Iterative approach to the discovery of novel degarelix analogues: Substitutions at positions 3, 7, and 8. Part II

Article abstract of DOI:10.1021/jm050134t

Degarelix(FE200486, Ac-D-2Nal¹-D-4Cpa²-D-3Pal ³-Ser⁴-4Aph(L-Hor)⁵-D-4Aph(Cbm) ⁶-Leu⁷-ILys⁸-Pro⁹-D-Ala ¹⁰-NH₂) is a potent and very long acting antagonist of gonadotropin-releasing hormone (GnRH) after subcutaneous administration in mammals including humans. Analogues of degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. The duration of action was also determined in the castrated male rat assay to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release. Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and N^α-methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptor's cavity and to map the steric and ionic boundaries. Some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitutions at positions 3 ([N^β-(2-pyridyl-methyl)D-Dap³]degarelix, IC₅₀ = 2.71 nM) (5), 7 ([Pra⁷]-degarelix, IC₅₀ = 2.11 nM) (16), and 8 ([N^δ-(IGly)Orn⁸]degarelix, IC₅₀ = 1.38 nM) (20) and N-methylation ([N^α-methyl- Leu⁷]degarelix, IC₅₀ = 1.47 nM) (32) yielded analogues that were equipotent to degarelix (2) in vitro (IC₅₀ = 1.64 nM) but shorter acting in vivo. Out of the 33 novel analogues tested for the duration of action in this series, two analogues ([N^ε-cyclohexyl-Lys ⁸]degarelix, IC₅₀ = 1.50 nM) (23) and ([N ^β-(IβAla)Dap⁸]degarelix, IC₅₀ = 1.98 nM) (26) had antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%) release for >72 h after sc injection to castrated male rats at a standard dose of 50 μg/rat in 5% mannitol}. Under similar conditions analogues ([N^γ-(IGly)Dab⁸] degarelix, IC₅₀ = 1.56 nM) (21) and ([IOrn⁸]degarelix, IC₅₀ = 1.72 nM) (18) had a longer duration of action {inhibited LH (>96 h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of these analogues, a potential measure of their ability to be formulated for sustained release, was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer retention times. No correlation was found between retention times and antagonist potency or duration of action.

Full text of DOI:10.1021/jm050134t

J. Med. Chem. 2005, 48, 4851-4860
4851
Iterative Approach to the Discovery of Novel Degarelix Analogues:
Substitutions at Positions 3, 7, and 8. Part II
Manoj P. Samant,† Jozsef Gulyas,† Doley J. Hong,‡ Glenn Croston,‡ Catherine Rivier,† and Jean Rivier*,†
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, California 92037, and Ferring Research Institute Inc.,
3550 General Atomics Court, San Diego, California 92121
Received February 11, 2005
Degarelix (FE200486, Ac-D-2Nal1-D-4Cpa2-D-3Pal3-Ser4-4Aph(L-Hor)5-D-4Aph(Cbm)6-Leu7-ILys8-
Pro9-D-Ala10-NH2) is a potent and very long acting antagonist of gonadotropin-releasing hormone
(GnRH) after subcutaneous administration in mammals including humans. Analogues of
degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced
response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor.
The duration of action was also determined in the castrated male rat assay to measure the
extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release.
Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and NR-
methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were
designed to probe the spatial limitations of the receptor’s cavity and to map the steric and
ionic boundaries. Some functional groups were introduced that were hypothesized to influence
the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or
intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitu-
tions at positions 3 ([Nâ-(2-pyridyl-methyl)D-Dap3]degarelix, IC50 ) 2.71 nM) (5), 7 ([Pra7]-
degarelix, IC50 ) 2.11 nM) (16), and 8 ([Nδ-(IGly)Orn8]degarelix, IC50 ) 1.38 nM) (20) and
N-methylation ([NR-methyl-Leu7]degarelix, IC50 ) 1.47 nM) (32) yielded analogues that were
equipotent to degarelix (2) in vitro (IC50 ) 1.64 nM) but shorter acting in vivo. Out of the 33
ꢀ
novel analogues tested for the duration of action in this series, two analogues ([N -cyclohexyl-
Lys8]degarelix, IC50 ) 1.50 nM) (23) and ([Nâ-(IâAla)Dap8]degarelix, IC50 ) 1.98 nM) (26) had
antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%)
release for >72 h after sc injection to castrated male rats at a standard dose of 50 µg/rat in 5%
mannitol}. Under similar conditions analogues ([Nγ-(IGly)Dab8]degarelix, IC50 ) 1.56 nM) (21)
and ([IOrn8]degarelix, IC50 ) 1.72 nM) (18) had a longer duration of action {inhibited LH (>96
h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of
these analogues, a potential measure of their ability to be formulated for sustained release,
was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer
retention times. No correlation was found between retention times and antagonist potency or
duration of action.
Introduction
dotropin-gonadal axis, leads to rapid and predictable
recovery, achieves more profound inhibition of gonado-
tropins, and can be used as a diagnostic test of gona-
dotropin-dependent gonadal dysfunction.
Gonadotropin-releasing hormone (GnRH) antagonists
are used in the protocols for ovulation induction and are
recognized as potential drugs for the management of sex
steroid-dependent pathophysiologies, such as hormone-
responsive prostate cancers and, in females, the man-
agement or treatment of breast and gynecological
cancers, endometriosis, precocious puberty, uterine
myoma, ovarian hyperandrogenism, and premenstrual
syndrome.1-4
Most of these disorders can be treated with long-
acting preparations of the superagonists, which desen-
sitize the gonadotrophs after approximately two weeks
of treatment. An optimized antagonist will likely become
the preferred choice over the agonist in the clinic
because it avoids the initial up-regulation of the gona-
The present study of the effect of substitutions at
position 3 of degarelix is an extension of an earlier
study,5 which demonstrated that the nonaromatic sub-
stitution of D3Pal by D-glutamine in Ac-D-2Nal1-D-4Cpa2-
D-3Pal3-Ser4-4Aph(L-Hor)5-D-4Aph(Ac)6-Leu7-ILys8-Pro9-
D-Ala10-NH2 was compatible with high potency as well
as very long duration of action (i.e., >80% inhibition of
LH in the castrated male rat after sc administration of
50 µg in 5% mannitol for >96 h), whereas the equivalent
D-asparagine-containing analogue was comparatively
shorter acting. This suggested that very minor modifi-
cations at position 3 might possibly have an unexpected
impact on duration of action with minimal effect on pA2
(the x intercept in a Schild analysis of receptor antago-
nism, the concentration of antagonist in log terms,
which right-shifts agonist dose response curves 2-fold).
Substitutions at position 7 explore the effect of the
* Corresponding author. Tel: 858-453-4100, ext 1350. Fax: 858-
552-1546. E-mail: jrivier@salk.edu.
† The Salk Institute for Biological Studies.
‡ Ferring Research Institute Inc.
10.1021/jm050134t CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/28/2005

4852 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Samant et al.
introduction of functional groups such as a primary
amide (in asparagine), imidazole (in histidine), triple
bonds (in propargylglycine), phenol (in tyrosine), and
sulfhydryl (in cysteine) on the antagonist potency and
the duration of action in inhibition of luteinizing hor-
mone (LH) release. Early studies6 suggested that a
hydrophobic moiety would be preferable, yet the parent
analogues were structurally quite different from de-
garelix, and the question remained whether side chains
prone to hydrogen bonding could be tolerated in de-
garelix for intramolecular stabilization.
