Lipidated gonadotropin releasing hormone analogues

Abstract

Described are lipidated GnRH analogues, wherein the GnRH analogues are lipidated at one or more of amino acid positions, such as amino acid position 1, 5, 6 and / or 8 of a GnRH analogue peptide backbone. Also described are methods of making the lipidated compounds, pharmaceutical compositions comprising the lipidated compounds, and their use in methods of treatment.

Description

LIPID ATED GONADOTROPIN RELEASING HORMONE ANALOGUESFIELD

[0001] Described are lipidated forms of GnRH analogues. Also described are methods of making the lipidated compounds, pharmaceutical compositions comprising the lipidated compounds, and their use in methods of treatment.BACKGROUND

[0002] Gonadotropin releasing hormone (GnRH) is a decapeptide comprised of 10 amino acid residues that is produced in the hypothalamus and acts upon GnRH receptors in the pituitary gland. GnRH stimulates release of luteinizing hormone (LH) and follicular stimulating hormone (FSH) which in turn stimulate production and release of testosterone (by male testes) or estrogen (by female ovaries). Peptide analogues of GnRH have been identified and developed as GnRH agonists or GnRH antagonists in order to inhibit estrogen or androgen synthesis. GnRH agonists (such as leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, and deslorelin) and antagonists (such as degarelix and degarelix analogues) have primarily been developed for androgen deprivation therapy to treat prostate cancer, including advanced prostate cancer, but also have been used in the treatment of infertility (e.g., to promote ovarian stimulation in assisted reproduction technologies), endometriosis, uterine fibroids, and precocious puberty, and in combination with testosterone to induce azoospermia. Currently available products are formulated for parenteral administration by subcutaneous or intramuscular injection.

[0003] There remains a need for alternative forms of GnRH analogues, such as alternative forms of GnRH analogues that may offer one or more advantages with regard to physical stability, solubility, and pharmacokinetic parameters such as circulating half-life.SUMMARY

[0004] Provided herein are lipidated gonadotropin releasing hormone (GnRH) analogues that comprise a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone, such as atone or more of amino acid positions 1 (AA1), 5 (AA5), 6 (AA6), and 8 (AA8) of the peptide backbone, including particularly at position 6 (AA6).

[0005] In accordance with some aspects, the lipidated GnRH analogue exhibits GnRH antagonist activity. In accordance with some aspects, the GnRH analogue peptide backbone has the amino acid sequence of a GnRH antagonist selected from degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, or an amino acid sequence having one, two, three, four, five, six, or seven, or more, amino acid substitutions relative thereto.

[0006] In accordance with some aspects, lipidated GnRH analogue exhibits GnRH agonist activity. In accordance with some aspects, the GnRH or GnRH analogue peptide backbone has the amino acid sequence of a GnRH agonist selected from GnRH, leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, and deslorelin or an amino acid sequence having one, two, three, four, five, six, or seven, or more, amino acid substitutions relative thereto.

[0007] In accordance with some aspects, the lipidated GnRH analogue comprises one or more amino acid substitutions selected from:(i) an amino acid substitution that introduces an amino acid amenable to coupling with a lipid side chain, optionally at one or more of AA5, AA6, and AA8;(ii) an amino acid substitution that introduces an aromatic amino acid at one or more of AA5 and AA6;(iii) an amino acid substitution that increases hydrophilicity of the GnRH analogue; and / or(iv) an amino acid substitution that introduces Lys(iPr) at AA8.

[0008] In accordance with some aspects, the lipidated GnRH analogue comprises a peptide backbone (optionally a decapeptide backbone) that has any of the following amino acids at AA1- AA10:

[0009] In accordance with some aspects, the lipidated GnRH analogue comprises a peptide backbone (optionally a decapeptide backbone) that has any of the following amino acids at AA1- AA10:

[0010] In accordance with some aspects, the lipidated GnRH analogue comprises a peptide backbone (optionally a decapeptide backbone) that has any of the following amino acids at AA1- AA10, wherein the peptide backbone is lipidated at position 6 (AA6):

[0011] In accordance with some aspects of any other aspects described herein, the GnRH analogue peptide backbone is not lipidated at amino acid position 4 (AA4).

[0012] In accordance with some aspects of any other aspects described herein, the lipid side chain is coupled to amino acid position 6 (AA6) of the peptide backbone. In accordance with some aspects of any other aspects described herein, the lipid side chain is coupled to amino acid position 1 (AA1) of the peptide backbone. In accordance with some aspects of any other aspects described herein, the lipid side chain is coupled to amino acid position 5 (AA5) of the peptide backbone. In accordance with some aspects of any other aspects described herein, the lipid side chain is coupled to amino acid position 8 (AA8) of the peptide backbone.

[0013] In accordance with any aspects, the lipid side chain may comprise a lipid moiety, and optionally may further comprise one or both of a linker and a spacer. In accordance with some aspects of any other aspects described herein, the lipid side chain comprises a lipid moiety, a linker, and a spacer.

[0014] In accordance with some aspects, the lipid side chain may comprise a lipid moiety comprising a C8-C20 fatty acid, including a C14-C20 fatty acid. The lipid side chain may comprise a saturated C8-C20 fatty (di)acid; a saturated C8-C20 fatty (mono)acid; a saturated C8-C20 fatty acid tetrazole, or a saturated C8-C20 fatty acid sulfonic acid.

[0015] In accordance with some aspects, the linker may comprise one or more selected from (gGlu), (gGlu-gGlu), (aGlu), andwherein ** denotes the point of attachment to the spacer or to the peptide backbone if the lipid side chain does not include a spacer and *** denotes point of attachment to the fatty acid.

[0016] In accordance with some aspects, the spacer may comprise one or more selected from the following, wherein * denotes point of attachment to the peptide backbone and ** denotes point of attachment to the remainder of the lipid side chain, optionally wherein the lipid side chain comprises a linker and ** denotes point of attachment to the linker:

[0017] In accordance with specific aspects, the lipidated GnRH analogue comprises a lipid side chain comprising a lipid moiety, linker and spacer selected from Series A-Ab, wherein in each series the lipid moiety may be a saturated C8-20 fatty (di)acid:

[0018] In accordance with specific aspects, the lipidated GnRH analogue is selected from Compounds 1-561, optionally wherein the lipidated GnRH analogue is selected from LiGA5, L1GA15, L1GA21, L1GA22, L1GA23, L1GA27, L1GA28, L1GA29, L1GA31, L1GA35, L1GA36,L1GA43, L1GA44, L1GA45, L1GA54, L1GA55, L1GA6O, L1GA6I, L1GA62, L1GA69, L1GA70,L1GA72, L1GA74, L1GA86, L1GA91, L1GA92, L1GA93, L1GA94, L1GA96, L1GA97, L1GA98,L1GA99, LiGAlOO, L1GA102, L1GA103, LiGAl lO, LiGAl l l, L1GA112, L1GA113, L1GA114, L1GA115, L1GA119, L1GA124, L1GA125, L1GA129, L1GA135, L1GA151, L1GA152, L1GA153, L1GA157, L1GA158, L1GAI6I, L1GA162, L1GA164, L1GA165, L1GAI66, L1GAI68, L1GA169, L1GA171, L1GA172, L1GA175, L1GA176, L1GA177, L1GA178, and L1GA182, further optionally wherein the lipidated GnRH analogue is selected from LiGA5, LiGA28, LiGA43, LiGA151, and L1GA152.

[0019] In accordance with some aspects of any other aspects described herein, the lipidated GnRH analogue may exhibit a modified circulating half-life in vivo as compared to its non-lipidated counterpart, such as an extended circulating half-life in vivo as compared to its non-lipidated counterpart.

[0020] Also provided herein are pharmaceutical compositions comprising any of the lipidated GnRH analogues described herein and a pharmaceutically acceptable excipient. In accordance with some aspects, the composition is in a form selected from (i) a liquid pharmaceutical composition formulated for parenteral administration and (ii) a freeze-dried pharmaceutical composition formulated for reconstitution for parenteral administration. In accordance with some aspects, the composition is a liquid composition and the lipidated GnRH analogue exhibits one or both of a lower aggregation potential and reduced propensity for fibrillation in the composition than degarelix.

[0021] Also provided herein are processes for preparing any of the lipidated GnRH analogues described herein, comprising coupling a lipid side chain as described herein to an unprotectedamino group of an amino acid of a peptide backbone as described herein or an amino acid side chain thereof at one or more of amino acid positions 6 (AA6), 5 (AA5), 1 (AA1), and 8 (AA8) of the peptide backbone.

[0022] In accordance with some aspects, the process comprises:(i) providing the peptide backbone on a solid support;(ii) providing a solution comprising the lipid side chain, wherein the lipid side chain is protected with a protecting group;(iii) coupling the protected lipid side chain to the peptide backbone on the solid support, such that a carboxylic acid group of the protected lipid side chain forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof; and(iii) releasing the lipidated GnRH analogue from the solid support.

[0023] In accordance with some aspects, the lipid side chain comprises a spacer, linker, and lipid moiety, and the process further comprises preparing protected lipid side chain on a solid support by a process comprising:(i) providing solutions of the spacer, linker and lipid moiety, respectively;(ii) coupling the spacer to a solid support such that a carboxylic acid group of the spacer forms an ester bond with the solid support (spacer — support);(iii) coupling the linker to the spacer — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer on the solid support (linker — spacer — support);(iv) coupling the lipid moiety to the linker — spacer — support such that a carboxylic acid group of the lipid forms an amide bond with an amino group of the linker on the solid support (lipid — linker — spacer — support); and(v) releasing the protected lipid side chain from the solid support.

[0024] In accordance with some aspects, the lipid side chain comprises a spacer, linker, and lipid moiety, and the process comprises:(i) providing the peptide backbone on a solid support;(ii) providing solutions of the spacer, linker and lipid moiety, respectively;(iii) coupling the spacer to the solid support carrying the peptide backbone such that a carboxylic acid group of the spacer forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof (spacer — peptide — support);(iv) coupling the linker to the spacer — peptide — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer (linker — spacer — peptide — support);(v) coupling the lipid moiety to the linker — spacer — peptide — support, such that a carboxylic acid group of the lipid moiety forms an amide bond with an amino group of the linker (lipid — linker — spacer — peptide — support); and(vi) releasing the lipidated GnRH analogue from the solid support.

[0025] In accordance with some aspects of any other aspects of the processes described herein, the unprotected amino group comprises an a-amino group at position AA1 of the peptide backbone of the GnRH analogue and / or an s-amino group of a lysine residue at position AA5 or AA6 or AA8 of the peptide backbone of the GnRH analogue or an amino acid side chain thereof.

[0026] Also provided herein are methods of treating a condition amenable to treatment by a GnRH analog, comprising administering any of the lipidated gonadotropin releasing hormone (GnRH) analogues described herein to a subject in need thereof. Thus, in accordance with some aspects, the lipidated gonadotropin releasing hormone (GnRH) analogues described herein are for treating a condition amenable to treatment by a GnRH analog. Also provided herein are uses of a lipidated gonadotropin releasing hormone (GnRH) analogue as described herein in the preparation of a medicament for treating a condition amenable to treatment by a GnRH analog.

[0027] In accordance with some aspects, the condition is one or more of prostate cancer, advanced prostatic carcinoma (urologic oncology), ovarian cancer, breast cancer, endometriosis, myoma, infertility, menorrhagia, premenstrual dysphoric disorder (PMDD), severe PMS, benign prostatichyperplasia, uterine fibroids, central precocious puberty, or the use is as a puberty blocker such as for transgender or gender diverse youth, or for chemical castration. In accordance with some aspects, the condition is advanced prostate cancer or metastatic stage prostate cancer, further optionally wherein the condition is advanced hormone-dependent prostate cancer in adult males, further optionally wherein the advanced hormone-dependent prostate cancer is high-risk localized or locally advanced hormone-dependent prostate cancer. In accordance with some aspects, the treatment or use is in combination with radiotherapy. In accordance with some aspects, the lipidated GnRH analogue is used as neo-adjuvant treatment prior to radiotherapy. In some accordance with some aspects, the condition is infertility, and the treatment is for promotion of controlled ovarian stimulation in an assisted reproduction protocol. In accordance with other aspects, the lipidated GnRH analogues described herein are used in a veterinary context, such as for hormone suppression (e.g. chemical castration / neutralization) of pets or livestock, or in an in vitro fertilization protocol for pets or livestock.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows the amino acid sequences of GnRH and select GnRH agonists and GnRH antagonists. This figure is modified from Millar et al., Endocr Rev 2004, 25:235-275.

[0029] FIG. 2 shows the theoretical net charge of a series A LiGA compound as described herein and degarelix, over pH 0-12. The magnitude of the absolute charge is associated with solubility of the compounds; the width of the net charge interval for the LiGA compounds across the pH range may be advantageous in a formulation context, e.g., providing more formulation options. The net negative charge of the LiGA compounds may be associated with less discomfort upon injection.

[0030] FIG. 3 shows results of a ThT fibrillation assay on Varioskan microplate reader, demonstrating that LiGAl, LiGA5 and LiGA6 exhibited less propensity for fibrillation than degarelix.

[0031] FIG. 4 shows results of a ThT fibrillation assay onBioTek Synergy Neo2 microplate reader demonstrating that LiGAl, LiGA8, LiGA9, and Li GAI 0 exhibit less propensity for fibrillation than degarelix.

[0032] FIG. 5 shows results of a ThT fibrillation assay BioTek Synergy Neo2 microplate reader demonstrating that LiGA20, LiGAl 5 and LiGAl 7 exhibited more propensity for fibrillation than degarelix.

[0033] FIG. 6A, FIG. 6B, and FIG. 6C illustrate the chemical stability of LiGAl -LiGA6 formulated at 0.1 mM stored at 40°C over 8 weeks. FIG. 6A reports results for LiGAl -LiGA6 at pH 8.5; FIG. 6B and FIG. 6C report for LiGA6 formulated at pH 5 - pH 8.5, as compared to degarelix formulated at pH 5.0.

[0034] FIG. 7A and FIG. 7B show results of an hGnRH receptor affinity assay.

[0035] FIG. 8A and FIG. 8B show results of a pharmacokinetic study conducted in castrated rats subcutaneously injected with LiGAl -LiGA6 as compared to ganirelix, as reported in Example 2.

[0036] FIG. 8C shows the testosterone lowering effect of LiGAl, LiGA5, and LiGA6 as compared to ganirelix in intact male Sprague Dawley rats up to 24 hours following subcutaneous injection of doses varying from 0.0025 to 2.5 mg per rat (n=5).

[0037] FIG. 9 sets forth a chromatogram for LiGAl 22 after 2 weeks incubation at 40°C.

[0038] FIG. 10 shows results of a pharmacokinetic study conducted in castrated rats subcutaneously injected with LiGA5, LiGA28, LiGA43, LiGA151, and LiGA152, as reported in Example 4.

[0039] FIG. 11 shows the testosterone lowering effect of LiGA5, LiGA28, LiGA43, LiGA151, and LiGAl 52, as compared to PBS in intact male Sprague Dawley rats up to 48 hours following subcutaneous injection, as reported in Example 4.DETAILED DESCRIPTION

[0040] Described are lipidated GnRH analogues, comprising a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone. As used herein, a “GnRH analogue peptide backbone” includes peptide backbones based a GnRH antagonist (e.g., based on degarelix, abarelix, cetrorelix, ganirelix, acyline, teverelix, Nal-Glu, orntide, antide, or another degarelix analogue or another decapeptide (as “decapeptide” is defined herein below) exhibiting GnRH antagonist activity) and peptide backbones based on a GnRH agonist (e.g., based on leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, or deslorelin or another decapeptide (as “decapeptide” is defined herein below) exhibiting GnRH agonist activity). Also described are methods of making the lipidated compounds, pharmaceutical compositions comprising the lipidated compounds, and their use in methods of treatment. The lipidated GnRH analogues described herein may be referred to herein as LiGAs, for “lipidated gonadotropin-releasing hormone analogues.”1. Definitions

[0041] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined.

[0042] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0043] As used herein, the term “about” when qualifying a number or range means that the number or range is not limited to the exact number or range set forth, but encompass values around the stated number or range as will be understood by persons of ordinary skill in the art depending on the context in which the number or range is used. When not otherwise apparent from the context or convention in the art, “about” means up to plus or minus 10% of the particular term (± 10%).

[0044] The terms “administer,” “administration,” or “administering” as used herein refer to providing, giving, dosing and / or prescribing, such as by either a health professional or his or herauthorized agent or under his or her direction, and putting into, taking or consuming, such as by a health professional or the subject or patient.

[0045] The terms “subject” and “patient” as used herein refer to any mammal, including, but not limited to, humans, pets and laboratory animals (e.g., dogs, cats, rodents, rabbits, guinea pigs, primates, etc.}, and farm animals and livestock (e.g., horses, camels, donkeys, cattle, sheep, pigs, goats, etc. .

[0046] As used herein, the phrase “therapeutically effective amount” refers to a dose that provides or has been determined to provide the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, such as testosterone suppression or treatment of endometriosis. However, a “therapeutically effective amount” may not always be effective in treating the condition in a given subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. Exemplary doses and therapeutically effective amounts are provided below with reference to adult human subjects. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject and / or condition.2. Abbreviations

[0047] The following table explains various abbreviations used herein. Other abbreviations used herein have their ordinary meaning in the field.3. LiGA Peptide Backbones

[0048] As noted above, described herein are lipidated GnRH analogues (also referred to herein as LiGAs). The lipidated GnRH analogues described herein comprise a lipid side chain coupled to a GnRH or GnRH analogue peptide backbone (as defined herein) at one or more amino acid positions, such as at one or more of amino acid positions 6 (AA6), 1 (AA1), 5 (AA5) and / or 8 (AA8) of the peptide backbone (e.g., a decapeptide backbone as described herein), for example. The lipidated compounds described herein have a peptide backbone based on GnRH or a GnRH analogue, such as a peptide backbone based on a GnRH antagonist (such as degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, antide, or another degarelix analogue, or another decapeptide (as “decapeptide” is defined herein below) exhibiting GnRH antagonist activity) or a peptide backbone based on a GnRH agonist (such as leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, or deslorelin, or another decapeptide (as “decapeptide” is defined herein below) exhibiting GnRH agonist activity). Non-limiting examples of GnRH analogue peptide backbones that can be lipidated as described herein are described in this section.GnRH-Based Peptide Backbones

[0049] Human GnRH is a decapeptide having the following amino acid sequence where pGlu is pyroglutamate: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

[0050] Without being bound by theory it is believed that amino acids 1-3 play a role in receptor binding and activation, while amino acids 8-10 play a role in receptor binding (only). Additionally, it has been found that substitutions at amino acid position 6 (Gly) can enhance activity, as seen with the known GnRH agonists described below.

[0051] FIG. 1 (modified from Millar et al., Endocr Rev 2004, 25:235-275) aligns amino acid sequence of GnRH and certain GnRH agonists and GnRH antagonists.

