Design and synthesis of potent hexapeptide and heptapeptide gonadotropin-releasing hormone antagonists by truncation of a decapeptide analogue sequence

Article abstract of DOI:10.1021/jm990433g

A novel strategy for designing reduced-size analogues of the decapeptide gonadotropin-releasing hormone (GnRH) was developed. As opposed to previous attempts to delete residues from either of the peptide's termini, our approach is based upon the known imp

Full text of DOI:10.1021/jm990433g

J . Med. Chem. 2000, 43, 2831-2836
2831
Design a n d Syn th esis of P oten t Hexa p ep tid e a n d Hep ta p ep tid e
Gon a d otr op in -Relea sin g Hor m on e An ta gon ists by Tr u n ca tion of a Deca p ep tid e
An a logu e Sequ en ce
Dror Yahalom,†,‡ Shai Rahimipour,†,‡ Yitzhak Koch,‡ Nurit Ben-Aroya,‡ and Mati Fridkin*,†
Departments of Organic Chemistry and Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
Received August 24, 1999
A novel strategy for designing reduced-size analogues of the decapeptide gonadotropin-releasing
hormone (GnRH) was developed. As opposed to previous attempts to delete residues from either
of the peptide’s termini, our approach is based upon the known importance of both C- and
N-terminals of GnRH analogues for receptor recognition, whereas the central part of the
molecule is replaced by a short spacer. The present truncation strategy was successful for
generation of reduced-size hexapeptide and heptapeptide antagonists possessing potent
antagonistic capacity. The same methodology was not suitable for the generation of reduced-
size agonists, suggesting different conformational characteristics for GnRH agonists and
antagonists. A heptapeptide antagonist designed by this method was shown to inhibit serum
levels of luteinizing hormone in castrated rats in vivo. Structure-activity studies suggested
that the structural preferences for GnRH receptor recognition are similar to those reported for
decapeptide antagonists. Our studies resulted in a heptapeptide GnRH antagonist (Ac-D-Nal2-
D-Cpa-D-Pal-Gly-Arg-Pro-D-Ala-NH2) with high receptor binding affinity (IC50 ) 7 nM), as
compared to that of GnRH itself (IC50 ) 2 nM). The highest affinity of a hexapeptide antagonist
that we have synthesized was somewhat lower (IC50 ) 45 nM).
In tr od u ction
antagonist, which is based solely on the central 5-8
residues.8 These strategies are not in line with numer-
ous studies which suggested that both termini of the
GnRH molecule are involved in direct interactions with
the GnRH receptor (for review, see refs 9 and 10). On
the basis of conformational energy calculations11 and
various physicochemical methods,9 it was suggested that
the bioactive conformation of GnRH include a type II′
â turn involving residues 5-8 {Tyr5-Gly6-Leu7-Arg8}.
There are indications that the dominant conformation
of GnRH antagonists is similar, but not identical, to that
of GnRH.12
Our approach for developing reduced-size GnRH
analogues is based on replacing several residues from
the central part of the peptide by a short spacer, while
the terminal residues remain unchanged. A similar
truncation strategy was employed successfully to form
potent truncated analogues of the 36-amino acid neu-
ropeptide Y (NPY), e.g., NPY (1-4)-Aca-(25-36) (Aca
) amino caproic acid).13 The success of this strategy for
the establishment of novel GnRH antagonists was
followed by structure-activity studies.
Gonadotropin-releasing hormone (GnRH), pGlu-His-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, is secreted from
the hypothalamus in a pulsatile pattern and regulates
the reproductive system by controlling the secretion of
the gonadotropic hormones, luteinizing hormone (LH),
and follicle stimulating hormone (FSH) from the ante-
rior pituitary.1,2 These hormones, in turn, stimulate
gonadal steroidogenesis and gametogenesis. Continuous
administration of GnRH results in down-regulation and
receptor desensitization, leading to an inhibition of
pituitary gonadotropin secretion. Therefore, synthetic
GnRH analogues, both agonists and antagonists, may
be used for contraception and for the treatment of
various hormone-dependent diseases, including prostate
and breast cancer.3
GnRH agonists which were approved for clinical use
are deca- or nonapeptides, whereas all the antagonists
in clinical trials are decapeptides. Development of
reduced-size GnRH analogues is important for the
characterization of the interactions of the natural pep-
tide with its receptor. Furthermore, such analogues may
also be useful as drugs or as intermediates in the search
for non-peptide ligands.