Additionally, we investigated the effect of novel
substitutions at position 8 of degarelix. It is well
documented that substitution of arginine by isopropyl-
lysine at position 8 in GnRH antagonists yields potent
analogues in antiovulatory and GnRH receptor assays
with low potency to release histamine.7 It was therefore
hypothesized that other closely related structures to
that of ILys might be even more favorable. Most
substitutions are based on acylation of the side chains
of dibasic residues of R,â-diaminopropionic acid (Dap)
and R,γ-diaminobutyric acid (Dab), as well as ornithine,
reminiscent to our work on betidamino acids (acylated
R,R-diaminoacetic acids or amino-glycines).8
peptide resin, [Ac-D-2Nal-D-4Cpa-D-Dap/Dab(Fmoc)-Ser-
(Bzl)-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-ILys(N -Cbz)-Pro-
ꢀ
D-Ala]MBHA-resin. A number of reductive alkylation
protocols on solid phase have been reported in the
literature.15-18 Hocart et al.15,16 have described the
reductive alkylation of the side chain of D-Lys with
various aldehydes and ketones in the presence of sodium
cyanoborohydride (NaCNBH3) at position 6 of the GnRH
antagonist [Ac-D-2Nal1-D-Phe2-D-Phe3-Ser4-Tyr5-D-Lys6-
Phe7-Leu8-Pro9-D-Ala10-NH2] in an attempt to reduce the
histamine-releasing activity. In our hands, the reductive
alkylation of the free amino group on the resin with an
aromatic aldehyde in the presence of NaCNBH3 gave
the mixture of mono- and dialkylated products. The
formation of the dialkylated product was observed even
when the reaction time was shortened to 20 min. The
Nâ, Nâ- or Nγ, Nγ-dialkylated peptides 3 and 4 were
prepared by exhaustive reductive alkylation (2 × 3 h)
of the deprotected amino group of D-Dap and D-Dab,
respectively, with an excess of 2-pyridinecarboxaldehyde
and NaCNBH3 in DMF on the resin (see Experimental
Section).
In the synthesis of analogues 5-10, we used the
stepwise or indirect reductive alkylation19 involving the
preformation of the intermediate imine on the resin-
bound peptide followed by reduction in a separate step.
The synthesis of analogues 5-10 involved the reaction
of an excess of aromatic aldehydes [2-pyridinecarbox-
aldehyde (in 5 and 6), 2-quinolinecarboxaldehyde (in 7
and 8), and 2-imidazolecarboxaldehyde (in 9 and 10)]
in DMF with the unprotected orthogonal amino function
of amino acids (D-Dap/D-Dab) on the otherwise fully
protected and assembled peptide resins resulting in
aldamines. A significant reaction time (4 h) was re-
quired, and in the second step, the reduction of the
aldimine was carried out directly in DMF with an excess
of NaCNBH3 to give the desired secondary amine. The
formation of the secondary amine on the resin was
qualitatively determined by the chloranil test.20
Finally, the key residues encompassing a â-turn9 at
positions 6, 7, and 8 of degarelix were NR-methylated
to confer enzymatic stability,10 increasing solubility,11
and to induce or disrupt sterical constraints with
possible effects on activities.
Results and Discussion
Synthesis, Purification, and Chemical Charac-
terization. Analogues were synthesized by the solid-
phase peptide synthesis (SPPS) methodology on a
p-methylbenzhydrylamine resin (MBHA-resin, tert-bu-
tyloxycarbonyl (Boc) strategy) using protocols previously
described12 or shown below. In most cases, analogues
were obtained by the introduction of each of the side
chain substituents at positions 3, 5, 6, and 8 on the
partially deprotected peptido-resin obtained after the
removal of the 9-fluorenylmethyloxycarbonyl (Fmoc)
protecting group with 30% piperidine in N-methylpyr-
rolidinone (NMP) (for example, after the introduction
of Boc-D-Dab/D-Dap(Fmoc) at position 3, Boc-D-4Aph-
(Fmoc) at position 5, Boc-4Aph(Fmoc) at position 6, and
Boc-Dab/Dap/Orn/Lys(Fmoc) at position 8).
Most of the unusual amino acids substituted at
positions 7 and 8 of degarelix were obtained com-
mercially or synthesized as described in the literature.
However, the synthesis of two new amino acids NR-Boc-
ꢀ
ꢀ
Lys(N -cyclohexyl, N -Cbz) and IâAla(NR-Cbz) substi-
tuted at position 8 of degarelix deserves further com-
ꢀ
ꢀ
ment. The NR-Boc-Lys(N -cyclohexyl, N -Cbz) was pre-
pared via reductive alkylation of NR-Boc-Lys(N -Cbz)
ꢀ
The reference compounds azaline B (1) and degarelix
(2) were synthesized using published protocols.13,14 The
synthesis of all other analogues deserves further com-
ment.
with cyclohexanone under Pd/C-H2 conditions, followed
by secondary amine protection using Cbz-Cl. The syn-
thesis of IâAla(NR-Cbz) involved direct nucleophilic
displacement reaction of 3-bromopropionic acid with
isopropylamine to give isopropyl-âAla (IâAla) followed
by Cbz protection (see Experimental Section).
Analogues 11-18 and 23 were synthesized by intro-
ducing different amino acids at position 7 or 8 during
the elongation of the peptide chain and using the
strategy reported for the synthesis of degarelix.14
The synthesis of analogues 19-22, 24-26, and 30
was accomplished by DIC and HOBt-mediated coupling
reaction of the resin-bound deprotected orthogonal
amino group at position 8 with Gly(NR-isopropyl, NR-
Cbz) (in 19-22), Gly(NR-cyclohexyl, NR-Cbz) (in 24 and
25), âAla(NR-isopropyl, NR-Cbz) (in 26), and L-Hor (in
30). Further elongation of the peptide chain, HF cleav-
In all of the analogues, the side chain substitutions
at position 5 and 6 were carried out during the elonga-
tion of the peptide chain on the resin. The carbamoyla-
tion14 at position 6 was carried out by the reaction of
tert-butyl isocyanate in dimethylformamide (DMF) with
the free ω-amino function of D-p-aminophenylalanine (D-
4Aph) on the otherwise fully protected resin-bound
peptides. Coupling of L-hydroorotic acid (L-Hor) to the
resin-bound deprotected ω-amino group of L-4Aph at
position 5 was mediated by N,N′-diisopropylcarbodiim-
ide (DIC) and 1-hydroxybenzotriazole (HOBt) in NMP.
The synthesis of analogues 3-10 involved the reduc-
tive alkylation of the deprotected side chain amino group
of D-Dap/D-Dab at position 3 on the fully assembled

Iterative Approach to Novel Degarelix Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4853
Table 1. Physicochemical and Biological Characterization of GnRH Antagonists
MSd
e
purity
(M + H)+
pIC50
c
f
tR
IC50
duration
of actiong
no.