[0052] For convenience, in the discussion that follows, the 10 amino acids of GnRH and the corresponding amino acids of GnRH analogues and the LiGA peptide backbones disclosed herein are referred to as AA1-AA10.

[0053] In some embodiments, the peptide backbone of a LiGA as described herein is identical to the amino acid sequence of GnRH, e.g., consists of AA1-AA10 of GnRH. In some embodiments, the peptide backbone of a LiGA as described herein is a GnRH analogue peptide backbone that has one or more amino acid substitutions relative to the amino acid sequence of GnRH, optionally wherein the peptide backbone is a decapeptide having one or more amino acid substitutions relative to GnRH. Typically, a substitution introduces a D-amino acid at AA6 which may enhance activity relative to GnRH.

[0054] Additionally or alternatively, one or more of the following types of amino acid substitutions may be introduced in the peptide backbone:• An amino acid substitution that introduces an amino acid amenable to coupling with a lipid side chain (as described herein), such as any suitable natural amino acid (e.g., lysine or cysteine), or any suitable unnatural amino acid (e.g., an unnatural amino acid having a suitably reactive side chain, such as s-azidolysine or propargylglycine). Such substitutions may be introduced at one or more of AA5, AA6 and AA8, for example.• An amino acid substitution that introduces an aromatic natural or unnatural amino acid (e.g., tyrosine, tyrosine derivatives, p-ureido-phenylalanine, etc.) at one or more of AA5 and AA6.• An amino acid substitution that increases the hydrophilicity of the lipidated compound, such as introduction of Tyr or Hyp (e.g., trans-Hyp).• An amino acid substitution that replaces Arg at AA8 with (Ns-Isopropyl)Lys (also referred to as Lys(iPr)) to reduce histamine release.• An amino acid substitution that replaces an amino acid present in one GnRH analogue with an amino acid present at the corresponding position of another GnRH analogue.

[0055] In specific embodiments of these embodiments, in the LiGA peptide backbone, AA4 is Ser. In further specific embodiments, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in GnRH). In further specific embodiments, AA2 is D-Cpa, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in GnRH).

[0056] As used herein, an amino acid to which a lipid side chain is coupled is considered to be “the same” as (or “conserved” relative to) the amino acid of a reference peptide. For example, a LiGA with a lipid side chain coupled to the Glu residue at AA1 of GnRH is considered to have “the same” amino acid at AA1, notwithstanding the presence of the lipid side chain. In contrast, a LiGA with a lipid side chain conjugated to a Lys residue at AA5 or AA6 would not be considered to have “the same” amino acid as AA5 or AA6 of GnRH; rather, the Lys residue would represent an amino acid substitution relative to the GnRH sequence.GnRH Agonist-Based Peptide Backbones

[0057] As noted above, a LiGA as described herein may have a GnRH analogue peptide backbone based on the amino acid sequence of a GnRH agonist and, in some embodiments, may exhibit GnRH agonist activity.

[0058] Many of the known GnRH agonists differ from GnRH at AA6 or at AA6 and AA10, as illustrated below with reference to leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, and deslorelin.GnRH (Gonadorelin) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2Leuprolide: pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEtGoserelin: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-AzaGly-NH2Triptorelin: pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2Nafarelin: pGlu-His-Trp-Ser-Tyr-D-2Nal-Leu-Arg-Pro-Gly-NH2Buserelin: pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEtHistrelin: pGlu-His-Trp-Ser-Tyr-D-His(Bzl)-Leu-Arg-Pro-NHEtF ertirelin : pGlu-His -Trp- S er-Tyr-Gly-Leu- Arg-Pr o-NHEtDeslorelin: pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEtFor the GnRH agonists comprised of 9 amino acids (e.g., leuprolide, buserelin, histrelin, fertirelin, and deslorelin), the terminal NHEt group is treated as AA10 in the present disclosure; such peptides and other GnRH analogue peptides having such a terminal NHEt group are included in the term “decapeptide” as used herein.

[0059] Like these GnRH agonists, in some embodiments, the GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that comprises one or more of the following changes relative to GnRH (with the stereochemistry of the amino acid conserved relative to GnRH unless specified): AA6: D-Leu; D-Ser(tBu), D-Trp, D-2Nal, or D-His(Bzl); AA10: AzaGly or is NHEt (e.g., wherein a terminal NHEt is treated as AA10, as in leuprolide, busrelin, histrelin, fertirelin, and deslorelin). Typically, the amino acid of AA6 is a D-amino acid which may enhance activity relative to GnRH. In further specific embodiments of LiGA peptide backbones, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in GnRH). In further specific embodiments, at least AA1-AA4 and AA7-AA9 or AA1-AA5 and AA7-AA9 are the same as in GnRH.

[0060] In some embodiments, the GnRH analogue peptide backbone of a LiGA as described herein is identical to the amino acid sequence of leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, or deslorelin, e.g., is a decapeptide that consists of AA1-AA10 of leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, or deslorelin. In some embodiments, the GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that comprises one or more amino acid substitutions relative to theamino acid sequence of leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, or deslorelin, such as one, two, three, four, five, six, or seven, or more amino acid substitutions. For example, one or more of the following types of amino acid substitutions may be introduced in the peptide backbone:• An amino acid substitution that introduces an amino acid amenable to coupling with a lipid side chain (as described herein), such as any suitable natural amino acid (e.g., lysine or cysteine), or any suitable unnatural amino acid (e.g., an unnatural amino acid having a suitably reactive side chain, such as s-azidolysine or propargylglycine). Such substitutions may be introduced at one or more of AA5 and AA6, for example. Optionally, in some such embodiments, at least AA1-AA4 and AA7-AA9 or AA1-AA5 and AA7-AA9 are the same as in GnRH.• An amino acid substitution that introduces an aromatic natural or unnatural amino acid (e.g., tyrosine, tyrosine derivatives, p-ureido-phenylalanine, etc.) at one or more of AA5 and AA6. Optionally, in some such embodiments, at least AA1-AA4 and AA7-AA9 are the same as in GnRH.• An amino acid substitution that increases the hydrophilicity of the lipidated compound, such as introduction of Tyr or Hyp (e.g., trans-Hyp). Such substitutions may be introduced at one or more of AA5 and AA6, for example. Optionally, in some such embodiments, at least AA1-AA3 and AA7-AA9 or AA1-AA4 and AA7-AA9 of the LiGA peptide backbone are the same as in GnRH.• An amino acid substitution that replaces Arg at AA8 with Lys(iPr) to reduce histamine release. Optionally, in some such embodiments, at least AA1-AA4, AA7, and AA9 or AA1-AA5, AA7, and AA9 of the LiGA peptide backbone are the same as in GnRH.• An amino acid substitution that replaces an amino acid present in one GnRH agonist with an amino acid present at the corresponding position of another GnRH agonist. Withoutbeing bound by theory, it is contemplated that such substitutions will not undermine GnRH agonist activity.

[0061] As noted above, a LiGA as described herein having a peptide backbone based on a GnRH agonist (e.g., as described in this section) may exhibit GnRH agonist activity.Degarelix and Degarelix Analogue-Based Peptide Backbones

[0062] As noted above, a LiGA as described herein may have a GnRH analogue peptide backbone based on the amino acid sequence of degarelix or a degarelix analogue and, in some embodiments, may exhibit GnRH antagonist activity. Degarelix is a decapeptide that contains seven unnatural amino acids, five of which are D-amino acids. It has ten chiral centers in the backbone of the decapeptide and an additional chiral center in the side chain of AA5, for a total of eleven chiral centers. It also can be referred to as:Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2For convenience, each of the 10 amino acids (AA1-AA10) of degarelix is given a shorthand notation as follows: AA1: D-2Nal; AA2: D-4Cpa; AA3: D-3Pal; AA4: Ser; AA5: 4Aph(L-Hor); AA6: D-Aph(Cbm); AA7: Leu; AA8: Lys(iPr); AA9: Pro; AA10: D-Ala.

[0063] The chemical structure of degarelix is set forth below.

[0064] Alternatively, a LiGA as described herein may have a GnRH analogue peptide backbone based on a degarelix analogue such as abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, antide, or another degarelix analogue.Abarelix: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-N(Me)Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2Cetrorelix: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2Ganirelix: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Tyr-D-hArg(Et2)-Leu-hArg(Et2)-Pro-D-Ala-NH2Prazarelix: Ac-D-2Nal-D-Cpa-D-3-Pal-Ser-4-(5-AT)Phe-D-4-(5-AT)Phe-Leu-Lys(iPr)-Pro- D-Ala-NH2Acyline: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Aph(Ac)-D-Aph(Ac)-Leu-Lys(iPr)-Pro-D-Ala-NH2Teverelix: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Tyr-D-Hci-Leu-Lys(iPr)-Pro-D-Ala-NH2Nal-Glu: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Arg-D-Glu(AA)-Leu-Arg-Pro-D-Ala-NH2Orntide: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-PicLys-D-(6Anic)Orn-Leu-Lys(iPr)-Pro-D-Ala-NH2Antide: Ac-D-2Nal-D-Cpa-D-3Pal-Ser-Lys(Nic) -D-Lys(Nic)-Leu-Lys(iPr)-Pro-D-Ala-NH2

[0065] In the description of peptide backbones herein, to the extent a peptide backbone is presented as an amide (with a C-terminal -NH2 moiety), it should be understood that a corresponding peptide backbone that is an acid (with a C-terminal -OH moiety) also is contemplated and described. Further, to the extent a compound is presented as acetylated (having an N-terminal acetyl moiety (Ac)), it should be understood that a corresponding “free” compound lacking such a moiety also is contemplated and described.

[0066] In some embodiments, the GnRH analogue peptide backbone has substitutions relative to degarelix or a degarelix analogue such as abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, at one of the following sets of positions:AA6;AA5 and AA6;AA6 and AA8;AA5, AA6, and AA10;AA3, AA5, and AA6;AA1, AA3, AA5, and AA6;AA5, AA6, and AA7;AA5, AA6, and AA8;AA5, AA6, and AA9;AA3, AA5, and AA9;AA3, AA5, AA6, and AA10;AA5, AA6, AA7, and AA10;AA5, AA6, AA9 and AA10;AA5, AA6, AA7, AA8 and AA10;AA3, AA5, AA6, AA9 and AA10;AA1, AA3, AA5, AA6, and AA8;AA1, AA3, AA5, AA6, and AA10;AA1, AA3, AA5, AA6, AA7 and AA10; orAA1, AA3, AA5, AA6, AA7, AA8 and AA10, optionally wherein the substitutions are relative to degarelix.

[0067] In some embodiments, a GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that has any of the following amino acids at AA1-AA10. The first amino acid listed for each position is that of degarelix, which may be present, although it should be understood that the LiGA may have a peptide backbone based on any GnRH analogue as discussed above.

[0068] A GnRH analogue peptide background in which AA10 is absent (e.g., “des Ala”) is encompassed by the term “decapeptide” as used herein.

[0069] In some embodiments, a GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that has any of the following amino acids at AA1-AA10.The first amino acid listed for each position is that of degarelix, which may be present, although it should be understood that the LiGA may have a peptide backbone based on any GnRH analogue as discussed above.

[0070] In some embodiments, a GnRH analogue peptide backbone of a LiGA as described herein has substitutions relative to degarelix selected from the following sets (Lys*denotes point of attachment of lipid side chain):AA6: D-Lys*AA5 and AA6: AA5 is Tyr, Lys*, Tic, Phe(4-F), Phe(4-CN), Phe(4-Cl), or N-Me-Tyr, andAA6 is D-Lys* or trans-D-HypAA3, AA5, and AA6: AA3 is D-3-Pal, D-Tyr, D-Trp, D-Gln, D-Gln(Me2), or D-Asn, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Ser, Sar, N- Me-D-Ala, D-Thr, D-Ser, D-Hse, beta- Ala, Ala, D-Ala, Ado, 6-Ahx, or absentAA5, AA6, and AA7: AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-Leu or NleAA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* or D-Lys, and AA8 is Lys*, Lys(Mes) or ArgAA5, AA6, and AA9: AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-HypAA3, AA5, AA6,and AA9: AA3 is D-Asn, AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-HypAA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D- Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*AA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr or Tyr, AA6 is D-Lys*, and AA10 is Gly or N-Me-D-Ala or absentAA5, AA6, AA9 and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, AA9 is Pip, cis-D-Hyp, trans-Hyp, or Pro, and AA10 is absentAA3, AA5, AA6, AA9 and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr, AA6 is D- Lys*, AA9 is trans-Hyp, and AA10 is N-Me-D-Ala or absentAA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is N-Me-D-Ala, Sar, or absent.

[0071] In some embodiments, LiGA as described herein is lipidated at position 6 (AA6) and has a GnRH analogue peptide backbone that is a peptide (optionally a decapeptide) that has any of the following amino acids at AA1-AA10 (except for AA5 and AA6 the first amino acid listed for each position is that of degarelix, which may be present):

[0072] In some embodiments, a GnRH analogue peptide backbone of a LiGA as described herein lipidated at position 6 (AA6) has substitutions relative to degarelix selected from the following sets (Lys* denotes point of attachment of lipid sidechain):AA5 and AA6: AA5 is Tyr or Tyr(SC>2F) and AA6 is D-Lys*AA3, AA5, and AA6: AA3 is D-Gln(Me2), D-Trp, or D-Tyr, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*AA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D- Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Sar, Ala, beta-Ala, N-Me-D-Ala, D-Ser, D-Hse or absentAA5, AA6, and AA7: AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-LeuAA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* and AA8 is Lys(Mes) or ArgAA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is absentAA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly or absentAA5, AA6, AA9 and AA10: AA5 is N-Me-Tyr, AA6 is D-Lys*, AA9 is trans-Hyp, and A10 is absent

[0073] In accordance with any of the foregoing embodiments, additional amino acid substitutions that replace an amino acid present in one GnRH antagonist (e.g., degarelix) with an amino acid present at the corresponding position of another GnRH antagonist (e.g., cetrorelix) are specifically contemplated. Without being bound by theory, it is contemplated that such substitutions will not undermine GnRH antagonist activity.

[0074] In some embodiments, a GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that comprises one or more of the following changes relative to degarelix (with the stereochemistry of the amino acid conserved relative to degarelix unless otherwise noted), optionally wherein the backbone is lipidated at a lysine moiety:AA5: Lys, Tyr, Aph(Hor), N(Me)Tyr, Aph(Ac), Arg, PicLys, or Lys(Nic)AA6: D-Lys, D-Tyr, D-Asn, D-Cit, D-hArg(Et2), D-Aph(Ac), D-Hci, D-glu(AA), D-(6Anic)Orn) or D-Lys(Nic)AA8: hArg(Et2), Lys or Arg

[0075] Typically, the amino acid of AA6 is a D-amino acid to conserve the stereochemistry relative to degarelix. For example, peptide backbones comprising the following combinations of changes relative to degarelix can be lipidated as described herein:AA5: N(Me)Tyr and AA6: D-Asn (e.g, abarelix); orAA5: Tyr; AA6: D-Cit; and AA8: Arg (e.g., cetrorelix); orAA5: Tyr; AA6: D-hArg(Et2), AA8: hArg(Et2) (e.g., ganerelix); orAA5: 4-(5-AT)Phe; AA6: D-4-(5-AT)Phe (e.g., prazarelix); orAA5: Aph(Ac); AA6: D-Aph(Ac) (e.g., acyline); orAA5: Tyr; AA6: D-Hci (e.g., teverelix); orAA5: Arg; AA6: D-glu(AA), AA8: Arg (e.g., Nal-Glu); orAA5: PicLys; AA6: D-(6Anic)0rn (e.g., orntide); orAA5: Lys(Nic); AA6: D-Lys(Nic) (e.g., antide); orAA5: N(Me)Tyr and AA6: D-Aph; orAA5: N(Me)Tyr; and AA6: D-Asn; orAA5: Tyr; and AA6: D-Lys (e.g, LiGA5 described herein); orAA5: Lys; and AA6: trans-D-Hyp (e.g., LiGA6 described herein); orAA4: Lys (e.g., LiGA4 described herein); orAA5: Lys (e.g., LiGA3 described herein); orAA6: D-Lys (e.g., LiGA2 described herein).

[0076] Thus, in some embodiments, the GnRH analogue peptide backbone of a LiGA as described herein is identical to the amino acid sequence of degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, or antide e.g., is a decapeptide that consists of AA1- AA10 of degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, or antide. In some embodiments, the GnRH analogue peptide backbone of a LiGA as described herein is a peptide (optionally a decapeptide) that has one or more amino acid substitutions relative to the amino acid sequence of degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, or antide, such as one, two, three, four, five, six, or seven, or more, amino acid substitutions. For example, one or more of the following types of amino acid substitutions may be introduced in the peptide backbone:• An amino acid substitution that provides the peptide backbone with an amino acid amenable to coupling with a lipid side chain (as described herein), such as any suitablenatural amino acid (e.g., lysine or cysteine), or any suitable non-natural amino acid (e.g., having a suitably reactive side chain, such as s-azidolysine or propargylglycine). Such substitutions may be introduced at one or more of AA5, AA6, AA7, and AA8, for example. Optionally, in some such embodiments, at least AA1-AA4 and AA9-AA10 are the same as in degarelix, although in other embodiments one or more of positions AA1-AA4 and AA9-AA10 may be substituted relative to degarelix, as discussed above.• An amino acid substitution that introduces an aromatic natural or unnatural amino acid (e.g., tyrosine, tyrosine derivatives, p-ureido-phenylalanine, etc.) at one or more of AA5 and AA6. Optionally, in some such embodiments, at least AA1-AA4 and AA9-AA10 are the same as in degarelix, although in other embodiments one or more of positions AA1- AA4 and AA9-AA10 may be substituted relative to degarelix, as discussed above.• An amino acid substitution that increases the hydrophilicity of the lipidated compound, such as introduction of Tyr or Hyp (e.g., trans-Hyp). Such substitutions may be introduced at one or more of AA5 and AA6, for example. Optionally, in some such embodiments, at least AA1-AA4 and AA9-AA10 of the LiGA peptide backbone are the same as in degarelix, although in other embodiments one or more of positions AA1-AA4 and AA9- AA10 may be substituted relative to degarelix, as discussed above.• Where not already present, Lys(iPr) may be introduced at AA8 (e.g., to replace the Arg of GnRH) to reduce histamine release. Optionally, in some such embodiments, at least AA1- AA4 and AA9-AA10 are the same as in degarelix, although in other embodiments one or more of positions AA1-AA4 and AA9-AA10 may be substituted relative to degarelix, as discussed above.• An amino acid substitution that replaces an amino acid present in one GnRH antagonist with an amino acid present at the corresponding position of another GnRH antagonist. Without being bound by theory, it is contemplated that such substitutions will not undermine GnRH antagonist activity.

