedure 1). Y$eld 63~ of white oil.
T3C ~Method A) ~f C.65-0.72 lone Ypot) 35 NM~ ~CDCl,) in a~L~ t with the title r~,,vu wo gS/3376s ~ ~ 9~3 05 . ~

CQMPQU~D E
N-Boc N-Bzl 1.6 dlamlno hPYAn~
A solution of 10.82 g of N-Boc 1,6 diamino hexane ~r~rOun~ 4) was u6ed ~Procedure 11. The ethyl acetate 5 ~olution after extraction was dried over Na~SO~ ~nd evaporated under vacuum to dryne~s. The ~ ;n;r~ crude product wa~
dissolved in 400 m~ chloroform and washed with 0.5 N H~l 13 X
80 m~, 0.12 molè~, water (~ X 100 m~) dried over Na,SO, and evaporated to dryness. Then 200 m~ of ether was added. The 10 precipitate was fLltered, wa~hed with ether t3 X 50 m~) and dried under vacuum.
Yicld 70~ of white ~olid mp 150-lS2~C.
T~C (Method A) Rf 0. e ~one spot) To remo~e HCl from product with n 6, the HCl ~alt was 15 dis~olved ln CXCl~, wa~hed with an alkali solution ~0.5 NH,OH~, dried over Na,SO~ and evaporatet to dryne~s.
NMR ~CDCl~ in hy~ ~_ t with the title compou~.

rc~blpo~r~n g 20 (S)-3-PhenYlacetic acid methYl esr~r ~k~wn co~o~n~) A ~uspens~on of 10.& g of (S)-3-Phenylacetic acid in 100 m~ ether was treated with diazomethane ~Procedure 4).
Yield 8S~.
T~C ~Method B) Rf 0.6-0.65 ~one spot~
a5 (~)D' +3,3 (c-1, MeO8) NMR ~CDCl,) in ay ~ r with the title r,l~, INI~ 10 (R)-~-Ph-nvlacetic acid methvl ester ~known ~_ d).
A sn~p~r~ion o~ 10. a g ~R)-3-Phenylacetic acid in lOC
m~ ether WA5 treated with diazomethane ~Procedure 4). Yield 8~
T~C ~Method B) Rf 0.6-0.65 (one ~pot) ~) D~ - 3,3 ~c.1, MeOX) 15 NMR ~CDCl~) in aSreement with the title _ . _ '.

2i 92305 WO 95/33765 . ~I

CO.I~IJUNU 11 ~S)-O-Trf-3-Ph~nvlacetic acid methYl e~ter To ~ cooled solution of Trf,o and pyridine in dry DCM
(PLOCe 1Ure 5~, a ~01UtiOn of 5 4 g of tS~-3-phenylacetic acid 5 methyl e~ter wa~ added A~ter ~he wor~up ~P~ccdu . 5~ the yield was 74S The product was u~ed i -~ia~ely or kept in a cold desiccaeor under Ar ~V..:"./UNL~ 12 10 ~R)-O-Tr~-3-PhenvlAcetic acld methvl e~ter To ~ cooled oolution o~ Tr~O and pyrldine in dry DCM
(Procedure 5), a solution o~ 5 4 g of (R)-3-Phenylacetic acid ~ethyl c~ter wau added After the workup ~Procedure 5~ the yield waa 74S The product wa~ used immediately or kept in a 15 cold de~iccator under Ar CC. ,~'O~iNl~ 13 (Bzl)r2-Bo -~min~ eth~len~) (R)PhenYlal~;n~ m thyl e~ter A ~olutlon of 6 24 g of (S)-O-~rf-3-Phenyllactic ncid 20 m-thyl e~ter in dry DCM ~rnm~o n~ was added to a oolution of 5 51 g Or N-Boc, N-Bzl-diamino ethane ~C ~n~ 5~ in dry DCM (Procedure 6~ Yield 6g 2S
~~) D ~ +64 0 (c.l, MeOH~
TLC (Method C) Rf .0 41 (one spot) NMR (CDCl~) in ay . t with the title u UU.~J 14 Nr ~Bzl)(3-Boc-amlno Dro~vlene) (s~ph~nylAl~nin~ methvl ester A ~lution o~ 6 24 g of (R)-o-Tr~-3-phenyllactic acid 30 methyl eater in dry DCM ~Compound la~ wa~ adted to a solu~ion of 5 82 g of N-Boc, ~-Bzl-diamino propane (C ~u d 6~ in dry DCM (Procedure 6) Yield 67 7%
~ 55 8 Ic-l, MeOX) - T~C (Method C~ R~0 38 tone spot~
35 NMR tCDCl,~ in agLe~ t with the title ~c~,ouAd ~ ~ 9 ~

rnr.~PoUND 1 5 NI(Bzl~4-~oc-amino butvlene~ (S)Ph~nvlalanine methvl ester A solueion of 6 ~ 24 g of 0-Trf-(R)-3-Phenyllactic acid methyl ester in dry DCM (C ~ 12) was adt to a solution of S 6.12 g of N-30c, N-Bzl-diamino butane ~Compound 7) in dry DCM
(Procedure 6). Yield 58.6 1Q)~ ~ -62.6 (c-l, MeOH) Rf (Method C ) 0.42 ~one spoe) NMR (CDC1~) in agreement wieh the title Nr ~Bzl~ t6-30C-am~n~ hexYlene) (S)Phenvl~lanin~ methvl ester A colution of 6. 24 g of 0-Trf-IR)-3-Phenyllactic acid methyl ester in dry DCM ~Compound 12) was add to a solution of 15 6.74 g of N-Boc, N-Bzl-diamino hexane ~Compound 8) in dry DCM
~Procedure 6~ . Yield 78.9 (~)D' -60.C ~c~l, MeOH) TLC ~Method C) ~f-0.47 (one 6pot) NMR (CDCl~) in ~y~ ~ with the title ao ~u.I~uu~ 17 NIIBzl)~3-Boc-amino ~ro~vlene~ (R~Phenylalanine methYl ester A solution of 6.24 g of 0-Trf-~S)-3-Phenyllact$c acid methyl ester in dry DCM ~Co~pound 11) was add to a solution of 25 5.82 g of N-Boc, N-Bzl-diamino propane ~ .round 6) in dry DCM
~PLOC~iU~ 6) . Yield 51.5 (Q)D~ 158.8 ~c=l, MeOH) T~C (Method C) Rf-0.35 (one spot) NMP~ (CDCl3) in ay~ t with the title c . _ COMPOUND lB
Nr (szl~t4-Boc-~1no butvlene) (~)Phenvlalanine methYl e~t~r A /301ution Of 6.24 g of 0-Trf-(5)-3-Phenyllactic acid methyl ester in dry DCM (C un~ 11) was add to a solution of 35 6.12 g of N-Boc, N-Bzl-diamino butane ~Co-.ro~n~ 7) in dry DCM
(Procedure 6). Yield 66.8 (Q)D~ +59.0 (c-1, MeOH) 21 92~0~
09s~3765 T~C (Method C) Rf~0.33 (one ~pot) NMR ~CDCl~) in agreement with the title co-poun~.
.

5 N~(Bzl) (3-Boc-~;nn ~roDvlene~ (5~ Phenvlalanine A solution of 6.61 g of N~ ~Bzl~ 13-Boc-amino propylene) (S)Phenyla~nine methyl ester in MeOH (Co-,rol~n~ 14) wa~
hydrolyzed by NaOH 7.5N ~P.~ce~c 7). Yield 89.5S
(~)D~ -24-0 (c-1, MeOH) 10 TLC (Method B) Rf'0.16 (one 6pot) NMR (CDCl~) in ~yL~-r--- with the title r~ o~NI- 20 N~ (8z1~4-30c-A-~n~ butvlene) (S)Phenvlalanine A oolution o~ 6.61 g of N-(Bzl)(4-Boc-amino butylene) ~S)Phenyl~lAn;ne methyl ester in MeOH ~r~mroun~ 15) wa~
hydrolyzed by NaOH 7.5N ~Procedure 7). Yield 73.5S
~)D' - 12.0 ~c-l, MeO~) TLC ~Method D) R~0.6 ~one spot) 20 NMR ICDCl,) in agreement with the title c~ v N~ ~zl)(3-30c-amino ~ro~vlene) ~R~PhenYlalanine A aolution of 6.40 q of N'(8zl)(3-80~-amino propylene) 25 tR)phenyl~l~n~ methyl ester in MeOH IC , ' 17) wa~
hydrolyzed by NaOH 7.5N IProeedure 7). Yi-ld lOoS
~)D' +15-33 ~c-1, MeOH) TLC ~Method B) Rf-0.38 ~one rpot) NMR ~CDCl~) in ayrc~er~ with the title c , '.