compound
HPLCa CZEb (min)
calcd
obsd
avg ( SEM
(nM)
1
[Ac-D-2Nal1,D-4Cpa2,D-3Pal3,4Aph(Atz)5,
D-4Aph(Atz)6,Ilys8,D-Ala10] GnRH azaline B
[Ac-D-2Nal1,D-4Cpa2,D-3Pal3,4Aph(L-Hor)5,
D-4Aph(Cbm)6,Ilys8,D-Ala10]GnRH degarelix
[Nâ, Nâ-di-(2-pyridyl-methyl)D-Dap3]degarelix
[Nγ, Nγ-di-(2-pyridyl-methyl)D-Dab3]degarelix
[Nâ-(2-pyridyl-methyl)D-Dap3]degarelix
[Nγ-(2-pyridyl-methyl)D-Dab3]degarelix
[Nâ-(2-quinolyl-methyl)D-Dap3]degarelix
[Nγ-(2-quinolyl-methyl)D-Dab3]degarelix
[Nâ-(2-imidazolyl-methyl)D-Dap3]degarelix
99
99
27.4 1612.8 1612.7 8.9 ( 0.07
1.4
long
2
96
98
27.9 1631.8 1631.9 8.8 ( 0.03
1.64 very long
3
4
5
6
7
8
9
97
89
95
95
94
95
95
98
97
97
98
95
95
96
97
99
99
98
97
95
98
98
99
96
98
98
99
98
97
98
99
99
93
98
97
96
95
96
97
97
98
98
99
99
97
96
99
98
99
98
98
97
98
97
98
98
97
98
98
98
99
96
33.4 1751.8 1751.6 8.3 ( 0.01
33.5 1765.8 1765.8 7.6 ( 0.01
29.5 1660.8 1660.8 8.6 ( 0.05
28.8 1674.8 1674.8 8.4 ( 0.18
35.1 1710.8 1710.6 8.4 ( 0.07
34.8 1724.8 1724.6 8.3 ( 0.02
27.6 1649.8 1649.6 8.5 ( 0.06
26.7 1663.8 1663.8 7.9 ( 0.03
22.5 1632.7 1632.6 8.0 ( 0.03
23.2 1646.7 1646.8 7.6 ( 0.01
24.2 1660.7 1660.7 7.3 ( 0.08
22.7 1655.7 1655.7 8.0 ( 0.10
25.1 1681.7 1681.7 8.5 ( 0.01
24.9 1613.7 1613.7 8.7 ( 0.03
5.31 short
22.58 short
2.71 short
3.79 short
4.25 short
5.52 short
3.50 intermediate
13.47 short
9.18 short
10 [Nγ-(2-imidazolyl-methyl)D-Dab3]degarelix
11 [Asn7]degarelix
12 [Nγ-methyl-Asn7]degarelix
13 [Nγ, Nγ-dimethyl-Asn7]degarelix
14 [His7]degarelix
25.79 short
48.91 short
10.64 short
2.85 intermediate
2.11 short
15 [Tyr7]degarelix
16 [Pra7]degarelix
17 [Cys7]degarelix
25.3 1621.7 1621.7 6.7 ( 0.01 178.19 short
18 [IOrn8]degarelix
28.1 1617.7 1617.7 8.8 ( 0.14
30.6 1688.8 1688.7 8.7 ( 0.11
30.1 1674.8 1674.7 8.9 ( 0.12
30.3 1660.7 1660.7 8.8 ( 0.09
30.6 1646.7 1646.8 8.8 ( 0.02
31.7 1671.8 1671.7 8.8 ( 0.08
35.8 1700.8 1700.7 8.8 ( 0.15
36.0 1686.8 1686.7 8.7 ( 0.14
28.3 1660.7 1660.7 8.7 ( 0.25
29.0 1674.8 1674.7 8.7 ( 0.19
31.7 1632.7 1632.7 8.8 ( 0.16
31.8 1646.7 1646.7 8.6 ( 0.06
27.2 1715.7 1715.7 8.8 ( 0.16
30.2 1645.8 1645.6 8.7 ( 0.15
30.3 1645.8 1645.7 8.8 ( 0.11
29.1 1645.8 1645.8 7.3 ( 0.01
1.72 long*
1.83 short
1.38 intermediate
1.56 long*
19 [N -(IGly)Lys8]degarelix
ꢀ
20 [Nδ-(IGly)Orn8]degarelix
21 [Nγ-(IGly)Dab8]degarelix
22 [Nâ-(IGly)Dap8]degarelix
1.54 intermediate
1.50 long
23 [N -cyclohexyl-Lys8]degarelix
ꢀ
24 [Nγ-(NR-cyclohexyl-Gly)Dab8]degarelix
25 [Nâ-(NR-cyclohexyl-Gly)Dap8]degarelix
26 [Nâ-(IâAla)Dap8]degarelix
1.77 short
2.01 short
1.98 long
27 [Nâ-(IâAla)-Nâ-methyl-Dap8]degarelix
28 [Nâ-(ICbm)Dap8]degarelix
1.97 intermediate
1.62 short
29 [Nγ-(ICbm)Dab8]degarelix
2.24 short
30 [Nδ-(L-Hor)Orn8]degarelix
1.77 short
31 [NR-methyl-D-4Aph(Cbm)6]degarelix
32 [NR-methyl-Leu7]degarelix
1.85 short
1.47 short
33 [NR-methyl-Ilys8]degarelix
52.24 short
a Percentage purity determined by HPLC using buffer system A (TEAP, pH 2.30) and buffer system B (60% CH3CN/40% A) under
gradient conditions (30%-80% B over 50 min) at a flow rate of 0.2 mL/min on a Vydac C18 column (0.21 cm × 15 cm, 5 µm particle size,
300 Å pore size). Detection at 214 nm. b Percentage purity determined by capillary zone electrophoresis (CZE) using a Beckman P/ACE
System 2050 controlled by an IBM Personal system/2 model 50Z; field strength of 15 kV at 30 °C; buffer, 100 mM sodium phosphate
(85:15, H2O/CH3CN), pH 2.50, on a Agilent µSil bare fused-silica capillary (75 µm i.d. x 40 cm length). Detection at 214 nm. c Retention
times under gradient conditions (30%-80% B over 50 min); buffer system A, TEAP, pH 7.0; buffer system B, 60% CH3CN/40% A. d Mass
spectra (MALDI-MS) were measured on an ABI-Voyager DESTR instrument using saturated solution of R-cyano-4-hydroxycinnamic
acid in 0.3% trifluoroacetic acid and 50% acetonitrile as matrix. The calculated [M + H]+ of the monoisotope is compared with the observed
[M + H]+ monoisotopic mass. e The pIC50 is the negative log of the IC50 in molar, as determined in the GnRH reporter gene assay. f IC50
is the concentration of antagonist required to repress the GnRH induced response by 50% in the reporter gene assay in HEK-293 cells
expressing the human GnRH receptor and a GnRH-responsive stably integrated luciferase reporter gene. g Castrated male rat assay.
Duration of action: very long ) over 80% inhibition of LH release for more than 120 h; long* ) over 80% inhibition of LH release at 96
h but not at 120 h; long ) over 80% inhibition of LH release at 72 h but not at 96 h; intermediate ) partial inhibition of LH release at
72 h; short ) over 80% inhibition of LH release at 3 h but not at 72 h.
age, and purification afforded the desired analogues.
The synthesis of 28 and 29 involved the reaction of
isopropyl isocyanate with the unprotected orthogonal
amino function of Dap (in 28) or Dab (in 29) at position
8 on the otherwise fully protected and assembled
peptide resins.
solvent systems (acidic and neutral) and capillary zone
electrophoresis (CZE). With very few exceptions, all of
the analogues were greater than 98% pure. Mass
spectrometric analysis supported the identity of the
intended structures, Table 1.
Biological Characterization. Analogues were tested
in vitro for their antagonism on the GnRH receptor in
a reporter gene assay in HEK-293 cells expressing the
human GnRH receptor and a stably integrated lu-
ciferase reporter gene.14,22 The antagonism of the GnRH
agonist-induced response by each analogue was deter-
mined at several concentrations and reported as IC50,
the concentration required to suppress the response in
the reporter gene assay by 50%, Figure 1.
The introduction of the Nâ-methyl group in 27 and
NR-methyl group in analogues 31-33 was achieved
during the elongation of the peptide chain on the
partially assembled peptide resin using the published
procedure of Kaljuste et al.21
The protected peptido-resins were cleaved and depro-
tected in anhydrous HF (1.5 h at 0-5 °C) in the presence
of a scavenger (anisole). The crude peptides were
purified by reversed-phase HPLC (RP-HPLC) in two
steps and isolated as their trifluoroacetate (TFA) salts.