Resu lts
To test the truncation strategy, we have synthesized
the two agonists and two antagonists that are listed in
Table 1. The residues 4-8 or 4-7 of the parent corre-
sponding analogues, GnRH and antide (a potent GnRH
antagonist), were replaced by D-Ala as a spacer. Thus,
in each pair of analogues (peptides 1,2 and peptides 3,4),
there is one in which the original Arg residue in position
8 of GnRH is conserved and one which lacks this
residue. The side chain of Arg8 of GnRH was previously
suggested to stabilize the active conformation of the
The reported studies of reduced-size GnRH analogues
were traditionaly based on the gradual deletions of
residues from either the C- or the N-termini of GnRH
or its decapeptide/nonapeptide analogues.4-7 There is
also a report of a cyclic hexapeptide, a weak GnRH
* Corresponding author: Mati Fridkin, Department of Organic
Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Tel: 972-8-9342505. Fax: 972-8-9344142.
† Department of Organic Chemistry.
‡ Department of Neurobiology.
10.1021/jm990433g CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/30/2000

2832 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yahalom et al.
Ta ble 1. Sequences and Mass Spectra Analysis of GnRH, of Antide, and of Their Reduced-Size Analogues
MH+
peptide
sequencea
obsd (calcd)b
GnRH
1
p Glu 1-His2-Tr p 3-Ser-Tyr-Gly-Leu-Ar g8-P r o9-Gly10-NH2
p Glu -His-Tr p -D-Ala-Ar g-P r o-Gly-NH2
834.2 (833.9)
677.7 (677.4)
2
p Glu -His-Tr p -D-Ala-P r o-Gly-NH2
antide
3
4
Ac-D-Na l21-D-Cp a 2-D-P a l3-Ser-Lys(Nic)-D-Lys(Nic)-Leu-Lys(Isp)-P r o9-D-Ala 10-NH2
Ac-D-Na l2-D-Cp a -D-P a l-D-Ala-Arg-P r o-D-Ala -NH2
Ac-D-Na l2-D-Cp a -D-P a l-D-Ala-P r o-D-Ala -NH2
983.6 (982.6)
827.4 (826.4)
a
Bold letters represent the residues of GnRH or of antide, which were preserved in the respective reduced-size analogues. D-Nal2 )
â-[2-naphthyl]-D-Ala; D-Cpa ) D-p-chloro-Phe; D-Pal ) â-[2-pyridyl]-D-Ala; Lys(Nic) ) Nꢀ-[nicotinoyl]; Lys(Isp) ) Nꢀ-[isopropyl]Lys.
Observed (obsd) and calculated (calcd) m/z values of MH+ monoisotopes. Purities of the reduced-size analogues 1-4 were >99%, according
b
to two analytical HPLC solvent systems that are detailed in Table 2.
F igu r e 2. Induction of LH release from dispersed rat pituitary
F igu r e 1. Dose-response curves for the displacement (%) of
specific binding of 125I-[D-Lys6]GnRH to pituitary membranes
of proestrous rats, by GnRH and by reduced-size analogues.
Membranes were incubated for 90 min at 4 °C with 125I-[D-
Lys6]GnRH and with the unlabeled peptides. Nonspecific
binding was determined by the presence of 1 µM of [D-Lys6]-
GnRH, and it was subtracted from the total binding for the
calculation of specific binding. Results are the mean ( SEM
of two experiments carried out in triplicate.
cells by GnRH and its reduced-size analogues. At 48 h after
dispersion, the medium was changed, and the cells were
incubated for 4 h at 37 °C with the examined peptides. The
medium was collected, and LH concentration was determined
by RIA. Results are the mean ( SEM of LH concentrations in
two experiments (four wells/experimental group). The LH
concentration in each well was determined using triplicates.