COMPO~ ~ 22 N' ~Bzl)~4-Boc-amino butylene) (R)phenylal~nine A solution of 6.61 g of N~~}3zl)~4-80c-amino butylene) - (R)Phenylalanine meehyl ester in MeOH (Ccmro~n~ 1~) wa~
35 hydrolyzed by NaOH 7.5N ~P~ocedure 7). Yield 100 (~) D" +lZ ~ O ~c'l, ~eOH) ~LC (Method D) Rf~0.54 ~one apot) ~192~i05 woss/3376s r~

NMR ~CDCl~) in agreemenc wi~h the title compound r~ I~UUNJ 23 N~ 13-Boc-amino o~oDvlene) (S)Phenylal~n;n! HCl 5 A fiolution of 5 39 g o~ N ~Bzl)t3-Boc-amino propylene) ~S)Phenylalanine ~Compound 19) in MeOH-DMF wa6 hydrogenated on Pd/C ~Procedure 8) Yield 86 1~
TLC tMethod ~)Rf ~ 0 51 (one ~3pot) NMR tD20+Na~CO~) in a~.c -t with the title c~ , C~ UI-N U ~ 4 N7t4-Boc-amino butvlene) ~S)Phenvlal~ine H~l A solution of 5 56 g o~ ~ ~Bzl)~4-Boc-amino ~utylene) ~S)Phenylalanine (r~ ~ ~ ' 20) in MeOH-DMF was hydrogenated on 15 Pd/C ~Procedure 8) Yield 79 25~
TLC ~Method A) R~ ~ 0 50 ~one spot) ~MR ~D~ûlNa2CO~) ln ayL ----e with the title ~IPUUN~ 25 20 N7(3-Boc-~minA oro,oylene) (R)Ph~nylal~1ne XCl A solutlon of 5 39 5 o~ ~ ~3zl)~3-Boc-amino propylene) ~R)Pheny]~l~nin~ t~ , 1 21) in MeOH-DMF wa~3 hydrogenated on Pd/C ~Procedure 8) Yielt 75 5~
TLC ~Method A)Rf - 0 51 (one spot) 25 NMR ~D,OIN~,CO,) in a ~e- --t with the title c ,:

Q~pOtt n a 6 N~ (4-Boc-~mi rA ~utylene) (R)PhenvlalA~
A ~olution of 5 56 g o~ 3zl)~4-Boc-amino butylene) 30 ~R)Phenylalanine ~r~ 22) in MeOFt-DMF wa8 hydrogenated on Pd/C ~Procedure ~) Yield 73 85~
TLC IMethod ~) Rf ~ 0 50 ~one 8pOt) NMR ~DlO+Na2CO3) in ag~. t with the t~ tle eC:.L~J "~.

35 ~Y--~T.Y 5~
C~ ~uu~L~ 27 N~ ~Fmoc)~3-Boc-amino t~ropylen~) tS)Phen~lalanine WO 95/33765 P~

A solution of 2.51 g of N ~3-Boc-amino propylene) (S)Phenylalanine.~Cl tC - ~nd 23) in H~O-ACN was reaceed with FmocOSu (P~uc6iu~e 9). Yield 64.84 TLC (Method D) Rf ~ 0.74 (one 8POt) 5 (~Yja~ -87 (C~1~ MeOEI) HPLC (Method G) ga %
NM~ (CDCl3) in aylecm~n~ with the title - _ In~, ~vPL~ 6~
0 ~'M~VUNU 2B: N~(Fmoc)(3-Boc-~m;nn ~ro~ylene) (R)Phen~lalanine A 801ution cf 2.51 g of N'l~-Boc-amino propylcne) (R)Phenylal~nine.HCl (C ' 25) in H~O-ACN w~ reacted with FmocOSu (PLOCCd~Le 9) . Yield 61.56$
TLC (Method D) Rf e 0.62 (one spot) 15 (~) D- i79.6 (c-~, MeO~) ~PLC (Method G) 94 NM~ (cDcl~) in a~ccment with the tltle ~ _ ~1nvpr~ 7:
20 UU..fUU~ 2g: N~(Fmoc)(4-Boc-amino ~utylPn~) ~S)Phenvlalanine A solution of 2.61 g of N~(4-30c-a~ino ~utylene) (S)Phenylalaninc.XCl (C _- 24) in ~û-ACN was reaceed with FmocûSu ~Procedure 9). Yield 56%
T~C IMethod D) Rf - 0.64 (one spot) 25 XPLC (Method G) 89 ~
NMR ~CDCl~) in ay~e~r~nt with the title ~ uu~u 30 O-Trf-~S)-L~tlc acid met~yl ester To a cooled solution of Trf~O ~nd pyridine in DCM
~P.ocel~e 5) a uolution of 2.9 mL of (S)lactic acid methyl ester was add-d. Af~er the workup (FL~C-dU~e 5), the yield was 70%. The produc~ was used i i~tely or kept in a cold desiccator under ~r.
~C.~ 31 O-Tr~-~R)-L~ctic Acid mcthyl ester - g5 .

2~ q2~05 W095/3376~

To a coolcd 601ution of Trf20 and pyrldine in DCM
~Procedure 5), a ~olution of 2 9 mh of (R~ lactic acid me~hyl ester wa~ adaed After the workup (Pro~edure 5), the yicld wa~ 70~ The producc wa5 u6ed lmmediately or kept in a cold 5 de~iccator under Ar .~ 32 zllt3-Boc-~mino oro~Ylenel (S)Alanlne ~ethYl ecter ~ eolution of 4 72 g o~ O-Tr~-(R)-lactic acid methyl 10 ester ln dry DCM (C ,-un~ 31) was add to a ~olution of 5 E2 g of N-Boc, N-Bzl-dlamino prop~ne ~Oompound 5) in dry DCM
(Procedure 6~ Yield 69 5 %
~)D - -6 6 (C-1, MeOH) 5hC (Method C) Rf - O.42 tone ~pot~
15 5LC (Method D) Rf ~ 0 92 (one ~pot) TLC (Method ~) Rf ~ 0 13 lone spot) NMR ~CDCl~) in a~ t with the title c~

r~ NI~ 33 20 N~ rBz1)(3-Boc-A~n~ ~ro~vlene~ ~R)Al~n~ne me~hyl e~ter A 601utLon of 4 72 g of O-Tr~-(S-lactic acid methyl ester in dry DCM ~C ,_ ~ 30) wa~ add to a solution of 5 82 g of N-30c, N-Bzl-diamino propane tC ,_~ 6) in dry DCX
~Procedure 6) Yield 71 (~)~ c l6 53 tC~1 MeOX) TLC ~Method C) Rf ~ 0 42 (one 6pot) ~LC (Method D) Rf ~ O g3 (one spot) NMR (CDCl,) in ay G- t wieh the title c~ ~iv A

30 r ~ 34 N~3zl~t6-Boc-~no hexYlenel ~S~AlaninP methyl e~ter A colutlon of 4 72 ~ of C-Trl-(R~-lactlc acid~ methyl o-ter in dry DCM ~C~o~n~ 31) wa~ add to a ~olution of 6 74 g of N-Boc, N-Bzl-diamino hexane tC ,_a ~ E) in dry DCM
35 tP~oc,d~ ~ 6) Yield el.
~ -6,76 ~C-l, MeOH) TLC ~Method D) Rf - O g5 tone ~pot) ... _ ... . .. . _ _ . _ _ _ _ _ 21 q2305 095l33765 r_ .,~,s ~ .

TLC (Method E) Rf = 0.26 (one spot) NMR (CDCl,) in agreement with the title ~.

CQMPOUND 35 _ _ 5 Nr (Bzl~(2-Boc-amino DroDvl~nP~ (S)Alanine A solution of 5.25 g of N~(Bzl~t2-Boc-amino ethylene) (S)Alanine methyl ester in MeOX ~r onn~ 32) was hydrolyzed by NaOX 7.5N (PLvcel~e 7). Yield 100 ~ of white solid, mp 0 t~)D ' +0.5 (C~l MeOH) T~C (Method ~) Rf ~ 0.64 (one spot) TLC (Method D) Rf - 0.47 (one spot) NMR (CDCl~) in agreement with the title _ ~.