The analytical techniques used for the characterization
of the analogues included RP-HPLC with two different
Since there is strong evidence showing that most of
the GnRH antagonists (except for those that are very
short acting) inhibit LH secretion maximally to about
the same level in the highly reproducible in vivo

4854 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Samant et al.
more than 120 h. Under the same conditions, an
analogue that is long* acting (see Table 1) will inhibit
LH (>80%) secretion for at least 96 h, an analogue that
is long acting will inhibit LH (>80%) secretion for at
least 72 h, an analogue that is intermediate acting will
inhibit LH secretion only partially at 72 h, and an
analogue that is short acting will inhibit LH (>80%)
secretion at 3 h but not at 72 h.
The overall rationale for the synthesis of the different
analogues of degarelix was presented in our introduction
and consists of probing the steric boundaries, ionic
properties, and hydrogen bond forming capacity of the
pharmacophores at positions 3, 7, and 8, known to be
critical yet not necessarily optimized in the analogues
reported so far.6,25 Additionally, some functional groups
were introduced that were hypothesized to influence the
phamacokinetic properties of the analogues through
altered solubility, stability, and ability to bind to carrier
proteins. There is precedence for favorable substitutions
at position 3; it includes glutamine and carbamoylation
of Dap and Dab.5 Another favorable substitution at
position 3 includes the aminotriazole functionality.13 Yet
there is no available data on the effect of alkylation and
dialkylation of an ω-NH2 at this position. To mimic the
functionality of the favored D-3Pal3 found in azaline B,26
acyline,27 degarelix14 and many other analogues, we
carried out reductive alkylation on D-Dap/D-Dab to
generate a variety of amino acids that can provide a
combined modulation of steric effects, basicity, aroma-
ticity, and hydrophobicity at position 3. The reductive
alkylation with 2-pyridinecarboxaldehyde under two
different experimental conditions resulted in the forma-
tion of the mono- and dialkylated products (5-6 and
3-4, respectively). Comparison of the antagonist po-
tency data indicated that increasing the length of the
side chain from Dap (in 3 and 5) to Dab (in 4 and 6)
resulted in ca. 2-4-fold loss of potency (compare IC50’s
of 4 ) 22.58 nM versus 3 ) 5.31 nM and of 6 ) 3.79
nM versus 5 ) 2.71 nM). All four analogues are short
acting. A similar set of derivatives (7-10) was signifi-
cantly less potent in vitro, likely due to increased steric
hindrance (7 and 8) and charge distribution (9 and 10).
Interestingly, 9 (with intermediate duration of action)
showed some extended duration of action over 10 (with
short duration of action).
Figure 1. Antagonism of GnRH by selected antagonists in a
reporter gene assay in HEK-293 cells expressing the human
GnRH receptor and a GnRH-responsive stably integrated
luciferase reporter gene: (9) degarelix 2 , (2) 9, (1) 10, ([)
15, (b) 17, (0) 19, and (4) 26.
Figure 2. Inhibition of LH secretion after sc administration
of analogues. Total dose was 50 µg in 50 µL of 5% mannitol
containing 0.1% DMSO. Blood samples were collected at the
times shown on the abscissa. Results are mean plasma LH
levels (n ) 5-8 rats) ( SEM. The SEM, where not appearing,
are encompassed within the size of the symbols in the graph:
(O) vehicle; (b) azaline B (1); (9) degarelix (2); (2) 9; (0) 16;
(4) 18; (3) 21; (1) 23; ([) 26.
Several different amino acids have been introduced
at position 7 of GnRH agonists and antagonists in the
past.6 Interestingly, leucine, found in mammalian GnRH,
is not a conserved amino acid in many other species:
tryptophan is found in salmon GnRH,28 dogfish GnRH,29
chicken II GnRH,30 lamprey I GnRH,31 and lamprey III
GnRH,32 phenylalanine is found in tunicate GnRH-133
and tunicate GnRH-3,34 histidine is found in tunicate
GnRH-2,33 and tyrosine is found in tunicate GnRH-5
and -6.34
In the present study, we have introduced amino acids
such as Asn (in 11), Asn(Me) (in 12), Asn(Me)2 (in 13),
Pra (in 16), or Cys (in 17), along with His (in 14) and
Tyr (in 15) found in GnRHs of other species (see above).
It is noteworthy that all of these diverse substitutions
with the exception of [Tyr7] and [Pra7] yielded analogues
(15 and 16, respectively) with increased IC50 and
significantly shorter duration of action than degarelix.
castrated male rat assay,23,24 the most efficient ana-
logues are those with the longest duration of action,
suggesting the use of this assay for screening purposes
as validated by the discovery of degarelix (FE200486).14
In short, 10 days after castration, rats (five or more per
group) were injected sc on a Friday with the excipient
(50 µL of 5% mannitol), degarelix, and azaline B (50 µg/
50 µL of 5% mannitol) as an internal standard, or the
novel analogues (50 µg/50 µL of 5% mannitol). Blood
sampling was performed predose, then 3, 72, 96, 120,
144, 168, and occasionally 192 h post-sc administration.
The effects of the test compounds on the gonadotropic
axis were determined by measurement of plasma LH
levels by radioimmunoassay24 (Figure 2).
By our definition, an analogue is very long acting
when 50 µg in 5% mannitol in a volume of 50 µL injected
sc results in an inhibition of LH (>80%) that lasts for

Iterative Approach to Novel Degarelix Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4855
The rationale for the introduction of Asn, Asn(Me) and
Asn(Me)2 was to increase the potential for hydrogen
bond formation. While Pra in 16 could induce an intra-
or intermolecular π-π interaction with aromatic groups
in degarelix or the receptor, respectively, the sulfhydryl
of cysteine in 17 could be used for further derivatization
at position 7. While 16 was the most potent analogue
in vitro of this series (IC50 ) 2.11 nM), the antagonist
potency of 17 was almost 100 times less (IC50 ) 178.2
nM). The poor affinity of 17 was confirmatory of an
earlier study (unpublished results) whereby a partial
cysteine scan of degarelix had shown loss of activity
when introduced at positions 1, 2, and 3. Overall, our
observations are complementary to those of Millar et
al.35 and Hocart et al.36 who observed a limited prefer-
ence for hydrophobic residues at position 7.
Isopropyl-ornithine (IOrn) and isopropyl-lysine (ILys)
were first introduced in GnRH antagonists at position
8 by Ljungqvist et al.7 to yield [Ac-D-2Nal1,D-4Cpa2,D-
3Pal3,D-Lys(Nic)6,IOrn/ILys8,D-Ala10]GnRH. They found
that the IOrn-containing peptide was less potent than
the ILys-containing one, both in an antiovulatory assay
and in a histamine release assay. Whereas these two
substitutions, that is, IOrn8 in 18 and ILys8 in degarelix
(2) have nonsignificant effect on antagonist potency
(IC50’s ) 1.72 and 1.64 nM, respectively), the former is
not as long acting as degarelix. However, analogue 18
is longer acting than azaline B, Figure 2. It is rather
intriguing that such a subtle modification (shortening
the chain length by an additional methylene) in 18 and
2 may have an effect on their ability to release hista-
mine (by analogy with Ljungqvist’s observation, see
above), and to influence those parameters responsible
for the duration of action (present investigation). This
is further documented in another series whereby the
side chains of Lys, Orn, Dab, and Dap are each extended
by an isopropyl-glycine residue to yield analogues 19-
22. The antagonistic potency for all of these analogues
19-22 is equal to that of degarelix; however, the
duration of action is extended from short (for 19) to
intermediate (for 20) to long* (for 21) to revert back to
intermediate (for 22), as if an optimal chain length had
been reached in 21 somewhat independently from the
actual hydrophobicity of the peptide as measured by the
retention time on HPLC at physiological pH (retention
time, tR ) 30.6, 30.1, 30.3, and 30.6 min for 19-22,
respectively). In this series, 21 had a longer duration
of action than azaline B and inhibited LH (>80%)
release for >96 h while still being shorter acting than
degarelix.