*LH release significantly different (p < 0.01) from the control
group (basal).
peptide by hydrogen bonding with the side chains of
His2 and Tyr5.14 We have hypothesized that, in reduced-
size analogues lacking the Tyr residue, the Arg residue
may be less important for attaining GnRH receptor
binding affinity. A representative sequence of an an-
tagonist that is now in clinical trials, antide,15 is shown
in Table 1. All of the currently used decapeptide GnRH
antagonists possess identical substitutions at residues
1, 2, 3, and 10, while Pro9 is conserved. Residue Arg8 is
generally replaced by a polar, noncharged residue
(except for cetrorelix, which contains Arg8).16 Peptides
3 and 4 are therefore the reduced form of antide or of
any of the other clinically tested GnRH antagonists. The
GnRH receptor binding affinity of antide is about 1
order of magnitude higher than that of GnRH (in our
system, IC50 values are 0.3 and 2 nM, respectively).
The two reduced-size GnRH agonists (peptides 1 and
2) were found to have very low GnRH receptor binding
affinities (Figure 1). Consequently, the agonistic activity
of these peptides was weak or negligible, even at 10 µM
(Figure 2). On the other hand, peptide 3 is a high
affinity GnRH analogue (Figure 1). Thus, the size-
reduction strategy employed, i.e., the elimination of the
central region of the decapeptide GnRH analogue, is
successful only for the generation of potent antagonists.
The presence of an Arg residue increases the binding
affinity of agonists (peptide 1 versus peptide 2) and,
more crucially, of antagonists (peptide 3 versus peptide
4) (Table 1). To exclude the option that peptide 3 is a
partial agonist, we performed in vitro studies using
dispersed cells of rat pituitaries. At 1 µM, peptide 3 was
not only inactive as an agonist but like antide it actually
inhibited basal release of LH (Figure 2). No agonistic
activity was observed at lower concentrations of peptide
3 (data not shown).
To further demonstrate the antagonistic activity of
peptide 3, we have tested it for its in vivo inhibition of
LH release in castrated rats (Figure 3). While the
inhibition of LH release by peptide 3 is significantly
weaker than that of antide, this experiment suggests
that peptide 3 is a rather long-acting GnRH antagonist.
Structure-activity studies of the reduced-size an-
tagonists, peptide 3 and peptide 4, are summarized in
Table 2. The results of the GnRH receptor binding
studies suggest that (I) Arg in position Y (peptide 3,
Table 2) can be replaced by the positively charged Lys
(peptide 5) or by polar residues (peptides 7, 8) without
substantial change in GnRH receptor affinity, while its
substitution by a hydrophobic residue results in much
lower receptor affinity (peptides 6, 9). These preferences
are very similar to those of the respective position in
decapeptide agonists17 and antagonists.18 It seems that
there is not much preference for L-configuration over
D-configuration (e.g., peptide 11 versus peptide 22). (II)
Position X (the spacer residue, see Table 2) is very

Hexapeptide and Heptapeptide GnRH Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2833
to have very low GnRH receptor binding affinities (data
not shown), similar to the affinities of peptides 1 and 2,
respectively. These results suggest that the low affini-
ties of peptides 1 and 2 are not the result of the use of
a specific spacer (D-Ala) but rather a consequence of
fundamental conformational attributes of GnRH ago-
nists.
To test the preferences in the N-terminal tripeptide
of heptapeptide antagonists, we have modified these
residues, using peptide 11 as a template (Table 3).
Substitution of positions 2 and 3 by D-Phe resulted in a
fairly potent antagonist (peptide 27, Table 3). However,
while the peptides shown in Tables 1 and 2 are all
highly water-soluble, this peptide has a much lower
solubility. Thus, solubility can be varied according to
needs, without major changes in receptor binding af-
finity. Various aromatic D-amino acid substitutions in
position 1 of peptide 27 resulted in immensely decreased
binding affinity (peptide 28-32, Table 3). These include
substitution of the D-Nal2 residue (D-Nal2 ) â-[2-
naphthyl]-D-Ala) by the related D-Nal1 residue (D-Nal1
) â-[1-naphthyl]-D-Ala, peptide 29), as well as deletion
of the terminal acetyl (peptide 31) or its replacement
by an Fmoc group (peptide 32). The preferences in the
N-terminal tripeptide of the heptapeptide antagonists
are therefore similar to those of the relevant decapeptide
antagonists,19 i.e., positions 2 and 3 may be accom-
modated by various aromatic D-amino acids (polar or
nonpolar), while Ac-D-Nal2 in position 1 is clearly
superior to other aromatic D-amino acids, including
residues as similar as Ac-D-Nal1.
