CA2112566A1 - Non-desensitizing analogs of gnrh and other biologically active ligands - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention is directed to novel non-desensitizing analogs of a biologically active ligand having at least one 6-membered ring or 5-membered ring and a method for selecting non-desensitizing analogs of a biologically active ligand having at least one 6-membered ring or 5-membered ring. Additionally, the present invention is directed to a novel method for treating a patient in a non-desensitizing fashion with a non-desensitizing analoq of a kiologically active ligand having at least one 6-membered ring or 5-membered ring. Moreover, the present invention is directed to a novel method for making new and useful non-desensitizing analogs of GnRH and opiate peptides as well as the novel non-desensitizing compounds and compositions prepared thereby.

Description

~.V093~0.~582 1 1 ~ ~ ~ 6 PCT/C~/OO~I

NoN-DE8EN8ITI8INa ANALOG8 OF GnR~
~ND OT~ER B~OLOG~CAL~Y ACTIVE LIaAND8 BACKGROUND OF THE INVENTION

Field of the In~a~ion i 5The present invention is directed to novel non-desensitizing snalogs of a biologically active ligand containing at least one 6-membered aromatic ring or 5-membered ring and a method for selecting non-desensitizing analogs of a biologically active ligand `' 10 containing at least one 6-membered aromatic ring or S-membered ring. Additionally, the present invRntiOn is directed to a novel method for treating a patient in a non-desensitizing fashion with a non-desensitizing analog of a biologically active ligand containing at least one 6-membered aromatic ring or S-membered ring.
~oreover, the present invention is directed to a novel method for ~alcing new and usef-ul non-desensitizing analogs of G~ and opiate peptides as well as the novel non-desensitizing compounds prepared thereby.

8tate o t~e ~rt In living organisms, the interaction af a biologically active ligand with a receptor to produce a biological response is often accompanied by desensitization of the receptor, such that a subseguent interaction of the ligand with the receptor results in attenuation of the subseguent biological response to the ligand. Prior to the present invention, however, very little was understood about the causes and effects ,' ~''', . ... . .

W093/0305X ~ ,i f, fi ~T/CA92/~281 surrounding the desensitization proce~ or how 6uch detrimental desen~itization could be avoided. Thu6, the primary focu~ up to thi~ point h~s been ~tte~pts to develop agonistic and antagoni~tic analog6 of biologically active ligands with l~ttle or no regArd to their poten~ial desensitizing effect~.

For example, Gonadotropin-Relea6ing Hormone (GnRH~ is one such biolog~cally actlve co~pound which has been modified to produce both agoni~tic and antagoni~tic analogs thereof. GnRH stimulates the release of gonadotropins through interaction wlth membrane-associated high affinity receptors on the pituitary gonadotropes. Subsequently, these gonadotropins act on the gonad~ to 6timulate the synthesi~ of steroid 6ex hormonec. The pulsatile release of GnRH, and thereby the release of gonadotropins, controls the reproductive cycle in domestic animals and humans.

Acute doses of GnRH agonists administered in pulsatile fashion can increase the levels of ~uteinizing Hor~one (LH) and s~eroid 6ex hormones in both animals and humans. Paradoxically, acute or chronic doses of these agonists can 6uppress the levels of LH and steroid hormones. Additionally, the effect of multiple doses of GnRH agonists can be to suppress estrogen formation in the female and suppress testosterone formation in the male. Accordingly, knowledge of the molecular process by which desensitization occurs would be extremely useful in the design of analogs of biologically active ligands in which said receptor desensitization effects are reduced.

.

~' ~ ' ~93/0~5X ~ 2 ~i ~; f) p( r/c~92/~o~, Thus, it is an object of the pre~ent invention to provide novel non~de~en6itizing ~nAlog~ of ~- ~ a biologically active ligand containing at lea~t one 6-membered aromatic ring or 5-me~bered ring.

` 5 Yet another object of the present invention - is to provide a novel method for selecting non-; de6ensitizing analogs of a biologically active;lig nd which cont~in at least one 6-membered aromatic ring or 5-membered ring.

still another object of the present invention is to provide a novel method for treating a patient in a non-desensitizing fashion with a non-desensitizing ~, analog of a biologically active ligand containing at least one 6-membered arQmatic ring or 5-membered ring.

A further object of the present invention is to provide a novel method for producing new and u~eful analogs of Gnl~ and opiate peptides as well as the novel analogs produced thereby.

~XaRY OF T9~ INVE~TION

,Jl 20 In accordance with the foregoing objectives, ~ there is provided a method for selecting non-!~ ' desensitizing analogs of a biologically active ligand ¢ containing at least one 6-membered aromatic ring or 5-membered ring. The method comprises selecting an ',25 analog of a biologically active ligand containing a 6-; membered aromatic ring according to Formula I or selecting a biologically active ligand containing a 5-membered ring according to Formula II:

~5 ~, ':~

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W093/030~8 ~ r~ rr/~92/no~l S ~ R I R~ C C - ~ II

R~ 7 C
I
-N ~_ C~ R~ B R7 RP R9 N~ v~-~ R9 In Formulafs I and II, above, X is selected from H, R~, -ORI, h~lide, -CN, -CHO, C(halide)l, -alk-OH, -alk-OR~, -aIk~ lk-CO~RI, -alk-SH, -alk-S~I, -dlk-coNH2, -CO2H, -CO2RI, --CORI, --OCONH2, -OCH20H, --OCH20RI,--OCORI,--N3,--N2,--NHCORI,--NO2,--NH2,--NHRI, -NRI2, -Sf~H, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2, -SO~nERI, -Sf~RI2, -SO4H, -PO3H, -PO~H2, -SH, -SRI, -N-N-RI, 'O, =NH, -N-RI, =N-OH, ~N-ORI, -CONH2, -CONHRI, -CONRI2, -NHSO2RI, -SiRI3, tetrazole, imidazole, or 6ubstituted or f 10 unsub~tituted phenyl, -alkylphenyl, -O-phenyl, -f~-alkylphenyl, -O-benzyloxy-cnrbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, alkenyl or alkynyl of 2-~7 carbon atoms or cycloalkyl of 3-7 carbon : atoms, optionally halogenated at one or more hydrogen.
15 Likewise, R2, R3, ~, Rs, ~, R7, R~ and R9 are groups selected from X or -OH. In the above, ~ is selected from P, N, S, or C being in either the L- or D-configuration and ~ can be deleted or extended by 1-2 carbon ~toms, substituted or unsubstituted. Moreover, 20 the nitrogen atom attached to ~ can be substituted or unsubstituted C, S, O or P and the carbon atom attached to ~ and oxygen can be substituted or unsubstituted N, S, O, P or C. The aromatic ring of Formula I or the ring of Formula II can contain 0-4 N, S or O atoms.

''.'~' 21.~ 6 pr/cAs2/oo2*l Likewise, the aromatic ring cf Formul~ I or ring o~
Formula II can be fu~ed with a 6-membQred ~romat1a rlng which can contain 0-4 N, S or 0 ~tom~ and which can be substituted in the ~ame manner as the nromat~c r~ng o~
- S Formula I. Also included within Formulns I and II are D-tyrosine ~nd D-histidine.

Al~o provided is a method of treating a patient in a non-de6ensitizing fashisn with a non-desensitizing analog of a biologically active ligand containing at least one 6-membered aromatic ring or S-~embered ring. The method comprises ~electing an analog of a biologically active ligand containing a 6-membered aromatic ring according to Formula I, a8 defined above, or selecting a biologically active ligand containing a 5-membered ring according to Formula II, ~s defined above. ~he next step in the ~ethod co~prises ~dministering to a patient in need thereof a composition comprising a pharmaceutically effective amount of the ~elected analog together with a pharmaceutically acceptable carrier.

Also provided ~re new, non-desensitizing analogs of a biologically active ligand containing at least one 6-membered aromatic ring according to Formula . I or 5-membered ring according to Formula II, as defined above, but excluding the following compounds:
(13 antagonist analogs obtained by substituting D-amino acids at positions 1, 2 or 3, and analogs which have position 2 deleted;

(2) the following agonist analogs based on human GnRH:

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(A) [D-Phe6~GnRH in which the nydroxyl - gxoup of tyrosine i6 pro~cted with benzyl, acQtyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl;
(B) tPhe~]GnRH, [Ala4Phe~]GnRH and tPhe(N3)s]GnRH;
(C) tDes~he5-D-Ala~ProNHR9]GnRH in which position 2 i8 Hi~, Tyr, Trp or Phe(4'-NH2), position 7 i~ Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH or CHMe2;
(D) tDes~5Pro-B9]GnRH in which position 3 is Trp, Phe or 3tl-napthylalanine); X is His, Phe or ~ Phe(F5); position 6 i8 D-Ala(Y) wherein Y i8 naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, . perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-nlkylated alkylanine or cycloalkylamine, or NHCHt(CH2)~HC(~NR2)RI~CO wherein: n is l-S; Rl is alkyl or NRR3, wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, ;~ ~lkyl-morpholino or (CH2).(RJ)2, wherein n is 1-5 ~nd R, i8 alkyl; R2 is H or R3; and wherein C~NR~ cnn be a ring; position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; B is selected from GlyNH2, NHE~
~ . wherein B2 is alkyl, cycloalkyl or haloalkyl, and ! NHCONHB3, wherein ~ is H or alkyl;
; (E) analogs containing a gamma (~) lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C~7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; position 5 is an aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is an imino acid or aliphatic amino acid; po~ition 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and . ~
~ ~ ' ' ,.~
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~093/n~5~ l'(l/CA92/~ ~l h~
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~ F) analogs in which po6ition 5 i~ Phe, Tyr(Me), N-alkyl~Phe, N-alkyl-Tyr(Me) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is Alkylated.

Also provided 18 a novel methc~ for synthesizing new and u~e~ul ~nalogs of GnRH and opinte peptides a6 well a8 the novel analc~6 produc~d thereby.

Also provided are the distinctly novel non-de ensitizing ~nalogs having the For~ula III:
X~-X2~X3~X~~X5~X6~X7~~~xs~xlo III.
In Formula III, above, Xl is L- or D-Pyr, N-acyl-~oino acid, ~-C-alkyl-Pyr, or N-~cyl-~-C-alkyl-~mino acid; X2 X3, ~, ~, X7 and X~ are independently selected frc~m natural, synthetic, protected or ~-C-alkyl- amino acids, or ~-X~ optionally contains a ~-lact~; ~ is an imino acid or ~-C-alkyl-imino acid; X~0 is -N(H)R
wherein R is GlyNH2, azaGlyNH2, alkyl or alkenyl, cycloalkyl, haloalkyl, hydroxyalkyl or aryl; and X5 is selected from Formulas I or II, as defined above. The compounds excluded from the compounds according to - Formula~ I an~ II are likewise-excluded from the compounds according to Formula III

: Also provided are novel agonist analogs according to Formula III, wherein X~ is Pyr, N-acyl-amino acid, ~-C-alkyl-Pyr, N-acyl-~-C-alkyl amino acid;
X2 and X3 are aromatic L-amino acids; X~, X~ and X~ are independently natural, synthetic, protected or ~-C-alkyl- amino ac:ds; ~ is a natural, synthetic or protected D-amino acid, Gly, ~-C-alkyl-amino acid; or ~-X~ optionally contains a ~-lactam; X9 is imino Acid or ~-C-alkyl-imino acid; XJO is -N(H)R wherein R is GlyNH2, azaGlyNH2, alkyl, alkenyl, cycloalkyl, haloalkyl, hydroxyalkyl or aryl; and Xs is according to For~ulas I

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or II. The compounds excluded fro~ the compounds according to Formulas I and II nre likewi~e excludQd from thege compounds according to Formuln III.