[0077] In some embodiments the GnRH analogue peptide backbone of a LiGA as described herein may be a peptide (optionally a decapeptide) wherein AA4 is Ser. In some embodiments, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in degarelix). In some embodiments, AA2 is D-Cpa, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in degarelix). In some embodiments, AA1 is D-Nal, AA2 is D-Cpa, and AA3 is D-Pal, such as wherein AA1 is D-2Nal, AA2 is D-4Cpa, and AA3 is D-3Pal (as in degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, for example). In further specific embodiments of GnRH analogue peptide backbones, AA1 is D-2Nal, AA2 is D-4Cpa, AA3 is D-3Pal, AA4 is Ser, AA7 is Leu, and AA9 is Pro (as in degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, for example). In some specific embodiments, at least AA1-AA4 and AA9-A10 are the same as in degarelix. In some specific embodiments, at least AA1-AA4 and AA7-A10 are the same as in degarelix. In some specific embodiments, at least AA1-AA2, AA4 and AA7-A10 are the same as in degarelix. In some specific embodiments, at least AA2, AA4 and AA7-A10 are the same as in degarelix.

[0078] As noted above, a LiGA as described having a GnRH analogue peptide backbone based on degarelix or a degarelix analogue (e.g., as described in this section) may exhibit GnRH antagonist activity.Other Peptide Backbones

[0079] It should be understood that LiGAs as described herein may have other GnRH analogue peptide backbones than those specifically disclosed above, including peptide backbones having amino acid sequences with additional amino acid substitutions, deletions, or additions, including substitutions or additions of unnatural amino acids, and / or modification of one or more amino acids or coupling of one or more amino acids with another moiety.

[0080] As discussed above, advantageously, in some embodiments, the GnRH analogue peptide backbone of the lipidated GnRH analogues described herein have one or more of the following features: (a) the GnRH antagonist or agonist pharmacophore is preserved (for example, AA1-AA4 and AA9-AA10 of degarelix may be relevant (but not necessarily required) for antagonist activitywhile AA1-AA4 and AA7-AA9 or AA1-AA5 and AA7-AA9 or AA1-AA2, AA4 and AA7-AA9 or AA2, AA4 and AA7-AA9 of GnRH may be relevant (but not necessarily required) for agonist activity), (b) Arg at AA8 of GnRH is replaced with Lys(iPr) to reduce histamine release, (c) and an aromatic amino acid is present at one or more of AA5 and AA6 (e.g., an aromatic natural or unnatural amino acid such as tyrosine, tyrosine derivatives, p-ureido-phenylalanine, etc.).4. Lipidated GnRH Analogues

[0081] The lipidated GnRH analogues disclosed herein comprise one or more lipid moieties at one or more positions of the GnRH analogue peptide backbone. The number, type, and site(s) of lipidation may vary depending on the analogue being lipidated, and the target properties of the lipidated GnRH analogue.

[0082] The lipid moiety may be conjugated to the peptide backbone directly or through one or both of a linker and a spacer. In the discussion that follows, the term “lipid side chain” is used to refer to the lipid moiety and any linker(s) and any spacer(s). For instance, the “lipid side chain” in some examples herein comprises the following moieties: lipid moiety — linker — spacer, wherein the spacer is the moiety attached to the peptide backbone, and the linker links the fatty acid (lipid moiety) and spacer. Thus, a lipidated compound as described herein may comprise a lipid side chain and peptide backbone.

[0083] The lipid moiety typically is a fatty acid, such as a C8-C20 fatty acid. Non-limiting examples include C8-C20 fatty (di)acids, such as saturated C8-C20 fatty (di)acids. Other nonlimiting examples include C8-C20 fatty (mono)acids, such as a saturated C8-C20 fatty (mono)acids, Other non-limiting examples include C8-C20 fatty acids comprising a terminal tetrazole or sulfonic acid moiety (in place of a fatty acid moiety), such as a saturated C8-C20 fatty acid comprising a terminal tetrazole or sulfonic acid moiety.

[0084] The structures of representative C8-C20 fatty (di)acids are set forth below; it should be understood that any C8-C20 fatty (di)acid may be used.

[0085] The structures of representative fatty (mono)acids are set forth below; it should be understood that any C8-C20 fatty (mono)acid may be used.***acid)

[0086] The structures of representative fatty acids comprising a terminal tetrazole or sulfonic acid moiety (in place of a fatty acid moiety) are set forth below; it should be understood that any C8- C20 fatty acid comprising a terminal tetrazole or sulfonic acid moiety may be used.y cid)

[0087] In the foregoing structures, *** denotes point of attachment to the linker (if present in the lipid side chain) or to a spacer (if present in a lipid side chain lacking a linker) or to the peptide backbone (if no linker or spacer are present, e.g., if the lipid side chain consists of the lipid moiety).

[0088] When a spacer is used, the spacer may be attached to the peptide backbone through, for example, the Ns in the side chain of a lysine moiety. Examples of suitable spacers include those comprising one or more Ado moieties and / or one or more gGlu or aGlu moieties, such as the following:(* * Ado-Ado-Lys(Me3)-Ado*),(**Ado-NH-(CH2CH2O)3-CH2CH2-N3*) wherein * denotes point of attachment to the peptide backbone and ** denotes point of attachment to the linker (if present in the lipid side chain) or to the lipid moiety (if the lipid side chain does not include a liker).

[0089] The spacers Ado-Ado, Ado-Ado-gGlu, Ado-Ado-gGlu-Ado-Ado, and Ado-Ado-gGlu- Ado-Ado-gGlu and other spacers wherein the moiety at the point of attachment to the peptide backbone is an Ado, gGlu, or Lys moiety may be conveniently used for attachment via a lysine residue in the peptide backbone. The spacers Ado-Ado-Lys-C(=O)CH2S- and Ado-Ado-Lys- C(=O)CH2- (and other spacers with a similar moiety at the point of attachment to the peptide backbone) may be conveniently used for attachment via a cysteine residue in the peptide backbone. The spacer Ado-Ado-NH-CH(COOH)-CH2-C=CH- (and other spacers with a similar moiety at the point of attachment to the peptide backbone) may be conveniently used for attachment via an azido group in a side chain of an amino acid residue (e.g., azido lysine) in the peptide backbone. The spacer Ado-NH-(CH2CH2O)3-CH2CH2-N3 (and other spacers with a similar moiety at the point of attachment to the peptide backbone) may be conveniently used for attachment via an alkynyl group in the side chain of an amino acid residue (e.g., lysine where the amino group of the lysine side chain is mono-substituted with a functional group containing a terminal alkyne, such as -C(=O)- CH2CH2-C=CH) in the peptide backbone.

[0090] Additionally or alternatively, the spacer may be or comprise an amino acid spacer, such as a flexible spacer comprised of a short amino acid sequence, such as repeating units of one or more of glycine (polyglycine), serine (polyserine), glycine and serine (sometimes referred to as glycineserine linkers), beta-alanine (beta-Ala), and / or 6-aminohexanoic acid (Ahx). Such a spacer may be of any suitable amino acid sequence length. In some aspects, such a spacer may comprise 2-6 amino acids, such as Gly-Gly-Gly-Gly-Ser.

[0091] When a linker is used, the linker may be any suitable linker. In some embodiments a linker is one or more selected from:wherein ** denotes the point of attachment to the spacer (if present in the lipid side chain) or to the peptide backbone (if no spacer is present in the lipid side chain), and *** denotes attachment to the fatty acid.

[0092] Without being bound by theory, it is noted that the acid moiety of the linker (e.g., gGlu in the examples above or the sulfonamide linker above) may facilitate interaction with albumin in vivo and increase circulating half-life of the lipidated compounds described herein, e.g., after administration by injection.

[0093] It should be understood that a lipid side chain as described herein may include any combination of lipid and optional spacer(s) and / or linker(s) as disclosed herein, such as any lipid moiety, any one or more linkers or no linker, and any one or more spacers or no spacer. It also should be understood that a lipidated compound as described herein may include one or more lipid side chains (e.g., with each side chain coupled to a different amino acid moiety of the peptide backbone), wherein each lipid side chain may be the same or different, and may be independently selected from any combination of lipid and optional spacer(s) and / or linker(s) as disclosed herein.

[0094] As noted above, the number, type, and site(s) of lipidation may vary depending on the peptide being lipidated, and the target properties of the lipidated compound.

[0095] In the lipidated GnRH analogues described herein, the peptide backbone (e.g., the decapeptide backbone) may be lipidated at any one or more of AA6, AA1, AA5, AA7 and AA8. In specific embodiments, the peptide backbone is lipidated at AA6 (only). In other specific embodiments, the peptide backbone is lipidated at AA1 (only). In other specific embodiments, the peptide backbone is lipidated at AA5 (only). In other specific embodiments, the peptide backbone is lipidated at AA8 (only). In other specific embodiments, the peptide backbone is lipidated at AA7 (only). In other embodiments, the peptide backbone is lipidated at AA9. In other embodiments, the peptide backbone is lipidated at AA10. In specific embodiments, the peptide backbone is not lipidated at AA4. In other specific embodiments, the peptide backbone is lipidated at AA1 and at one or more of AA5 and AA6, such as being lipidated at AA1 and AA5, at AA1 and AA6, or at AA1, AA5, and AA6. In accordance with any of these embodiments, the lipidated GnRH analogues may have (unlipidated) Ser at AA4.

[0096] For peptide backbones based on degarelix or degarelix analogues (as discussed in more detail above), the peptide backbone (e.g., the decapeptide backbone) may be lipidated at any one or more of AA1, AA4, AA5, AA6, AA7, and AA8. In specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA6 (only). In specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA1 (only). In other specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA5 (only). In specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA8 (only). In specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA7 (only). In other specific embodiments, the peptide backbone is lipidated at AA7 (only). In other embodiments, the peptide backbone is lipidated at AA9. In other embodiments, the peptide backbone is lipidated at AA10. In specific embodiments, peptide backbones based on degarelix or degarelix analogues are not lipidated at AA4. In other specific embodiments, peptide backbones based on degarelix or degarelix analogues are lipidated at AA1 and at one or more of AA5 and AA6, such as being lipidated at AA1 and AA5, at AA1 and AA6, or at AA1, AA5, and AA6. In accordance with any of these embodiments, the lipidated peptide backbones based on degarelix and degarelix analogues may have (unlipidated) Ser at AA4.

[0097] It was surprisingly found that lipidation of peptide backbones as described herein is particularly well-tolerated at one or more of AA5 and AA6. In order to increase the hydrophilicity of the lipidated compounds, an additional amino acid substitution may be introduced at AA5 and / or AA6, such as Tyr or Hyp (e.g., trans-Hyp). For example, in the lipidated compounds set forth below, LiGA5 differs from LiGA2 by having Tyr at AA5, while LiGA6 differs from LiGA3 by having trans-D-Hyp at AA6. LiGA7 has 3-amino-L-Tyr at AA5 (its lipidation site) and trans-D- Hyp at AA6.

[0098] In some embodiments, modifying one or more of the components of the lipid side chain (lipid, linker, and / or spacer) can have an advantageous impact on potency / binding, half-life, solubility, and / or stability of the resulting lipidated GnRH analogue. For instance, lipid moieties having a longer fatty acid chain may exhibit increased binding to albumin and increased half-life(e.g., C20 > C18 > C16 > C14). On the other hand, lipid moieties having a shorter fatty acid chain (e.g., C8-C12) may be suitable when a long half-life is not required. Longer spacers may exhibit improved binding / potency. Spacers with a greater charge may exhibit improved solubility (e.g., Ado-Ado-gGlu > Ado-Ado). Using a linker such as gGlu also may increase solubility.

[0099] Such variations in the lipidated side chains are illustrated in the different series of lipid side chains discussed below and illustrated in the examples. The table below describes 31 series of lipid side chains with different linker and spacer moieties (Series A-Series Ab). For each series, it should be understood that any lipid moiety described above may be used. In specific embodiments, the lipid moiety is a saturated C8-C20 fatty (di)acid. While C14-C20 saturated (di)acids are present in many of the examples described herein, it should be understood that other lipid moieties, including other saturated C8-C20 fatty (di)acids may be used. For example a relatively longer fatty acid moiety may impart a longer half-life and / or desired solubility properties, while a relatively shorter fatty acid moiety may impart increased cell membrane permeability and / or a shorter half-life and / or a faster onset of action.

[0100] The lipidated GnRH analogues described herein may exhibit one or more improved properties (e.g., relative to degarelix) including improved physical stability (e.g., lower aggregation potential and / or reduced fibrillation in liquid formulation, and lower tendency to form a depot in vivo after subcutaneous injection, which may provide more predictable pharmacokinetic properties, and also permit injection with a thin needle — for improved patient comfort — as discussed in more detail below). In some embodiments, the lipidated GnRH analogues described herein may exhibit increased circulating half-life (e.g., relative to degarelix) while still exhibiting desired solubility properties (e.g., low solubility at physiological pH, which may advantageously provide a low Cmaxand high mean residence time and thus optimize the therapeutic window). Without being bound by theory, it is believed these improved properties are achieved by the combination of lipidation at one or more specific positions (e.g., AA6, AA5, AA1, AA8, and / or AA7), specific amino acid substitutions in the peptide backbone, and specific components of the lipid side chain (as discussed above).

[0101] Specific examples of lipidated GnRH analogue peptide backbones based on degarelix and degarelix analogues provided herein include LiGAl, LiGA2, LiGA3, LiGA4, LiGA5, LiGA6, and LiGA7. Further specific examples include LiGA28, LiGA43, LiGAl 51, and LiGAl 52, which are described further below. Still further specific examples are set forth in the tables below and representative example structures.

[0102] LiGAl has a lipidated degarelix peptide backbone (lipidated at AA1) and the following structure:

[0103] LiGAl may be represented as *D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)- Leu-Lys(iPr)-Pro-D-Ala-NH2, where * denotes the point of attachment of the lipid side chain to the peptide backbone (e.g., the lipidation site of LiGAl). The lipid side chain is from Series A with a saturated Cl 8 fatty (di)acid lipid moiety.

[0104] Relative to degarelix, LiGA2 is lipidated at AA6, which has been replaced with D-Lys. LiGA2 has the following structure:

[0105] LiGA2 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-Lys*- Leu-Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain to the peptide backbone.

[0106] Relative to degarelix, LiGA3 is lipidated at AA5, which has been replaced with L-Lys.LiGA3 has the following structure:

[0107] LiGA3 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-L-Lys*-D-4Aph(Cbm)- Leu-Lys(iPr)-Pro-D-Ala-NH2, wherein L-Lys* denotes point of attachment of the lipid side chain to the peptide backbone.

[0108] Relative to degarelix, LiGA4 is lipidated at AA4, which has been replaced with L-Lys.LiGA4 has the following structure:

[0109] LiGA4 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-L-Lys*-4Aph(L-Hor)- D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, where L-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. LiGA4 is outside the scope of some aspects of the present disclosure.

[0110] Relative to degarelix, LiGA5 is lipidated at AA6 which is replaced with D-Lys, and AA5 is replaced with Tyr. LiGA5 has the following structure:

[0111] LiGA5 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-L-Tyr-D-Lys*-Leu-Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain tothe peptide backbone. The lipid side chain is from Series A with a saturated Cl 8 fatty (di)acid lipid moiety.

[0112] Relative to degarelix, LiGA6 is lipidated at AA5 which is replaced with Lys, and AA6 is replaced with trans-DHyp. LiGA6 has the following structure:

[0113] LiGA6 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-L-Lys*-trans-D- Hyp-Leu- Lys(iPr)-Pro-D-Ala-NH2, wherein L-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. The lipid side chain is from Series A with a saturated Cl 8 fatty (di)acid lipid moiety.

[0114] Relative to degarelix, LiGA7 is lipidated at AA5 which is replaced with 3-Amino-L-Tyr, and AA6 is replaced with trans-D-Hyp. LiGA7 has the following structure:

[0115] LiGA7 may be represented as Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-3-Amino-L-Tyr*-trans-D- Hyp-Leu-Lys(iPr)-Pro-D-Ala-NH2, wherein 3-Amino-L-Tyr* denotes point of attachment of the lipid side chain to the peptide backbone.

[0116] Relative to degarelix, LiGA28 is lipidated at AA6 which is replaced with D-Lys, and AA5 is replaced with Tyr. LiGA28 has the following structure (peptide backbone — lipid side chain):

[0117] LiGA28 may be represented as Ac-D-2-Nal-D-4Cpa-D-3Pal-Ser-Tyr-D-Lys*-Leu- Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. The lipid side chain is from Series C with a saturated C20 fatty (di)acid lipid moiety.

[0118] Relative to degarelix, LiGA43 is lipidated at AA6 which is replaced with D-Lys, and AA5 is replaced with Tyr. LiGA43 has the following structure (peptide backbone — lipid side chain):

[0119] LiGA43 may be represented as Ac-D-2-Nal-D-4Cpa-D-3Pal-Ser-Tyr-D-Lys*-Leu- Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. The lipid side chain is from Series E with a saturated Cl 8 fatty (di)acid lipid moiety.

[0120] Relative to degarelix, LiGA151 is lipidated at AA6 which is replaced with Lys, and AA3 is replaced with D-Gln(Me2) and AA5 is replaced with Tyr. LiGA151 has the following structure (peptide backbone — lipid side chain):

[0121] LiGA151 may be represented as Ac-D-2-Nal-D-4Cpa-D-Gln(Me2)-Ser-Tyr-D-Lys*-Leu- Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. The lipid side chain is from Series A with a saturated Cl 8 fatty(di)acid lipid moiety.

[0122] Relative to degarelix, LiGAl 52 is lipidated at AA6 which is replaced with D-Lys, and AA1 is replaced with D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is replaced with D-Gln(Me2) and AA5 is replaced with Tyr. LiGAl 52 has the following structure (peptide backbone — lipid side chain):

[0123] LiGAl 52 may be represented as Ac-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-D- 4Cpa-D-Gln(Me2)-Ser-Tyr-D-Lys*-Leu-Lys(iPr)-Pro-D-Ala-NH2, wherein D-Lys* denotes point of attachment of the lipid side chain to the peptide backbone. The lipid side chain is from Series A with a saturated Cl 8 fatty di (acid) lipid moiety.

[0124] Also encompassed by the present disclosure are lipidated forms of stereoisomers of the compounds described herein, as well as salts and solvates of the lipidated compounds describedherein, including acetate salts, sodium salts, and citrate salts of the lipidated compounds described herein.5. Pharmaceutical Compositions, Uses, and Methods

[0125] Also provided herein are pharmaceutical compositions comprising a lipidated GnRH analogue as described herein, and therapeutic uses thereof.

[0126] In some embodiments, the pharmaceutical composition comprises a lipidated GnRH analogue as described herein and one or more pharmaceutically acceptable excipients for the intended route of administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration, e.g., for administration by subcutaneous or intramuscular injection, and comprises one or more pharmaceutically acceptable excipients for parenteral administration. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition, optionally a liquid pharmaceutical composition formulated for parenteral administration.

[0127] The lipidated compounds and pharmaceutical compositions described herein may be used for treating any condition for which the compound that is lipidated is useful for treating.