insufficient release (due to gel formation) after an initial
burst resulting in the retention of a fraction of the
material at the site of injection. Although degarelix
levels were measured and shown to be above 1.6 ng/
mL for the observed duration of gonadotropin inhibition
in ovariectomized rhesus monkeys, similar studies were
not carried out on shorter acting analogues thus pre-
cluding us from any conclusion.37 Additional analogues
(24 and 25) with the cyclohexyl substitution and oth-
erwise analogous to 21 and 22 are significantly shorter
acting. In 26 and 27, we have two analogues differing
by one methyl group on the â-nitrogen of Dap8. Whereas
both analogues are potent (IC50 ca. 2.0 nM each), the
Nâ-methylated 27 is shorter acting than its correspond-
ing nonmethylated analogue 26. Another subtle struc-
tural difference such as that found in 21 (with (IGly)-
Dab8) as compared to 26 (with (IâAla)Dab8) with the
same composition yield compounds with different and
difficult to understand in vivo activities; while analogue
21 is active for >96 h, analogue 26 is shorter acting by
approximately 24 h. Does this reflect yet to be identified
inter- or intramolecular interactions that significantly
alter one or several physicochemical parameters such
as enzymatic resistance, solubility, ability to gel, or
clearance rates, among others? On the other hand, two
analogues with the same composition but with a quite
different side chain configuration at position 8 such as
20 (Nδ-(IGly)Orn8) and 27 (Nâ-(IâAla)-Nâ-methyl-Dap8)
both have an intermediate duration of action. NMR
studies in progress will try to address such observations
that may be critical for a rational design of optimized
structures.
In an attempt to further increase the number of inter-
or intramolecular hydrogen bonding opportunities, three
additional analogues, 28 (with Nâ-(ICbm)Dap8) and 29
(with Nγ-(ICbm)Dab8), which contain an alkylated car-
bamoyl function, and 30 with an L-hydroorotyl group
(Nδ-(L-Hor)Orn8) at position 8, were synthesized; all
showed antagonist potency similar to that of degarelix
in vitro and short duration of action in vivo.
In our previous paper describing the design of de-
garelix (FE200486), we investigated the effect of NR-
methylation at position 5, which was deleterious (loss
of duration of action). Here we scanned positions 6, 7,
and 8 with the introduction of an NR-methyl group in
analogues 31-33. Whereas it was well documented that
NR-substitution at position 7 is well tolerated in both
agonists and antagonists of GnRH,38,39 NR-methylation
of ILys in azaline B was reported to be detrimental to
biological activity in a rat antiovulatory assay.27 As
shown here, such a substitution resulted in a shortened
duration of action when introduced at position 6 of
degarelix and reduced antagonistic potency significantly
when ILys (IC50 ) 52.24 nM) was substituted at position
8.
To further probe the steric environment of the phar-
macophore at residue 8, we introduced the cyclohexyl
substituent instead of the isopropyl group in 23. Whereas
the antagonistic potency of 23 is identical to that of
degarelix, the duration of action was significantly
altered. Analogue 23 has duration of action similar to
that of azaline B, but it is about half as long acting as
degarelix. Interestingly, 23 may be significantly more
hydrophobic than degarelix (measured by the retention
time on HPLC, tR for 23 ) 31.7 min versus 27.9 min for
degarelix). Short duration of action may be due to
several factors among which one should consider the
rapid release of the peptide from the injection site
exhausting all bioactive material or, alternatively,
Conclusions
As we probe closely related structures of degarelix, a
promising drug candidate that can be administered sc
to achieve long duration of action (>30 days in hu-
man),40 we modulated the steric boundaries, ionic
properties, and hydrogen bond forming capacity of the
pharmacophore at positions 3, 7, and 8 of degarelix
known to be critical yet not necessarily optimized in

4856 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Samant et al.
analogues reported so far.14,22,27,41 To generate degarelix
analogues mono-N-alkylated at position 3, we developed
an indirect two-step reductive alkylation protocol of the
orthogonal amino group of D-Dap/D-Dab residues in
otherwise protected resin-bound peptides with various
aldehydes thereby generating a wide range of basic
amino acids having unique aromatic, steric, and elec-
tronic characteristics. Concomitantly, we probed the
effect of novel substitutions to influence solubility
characteristics favorable for slow release formulations
comparable to those used for GnRH agonists, which may
depend primarily on the hydrophilic character of the
analogue. Interestingly, some early generation ana-
logues of degarelix14 including azaline B11 and acyline27
had a significant tendency to gel upon standing in
aqueous buffers such as 5% mannitol at concentrations
as low as 2.5 mg/mL. Very few of the analogues (10,
16, and 23) reported here show such tendency.
Prep LC 4000 preparative chromatograph system with a
Waters 486 tunable absorbance UV detector and Omniscribe
(Houston Instrument) chart recorder. Matrix-assisted laser
desorption-ionization mass spectroscopy (MALDI-MS) of the
peptide analogues was measured on an ABI-Perseptive DE
STR instrument. The instrument employs a nitrogen laser (337
nm) at a repetition rate of 20 Hz. The applied accelerating
voltage was 20 kV. Spectra were recorded in delayed extraction
mode (300 ns delay). All spectra were recorded in the positive
reflector mode. Spectra were sums of 100 laser shots. Matrix
R-cyano-4-hydroxycinnamic acid was prepared as saturated
solutions in 0.3% trifluoroacetic acid in 50% acetonitrile. The
observed monoisotopic (M + H)+ values of each peptide
corresponded with the calculated (M + H)+ values. The
capillary zone electrophoresis (CZE) analysis of peptides was
performed on a Beckman P/ACE System 2050 controlled by
an IBM Personal system/2 model 50Z, field strength of 15 kV
at 30 °C using buffer, 100 mM sodium phosphate (85:15, H2O/
ACN), pH 2.50, on a Agilent µSil bare fused-silica capillary
(75 µm i.d. x 40 cm length).
Starting Materials. Most amino acid derivatives were
obtained from Reanal Finechemical Co. (Budapest, Hungary),
including Boc-D-Ala, Boc-Asn(Xan), Boc-Cys(4-MeOBzl), Boc-
His(Dnp), Boc-Leu, Boc-Lys(Cbz), Boc-Lys(Fmoc), Boc-Orn-
(Fmoc), Boc-Pro, Boc-Ser(Bzl), and Boc-Tyr(2Br-Cbz). Boc-Pra
was purchased from Neosystem Groupe SNPE (France). Fmoc-
Dap(Boc) was obtained from Bachem Inc. (Torrance, CA). Boc-
D-4Cpa, Boc-D-2Nal, and Boc-D-3Pal were synthesized at the
Southwest Foundation for Biomedical Research (under NIH
Contract NO1-HD-6-2928) and made available by the Contra-
ceptive Development Branch, Center for Population Research,
NICHD. Boc-L- and Boc-D-4Aph(Fmoc) were synthesized ac-
cording to the published procedure.13 Boc-L- and Boc-D-Dab-
(Fmoc) and Boc-L- and Boc-D-Dap(Fmoc) were synthesized
according to the published procedure.42 The L-isomer of hy-
droorotic acid was prepared using a published procedure.43
Boc-Asn(Me) and Boc-Asn(Me)2 were prepared according to the
procedure reported earlier.44-46 The benzylchloroformate (Cbz-
Cl), 3-bromopropionic acid, cyclohexanone, 2-imidazolecarbox-
aldehyde, isopropylamine, isopropyl isocyanate, 10% Pd/C,
2-pyridinecarboxaldehyde, 2-quinolinecarboxaldehyde, tert-
butyl isocyanate, and sodium cyanoborohydride were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI). NR-
The highlight of this work is the observation that very
minor modifications at positions 3, 7, or 8 of degarelix
aimed at modifying the steric properties, the hydrogen
bonding potential, or the ability to form π-π interac-
tions resulted in all cases in loss of duration of action
with retention in most cases of antagonistic potency
thus paving the way for promising structural studies.