DETA~LED D~8CRIP~ION OF ~R~ INV~NTION

Although biologically ~ctive lig~nds have been the subject of numeroua ~tudies in which thore have been a multitude of agonistic and antagonistic analogs crea~ed, there has been no succe6sful atte~pt~
at developing criteria by which one can 6elect agoni~tic and antagonistic analogs of biologically active ligands which are non-desen~itizing in natuxe.
For thi~ rea~on, the discovery by the pre6ent inventors is potentially very falr reaching in 6cope. That i8, the pre~ent inventor~ have di6covered that by ~electively ~odifying a biologically active ligand containing ~t least one 6-membered aromatic ring or 5-membered ring in the manner ~ccording to the pre6ent invention, that it is po~sible to produce analog6 of a biologically alctive ligand which are non-desensitizing in nature. It is to be understood that the term ~non-desensitizing" de~cribes n biQlogically ~ctive ligand with at least partially reduced desensitizing properties.

As used herein, the term "biologically active ligand" refer~ to a molecule which binds to a biologically active receptor molecule and which directly or indirectly affects the activity of the receptor molecule. The binding of such ligands to the receptor (acceptor) molecule is accordingly a necessary precondition for initiating, terminating, altering or preventing the biological activity in the receptor ; molecule. Any ligand which effects the biological activity of the receptor molecule is said to be a 2 ~ , '1 r(~r/~2/oo2~l biologically active ligand. The biologic~lly ~ct~ve ligand can be a substrAte, an agoni~t, an ~ntagonlst, an activntor, an inhibltor, etc. Example~ of biologically active lig~nd~ according to the present S .invention include GnRH, al60 known ns Luteinizing Hormone-Releasing Hormone ~LH-RH), and Angiotensln ~II
or III), and the like. Additional examples of biologically active ligands are well documented in the art.

Moreover, the biologically active ligand can be either peptidic or non-peptidic in nature. Such ligands can be indigenou~ to the organism where the biologically active receptor i~ found. When the ligand is one which is naturally occurring in that organism, then that ligand is referred to as a naturally occurring biologically active ligand. On the other hand, the biologically ~ctive ligands can be synthetic molecules which are complementary to the biologically active receptor and which ~ffect the biological activity of the receptor. Thus, any molecule which is complementnry to n biologically active receptor and which affects the biologiGal ~ctivity of the receptor, is a biologically active ligand.

~The term n ligand" according to the present ;25 invention refers to any organic compound for which a receptor naturally exists or can be prepared. The term "receptor" according to the present invention refers to a molecule which binds the ligand.

When binding of the biologically active ligand to the biologically active receptor and the activation of the active site results in an alteration -of the biological activity of the receptor, e.g., initiates, increases, decreases or terminates the ;

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`~VO 93/0305X ~ PCr/CA92/0~

biological activity of the receptor, the llgand 18 ~ld to directly affect the activity of the receptor. On the other hand, a biologically active ligand lndlrectly affects the activity of the biologically actlve receptor when the binding of the ligand to the receptor results in an inability to activate the receptor.

Activation of the active site of the naturally occurring biologically active ligand/receptor complex is generally nccomplished by some sort of chemical interaction between the ligand and the r~ceptor. When the chemical interaction involve~ the transfer of charge from one re6idue to another wherein one of the residues is either a phenol or phenolate re~idue, the ~nteraction is termed a charge-transfer interaction. Such charge-transfer interactions are believed to result in the alteration of the structure of the ligand or ligand/receptor complex. Becau~e such charge-transfer interactions can now be detected by the techniques employed in the present invention, it is now possible to incorporate such interactions into the model created for the naturally occurring biologically active ligand ~nd to create agonists and antagoni~ts which have reduced desensitizing properties at the complementary receptor.

Any biologically active ligand containing a phenolic group, such as tyrosine, may utilize the phenolic group to desensitize a complementary receptor.
Methods for determining biologically active phenolate ligands using spectroscopic techniques have been described in detail in U.S. Ser No. 07/458,926 filed December 29, 1989, which is incorporated herein by reference.

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W093/0305~ PCr/CA92/~2~l 2 1 1 ~~ ri ~i tj : In one aspect of the method according to the present invention, the presence of a phenolate spec1e~
~ in a biologically active ligand i6 a~certained by mea~uring the fluorescence lifetime due to 6aid : ` 5 phenolate species in a suitnble environ~ent ~uch n6 propylene glycol. The pre~ence of long lifetime phenolate (tyrosinate) fluorescence is diagno~tic of the occurrence of the active phenolate ~pecies which may cause receptor desensitization. The long 10 fluorescence lifetime is produced by intra~olecular interactions within the biologically active ligand when the ligand is dissolved in a receptor-si~ulating solvent such a8 propylene glycol.

In ~nother method aspect, the lability of the 15 phenolic OH proton may be determined by NMR
~pectroscopy of the biologically acti~e ligand in a ~uit~ble ~olvent such aæ DMSO. The absence of a NMR
signal characteristic of the phenolic OH proton, at or - ~bout 9.2 ppm, is also an indication of the presence of 20 n phenolate species which may be involved in a receptor J desensitization process. Furthermore, and as described in the pre~ent disclosure, modification or ~ubstitution of the phenolic OH group of ~ biologically active ligand provides a method for establishing the ~ 25 involvement of the phenolic OH group in a receptor ¦ desensitizing process, particularly when such :~ modification results in a ligand with reduced receptor ~j desensitizing properties, as exemplified herein for : peptides based on Angiotensin II and GnRH.
The term "agonist" according to the present invention refers to a biologically active ligand which binds to its complementary biologically active receptor and activates the latter either to cause a biological 35 response in the receptor or to enhance pre-existing :
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~1VO g3/03058 PC.~/C~92/0028 1 ~12.'~

biological activity of the receptor. The ~gonist can be the naturally occurring biologically active ligand or it can be 2 synthetic molecule which can also activate the receptor. For ex~mple, it i6 known in the art that Angiotensin II acts a8 an agoni6t for it6 complementary receptor, the Angiotensin II receptor.
OthOE examples of ~goni6ts for the Angiotensin II
receptor include ts~r~] Angioten~in II and the like. A
common characteristic of all ligands in this invention is that the ~harge-transfer interaction in the ligand which is necessary to desensitize the biologically active receptor i8 compromised. Thnt is to s~y that the charge-transfer interaction i~ es6entially inoperable in the ligand.

The term "antagonist~ refer~ to a biologically active ligand which binds to its complementary biologically active receptor and either prevents the activation of the latter or deactivates the latter 80 as to either prevent or diminish the biological activity of the receptor. For example, it i6 known in the art that the non-peptides 2-n-butyl-1-4-c~rboxybenzyl]-4-chloroimidazole-5-acetic acid) and (methyl 2-n-b~ltyl-1-[4-(2-carboxybenzamido)-benzyl]-4-chloroimidazole-5-acetate) sodium salt act as ~ 25 antagonists of the Angiotensin II receptor. Other j examples of art-recognized antagonists to other biologically active receptors include propranolol for the ~-adrenergic receptor, cimetidine for the Histamine-H2 receptor and the like.

The term "Angiotensin II" refers to the biologically active ligand which is an octapeptide represented by the amino acid seguence of: -Asp-Arg-Val-Tyr-Ile-His-Pro-Phe. Similarly, the term "Angiotensin III" refers to the biologically active ~93~0~58 ~'C~'/C~92/~28l ri ,) ligand which i6 a heptapeptide represented by the amino acid sequence of: Arg-V~l-Tyr-Ile-Hi~-Pro-Phe.
, ~
It is to be understood that the disclo~ure of the present invention employs the conventional abbreviations for the v~riou~ common amino acid~ as gener~lly accepted in the peptide art and ns recommended by the IUPAC-IUB Commission on Biochemical Nomencl~ture, Biochemistry II, 1726 (1972). Many other terms used throughout the present di~closure are believed to be readily understood in the art but a definition of some of these terms is provided in Table 1 for convenience sake.

DEFINITION OF VARIOU8 T~XM8 Term Definition Aib 2-aminoi~obutyryl Alk Cl-C~ alkyl or alkenyl group;
Bzl benzyl group Cha L-cyclohexylalanyl DCC N,N~Dicyclohexylcarbodiimide DMF dimethylformamide Et ethyl group Halide F, Br, Cl, or I;
HBT l-hydroxybenzo~riazole HPLC high-pressure liquid chromatography Me ~ethyl group Mtr 4-methoxy-2,3,6-Trimethylbenzenesulfonyl;
Pmc 2,2,5,7,8-pentamethylchromzn-6-sulfonyl;
Pr propyl t-Boc t-Butoxycarbonyl TFA trifluoroacetic acid Tos tosyl group Trt trityl qroup :;". - - ''""' ' , .: , '~: ' `, ~ '., .' '' ~' . '" ' ~ '; ` '' '' ,. ' '' , ', ~g3/03058 P-'l'/C~92/~ ~l rj ~, 6 As noted pxe~iously, the chemic~1 interaction between the ligand and the receptor can cause th~
desenæitization of the receptor such that a subsequent interaction of the ligand on the receptor results ln attenuation of the subseguent biological response to the ligand. One ~uch biologic~lly active liqand which 6erves to illu~trate the effects of desensitization of a receptor i8 GnRH.

As mentioned earlier, GnRH is released from the hypothalamus and binds to a receptor on the pituitary gl~nd, causing the release of LN (Luteinizing Hormone) and FSH (Follicle-Stimulating Hormone). GnRH
has been purified and identified in mammals to be a decapeptide having the amino ~cid fiequence: Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. The following are other naturally occurring analogs of GnRH:
Salmon GnRH: Pyr-His-Trp-Ser-Tyr-Gly-~E~-Iç~-Pro-~ Gly-NH2;
', Chicken GnRH-II: Pyr-His-Trp-Ser-His-Gly-~L~-~yE-Pro-i 20 Gly- ~ ; and Chicken GnRH-I: Pyr-His-Trp-Ser-Tyr-Gly-Leu-Çln-Pro-Gly-NH2 .
~ . .
How~ever, initial attempts at using GnRH to induce ovulation were unsuccessful because of the failure to rec~gnize the necessity of pulsatile administration. Upon recognizing the therapeutic potential of GnRH, biochemists began synthesizing both agonistic and antagonistic analogs of GnRH.
.1 .
one approach to this goal has been the replacement of an amino acid residue of GnRH with -' another amino acid. Although in a few instances decapeptides containing such a replacement have been found to be more active than GnRH, due primarily to an "

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~''- ' ' ' ' - ' , , ' .
- , W0~3/O~SX P(~/CA92/nn~l ~ J ti increase in re~i6tance to degr~dAtion by enzymes i~
vitro and in vivo, e.g., ~D-Aln6]-GnRH, A. Arimura, et al., Endocrinology, 95, 1174 (1971) and [D-Leu~]-GnRH, J. A. Vilchez-Martinez et al., Blochem. Blophys. Re~.
Commun, 59, 1226 (1974), for the most part the replacement containing decapeptides have been less active.

Thus, GnRH analogs fiynthesiz~d previou~ly have been designed on the basis of resistance to metabolic degradation, and have been particularly concerned with the incorporation of a D-amino acid into the GnRH peptide (as mentioned above), or with N-methylation of one or more of the peptide bonds in order to invoke intestinal stability to enzymes (European Patent No. 0 328 090). The criteria for selection of the analogs described herein is distinctly different from previous work such as that of EP-0,328,090 in that ~election is ba~ed on GnRH analogs with receptor desens~tizing properties, achieved 2D principally by modifying the Tyr5 or His5 residue of G~RH peptides iD the manner according to Formulas I and II. -. .
Contrary to the initial intent of theprevious workers, even when more active agonists of GnRH were synthesized using the criteria for developing agonists and antagonists with increased resistance to degradation by enzymes, many were found to have disadvantageous long-term effects. More specifically, these analogs of GnRH stimulated the release of LH and FSH initially, but continuous administration resulted in desensitization of GnRH receptors on gonadotropes and decreased gonadotropin secretion. Accordingly, prolonged u6e of high concentrations of GnRH agonists has caused reversible ovarian and uterine atrophy in . , , ~' ~:

~ ~093/03058 P~T/CA92/00~l 21 ~2~ f-i ani~als and has prevented implantation of ova ~nd gestation. Likewise, high concentrations of previously ~ynthesi~ed GnRH agoni~ts in malQs has inhlbited te~ticular synthe~is of te~to6terone.