[0128] Thus, for example, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used for treating prostate cancer, including advanced prostate cancer, or metastatic stage prostate cancer, including the treatment of adult male patients with advanced hormone-dependent prostate cancer. Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used for treating high-risk localized or locally advanced hormone-dependent prostate cancer, in combination with radiotherapy. Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical compositions comprising it may be used as neo-adjuvant treatment prior to radiotherapy in patients with high-risk localized or locally advanced hormone dependent prostate cancer. Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical compositions comprising it may be used in the treatment of advanced prostatic carcinoma (urologic oncology). Additionally or alternatively, a lipidated GnRH analogueor pharmaceutical compositions comprising it may be used in the treatment of ovarian cancer or breast cancer. For example, a composition (e.g., a pharmaceutical composition) comprising any one of the lipidated GnRH analogues described herein may be for use in the treatment of prostate cancer, including advanced prostate cancer, or metastatic stage prostate cancer, including a composition for use in the treatment of adult male patients with advanced hormone-dependent prostate cancer. Additionally or alternatively, a composition (e.g., a pharmaceutical composition) comprising any one of the lipidated GnRH analogue described herein may be for use in the treatment of high-risk localized or locally advanced hormone-dependent prostate cancer, in combination with radiotherapy. Additionally or alternatively, a composition (e.g., a pharmaceutical composition) comprising any one of the lipidated GnRH analogues described here may be for use in the neo-adjuvant treatment prior to radiotherapy in patients with high-risk localized or locally advanced hormone dependent prostate cancer. Additionally or alternatively, a composition (e.g., a pharmaceutical composition) comprising any one of the lipidated GnRH analogues described herein may be for use in the treatment of advanced prostatic carcinoma (urologic oncology). Additionally or alternatively, a composition (e.g., a pharmaceutical composition) comprising any one of the lipidated GnRH analogues described herein may be for use in the treatment of ovarian cancer or breast cancer.

[0129] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used for the treatment of endometriosis. For example, the lipidated GnRH analogue or pharmaceutical composition comprising it may be for use in the treatment of endometriosis.

[0130] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used for the treatment of myoma. For example, the lipidated GnRH analogue or pharmaceutical composition comprising it may be for use in the treatment of myoma.

[0131] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used for the treatment of infertility, such as to promote controlled ovarianstimulation in an assisted reproduction protocol. For example, the lipidated GnRH analogue or pharmaceutical composition comprising it may be for use in the treatment of infertility.

[0132] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used in the treatment of one or more of benign prostatic hyperplasia, uterine fibroids, endometriosis, menorrhagia, premenstrual dysphoric disorder (PMDD), or severe premenstrual syndrome (PMS). For example, the lipidated GnRH analogue or pharmaceutical composition comprising it may be for use in the treatment of one or more of benign prostatic hyperplasia, uterine fibroids, endometriosis, menorrhagia, premenstrual dysphoric disorder (PMDD), and severe PMS. In accordance with some aspects, a lipidated GnRH analogue described herein that exhibits GnRH antagonist activity may be used in combination treatments, such as in combination with one or both of estradiol and norethindrone acetate, such as in the treatment of premenopausal women to control heavy menstrual bleeding due to uterine fibrids (uterine leiomyomas), or to manage moderate to severe pain associated with endometriosis.

[0133] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used in the treatment of central precocious puberty, or as a puberty blocker such as for transgender or gender diverse youth, or for chemical castration. For example, the lipidated GnRH analogue or pharmaceutical composition comprising it may be for use in the treatment of of central precocious puberty, or as a puberty blocker such as for transgender or gender diverse youth, or for chemical castration.

[0134] Additionally or alternatively, a lipidated GnRH analogue or pharmaceutical composition comprising it may be used in a veterinary context, such as for hormone suppression (e.g. chemical castration / neutralization) of pets or livestock, or in an in vitro fertilization protocol for pets or livestock.

[0135] Thus, also provided are methods of treatment comprising administering a lipidated GnRH analogue as described herein or pharmaceutical composition comprising it to a subject in need thereof, such as a subject in need of treatment for one or more of the diseases and conditionsdiscussed above. Also provided are uses of a lipidated compound as described herein in the manufacture of a medicament. Optionally, the use of a lipidated compound in the manufacture of medicament is for the treatment of one or more of the diseases and conditions discussed above. Also provided is a composition comprising any one of the lipidated GnRH analogues described herein for use as a medicament. The composition may be a pharmaceutical composition.

[0136] As discussed above, the lipidated compounds described herein and compositions comprising them may offer one or more advantages with regard to physical stability, desired solubility, and pharmacokinetic parameters such as circulating half-life, as illustrated in the examples below. For example, with regard to desired solubility, lipidated compounds as described herein may exhibit low solubility at physiological pH, which may advantageously provide a low Cmax and high mean residence time which permits optimization of the therapeutic window with each dosing, thereby permitting minimizing dosing frequency and maximizing drug utilization (e.g., not overtreating the patient beyond profound / full castration). Further lipidated compounds as described herein exhibit a lower propensity for fibrillation than degarelix, as illustrated in the examples below. This is an advantageous property of the lipidated compounds as described herein. For example, a lower fibrillation potential enables the preparation and use (injection) of ready -to- use parenteral solutions with a thin needle (e.g., 31G), and provides better control (predictability) of pharmacokinetic properties than seen with degarelix, including the absorption phase and the elimination phase, since the lipidated compounds described herein do not form a depot in subcutaneous tissue like degarelix does.6. Methods of Preparing Lipidated GnRH Analogues

[0137] In a further aspect, there are provided methods of preparing a lipidated GnRH analogue as described herein. In the description that follows, methodology is described in the context of a LiGA having a degarelix peptide backbone as a representative lipidated GnRH analogue. Those skilled in the art will be able to adopt the methodologies described herein to LiGAs having a different peptide backbone.Synthesis of Peptide Backbones

[0138] Peptide backbones suitable for lipidation as described herein, including peptide backbones based on GnRH, GnRH agonists, or GnRH antagonists such as degarelix and degarelix analogues, may be synthesized by one or both of Solid Phase Peptide Synthesis (SPPS) (such as described WO 1998 / 46634, W02010 / 121835 and WO 2011 / 066386) and Liquid Phase Peptide Synthesis (LPPS) (such as described in W02012 / 055903 and W02012 / 055905), which are incorporated herein by reference.

[0139] SPPS methods may be particularly suitable in the context of the present disclosure, because coupling the lipid side chain to the peptide backbone may be carried out while the peptide backbone is attached to a solid support, for example following SPPS synthesis of the peptide backbone.

[0140] SPPS typically comprises the steps of loading a first amino-acid whose alpha-amino group is protected by a protecting group or a peptide whose alpha-amino group is protected by a protecting group onto a solid support, cleaving the protecting group and then coupling the unprotected alpha-amino group to the carboxylic acid group of a second amino acid whose alphaamino group is protected by a protecting group or peptide whose alpha-amino group is protected by a protecting group, such that a peptide bond is formed and the peptide chain attached to the support is elongated. The steps are repeated until the desired peptide backbone is prepared. Suitable protecting groups for the alpha-amino group include 9-fluorenylmethoxycarbonyl (Fmoc), which is base labile, and tert-butyloxycarbonyl (Boc), which is acid labile. Any other functional groups present on the amino acids or peptide may be protected with suitable protecting groups, for example tert-butyl (tBu) in case of the Fmoc or for example benzyl (Bzl) in case of Boc. Typically, the alpha-amino groups of the amino acids or peptide are protected by Fmoc, because Fmoc chemistry typically employs milder reaction conditions than Boc chemistry. In the discussion that follows, the “peptide” whose alpha-amino group is protected may be a fragment of peptide backbone of a LiGA as described herein. For example, for a LiGA having a decapeptide peptide backbone, a protected peptide fragment may be 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s) inlength. In the final step, the desired peptide backbone can be released from the solid support and the protecting groups are cleaved off in a cleavage step using a suitable reagent, for example trifluoroacetic acid in case of the Fmoc / tBu, or and anhydrous hydrogen fluoride in case Boc / Bzl.

[0141] Typically, a peptide backbone based on GnRH or a GnRH analogue may be synthesized stepwise on a solid support comprising an amino group linked to the support, by a process that comprises: (i) providing a solution of an amino acid or peptide whose alpha-amino group is protected, e.g., by an Fmoc or Boc group; (ii) contacting the solid support with the solution of step (i) and a coupling agent, such that the carboxylic acid group of the amino acid or peptide of the solution of step (i) and the amino group linked to the support form an amide bond; and (iii) removing the protecting group, e.g., removing the Fmoc group of the amino acid or peptide by treating the solid support with a base in an organic solvent or removing the Boc group of the amino acid or peptide by treating the solid support with an acid in an organic solvent, and repeating steps (i) - (iii) as necessary to obtain the peptide backbone on the solid support. Optionally, the peptide backbone can be recovered from the solid support, or it can be retained on the solid support for coupling to the lipid side chain. Optionally, the solid support may be selected from a Rink amide or Ramage amide resin.

[0142] Typically, the side chains of each amino acid are protected during the SPPS process. Typically, the protecting groups are stable during the removal of Fmoc (where relevant), the coupling reactions, and removable under acidic conditions (or alternatively other orthogonal conditions relative to the alpha-amino group). For example, the hydroxyl group of serine (Ser) may be protected by trityl (Trt), tertbutyldimethylsilyl (TBDMS) or tertbutyl (tBu); the e-amino group of lysine (Lys(iPr)) may be protected by 4-methyltrityl (Mtt), allyloxycarbonyl (Alloc), tertbutyloxycarbonyl (Boc) or benzyloxycarbonyl (Cbz). In some embodiments, the carbamoyl group (Cbm) of D-Aph(Cbm) may be free or optionally protected with tert-butyl (tBu); the p-amino group of p-amino-phenylalanine (Aph) may be free or optionally protected by tertbutyloxy carbonyl (Boc), formyl (For), allyloxy carbonyl (Alloc) or benzyloxy carbonyl (Cbz).

[0143] Fmoc protected amino acids used in the synthesis of the GnRH analogue may comprise, for example, Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys(iPr, PG)-OH, Fmoc-Leu-OH, Fmoc-D- Aph(Cbm)-OH, Fmoc-D-Aph(Cbm, PG)-OH, Fmoc-Aph(Hor)-OH, Fmoc-Ser(PG)-OH, Fmoc- D-Pal-OH, Fmoc-D-Cpa-OH, Fmoc-D-Nal-OH, wherein PG is a protecting group, such as those described above. The Fmoc protecting group of an amino acid may be cleaved using any secondary amine base, for example tert-butyl amine, pyrrolidine, piperazine, DBU or piperidine, , or using 3- (diethylamino)propylamine (DEAPA), typically in an organic solvent, such as dimethylformamide (DMF).

[0144] After synthesis of the full peptide backbone, a further step of treating the peptide backbone with an acylating agent to acetylate the N-terminus may be carried out. For example, in the context of degarelix and degarelix analogues, the phrase “acetylate the N-terminus” refers to the addition of an CH3C(O)- group to the N-terminal amine of the amino acid D-Nal. The acylating agent may be glacial acetic acid. To aceylate the N-terminus, the peptide backbone may be treated with glacial acetic acid, ethyl cyano(hydroxyimino)acetate (Oxyma Pure) and diisopropylcarbodiide (DIC). Prior to acetylating the N-terminus, any protecting group (such as Fmoc group) on the N-terminal amino acid is removed.Lipidation of Peptide Backbones

[0145] Typically, the lipid side chain is coupled to or synthesized directly on an un-protected amino group of an amino acid of the peptide backbone, such as an amino group at position AA1, AA5, AA6, AA7, and / or AA8 of the peptide backbone, such as at an a-amino group at position AA1 of the peptide backbone and / or in embodiments, when the lipidation site is AA5 and / or AA6 and / or AA7 and / or AA8, at an un-protected (or free) s-amino group of a lysine residue of the peptide backbone.

[0146] As noted above, it can be convenient to carry out the step of coupling the lipid side chain to the peptide backbone while the peptide backbone is attached to a solid support, for example following SPPS synthesis of the peptide backbone. As described above, the lipid side chaincomprises a lipid (such as a fatty acid) and optionally (e.g., typically) one or more linker(s) and / or one or more spacer(s). In any embodiments, the spacer, linker, and lipid may independently be any spacer(s), linker(s), and lipid(s) described above.

[0147] In some embodiments, the coupling of the lipid side chain to the peptide backbone is carried out by a convergent approach, wherein the partially protected lipid side chain is synthesized stepwise on a solid support, cleaved from the solid support, and then coupled to the peptide backbone on-resin. In other embodiments, the synthesis of the lipid side chain is carried out directly on the peptide backbone by a sequential approach, wherein the lipid side chain is synthesized stepwise directly onto the peptide backbone on-resin.Convergent Approach

[0148] When the lipid side chain comprises a lipid, linker(s) and spacer(s), the lipid side chain may be synthesized stepwise, for example by sequential coupling of the linker to the spacer, and the lipid (e.g., fatty acid) to the linker-spacer, on a solid support. This may occur prior to coupling the lipid side chain to the peptide backbone. For synthesis of a protected lipid side chain on a solid support, the solid support may be a 2-Chlorotrityl chloride resin (2-CTC resin) or Rink acid resin.

[0149] The stepwise synthesis of the lipid side chain on a solid support (e.g., 2-CTC resin) may comprise: (i) providing solutions of the spacer, linker and lipid, respectively; (ii) contacting the solid support with the spacer solution and a base (e.g., DIPEA) in an organic solvent for coupling to a 2-CTC resin (or a coupling agent if needed for a different resin), such that the carboxylic acid group of the spacer forms an ester bond with the solid support (spacer — support); (iii) contacting the solid support with the linker solution and a coupling agent, such that the carboxylic acid group of the linker forms an amide bond with an amino group of the spacer on the solid support (linker — spacer — support); and (iv) contacting the solid support with the lipid (fatty diacid) solution and a coupling agent, such that a carboxylic acid group of the lipid forms an amide bond with an amino group of the linker on the solid support (lipid — linker — spacer — support). The protected lipid sidechain can be released from the solid support by contacting the solid support with any suitable acid and solvent.

[0150] When the protected lipid side chain is synthesized stepwise on a suitable solid support, the solid support may comprise a 2-CTC resin. For example, following the initial coupling, the solid support may be treated with an excess of alcohol, for example MeOH in DCM / DIPEA, to block un-reacted sites on the solid support. After synthesis of the protected lipid side chain on a solid support, the lipid side chain containing a free carboxylic acid functionality can be released from the solid support by treating the solid support with any suitable mild acid and solvent, for example HFIP in DCM.

[0151] Coupling a protected lipid side chain to the peptide backbone may be conducted on-resin, e.g., by (i) providing the peptide backbone of the GnRH analogue on a solid support; (ii) providing a solution of the protected lipid side chain; (iii) contacting the peptide backbone with the protected lipid side chain solution and a coupling agent, such that a carboxylic acid group of the protected lipid side chain forms an amide bond with a free amino group of an amino acid and / or an amino acid side chain of the peptide backbone (such as an alpha-amino group at position AA1 and / or the s-amino group of AA5 and / or AA6 of the peptide backbone, such as an un-protected lysine residue at position AA5 and / or AA6).

[0152] In some embodiments, coupling the lipid side chain to the peptide backbone is carried out while the peptide backbone is attached to a solid support, for example following the SPPS stepwise synthesis of the peptide backbone.Sequential Approach

[0153] Alternatively, the lipid side chain may be synthesized stepwise, for example by the sequential coupling of the spacer, linker, and lipid (e.g., fatty acid) directly onto the peptide backbone, e.g, to an amino acid of the peptide backbone, such as an amino group at position AA1, AA5, and / or AA6 of the peptide backbone, such as an un-protected s-amino group of a lysine residue, typically while the peptide backbone is on a solid support.

[0154] The stepwise synthesis of the lipid side chain directly onto to a peptide backbone on a solid support may comprise (i) providing solutions of the spacer, linker and lipid; (ii) contacting the solid support carrying the peptide backbone with the spacer solution and a coupling agent such that a carboxylic acid group of the spacer forms an amide bond with an amino group of the peptide backbone (spacer — peptide — support); (iii) contacting the solid support with the linker solution and a coupling agent, such that the carboxylic acid group of the linker forms an amide bond with an amino group of the spacer (linker — spacer — peptide — support); and (iv) contacting the solid support with the lipid (fatty (di)acid) solution and a coupling agent, such that a carboxylic acid group of the lipid forms an amide bond with an amino group of the linker (lipid — linker — spacer — peptide — support).

[0155] In any embodiments, the alpha-amino group of the spacer may be protected by a Fmoc group and, prior to a coupling step, the Fmoc group may be removed from the spacer by treating with a base in an organic solvent. Likewise, in any embodiments, the s-amino group of the linker may be protected by a Fmoc group and, prior to a coupling step, the Fmoc group may be removed by treating with a base in an organic solvent. Alternatively, analogous protocols suitable for using the Boc protecting group may be used.

[0156] In any embodiments, prior to coupling the lipid side chain to the peptide backbone (e.g., by the convergent or sequential approaches described above), any protecting group on the target amino acid of the peptide backbone may be removed. For example, a Fmoc protecting group (e.g., present on an N-terminal amino acid of the peptide backbone) may be removed by treating with a base in an organic solvent or a Boc protecting group (e.g., present on an N-terminal amino acid of the peptide backbone) may be removed by treating with an acid in an organic solvent. When the lipid side chain is coupled to an internal amino acid of the peptide backbone (e.g., AA5 or AA6), such as the s-amino group of a lysine residue, the amino acid may have a protecting group such as Mtt or Alloc, which may be removed by treating with a suitable deprotecting agents (e.g. HFIP in DCM for Mtt and Pd(PPh3)4 / PhSiH3in DCM for Alloc).

[0157] In any embodiments, after the lipid side chain has been coupled to the peptide backbone (e.g., by the convergent or sequential approaches described above), the lipidated GnRH analogue (LiGA) may be released from the solid support by contacting the solid support with an acid.

[0158] The invention is further illustrated by the following discussion of specific embodiments and examples, which do not in any way limit the scope of the invention.Preparation of Fmoc-D-Asp(E2.3.4-tetrahydroisoquinoline amide)-OH Building Block

[0159] For embodiments comprising a D-Asp(l,2,3,4-tetrahydroisoquinoline amide) moiety in the peptide backbone, the following methodology may be used to prepare a Fmoc-D-Asp(l, 2,3,4- tetrahydroisoquinoline amide)-OH building block for use in a method of preparing a lipidated GnRH analog as outlined above.1. Synthesis of Fmoc-D-Asp(1.2.3.4-tetrahydroisoquinoline amide)-OtBu

[0160] Fmoc-D-Asp-OtBu (1.0 equiv.), HOBt (2.0 equiv.), EDC (2.0 equiv.), and DIPEA (3.0 equiv.) are combined in Me-THF and stirred. 1,2,3,4-tetrahydroisoquinoline (1.1 equiv.) is added with further stirring at room temperature. A reaction mixture comprising a yellow solution with white precipitate may be obtained.