F igu r e 3. Effect of peptide 3 or antide on serum LH levels in
castrated rats following intraperitoneal administration of the
peptides. Blood samples were taken from each rat at the
indicated time. Control animals were injected 0.1 M PBS,
which was also used for dissolving the examined peptides. The
serum samples were assayed for LH using RIA. Results are
the mean ( SEM of LH concentrations in the serum of five
animals/experimental group. The LH concentrations were
determined using three different dilutions of each serum
sample. LH release in the presence of both peptides is
significantly lower (p < 0.05) than in the respective vehicle
control group (at all time intervals except for time 0).
Discu ssion
Reduced-size high affinity GnRH antagonists were
rationally designed based on previous structure-activ-
ity studies of GnRH analogues. Substitution of the four
central residues by a single amino acid resulted in
heptapetides with GnRH receptor binding affinities in
the nanomolar range. These results emphasize the role
of the two termini of GnRH antagonists for receptor
recognition. When the same methodology was employed
for the GnRH molecule itself, it resulted in peptides
which recognize the GnRH receptor only at the micro-
molar range. These findings reflect fundamental differ-
ences between the conformations of GnRH agonists and
antagonists. It was previously suggested by Nikiforovich
and Marshall12 that the â-II′ turn in the central region
of GnRH agonists maintains the proper spatial arrange-
ment of the N-terminal tripeptide, which presumably
participates in a direct interaction with the GnRH
receptor. This model explains the loss of receptor affinity
in the reduced-size agonists described above, which lack
the central â-II′ turn region. As for GnRH antagonists,
it was suggested that they differ from GnRH agonists
with respect to the conformation of the central 5-8
residues, while the spatial arrangement of the N-
terminal tripeptide is very similar to that of the ago-
nists.12 Our detailed structure-activity studies, directed
at the spacer connecting the two termini of the reduced-
size antagonists, demonstrate high tolerance of this
position. Substitution by Ala, D-Ala, D-Tyr, Sar, âAla,
and even aminocaproic acid (Aca) and 1-amino cyclobu-
tane-1-carboxylic acid (MePro) all resulted in fairly
similar affinities. The latter two substitutions indicate
tolerant for a variety of residues (peptides 10-20).
Substitution by the Gly residue, enabling maximal
freedom of bond angles, resulted in the peptide with the
highest GnRH receptor affinity in this series (IC50 ) 7
nM), only 3-4 times lower than that of GnRH. The
analogue containing the stretched aminocaproic acid
(Aca) residue in the X position has high GnRH receptor
affinity (peptide 18), which suggests that the distance
between the two parts of the molecule can be varied
without disturbing the GnRH receptor binding affinity.
(III) High affinity hexapeptides can also be produced
by the size-reduction strategy (the last five peptides
shown in Table 2 are hexapeptides). The preferences for
the residue in position X of these hexapeptides (Table
2) are not clear from our limited results. A Gly residue
substitution (peptide 24) resulted in the hexapeptide
with the highest GnRH receptor affinity, similar to the
results concerning the heptapeptides (peptide 11). The
preferences as to D- versus L-configurations are un-
clear: while D-Arg is preferred over Arg or Lys, D-Ala
seems to be much inferior (peptides 23, 25, 26, and 4,
respectively).
The employment of Gly as a spacer was preferred in
both hexapeptide (peptide 24) and heptapeptide (peptide
11) antagonists. To apply this substitution for agonists,
we have synthesized two reduced-size agonists: pGlu-
His-Trp-Gly-Arg-Pro-Gly-NH2 and pGlu-His-Trp-Gly-
Pro-Gly-NH2. These peptides are based on the sequences
of peptides 1 and 2 (Table 1), respectively, with D-Ala
substituted by Gly. Both of these peptides were found

2834 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Yahalom et al.