; 5 However, through exten~ive rosQarch, the present in~entors have di~covared a method for 6electing non-desen6itizing analog6 o~ a biologic~lly active ligand containing at least one 6-me~bered aromatic ring or 5-~embQred ring. ~he m-thod co~prise~
~electing an analog of a biologically active ligand containing a 6-membered aromatic ring according to Formula I or selecting an analog of a biologically active ligand containing a 5-m~mbered ring according to ~ormula II:

; ~ ~ R I R~ C C-----R3 II
R6 1 ~ \IcD
~ --N~ CO-- R6 ~----------R~
' R R -N f co - -Rt R9 In Formulas I and II, above, X is selected i from H, Rl, -ORI, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-ORI, -alk-CO2H, -alk-CO2RI, -alk-SH, -alk-SRI, -alk-CONH2, -CO2H, -C4RI, -CORI, -OCONH2, -OCH20H, -OCH2ORI, -OCORI, -N3, -N2, -NHCORI, -NO2, -NH2, -NHRI, -NRI2, -SO3~, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2, -SO2NHRI, -SO2NRIz, -S04H, -PO3H, -PO4H2, -SH, -SRI, -N~N-RI, =O, =NH, =N-RI, =N-OH, =N-ORI, -CONH2, -CONHRI, -CONRI2, ~ , ~'VI) 93/0305X PC'r/CA92/002~ 1 ,, .1 1 ,~ .i ~; ~, -NHSO2RI, -SiRI3, tetrazole, imidAzole, or substltuted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl. Addition~lly, Rl i~ selected from alkyl of 1-7 carbon atom~, alkenyl or alkynyl of 2-7 cnrbon S atoms or cycloalkyl of 3-7 carbon atoms, option~lly halogenated at one or more hydrogen. Likewise, R2, R3, ~, Rs, R6, R7, R~ and R9 are groups ~elected from X or -OH. In the above, ~ i8 selected from P, N, Sj or C
being in either the L- or D-configuration and B can be deleted or extended by 1-2 carbon atoms, substituted or unsubstituted. Moreover, the nitrogen atom attached to can be ~ubstituted or un6ubst$tuted C, S, O or P and the carbon ~tom attached to a and oxygen can be 6ubstituted or unsubstituted N, S, O, P or C. The aroma~ic ring of Formula I or the ring of Formula II
can contain 0-4 N, S or O atoms. Likewise, the aromatic ring of Formula I or ring of For~ula II can be fused with a ~-me~bered aro~atic ring which can contain 0-4 N, S or O atoms nnd which can be substituted in the s2me ~anner as the aromatic ring of Formula I. Also ' included within Formulas I and II are D-tyrosine and D-Histidine.
..
! The analogs of a bioiogically active ligand are selected 80 as to contain a ring modified according to Formulas I or II and so that the resulting biologically active ligand has reduced desensitizing properties, i.e., the biologically active ligand i8 such that the subsequent interaction of the ligand with the receptor does not substantially attenuate the biological responce. The di~covery and use of analogs according to Formulas I or II, and especially GnRH
analogs according to Formulas I or II, with reduced desensitizing properties is new.

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: ~

'. ~

~'093/0.~0~ 2/00~l ,~ I 1. ~ .i ~i ii The an~log of a biologlcally ~ctive lig~nd containing at least one 6-mQmberQd aromatic ring can be ~elected, for exnmple, from the peptides Angiot~nsin (II or III) or GnRH. Thu6, one method according to the 5 pre~ent invention is the ~election of a non- -de~ensitizing analog of tbe blologically active ligand GnRH wherein the tyro~ine residue At the 5-position ha~
been modified according to Formula I.

More speciSically, some preferred analog6 6elected by the present method include GnRH analogs represented by the formula III:
X~-X2-X3-X4-~-~-Xrx~~xs~xlo III.
In Formula III, above, X~-X~0 (positions 1-10) are selected from the following: X~ i6 Pyr or N-acyl-amino acid; X2 i5 an aromatic amino acid; X3 is an aromatic amino acid, X4 i6 Ala, Thr, Ser, H6e, Ser(Me), ~-C-methyl-Ser(Me), Aib; Xs is Tyr(0-alkyl), Phe(4'-halogen); Phe(4'-alkyl); }4 i6 Gly, Aib, or D-amino acid (natural or 6ynthetic, including an amino acid bearing a protecting group), ~-C-alkyl-amino acid;
X7 is Leu, Ile, Trp, Cha, ~-C-methyl-~mino acid, Val; X~
is Arg, Gln, Tyr, Leu; X9 i6 P~o, pipecolic acid, nipecotic acid, azetidine-2-carboxylic acid, dehydroproline, thioproline, hydroxyproline; and X~0 is N(H)R wherein ]R i8 GlyNH2, azaGlyNH2, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl or aryl, wherein each alkyl or aryl group has from 1 to 7 carbon atoms.

Another particularly preferred embodiment is one in which the ~-carbon atom of at least one of the amino acids is alkylated, in which ca6e position 5 may be as described by Formulas I or Il or may be ~-C-methyl-histidine or ~-C-methyl-tyro6ine, or the like. The modifications illustrated for the tyrosine ~V093/03058 .Jl~ ~r;~; fi PC r/cA92/~2#l - ~ 9 -residue of GnRH are also applicable to other Tyr-containing peptides.
.
In a similar fashion, the role of ~yr in receptor desensitization can ~160 b~ carried out by Hi~tidine in certain peptides, sf which chicken GnRH-II
i~ an example. Therefore, modifications to the imidazole group which remove the hydrogen bond donor capability of imidazole, for exaDple alkylation at N~, or N3, can be expected to dimini~h the desensitizing ~ 10 effects of certain ligands containing imidazole.

- More generally, selective ~odification or sub~titution of the hydroxyl groups in a biologically sctive ligand containing a 6-membered zlromatic ring or S ne~bered ring according to Formulas I and II, re~pectively, may produce zmalogs with reduced desen~itizing effects. Such low-desensitizing analogs may ~160 be used to attenuate the de6ensitization evoked by a high-desensitizing ligand, as illu~trated for GnRH in ~camples 4 nnd 5, infra.

Additionally, selection of a bioloqicz~lly ~ctive ligand having a phenolate group zlccording to Formula I or a 5-membered ring according to Formula II
1 may likewise favorably alter the duration of zlction of ¦ the ligand. Thus, in the case of an zlgonistic ligand, the duration of the biological response is increased.
For example, tTyr(Me)s]-GnRH has a longer duration of action than GnRH. This increase in duration of action and the non-desensitizing nature of the analog ; tTyr(Me)5]-GnRH iG illustrated in Example 2 and, infra.

The same method is al~o applicable to antagonist analogs of a ligand. In the case of an antagonist ligand, however, the duration of action is . ~; `

~'~93/0305~ PCr/C~2/00 decreased. An example i6 seen with ~ngiotensin, wherein methylation of the tyro~ine OH of the angiotensin antagoni6t tSarl Ile~] ANG II to give tsar' Tyr(Me)4 Ile~] ANG II result~ in a reduction of the desensitization properties and duration of action. The decrease in the duration of action and the non-desensitizing nature of the above antagoni6tic analogs is illustr~ted in Ex~mple 1, infra.

It i6 important to understand, however, that the present invention i8 not limited to peptides such as GnRH and Angiotensin II or III, but m~y be applied to any desensitizing b~ologically active ligands having a 6-membered aromatic rlng or 5-membered ring.
Accordingly, other example6 demonstrating the exten6ive applicability of the ~election method according to the present invention are as follows.

Additional examples of non-desensitizing analogs of a biologically active ligand selected according to the present invention include, but are not limited to, the opioid ligands, wherein de~ensitization, tolerance, dependence, and withdrawal may be effectively reduced by modification of the phenolic group or conjugated hydroxyl group of an -opiate peptide (which have the N-terminal sequence Tyr-Gly-Gly-Phe-Met/Leu such as enk~phalins, dynorphins and endorphins) or non-peptide ligands. Thuc, methylation of a phenolic hydroxyl group of morphine produces the less active analog codeine. Furthermore, deletion of both hydroxyl groups in morphine can result in the agonistic mimetic metbadone which di6plays increased potency and decreased withdrawal symptoms due to the blockade of morphine dependence. Accordingly, it i6 understood in the present disclosure that GnRH is u~ed as an example only, and that tbe ~tructural ~
~.,.~.

~' ~YO93/0305X PCT/CA~/0028l ,''ii.~,jfj,i modific~tion6 to the 6-~ember aro~atlc rlng in GnPH c~n likewise be applied to ~ny bloloqic~lly active ligand`
having a 6-membered aromatio ring or 5-memb4red ring which can be modified according to Formula~ I or Il.

~8 wa6 mentioned, such biologically actlve ligands are likewise not limlted to mQrely the peptidic ligands but al~o include non-peptidic ligands.
Consequently, the present invention i6 u6eful in selecting agonistic non-peptidic analogs of a lo biologically active ligand containing at least ane 6-membered aromatic ring according to Formula I or 5-me~bered ring accord~ng to Formula II in which the interrelnted properties of desensitization, tolerance, dependence, withdrawal and addiction are reduced.

As used herein, the term "non-peptidic ligands" ~8 ~pplied to the Formulas I and II refer to the ring structures depicted in Formulas I and II
wherein the peptide backbone has been removed. For example, representative example~ of non-desensitizing analogs of a hiologically active lig~nd ~elected according to the prefient invention include, but are not limited to, certain steroid hormone6 and catechol~cine hormones. Such modifications where applicable, may also be applied to a biologically active ligand containing a conjugated hydroxyl group, of which -C=C-C-OH is an example.

To further illustrate the far-reaching effects of the present invention, GnRH will once again be used as a representative example to di6play the numerous advantages which can be nchieved by the practice of the present invention. GnRH analogs are useful for increasing 6exual activity, fertility And egg yield and inducing ovulation in animals, and are ~, .,': .
, .. . .
~`
~'' .

~093/O~S8 PCr/CA~2/00~l 21~2ri~ fi useful for increasing productivity in the farm1ng of fifih, poultry and mammals, as well as for incre~elng fertility in humans. GnRH analogs can also increase lifetime gamete production, and permlt collection of gametes from pre- and poQt-natal, juvenile, prepubertal and mature mammals for natural and artificial fertilization. GnRH analog6 are also effective in the treatment of gonadotropin-related disease6 suah a8 prostatic cancer, breast cancer, endometriosis and ~0 fibroid shrinkage.

However, as was mentioned, agonistic and super agonistic analogs of GnRH often reguire continuous or rapid pulsatile dosing ~n order to avoid the potent desensitizing effects associated with these peptides. The development of GnRH agonist analogs with reduced receptor desen~itizing properties is advantageous because such analogs obviate the need for continuous or rapid pulsatile dosing methods and may be administered in a single dose or witb considerably lower dose frequency. The advantage of eliminating the need for pulsatile dosing likewi~e extends to all ~ analogs of a biologically active ligand according to .~ the present invention.
i~ .
~; The use of tbe present metbod of selecting a -non-desensitizing analog of a biologically active ligand quite surprisingly possesses tbe additional advantage of not requiring tbe continued use of solely the non-desensitizing analog but allows for the use of "normallyH desensitizing analogs witbout tbe expected desensitizing effects upon tbe biologically active receptor. For example, a non-desensitizing GnRH analog may be administered alone or in combination with another normally desensitizing GnRH agonist since the desensitizing properties of the latter are suppressed ~, ,'' ' '' ~' S~

W093/0~s8 , r~/CA92/~28l by the pre6ence of, or pretreatment with, the ~or~er.
Stated in another wny, by treating a pntient with ~
non-desensitizing GnRH analog ~ccording to the present invention, it is po66ible to sub~equently or simultaneou61y treat the p~tient with an normally desen6itizing analog of GnRH becau~e tho pretreatment with, or pre~encQ of, the non-desen~itizing GnRH analog ~ccording to the present invention act6 to eliminate or suppres6 the de6en6itizing effect of the normally de6ensitizing GnRH analog. A "normally" desen6itizing analog is an analog of GnRH which has not been modified in a manner which reduce6 the de6ensitizing properties as illu~trated in the present invention. The use of a non-desQnsitizing GnRH analog in combin~tion with normally de6ensitizing GnRH or 6ynthetic analogs thereof i8 a preferred method a6pect of the present invention.