N~ (Bzl~ (6-30c-amino hexvlene~ (S)~l~n;n~
~ solution of 5.88 g o~ N~ (Bzl) (6-Boc-amino hexylene) (S)AlAnine methyl ester (~o-ponnd 34) in MeOH was hydrolyzed by NaOH 7.5N (P oceluLe 7). Yield 100 20 (~)D - +0.7 ~C-l, MeOH) TLC (Method D) Rf ~ 0.51 (one spot) NMR (CDCl,) in agreement with the title compound.

COMPOT~ 37 25 N~ (Bzl~(3-Boc-~m;nn DroDvlenc~ (R)~l~n;r~
A snllltinn af 5.25 g of N~ (Bzl)(2-Boc-amino ethylene) (R)Alanine methyl ester (Compound 3S) in MeOX was hydrolyzed by NaO~ 7.5N (Procedure 7). Yield 100 (~) D ~ ~0-5 (C-1, MeOX) 30 T~C (Method D) Rf ~ 0.51 (one spot) NMR (CDCl~) in agreement with the title _ ~"d.

COMpOT~Nn 38 Nr ~3-Boc-amino ~roDvlene ~ (S~Alanine.HC1 A solution of 4.~7 g of N~ (Bzl)t3-Boc-amino propylene) ~S)Alanine tC _1-n~ 35) in MeOH was }~yi~oy~nated on Pd/C
tProcedure 8). Yield 75 ~

_ 97 _ wO gsl3376s2 1 9 2 3 ~) S p TLC (Method A) Rf ~ 0.42 (one spot) NMR ICDCl,) in agreement with the title c ~.

COMPQr~ 39 N~ (6-Boc-~m;nn hexvlene) ~S~Alznine .HCl A solution of 5 g of N (Bzl)t4-Boc-amino hexylene) (S)AlAnine (Compound 36) in MeOH-DMF was hydrogenated on Pd/C
~PL~CedULe 8). Yield 64.5 ~ of white solid, mp 134-136 ~C
TLC ~Method A) R~ - 0.39 ~one spot) 10 NMR (cDcl~) ln ay. e~~ ~ with the title _ u~ld.

~Q (3-Boc-Ami n~ T~rosvlPnp) ~R) ~1 ~n; n~ .~Cl A solution of 5 g of Nr (Bzl)~-Boc-amino propylene) 15 ~R)Alanine ~Compound 37) in MeOH-DM~ 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 ~ ~ ~.

20 FY~T~! 8 CO..~UUN~ 41: N~Fmo~)~3-Boc-am~n~ ~ropylene) ~S)Alanine A solution of 2.82 g of N' ~Bzl)~4-Boc-amino propyiene ) (S)Alanine .HCl ~C ' 38) in H~O-ACN was reacted with FmocOSu (Procedure 9). Yield 75 ~ of white solid, mp 70-72CC
25 TLC (Method D) Rf = 0.65 (one spot) NM~ (CDCl,) in a~L. nt with the title c~
Analysis: % C ~ ~ ~ N
Found: 66.40 6.78 5.63 Calc: 66.65 6.88 5.93 ~YaMPLE 9: ' uu~u 42: N~ (~moc~6-Boc-am;n~ hexYlene~ (S)~l~n;nP
A solution of 3.25 g of N~ (Bzl)(4-Boc-amino hexylene) (S)Alanine .HCl (Compound 39) in H,O-ACN was reacted with 35 FmocOSu (Procedure 9). Yield 72.8 ~ of white solid, mp 70-72~C
TLC (Method D) Rf - 0.7 (one ~pot) woss/3376s r "~ ~ ( r NMR (CDCl~) in a~L.- nt with the title ro~po~ m Elemental Analy~ % C t H % N
Found: 68.31 7.40 5.23 Calc: 68.21 7.50 5.49 5 HPLC ~Method G) 90 ~V~Ll~ 10 t ~u,l~vu~.~ 43: N~~Rmoc)(3-BQc-~m;nr. Dropylrn~)~R)~ n~
A solution of 2.82 g of N~Szl)(4-Boc-smino prcpylene) 10 ~S)Alanine .HCl ~Co~s~pound 40) in H O-ACN wa3 reacted wlth FmocOSu ~Procedure 9). Yield 75.9 % of white ~olld, mp 70-72~C
TE.C (Method D) Rf . 0.5 ~onc spot) N~R ~C~Cl,) in agreement with the title ~
15 Elemental Analysis: ~ C % H '~ N
Found: 66.4 6.7a 5.63 Calc: 66.65 6.88 5.g3 ~XA~PLE 11:
20 CO.I~uu..l~ 44- N-~2-(benzvl7~h;o~ethylene)alycine ethvl e~ter The title ~ ' was prepared according to procedure 13 from sthyl bromo acetate.
YLeld 75% of colorle-~ oil.
NMR ~CDCl~) in a.,,~ t with the title ~
25 Elemental analy~is-c~lculated: C-61.16, H-7.70, N-3.96 ;
~ound: C-61.45, H-8.03, N-3.4g.

7''~ 07UNV 45 N-(3-tbenzylt~ )nrQ~ovlene)glycine methvl eeter The tLtle : ~__ ' waa prepared accordins to y edu-.
13 from methyl bromo acetate.
YLeld 74% of colorle~3e oil.
N,~R ~CDCl~) ln a, .-- rt with the title -35 ~SY~ E 12:
~u.l~uu~u 46: N-~2-~benzvl~h;o~ethYl~ S)leUCir~e methyl e~ter Wogsl3376s 21 9 Z 3 05 F~,5~

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

10 EXAMPLE 13:
COMPOUND 47: N-(3-(benzYlthio)pro~ylene)~S)leucine methYl The title compound was prepared according to procedure 13 from the Triflate of (R)leucine methyl ester (Procedure 5).
15 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:
~-(2-(benzvlthio~ethYlene)(S)~henYl~l~n;n~ methvl ester The title comro~ln~ was prepared ar~r~; ng to procedure 13 from the Triflate of (R)phenyl lactic acid methyl ester 25 (Procedure 5).
Yield 82~ of white crystals.
m.p.=48-49~C.
NMR (CDC13) in agreement with the title ~ oul.d.
Elemental analysis- calculated: C-69.27, H-7.04, N-4.25 ;
30 found: C-69.55, H-7.21, N-4.08.
(a)D"=-23.3~ (C =1.01, DCM).

N-(3-(benzvlthio)~ro~Ylene)(S)~henYlalanine methvl ester The title compound was prepared according to procedure 13 from the Triflate of (R)phenyl lactic acid methyl ester (Procedure 5).

21 ~2~a5 095/33765 r~ 3c[~

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 ;
5 found: C-69.66, H-7.39, N-4.37.
(a)D26=+2.0~ (C 1~00, DCM).

10 N-(4-~benzYlthio)butvlene~(S)~henvlalanine methYl ester The 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.
15 NMR (CDCl3) in ayL~- - t 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).
EX~MPLE 14:
CQMPOUND 51: Boc-N-~2-(benzvlthio)ethvlene)qlYcine The title u~.u~uu.-d was prepared from Compound 44 by hydrolysis according to Procedure 11.
25 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.
~ E 15:
COMPOUND 52: Boc-N-(2-(benzYlthio)ethvlene)(S)~henYlalanine The title compound was prepared from Compound 48 by hydrolysis according to Procedure 11.
35 Yield 78% of white crystals.
m.p.=82-83~C.
NMR (CDCl3) in agreement with the title compound.

2 1 923~5 W095/33765 P~ 5'~ ''' (a)D25=-105.9~ (C 1.01, DCM).

EXAMPLE 16~
COMPCUND 53: Boc-N-(3-(benzvlthio)~roPvlene)(S)~henvlalanine The title compound was prepared ~rom rnmrolln~ 4-9 by hydrolysis according to Procedure 11.
Yield 99~ of white crystals.
m.p.=63-64~C.
NMR (CDCl3) in agreement with the title compound.
10 (a)D5=-87.4~ (C 1.01, DCM).

~XA~PLE 17:
COMPO~ND 54: Boc-L-~henYlalanvl-N-(2-(benzylthiQ)-ethvlene)qlYcine ethvl ester Boc-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.
20 Elemental analysis- calculated: C-64.77, H-7.25, N-5.60 ;
found: C-64.39, H-7.02, N-5.53.
~a)D~6=+4.5~ (C 0.88, DCM).