[0161] Purified Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu can be obtained by an extraction process using an aqueous solution of 10% HC1, separating the phases, washing the organic phase with saturated NaHCCF, and drying. For further purification, the obtained solid may be re-dissolved in DCM and loaded onto a silica column for purification by flash chromatography using a Heptane / EtOAc eluent system. The product obtained is the intermediate Fmoc-D- Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu.2, Synthesis of Fmoc-D-Asp(1.2.3.4-tetrahydroisoquinoline amide)-OH

[0162] Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu is dissolved in DCM (approx.10 mL / g) and TFA is added (approx. 10 mL / g) and the reaction mixture is stirred, e.g., for 3 hours.The reaction mixture is concentrated in vacuo and the resulting residue co-concentrated with toluene to obtain an oily residue which can be dried under vacuum, e.g., at 30 °C, e.g., overnight. The dried residue can be re-dissolved in MeCN (approx. 20 mL / g) and water added (approx. 20 mL / g) to obtain an emulsion. The emulsion can be stirred vigorously immediately before flash freezing in a dry ice / acetone bath and lyophilization. Following lyophilization, the product, obtained as an orange-white powder, can be re-crystallized . The product obtained is Fmoc-D- Asp(l,2,3,4-tetrahydroisoquinoline amide)-OH.7. Examples of Lipidated GnRH Analogues

[0163] The following tables summarize illustrative examples of lipidated GnRH analogues according to the present disclosure, grouped by peptide backbone and showing the lipid side chain (lipid, linker, and spacer) and peptide backbone moieties, wherein the * indicates denotes the point of attachment to the peptide backbone (e.g., the lipidation site). The full chemical structures of selected LiGA compounds are set forth following the tables in which the compounds are described.

[0164] As noted above, to the extent a peptide backbone is presented as an amide (with a C- terminal -NH2 moiety), it should be understood that a corresponding peptide backbone that is an acid (with a C-terminal -OH moiety) also is contemplated and described. Further, to the extent a compound is presented as acetylated (having an N-terminal acetyl moiety (Ac)), it should be understood that a corresponding “free” compound lacking such a moiety also is contemplated and described. In the following tables, many lipid side chains are from Series A-E. The structures of the Series A-E lipid side chains are shown below, depicted with a saturated C8-C20 fatty (di)acid lipid moiety (n= 5-17, respectively) . In all structures, * denotes point of attachment to the peptide backbone.

[0165] Series A (faty acid-gGlu-Ado-Ado):Senes A (C8-C20)

[0166] Series B (faty acid-gGlu-Ado-Ado-gGlu):Series B (C8-C20).

[0167] Series C (faty acid-gGlu-Ado-Ado-gGlu-Ado-Ado):

[0168] Series D (faty acid-gGlu-Ado-Ado-gGlu-Ado-Ado-gGlu):Senes D (C8-C20).

[0169] Series E lipid (faty acid-gGlu-Ado-Ado-Ado-Ado):Series E (C8-C20)LiGA22 (lipid side chain Series A (C16))LiGA5 (lipid side chain Series A (Cl 8))LiGA27 (lipid side chain Series B (C20))LiGA29 (lipid side chain Series D (C20))LiGA31 (lipid side chain Series A (C18))

[0170] Additional representative lipidated GnRH analogues as described herein were prepared and subjected to in vitro characterization as summarized in the examples below. In the tables below, all compounds are lipidated at AA6 with a lipid side chain selected from Series A-Ab described above.LiGA175 (lipid side chain Series S (C20))LiGA165 (lipid side chain Series P (C20))LiGA60 (lipid side chain Series E (C20))LiGA162 (lipid side chain Series M (C20))LiGA61 (lipid side chain Series F (C20))LiGA158 (lipid side chain Series P (C18))LiGA62 (lipid side chain Series G (C20))LiGA43 (lipid side chain Series E (C18))LiGA172 (lipid side chain Series X (C18))LiGA45 (lipid side chain Series G (C18))LiGA178 (lipid side chain Series V (C20))LiGA124 (lipid side chain Series A (C18))LiGA72 (lipid side chain Series A (C18))LiGA35 (lipid side chain Series A (C18))LiGA102 (lipid side chain Series E (C18))LiGA86 (lipid side chain Series A (C18))LiGA115 (lipid side chain Series E (C18))LiGAllO (lipid side chain Series A (C18))LiGAlll (lipid side chain Series E (C18))LiGA36 (lipid side chain Series A (C18))LiGA93 (lipid side chain Series E (C20))LiGAlll (lipid side chain Series A(C18))LiGA182 (lipid side chain Series A (C18))LiGA94 (lipid side chain Series A (C18))LiGA135 (lipid side chain Series A (C18))LiGA129 (lipid side chain Series A (C18))LiGA54 (lipid side chain Series Aa (C18))LiGA97 (lipid side chain Series A (C20))LiGA98 (lipid side chain Series E (C18))LiGA119 (lipid side chain Series A (C18))LiGAlOO (lipid side chain Series A (C18))LiGA55 (lipid side chain Series Aa(C18))LiGA153 (lipid side chain Series A(C18))

[0171] Additional LiGA compounds lipidated at AA6 or AA8 were prepared as set forth in the table below. In the table below, the lipid side chains are described by nomenclature “(FA{LetterNumber})” where Letter denotes the lipid side chain series and Number denotes the length of the saturated fatty di(acid) lipid moiety. For example, for compound 434 (LiGA47), “(FA{A18})” denotes a lipid side chain of series A with a saturated Cl 8 fatty (di)acid lipid moiety; for compound 435 (LiGA68), “(FA{J20})” denotes a lipid side chain of series J with a saturated C20 fatty (di)acid lipid moiety.

[0172] Additional LiGA compounds were prepared as set forth in the table below using the same general nomenclature as above. Note that the lipidation position is AA5 or AA6.

[0173] Additional series of LiGA compounds similar to LiGA5, LiGA28, LiGA43, LiGA151, and LiGA152 are set forth below.

[0174] LiGA compounds similar to LiGA5, LiGA28, or LiGA43 (peptide backbone Ac-D-2-Nal- D-4Cpa-D-3Pal-Ser-Tyr-D-Lys*-Leu-Lys(iPr)-Pro-D-Ala-NH2; Series A (R1), Series C (R2), or Series E (R3) lipid side chain attached at D-Lys*) wherein the lipid moiety is a saturated C8-C20 fatty (di)acid (L1GA5: R=R\ n’=15; L1GA28: R=R2, n”=17; L1GA43: R= R3, n”’= 17):n"' = 5 - 17

[0175] LiGA compounds similar to LiGA151 (peptide backbone Ac-D-2-Nal-D-4Cpa-D- Gln(Me2)-Ser-Tyr-D-Lys*-Leu-Lys(iPr)-Pro-D-Ala-NH2; Series A (R1), Series C (R2), or Series E (R3) lipid side chain attached at D-Lys*) wherein the lipid moiety is a saturated C8-C20 fatty (di)acid (LiGAl 51 : R=R' with n’ = 15) are set forth below:n"' = 5 - 17

[0176] LiGA compounds similar to LiGA152 (peptide backbone Ac-D-Asp(l, 2,3,4- tetrahydroisoquinoline amide)-D-4Cpa-D-Gln(Me2)-Ser-Tyr-D-Lys*-Leu-Lys(iPr)-Pro-D-Ala- NH2,; Series A (R1), Series C (R2), or Series E (R3) lipid side chain attached at D-Lys*) wherein the lipid moiety is a saturated C8-C20 fatty (di)acid (LiGA152” R= R1with n’ = 15):n"' = 5 - 17

[0177] Further additional series of LiGA compounds based on LiGA5, LiGA28, LiGA43, LiGA151, and LiGA152 with different lipid side chains are set forth in the table below.Relative to Degarelix AA1 and AA3 are indicated as below, AA5 is Tyr, and AA6 is D-Lys* which is the point of attachment for the lipid side chain

[0178] Additional LiGA compounds are set forth in the table below using the same general nomenclature as above, e.g., where the lipid side chains are described by nomenclature “(FA{LetterNumber})” where Letter denotes the lipid side chain series and Number denotes the length of the saturated fatty di(acid) lipid moiety.Additional LiGA compounds are set forth in the table below using the same general nomenclature as above, e.g., where the lipid side chains are described by nomenclature “(FA{LetterNumber})” where Letter denotes the lipid side chain series and Number denotes the length of the saturated fatty di(acid) lipid moiety.

[0179] LiGA compounds having a peptide backbone based on a GnRH agonist also were prepared, with lysine substitutions at position AA5 or AA6 for lipidation (Lys*): pGlu-His-Trp-Ser-Tyr-Lys*-Leu-Arg-Pro-Gly-NH2 (GnRH backbone) pGlu-His-Trp-Ser- Lys*-Gly-Leu-Arg-Pro-Gly-NH2 (GnRH backbone) pGlu-His -Trp- S er-Lys * -D -T rp-Leu- Arg-Pro-Gly-NH2 (triptorelin backbone)These LiGA compounds were lipidated with a Series A side chain having a saturated Cl 8 fatty acid lipid moiety.8. Representative Embodiments

[0180] The following embodiments are included in the present disclosure.

[0181] Embodiment 1. A lipidated gonadotropin releasing hormone (GnRH) analogue, wherein the lipidated GnRH analogue comprises a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone at one or more of amino acid positions 6 (AA6), 5 (AA5), and 1 (AA1) of the peptide backbone.

[0182] Embodiment 2. A lipidated gonadotropin releasing hormone (GnRH) analogue, wherein the lipidated GnRH analogue comprises a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone at one or more of amino acid positions 6 (AA6), 5 (AA5), 1 (AA1), and 8 (AA8) of the peptide backbone.

[0183] Embodiment 3. The lipidated GnRH analogue of Embodiment 1 or Embodiment 2, wherein the lipidated GnRH analogue exhibits GnRH antagonist activity.

[0184] Embodiment 4. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of a GnRH antagonist selected from degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, or an amino acid sequence having one, two, three, four, five, six, or seven amino acid substitutions relative thereto.

[0185] Embodiment 5. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of degarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0186] Embodiment 6. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of abarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0187] Embodiment 7. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of cetrorelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0188] Embodiment 8. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of ganirelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0189] Embodiment 9. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of prazarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0190] Embodiment 10. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of acyline, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0191] Embodiment 11. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of teverelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0192] Embodiment 12. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of Nal-Glu, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0193] Embodiment 13. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of orntide, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

[0194] Embodiment 14. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has the amino acid sequence of antide or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.Embodiment 15. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10 (AA1-AA10):

[0195] Embodiment 16. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10(AA1-AA10):

[0196] Embodiment 17. The lipidated GnRH analogue of any one of Embodiments 1-3, wherein the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10(AAl-AAlO):

[0197] Embodiment 18. The lipidated GnRH analogue of any one ofEmbodiments 15-17, wherein the GnRH analogue peptide backbone has an amino acid sequence with one of the following sets of amino acids present in degarelix:(i) AA1: D-Nal, AA2: D-Cpa, and AA3: D-Pal;(ii) AA4: Ser, AA7: Leu, and AA9: Pro;(iii) AA2: D-Cpa; AA4: Ser, AA7: Leu, and AA9: Pro;(iv) AA1: D-Nal, AA2: D-Cpa; AA3: D-Pal; AA4: Ser, AA7: Leu, and AA9: Pro.

[0198] Embodiment 19. The lipidated GnRH analogue of any one of Embodiments 1-18, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix or a degarelix analogue such as abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, at one of the following sets of positions:AA6;AA5 and AA6;AA6 and AA8;AA5, AA6, and AA10;AA3, AA5, and AA6;AA1, AA3, AA5, and AA6;AA5, AA6, and AA7;AA5, AA6, and AA8;AA5, AA6, and AA9;AA3, AA5, and AA9;AA3, AA5, AA6, and AA10;AA5, AA6, AA7, and AA10;AA5, AA6, AA9 and AA10;AA5, AA6, AA7, AA8 and AA10;AA3, AA5, AA6, AA9 and AA10;AA1, AA3, AA5, AA6, and AA8;AA1, AA3, AA5, AA6, and AA10;AA1, AA3, AA5, AA6, AA7 and AA10; orAA1, AA3, AA5, AA6, AA7, AA8 and AA10, optionally wherein the substitutions are relative to degarelix.

[0199] Embodiment 20. The lipidated GnRH analogue of Embodiment 19, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix selected from the following sets, wherein Lys* denotes point of attachment of lipid side chain):AA6: D-Lys*AA5 and AA6: AA5 is Tyr, Lys*, Tic, Phe(4-F), Phe(4-CN), Phe(4-Cl), or N-Me-Tyr, andAA6 is D-Lys* or trans-D-HypAA3, AA5, and AA6: AA3 is D-3-Pal, D-Tyr, D-Trp, D-Gln, D-Gln(Me2), or D-Asn, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Ser, Sar, N-Me-D-Ala, D-Thr, D-Ser, D-Hse, beta-Ala, Ala, D-Ala, Ado, 6-Ahx, or absentAA5, AA6, and AA7: AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-Leu or NleAA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* or D-Lys, and AA8 is Lys*, Lys(Mes) or ArgAA5, AA6, and AA9: AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-HypAA3, AA5, AA6, and AA9: AA3 is D-Asn, AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-HypAA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*AA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr or Tyr, AA6 is D-Lys*, and AA10 is Gly or N-Me-D-Ala or absentAA5, AA6, AA9 and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, AA9 is Pip, cis-D-Hyp, trans-Hyp, or Pro, and AA10 is absentAA3, AA5, AA6, AA9 and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr, AA6 is D-Lys*, AA9 is trans-Hyp, and AA10 is N-Me-D-Ala or absentAA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is N-Me-D-Ala, Sar, or absent.

[0200] Embodiment 21. The lipidated GnRH analogue of Embodiment 19, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix selected from the following sets, wherein Lys* denotes point of attachment of lipid side chain):AA5 and AA6: AA5 is Tyr or Tyr(SC>2F) and AA6 is D-Lys*AA3, AA5, and AA6: AA3 is D-Gln(Me2), D-Trp, or D-Tyr, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*AA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D- Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Sar, Ala, beta-Ala, N-Me-D-Ala, D-Ser, D-Hse or absentAA5, AA6, and AA7: AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-LeuAA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* and AA8 is Lys(Mes) or ArgAA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is absentAA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly or absentAA5, AA6, AA9 and AA10: AA5 is N-Me-Tyr, AA6 is D-Lys*, AA9 is trans-Hyp, and AA10 is absent

[0201] Embodiment 22. The lipidated GnRH analogue of Embodiment 1 or Embodiment 2, wherein the lipidated GnRH analogue exhibits GnRH agonist activity.

[0202] Embodiment 23. The lipidated GnRH analogue of Embodiment 22, wherein the GnRH or GnRH analogue peptide backbone has the amino acid sequence of a GnRH agonist selected from GnRH, leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, and deslorelin or an amino acid sequence having one, two, three, or four amino acid substitutions relative thereto.

[0203] Embodiment 24. The lipidated GnRH analogue of Embodiment 22, wherein the GnRH analogue peptide backbone has the amino acid sequence of leuprolide, or an amino acid sequence having one, two, three, or four amino acid substitutions relative thereto.

[0204] Embodiment 25. The lipidated GnRH analogue of Embodiment 22, wherein the GnRH analogue peptide backbone has the amino acid sequence of triptorelin, or an amino acid sequence having one, two, three, or four amino acid substitutions relative thereto.

[0205] Embodiment 26. The lipidated GnRH analogue of any one of Embodiments 22-25, wherein the GnRH analogue peptide backbone has an amino acid sequence with the following amino acid substitutions relative to GnRH:AA6: D-Leu; D-Ser(tBu), D-Trp, D-2Nal, or D-His(Bzl) andAA10: AzaGly or absent, optionally wherein the terminal NH2 group is replaced by NHet, optionally wherein AA1-AA5 and AA7-AA9 are the same as in GnRH.

[0206] Embodiment 27. The lipidated GnRH analogue of any one of the preceding Embodiments, comprising or further comprising one or more amino acid substitutions selected from:(i) an amino acid substitution that introduces an amino acid amenable to coupling with a lipid side chain, optionally at one or more of AA6, AA5, and AA8;(ii) an amino acid substitution that introduces an aromatic amino acid at one or more of AA5 and AA6;(iii) an amino acid substitution that increases hydrophilicity of the GnRH analogue;(iv) an amino acid substitution that introduces Lys(iPr) at AA8; and / or;(v) an amino acid substitution that introduces an amino acid present at a corresponding position in another GnRH analogue.

[0207] Embodiment 28. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain is coupled to amino acid position 1 (AA1) of the peptide backbone.

[0208] Embodiment 29. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain is coupled to amino acid position 5 (AA5) of the peptide backbone.

[0209] Embodiment 30. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain is coupled to amino acid position 6 (AA6) of the peptide backbone.

[0210] Embodiment 31. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain is coupled to amino acid position 8 (AA8) of the peptide backbone.

[0211] Embodiment 32. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain comprises a lipid moiety, and optionally further comprises one or both of a linker and a spacer.

[0212] Embodiment 33. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain comprises a lipid moiety, a linker, and a spacer.

[0213] Embodiment 34. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain comprises a lipid moiety comprising a C8-C20 fatty acid, optionally comprising a C14-C20 fatty acid.

[0214] Embodiment 35. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipid side chain comprises a lipid moiety selected from a saturated C8-C20 fatty (di)acid; a saturated C8-C20 fatty (mono)acid; a saturated C8-C20 fatty acid tetrazole, and a saturated C8-C20 fatty acid sulfonic acid.

[0215] Embodiment 36. The lipidated GnRH analogue of any one of Embodiments 32-35, wherein the linker comprises one or more selected from (gGlu), (gGlu-gGlu), (aGlu) andwherein ** denotes the point of attachment to the spacer or to the peptide backbone if the lipid side chain does not include a spacer and *** denotes point of attachment to the fatty acid.

[0216] Embodiment 37. The lipidated GnRH analogue of any one of Embodiments 32-36, wherein the spacer comprises one or more selected from:wherein * denotes point of attachment to the peptide backbone and ** denotes point of attachment to the remainder of the lipid side chain, optionally wherein the lipid side chain comprises a linker and ** denotes point of attachment to the linker.