A "biologically active receptor" according to the present invention is a molecule, having a specific binding 6ite for its complementary ligand, and includes classical hormon~l receptors, binding and/or transport proteins, enzyme~, antibodies and the like. One embodiment of a biologically active receptor includes membrane-bouncl proteins which control certain cellular proces6es in which themselves Are regulated by the binding, or lack of binding of a complementary naturally occurring biologically active ligand.
Because such membrane bound biologically active receptors are bound to membrane, it i6 believed that the conformation of the biologically active ligand necessary to activate such receptors are lipid induced.
On the other hand, there are other biologically active - receptor6 which are not membrane bound. In 6uch cases, cuch receptors may not require a lipid induced conformation of the biologically active ligand and, in ~' ' `' .
i~.'' ~' .

~!O 93/0305X I'CI /~,A92/01)2~ 1 fact, may require an aqueous induced conformation of the complementary biologically active lig~nd in order to activate such receptors.

Examples of biologically active receptors have been well documented in the art. Specific examples include insulin receptor (wherein the complementary ligand i~ insulin) and the Angioten6in II
receptor (wherein the complementary ligand i6 Angiotensin II~, and the like.

A6 de6cribed above, the non-desen6it$zing analogs of a biologically active ligand ~elected according to the present invention have a multitude of uses in hu~ans and animals, either of which are considered "patients~ as that term is employed in the present disclosure or claims. Additional exa~ples of desired use6 of non-de~ensitizing GnRH analogs of a biologically active lig~nd selected according to the present invention include, for example, the ability to more readily ~ynchronize estrus in live6tock, e.g., cattle, sheep or 6wine, either in order to be able to ~ mate all the female6 in a given group with a male of i the desixed genetic quality, or so as to be able to ¦ perform artificial insemination OD a ~aximum number of females, both within a comparatively short period of time. It will be appreciated that 6uch a method is of particular importance for breeders of race horse6 and show animals, where the fee is paid for the services of an exceptional male animal sften amount to considerable sums of money.

Thus, in accordance with the multitude of potential uses for non-desensitizing analogs of biologically active ligands, the present invention i5 also directed to a method of treating a patient in a ~ ' ' ' ~, ' ''"''' ;' ' . '' , , ~ ''' ' '' , , "`-, ~. .. . . ..

W0 93/0305X ~ rj r~ . j P(~l /CA92/0028 1 non-desensitizing fashion with a non-de~Qnsitizlng analog of a biologically active ligand containing at least one 6-membered aromatic ring or 5-~e~bered ring.
The method compri6es first selecting ~n nnalog Or a biologically active lig~nd cont~ining a 6-membered aromatic ring according to Formula I or selectlng an analog of ~ biologically ~cti~e ligand containing a 5-me~bered ring According to Formula II:

R~ ~,,R2 C~C

~5 ~ R3 I R~ C C ~ II
\~/

R~-----~ R~ C
----N_ F~-- B~ --R7 Rt RP --N Co----In Formula~ I and II, above, X is selected from H, R~, -ORI, halide, -CN, -CHO, C(balide)3, -alk-OH, -alk-ORI, -alk-C ~ , -alk-C~R~, -alk-SH, -alk-SR~, -alk-CONH2, -CO2H, - ~ Rl, -CORI, -OCONH2, -OCH20H, -OCH20RI, -OCORI, -N3, -N2, -NHCORI, -NO2, -NH2, -NHRI, -NRI2 ~ -SO3H, -SCiRI, -SCORI, -NCS, -SCSRI, -SO~NH2 ~
-SO~nERI, -SO2NRI2, -SO4H, -PO3H, -PO4H2, -SH, -SRI, -N~N-RI, =0, =NH, -N-RI, =N-OH, ~N-ORI, -CONH2, -CONHRI, -CONRI2, -NHS~RI, -SiRI3, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-aIkylphenyl, -O-benzyloxy-carbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, alkenyl or . alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated at one or more hydrogen.
Li~ewi~e R2 R3 R4, R5, R6, R7, R3 and R9 are groups ;

~ .
" ~ .
, . ~ .
.

WO 93/0305X ~ I'CI'/C'~92/00~#1 selected from X or -OH. In the above, ~ i8 ~elected from P, N, S, or C being in either the L- or D-configuration and B can be deleted or extended by 1-2 carbon atoms, sub6tituted or unfiubstituted. Moreover, S the nitrogen atom attnc~ed to ~ can be ~ubstituted or ~nsubætituted C, S, O or P and the carbon atom attached to ~ and oxygen can be ~ub~tituted or un~ub~tituted N, S, O, P or C. The ~romatic ring of Formula I or the ring of Formula II can contain 0-4 N, 8 or O ato~s.
Likewise, the aromatic ring of Formula I or ring of Formula II can be fu6ed with a 6-membered aromatic ring which can also contain 0-4 N, S or O atom6 which can be substituted in the ~ame manner as the aromatic ring of Formula I. Al60 included within Formulas I ~nd II, above, are D-tyro6ine and D-histidine.

Step two of the method compri6es administering to a patient in need thereof the ~elected analog of a biologically active ligand a6 a compo6ition compri6ing (i) a pharmaceutically effective amount of the selected analog and (ii) a pharmaceutically acceptable carrier for the sQlected analog. While it is possible for the particular active ingredient to be administered as the raw chemical, it i~ preferable, in view of the potency thereof, to present it a8 pharmaceutical formulation containing an acceptable carrier.

In a preferred method, at least one non-desensitizing an~log of GnRH i6 given in combination with at least one normally desensitizing GnRH or synthetic analog thereof. The non-desen6itizing analog(s) of GnRH may be given before a normally desenstizing GnRH analog, simultaneously therewith, or both.

, ~J~O 93/0305~ 1'( '1/( `A92/1~2S~ 1 i~ :1 1. ,' ,,, ~, For each of t~e utilities mentioned nbov¢, the amount of the analog of the biologic~lly ~ctlv~
ligand will, of course, vary both with the p~rtlcular active ingredient and with the route of adminifitratlon.
In general, however, for e~ch of t~e~e utilities the dosage for na~al or parental ad~inistration will be in the rAnge of about 0.005 to 200~g per kilogr~m body weight of the patient, preferably between about 0.01 and 100 ~g/kg. For ornl or vaginal administr4tion the dosage would generally be in the range of about 0.005 to lOOO~gJkg, preferably about 0.05 to 20~g/kg. It i6 under tood that all dos~ges are calculated with reference to the base peptide. These dose~ apply to both a non-desensitizing and normally desensitizing analog of GnRH when ~dministered in one of the combination protocols described above.

It should be recognized that by "non-desensitizing fashion", it is understood that the present method treats a patient in a manner such that the patient does not need to be treated with the type of pulsatile dosing that was heretofore required in order to successfully ~chieve the hormonal changes in the body of ani~als and humans. ~ather, the analogs, alone or in combination with normally desensitizing analogs, according to the present invention obviate the need for continuous or rapid pulsatile dosing methods and thus may be administered in a single dose or with considerably lower dose frequency.

The formulations of the present invention comprise an active ingredient or combination thereof, as described above, together with one or more pharmaceutically acceptable carriers as well as other optional therapeutic ingredients. The carriers must be pharmaceutically "acceptable" in the sense of being .
~. ' '' ~. '' ~"'~ ' ' .

~093tO~SX ~ ,it;lj P~ A92/oo~l compatible with the other ingredient~ of the formulation and not deleterious to thQ reciplent thereof. Desirably, the formulatione ~hould not include oxidizing agents and other substances with S which peptides, or non-peptides, Are known to be incompatible.

The foruul~tions include those suitable for oral, rectal, nasal, topical, vaginal or parenteral (including subcutaneous, intr~muscul~r, and intravenous) administration, although the more suitable route in any given case will depend upon the sel~cted active ingredient.

A6 another preferred route of adcinistration, an active ingredient(s) may be presented as a depot formulation having a slow-release characteristic suiting it for implantation in the body of the recipient, for example, sub-cutaneously, intramuscularly, intraperitoneally or intravaginally.
For example, injections may be given in depot form with a slow rele~se agent such as an emulsion, sesa~e oil, a 10% ~queous polyvinylpyrolido~e, or in biodegradable microspheres ~uch ~s polylactic/glycolic acid.
Alternatively, administration may be given with the aid of a mini osmopump, located internally or externally.
In animals such as fish, administration may be optionally given with a dopamine antagonist such as domperidone.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
Typical methods include the step of bringing into association the active ingredient with the carrier which optionally contains one or more accessory ~X ~ :
~'.. :..... - ~ ,~, . ....
.' . ...
~ .
:~
.

W~93/03~5X ,~ 92/~2~l ingredient~. In gener~l, the formulatlon~ a~e prepared by uniformly and intimately bringlng into ~seoci~tion the active ingredient with liquid carrierG or finely divided solid carriers or both, and then, if neces~ry, ~haping the product into the desired formulation.

Pormulations of the present invention suitable for oral administrntion may be presented as discrete units such as capsule , cnchets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; or as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or a5 an oil-water liquid emulsion or a water-oil liquid emulsion. The active ingredient ~ay also be presented as a bolus, electuary or pa6te.

A tablet ~ay be made by compres6ing or ~olding, optionally with one or morç accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or grnnules, optionally mixed with a binder, lubricant, inert dilution, lubricating, surface active or disper6ing agent. Molded tablet6 ~ay be made by molding in a suitable machine a mixture of the powder compound moistened with an inert liquid diluent.

For~ulations for rectal administration may be presented as a suppository with the usual carriers such as coco-butter, while a suitable formulation for nasal administration is nasal drops comprising an active ingredient in an aqueous or oily solution.

Formulations suitable for oral administration include lozenges comprising the active ingredient in a flavored basis, usually sucrose or acacia or ~'' "
~' W093/03058 2 11 ~ rj !~ r~ Ag2/~l tragacanth; and pa6tilles comprising the activ~
ingredient in an inert basiG such as gelatln, glycerlne or sucrose and acacia.

Formulations suitable for vaginal administration may be presented as pessaries, creams, pastes, or sprny formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.

Formulations suitable for parentnl ~dministration conveniently comprise sterile aqueous solutions of the active ingredient, which solutions are preferably isotonic with the blood of the recipient.
Such formulations may be conveniently prep~red by dissolving a solid active ingredient in water to produce an agueous solut~on, and rendering said solution sterile and isotonic with the blood of the recipient.

Also provided herein are new, non-desensitizing analogs of biologically active ligands containing at least one 6-m~mhered aromatic ring or 5-membered ring. The non-desensitizing analogs of a biologically active ligand contain at least one 6-membered aromatic ring according to Formula I or 5-membered ring according to Formula II:
X X RZ

3 I~ C o~ 5 II

l i - N C0-- R4-~ R7 R~ R~ -N~ 0--, ,, ' ,~

WO93/0305R ,)i l~'JrjJj;; r(~r/cA92/~28 In Formulas I ~nd II, ~bove, X le ~e~ected from H, Rl, -ORI, hnlide, -CN, -CHO, C~h~1ido)3, -~lk-OH, -alk-OR~, -Alk-CO2H, -a1k-CO2R~, -alk-SH, -alk-8R~, -alk-CONH2, -CO2M, -CO2RI, -CORI, -OCONH2, -OCH20H, -OCH2ORI, -oCORI, -N3, -N2, -NHCORI, -NO2, -NH2 ~ -NHRI, -NRI2, -SO~H, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2, -SO2NHRI, -SO2NRI2~ -S04H, -PO3H, -PO4H2, -SH, -SRI, -N-N-RI, -O, =NH, -N-RI, ~N-OH, -N-ORI, -CONH" -CONHRI, -CONRI2, -N~SO2RI, -SiRI3, tetrazole, i~idazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxy-carbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, ~lkenyl or alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated at one or more hydrogen.
Likewise, R2, R3, R4, R5, R6, R7, R~ and R9 are groups selected from X or -OH. In the above, ~ is selected from P, N, S, or C being in either the L- or D-configuration and B can be deleted or extended by 1-2 carbon atoms, 6ubstituted or unsubstituted. Moreover, the nitrogen atom nttached to ~ can be substituted or unsubstituted C, S, O or P and the carbon atom attached to ~ and oxygen can be 6ub6tituted or unsub6tituted N, S, O, P or C. The ~ro~atic ring of Formula I or the ring of Formula II can contain 0-4 N, S or O atom6.
Likewise, the aromatic ring of Formula I or ring of Formula II can be fused with a 6-membered aromatic ring which can also contain 0-4 N, S or O atoms which can be substituted in the same manner as the aromatic ring of Formula I. Also included within the Formulas I and II, above, are D-tyrosine and D-histidine.