~XA~P~ 18: _ _ __ _ 25 COMPO~ND 55: Boc-L-~henYlalanYl-N-(2-(benzYlthio)- __ ethvlene)(S)~henvlalanine methvl ester Boc-L-Phe was coupled to N-~2-~benzylthio)ethylene) (S)phenylalani~e methyl ester (Cnm~olln~ 48) according to Procedure 12.
30 Yield 46% of colorless oil.
NMR (CDCl3) in agreement with the title compound.
(a)D,6=-115.9~ (C 1.0, CHCl3).

35 N-Bzl-~- alanine t-butvl ester 21 9230~
WO95/3376s r~

A solution of 6.16 g of ~- alanine t-butyl ester acetate in 150 mL water wag reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5~ yield TLC (Method A) Rf=0.78 (one spot) 5 NMR (CDCl3) in agreement with the title compound.

CoMpouND 57 N-Bzl-~- amino butvric acid t-butvl ester A solution of 6.58 g of ~- aminobutyric acid t-butyl 10 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.

15 COMPOVND 58 =~
N~ (Bz1)(2-t-butvl carboxy ethvlene~qlvcine benzvl ester A solution of 3.53 g of N-Bzl-~- alanine t-butyl ester (C~rolln~ 56) in DMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17). Yield 86.9 20 TLC (Method F) Rf=0.95 ~ne spot) NMR (CDCl3) in agreement with the title compound.

N~ (Bz1)(3-t-butvl carboxv ~ro~vlene)clvcine benzvl ester A solution of 3.S3 g of N-Bzl-~- 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.

COMPOUN~ 60 _~(2-t-butvl carboxv ethvlene)qlvcine A solution of N~ (Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester (Compound 58) in MeOH was 35 hydrogenated (Procedure 8). Yield 87.8 TLC (Method A) Rf=0.56 (one spot) NMR (CDCl~) in agreement with the title compound.

wOss/33765 2 ~ 9 ~3 0 5 1 3; r 1 rr N~(3-t-butvl carboxv ~ropvlene)qlvcine A solution of N~ (Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester (Compound 59) in MeOH was 5 hydrogenated (Procedure 8). Yield 94~ -T~C tMethod A) Rf=0.3 (one spot) NMR (CDCl~) in agreement with the title compound.

,~PLE 19: COMPOUND 62 10 N~(Fmoc) (2-t-butvl carbgxv ethvlene)qlvcine A solution of N~ (2-t-butyl carboxy ethylene)glycine (Compound 60) in H2O:Et3N was reacted with FmocOSu (Proc~iure 9). Yield 90~
TLC (Method D) Rf=0.5 (one spot) 15 NMR (CDCl3) in agreement with the title compound.
Elemental Analysis: ~ C ~ H ~ N
Found: 67.38 6.34 3.11 Calc: 67.75 6.40 3.29 20 EXANLPLE 20:
COMpOUN~ 63: N~(Fmoc)(3-t-butvl carboxv ~ro~vlene)qlvcine A solution of N~(3-t-butyl carboxy propylene)glycine (Compound 61) in H2O:Et3N was reacted with FmocOSu (Procedure 9). Yield a2~
25 TLC (Method D) Rf=0.58 (one spot) NMR (CDC13) in agreement with the title compound.
Elemental Analysis: ~ C ~ H ~ N
Found: 68.29 6.83 3.88 Calc: 68.32 6_65 3.19 COMPOUND 64 __ _ _ _ (R)-O-Trf-3-Phe~vllactic acid benzvl ester To a cooled solution of Irf2~ and pyridine in dry DCM
(Procedure 5), a solution of 5.3 g of (~)-3-Phenyllactic acid 35 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.

.. . .. . . . . _ _ 2~ 923~5 Woss/3376s r~"~ ~c ~l55 N~(BZl)~2-t-butyl carboxv ethvlene)(S~ PhenYlalanine benzvl ~E
A solution of 5.48 g of N-Bzl-~- alanine t-butyl ester 5 (Compound 56) in DCM was reacted with 7.35 g o~ (R)-O-Trf-3-Phenyllactic acid benzyl ester (COMPOUND 64) in dry DCM
(Procedure 6). Aiter workup the crude product was purified by flash chromatography. PE:EtOAc (4:1) 1.5 ~. After solvent evaporation under vacuum, the product was dried under vacuum.
10 Yield 71.5~
TLC (Method C) Rf=0.77 (one spot) (~)D = -62-7 (C=1, MeOH) NMR (CDCl3) in agreement with the title compound.

N~(2-t-butvl carboxY ethvlene)(S) PhenYlal;ln;np A solution of 6.3 g of N~(Bzl)(2-t-butyl carboxy ethylene)(S) phenylAl An; n~ benzyl ester (Compound 65) in MeOH
was hydrogenated (Procedure 8). Yield 48.6%
20 TLC (Method A) Rf=0.52-0_54 (one spot) NMR (CDCl3) in agreement with the title compound.

~E 21:
COMPOUND 67: N~(Fmoc)(2-t-butvl carboxY ethYlene)(S) 25 phenvlAl~n; n~
A solution of 2.13 g of N~(2-t-butyl carboxy ethylene)(S) PhenylAlAn;n~ (Compound 66) in H2O:Et3N was reacted with FmocOSu (Pl~eduLe 9). Yield 38 TLC (Method D) Rf=0.77 (one spot) 30 NMR (CDCl3) in agreement with the title compound.
Elemental Analysis: ~ C ~ H ~ N
Found: 71.92 639 2.87 Calc: 72.21 6.45 2.72 HPLC (Method G) 93 COMPO~ND 68 ~
N~(Bzl)(2-Boc Am;no ethvlene)qlYcine benzYl ester . _ _ _ . _ _ _ _ _ .... .. . . . . ... ... . _ .. _ . .. . _ _ . _ .

W095/33765 ~1 9 2 3 0 5 P~ I /~s ~ r A solution of 0.0325 mole o~ N-Boc, N-Bz1,1,2 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) 5 NMR (CDCl3) in agreement with the title compound.

COMPQUNP 69 ~ ~ _ N' (Bzl)~3-Boc amino ~ro~vlene)qlvcine benzvl ester A solution of 0.0325 mole of N-Boc, N-Bz1,1,3 10 diaminopropane ~Compound 6) in DMF was reacted with 5.15 mL
benzyl bromoacetate (Procedure 17). Yield 98.2%
TLC (Method F) Rf=3~78 (one spot) NMR (CDCl3) in agreement with the title compound.

15 COMPQUND 70 _ _ _ _ N'(Bz1~(4-Boc Am;nn butvlene)clvcine benzvl e~ter A solution of 0.0325 mole of N-Boc, N-Bz1,1,4 ~;Am;~ohutane (Compound 7) in DMF was reacted with 5.15 mL
benzyl bromoacetate (Procedure 17). Yield 98.8%
20 TLC (Method F) Rf=0.82(cne spot) NM~ (CDCl3) in agreement with the title compound.

COMPOUND 71 _ ____ _ _ ___ _ _ _ _ _ _ _ N~Bz1)(6-Boc amino hexvlene)clvcine benzvl ester A solution of 0.0325 mole of N-Boc, N-Bz1,1,6 di~m;nnh~YAnP ~Compound 8) in DMF was reacted with 5.15 mL
benzyl brn~~a~Ate (Procedure 17). Yield 98.8%
TLC ~Method F) Rf=0.79(one spot) NMR (CDCl,) in agreement with the title compound COMPOUND 72_ N~(2-Boc amino ethvlene)clvcine A solution of 0.025 mole of N'(Bzl)~2-Boc amino ethylene)glycine benzyl ester (Compound 68) in 60 mL MeO~ was 35 hydrogenated (Procedure 8). Yield 85~ of white solid. mp 200- 2~C
TLC (Method A) Rf=0_22 ~one spot) , _ _ _ 21~2305 WO 95/33765 ~ ~, r~ Z55 NMR ~CDC13) in agreement with the title compound.

NQ(3-Boc amino ~ro~vlene)qlvcine A solution of 0.025 mole of N~ (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 lone spot) 10 NMR (CDCl3) in agreement with the title compound.

COMPO~ND 74 N3(4-Boc amino butvlene)clvcine A solution of 0.025 mole of NQ (Bzl)(4-Boc amino 15 butylene)glycine benzyl ester (Compound 70) in 60 mL MeOH was hyd.~g~llated(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.