[0217] Embodiment 38. The lipidated GnRH analogue of any one of Embodiments 32-37, wherein the lipid side chain comprises a saturated fatty acid lipid moiety, linker, and spacer and is selected from Series A - Aa in the table below:

[0218] Embodiment 39. The lipidated GnRH analogue of Embodiment 1, selected from Compounds 1-561, optionally wherein the lipidated GnRH analogue is selected from LiGA5, L1GA15, L1GA21, L1GA22, L1GA23, L1GA27, L1GA28, L1GA29, L1GA31, L1GA35, L1GA36,L1GA43, L1GA44, L1GA45, L1GA54, L1GA55, L1GA60, L1GA61, L1GA62, L1GA69, L1GA70,L1GA72, L1GA74, L1GA86, L1GA91, L1GA92, L1GA93, L1GA94, L1GA96, L1GA97, L1GA98,L1GA99, LiGAlOO, L1GA102, L1GA103, L1GA110, LiGAl l l, L1GA112, L1GA113, L1GA114, L1GA115, L1GA119, L1GA124, L1GA125, L1GA129, L1GA135, L1GA151, L1GA152, L1GA153, L1GA157, L1GA158, L1GA161, L1GA162, L1GA164, L1GA165, L1GA166, L1GA168, L1GA169, L1GA171, LiGA 172, L1GA175, L1GA176, L1GA177, L1GA178, andLiGA182, further optionally wherein the lipidated GnRH analogue is selected from LiGA5, LiGA28, LiGA43, LiGA151, and L1GA152.

[0219] Embodiment 40. The lipidated GnRH analogue of Embodiment 39, further comprising an additional amino acid substitution that introduces an amino acid present at a corresponding position of a GnRH antagonist, optionally wherein the GnRH antagonist is selected from degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide.

[0220] Embodiment 41. The lipidated GnRH analogue of Embodiment 39 or 40, comprising or further comprising an amino acid substitution that introduces an amino acid at position 8 (AA8) selected from hArg(Et2), Lys, Arg, and Lys(iPr).

[0221] Embodiment 42. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the lipidated GnRH analogue exhibits a modified circulating half-life in vivo as compared to its non-lipidated counterpart, optionally wherein the lipidated GnRH analogue exhibits an extended circulating half-life in vivo as compared to its non-lipidated counterpart.

[0222] Embodiment 43. The lipidated GnRH analogue of any one of the preceding Embodiments, wherein the GnRH analogue peptide backbone is not lipidated at amino acid position 4 (AA4).

[0223] Embodiment 44. A pharmaceutical composition comprising the lipidated GnRH analogue of any one of the preceding Embodiments and a pharmaceutically acceptable excipient.

[0224] Embodiment 45. The pharmaceutical composition of Embodiment 44, wherein the composition is in a form selected from (i) a liquid pharmaceutical composition formulated for parenteral administration and (ii) a freeze-dried pharmaceutical composition formulated for reconstitution for parenteral administration.

[0225] Embodiment 46. The pharmaceutical composition of Embodiment 45, wherein the composition is a liquid composition and wherein the lipidated GnRH analogue exhibits one or both of a lower aggregation potential and reduced propensity for fibrillation in the composition than degarelix.

[0226] Embodiment 47. A process for preparing the lipidated GnRH analogue, of any one of Embodiments 1-43, comprising coupling the lipid side chain to an unprotected amino group of an amino acid of the peptide backbone or an amino acid side chain thereof at one or more of amino acid positions 1 (AA1), 5 (AA5), 6 (AA6), and 8 (AA8) of the peptide backbone.

[0227] Embodiment 48. The process of Embodiment 47, comprising:(i) providing the peptide backbone on a solid support;(ii) providing a solution comprising the lipid side chain, wherein the lipid side chain is protected with a protecting group;(iii) coupling the protected lipid side chain to the peptide backbone on the solid support, such that a carboxylic acid group of the protected lipid side chain forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof; and(iv) releasing the lipidated GnRH analogue from the solid support.

[0228] Embodiment 49. The process of any one of Embodiments 47-48, wherein the lipid side chain comprises a spacer, linker, and lipid moiety, wherein the process further comprises preparing protected lipid side chain on a solid support by a process comprising:(i) providing solutions of the spacer, linker and lipid moiety, respectively;(ii) coupling the spacer to a solid support such that a carboxylic acid group of the spacer forms an ester bond with the solid support (spacer — support);(iii) coupling the linker to the spacer — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer on the solid support (linker — spacer — support);(iv) coupling the lipid moiety to the linker — spacer — support such that a carboxylic acid group of the lipid forms an amide bond with an amino group of the linker on the solid support (lipid — linker — spacer — support); and(v) releasing the protected lipid side chain from the solid support.

[0229] Embodiment 50. The process of Embodiment 47, wherein the lipid side chain comprises a spacer, linker, and lipid moiety, wherein the process comprises:(i) providing the peptide backbone on a solid support;(ii) providing solutions of the spacer, linker and lipid moiety, respectively;(iii) coupling the spacer to the solid support carrying the peptide backbone such that a carboxylic acid group of the spacer forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof (spacer — peptide — support);(iv) coupling the linker to the spacer — peptide — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer (linker — spacer — peptide — support);(v) coupling the lipid moiety to the linker — spacer — peptide — support, such that a carboxylic acid group of the lipid moiety forms an amide bond with an amino group of the linker (lipid — linker — spacer — peptide — support); and(vi) releasing the lipidated GnRH analogue from the solid support.

[0230] Embodiment 51. The process of any one of Embodiments 47-50, wherein the unprotected amino group comprises an a-amino group at position AA1 of the peptide backbone of the GnRH analogue.

[0231] Embodiment 52. The process of any one of Embodiments 47-50, wherein the unprotected amino group comprises an s-amino group of a lysine residue at position AA5 or AA6 or AA8 of the peptide backbone of the GnRH analogue or an amino acid side chain thereof.

[0232] Embodiment 53. A method of treating a condition amenable to treatment by a GnRH analog, comprising administering the lipidated gonadotropin releasing hormone (GnRH) analogue of any one of claims 1-43 to a subject in need thereof.

[0233] Embodiment 54. The lipidated gonadotropin releasing hormone (GnRH) analogue of any one of Embodiments 1-43 for treating a condition amenable to treatment by a GnRH analog.

[0234] Embodiment 55. Use of the lipidated gonadotropin releasing hormone (GnRH) analogue of any one of Embodiments 1-43 in the preparation of a medicament for treating a condition amenable to treatment by a GnRH analog.

[0235] Embodiment 56. The method, lipidated GnRH analogue for treating, or use of any one of Embodiments 53-55, wherein the condition is one or more of prostate cancer, advanced prostatic carcinoma (urologic oncology), ovarian cancer, breast cancer, endometriosis, myoma, infertility, benign prostatic hyperplasia, uterine fibroids, menorrhagia, premenstrual dysphoric disorder (PMDD), severe PMS, central precocious puberty, or the use is as a puberty blocker such as for transgender or gender diverse youth, or for chemical castration, or the use is in a veterinary context, such as for hormone suppression (e.g. chemical castration / neutralization) of pets or livestock, or in an in vitro fertilization protocol for pets or livestock.

[0236] Embodiment 57. The method, lipidated GnRH analogue for treating, or use of any one of Embodiments 53-55, wherein the condition is advanced prostate cancer or metastatic stage prostate cancer, further optionally wherein the condition is advanced hormone-dependent prostate cancerin adult males, further optionally wherein the advanced hormone-dependent prostate cancer is high-risk localized or locally advanced hormone-dependent prostate cancer.

[0237] Embodiment 58. The method, lipidated GnRH analogue for treating, or use of any one of Embodiments 56-57, wherein the treatment is in combination with radiotherapy.

[0238] Embodiment 59. The method, lipidated GnRH analogue, or use of Embodiment 58, wherein the lipidated GnRH analogue is used as neo-adjuvant treatment prior to radiotherapy.

[0239] Embodiment 60. The method, lipidated GnRH analogue, or use of any one of Embodiments 52-54, wherein the condition is infertility, and the treatment is for promotion of controlled ovarian stimulation in an assisted reproduction protocol.9. Characterization of Representative Lipidated GnRH AnaloguesExample 1 - 7 / 7 vitro Characterization

[0240] Representative lipidated GnRH analogues LiGAl-LiGA6 as described herein were prepared and subjected to in vitro characterization as summarized below.Solubility

[0241] Kinetic solubility was tested by dissolving each LiGA compound referenced in Table 1 in DMSO and further performing a 20-fold dilution in each of the following aqueous buffers: (A) 10 mM PBS at pH 7.4 and (B) 10 mM glycylglycine at pH 8.5. The test samples were then stored up to 3 days at room temperature or refrigerated and assessed for turbidity both visually and microscopically.

[0242] Surprisingly, LiGAl, LiGA2, LiGA3, LiGA4, and LiGA5 exhibited low solubility at pH 7.4 (e.g. « 1 mM). This low solubility at physiological pH may advantageously provide a low Cmax and high mean residence time after parenteral administration, which optimizes the therapeutic window with each dosing thereby permitting minimizing dosing frequency and maximizing drugutilization (e.g. not overtreating the patient beyond profound / full castration). Also as shown in Table 1, all LiGA compounds are soluble at pH > 8.5.Table 1 Purity and Solubility

[0243] Additional solubility studies showed that LiGA 6 is soluble (> 20 mg / ml) at pH 4.4 and pH 7.1 and less soluble (<10 mg / ml) at pH 5 to 7.Self-Association / Fibrillation

[0244] A ThT fibrillation assay was conducted with LiGAl-LiGA6 on a fluorescence microplate reader (Varioskan / BioTek Synergy Neo2) to assess self-association (aggregation / fibrillation). Test compositions of LiGA compounds in 5% DMSO, glycylglycine (“glygly”) buffer at pH 8.5 were prepared, and a 15 minute shake was performed between each measurement. ThT fluorescence for LiGAl-LiGA6 was compared against degarelix (e.g., unlipidated degarelix). As shown in FIG. 3 and Table 2, LiGAl, LiGA5 and LiGA6 exhibited less propensity for fibrillation than degarelix (“Dega”). This is an advantageous property of the lipidated compounds as described herein. For example, a lower fibrillation potential enables the preparation and use (injection) of ready -to-use parenteral solutions with a thin needle (e.g., 31G), and provides better control (predictability) of pharmacokinetic properties than seen with degarelix, including the absorption phase and the elimination phase, since the lipidated compounds described herein do not form a depot in subcutaneous tissue like degarelix does.Table 2 Fibrillation propensity, impurities, and receptor affinity*As reported in Jiang, et al., J. Med. Chem. 2001, 44, 453-467.

[0245] ThT fibrillation assays were conducted with additional LiGA compounds on a Varioskan microplate reader to assess self-association (aggregation / fibrillation). Test compositions of LiGA compounds in PBS buffer at pH 8.5 were prepared, and a 15 minute shake was performed between each measurement. In one assay, the ThT fluorescence for Li GAI, LiGA8, LiGA9, and Li GAI 0 (all lipidated at AA1) was compared against degarelix (e.g., unlipidated degarelix). As shown in FIG. 4 and consistent with the results reported above, LiGAl, LiGA8, LiGA9, and LiGAlO exhibited less propensity for fibrillation than degarelix. In another assay, the ThT fluorescence for LiGAl 3-LiGA20 was compared to unlipidated LiGA5. As shown in FIG. 5, LiGA20 (having an acyline peptide backbone with a series A Cl 8 lipid side at AA6), LiGAl 5 (having the same peptide backbone as LiGA5 but having a series B C18 lipid side chain), and LiGAl 8 (having an abarelix peptide backbone with a series A Cl 8 lipid chain at AA1 ) exhibited more propensity for fibrillation than the others.Chemical Stability

[0246] Chemical stability of the compounds formulated at 0.1 mM was determined at 40°C in 50 mM glygly buffer pH 8.5. The results are shown in FIG. 6A. LiGAl and LiGA4 showed lessstability, which may be associated with known chemically-labile hotspots of the degarelix backbone (AA5, AA6, AA8, and the C-terminal group). Chemical stability of LiGA6 formulated at 0.1 mM and pH 5 - pH 8.5 (10 mM PBS + 10 mM citrate) was determined at 40 °C and compared to degarelix formulated at 0.1 mM pH 5.0. The results are shown in FIG. 6B and FIG. 6C. hGnRH Receptor Affinity

[0247] hGnRH receptor affinity was determined at Eurofins (in Chem-1 cells with 0.2% albumin) according to the method described in Halmos et al., 1996, Proc Natl Acad Set USA. 93(6): 2398- 2402. The results are shown in FIG. 7A and FIG. 7B. The receptor affinity / half maximal inhibitory concentration (IC50) of LiGA compounds was in the range of 10'8-l O'7M (pIC50 ranging from 6.9-7.3). This represents a 10-100-fold reduction as compared to marketed GnRH antagonists (pIC50 = 9.1 for degarelix and 9.3 for ganirelix, as reported by Jiang et al., J. Med. Chem., 2001, vol. 44[3]). Without being bound by theory, this could be attributed to lipidation leading to tight but reversible binding to albumin present in the receptor binding assay, thereby reducing the free, unbound fraction to < 1%. Since it is possible lipidated GnRH analogues could exhibit receptor binding activity in a bound state, these results highlight the potential importance of lipidation on site(s) that preserve receptor binding ability of the lipidated molecule. Additionally, the reduced receptor potency of lipidated GnRH analogues indicated by these results may be balanced by a significantly higher plasma half-life (10-100 fold higher), higher relative bioavailability (1-3 fold higher) and higher plasma exposure via an estimated reduced volume of distribution (5-10 fold).Example 2 - Pharmacokinetic and Pharmacodynamic Studies in Male Rats

[0248] A pK study was conducted in adult male rats after subcutaneous administration of compounds listed in Table 3. The pK profiles are shown in FIG. 8A for the 0.25 mg dose and in FIG. 8B for the 2.5 mg dose. Table 4 reports the Tmaxand Cmaxdata.Table 3 pK StudyTable 4 T max and C max

[0249] The results indicate that the LiGA compounds exhibit a significantly longer plasma halflife (especially LiGAl and LiGA6) as compared with semaglutide, as reported in Lau, J. et al., J. Med. Chem. 2015, 58, 18, 7370-7380. More specifically, the half-life of LiGAl and LiGA6 was twice as long as that reported for semaglutide.

[0250] FIG. 8C shows the testosterone lowering effect of LiGAl, LiGA5, and LiGA6 as compared to ganirelix in intact male Sprague Dawley rats up to 24 hours following subcutaneous injection of doses varying from 0.0025 to 2.5 mg per rat (n=5). Plasma concentration of testosterone was determined by a LC / MS method. In one group dosed with 2.5 mg LiGA5, the plasma concentration of testosterone after 24 hours was below the detection limit.

[0251] A plasma protein binding study was conducted with human plasma and rat plasma with LiGAl-LiGA6. The tested compounds were added individually or as a mixture of multiple compounds to human or rat plasma at a single concentration. After incubation to allow binding between the test compounds and plasma protein, ultracentrifugation was used to separate plasma protein-bound and unbound test compound. The concentration of unbound test compound was determined by LC / MS analytical methods. Test compound concentrations were calculated from standard calibration curves generated in the same plasma matrix to negate possible compound loss from nonspecific binding to the experimental apparatus. Table 5 reports the percentage of unbound compound in human plasma and rat plasma. As seen in the table, for all LiGA compounds tested, < 1% free compound was detected, indicating that the compounds are strongly bound to plasma proteins hereby providing basis for prolonged plasma circulation time.Table 5 Free amount in human and rat plasmaExample 3 - Additional In Vitro Studies

[0252] Physicochemical screening of additional LiGA compounds was conducted to assess kinetic solubility in PBS (20 mM phosphate, 145 mM NaCl, pH 7.4), fibrillation propensity, and accelerated chemical stability at 40°C.

[0253] Liquid LiGA peptide samples were prepared by dissolving neat LiGA peptide powder with pure DMSO to a concentration of 20 mg / ml. The peptide / DMSO stock solution was diluted to a final peptide concentration of 1 mg / ml with a phosphate buffer solution pH 7.4 (corresponding toa 20-fold dilution). The 1 mg / ml LiGA solutions were assessed by visual examination and / or via light scattering measured at 350 nm in a plate reader instrument. UV absorbance signals higher than 0.05 (corrected from background signal) were considered as turbid samples and indicative of a LiGA solubility below 1 mg / ml in PBS at room temperature, while clear samples or UV350 nm below 0.05 were indicative of a peptide solubility above 1 mg / ml.

[0254] Thioflavin T (ThT) fluorescence was used to characterize fibrillation propensity. Samples of 1 mg / ml LiGA peptide in PBS and 5% w / w DMSO were mixed with Thioflavin T (ThT) to a final concentration of 4 pM ThT and filled into 96 well black titer plates and sealed. The samples were incubated at 40°C and further stressed by shaking the plate for a minimum of 48 hours. Fluorescence was measured every 15-20 minutes for a minimum of 48 hours by exciting the ThT at 450 nm and measuring the emission at 480 nm. Fibrillation propensity in % relative to a degarelix reference was measured as the maximum ThT fluorescence signal in the LiGA sample normalized with the maximum ThT fluorescence signal from a 1 mg / ml degarelix reference. A value below 10% is considered to reflect a low fibrillation propensity.

[0255] For chemical stability, an RP-HPLC method using an Agilent or Waters HPLC instrument equipped with an Kinetex 2.6 um, Cl 8 100 A column, 150x4.6 mm was employed. Samples of Img / ml LiGA peptide in PBS were further diluted to 0.1 or 0.2 mg / ml in PBS, pH 7.4, and incubated at 40°C for 2 or 4 weeks. The total amount of various peptide related impurity species was determined as area relative to total area in percentage and the increase (A) in impurities relative to T=0 was reported. Results are set forth in the table below, where “nd” means not determined. FIG. 9 sets forth a chromatogram for LiGA 122 after 2 weeks incubation at 40°C as an example.Human GnRH Receptor Inositol phosphate Assay

[0256] An assay was conducted to screen LiGA compounds as described herein for GnRH antagonist activity, e.g., to identify potent gonadotropin-releasing hormone receptor (GnRHR) antagonists. A Chem-1 rat cell line, genetically modified to express a cloned human GnRH receptor, was used in the assay. Ready -to-assay cells were plated at 15000 cells / well in a 384- well plate format and incubated for 15 minutes with a titration of 5-fold diluted LiGA compound. The cells were stimulated with luteinizing hormone-releasing hormone (LHRH) at a concentration of 15 nM, corresponding to EC90 of LHRH, for 2 hours at 37°C. To measure accumulated inositol phosphate (IP1 ) in the cells, a competitive homogeneous time resolved fluorescence (HTRF) IP- One Gq immunoassay from PerkinElmer was used. LiGA5 was used as an assay control to compare results across assay runs, and cells with no treatment (media only) and cells stimulated with LHRH at EC90 were used to normalize the data. To calculate IC50, normalized values were plotted against the log of the concentration, and an IC50 value was derived from nonlinear regression to a four-parameter logistic equation. The geometric mean of the IC50 from at least two independent experiments is reported for each compound in the table below.