Excluded from the compounds according to this aspect of the present invention are those antagonist analogs of mammalian GnRH obtained by substituting ~ ~ ' ` ~;~93tO3058 2 L i ~ j ~ fJ I'CI/CA92/00281 D-amino acids at positions 1, 2 or 3, and analogs whlch have position 2 deleted. Also excluded are the following agonist analog6 based on mammalian GnRH:
(A) ~D-Phea]GnRH ~n which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl;
(B) lPhe5]GnRH, tAla~Phe51GnRH and 10 tPhe(N3)5~GnRH;
(C) tDesl~hes-D-Ala~roNHR9~GnRH in which position 2 is Hi~, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R i6 Et, Pr, CH2CH20H or CHMe2;
(D) tDes~qKsPro-B93GnRH in which position 3 is Trp, Phe or 3(1-napthylal~nine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, ~nthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCHt(CH2)~NHC(eN~0 RI~CO wherein: n is 1-5; Rl is ~lkyl or NRR3 whe~ein R i8 H or aIkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl-morpholino or (CH2).(~)2 wherein n is 1-5 and RJ is alkyl; R2 is H or R3; or wherein C=NR~R~ can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is selected from GlyNH2, NHB2 wherein 82 is alkyl, cycloalkyl or haloalkyl, and NHCONH~ wherein ~ i~ H or alkyl;
(E) analogs containing a ~-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C~7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; posit:ion 5 is aromatic amino acid His, Trp or Phe; position B is an amino acid with a ~'" ', ,.., ~' , '~"'', '"

~; .,.~ : .' ,., W093/0~58 PCr/CA92/~ ~l basic sidechain; position 9 i6 imino ~cid or aliphatic amino acid; position 10 ~8 GlyNH2, Al~NH2, NHEt, NHPr, NHCH2CH20H; nnd (F) analog~ in which po~ition 5 i~ Phe, Tyr~Ne), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et~, wherein the nitrogen atom of at least one of the amide bonds is alkylated.

Some preferred novel analogs selected by the present invention include GnRH analogs having the Formula III:
Xl~X2~X3~X~~X~~X6~X7~x~~xs-xlo III.
In Formula III, above, X~-X~0 (positions 1-10) are ~elected from the following: X~ is Pyr or N-acyl-amino acid; X2 is an aromatic amino acid; X3 is an aromatic amino acid, ~ is Ala, Thr, Ser, Hse, Ser(Me), ~-C-methyl-Ser(Me) or Aib; X5 is Tyr(0-alkyl), Phe(4'-halogen) or Phe(4'-alkyl); X6 is Gly, Aib, or D-amino acid (natural or synthetic, including an amino ,~ acid bearing a protecting group) or ~-C-alkyl-~mino acid; X~ is Leu, Xle, Trp, Cha, Val or ~-C-methyl-a~ino acid; X~ is Arg, Gln, Tyr or Leu; Xg is Pro, pipecolic acid, nipecotic acid, azetidine-2-carboxylic acid, dehydroproline, thioproline or hydroxyproline; and XlO is N(H)R wherein R is GlyNH2, 25 : azaGlyNH2, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl or aryl, wherein each alkyl or aryl group has from 1 to 7 carbon atoms.

Another preferred embodiment is one in which the ~-carbon atom of at least one of the amino acids is alkylated, in which case position 5 may be as described by For~ulas I or II or may be ~-C-methyl-histidine or ~-C-methyl~tyrosine. GnRH analogs and other analogs of biologically active ligands containing ~-C-alkyl-amino acids are new. A particularly preferred embodiment ,, ~09?~/030~ P~'r/C~92/0021~1 2 I l ~ r) !; ~;

includes GnRH analogs in wh~ch at lea6t one o~
po~itions 4, 5, 6 and 7 contaln an ~-C-alkyl-amlno acid. Such modifications are advantageous for promoting and stabilizing ~urns occurring in thi~
region of the peptide backbone, and for lim~ting proteolysis of the peptide.

M~ny of the analogs of a b~ologically ~ctive ligand according to the pre~ent invention can be synthesized by the methods well-known to one skilled in the art. For example, compounds can be synthesized by the solid phase method by one or more str~tegies Xnown to one skilled in the nrt (J. Stewart and J. Young, Solid Phase Peptide Svnthesis, 2nd Ed. (1984), Pierce Chemical Co.). In one such example, chloromethylated polystyrene or benzhydrylamine resin can be used with N-t-butyloxycarbonyl protected amino acids and sidechain protection such as His(Tos), Ser(Bzl) and Arg(Tos). The peptide-resin bond may be cleaved and protecting groups removed by treatment with ~nhydrous HF, or the peptide-resin bond may be cleaved and a~inoalkylated with alkylamine and the protecting groups subse~ently removed with HF. The peptide may then be purified by reversed-phase HPLC.

Purification of peptides can be accomplished, for example, with the use of a Varian HPLC system equipped with a Vista 401 micro-processor controller.
Separations can be achieved, for example, on a Bio-Rad ~i-Pore 313 reverse-phase preparative column (25.0 x 2.15 cm) at 25C with a stepped linear gradient of acetonitrile in 0.1~ CF3CO~ at a flow rate of 7.5 ml/min. Automated repetitive injections of peptides (5 x 5 mg) can then be made from a nitrogen pressurized Rheodyne injector with a 2.0 ml sample loop. One-fifth ~";, :

93/03~ 3 , i` ~ l/<A92/~2~l of the total sa~ple may then be injected durlng each run by lowering the flow r~te to 4.0 ml/min for a O.l-min Hinject" period. One cycle could thus con~i~t of the following event6: o - lo min, 7.5 ml/min, 90%
H20/l0% of 1% ~queous CF3C02H; lo - 11 min, 4.0 ml/min;
11 - 11.1 ~in, "in~ectn; 11.1 - 13 min, 7.5 ml/mln, 70%
H20/20% CR3CN/lOS of 1% CF3C~; 13 - 30 m~n, 45% ~0/45%
CH3CN/10% of 1% CF3CO~; 30 - 42 min, so% ~HlCN/io% of 1%
CF3CO2H; 42-50 min, 100% H20.
In a novel solid phase peptide ~ynthes~s method according to the present invention, synthesis i~
carried out at room temperature, the resin is a novel chloro-(orthochloro)-trityl-resin to which the FMOC-lS protected, terminal amino acid is attached in the presence of diethylpropylamine (1.1 equivalents) in methylene chloride for 1 hour.

Traditionally, two solid phase peptide synthesis strategies using Boc-amino..acids and FMOC-amino acids, respectively, have been used. In the Bocstrategy, the peptide-resin bond i5 cleaved with the use of strong acid conditions,:such as anhydrous HF, ~uch that the protecting groups are simultaneously removed. In the FMOC-strategy, the peptide-resin i5 25 .- cleaved with the use of intermediate acid conditions, such as TF~, ~uch that most or all of the protecting : groups are simu Naneously removed. ~sing the novel (or~hochloro)trityl resin, the conditions for cleaving , the peptide-resin bond is so mild that even the most ¦ 30 acid-sensitive protecting groups commonly used in solid phase peptide synthesis remain attached to the peptide.
. Consequently, the ability to retain the acid-sensitive protecting groups has thus allowed the present inventors to synthesize new and useful analogs ` ~0 93/030s~s P(~/C~92/0028 1 biolo~ically active ligand~ such ~s GnRH and opiate peptides.

Using the (orthochloro)trltyl resln, ~tepwise synthesis of the peptide with FMOC-~mino acid6 (2.5 equivalents) is carried out wlth DCC/N~T
(2.5 equivalents) as coupling agent(s) 1n a ~ultable solvent such a~ DMF for 1-2 hour6, and the FMOC groups removed by treatment with 20~ piperidine in DMF for 10-30 minutes. Acid-sen6itive sidechain protecting group~ such as His(trityl), Ser(t-butyl) and Cys(t-butyl), and the like, remain intact when the peptide-resin bond is cleaved with trifluoromethanol/acetic acid/methylene chloride in a ratio of 7/1/2, re~pectively.

Additionally, a peptide ~mide may be prepared by treating the liberated peptide with meth~nolic HCl for 1-4 hour~ to yield the methyl ester followed by - aminolysis in methanolic ammonia for 4-24 hours.
Alternatively, treatment of the received peptide with ~onoalkylamine in the presence of a suitable coupling agent such as DCC/HBT in a suitable ~olvent such as DMF
for 1-4 hours affords the peptide N-alkylamide.
Removal of protecting groups may be accomplished with 50% TFA in chloroform for 30 min. When an acid-sensitive group is to be maintained, such asD-His( m ), the synthesis i~ carried out with ~limitedH
sidechain protection, for example, Pyr-His-Trp-Ser(Me)-Tyr(Me)-D-His( m )-Leu-Arg-Pro-resin, or alternatively by fragment condensation of Pyr-His-Trp-Ser(Me)-Tyr(Me)-D-Hi~(Trt)-LieuOH (prepared using the (orthochloro)trityl resin) to Arg-Pro-resin or Arg-Pro-NHEt using a suitable coupling agent such as DCC/HBT
and a suitable solvent such as DMF.

J

~.

W093/0~58 ~; PC~/CA~/~ ~l Synthesis of tSer(Me)~Tyr(Me)5DHis(Trt)~-ProNHEt9]-GnR~ i8 accomplished using FMOC-amino ~cid~
and (orthochloro)trityl resin by the protocol outllned above to give: Pyr-Nis-Trp-Ser(He)-Tyr(Me)-D-His(Trt)-Leu-Arg-Pro-re6in which i6 treated with F3COH/CH3C~N/CH2Cl, (7:1:2) for 1 hour ~nd filtered. The peptide i8 precipitated from the filtrate with ether, filtered, dis~olved in DMF and treated with NH~Et and DCC/HBT (2 equiv. of eaoh) for 2 hours. The desired product i5 precipitated with ether and puri~ied by reverse-pha6e HPLC.

The novel compounds produced according to the novel process described above are an~logs of GnAH, and other hor~onal peptides, in which an acid-sensitive protecting group is maintained at the end of the synthesi~ as ~ result of the use of an extremely labile peptide-resin bond provided by the (orthochloro)trityl-resin bond. Such analogs include, but are not limited to, compounds having the Formula III:

Xl-X2-X3-~-X5-~-X~-X~-X9-X~O III.

In Formula III, above, X6 is D-His having the protecting group trityl, tosyl, dansyl, dinitrophenyl, benzyloxymethyl or optionally substituted benzyl;
D-Ser/Thr having the protecting group trityl, t-butyl, acyl, optionally substituted benzyl or benzyloxycarbonyl; D-~ys having the protecting group trityl, t-butyl, tosyl, or optionally substituted benzyl; D-Tyr having the protecting group tosyl, trityl, acyl, t-butyl, optionally substituted benzyl or benzyloxycarbonyl; D-Arg having the protecting group Tos, Mtr or Pmc; D-Orn/Lys having the protecting group t-Boc, trityl or optionally substituted benzyloxycarbonyl; D-Asp/Glu having the protecting ~' ' '' ' . .