Experimental Section
Abbreviations. IUPAC rules are used for nomenclature
except for the following: Ac, acetyl; ACN, acetonitrile; 4Aph,
4-aminophenylalanine; Atz, 5′-(3′-amino-1H-1′,2′,4′-triazolyl);
Boc, tert-butyloxycarbonyl; 2Br-Cbz, 2-bromocarbobenzoxy;
Bzl, benzyl; Cbm, carbamoyl; Cbz, carbobenzoxy; 4Cpa, 4-chlo-
rophenylalanine; CZE, capillary zone electrophoresis; Dab, R,γ-
diaminobutyric acid; Dap, R,â-diaminopropionic acid; DCM,
dichloromethane; DIC, N,N′-diisopropylcarbodiimide; DIPEA,
N,N′-diisopropylethylamine; DMF, dimethylformamide; Fmoc,
9-fluorenylmethyloxycarbonyl; GnRH, gonadotropin-releasing
hormone; HF, hydrofluoric acid; HOBt, 1-hydroxybenzotria-
zole; Hor, L-hydroorotyl; IâAla, NR-isopropyl-âalanine; ICbm,
ꢀ
cyclohexyl-Gly(NR-Cbz),47 IGly(NR-Cbz),48 Boc-Ilys(N -Cbz)6 and
ꢀ
isopropylcarbamoyl; IGly, NR-isopropylglycine; ILys, N -iso-
Boc-IOrn(Nδ-Cbz)6 were synthesized according to the published
procedure. The methylbenzhydrylamine (MBHA) resin49 with
substitution 0.33 mequiv/g was obtained according to the
published procedure of Rivier et al. using p-toluoyl chloride
in lieu of benzoyl chloride in the Friedel and Crafts step.50 All
solvents were reagent grade or better.
propyllysine; IOrn, Nδ-isopropylornithine; LH, luteinizing
hormone; MBHA, p-methylbenzhydrylamine; NaCNBH3, so-
dium cyanoborohydride; 2Nal, 3-(2-naphthyl)-alanine; NMP,
N-methylpyrrolidinone; 3Pal, 3-(3-pyridyl)-alanine; Pra, pro-
pargylglycine; PyBrOP, bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate; RP-HPLC, reversed phase high-perfor-
mance liquid chromatography; RGA, reporter gene assay; sc,
subcutaneous; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium tetrafluoroborate; TFA, trifluoroacetic acid.
Instruments. The optical rotation was measured on a
Perkin-Elmer model 241 polarimeter in a 1 dm microcell at
25 °C at the concentration indicated (w/v %). Thin-layer
chromatography (TLC) was performed in a solvent-vapor-
saturated chamber on Merck silica gel 60 F254 plates using the
following solvent systems: (A) EtOAc/hexane ) 1:1, (B) CHCl3/
MeOH ) 9:1, (C) n-butanol/AcOH/H2O ) 4:1:2, (D) CHCl3/
MeOH/AcOH ) 95:5:3. The plates were visualized by UV, I2,
and ninhydrin spray. 1H NMR spectra were recorded on a
Varian Mercury 200 MHz spectrometer with CDCl3 as a
solvent. Chemical shifts (δ) are expressed in parts per million
relative to internal standard tetramethylsilane (TMS). Mass
spectra of the amino acids were recorded on a Bruker Esquire
3000 plus instrument using nitrogen/helium gas ESI-MS.
Analytical RP-HPLC was run on a system using two Waters
501 HPLC pumps, a Schimadzu SPD-6A UV detector, Rheo-
dyne model 7125 injector, Omniscribe (Houston Instrument)
chart recorder, and a Vydac C18 column (0.46 cm × 25 cm, 5
µm particle size, 300 Å pore size). Preparative RP-HPLC was
accomplished on a Vydac C18 preparative cartridge (5 cm ×
30 cm, 15-20 µm particle size, 300 Å pore size) on a Waters
ꢀ
Synthesis of Novel Amino Acids. L-NR-Boc-Lys(N -
ꢀ
ꢀ
cyclohexyl, N -Cbz). L-NR-Boc-Lys(N -Cbz) (15.20 g, 40 mmol),
cyclohexanone (7.85 g, 80 mmol), MeOH (100 mL), and
molecular sieves (6.0 g, 4 Å) were added in a 500 mL Parr
hydrogenation vessel. The mixture was purged with N2 for 10
min, and then Pd/C 10% (600 mg) was added. The reductive
alkylation under H2 at 40 psi was monitored by HPLC for 26
h. After the filtration of the catalyst and molecular sieves and
the evaporation of the solvent in a vacuum, the desired
ꢀ
intermediate l-NR-Boc-Lys(N -cyclohexyl) was obtained as an
ꢀ
oil. The L-NR-Boc-Lys(N -cyclohexyl) was cooled to 0 °C and
then Cbz-protected using benzyl chloroformate (Cbz-Cl; 8.6
mL, 60 mmol) in a mixture of THF/H2O (1:1, 200 mL)
maintained at pH 9.5 by the use of an autotitrator delivering
0.5 N NaOH. The mixture was then stirred at room-temper-
ature overnight. The mixture was concentrated under vacuum
to remove THF and then extracted with petroleum ether (3 ×
100 mL) to remove excess Cbz-Cl. The pH of the aqueous layer
was brought to 2.5 with saturated sodium hydrogen sulfate
and then extracted with EtOAc (3 × 150 mL). The combined
organic layers were washed with a saturated sodium chloride
solution (2 × 100 mL) and dried over anhydrous sodium
sulfate. After evaporation of EtOAc under vacuum L-NR-Boc-
ꢀ
ꢀ
Lys(N -cyclohexyl, N -Cbz) was obtained as a foam. Yield )

Iterative Approach to Novel Degarelix Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4857
11.47 g (24.80 mmol, 62%); [R]25D ) -5.5° (c ) 1, MeOH); ESI-
MS (M + H)+ ) 463.20; (M + Na+) ) 485.20. HPLC assay:
column (C18 0.21 cm × 15 cm); buffer A, 0.1% TFA in H2O;
buffer B, 0.1% TFA in 60% ACN/40% H2O; gradient condition,
40-90% buffer B over 50 min at a flow rate of 0.2 mL/min;
UV detection, 0.1 AUFS at 214 nM; tR ) 44.3 min; purity )
98%. 1H NMR (CDCl3) δ 1.15-1.40 (m, 15H, CH3s + CH2s-
cyclohexyl), 1.45-1.56 (m, 4H, CH2-Lys + CH2-Lys), 1.58-
1.92 (m, 6H, âCH2-Lys + CH2s-C-cyclohexyl), 3.10 (t, 2H,
δCH2-Lys), 3.68 (m, 1H, CH-cyclohexyl), 4.28 (m, 1H, RCH-
Lys), 5.12 (s, 2H, CH2-Cbz), 6.19 (br, 1H, NH), 7.32 (br s, 5
H, ArHs-Cbz), 9.30 (br, 1H, COOH).
drous) at 0-5 °C in the presence of anisole (10% v/v) yielded
the desired crude analogue after elimination of HF under
vacuum, diethyl ether wash, extraction with 0.1% TFA in 40%
acetonitrile (ACN)/H2O and lyophilization. The crude peptide
(0.45 g) was purified by preparative RP-HPLC.52 The peptide
was dissolved in 0.25 M triethylammonium phosphate (200
mL), pH 2.25 (TEAP 2.25), and loaded onto the cartridge
described earlier. The peptide was eluted using a flow rate of
100 mL/min with a mixture of A (TEAP 2.25) and B (60% ACN,
40% A) and an appropriate gradient (35% B for 10 min followed
by a 90 min linear gradient to 65% B). The collected fractions
were screened by analytical RP-HPLC under isocratic condi-
tions, with a mixture of A (0.1% TFA) and B (60% ACN, 40%
A) at a flow rate of 1.0 mL/min. Appropriate fractions were
then combined (diluted 1:2 with water) and desalted on
preparative HPLC with a mixture of A (0.1% TFA) and B (60%
ACN, 40% A) using a gradient: 35% B (10 min) followed by a
40 min gradient to 75% B. Yield of 18 after purification was
80 mg (47.88 µmol, 14.52%).