Ta ble 2. Analytical Data and GnRH Receptor Binding Affinities of Peptides of the Form: Ac-D-Nal2-D-Cpa-D-Pal-X-Y-Pro-D-Ala-NH2
purity (%)
MH+
peptide
X
Y
HPLC-1a
HPLC-2b
obsd (calcd)c
IC50 (nM)d
3
5
6
7
8
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
Ala
Gly
Sar
âAla
Leu
D-Leu
Pro
D-Tyr
Aca
MePro
Ana
D-Arg
Gly
D-Ala
D-Arg
Gly
Arg
Lys
Arg
>99
>99
>99
>99
>99
>97
>97
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>97
>99
>99
>95
>98
>98
>98
>99
>99
>99
>99
>98
>99
>99
>99
>99
>99
>99
>99
>97
>99
>95
>99
>99
>95
>99
>99
>98
>99
983.6 (982.6)
955.6 (954.6)
1001.6 (1000.6)
1059.7 (1058.0)
981.6 (981.2)
942.1 (941.6)
982.6 (981.5)
968.6 (967.4)
982.6 (981.4)
982.6 (981.5)
1024.7 (1023.1)
1024.7 (1023.3)
1008.6 (1008.5)
1074.7 (1074.5)
1024.7 (1023.5)
1008.6 (1007.5)
1030.6 (1029.4)
1065.7 (1065.6)
968.6 (967.4)
827.4 (825.3)
911.5 (910.5)
812.5 (811.3)
911.5 (911.1)
883.5 (883.2)
17 ( 3
20 ( 3
Lys
Lys(Isp)
Lys(Nic)
Cit
130 ( 40
14 ( 3
23 ( 7
9
Nle
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Cit
88 ( 28
28 ( 7
10
11
12
13
14
15
16
17
18
19
20
21
22
4e
23
24
25
26
7 ( 2
31 ( 11
38 ( 15
81 ( 17
74 ( 21
110 ( 25
25 ( 2
11 ( 2
42 ( 13
140 ( 40
72 ( 20
16 ( 4
D-Arg
-
-
-
-
1500 ( 600
70 ( 5
45 ( 5
130 ( 50
550 ( 70
-
a
The purity of the peptides was assessed by two HPLC systems. (1) A 0.1% TFA in water, B 0.1% TFA in 75% acetonitrile: 20% B to
80% B at 1%/min. (2) A 0.1% TFA in water, B 0.1% TFA in 75% 2-propanol: 0% B to 50% B at 1%/min, using Lichrosorb RP-18 column.
b
Elution system: A 0.1% TFA in water, B 0.1% TFA in 75% acetonitrile: 0% B to 100% B at 2.5%/min employing a wide pore butyl (C4)
column. c Observed and calculated m/z values of MH+ monoisotopes are reported. IC50 ) Concentration of peptide which displaces 50%
d
of 125I-[D-Lys6]GnRH bound to rat pituitary membranes; values ( SEM are based on displacement curves obtained by incubating rat
pituitary membranes for 90 min at 4 °C with 125I-[D-Lys6]GnRH and increasing concentrations of the indicated unlabeled peptides. The
IC50 of GnRH at the same experimental conditions was found to be 2 nM. e This peptide and the following peptides 23-26 are hexapeptide
(no Y residue). Aca ) aminocaproic acid; Ana ) anthranilic acid; Cit ) citrulline; Isp ) isopropyl; MePro ) 1-amino cyclobutane-1-
carboxylic acid; Nle ) norleucine; Sar ) sarcosine.
Ta ble 3. Analytical Data and GnRH Receptor Binding Affinities of Peptide 11 and Its N-Terminal Modified Analoguesa
purity (%)
MH+
peptide
sequence
HPLC-1
HPLC-2
obsd (calcd)
IC50 (nM)
11
27
28
29
30
31
32
Ac-D-Nal2-D-Cpa-D-Pal-Gly-Arg-Pro-D-Ala-NH2
Ac-D-Nal2-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
Ac-D-Phe-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
Ac-D-Nal1-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
Ac-D-Trp-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
D-Nal2-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
Fmoc-D-Nal2-D-Phe-D-Phe-Gly-Arg-Pro-D-Ala-NH2
>99
>99
>99
>98
>99
>99
>98
>99
>98
>99
>99
>99
>98
>97
968.6 (967.4)
933.4 (933.4)
880.6 (880.5)
933.4 (933.2)
918.6 (918.1)
892.0 (891.9)
1113.4 (1112.8)
7 ( 2
28 ( 7
230 ( 20
370 ( 90
450 ( 100
280 ( 70
2200 ( 400
a
For abbreviations and reference to HPLC systems, see legend to Table 2. D-Nal1 ) â-[1-naphthyl]-D-Ala; D-Nal2 ) â-[2-naphthyl]-
D-Ala.