~ r~
l~:

~W~93/O~X ~ CI`/C~92/00~l group t-butyl, trityl or optionally 6ubstituted benzyl;
D-Asn/Gln having the protecting group trityl or xanthyl; D-Trp having the protecting group formyl or t-sOc; X~ is Pyr, N-acyl-amino acid, N-acyl-~-C-alkyl-amino acid, ~-C-alkyl-Pyr; X2, X3, ~, X7, X~are independently natural, synthetic, protected or ~-C-alkyl- amino acid6; ~ i~ imino Acid; X~0 is -N(H)R
wherein R i6 GlyNH2, azaGlyNH2, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, hnloalkyl, nryl, wherein each alkyl or alkenyl group has from 1-7 carbon atoms; and X5 is according to Formulas I or II, as described ~bove, with the proviso that the same compounds excluded from ~ormulas I and II are likewise excluded from the compounds ~ccording to Formula III.

Additionally, analogs of opiate peptides j containing ~he acid-sensitive groups listed above together with modification of Tyr or His according to Formulas I and II are included.

A particularly preferred set of compounds prepared by the novel process according to the present ~ invention are the compounds according to Formula III, r~ wherein ~ comprises an amino acid which contains an acid-sensitive sidechain protecting group. The amino - acids containing acid-sensitive protecting groups are a particularly preferred set of novel compounds which can be prepared by the novel synthesis method of the present invention as a result of the use of the ex*remely labile peptide-resin bond provided by the ~ (orthochloro~trityl-resin bond. Thus, the particularly i3, 30 preferred acid-sensitive protecting groups which are uniquely useful in the novel method described above are, for example, trityl, t-Boc, t-butyl, Mtr, Pmc or formyl. The terms "acid-sensitive" and "sidechain protecting group" are used in accordance with their art ~, ' :, ., ~ .
,......

W1~93/0305~ l`/(A92/~2~l recognized definition, to the extent not inconsietent with the principles of the present invention.

The novel method of the pre~ent invention i~
also particularly well suited for the preparation of S nnalogs of a biologic~lly active ligand having the Formula III, wherein X~ i8 Pyr (L or D), N-acyl-~ino - acid, ~-C-Alkyl-Pyr or N-acyl-~-C-alkyl-~mino ~c~d; X2, X3, X4, ~, X7 and X~ are independently n~tural, synthetic, protected or ~-C-alkyl- amino acid, or ~-X~
optionally contains a ~-lactam; ~ i5 imino acid or ~-C-alkyl-i~ino acid; X~0 is -N(H)R wherein R is GlyNH2, azaGlyNH2, alkyl, alkenyl, haloalkyl, hydroxyalkyl, cycloalkyl or aryl, wherein each alkyl, alkenyl or aryl group hac from 1 to 7 carbon atoms; and X5 iS selected according to Formulas I or II, as described above, with the proviso that the same compounds excluded from Formula~ I and II are likewise excluded from the compounds according to Formula III.

The invention will be illustrated in qreater detail by the fsllowing specific examples. It i8 understood that these example~-are given by way.of illustration and are not meant to limit the disclosure of the claims to follow.

- Example l Desensitization effects of anqiotensin antagonists in the rat isolated uterus assay:

Analog Recovery Time [Sar~ Iles]ANG II 180 min Sarl Tyr(0-CH3)4 Ile8]ANG II 20 min 30[Sar~ Phe(~'-F)4 Ile8]ANG II 20 min ~':
, :
~,,.j ~"~ 93/0305X ~ 2/002~ 1 Ti~sues were treated with the particular an~log (lo ~) for 2 min followed by washing out; tlme for recovery of the response to an ED50 dose of ANG II wa~ determined.
.

Example 2 Desensitization propertie~ of GnRH ngonist~
continuously ~uperfused in goldfish pituitaries in vitro.

Pe.ptide Response (na LH/ml~
5 min 10 min 20 min 40 min 60 min 10 GnRH (lO~M) 50 12 15 14 13 tTYr(Me)53GnRH 50 50 47 47 46 (5xlO~M) Methods were as described by Habibi, 1991 (Biology of Reproduction 44, 275-283)~

More specifically, the relative responsiveness of goldfish pituitary fragments to various salmon and mammalian GnRH snalogs were determined by using a superfusion system based on that described previously (Mackenzie et al., 1984; Chang et al., 1984; Marcha~t et al., 1387). Briefly, goldfish pituitaries were removed, pars distalis were sep~rated, and fragments were prepared (<0.5 mm2) and placed : between two l90~ layers of Cytodex carrier beads in a 300-~1 superfusion chamber (3 pituitary equivalents per chamber). The fragments were superfused (5 ml/h) overnight (10-12 h) with Medium 199 containing Hanks' basic salts (Gibco Laboratories, Grand Island, N.Y.) 25 mM 4-(1-hydroxyethyl)-1-piperazineethanesulfonic acid ~HEPES) and 56 U/ml nystatin. Two hours prior to the experiment, the medium was switched to Hanks' basic salts solution, supplemented with 25 mM HEPES buffer and 0.1% BSA (HBSS), and the flow rate was increased to 15 ml/h. Using a 3-way valve system, we exposed pituitary fragments to 2-min pulses of various ~,.j",. . .

~;.' , ' ' ~;
,:

!. . ' -~ WO 93/03058 PC'r/CA92/0028 1 251 ~r,f~;

concentrations of various GnRH analog~ (10~, 10~, And s x 105 M~ everv 60 min. All expQriments lnvolv~d .. running 4 or 8 c01umn8 ~i~ultaneously, with 5-min fractions collected automAtically; samples were ~rozen 5 at -25C until determination of GTH by a A radioimmunoa6say, as described previou~ly (Peter et al., 1984).

Example 3 Desensitization propertie6 of GnRH agonist~
10 aPter 2 ~in pulses every 20 min in superfused goldfish pituitaries n vitro according to the methods de~cribed ~! in Example 2.

Peptide ResDonse rna LH/ml) . Pulse ~1 ~2 ~4 ~7 GnRH (104M) 60 14 13 10 ~I tTyr~Me)5~GnRH 36 32 33 38 ;;~ (5xlO~M) ~, Exa~Dle 4 Release of LH during superfusion of goldfish pituitaries n vitro induced by tTyr(Me)s]GnRH in the absence (fiolid bar; S ~in) and presence (open bar; 3 min) of GnRH. Methods were as described by Habibi, ' 1991.
.~ _ .
' ,~ 25 .
~ _ lS .

¦ h lo --o~
r ~ 211 ~0 IID ~¦0 : ~ ` :

~.

W093/0305~ 'C'r/C'A~2/~ ~l Exam~le 5 Effect of subcut~neous in~Qct~on of l.S ~g/ln~ctlon o~
GnRH, [Tyr(Me)5]G~, or Gn~H+[Tyr(Me}~]GnRH on ~erum LH
levels in rats determ~ned by radiolmmuno~say LH (Units/S0 ~1 ~erum -Tlme fter ln~ectlon CnRB ~Tyr(l~c)~]CnRH CnRH~ITyr(~)~lCnRB
120 min 0.7 0 2.5 150 min 0.1 0 1.9 180 min 1.2 0 3.6 225 min - o 3.3 240 min - o 3,9 __ Rats were injected every 30 min with 1.5 ~g of GnRH, tTyr(Me)5~GnRH, or GnRH+tTyr(Me)5]GnRH. The values are corrected ~or basal levels (1.1 units/50 ~1) by subtraction.

It w:ill be noted that the analog of GnRH, tTyr(Me)5]GnRH~, administered alone was not effective due to the low dosaqe administered.

7: - - .
~ . , : .
.
.

Claims (81)

What is claimed is:

1. An analog of a biologically active ligand comprising at least one 6-membered ring replaced by Formula 1 or 5-membered ring replaced by Formula II:
I II
wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, -NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxycarbonyl;
R1 is an alkyl of 1-7 carbon atoms, alkenyl or alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R4, R6, R7 and R8 are the same or different and are selected from X or -OH;
.alpha. is P, N, S, or C being in either the L- or D-configuration and .beta. may be deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;
the nitrogen atom attached to the .alpha. atom may be substituted or unsubstituted C, S, O or P and the carbon atom attached to the .alpha. and the oxygen atoms may be substituted or unsubstituted N, S, O, P or C;
said aromatic ring of Formula I or said ring of Formula II may contain 0-4 N, S or O atoms;
said aromatic ring of Formula I or said ring of Formula II may be fused with a 6-membered ring which may also contain 0-4 N, S or O atoms and which may be substituted in the same manner as said aromatic ring of Formula I;
Formula I may be D-tyrosine and Formula II
may be D-histidine; and wherein R9 is:
(i) X or OH, wherein X is not H; or (ii) H, provided that the analog is not one of the following analogs based on mammalian GnRH:
(A) [D-Phe6]GnRH in which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2- bromobenzyloxycarbonyl;
(B) [Phe5]GnRH, [Ala]Phe5]GnRH and [Phe(N3)5]GnRH;
(C) [Des10Phe5-D-Ala6ProNHR9]GnRH in which position 2 is His, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH or CHMe2;
(D) [Des10X5Pro-B9]GnRH in which position 3 is Trp, Phe or 3(1-napthylalanine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCH[(CH2)aNHC(=NR2)R1]CO wherein: n is 1-5; R1 is alkyl or NRR3 wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl-morpholino or (CH2)a(R4)2 wherein n is 1-5 and R4 is alkyl; R2 is H or R3; or wherein C=N2R1 can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is GlyNH2, NHB2 wherein B2 is alkyl, cycloalkyl or haloalkyl, and NHCONHB3 wherein B3 is H or alkyl;
(E) analogs containing a .gamma.-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C3-7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; position 5 is aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is imino acid or aliphatic amino acid; position 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and (F) analogs in which position 5 is Phe, Tyr(Me), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is alkylated.

2. The analog of Claim 1, wherein R9 is R1.

3. The analog of Claim 2, wherein Formula I
comprises Tyr(alkyl), Phe(alkyl), or Phe(halide).

4. The analog of Claim 2, wherein Formula I
comprises .alpha.-C-alkyl-Tyr or Formula II comprises .alpha.-C-alkyl-His.

5. The analog of Claim 1, wherein said analog is selected from the group consisting of analogs of opiate peptides and analogs of Gonadotropin Releasing Hormone.

6. The analog of Claim 5, wherein the tyrosine residue of said analog of said opiate peptides or said analog of said Gonadotropin Releasing Hormone has been replaced by an analog according to Formula I.

7. The analog of Claim 1, wherein said analog is further combined with at least one normally desensitizing analog of Gonadotropin Releasing Hormone or opiate peptide or synthetic analogs thereof.

8. A composition comprising an analog according to Claim 6 combined with at least one normally desensitizing analog of Gonadotropin Releasing Hormone or synthetic analog thereof.