γ
δ
âAla(NR-isopropyl, NR-Cbz). Isopropylamine (11.90 g, 200
mmol) in absolute ethanol (100 mL) was added dropwise to
the stirred solution of 3-bromopropionic acid (15.3 g, 100 mmol)
in absolute ethanol (100 mL). After the addition, the mixture
was slowly heated and refluxed for 2 h. The reaction mixture
was cooled to room temperature, and then NaOH (250 mmol)
in H2O (100 mL) was added. The excess of isopropylamine and
ethanol was distilled off in a vacuum. The residue was
dissolved in a mixture of THF/H2O (1:1, 200 mL) and Cbz-
protected using the procedure similar to that used for L-NR-
Analogues 11-17 and 23 were obtained in comparable
yields using this general procedure with the appropriate amino
acids introduced at position 7 or 8 during the chain elongation
step of the resin-bound peptide. The dinitrophenyl (Dnp)
protection of the imidazole of histidine in 14 was removed with
thiophenol/NMP at the end of the synthesis and prior to HF
cleavage.
ꢀ
ꢀ
Boc-Lys(N -cyclohexyl, N -Cbz) to yield the desired âAla(NR-
isopropyl, NR-Cbz) as a thick oil. Yield ) 19.90 g (75 mmol,
75%); ESI-MS (M + H)+ ) 266.10, (M + Na+) ) 288.10. HPLC
assay: column (C18 0.21 cm × 15 cm); buffer A, 0.1% TFA in
H2O; buffer B, 0.1% TFA in 60% ACN/40% H2O; gradient
condition, 40-90% buffer B over 50 min at a flow rate of 0.2
mL/min; UV detection, 0.1 AUFS at 214 nM; tR ) 13.6 min;
Synthesis of [Nâ, Nâ-di-(2-pyridyl-methyl)D-Dap3]de-
garelix (3). The fully protected [Ac-D-2Nal-D-4Cpa-D-Dap-
ꢀ
(Fmoc)-Ser(Bzl)-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-ILys(N -Cbz)-
1
purity ) 99%. H NMR (CDCl3) δ 1.15 (d, 6H, CH3s), 2.64 (t,
Pro-D-Ala]MBHA resin was synthesized manually on methyl-
benzhydrylamine resin (1 g, 0.33 mmol/g substitution). The
Fmoc side chain protecting group of D-Dap was then removed
with 30% piperidine in NMP (20 min). The exposed D-Dap
amino group was reductively alkylated with 2-pyridinecar-
boxaldehyde (16.5 mmol) in the presence of NaCNBH3 (3.3
mmol) in DMF (25 mL) at room temperature for 3 h. The cycle
was repeated with fresh reagents for an additional 3 h. The
progress of the reaction was monitored by chloranil test20 (used
for detection of secondary amine on resin) and ninhydrin test.51
The completed resin-bound peptide (1.45 g) was then cleaved,
washed, extracted, and purified by preparative RP-HPLC
using the same conditions described for analogue 18. Yield of
3 after purification was 68 mg (38.80 µmol, 11.77%).
2H, CH2-CO), 3.44 (t, 2H, CH2-N), 4.30 (br, 1H, CH-(CH3)2),
5.15 (s, 2H, CH2-Cbz), 7.32 (br, 5H, ArHs-Cbz), 11.20 (br s,
1H, COOH).
Peptide Synthesis. All of the peptides were synthesized
manually by solid-phase peptide synthesis (SPPS) methodol-
ogy12 using previously described tert-butyloxycarbonyl (Boc)
strategy on methylbenzhydrylamine (MBHA) resin (approxi-
mately 1 g of starting resin per peptide). Trifluoroacetic acid
(TFA) treatment was used for Boc removal for 20 min. Two-
to three-fold excess of protected amino acid based on the
original substitution of the resin was used for coupling for 90-
120 min. N-terminal acetylation was performed by using
excess acetic anhydride in DCM for 15 min. Compounds 1
and 2 were synthesized and purified as previously de-
scribed.13,14
Analogue 4 was obtained in comparable yield using this
procedure and D-Dab at position 3.
Synthesis of [IOrn8]degarelix (18). Analogue 18 was
derived from the fully protected [Boc-D-4Aph(Nω-Fmoc)-Leu-
IOrn(Nδ-Cbz)-Pro-D-Ala]-MBHA resin (1 g; 0.33 mmol/ sub-
stitution). The individual amino acids were incorporated in a
sequential manner utilizing either DIC (N,N′-diisopropylcar-
bodiimide) or TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-hexa-
methyluronium tetrafluoroborate) mediated activation of the
carboxyl group. The extent to which individual couplings had
proceeded was qualitatively determined by the ninhydrin test
as described by Kaiser et al.51 The NR-Boc group was removed
after each coupling cycle by treatment of the growing peptide
resin with 60% TFA in DCM in the presence of 1% m-cresol
for 20 min. The NR-Boc-protected, resin-bound pentapeptide
was treated with 30% piperidine in NMP (20 min) to selectively
liberate the ω-amino function of D-4Aph. Carbamoylation of
the free Nω-amino function of D-4Aph was then carried out
using tert-butyl isocyanate (2 mmol) in dry DMF (5 mL) at
room temperature for 12 h. The completion of reaction was
monitored by ninhydrin test. After the removal of NR-Boc from
D-4Aph, the synthesis on the resin was continued with the
coupling of Boc-4Aph(Nω-Fmoc). The ω-amino function of 4Aph
was freed with 30% piperidine in N-methylpyrrolidone (NMP)
and then L-hydroorotic acid (1 mmol) was coupled with DIC
(1 mmol) and HOBt (1 mmol) in NMP. The mixture was
agitated at room temperature for 2 h and a ninhydrin test
indicated the completed reaction. After the removal of NR-Boc
from 4Aph5 at position 5, chain elongation with the four
N-terminal amino acids and acetylation gave the fully pro-
tected resin-bound peptide precursor. HF treatment (anhy-
Synthesis of [Nâ-(2-pyridyl-methyl)D-Dap3]degarelix
(5). This analogue was synthesized the same way as analogue
3 except the deprotected amino group of the side chain of D-Dap
was first reacted with the excess of 2-pyridinecarboxaldehyde
(20 mmol) in DMF (10 mL) at room temperature for 4 h. All
of the solvents and reagents were drained, and the resin was
washed sequentially with methanol (2 × 25 mL) and dichlo-
romethane (2 × 25 mL). The resin bound peptide aldimine was
agitated for 30 min with solid NaCNBH3 (3.3 mmol) in DMF
(15 mL). The reduction step was repeated with fresh reagents
for an additional 30 min. The progress of the reaction was
monitored by chloranil test.20 The workup and purification of
the completed peptide was performed as described above. Yield
of 5 after purification was 42 mg (25.30 µmol, 7.66%).