NQ(6-Boc amino hexvlene)qlvcine A solution of 0.025 mole of NQ(Bzl)(6-Boc amino hexylene)glycine benzyl ester (Compound 71) in 60 mL MeOH was 25 hydrogenated (Procedure 8). Yield 80~ of white solid. mp 172- 4~C.
TLC (Method A) Rf=0.26 (one spot) ~MR (CDCl3) in agreement with the title compound.

30 EXAMPLE 22: =
COMPO~ND 76: NQ(Fmoc)(2-BQc amino ethvlene)qlvcine A solution of 0.02 mole of NQ (2-Boc amino ethylene)glycine (Compound 72) in H2O:Et3N was reacted with FmocOSu (Procedure 9). Yield ~0~ of white solid. mp 130-132 35 ~C. TLC (Method D) Rf=0.5 (one spot) NMR (CDCl3) in agreement with the title compound.
Elemental Analysis: ~ C ~ H ~ N

Wogs/33765 2 1 9 2 3 0 5 . ~I~.S ~ ~ ~ss Found: 65.18 6.11 5.91 Calc: 65.43 6.~0 6.63 ~MP~E 23:_ --- - -5 COMPOUND 77: N~(Fmoc)(3-Boc smin~ ~ro~vlene)qlvcine A solution of 0.02 mole of N~(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) 10 NMR (CDCl3) in agreement with the title compound.
Elemental Analysis: ~ C % H ~ N
Found: 66.05 6.65 6.00 Calc: 66.06 6_65 6.16 LE 24:
COMPOUND 78: N~(Fmoc)(4-Boc amino butvlene)qlvcine A solution of 0.02 mole of N~(Fmoc) (4-Boc amino butylene)glycine (Compound 74) in H2O:Et3N was reacted with 20 FmocOSu (Procedure 9). Yield 79.4~ of white solid. mp 150 -152 ~C.
TLC (Method D) Rf=Q_42-0.47 (one spot) NMR (CDCl3) in agreement with the title compound.
Elemental Analysis: % C ~ H ~ N
Found: 66.35 6.B4 5.77 Calc: 66.06 6,88 5.98 ExaM,PLE 25:
COMPOUND 79: ~(Fmoc)(6-Boc amino hexvlene)clvcine A solution of 0.02 mole of N~(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. T~C (Method D) Rf=0.7 (one spot) NMR (CDC13) in agreement with the title compound.~5 Elemental Analysis: % C ~ H ~ N
Found: 68.02 7.08 5 37 Calc_ 67 72 7.31 5.64 95/33765 r~ l55 ~,~lC ~XAMPLES

Two series of octapeptide somatostatin analogs of the 5 present invention were synthesized, characterized, and tested for biological activity.
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-Rll-Thr-NH2 wherein R6 and Rll are N~ ~-functionalized alkylene amino acid building units.
2) The second series of - ,~ullds corresponds to the general Formula (XVIc); this series comprises compounds of the 15 specific formula H-(D)Phe-R5-Phe-(D)Trp-Lys-Rl0-Thr-NH~
wherein R6 and Rl~ are N~ ~-functionalized alkylene amino acid building units.
The structures of these novel synthetic peptide analogs 20 into which N~ ~-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 bArkh~n~ were varied.
For the sake of simplicity, these two series are referred to herein as the SST GlyF,Glyll and SST Gly6,Glyl0 series, respectively.
In each series, the position of the cyclization points was co~stant, while the length and direction of the bridge was 30 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 barkhrn~ nitrogens involved in the bridge.
Peptide assembly was carried out either manually or with an automatic peptide synthesizer (Applied Biosystems Model 433A). Following peptide assembly, de-protection of WO9S/33765 r~,~ s~

bridging groups that form the cyclization arms was carried out with Pd(PPh3)~ (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-5 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 ~eren~; ng on the type of resin used, e.g., with TFA for Rink amide type resins and 10 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.

Table 7 -ST Gly~ Glyll Example Bridging Compound Method Crude No. Groups Number Yield 26 Cl,N2 Cyclic DE-3-32-4 1 NA**
27 Cl,N2 Linear DE-3-32-2 1 NA
28 Cl,N3 Cyclic PTR 3004 2 79 mg 29 Cl,N3 Linear PTR 3005 2 34 mg C2,N2 Cyclic PTR 3002 1 NA
31 C2,N2 Linear PTR 3001 1 NA
32 C2,N3 Cyclic PTR 3007 2 40 mg 33 C2,N3 Linear PTR 3008 2 40 mg 34 N2,C2 Cyclic YD-9-165-1 2 NA
N2,C2 Linear YD-9-168-1 2 NA
36 N3,C2 Cyclic PTR 3010 2 100 mg 37 N3,C2 Linear PTR 3011 2 NA
38 Linear~ PTR 3003 3 96 mg * Linear refers o the i~nt ~ ~1 sequenc- with Gly residues in place of R6 and R
** NA denotes not available.
Table 7 methods:
1) Manual synthesis on mBHA resin. HF cleavage.

2 1 ~2305 W095/33765 ~1/~7~ 55 2) Manual synthesis on Rapp tentagel resin. TFA
cleavage.
3) Rink amide resin; assembly in automated peptide synthesizer, 0.1 mmol scale.
Table 8 'ST Gly~,Gly10 Example Bridging Compound Method Crude No. Groups Number Yield 39 Cl,N2 Cyclic YD-9-171-3 1 20 mg Cl,N2 Linear YD-9-171-2 1 10 mg 41 Cl,N3 Cyclic YD-9-175-3 1 44.9 mg 42 Cl,N3 Linear YD-9-175-2 1 25.4 mg 43 C2,N2 Cyclic PTR 3019 1 40 mg 44 C2,N2 Linear PTR 3020 1 26 mg C2,N3 Cyclic YD-5-28-3 3 101.5 mg 46 C2,N3 Linear YD-5-28-2 3 48.3 mg 47 N2,C2 Cyclic PTR 3016 2 60 mg 48 N2,C2 Linear PTR 3017 2 40 mg 49 N3,C2 Cyclic YS-8-153-1 2 93 mg N3,C2 Linear YS-8-152-l 2 54 mg 51 * Linear PTR 3021 1 100 mg ** Acetylated Des-D-Phes 52 N3,C2 Cyclic PTR 3013 67 mg 53 N3,C2 Linear PTR 3014 48 mg * Linear refers ro the identi al sequenc with Gly residues in place of R6 and Rl~.
** Acetylated Des-D-Phes refers to the same sequence in which the N terminal D-Phes is absent and the N-terminus i8 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).

Zl 923~
WO95/33765 r~l,~7s,t~''-Synthesi~ of SST GlyC,Glyl~ N3,C2:

Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelled in N-methylpyrrolidone (NMP) in a reaction vessel 5 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 10 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, 15 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 20 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-~ys(Boc)-OH was coupled to the peptide 25 resin by reaction with HATU (3 equivalents) and DIEA (6 equivalents) at room temperature overnight and then at 50~C
for one hour. Additional D BA was added during reaction to r-;nt~;n a basic medium (as determined by pH paper to be about 9). This coupling was repeated. Coupling completion was 30 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). Emoc-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 35 way. Synthesis was continued with one-fifth o~ the peptide resin.

WO95~3765 P~l/~ C~ ~55 Eollowing 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 5 e~ual portions. Synthesis was c~ntlnnG~ with one of these portions. Boc-D-Phe-OH was coupled by reaction with XATU, as described above for Fmoc-Bys(Boc)-OH. Capping was carried out as above.
The Allyl and Alloc protecting groups were removed by 10 reaction with Pd(PPh3)~ 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 eo,uivalents, DIEA 5 eo,uivalents, in NMP, at room 15 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 (tri-isopropyl-silane) 1%, and 5~ methylene chloride, at 0~C for 15 minutes and 2 hours at room temperature under argon. The 20 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
25 white powder was obtained. This crude product was dried. The weight of the crude product was 93 mg.