that both a long-chain spacer (Aca) and a residue that
receptor or that it stabilizes the active conformation of
the peptide by hydrogen bonding, as was suggested for
GnRH itself.14
constrains the peptide conformation (MePro) can be
fairly tolerated. On the basis of the high affinity and
tolerability of the reduced-size antagonists, we suggest
that the spatial arrangement of the N-terminal tripep-
tide tested (Ac-D-Nal2-D-Cpa-D-Pal) may be significantly
based on stabilization by local interactions within this
tripeptide, rather than the assumed stabilization of a
similar conformation of the N-terminal tripeptide of
agonists by the central region of these peptides.12 This
notion may open new directions for the design of novel
GnRH antagonists.
The improved GnRH receptor affinities of peptide 3
over peptide 4 (Figure 1) and the structure-activity
studies of peptide 3 (peptides 5-9, Table 2) suggest an
important role for the Arg residue in GnRH receptor
recognition by GnRH antagonists. Nevertheless, these
results, as well as other observations following analo-
gous structure-activity studies in decapeptide GnRH
agonists17 and antagonists,18 still raise a debate as to
whether the Arg residue interacts directly with the
In all decapeptide GnRH antagonists that are cur-
rently in clinical trials, the positive charge of the eighth
residue is masked by various groups (e.g., isopropyl
attached to the Lys residue in antide, see Table 1). It
was previously shown that the combination of positive
charges in both the sixth and eighth residues, together
with the aromatic N-terminus, result in potent GnRH
antagonists. These basic antagonists, however, also
cause side effects due to histamine release.20 The potent
reduced-size antagonists presented herein, although
containing an Arg residue or a positively charged/polar
substitution, may, potentially, be of low potency in
inducing histamine release, due to the lack of a second
positively charged/polar residue (found at the sixth
position of most decapeptide antagonists).
In conclusion, our studies resulted in a novel class of
reduced-size GnRH analogues which may contribute to
the understanding of conformational differences be-

Hexapeptide and Heptapeptide GnRH Antagonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2835
The peptides were also analyzed by an LCQ mass spectrometry
system (Finnigan, Bremen, Germany) using a nanospray
ionization technique. The latter two analyses further confirmed
the composition and purity of the products. Pure peptides were
dissolved in water, to obtain 1 mM concentration, and aliquots
were kept frozen (-20 °C).
tween agonists and antagonists and to the structural
requirements of GnRH antagonists. This class of GnRH
antagonists may be further developed to form candidate
drugs, which may have advantages over the currently
available decapeptide antagonists.
Sid e Ch a in Mod ifica tion s. Two 10 mg aliquots of purified
Ac-D-Nal2-D-Cpa-D-Pal-D-Ala-Lys-Pro-D-Ala-NH2 (peptide 5)
were used for modifications of the Lys residue: one aliquot
was reacted with 5 equiv of nicotinic acid (BDH, Poole, U.K.)
and 5 equiv of DCC/HOBT in DMF, resulting in Ac-D-Nal2-
D-Cpa-D-Pal-D-Ala-Lys(Nic)-Pro-D-Ala-NH2 (peptide 7). The
other aliquot was subjected to reductive alkylation in solution
using 10 equiv of NaCNBH3 in a DMF/acetone/acetic acid (30:
10:1) solution, according to a reported procedure,25 resulting
in Ac-D-Nal2-D-Cpa-D-Pal-D-Ala-Lys(Isp)-Pro-D-Ala-NH2 (pep-
tide 6). Both peptides were purified by HPLC and analyzed
as described above.