Analogues 6-10 were obtained in comparable yields using
this procedure; different aldehydes (2-quinolinecarboxaldehyde
or 2-imidazolecarboxaldehyde) and D-Dab were substituted at
position 3.
ꢀ
Synthesis of [N -(IGly)Lys8]degarelix (19). First, we
ꢀ
synthesized [Boc-Lys(N -Fmoc)-Pro-D-Ala]-MBHA resin on 1
g of methylbenzhydrylamine resin with 0.33 mmol/g substitu-
tion. This protected, resin-bound peptide was treated with 30%
ꢀ
piperidine in NMP (20 min) to free the N -amino group of
lysine and then IGly(NR-Cbz) (1 mmol) was coupled by using
DIC (1 mmol) and HOBt (1 mmol) in NMP. After the removal
of NR-Boc from Lys, the chain elongation with the seven
N-terminal amino acids and acetylation gave the completed
peptide resin (1.60 g). The workup and purification of the

4858 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Samant et al.
completed peptide was performed as described above. Yield of
19 after purification was 120 mg (71.80 µmol, 21.78%).
radioimmunoassay using kits purchased from Diagnostic
Systems Laboratories (Webster, TX).
Analogues 20-22, 24-26, and 30 were obtained in compa-
rable yields using this procedure and different amino acid
derivatives: NR-isopropyl-Gly(NR-Cbz); NR-cyclohexyl-Gly(NR-
Cbz); NR-isopropyl-âAla(NR-Cbz); L-Hor coupled on the orthogo-
nal amino group of Orn/Dap/Dab at position 8 during the chain
elongation of the peptide resin.
Synthesis of [Nâ-(ICbm)Dap8]degarelix (28). This ana-
logue was synthesized the same way as analogue 19 except
the deprotected amino group of the side chain of Dap intro-
duced at position 8 was reacted with isopropyl isocyanate (2
mmol) in dry DMF (5 mL) on the resin. The mixture was
agitated at room temperature for 12 h, and the ninhydrin test
indicated a complete reaction. After the removal of NR-Boc from
Dap, chain elongation with the seven N-terminal amino acids
and acetylation was carried out on the resin. The completed
peptide resin (1.65 g) was then cleaved and purified by
preparative RP-HPLC as described above. Yield of 28 after
purification was 110 mg (67.40 µmol, 20.41%).
Cell Culture. Human embryonic kidney cells (HEK293
cells) genetically modified to express a cloned human GnRH
receptor (Larry Jameson, Northwestern University, IL) and a
luciferase reporter gene under the control of LH R subunit
promoter56 were cultured in phenol red free DMEM containing
10% (v/v) FBS, G418 (0.4 mg/mL), penicillin/streptomycin
solution (100 U of penicillin and 100 µg of streptomycin per
mL of medium), and L-glutamine (2 mM). The cells were
harvested and plated at 5 × 104 cells per well in a volume of
80 µL per well in white 96 well culture plates. The cells were
incubated at 37 °C under 5% CO2 overnight for assay the next
day.
IC50 Determination Using the Reporter Gene Assay.
Each compound was assayed in duplicate at 11 descending
concentrations in half-log increments. Compounds in 1%
DMSO (10 µL) or 1% DMSO alone as a control was added to
the hGnRH receptor expressing HEK293 cells followed by
gentle mixing and incubation for an additional 10 min at 37
°C under 5% CO2. Following this, GnRH (10 µL) was added to
a final concentration of 1 nM. Plates were then incubated for
a minimum of 5 h at 37 °C under 5% CO2, after which 100 µL
of luciferase substrate mix was added to each well. Plates were
sealed with Packard Topseal film, and luminescence was
measured on a Molecular Devices Analyst after a 10 min
incubation at room temperature in the absence of direct light.
Analogue 29 was obtained in comparable yields using this
procedure, and Dab was substituted at position 8.
Nr-Methylation on the Resin. A general procedure for the
introduction of methyl group at the NR-amino function of a
nascent peptide chain on the resin is described in the litera-
ture.53 We have used this methodology for the synthesis of
some of the analogues (31-33) presented here. In short, the
analogue was built on the MBHA resin up to and including
the amino acid that would ultimately contain the NR-methyl
group. Removal of the NR-Boc group (60% TFA in DCM, 20
min) provided the unprotected NR-amino function. Alkylation
of this primary amino group with 4,4′-dimethoxybenzhydryl
chloride54 in the presence of triethylamine gave the corre-
sponding N-terminal secondary amino function now containing
the TFA-labile 4,4′-dimethoxybenzhydryl group. Methylation
of this secondary amine by treatment with 36% aqueous
formaldehyde in NMP (3:7) in the presence of excess sodium
cyanoborohydride (2 × 40 min), followed by treatment with
60% trifluoroacetic acid in DCM (2 × 20 min) to remove the
4,4′-dimethoxybenzhydryl group, provided the corresponding
NR-methylated peptide. The coupling of N-protected amino acid
to a N-methyl amino acid on the resin is difficult and usually
gives low to moderate yield of products, which are often
contaminated with unwanted diastereomers. We used bromo-
tris-pyrrolidino-phosphonium hexafluorophosphate55 (PyBrop)/
DIPEA mediated coupling in NMP to get the desired ana-
logues.
To derive the IC50, the test compound counts per second (cps)
values (minus blank cps) were expressed as a percentage of
the control cps values (minus blank cps). The percentage
values were plotted against the log of the concentration used,
and a curve was fitted to the data. An IC50 value was derived
by nonlinear regression to a 4-parameter logistic equation
[sigmoidal dose-response (variable slope)], using the Graph-
Pad Prism (version 2.01) curve fitting software package. The
geometric mean of the IC50 from at least two independent
experiments is reported for each compound.
Acknowledgment. We thank Ron Kaiser and Char-
leen Miller for technical assistance and William Low
and Dr. Wolfgang Fisher for MS analysis. Work at the
Salk Institute was supported by NIH Grant HD-39899.
In vitro biological assays were carried out at FRI Inc.
We thank Dr. Judit Erchegyi for critical reading of the
manuscript and Debbie Doan for manuscript prepara-
tion. J.R. is the Dr. Frederik Paulsen Chair in Neuro-
sciences Professor.
Analogue 27 was synthesized by introducing NR-Fmoc-Dap-
(Boc) at position 3 during the elongation of the peptide chain
followed by Nâ-methylation of Dap as described above on the
resin. âAla(NR-isopropyl, NR-Cbz) was then coupled to Nâ-
methyl of Dap using PyBrop/DIPEA in NMP. After the removal
of Fmoc group from NR of Dap, chain elongation was carried
out as described for 28.
Peptide Characterization. Purity of the peptides was
assessed using RP-HPLC and CZE under conditions reported
in the legend of Table 1. Composition of the analogues was
confirmed by mass spectrometric analysis.
Biological Testing. Castrated Male Rat Assays. Male
Sprague-Dawley rats (180-200 g at the beginning of experi-
ments, n ) 5-8) were castrated under ether anesthesia 10
days prior to the start of the experiment. The peptides (500
µg) were dissolved in a bacteriostatic water containing 5%
mannitol and 0.6% DMSO. The rats were injected sc with a
total dose of 50 µg/rat in 50 µL of aqueous buffer. Blood was
sampled from the tail tip (300 µL) at the given times. Plasma
LH was determined by radioimmunoassay (RIA) using re-
agents provided by the National Pituitary and Hormone
Distribution Program of the NIDDK (Bethesda, MD) with the
exception of the second antiserum. NIDDK anti-rat LH S11
serum was used. For each experiment, all plasma samples
(vehicle control and tested peptides) were measured in the
same RIA. Plasma testosterone levels were determined by
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