9. The analog of Claim 1, wherein said analog includes a histidine residue modified according to Formula II.

10. The analog of Claim 1, wherein said analog comprises a non-peptidic ligand.

11. A compound having the Formula III:

wherein:
X1 is Pyr, N-acyl-amino acid, .alpha.-C-alkyl-Pyr, or N-acyl-.alpha.-C-alkyl-amino acid;
X2, X3, X4, X6, X7 and X8 are independently selected from natural, synthetic, protected or .alpha.-C-alkyl- amino acids, wherein X6-X7 optionally contains a .gamma.-lactam;
X9 is an imino acid or .alpha.-C-alkyl-imino acid;
X10 is -N(H)R wherein R is GlyNH2, azaGlyNH2, alkyl or alkenyl, cycloalkyl, haloalkyl, hydroxyalkyl or aryl, wherein each alkyl, alkenyl or aryl group has from 1-7 carbon atoms; and X5 is selected from Formula I or II:
I II
wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, -NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxycarbonyl;
R1 is selected from alkyl of 1-7 carbon atoms, alkenyl or alkynyl, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R4, R6, R7 and R8 are the same or different and are selected from X or -OH;
.alpha. is selected from P, N, S, or C being in either the L- or D-configuration and .beta. is optionally deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;

the nitrogen atom attached to the .alpha. atom is optionally substituted or unsubstituted C, S, O or P
and the carbon atom attached to the .alpha. and the oxygen atoms is optionally substituted or unsubstituted N, S, O, P or C;
said aromatic ring of Formula I or said ring of Formula II optionally has 0-4 N, S or O atoms;
said aromatic ring of Formula I or said ring of Formula II is optionally fused with a 6-membered ring which optionally has 0-4 N, S or O atoms and which is optionally substituted in the same manner as said aromatic ring of Formula I;
Formula I is optionally D-tyrosine and Formula II is optionally D-histidine; and provided that when none of X1 - X9 is an .alpha.-C-alkyl- amino or imino acid, the compound is not:
(1) an antagonist analog of Gonadotropin Releasing Hormone obtained by substituting D-amino acids at positions 1, 2 or 3, or analogs which have position 2 deleted: or (2) one of the following analogs based on mammalian GnRH:
(A) [D-Phe6]GnRH in which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl;
(B) [Phe5]GnRH, [Ala4Phe5]GnRH and [Phe(N3)5]GnRH;
(C) [Des10Phe5-D-Ala6ProNHR9]GnRH in which position 2 is His, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH or CHMe2;
(D) [Des10X5Pro-B9]GnRH in which position 3 is Trp, Phe or 3(1-napthylalanine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCH[(CH2)aNHC(=NR2)R1]CO wherein: n is 1-5; R1 is alkyl or NRR3 wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl-morpholino or (CH2)a(R4)2 wherein n is 1-5 and R4 is alkyl; R2 is H or R3; or wherein C=N2R1 can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is GlyNH2, NHB2 wherein B2 is alkyl, cycloalkyl or haloalkyl, and NHCONHB3 wherein B3 is H or alkyl;
(E) analogs containing a .gamma.-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C3-7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; position 5 is aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is imino acid or aliphatic amino acid; position 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and (F) analogs in which position 5 is Phe, Tyr(Me), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is alkylated.

12. The compound of Claim 11, wherein at least one of said X1 - X9 of Formula III is an .alpha.-C-alkyl- amino or imino acid.

13. The compound of Claim 12, wherein X5 of Formula III is Tyr(Me) or Tyr(Et).

14. The compound of Claim 11, wherein X5 is .alpha.-C-alkyl-Tyr or .alpha.-C-alkyl-His.

15. The compound of Claim 11, wherein said compound includes a histidine residue modified according to Formula II.

16. The compound of Claim 11, wherein said compound is an antagonist and is further combined with testosterone.

17. A compound having the Formula III:

wherein:
X1 is Pyr, N-acyl-amino acid, .alpha.-C-alkyl-Pyr, or N-acyl-.alpha.-C-alkyl-amino acid;
X2 and X3 are aromatic L-amino acids;
X4, X7 and X8 are independently selected from natural, synthetic, protected or .alpha.-C-alkyl-amino acids X6 is a natural, synthetic, or protected D-amino acid, Gly or .alpha.-C-alkyl-amino acid, wherein X6 and X7 optionally contains a .gamma.-lactam;
X9 is an imino acid or a .alpha.-C-alkyl-imino acid;
X10 is -N(H)R wherein R is GlyNH2, azaGlyNH2, alkyl or alkenyl, cycloalkyl, haloalkyl, hydroxyalkyl or aryl, wherein each alkyl, alkenyl or aryl group has from 1-7 carbon atoms; and X5 is selected from Formula I or II:
I II

wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, -NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxycarbonyl;
R1 is alkyl of 1-7 carbon atoms, alkenyl or alkynyl, of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R4, R6, R7 and R8 are the same or different and are selected from X or -OH;
.alpha. is P, N, S, or C being in either the L- or D-configuration and .beta. is optionally deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;
the nitrogen atom attached to the .alpha. atom is optionally substituted or unsubstituted C, S, O or P
and the carbon atom attached to the .alpha. and the oxygen atoms is optionally substituted or unsubstituted N, S, O, P or C;
said aromatic ring of Formula I or said ring of Formula II optionally has 0-4 N, S or O atoms;
said aromatic ring of Formula I or said ring of Formula II is optionally fused with a 6-membered ring which optionally has 0-4 N, S or O atoms and which is optionally substituted in the same manner as said aromatic ring of Formula I;
Formula I is optionally D-tyrosine and Formula II is optionally D-histidine; and provided that when none of X1, or X4 - X9 is an .alpha.-C-alkyl- amino or imino acid, the compound is not:

(1) one of the following analogs based on mammalian GnRH:
(A) [D-Phe6]GnRH in which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl;
(B) [Phe5]GnRH, [Ala4Phe5]GnRH and [Phe(N3)5]GnRH;
(C) [Des10Phe5-D-Ala6ProNHR9]GnRH in which position 2 is His, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH or CHMe2;
(D) [Des10X5Pro-B9]GnRH in which position 3 is Trp, Phe or 3(1-napthylalanine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCH[(CH2)aNHC(=NR2)R1]CO wherein: n is 1-5; R1 is alkyl or NRR3 wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl-morpholino or (CH2)a(R4)2 wherein n is 1-5 and R4 is alkyl; R2 is H or R3; or wherein C=N2R1 can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is GlyNH2, NHB2 wherein B2 is alkyl, cycloalkyl or haloalkyl, and NHCONHB3 wherein B3 is H or alkyl;
(E) analogs containing a .gamma.-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C3-7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; position 5 is aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is imino acid or aliphatic amino acid; position 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and (F) analogs in which position 5 is Phe, Tyr(Me), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is alkylated.

18. The compound of Claim 17, wherein at least one of said X1 and X4 - X9 of Formula III is an .alpha.-C-alkyl-amino or imino acid.

19. The compound of Claim 18, wherein X5 of Formula III is Tyr(Me) or Tyr(Et).

20. The compound of Claim 17, wherein X5 of Formula III is Phe(halide), Phe(alkyl), .alpha.-C-alkyl-Phe or .alpha.-C-alkyl-Tyr(alkyl).

21. The compound of Claim 17, wherein X4 of Formula III is Ser(Me), Thr(Me), Aib, .alpha.-C-methyl-Ser(Me) or .alpha.-C-methyl-Thr(Me).

22. The compound of Claim 17, wherein X6 is .alpha.-C-alkyl-amino acid.

23. The compound of Claim 17, wherein X7 is .alpha.-C-alkyl-amino acid.

24. The compound of Claim 17, wherein X8 is .alpha.-C-alkyl-amino acid.

25. The compound of Claim 17, wherein X6 is a D-amino acid having a sidechain protecting group.

26. The compound of Claim 25, wherein said amino acid is His, Ser/Thr, Cys, Tyr, Arg, Lys, Orn/Lys, Asp/Glu, Asn/Gln or Trp.

27. The compound of Claim 25, wherein said protecting group of said amino acid of X6 is trityl, tosyl, dansyl, dinitrophenyl, benzyloxymethyl, t-butyl, t-Boc, acyl, Mtr, Pmc, xanthyl, formyl, or optionally substituted benzyl or benzyloxycarbonyl.

28. The compound of Claim 25, wherein X6 is D-His having the protecting group trityl, tosyl, dansyl, dinitrophenyl, benzyloxymethyl or optionally substituted benzyl; D-Ser/Thr having the protecting group trityl, t-butyl, acyl, optionally substituted benzyl or benzyloxycarbonyl; D-Cys having the protecting group trityl, t-butyl, tosyl, or optionally substituted benzyl; D-Tyr having the protecting group tosyl, trityl, acyl, t-butyl, optionally substituted benzyl or benzyloxycarbonyl; D-Arg having the protecting group Tos, Mtr or Pmc; D-Orn/Lys having the protecting group t-Boc, trityl or optionally substituted benzyloxycarbonyl; D-Asp/Glu having the protecting group t-butyl, trityl or optionally substituted benzyl; D-Asn/Gln having the protecting group trityl or xanthyl; D-Trp having the protecting group formyl or t-Boc.

29. The compound of Claim 25, wherein said protecting group is trityl, t-butyl, t-Boc, Mtr, Pmc or formyl.

30. The compound of Claim 17, wherein X9 is azetine-2-carboxyl.

31. The compound of Claim 17, wherein X9 is dehydroproline.

32. The compound of Claim 17, wherein X9 is nipecotyl.

33. A composition comprising an analog of Claim 17 in combination with at least one normally desensitizing analog of Gonadotropin Releasing Hormone or synthetic analog thereof.

34. A composition comprising an analog of Claim 29 in combination with at least one normally desensitizing analog of Gonadotropin Releasing Hormone or synthetic analog thereof.

35. A opiate peptide comprising an N-terminal sequence of the Formula IV:

wherein X2, X3, X4 and X5 are independently selected from natural, synthetic, protected and .alpha.-C-alkyl-amino acids wherein at least one of said X2 - X5 is an .alpha.-C-alkyl-amino acid;
X1 is an amino acid residue selected according to Formulas I or II:
I II
wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, -NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxycarbonyl;
R1 is an alkyl of 1-7 carbon atoms, an alkenyl or alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R4, R6, R7 and R8 are the same or different and are X or -OH;
.alpha. is P, N, S, or C being in either the L- or D-configuration and .beta. is optionally deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;
the nitrogen atom attached to the .alpha. atom is optionally substituted or unsubstituted C, S, O or P
and the carbon atom attached to the .alpha. and the oxygen atoms is optionally substituted or unsubstituted N, S, O, P or C;
said aromatic ring of Formula I or said ring of Formula II optionally has 0-4 N, S or O atoms;
said aromatic ring of Formula I or said ring of Formula II is optionally fused with a 6-membered ring which optionally has 0-4 N, S or O atoms and which is optionally substituted in the same manner as said aromatic ring of Formula I;
Formula I is optionally D-tyrosine and Formula II is optionally D-histidine.

36. The compound of Claim 35, wherein X1 of Formula IV is Tyr(Me) or Tyr(Et).

37. The compound of Claim 35, wherein X1 of Formula IV is Phe(halide), Phe(alkyl), .alpha.-C-alkyl-Phe or .alpha.-C-alkyl-Tyr(alkyl).

38. The compound of Claim 35, wherein at least one of X2 - X5 is an .alpha.-C-alkyl-amino acid.

39. The compound of Claim 36, wherein at least one of X2 - X5 is an amino acid having a sidechain protecting group.

40. The compound of Claim 39, wherein said amino acid is His, Ser/Thr, Cys, Tyr, Arg, Lys, Orn/Lys, Asp/Glu, Asn/Gln or Trp.

41. The compound of Claim 39, wherein said protecting group of said amino acid of X4 or X5 is trityl, tosyl, dansyl, dinitrophenyl, benzyloxymethyl, t-butyl, t-Boc, acyl, Mtr, Pmc, xanthyl, formyl, or optionally substituted benzyl or benzyloxycarbonyl.

42. The compound of Claim 39, wherein X4 or X5 is His having the protecting group trityl, tosyl, dansyl, dinitrophenyl, benzyloxymethyl or optionally substituted benzyl; Ser/Thr having the protecting group trityl, t-butyl, acyl, optionally substituted benzyl or benzyloxycarbonyl; Cys having the protecting group trityl, t-butyl, tosyl, or optionally substituted benzyl; Tyr having the protecting group tosyl, trityl, acyl, t-butyl, optionally substituted benzyl or benzyloxycarbonyl; Arg having the protecting group Tos, Mtr or Pmc; Lys having the protecting group t-Boc, trityl or optionally substituted benzyloxycarbonyl;
Orn/Lys having the protecting group t-Boc, trityl or optionally substituted benzyloxycarbonyl; Asp/Glu having the protecting group t-butyl, trityl or optionally substituted benzyl; Asn/Gln having the protecting group trityl or xanthyl; Trp having the protecting group formyl or t-Boc.

43. The compound of Claim 39, wherein said protecting group is trityl, t-butyl, t-Boc, Mtr, Pmc or formyl.

44. The compound of Claim 35, wherein said compound includes a histidine residue modified according to Formula II.

45. A composition comprising an analog according to Claim 35 in combination with at least one normally desensitizing compound of an opiate peptide or synthetic analog thereof.