Exp er im en ta l Section
Ma ter ia ls. Unless otherwise stated, all chemicals and
reagents were of analytical grade. Trifluoroacetic acid (TFA)
for high performance liquid chromatography (HPLC) was
obtained from Merck (Darmstadt, Germany). Commonly used
9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid de-
rivatives and Rink-amide resin were purchased from Nova-
biochem (Laufelfingen, Switzerland). Ac-â-[2-Naphthyl]-D-Ala
was purchased from Synthetech (Albany, OR). Fmoc-D-p-
chloro-Phe and Fmoc-â-[2-pyridyl]-D-Ala were obtained from
Bachem (Bubendorf, Switzerland). N,N′-disuccinimidyl car-
bonate, GnRH, and antide were acquired from Sigma (St.
Louis, MO). 1-Aminocyclobutane-1-carboxylic acid (termed
MePro; methano proline) was synthesized according to a
published procedure.21 The Fmoc derivative of this amino acid
was synthesized in our laboratory according to a published
procedure.22
An im a l Stu d ies. Wistar-derived rats were obtained from
the Weizmann Institute Animal Resource Center. Experiments
were carried out in compliance with the regulations of the
Weizmann Institute of Science.
Bin d in g to th e P itu ita r y Gn RH Recep tor . [D-Lys6]-
GnRH (synthesized in our laboratory) was iodinated by the
chloramine T method,26 and 125I-[D-Lys6]GnRH was purified
(1700 µCi nmol-1) by an analytic HPLC system as described
above. The binding assay was conducted as previously de-
scribed.26 In brief, pituitary membranes (25 µg protein/tube,
prepared from Wistar-derived proestrous rats) were incubated
for 90 min at 4 °C with 50 000 cpm (23.5 pM) 125I-[D-Lys6]-
GnRH, alone or in the presence of various concentrations of
the unlabeled peptides, in a total volume of 0.5 mL of the assay
buffer (10 mM Tris-HCl containing 0.1% bovine serum albumin
[BSA]). The reaction was terminated by rapid filtration
through Whatman GF/C filters. The filters were washed three
times with cold assay buffer and counted in an Auto-Gamma
Counting System (Packard, Meriden, CT). The experiments
were performed in triplicate. Nonspecific binding was defined
as the residual binding in the presence of excess of [D-Lys6]-
GnRH (1 µM). Specific binding was calculated by subtracting
the nonspecific binding from the maximal binding, determined
in the absence of any competing peptide. IC50 values were
calculated using the curve-fitting software program Enzfitter
(Elsevier Biosoft, Cambridge, U.K.).
LH Relea se fr om Cu ltu r ed , Disp er sed , P itu ita r y Cells.
Cells from 21-day-old Wistar-derived female rats were dis-
persed as previously described27 and incubated in 96-well
plates (50 000 cells/well) at 37 °C in M-199 medium containing
5% horse serum. After 48 h the cells were washed with M-199
medium containing 0.1% BSA and incubated for 4 h at 37 °C
with M-199/0.1% BSA (0.25 mL) containing the desired
concentrations of the various peptides (four wells/experimental
group). The incubation was terminated by removing the
medium and diluting it by an equal volume of 1% BSA in
phosphate buffered saline (PBS) solution. Three different
aliquots from each sample were analyzed for LH concentration
by radioimmunoassay (RIA)28 using the kit kindly supplied by
the National Institute of Arthritis, Metabolism and Digestive
Diseases (NIAMDD) Rat Pituitary Program. Results are
expressed in terms of the RP-3 reference preparation.
In Vivo Bioa ctivity of Gn RH An ta gon ists. A total of 0.5
mL of 0.1 M PBS containing peptide 3 or antide was injected
intraperitoneally to castrated rats (five per group). The control
group was injected with the vehicle (0.5 mL of 0.1 M PBS).
Blood samples were withdrawn by cardiac puncture under
light ether anesthesia at 0, 2.5, 5, and 10 h after drug
administration,. The serum samples were assayed for LH
content as described above.