~HYSIOLOGICAL EXAMPLES

30 EXAMPRE 54: RRADY~ININ ANTAGONIST ASSAY (Displacement o~
(3E)doPamine release from PC 12 cells) ovel backbone cyclized peptide analogs of the present invention were assayed in vitro for bradykinin antagonist - activity by protection of (3H)dopamine release from PC 12 35 cells that express bradykinin receptors. PCl2 cells were grown in Dulbecco Modified Eagle's medium with high glucose, supplemented with 10~ horse serum, 5% fetal calf serum, 130 ~ -Zl 9~3~5 WO95/33765 r~

units/ml penicillin and 0.1 mg/ml streptomycin. For experiments, cells were removed from the medium using 1 mmole EDTA and replated on collagen coated-12- well plates and assayed 24 hr later. Release of ('H)~np~;nP was determined 5 as follows: cells were incubated for 1.5 hr at 37 ~C with 0.5 ml of growth medium and 0.85 ml ~3H)DA ~41 Ci/mmole~ and 10 mg/ml pargyline followed by extensive washing with medium ~3Xl ml) and release buffer consisting of ~mM): 130 NaCl; 5 KCl; 25 NaHCO3; 1 NaH2PO~; 10 glucose and 1.8 CaCl2. In a typical 10 experiment, cells were incubated with 0.5 ml buffer for 5 consecutive incubation periods of 3 min each at 37 ~C.
Spontaneous ~3H)DA release was measured by collecting the medium released by the cells successively for the first 3 min period. Antagonists were added to the cells 3 min prior to 15 stimulation (at the second period), and stimulation of (3H)DA
release by 100 nmole of bradykinin are monitored during the 3 period by 60 mmole KCl. The r~;n;ng of the (3H)DA was extracted from the cells by over night incubation with 0.5 ml 0.1 N HCl. ~3H)DA release during~each 3 min period was ao expressed as a ~ of the total ~3H)DA content of the cells.
Net evoked release was calculated from ~3H)DA release during stimulation period after subtracting basal ~3H)DA release in the preceding baseline period i~ not indicated otherwise.
At 10-6 M, Example 1 showed 30 ~ inhibition of BK _ 25 activity, Example 4 showed 17 ~ inhibition of BK activity.
Note, the noncyclized ~control) peptide of example 2 showed 0 ~ inhibition of BK activity.

~MPLE 55 sRADyKININ ~NT~iONTST ASSAY (Guinea-~iq assaY) The ileum of the guinea-pig was selected as the preparation for the bioassay. This tissue contains pre~n~;n.~n~ly BK2 receptors. The preparation consists of the longitudinal muscle layer with the adhering mesenteric plexus.
The isolated preparation was kept in Krebs solution and~
35 contractions were measured with an isometric~force~transducer.
The guinea-pig ileum is highly sensitive to .3K, with ECso at 2x10-6 M. At least two control responses to BK (2x10-8 M) were ~1 92305 95/33765 r~l,~,s.~

measured previous ~o measuring the responseg of backbone cyclized peptides of the present invention. Atropine ~1 ~M) was always present.
At 10-~ M, Example 1 showed 24 % inhi~ition of BK
5 activity, Example 3 showed 10 ~ and Example 4 showed 17 ~
inhibition of BK activity. Note, the noncyclized (control) peptide of example 2 showed 0 ~ inhibition of BK activity.

EXAMPT~ 56: SOMATOSTATIN ASSAY ~Rece~tor based ~creeninq) Initial screening is conducted using ''sI-labeled SST
analogs and pituitary membrane preparations or cell lines.
The binding assay is described in Tran, V.T., Beal, M.F. and Martin, J.B. Sciençe, 228:294-495, 1985, which is incorporated herein by reference in its entirety and is optimized with 15 regards to membrane cnnc~ntration~ temperature and time. The assay is sensitive (nM range) and robust. Selectivity will be based on the recent cloning of the five human SST receptors.
The ability to screen the compounds with regard to binding and biological activity in mammalian cells should facilitate the 20 development of subtype-selective analogs. These compolln~
are useful in the treatment of specific endocrine disorders and therefore should be devoid of unwanted side effects.

EXAMPL3 57: SOMATOSTATIN (SST) ASSAY (In vivo assaY~) The biological effects of SST on growth hormone, insulin and glucagon release is conducted by measuring the levels of these hnrrnne~ using commercially available RIA test kits. Pharmacological effects of SST in patients with neuroendocrine tumors of the gut will reguire determination of 30 5-hydroxyindole acetic acid (for carcinoid) and VIP (for VIPoma). In vivo visualizaticn of SST receptor-positive - tumors is performed as described by ~ambert et al., New Encland J. Med., 323:1246-1249 1990, following i.v.
administration of radio-iodinated SST analogs.
EXAMPLE 58: Receptor binding ~pecificity o~ cyclic peptide analog~

WOgs/33765 Z~ 9 ~ 3 0 5 Y i/~ c Binding of representative pep~ides o~ Examples 39-~4 to different somatostatin receptors was measured in vitro, in Chinese Hamster Ovary (CHO) cells expressing the various receptors. An example of the selectivity obtained with the 5 cyclic peptides is presented in Table 9 The values presented are percent inhibition of radioactive iodinated somatostatin (SRIF-14) binding.
Table 9 Binding of peptide analoga to somatostatin receptor subtypea 0 Somatostatin Recep or SSTR) u'otype SSTR 2B SSTR~ 5 Conc. ~ 10-6 10-' 10-' 10-~ 10-' 10-' Compound Compound Xu~ber Deacr~ptlon PTR 3003 Linear 16 3 0 55 20 0 PTR 3004 Cyclic Cl,N3 0 0 0 14 0 0 PTR 3005 Linear Cl,N3 0 0 0 9 0 o PTR 3007 Cyclic C2,N3 0 0 0 19 9 0 PTR 3008 Linear C2,N3 0 0 0 15 6 0 PTR 3010 Cyclic N3,C2 0 0 0 63 26 9 PTR 3011 Cyclic N3,C2 0 0 0 27 66 27 Control Peptide~
BIM 3503 Pos Control ¦ 81 ¦ 33 ¦ 16 ¦ ¦ 92 ¦ 66 ¦ 27 PTR 4003 Neg Control ¦ 0 ¦ 0 ¦ 0 ¦ ¦ 0 ¦ 0 ¦ 0 EXANPLE 59: Resistance to biodegradation of SST analogs The in vitro biostability of a SST cyclic peptide analog, PTR 3002, was measured in human serum, and was 30 compared to the same sequence in a non-cyclic peptide analog (PTR 3001), to octreotide (Sandostatin), and to native somatostatin (SRIF). The results are shown in Figure l. In this assay, the cyclic peptide in accordance with the present invention is as stable as octreotide, is more stable than the 35 corresponding non-cyclic structure, and is much more stable than SRIF. The assay was based on HPLC determination of peptide degradation as a function of time at 37~C.

Woss/33765 P~

EXAMPLE 60: Inhibition o~ growth hormone releage by SST
ar,alogs In ViVQ detorm;nRt;on of the pharmacodynamic properties of cyclic peptide analogs was carried out Inhibition of 5 Growth Hormone (GH) release as a result of peptide administration was measured. Measurements were carried out in Sprague-Dawley male rats: peptide analog activity was compared in this study to SRIF or to octreotide (Sandostatin).
Each group consisted of 4 rats.i Time course profiles for GH
10 release under constant experimental conditions were measured.
Methods Adult male Sprague-Dawley rats, specific pathogen free (SPF), weighing 200-350 g, were mR;ntRin~d on a constant light-dark cycle (light from 8:00 to 20:00 h), temperature (21 15 + 3~C), and relative humidity (55 ~ 10~). Laboratory chow and tap water were available ad libitum. On the day of the experiment, rats were anesthetized with pentobarbitone (50 mg/kg). Rats anesthetized with pPntnhRrhitone exhibit low somatostatin levels in portal blood vessels. (Plotsky, P.M., 20 Science, 230, 461-463, 1985). A single blood sample (0.6 ml) was taken from the exposed cannulated jugular vein for the determ;nRt;nn of the basal GH levels (-15 min). Immediately thereafter the a~L~Liate peptide pretreatment was administered. The animals received 10 ~g/kg of either native 25 somatostatin (SRIF) or the synthetic analog octreotide (Sandostatin), or the cyclic peptide analog. A saline solution (0.9~ NaCl) was administered as a control. All peptides were administered subcutaneously in a final volume of 0.2 ml. Further sampling was carried out at 15, 30, 60, and 30 90 minutes after peptide administration. Immediately after the collection of each blood sample, an ~L~liate volume - (0.6 ml) of saline was administered intravenously. Blood samples were collected into tubes containing heparin (15 unites per ml of blood) and centrifuged immediately. Plasma 35 was separated and kept frozen at -20~C until assayed.
Rat growth hormone (rGH)[l2sI] levels were determined by iate radio;~mnnoRcs~y kit (Amersham). The s~andard in w095/3376s 2~ 9~305 r this kit has been calibrated against a reference standard preparaticn (NIH-RP2) obtained from the National Institute of Diabetes and Digestive and Kidney Diseases. All samples were .
measured in duplicate.
EXAMPL3 61: ~ack oi toxicity oi cyclized peptide analogs PTR 3007 at a dose of 1.5 mg/kg was well tolerated a~ter single intraperitoneal application. PTR 3013 was not toxic to the rats even with doses of 4 mg~kg. These two doses 10 are several orders of magnitude higher than those needed to elicit the desired endocrine effect. The peptides dissolved in saline produced no untoward side effects on the central nervous system, cardiovascular system, body temperature, nor on the periphery of the ani~als. Rats were observed for 4 15 hours post administration of the peptides. PTR 3007 and 3013 produced no respiratory distllrh~nc~l did not result in the appearance of stereotyped behavior, or produce any changes in muscle tone. After 3 hours, post~ortem examination did ~ot detect any abnormality in the liver, kidneys, arteries and 20 veins, gastrointestinal tract, lungs, genital system, nor the spleen.