[0257] In the table below, the peptide backbones are described by the changes relative to a reference peptide (e.g., degarelix), and the lipid side chains are described by nomenclature “(FA{LetterNumber})” where Letter denotes the lipid side chain series and Number denotes the length of the saturated fatty di(acid) lipid moiety. For example, for LiGA47, “[Tyr5, D-Lys6, Lys(FA{A18})8]Degarelix” denotes that the peptide backbone has substitutions at positions AA5 (Tyr), AA6 (D-Lys), and AA8 (Lys(FA{Al 8})) relative to degarelix, and is lipidated at AA8 with a lipid side chain of series A with a saturated Cl 8 fatty (di)acid lipid moiety. For LiGA31, “[D- Lys(FA{A18})6]Cetrorelix” denotes that the peptide backbone has a substitution at position AA6 (D-Lys(FA{Al 8}) relative to cetrorelix, and is lipidated at AA6 with a lipid side chain of series A with a saturated Cl 8 fatty (di)acid lipid moiety.Example 4 - Additional Studies in Rats

[0258] A PK / PD study in adult male Sprague Dawley rats was performed with LiGA5, LiGA28, LiGA43, LiGA151 and LiGA152. A single dose of each LiGA compound was administered subcutaneously to six rats at dose level of 450 mg / kg. Plasma samples were collected for up to 104 hours after the dosing and analyzed for LiGA (peptide) exposure (N=3) or testosterone level (N=3) by LC-MS. The pK parameters are presented in Table 6; plasma concentration vs. time profiles are shown in FIG. 10.Table 6. pK parameters in rats following SC administration of LiGA Compounds

[0259] FIG. 11 shows the testosterone lowering effect of LiGA5, LiGA28, LiGA43, LiGA151, and LiGA152 as compared to PBS in intact male Sprague Dawley rats up to 48 hours following subcutaneous injection of doses of 450 nmol / kg per rat (n=3). A testosterone level of 0.2 ng / ml (dashed line) is considered to reflect profound castration, while a testosterone level of 0.5 ng / ml is considered to reflect castration.10. Preparation of Representative Lipidated GnRH Analogues

[0260] In the description that follows, methodology is described in the context of a LiGA having a peptide backbone based on degarelix as a representative lipidated GnRH analogue. Those skilled in the art will be able to adopt the methodologies described herein to lipidated GnRH analogues having a different peptide backbone.Method Example A: General process for synthesis of peptide backbone on a solid support

[0261] A peptide backbone is synthesized stepwise, using Solid Phase Peptide Synthesis (SPPS) on a Rink-Amide resin, by the following process. Prior to use, the Rink- Amide resin is swelled in DMF, drained, and then washed in DMF before being drained again and the Fmoc protecting group is removed by treating the resin with a 20% (v / v) solution of piperidine in DMF.

[0262] Fmoc-protected amino acids of the peptide backbone are pre-activated with Oxyma (2 equivalents) and DIC (2 equivalents, followed by addition of another 2 equivalents during coupling) and sequentially coupled to the resin. After each amino acid coupling step, the peptide- loaded resin is washed with DMF and the Fmoc protecting group is removed by treating the peptide-loaded resin with a 20% (v / v) solution of piperidine in DMF.

[0263] Lipidation of the peptide backbone can be carried out by first treating the peptide-loaded resin with a 20% (v / v) solution of piperidine in DMF to remove the Fmoc protecting group from the N-terminal moiety and washing with DMF. Lipid side chain (e.g., 1.5 equivalents) synthesized according to general Method Example B below can be coupled to the peptide-loaded resin with, e.g., 1.5 / 3.0 equivalents of PyBOP / DIPEA in DMF relative to the peptide-loaded resin. An additional charge of the protected lipid side chain, PyBOP and DIPEA may be necessary to achieve acceptable coupling of the protected lipid side chain to the peptide-loaded resin.Method Example B: General process for synthesis of protected lipid side chain on a solid support

[0264] This method describes synthesis of a lipid side chain in a stepwise manner at a 5 mmol scale on a 2-CTC resin, for use in a convergent lipidation method as described herein.Loading of the 2-CTC resin

[0265] After swelling and washing of the 2-CTC resin in DCM, a loading solution is prepared by dissolving an Fmoc protected spacer (for example, Fmoc-Ado-OH) in DCM and adding DIPEA. The loading solution is added to the resin with agitation to couple the Fmoc-spacer-COOH to the 2-CTC resin.Capping of the 2-CTC resin

[0266] To cap the un-reacted sites on the 2-CTC resin (i.e., remaining chloride sites), a solution of DCM, MeOH and DIPEA is added to the resin.Coupling Step 1: Fmoc deprotection

[0267] The resin is flushed with nitrogen gas and washed with DMF. The Fmoc protecting group on the spacer is removed by treating the resin with a 20% (v / v) piperidine in DMF with stirring.Coupling Step 2: Spacer and fatty acid coupling

[0268] 1.5 to 2.0 equivalents of the spacer (for example, Fmoc-Ado-OH), ethyl 2-cyano-2- hydroxy imino-acetate (Oxyma Pure) (1.5 to 2 equivalents) and DIC (3 to 4 equivalents) in DMF are added to the resin. The resin is then washed with DMF. Similar steps are conducted to couple the linker (for example, Fmoc-L-Glu-OtBu) and the fatty acid (for example, octadecanedioic acid mono-tert-butyl ester, a mono-protected Cl 8 diacid)) to the link or spacer. After the final coupling, the resin is washed with DMF and DCM.Cleavage of the lipid side chain from the 2-CTC resin

[0269] The resin is swelled and washed in DCM. The resin is treated with 5 x 20 % HFIP in DCM and washed in DCM (collecting and pooling filtrates). The combined filtrates are concentrated in vacuo affording the crude lipid side chain which is isolated after drying in a vacuum oven for 48 to 72 hours.

[0270] The crude lipid side chain can be obtained with 90% yield with a HPLC purity of greater than 90% (214 nm). No further purification is required for the subsequent on-resin convergent lipidation.Method Example C: General Procedure for lipidation of peptide backbone on a solid support

[0271] This method describes lipidation of a peptide backbone carried out while the peptide backbone is on the solid support. This method can include sequential preparation of the lipid side chain on the peptide backbone, or can involve coupling of a pre-formed lipid side chain (e.g, prepared as described in Method Example B). In either approach, the lipid side chain is coupled to an un-protected amino group of the peptide backbone, such as an amino group at position AA1, AA5, and / or AA6 of the peptide backbone.Sequential lipidation

[0272] Using methodology similar to that outlined above, each of the spacer, linker, and fatty acid (3 equivalents), is dissolved in DMF with PyBOP (3 equivalents) and DIPEA (6 equivalents), and added to the peptide-loaded resin to sequentially couple the spacer, linker and fatty acid to the peptide-loaded resin (e.g., peptide backbone on the resin) with intermediate Fmoc-deprotection steps.Convergent lipidation

[0273] The protected lipid side chain synthesized as per Method Example B above (2.2 equivalents) and PyBOP (2.2 equivalents) and DIPEA (4.4 equivalents) are dissolved in DMF and added to the peptide-loaded resin. A second charge of the protected lipid side chain, PyBOP and DIPEA may be necessary to achieve acceptable coupling of the protected lipid side chain to the peptide-loaded resin.Cleavage of lipidated- peptide backbone from the resin

[0274] The lipidated peptide-loaded resin is treated with TFA / H2O (95:5) for global deprotection and cleavage from the resin and is then filtered. The resulting filtrate containing the crude lipidated peptide backbone is then neutralized using a neutralization solution containing NEUAc in H2O and EtOH and is purified in two chromatographic steps (RP-HPLC and SPE). The lipidated peptide is isolated as the corresponding acetate salt following lyophilization. The purified peptide may have an HPLC purity of greater than 90% (214 nm).Method Example D: Synthesis of LiGAl - LiGA6

[0275] Synthesis of LiGAl - LiGA6 was carried out in a parallel synthesis using a Fmoc Rink amide resin.SPPS Assembly

[0276] The common C-terminal tetrapeptide motif (amino acids AA10 to AA7) of LiGAl to LiGA6 was synthesized on a Rink-Amide resin on a 12 mmol scale as follows. After swelling and washing of the resin in DMF, the Fmoc protecting group of the Rink Amide resin was removed with a 20% (v / v) solution of piperidine in DMF and the resin was washed with DMF. Fmoc-D- Ala-OH (AA10), Fmoc-Pro-OH (AA9), Fmoc-Lys(iPr, Boc)-OH (AA8) and Fmoc-Leu-OH (AA7) were pre-activated with Oxyma (2 equivalents) and DIC (4 equivalents) and sequentially coupled to the resin. After each amino acid coupling step, the peptidyl-resin was washed with DMF and the Fmoc protecting group was removed by treating the peptidyl-resin with a 20% (v / v) solution of piperidine in DMF.

[0277] The Fmoc-Leu-Lys(z-Pr, Boc)-Pro-D-Ala-Rink Amide resin was then divided into 8 portions for preparation of the final peptide backbones by coupling amino acids AA7 to AA1. The six analogues were prepared with the same amino acids at AA3-AA1, with AA6-AA4 being as follows:LiGA 1 : AA6 = D-Aph(Cbm); AA5 = Aph(Hor); AA4 = Ser(tBu);LiGA 2: AA6 = D-Lys(Mtt) ; AA5 = Aph(Hor); AA4 = Ser(tBu);LiGA 3: AA6 = D-Aph(Cbm); AA5 = Lys(Mtt); AA4 = Ser(tBu);LiGA 4: AA6 = D-Aph(Cbm); AA5 = Aph(Hor); AA4 = Lys(Mtt);LiGA 5: AA6 = D-Lys(Mtt); AA5 = Tyr( / Bu); AA4 = Ser(tBu);LiGA 6: AA6 = D- / ra / ?.s-Hyp ; AA5 = Lys(Mtt); AA4 = Ser(tBu);LiGA 2’: AA6 = D-Lys(Alloc); AA5 = Aph(Hor); AA4 = Ser(tBu);LiGA 3’: AA6= D-Aph(Cbm); AA5 = Lys( Alloc); AA4 = Ser(tBu);

[0278] For LiGA2 and LiGA3, two variations were prepared using different protecting groups (Mtt or Alloc).

[0279] The resin was swelled, washed in DMF, and treated with a 20% (v / v) solution of piperidine in DMF to remove the Fmoc protecting group. After washing with DMF, AA6 to AA1 were preactivated with Oxyma (2 equivalents) and DIC (4 equivalents) and then sequentially coupled to the peptidyl-resin. After each amino acid was coupled to the peptidyl-resin, the peptidyl-resin was washed with DMF and the Fmoc protecting group was removed by treating the peptidyl-resin with a 20% (v / v) solution of piperidine in DMF.

[0280] The crude peptides had an HPLC purity of 60 to 86%, as shown in Table 6B below.Lipidation ofLiGAl

[0281] A degarelix peptide backbone (without the N-terminal acetyl group) was synthesized on a Rink amide resin as described above. Lipidation was carried out on the peptidyl-resin by treating the peptidyl-resin with a 20% (v / v) solution of piperidine in DMF to remove the Fmoc protecting group from the N-terminal D-Nal moiety. The peptidyl-resin was then washed with DMF.

[0282] The crude lipid side chain (1.5 equivalents) synthesized according to Method Example B was coupled to the peptidyl-resin with 1.5 / 3.0 equivalents of PyBOP / DIPEA in DMF relative to the peptidyl-resin. In order to obtain acceptable conversion to the desired product, a total of four treatments with 1.5 / 1.5 / 3.0 equivalents of the lipid side chain / PyBOP / DIPEA in DMF was required.Lipidation ofLiGA2 - LiGA5

[0283] The LiGA2-LiGA5 peptidyl-resins were treated with a 20% (v / v) solution of piperidine in DMF to remove the Fmoc protecting group from the N-terminal D-Nal moiety. The resin was then washed with DMF, followed by treatment with glacial acetic acid (4 equivalents), Oxyma Pure (4 equivalents) and DIC (8 equivalents) in order to acetylate the N-terminal moiety. The peptidyl- resin was then washed with DCM.

[0284] The lysine residue at position AA4, AA5 or AA6 (depending on the compound) was liberated by six successive treatments with 30% HFIP in DCMto remove the Mtt protecting group. The peptidyl-resin was then washed with DCM and DMF.

[0285] The lipid side chain was synthesized according to Method Example B and lipidation was carried out on the peptidyl-resin by treating the peptidyl-resin with 1.65 / 1.65 / 3.3 equivalents of lipid side chain / PyBOP / DIPEA in DMF relative to the peptidyl-resin for LiGA2 and 2.2 / 2.2 / 4.4 equivalents of lipid side chain / PyBOP / DIPEA in DMF relative to the peptidyl-resin for LiGA3- L1GA5.

[0286] In order to obtain acceptable conversion to the desired product, a total of two treatments of the lipid side chain / PyBOP / DIPEA in DMF was required to couple the lipid side chains.Lipidation ofLiGA6

[0287] Lipidation of LiGA6 was carried out by a sequential approach according to Method Example C above, wherein the lipid side chain was synthesized stepwise on the N-terminus of degarelix. N-terminal deprotection and acetylation as well as removal of the Mtt group on the lysine at AA5 was carried out in the same manner as for LiGA2 - LiGA5.

[0288] Upon liberation of the primary amine on the lysine side chain at AA5, the lipid side chain was synthesized in a step-wise manner as described in Method Example C using 3 equivalents of each of the spacer, linker, and 2 equivalents of the mono-protected fatty diacid, 2 - 3 equivalents of PyBOP, and 4 - 6 equivalents of DIPEA, all relative to the peptidyl-resin.Cleavage and global deprotection

[0289] Cleavage and global deprotection of the lipidated peptidyl-resins of LiGAl-LiGA6 was carried out using TFA / H2O (95:5), followed by filtration of the resin, to obtain crude lipidated analogues in solution.

[0290] The crude solutions were neutralized using a neutralization solution containing NHjAc in H2O and EtOH. The neutralized (pH = 3 to 4) crude solutions were stored cold (5 °C) until final purification.Purification

[0291] LiGAl-LiGA6 were purified from the crude neutralized solutions in two chromatographic steps (RP-HPLC and SPE) and isolated as their corresponding acetate salts following lyophilization. The purification steps are outlined below.

[0292] Crude LiGAl-LiGA6 were each dissolved in an acetonitrile aqueous solution with appropriate pH adjustments. Each obtained peptide solution was filtered through a 0.45 m microporous membrane and was subjected for chromatographic purification.

[0293] The first purification step was reverse phase high performance liquid chromatograph (RP- HPLC) with silica bound to Cl 8 resin (particle size of 15 pm) as the stationary phase. A mobile phase containing 0.1% trifluoroacetic acid (TFA) and acetonitrile in water was used, and elution was performed by gradually increasing the acetonitrile concentration in the mobile phase, while recovering the fractions containing LiGA at 290 nm UV.

[0294] Optionally, the fractions containing the pooled LiGA obtained in the first RP-HPLC step were subjected to a second reverse phase HPLC purification with silica bonded to Cl 8 resin (particle size of 5 pm) as the stationary phase. A mixture containing 0.1% trifluoroacetic acid (TFA) and acetonitrile in water was used as the mobile phase, and the elution was performed by gradually increasing the acetonitrile concentration in the mobile phase, while recovering the fraction containing the purified LiGA at 290 nm UV.

[0295] Optionally, the LiGA obtained in the previous steps was subjected to peptide salt exchange, by Solid Phase Extraction (SPE), in which silica bound to Cl 8 resin (particle size of 15 pm) was used as the stationary phase. Using a buffer containing pH adjusted ammonium acetate and acetonitrile in water, a salt exchange wash was performed. With a mobile phase containing 1%acetic acid (AcOH) and acetonitrile in water, elution was performed by gradually increasing the acetonitrile concentration, and the salt-exchanged LiGA peak was collected at 290 nm UV.

[0296] Prior to isolation, SPE pools were concentrated by evaporation in vacuo using a rotary evaporator. The pressure was slowly reduced while keeping the temperature at 38 °C and rotation speed at about 110 rpm.

[0297] The LiGA may be isolated by freeze-drying prior to packaging.

[0298] The pooled fractions obtained after each step were analyzed by analytical RP-UHPLC as a relative peak area using UV detection at 215 nm to evaluate the efficiency of each step. The purity of the final isolated powder was assessed > 90%.Characterization

[0299] The crude analogues of the LiGAl-LiGA6 acetate salts were analyzed by RP-HPLC and LC-MS / MS and submitted to ' D,JH and 2DJH-13C HSQC NMR for additional structural confirmation and content determination (including acetate content) as well as peptide content determination by nitrogen quantification.

[0300] Tables 6 A and 6B provide a summary of the synthesis and characterization data for LiGAl-LiGA6. Overall, the target purity of > 93% was reached, with individual impurities being < 2 % as determined by LC-MS (@ 215 nm), as shown in Table 6B below:Table 6A1HPLC at 214 nm.2HPLC at 215 nm.Table 6B1HPLC at 215 nm.2Peptide content reported in free base.Preparation of Fmoc-D-Asp(1.2,3.4-tetrahydroisoquinoline amide)-OH Building Block

[0301] For embodiments comprising a D-Asp(l,2,3,4-tetrahydroisoquinoline amide) moiety in the peptide backbone, the following methodology was used to prepare Fmoc-D-Asp(l, 2,3,4- tetrahydroisoquinoline amide)-OH building blocks for use in preparation of lipidated GnRH analogs.1. Synthesis of Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu

[0302] Fmoc-D-Asp-OtBu (20.017 g, 48.65 mmol, 1.0 equiv.) was weighed into a 2 L round bottom flask. Me-THF (500 mL) was prepared in a volumetric cylinder. HOBt (13.141 g, 97.25 mmol, 2.0 equiv.) was weighed and added to the round bottom flask containing Fmoc-D-Asp-OtBu and transferred fully with a portion of the prepared Me-THF. EDC (18.341 g, 97.24 mmol, 2.0 equiv.) was weighed and transferred to the round bottom flask by flushing with remaining portion of Me-THF. Stirring was applied. DIPEA (25.5 mL, 146.0 mmol, 3.0 equiv.) was added. 1,2,3,4-tetrahydroisoquinoline (7.046 mL, 53.47 mmol, 1.1 equiv.) was added and the reaction mixture turned yellow. The reaction mixture was left to stir at room temperature for 3.5 hours. The reaction mixture is a yellow solution with white precipitate.