46. A composition comprising an analog according to Claim 38 in combination with at least one normally desensitizing compound of an opiate peptide or synthetic analog thereof.

47. A method for synthesizing a peptide while retaining an acid-sensitive protecting group comprising employing a chloro-(orthochloro)trityl resin as the means for attaching the terminal amino acid thus allowing for the use of mild acid conditions for the cleavage of the peptide-resin bond.

48. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 1.

49. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 11.

50. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 17.

51. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 29.

52. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 35.

53. The method of claim 47, wherein the peptide synthesized by said method is according to Claim 43.

54. Method for selecting non-desensitizing analogs of a biologically active ligand containing at least one 6-membered ring or 5-membered ring, comprising selecting an analog of a biologically active ligand containing a 6-membered ring which has been replaced by the the Formula I or selecting a biologically active ligand containing a 5-membered ring which has been replaced by the Formula II:
I II
wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, -NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, -alkylphenyl, -O-phenyl, -O-alkylphenyl, -O-benzyloxycarbonyl;
R1 is an alkyl of 1-7 carbon atoms, an alkenyl or alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R4, R6, R7 and R8 are groups independently selected from X, alkyl or -OH
.alpha. is P, N, S, or C being in either the L- or D-configuration;
.beta. is optionally deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;
the nitrogen atom attached .alpha. is optionally substituted or unsubstituted C, S, O or P;
the aromatic ring of Formula I or the ring of Formula II optionally has 0-4 N, S or O atoms;
the aromatic ring of Formula I or ring of Formula II is optionally fused with a 6-membered ring which optionally has 0-4 N, S or O atoms which is optionally substituted in the same manner as the aromatic ring of Formula I;
the ring of Formula I is optionally D-tyrosine and or the ring of Formula II is optionally D-histidine; and wherein the analog is selected so that the resulting biologically active ligand is such that any subsequent interaction of said ligand with a biologically active receptor does not substantially attenuate said receptor.

55. The method of Claim 54, wherein R9 is:
(i) X or OH, wherein X is not H; or (ii) H, provided that the analog is not one of the following analogs based on mammalian GnRH:
(A) [D-Phe6]GnRH in which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2-bromobenzyloxycarbonyl;
(B) [Phe5]GnRH, [Ala4Phe5]GnRH and [Phe(N3)5]GnRH;
(C) [Des10Phe5-D-Ala6ProNHR9]GnRH in which position 2 is His, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH
or CHMe2;
(D) [Des10X5Pro-B9]GnRH in which position 3 is Trp, Phe or 3(1-napthylalanine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCH[(CH2)aNHC(=NR2)R1]CO wherein: n is 1-5; R1 is alkyl or NRR3 wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl-morpholino or (CH2)a(R4)2 wherein n is 1-5 and R4 is alkyl; R2 is H or R3; or wherein C=NR2R1 can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is selected from GlyNH2, NHB2 wherein B2 is alkyl, cycloalkyl or haloalkyl, and NHCONHB3 wherein B3 is H or alkyl;
(E) analogs containing a .gamma.-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C3-7 cycloalkyl)acyl;
positions 2 and 3 are aliphatic or aromatic amino acid;
position 4 is Ser, Thr or Ala; position 5 is aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is imino acid or aliphatic amino acid; position 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and (F) analogs in which position 5 is Phe, Tyr(Me), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is alkylated.

56. The method of Claim 54, wherein said analog of a biologically active ligand is selected from the group consisting of analogs of opiate peptides and analogs of Gonadotropin Releasing Hormone.

57. The method of Claim 56, wherein at least one of said analogs of Gonadotropin Releasing Hormone is used alone or in combination with at least one normally desensitizing Gonadotropin Releasing Hormone or synthetic analog thereof.

58. The method of Claim 57, futher comprising the use of domperidone in combination with said at least one analog of Gonadotropin Releasing Hormone.

59. The method of Claim 56, wherein at least one of said analogs of an opiate peptide is used alone or in combination with at least one normally desensitizing analog of an opiate ligand or synthetic analog thereof.

60. The method of Claim 56, wherein the tyrosine residue of said analog of said opiate peptides or said analog of said Gonadotropin Releasing Hormone has been selected according to Formula I.

61. The method of Claim 54, wherein lifetime fluorescence and/or NMR spectroscopy on said biologically active ligand in a suitable receptor-simulating environment i used to determine the non-desensitizing nature of said ligand.

62. The method of Claim 54, wherein said analog contains at least one 5-membered ring and is selected to contain a substituted or unsubstituted histidine residue modified according to Formula II.

63. The method of Claim 56, wherein the tyrosine of said Gonadotropin Releasing Hormone has been substituted with histidine modified according to Formula II and wherein said Gonadotropin Releasing Hormone is mammalian, salmon or chicken I Gonadotropin Releasing Hormone.

64. The method of Claim 54, wherein said analog of a biologically active ligand comprises a non-peptidic ligand.

65. The method of Claim 64, wherein said non-peptidic ligand is selected from the group consisting of substituted or unsubstituted steroids, substituted or unsubstituted catecholamines and substituted or unsubstituted nonpeptide opiate ligands.

66. The method of Claim 56, wherein said analog of said biologically active ligand is an antagonist and is used in combination with testosterone.

67. The method of Claim 57, wherein said analog of said biologically active ligand is an antagonist and is used in combination with testosterone.

68. Method of treating a patient in non-desensitizing fashion with a non-desensitizing analog of a biologically active ligand containing at least one 6-membered aromatic ring or 5-membered ring, comprising:
(A) selecting a non-desensitizing analog of a biologically active ligand containing a 6-membered ring which has been replaced by the Formula I or selecting an analog of a biologically active ligand containing a 5-membered ring which has been replaced by the Formula II:
I II
wherein:
X is selected from null, H, R1, -OR1, halide, -CN, -CHO, C(halide)3, -alk-OH, -alk-OR1, -alk-CO2H, -alk-CO2R1, -alk-SH, -alk-SR1, -alk-CONH2, -CO2H, -CO2R1, -COR1, -OCONH2, -OCH2OH, -OCH2OR1, -OCOR1, -N3, -N2, -NHCOR1, - NO2, -NH2, -NHR1, -NR12, -SO3H, -SO2R1, -SCOR1, -NCS, -SCSR1, -SO2NH2, -SO2NHR1, -SO2NR12, -SO4H, -PO3H, -PO4H2, -SH, -SR1, -N=N-R1, =O, =NH, =N-R1, =N-OH, =N-OR1, -CONH2, -CONHR1, -CONR12, -NHSO2R1, -SiR13, tetrazole, imidazole, or substituted or unsubstituted phenyl, - alkylphenyl, -O-phenyl, -O-alkylphenyl, -O- benzyloxycarbonyl;
R1 is an alkyl of 1-7 carbon atoms, an alkenyl or alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon atoms, optionally halogenated in place of one or more hydrogen;
R2, R3, R4, R5, R6, R7, R8 and R9 are X, alkyl or -OH;
.alpha. is P, N, S, or C being in either the L- or D-configuration;
.beta. is optionally deleted or extended by 1-2 carbon atoms, substituted or unsubstituted;
the nitrogen atom attached to .alpha. is optionally substituted or unsubstituted C, S, O or P;
the carbon atom attached to .alpha. and oxygen is optionally substituted or unsubstituted N, S, O, P or C;
the aromatic ring of Formula I or ring of Formula II optionally has 0-4 N, S or O atoms;
the aromatic ring of Formula I or ring of Formula II is optionally fused with a 6-membered ring which optionally has 0-4 N, S or O atoms which can be substituted in the same manner as the aromatic ring of Formula I;
the ring of Formula I is optionally D-tyrosine or the ring of Formula II is optionally D-histidine; and (B) administering to a patient said analog of a biologically active ligand selected in step (A) as a composition comprising:
(i) a pharmaceutically effective amount of said analog of a biologically active ligand selected in step (A); and (ii) a pharmaceutically acceptable carrier for said analog of a biologically active ligand.

69. The method of Claim 68, wherein R9 is:
(i) X or OH, wherein X is not H; or (ii) H, provided that the analog is not one of the following analogs based on mammalian GnRH:

(A) [D-Phe6]GnRH in which the hydroxyl group of tyrosine is protected with benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl, benzyloxycarbonyl or 2- bromobenzyloxycarbonyl;
(B) [Phe3]GnRH, [Ala4Phe5]GnRH and [Phe(N3)5]GnRH;
(C) [Des10Phe5-D-Ala6ProNHR9]GnRH in which position 2 is His, Tyr, Trp or Phe(4'-NH2), position 7 is Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH
or CHMe2;
(D) [Des10X5Pro-B9]GnRH in which position 3 is Trp, Phe or 3(1-napthylalanine); X is His, Phe or Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl, phenyl or cyclohexyl with 3 or more alkyl groups, perhydronaphthyl, adamantyl, perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl, heterocycle aryl, optionally N-alkylated alkylamine or cycloalkylamine, or NHCH[(CH2)?NHC(=NR2)R1]CO wherein: n is 1-5; R1 is alkyl or NRR3 wherein R is H or alkyl and R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl, alkyl- morpholino or (CH2)?(R4)2 wherein n is 1-5 and R4 is alkyl; R2 is H or R3; or wherein C=NR2R1 can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is Arg, Gln, Tyr or Leu; and B is GlyNH2, NHB2 wherein B2 is alkyl, cycloalkyl or haloalkyl, and NHCONHB3 wherein B3 is H or alkyl;
(E) analogs containing a .gamma.-lactam in the 6-7 position, in which position 1 is Pyr, N-acetyl, N-Pyr-imino acid, or (C3-7 cycloalkyl)acyl; positions 2 and 3 are aliphatic or aromatic amino acid; position 4 is Ser, Thr or Ala; position 5 is aromatic amino acid His, Trp or Phe; position 8 is an amino acid with a basic sidechain; position 9 is imino acid or aliphatic amino acid; position 10 is GlyNH2, AlaNH2, NHEt, NHPr, NHCH2CH2OH; and (F) analogs in which position 5 is Phe, Tyr(Me), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-Tyr(Et), wherein the nitrogen atom of at least one of the amide bonds is alkylated.

70. The method of Claim 68, wherein said analog of a biologically active ligand is selected from the group consisting of analogs of opiate peptides and analogs of Gonadotropin Releasing Hormone.

71. The method of Claim 70, wherein the tyrosine of said analog of said opiate peptides or said Gonadotropin Releasing Hormone has been selectively modified according to Formula I.

72. The method of Claim 71, wherein at least one of said analogs of Gonadotropin Releasing Hormone is used alone or in combination with at least one normally desensitizing Gonadotropin Releasing Hormone or synthetic analog thereof.

73. The method of Claim 72, futher comprising the use of domperidone in combination with said at least one analog of Gonadotropin Releasing Hormone.

74. The method of Claim 70, wherein at least one of said analogs of an opiate peptide is used alone or in combination with at least one normally desensitizing analog of an opiate ligand or synthetic analog thereof.

75. The method of Claim 68, wherein lifetime fluorescence and/or NMR spectroscopy on said biologically active ligand in a suitable receptor-simulating environment is used to determine the non-desensitizing nature of said ligand.

76. The method of Claim 68, wherein said analog containing at least one 5-membered ring is selected as a substituted or unsubstituted histidine residue selectively modified according to Formula II.

77. The method of Claim 68, wherein the tyrosine of said Gonadotropin Releasing Hormone has been substituted with histidine selectively modified according to Formula II and wherein said Gonadotropin Releasing Hormone is mammalian, salmon or chicken I
Gonadotropin Releasing Hormone.

78. The method of Claim 68, wherein said analog of a biologically active ligand comprises a non-peptidic ligand.

79. The method of Claim 78, wherein said non-peptidic ligand is selected from the group consisting of substituted or unsubstituted steroids, substituted or unsubstituted catecholamines and substituted or unsubstituted nonpeptide opiate ligands.

80. The method of Claim 70, wherein said analog of said biologically active ligand is an antagonist and is used in combination with testosterone.

81. The-method of Claim 71, wherein said analog of said biologically active ligand is an antagonist and is used in combination with testosterone.