Claims (61)

What is claimed is:

1. A backbone cyclized peptide analog comprising a peptide sequence that incorporates at least two building units, each of which contains one nitrogen atom of the peptide backbone connected to a bridging group comprising a disulfide, amide, thioether, thioester, imine, ether, or alkene bridge, wherein at least two of said building units are joined together to form a cyclic structure.

2. The backbone cyclized peptide analog of claim 1 that incorporates at least four building units, each of which contains one nitrogen atom of the peptide backbone connected to the bridging groups, wherein at least two pairs of said building units are joined together to form at least two cyclic structures within said peptide sequence.

3. The backbone cyclized peptide analog of claim 1 wherein at least one of said building units is not located at the end of the peptide sequence.

4. The backbone cyclized peptide analog of claim 1 wherein none of said building units is located at the end of the peptide sequence.

5. The backbone cyclized peptide analog of claim 1, having the general Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 or zero; d, e, and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or CO-E can be reduced to CH2-OH; each of R and R' is independently hydrogen or an amino acid side-chain optionally bound with a specific protecting group; and the lines designate a bridging group of the Formula:
(i) -X-M-Y-W-Z- ; or (ii) -X-M-Z-wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, amide, thioether, thioester, imine, ether, and alkene; and X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene.

6 The backbone cyclized peptide analog of claim 5 wherein -X-M-Y-W-Z- is:
- (CH2) x-M- (CH2)y-W- (CH2) Z-wherein M and W are independently selected from the group consisting of disulfide, amide, thioether, thioester, imine, ether, and alkene; x and z each independently designates an integer of from 1 to 10, and y is zero or an integer of from 1 to 8, with the proviso that if y is zero, W
is absent.

7. The backbone cyclized peptide analog of claim 5 wherein the group CO-E is CH2OH.

8. The backbone cyclized peptide analog of claim 5 wherein R is CH3-, (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, or methylimidazole.

9. The backbone cyclized peptide analog of claim 5 wherein R' is CH3, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, CH3S(CH2)2-, HOCH2-, CH3CH(OH)-, HSCH2-, NH2C(=O)CH2-, NH2C(=O) (CH2)2-, C(NH2)2 NH(CH2)3-, HOC(=O)CH2-, HOC(=O)(CH2)2-, NH2(CH2)2-, C(NH2)2 NH (CH2) 3-, HO-phenyl-CH2-, benzyl, methylindole, or methylimidazole.

10. A backbone cyclized peptide analog, wherein the backbone is cyclized to a side-chain of an amino acid of the general Formula (II):

wherein d, e and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting or an amino group, or CO-E can be reduced to CH2-OH; R is an amino acid side chain optionally bound with a specific protecting group; and the line designates a bridging group of the Formula:
(i) -X-M-Y-W-Z- ; or (ii) -X-M-Z-wherein M and W are independently selected from the group consisting of disulfide, amide, thioether, imine, ether, and alkene; X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene.

11. The backbone cyclized peptide analog of claim 10 wherein R is CH3-, (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, or methylimidazole.

12. The backbone cyclized peptide analog of claim 10 wherein -X-M-Y-W-Z- is:
- (CH2)x-M-(CH2)y-W-(CH2)z-wherein M and W are independently selected from the group consisting of disulfide, amide, thioether, imine, ether, and alkene; x and z each independently designates an integer of from 1 to 10, and y is zero or an integer of from 1 to 8, with the proviso that if y is zero, W is absent.

13. A bradykinin analog comprising the backbone cyclized peptide analog of claim 5.

14. A bradykinin analog comprising the backbone cyclized peptide analog of claim 10.

15. The backbone cyclized peptide analog of claim 13 of the general Formula (III):

wherein M is an amide bond, x and z are each independently an integer of 1 to 10, and K is H or an acyl group.

16. The backbone cyclized peptide analog of claim 13 of the general Formula (IVa):

wherein M is an amide bond, x and z are each independently an integer of 1 to 10, K is H or an acyl group, and R6 is Gly or Ser.

17. The backbone cyclized peptide analog of claim 14 of the general Formula (IVb):

wherein x is an integer of 1 to 10; K is H or an acyl group;
(R6) is selected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z is 1 or 2, with the proviso that when R5 is D-Asp or L-Asp, then z is 1.

18. The backbone cyclized peptide analog of claim 13 of the general Formula (V):

wherein M is an amide bond, x and z are independently an integer of 1 to 10, and K is H or an acyl group.

19. The backbone cyclized peptide analog of claim 13 selected from the group consisting of:
a) Ada-(D)Arg-Arg-cyclo(N.alpha.(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;
b) H-D-Arg-Arg-cyclo(N.alpha.(1-(4-propanoyl))Gly-Hyp-Phe-N.alpha.(3-mido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH; and c) H-D-Arg-Arg-cyclo(N.alpha.(4-propanoyl)Gly-Hyp-Phe-N.alpha.(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.

20. A somatostatin analog comprising the backbone cyclized peptide analog of claim 5.

21. A somatostatin analog comprising the backbone cyclized peptide analog of claim 10.

22. The backbone cyclized peptide analog of claim 20 having the general Formula (XIVa):
wherein m and n are 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is Gly, (D)- or (L)- Ala, Phe, Nal, and .beta.Asp(Ind); R6 and R11 are independently Gly or (D)- or (L)-Phe; R7 is Phe or Tyr;
R10 is absent or is Gly, Abu, Thr or Val; R12 is absent or is Thr or Nal; and Y2 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

23. The backbone cyclized peptide analog of claim 20 having the general Formula (XIVb):
wherein m and n are 1, 2 or 3; X is CH2OH or NH2; R6 and R11 are independently Gly or (D)- or (L)-Phe; R7 is Phe or Tyr; R10 is absent or is Gly, Abu, Thr or Val; and Y2 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

24. The backbone cyclized peptide analog of claim 20 having the general Formula (XVa):
wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr; and R11 is (D)- or (L)-Phe; R12 is absent or is Thr or Nal, and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

25. The backbone cyclized peptide analog of claim 20 having the general Formula (XVb):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH,; R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr;
and R11 is (D)- or (L)-Phe; R12 is absent or is Thr or Nal, and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

26. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIa):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr; and R11 is (D)- or (L)-Phe; R12 is absent or is Thr or Nal, and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

27. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIb):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R6 is (D) or (L)-Phe; R10 is absent or is Gly, Abu or Thr;
and R11 is (D)- or (L)-Phe; R12 is absent or is Thr or Nal, and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

28. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIc):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R6 is (D) or (L)-Phe; and R10 is absent or is Gly, Abu or Thr; R12 is absent or is Thr or Nal, and Yl is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

29. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIIa):

wherein i, j, m and n are independently 1, 2 or 3; X is CH2OH
or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R6 and R11 are independently Gly or (D)- or (L)-Phe;
R10 is absent or is Gly, Abu, Val or Thr; R12 is absent or is Thr or Nal; and Yl and Y2 are independently selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

30. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIIb):

wherein i, j, m and n are independently 1, 2 or 3; X is CH2OH
or NH2; R6 and R11 are independently Gly or (D)- or (L)-Phe; R10 is absent or is Gly, Abu, Val or Thr; and Y1 and Y2 are independently selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

31. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIIIa):

wherein i,j, m and n are independently 1, 2 or 3; X is CH2OH
or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R6 and R11 are independently Gly or (D)- or (L)-Phe;
R10 is absent or is Gly, Abu, Val or Thr; R12 is absent or is Thr or Nal; and Y1 and Y2 are independently selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

32. The backbone cyclized peptide analog of claim 20 having the general Formula (XVIIIb):

wherein i, j, m and n are independently 1, 2 or 3; X is CH2OH
or NH2; R6 and R51 are independently Gly or (D)- or (L)- Phe;
R10 is absent or is Gly, Abu, Val or Thr; and Y1 and Y2 are independently selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

33. The backbone cyclized peptide analog of claim 20 having the general Formula (XIXa):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind);
R10 is absent or is Gly, Abu or Thr; R12 is absent or is Thr or Nal; and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

34. The backbone cyclized peptide analog of claim 20 having the general Formula (XIXb):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

35. The backbone cyclized peptide analog of claim 20 having the general Formula (XXa):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind);
R10 is absent or is Gly, Abu or Thr; R12 is absent or is Thr or Nal; and Y1 is selected ~rom the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

36. The backbone cyclized peptide analog of claim 20 having the general Formula (XXb):

wherein i and j are independently 1, 2 or 3; X is CH2OH or NH2; R10 is absent or is Gly, Abu or Thr; R12 is absent or is Thr or Nal; and Y1 is selected from the group consisting of amide, disulfide, thioether, imine, ether, and alkene.

37. A method for the preparation of cyclic peptides of the general Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 or zero; d, e, and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or CO-E can be reduced to CH2-OH; R and R' each designates an amino acid side-chain optionally bound with a specific protecting group;
and the lines designate a bridging group of the Formula:
(i) -X-M-Y-W-Z- ; or (ii) -X-M-Z-wherein: one line may be absent; M and W are independently selected from the group consisting of disulfide, amide, thioether, thioester, imine, ether, and alkene; and X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene;
comprising the steps of incorporating at least one N.alpha.-.omega.-functionalized derivatives of amino acids of Formula (VI):

wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; R' is an amino acid side chain, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting group of G;
into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence or with another .omega.-functionalized amino acid derivative.

38. The method of claim 37 wherein both lines in Formula (I) are present and at least four N.alpha. .omega.-functionalized amino acid derivatives are incorporated into the peptide sequence, resulting in a bicyclic compound.

39. The method of claim 37 wherein E is covalently bound to an insoluble polymeric support.

40. The method of claim 37 wherein G is an amine, thiol or carboxyl group.

41. The method of claim 37 wherein R is selected from the group consisting of CH3-, (CX3)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.

42. The method of claim 37 wherein R' is selected from the group consisting of CH3-, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, CH3S(CH2)2-, HOCH2-, CH3CH(OH)-, HSCH2-, NH2C(=O)CH3-, 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.

43. A method for the preparation of cyclic peptides of the general Formula (II):

wherein d, e and f each independently designates an integer from 1 to 10; (AA) designates an amino acid residue wherein the amino acid residues in each chain may be the same or different; E represents a hydroxyl group, a carboxyl protecting or an amino group, or CO-E can be reduced to CH2-OH; R is an amino acid side chain optionally bound with a specific protecting group; and the line designates a bridging group of the Formula:
(i) - X-M-Y-W-Z- ; or (ii) -X-M-Z-wherein M and W are independently selected from the group consisting of disulfide, amide, thioether, thioester imine, ether, and alkene; X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo- or hetero-cycloalkylene and substituted cycloalkylene;
comprising the steps of: incorporating at least one .omega.-functionalized amino acid derivative of the general Formula (VI):

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; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters or alkyl halides and A is a protecting group thereof;
into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in said peptide sequence.

44. The method of claim 43 wherein G is a carboxyl group or a thiol group.

45. The method of claim 43 wherein R is CH3-, (CH3)2CH-, (CH3)2CHCH2-, CH3CH2CH(CH3)-, CH3S(CH2)2-, HOCH,-, 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, or methylimidazole.

46. The method of claim 43 wherein the peptide is covalently coupled to an insoluble polymeric support.

47. An .omega.-functionalized amino acid derivative of the general Formula:

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 aryl 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.

48. The .omega.-functionalized amino acid derivative of claim 47 wherein X is alkylene.

49. The .omega.-functionalized amino acid derivative of claim 47 wherein G is a thiol group.

50. The .omega.-functionalized amino acid derivative of claim 47 wherein G is a carboxyl group.

51. The .omega.-functionalized amino acid derivative of claim 47 wherein R is benzyl, methyl, or isobutyl.

52. The .omega.-functionalized amino acid derivative of claim 47 wherein G is an amine group with the proviso that R
is CH3-, (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, or methylimidazole.

53. The .omega.-functionalized amino acid derivative of claim 47 wherein R is protected with a specific protecting group.

54. The .omega.-functionalized amino acid derivative of claim 47 selected from the group consisting of:
a) N.alpha.-(Fmoc) (3-Boc-amino propylene)-(S)Phenylalanine;
b) N.alpha.-(Fmoc) (3-Boc-amino propylene)-(R)Phenylalanine;
c) N.alpha.-(Fmoc) (4-Boc-amino butylene)-(S)Phenylalanine;
d) N.alpha.-(Fmoc) (3-Boc-amino propylene)-(S)Alanine;
e) N.alpha.-(Fmoc) (6-Boc-amino hexylene)-(S)Alanine;
f) N.alpha.-(Fmoc) (3-Boc-amino propylene)-(R)Alanine;
g) N.alpha.-(2-(benzylthio)ethylene)glycine ethyl ester;
h) N.alpha.-(2-(benzylthio)ethylene)(S)leucine methyl ester;
i) N.alpha.-(3-(benzylthio)propylene)(S)leucine methyl ester:
j) Boc-N.alpha.-(2-(benzylthio)ethylene)glycine;
k) Boc-N.alpha.-(2-(benzylthio)ethylene)(S)phenylalanine;
l) Boc-N.alpha.-(3-(benzylthio)propylene)(S)phenylalanine;
m) Boc-L-phenylalanyl-N.alpha.-(2-(benzylthio)ethylene)glycine-ethyl ester;
n) Boc-L-phenylalanyl-N.alpha.-(2-(benzylthio)ethylene)-(S)phenylalanine methyl ester;
o) N.alpha.(Fmoc)-(2-t-butyl carboxy ethylene)glycine;
p) N.alpha.(Fmoc)-(3-t-butyl carboxy propylene)glycine;
q) N.alpha.(Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;
r) N.alpha.(Fmoc)(2-Boc amino ethylene)glycine;

s) N.alpha.(Fmoc) (3-Boc amino propylene)glycine;
t) N.alpha.(Fmoc) (3-Boc amino butylene)glycine; and u) N.alpha.(Fmoc) (6-Boc amino hexylene)glycine.

55. A method of making an .omega.-functionalized amino acid derivative of the general Formula:

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; A and B are protecting groups selected from the group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls;
said method comprising:
reacting a diamine compound of the general Formula:

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:

wherein A, B, E, R and X are as defined above and deprotecting the carboxyl to yield an N.alpha. .omega.-functionalized amino acid derivative, wherein the .omega.-functional group is an amine.

56. A method of making an .omega.-functionalized amino acid derivative of the general Formula:

wherein 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; 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, thio ether or aryl or substituted aryl thio ether;
comprising the steps of:
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:

ii) selectively removing the protecting group E, and protecting the free amino group to yield an N.alpha. (.omega.-functionalized) amino acid derivative, wherein the .omega.-functional group is a thiol.

57. A method of making an .omega.-functionalized amino acid derivative of the general Formula:

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; 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:
i) reacting a compound of the general Formula B-NH-X-CO-A with a triflate of the general Formula CF2SO2-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:

and selectively removing protecting group E, to yield an N.alpha.(.omega.-functionalized) amino acid derivative, wherein the .omega.-functional group is a carboxyl.

58. A pharmaceutical composition comprising a backbone cyclized peptide analog of claim 13 and a pharmaceutically acceptable carrier or diluent.

59. A pharmaceutical composition comprising a backbone cyclized peptide analog of claim 14, and a pharmaceutically acceptable carrier or diluent.

60. A pharmaceutical composition comprising a backbone cyclized peptide analog of claim 20, and a pharmaceutically acceptable carrier or diluent.

61. A pharmaceutical composition comprising a backbone cyclized peptide analog of claim 21, and a pharmaceutically acceptable carrier or diluent.