[0303] An aqueous solution of 10% HC1 (400 mL) was added and the reaction mixture was transferred to a separation funnel (exotherm). The phases were separated and the organic phase washed with saturated NaHCCF (400 mL). The organic phase was dried over MgSCL and concentrated in vacuo. The remaining yellow residue was dried over a flow of N2 for approximately 72 hours. The yellow residue was re-dissolved in DCM (60 mL) and loaded onto a silica column for purification by flash chromatography using a Heptane / EtOAc eluent system. Product-containing fractions were pooled and concentrated in vacuo affording the intermediate Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu (85 % yield).2. Synthesis of Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OH

[0304] Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OtBu obtained as described above was dissolved in DCM (approx. 10 mL / g) and TFA was added (approx. 10 mL / g) and the reaction mixture was stirred for 3 hours. The reaction mixture was subsequently concentrated in vacuo and the resulting residue co-concentrated twice with toluene obtaining an oily residue. The oily residue was dried under vacuum at 30 °C overnight. The dried residue was re-dissolved in MeCN (approx. 20 mL / g) and water was added (approx. 20 mL / g) obtaining an emulsion. The emulsion was stirred vigorously immediately before flash freezing in a dry ice / acetone bath and lyophilization. Following lyophilization, the product (obtained as an orange-white powder) was re-crystallized. The solids were dissolved / suspended MeCN (approx. 20 mL / g), stirred, and heated to 60 °C for 10 min. Heating was turned off and stirring was continued for another 1 hour obtaining a thick slurry to which water (approx. 22 mL / g) was added. Stirring was continued another 45 min before addition of another portion of water (approx. 65 mL / g). The solids were filtered off and theresulting filter cake was washed with water. The filtrates were dried in vacuo at 35 °C for 48 hours obtaining the product Fmoc-D-Asp(l,2,3,4-tetrahydroisoquinoline amide)-OH as a white to off- white powder (65 % yield).

[0305] The foregoing examples are illustrative of the invention described herein, but are not intended to limit the scope thereof in any way. Other aspects and advantages of the invention will be apparent to those skilled in the art to which the invention pertains.

Claims

1. WHAT IS CLAIMED IS:

1. A lipidated gonadotropin releasing hormone (GnRH) analogue, wherein the lipidated GnRH analogue comprises a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone at one or more of amino acid positions 6 (AA6), 5 (AA5), and 1 (AA1) of the peptide backbone.

2. A lipidated gonadotropin releasing hormone (GnRH) analogue, wherein the lipidated GnRH analogue comprises a lipid side chain coupled to GnRH or a GnRH analogue peptide backbone at one or more of amino acid positions 6 (AA6), 5 (AA5), 1 (AA1), and 8 (AA8) of the peptide backbone.

3. The lipidated GnRH analogue of claim 1 or claim 2, wherein the lipid side chain is coupled to amino acid position 6 (AA6) of the peptide backbone.

4. The lipidated GnRH analogue of claim 1 or claim 2, wherein the lipid side chain is coupled to amino acid position 1 (AA1) of the peptide backbone.

5. The lipidated GnRH analogue of claim 1 or claim 2, wherein the lipid side chain is coupled to amino acid position 5 (AA5) of the peptide backbone.

6. The lipidated GnRH analogue of claim 2, wherein the lipid side chain is coupled to amino acid position 8 (AA8) of the peptide backbone.

7. The lipidated GnRH analogue of any one of claims 1-6, wherein the lipidated GnRH analogue exhibits GnRH antagonist activity.

8. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of a GnRH antagonist selected from degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

9. The lipidated GnRH analogue of any one of claims 1 -7, wherein the GnRH analogue peptide backbone has the amino acid sequence of degarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

10. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of abarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

11. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of cetrorelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

12. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of ganirelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

13. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of prazarelix, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

14. The lipidated GnRH analogue of any one of claims 1-7, wherein the GnRH analogue peptide backbone has the amino acid sequence of acyline, or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto.

15. The lipidated GnRH analogue of any one of claims 1-14, wherein the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10 (AA1-AA10):

16. The lipidated GnRH analogue of any one of claim 1-2 and 7-15, wherein the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10 (AA1-AA10):

17. The lipidated GnRH analogue of any one of claim 1-3 and 7-15, wherein the lipid side chain is coupled to amino acid position 6 (AA6) of the GnRH analogue peptide backbone and the GnRH analogue peptide backbone has any of the following amino acids at positions 1-10 (AA1-AA10):

18. The lipidated GnRH analogue of any one of claims 1-17, wherein the GnRH analogue peptide backbone has an amino acid sequence with one of the following sets of amino acids present in degarelix:(i) AA1: D-Nal, AA2: D-Cpa, and AA3: D-Pal; or(ii) AA4: Ser, AA7: Leu, and AA9: Pro; or(iii) AA1: D-Nal, AA2: D-Cpa; AA3: D-Pal; AA4: Ser, AA7: Leu, and AA9: Pro or(iv) AA2: D-Cpa, AA4: Ser, AA7: Leu, and AA9: Pro; or(v) AA2: D-Cpa, AA4: Ser, AA7: Leu, and AA9: trans-Hyp; or(vi) AA2: D-Cpa, AA4:Ser, AA7: Leu, and AA9: Ado; or(vii) AA2: D-Cpa, AA4: Ser, AA7 N-Me-Leu, and AA9: Pro.

19. The lipidated GnRH analogue of any one of claims 1-18, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, or anti de, at one of the following sets of positions:AA6;AA5 and AA6;AA6 and AA8;AA5, AA6, and AA10;AA3, AA5, and AA6;AA1, AA3, AA5, and AA6;AA5, AA6, and AA7;AA5, AA6, and AA8;AA5, AA6, and AA9;AA3, AA5, and AA9;AA3, AA5, AA6, and AA10;AA5, AA6, AA7, and AA10;AA5, AA6, AA9 and AA10;AA5, AA6, AA7, AA8 and AA10;AA3, AA5, AA6, AA9 and AA10;AA1, AA3, AA5, AA6, and AA8;AA1, AA3, AA5, AA6, and AA10;AA1, AA3, AA5, AA6, AA7 and AA10; orAA1, AA3, AA5, AA6, AA7, AA8 and AA10, optionally wherein the substitutions are relative to degarelix.

20. The lipidated GnRH analogue of claim 19, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix selected from the following sets, where Lys* denotes point of attachment of lipid side chain:AA6: D-Lys*;AA5 and AA6: AA5 is Tyr, Lys*, Tic, Phe(4-F), Phe(4-CN), Phe(4-Cl), or N-Me-Tyr, and AA6 is D-Lys* or trans-D-Hyp;AA3, AA5, and AA6: AA3 is D-3-Pal, D-Tyr, D-Trp, D-Gln, D-Gln(Me2), or D-Asn, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*;AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Ser, Sar, N-Me-D-Ala, D-Thr, D-Ser, D-Hse, beta-Ala, Ala, D-Ala, Ado, 6-Ahx, or absent;AA5, AA6, and AA7 : AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-Leu or Nle;AA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* or D-Lys, and AA8 is Lys*, Lys(Mes) or Arg;AA5, AA6, and AA9: AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-Hyp;AA3, AA5, AA6, and AA9: AA3 is D-Asn, AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA9 is trans-Hyp;AA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*;AA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr or Tyr, AA6 is D-Lys*, and AA10 is Gly or N-Me-D-Ala or absent;AA5, AA6, AA9 and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, AA9 is Pip, cis-D-Hyp, trans-Hyp, or Pro, and AA10 is absent;AA3, AA5, AA6, AA9 and AA10: AA3 is D-Gln(Me2) or D-Asn, AA5 is N-Me-Tyr, AA6 is D-Lys*, AA9 is trans-Hyp, and AA10 is N-Me-D-Ala or absent; orAA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is N-Me-D-Ala, Sar, or absent.

21. The lipidated GnRH analogue of claim 19, wherein the GnRH analogue peptide backbone has substitutions relative to degarelix selected from the following sets, where Lys*denotes point of attachment of lipid side chain:AA5 and AA6: AA5 is Tyr or Tyr(SC>2F) and AA6 is D-Lys*;AA3, AA5, and AA6: AA3 is D-Gln(Me2), D-Trp, or D-Tyr, AA5 is Tyr or N-Me-Tyr, and AA6 is D-Lys*;AA1, AA3, AA5, and AA6: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is Tyr, and AA6 is D-Lys*;AA5, AA6, and AA10: AA5 is Tyr or N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly, Sar, Ala, beta- Ala, N-Me-D-Ala, D-Ser, D-Hse or absent;AA5, AA6, and AA7 : AA5 is Tyr, AA6 is D-Lys*, and AA7 is N-Me-Leu;AA5, AA6, and AA8: AA5 is Tyr, AA6 is D-Lys* and AA8 is Lys(Mes) or Arg;AA1, AA3, AA5, AA6, and AA10: AA1 is D-Asp(l,2,3,4-tetrahydroisoquinoline amide), AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is absent;AA3, AA5, AA6, and AA10: AA3 is D-Gln(Me2), AA5 is N-Me-Tyr, AA6 is D-Lys*, and AA10 is Gly or absent; orAA5, AA6, AA9 and AA10: AA5 is N-Me-Tyr, AA6 is D-Lys*, AA9 is trans-Hyp, and AA10 is absent.

22. The lipidated GnRH analogue of claim 1 or claim 2, wherein the lipidated GnRH analogue exhibits GnRH agonist activity, optionally wherein the GnRH or GnRH analogue peptide backbone has the amino acid sequence of a GnRH agonist selected from GnRH, leuprolide, goserelin, triptorelin, nafarelin, buserelin, histrelin, fertirelin, and deslorelin or an amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions relative thereto, optionally having one, two, three, or four amino acid substitutions relative thereto.

23. The lipidated GnRH analogue of any one of the preceding claims, comprising or further comprising one or more amino acid substitutions selected from:(i) an amino acid substitution that introduces an amino acid amenable to coupling with a lipid side chain, optionally at one or more of AA5, AA6, and AA8;(ii) an amino acid substitution that introduces an aromatic amino acid at one or more of AA5 and AA6;(iii) an amino acid substitution that increases hydrophilicity of the GnRH analogue;(iv) an amino acid substitution that introduces Lys(iPr) at AA8; and / or(v) an amino acid substitution that introduces an amino acid present at a corresponding position in another GnRH analogue.

24. The lipidated GnRH analogue of any one of the preceding claims, wherein the lipid side chain comprises a lipid moiety, and optionally further comprises one or both of a linker and a spacer.

25. The lipidated GnRH analogue of any one of the preceding claims, wherein the lipid side chain comprises a lipid moiety, a linker, and a spacer.

26. The lipidated GnRH analogue of any one of the preceding claims, wherein the lipid side chain comprises a lipid moiety comprising a C14-C20 fatty acid, optionally selected from a saturated C8-C20 fatty (di)acid; a saturated C8-C20 fatty (mono)acid; a saturated C8-C20 fatty acid tetrazole, and a saturated C8-C20 fatty acid sulfonic acid.

27. The lipidated GnRH analogue of any one of the preceding claims, wherein the lipid side chain comprises a lipid moiety selected from:,wherein *** denotes point of attachment to the remainder of the lipid side chain or to the peptide backbone if the lipid side chain consists of the lipid moiety, optionally wherein the lipid side chain comprises a linker and *** denotes point of attachment to the linker.

28. The lipidated GnRH analogue of any one of claims 24-27, wherein the linker comprises one or more selected from:wherein ** denotes the point of attachment to the spacer or to the peptide backbone if the lipid side chain does not include a spacer and *** denotes point of attachment to the fatty acid.

29. The lipidated GnRH analogue of any one of claims 24-28, wherein the spacer comprises one or more selected from:(* * Ado-Ado-Lys(Me3)-Ado*),(**Ado-NH-(CH2CH2O)3-CH2CH2-N3*) wherein * denotes point of attachment to the peptide backbone and ** denotes point of attachment to the remainder of the lipid side chain, optionally wherein the lipid side chain comprises a linker and ** denotes point of attachment to the linker.

30. The lipidated GnRH analogue of any one of claims 24-29, wherein the lipid side chain is selected from a lipid side chain of Series A - Series Ab comprising a lipid moiety, linker, and spacer selected from:

31. The lipidated GnRH analogue of claim 1, selected from Compounds 1-561, optionally wherein the lipidated GnRH analogue is selected from LiGA5, LiGA15, LiGA21, LiGA22, LiGA23, L1GA27, L1GA28, L1GA29, L1GA31, L1GA35, L1GA36, L1GA43, L1GA44, L1GA45, L1GA54, L1GA55, L1GA60, L1GA61, L1GA62, L1GA69, L1GA70, L1GA72, L1GA74, L1GA86, L1GA91, L1GA92, L1GA93, L1GA94, L1GA96, L1GA97, L1GA98, L1GA99, L1GA100, L1GA102, L1GA103, LiGAl lO, LiGAl l l, L1GA112, L1GA113, L1GA114, L1GA115, L1GA119, L1GA124, L1GA125, L1GA129, L1GA135, L1GA151, L1GA152, L1GA153, L1GA157, L1GA158, L1GA161, L1GA162, L1GA164, L1GA165, L1GA166, L1GA168, L1GA169, L1GA171, L1GA172, L1GA175, LiGA176, LiGA177, LiGA178, and LiGA182, further optionally wherein the lipidated GnRH analogue is selected from LiGA5, LiGA28, LiGA43, LiGA151, and LiGA152.

32. The lipidated GnRH analogue of claim 31, further comprising an additional amino acid substitution that introduces an amino acid present at a corresponding position of a GnRHantagonist, optionally wherein the GnRH antagonist is selected from degarelix, abarelix, cetrorelix, ganirelix, prazarelix, acyline, teverelix, Nal-Glu, orntide, and antide.

33. The lipidated GnRH analogue of claim 31 or 32, comprising or further comprising an amino acid substitution that introduces an amino acid at position 8 (AA8) selected from hArg(Et2), Lys, Arg, and Lys(iPr).

34. The lipidated GnRH analogue of any one of the preceding claims, wherein the lipidated GnRH analogue exhibits a modified circulating half-life in vivo as compared to its non-lipidated counterpart, optionally wherein the lipidated GnRH analogue exhibits an extended circulating halflife in vivo as compared to its non-lipidated counterpart.

35. The lipidated GnRH analogue of any one of the preceding claims, wherein the GnRH analogue peptide backbone is not lipidated at amino acid position 4 (AA4).

36. A pharmaceutical composition comprising the lipidated GnRH analogue of any one of the preceding claims and a pharmaceutically acceptable excipient.

37. The pharmaceutical composition of claim 36, wherein the composition is in a form selected from (i) a liquid pharmaceutical composition formulated for parenteral administration and (ii) a freeze-dried pharmaceutical composition formulated for reconstitution for parenteral administration.

38. The pharmaceutical composition of claim 36, wherein the composition is a liquid composition and wherein the lipidated GnRH analogue exhibits one or both of a lower aggregation potential and reduced propensity for fibrillation in the composition than degarelix.

39. A process for preparing the lipidated GnRH analogue, of any one of claims 1-35, comprising coupling the lipid side chain to an unprotected amino group of an amino acid of the peptide backbone or an amino acid side chain thereof at one or more of amino acid positions 6 (AA6), 5 (AA5), 1 (AA1) and 8 (AA8) of the peptide backbone.

40. The process of claim 39, comprising:(i) providing the peptide backbone on a solid support;(ii) providing a solution comprising the lipid side chain, wherein the lipid side chain is protected with a protecting group;(iii) coupling the protected lipid side chain to the peptide backbone on the solid support, such that a carboxylic acid group of the protected lipid side chain forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof; and(iii) releasing the lipidated GnRH analogue from the solid support.

41. The process of claim 40, wherein the lipid side chain comprises a spacer, linker, and lipid moiety, wherein the process further comprises preparing protected lipid side chain on a solid support by a process comprising:(i) providing solutions of the spacer, linker and lipid moiety, respectively;(ii) coupling the spacer to a solid support such that a carboxylic acid group of the spacer forms an ester bond with the solid support (spacer — support);(iii) coupling the linker to the spacer — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer on the solid support (linker — spacer — support) ;(iv) coupling the lipid moiety to the linker — spacer — support such that a carboxylic acid group of the lipid forms an amide bond with an amino group of the linker on the solid support (lipid — linker — spacer — support); and(v) releasing the protected lipid side chain from the solid support.

42. The process of claim 39 wherein the lipid side chain comprises a spacer, linker, and lipid moiety, wherein the process comprises:(i) providing the peptide backbone on a solid support;(ii) providing solutions of the spacer, linker and lipid moiety, respectively;(iii) coupling the spacer to the solid support carrying the peptide backbone such that a carboxylic acid group of the spacer forms an amide bond with the unprotected amino group of the amino acid of the peptide backbone or amino acid side chain thereof (spacer — peptide — support);(iv) coupling the linker to the spacer — peptide — support, such that a carboxylic acid group of the linker forms an amide bond with an amino group of the spacer (linker — spacer — peptide — support);(v) coupling the lipid moiety to the linker — spacer — peptide — support, such that a carboxylic acid group of the lipid moiety forms an amide bond with an amino group of the linker (lipid — linker — spacer — peptide — support); and(vi) releasing the lipidated GnRH analogue from the solid support.

43. The process of any one of claims 39-42, wherein the unprotected amino group comprises an a-amino group at position AA1 of the peptide backbone of the GnRH analogue.

44. The process of any one of claims 39-43, wherein the unprotected amino group comprises an s-amino group of a lysine residue at position AA5 or AA6 or AA8 of the peptide backbone of the GnRH analogue or an amino acid side chain thereof.

45. A method of treating a condition amenable to treatment by a GnRH analog, comprising administering the lipidated gonadotropin releasing hormone (GnRH) analogue of any one of claims 1-35 to a subject in need thereof.

46. The lipidated gonadotropin releasing hormone (GnRH) analogue of any one of claims 1-35 for treating a condition amenable to treatment by a GnRH analog.

47. Use of the lipidated gonadotropin releasing hormone (GnRH) analogue of any one of claims 1-35 in the preparation of a medicament for treating a condition amenable to treatment by a GnRH analog.

48. The method, lipidated GnRH analogue for treating, or use of any one of claims 45-47, wherein the condition is one or more of prostate cancer, advanced prostatic carcinoma (urologic oncology), ovarian cancer, breast cancer, endometriosis, myoma, infertility, benign prostatic hyperplasia, uterine fibroids , menorrhagia, premenstrual dysphoric disorder (PMDD), premenstrual syndrome (PMS), central precocious puberty, or the use is as a puberty blocker such as for transgender or gender diverse youth, or for chemical castration, or the use is in a veterinary context, such as for hormone suppression (e.g. chemical castration / neutralization) of pets or livestock, or in an in vitro fertilization protocol for pets or livestock.

49. The method, lipidated GnRH analogue for treating, or use of any one of claims 45-47, wherein the condition is advanced prostate cancer or metastatic stage prostate cancer, further optionally wherein the condition is advanced hormone-dependent prostate cancer in adult males, further optionally wherein the advanced hormone-dependent prostate cancer is high-risk localized or locally advanced hormone-dependent prostate cancer.

50. The method, lipidated GnRH analogue for treating, or use of any one of claims 48-49, wherein the treatment is in combination with radiotherapy, optionally wherein the lipidated GnRH analogue is used as neo-adjuvant treatment prior to radiotherapy.

51. The method, lipidated GnRH analogue, or use of any one of claims 45-48, wherein the condition is infertility, and the treatment is for promotion of controlled ovarian stimulation in an assisted reproduction protocol.

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