P ep tid e Syn th esis. All peptides, other than the above,
were prepared in our laboratory by solid-phase peptide synthe-
sis, with an AMS-422 multiple peptide synthesizer (ABIMED,
Langenfeld, GmbH) using Fmoc chemistry,22 following the
company’s protocols.23 The unnatural amino acid residues of
the GnRH antagonists were attached manually using 2 equiv
of N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazol (DCC/
HOBT) in N-methyl pyroliddone (NMP). The reactions were
performed in sintered plastic syringes which were shaken
overnight in a mechanical shaker at room temperature. The
solutions were than removed and the resin was washed three
times with N,N′-dimethylformamide (DMF) and dichloro-
methane. A negative ninhydrin test indicated the completion
of the reaction. Fmoc removal was achieved by 20% piperidine/
DMF (3 × 5 min). All synthesized peptides were deprotected
and cleaved from the resin using a solution of TFA:triethyl-
silane:anisole:water (17:1:1:1)23 or reagent K solution (TFA:
phenol:water:thioanisole:1,2-ethanedithiol; 33:2:2:2:1) for pep-
tides containing Arg and Trp.24 After 2 h at room temperature,
the cleavage mixtures were filtered, and the peptides were
precipitated with peroxide-free dry ether at 0 °C. Precipitated
peptides were washed with cold dry ether, dissolved in water
or water/acetonitrile solution, and lyophilized. Crude peptides
were then subjected to semipreparative HPLC purification,
performed on a Waters system composed of two model 510
pumps, a model 680 automated gradient controller, and a
model 441 absorbance detector (Waters, Milford, MA). The
column effluents were monitored by UV absorbance at 214/
254 nm. HPLC prepacked columns employed (Merck, Darm-
stadt, Germany) were LichroCART 250-10 mm containing
Lichrosorb RP-18 (7 µm) for semipreparative purifications, and
Lichrospher 100 RP-18, 250-4 mm (5 µm) and wide pore butyl
(C4, 5 µm), 250-4.6 mm (J . T. Baker Inc. Phillipsburg, NJ )
for analytical separations. Separations were achieved using
gradients of acetonitrile in water containing 0.1% trifluoro-
acetic acid (TFA). The homogeneity of the resulting peptides
was tested by analytical HPLC using two solvent systems: (1)
A 0.1% TFA in water, B 0.1% TFA in 75% acetonitrile; 20% B
to 80% B at 1%/min; A 0.1% TFA in water, B 0.1% TFA in
75% 2-propanol; 0% B to 50% B at 1%/min, using Lichrosorb
RP-18 column and (2) a gradient of 0% B to 100% B at 2.5%/
min employing a wide pore butyl (C4) column. The purity of
the peptides in both systems was usually higher than 98%
(Table 2). Solutions containing purified peptides were lyophil-
ized. Samples of each of the peptides were hydrolyzed (6 N
HCl, 110 °C, 22 h, in a vacuum) and analyzed with a Dionex
automatic amino acid analyzer. Quantification of â-[2-naph-
thyl]-D-Ala, D-p-Chloro-Phe, and â-[2-pyridyl]-D-Ala was achieved
by using the respective standards. These results were also used
for determination of the peptide content in each preparation.
Sta tistica l An a lysis. Results are expressed as the mean
( SEM. Comparisons were made using one-way analysis of
variance (Instat 2.01, GraphPad Software, CA). P < 0.05 was
taken as significant.
Abbr evia tion s. Abbreviations of common amino acids are
in accordance with the recommendations of IUPAC. Additional

2836 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
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abbreviations: Aca ) 6-aminocaproic acid; BSA, bovine serum
albumin; DMF, N,N′-dimethylformamide; DMSO, dimethyl
sulfoxide; D-Nal2, â-[2-naphthyl]-D-Ala; D-Pal ) â-[2-pyridyl]-
D-Ala; GnRH, gonadotropin-releasing hormone; HPLC, high
performance liquid chromatography; LH, luteinizing hormone;
IC50, concentration of ligand which displaces 50% of bound
tracer; Lys(Isp), Nꢀ-[isopropyl]Lys; Lys(Nic), Nꢀ-[nicotinoyl]Lys;
MePro, 1-aminocyclobutane-1-carboxylic acid; PBS, phosphate
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Ack n ow led gm en t. We thank Riri Kramer (Depart-
ment of Chemical Services, Weizmann Institute of
science, Rehovot, Israel) for performing amino acid
analysis, Ilana Navon (Protein Center, Technion, Haifa,
Israel) for performing the mass spectrometry analysis,
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Services, Weizmann Institute of Science, Rehovot, Is-
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Weizmann Institute of Science.
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