New active series of growth hormone secretagogues

Article abstract of DOI:10.1021/jm020985q

New growth hormone secretagogue (GHS) analogues were synthesized and evaluated for growth hormone releasing activity. This series derived from EP-51389 is based on a gem-diamino structure. Compounds that exhibited higher in vivo GH-releasing potency than hexarelin in rat (subcutaneous administration) were then tested per os in beagle dogs and for their binding affinity to human pituitary GHS receptors and to hGHS-R 1a. Compound 7 (JMV 1843, H-Aib-(D)-Trp-(D)-gTrp-formyl) showed high potency in these tests and was selected for clinical studies.

Full text of DOI:10.1021/jm020985q

J . Med. Chem. 2003, 46, 1191-1203
1191
New Active Ser ies of Gr ow th Hor m on e Secr eta gogu es
Vincent Guerlavais,† Damien Boeglin,† Delphine Mousseaux,† Catherine Oiry,† Annie Heitz,‡
Romano Deghenghi,§ Vittorio Locatelli,| Antonio Torsello,| Corrado Ghe´, Filomena Catapano,
Giampiero Muccioli, J ean-Claude Galleyrand,† J ean-Alain Fehrentz,*,† and J ean Martinez†
Laboratoire des Aminoacides, Peptides et Prote´ines (LAPP), UMR 5810, Universite´ Montpellier I et II, B.P. 14491,
Faculte´ de Pharmacie, 15 Avenue Charles Flahaut, 34093 Montpellier Ce´dex 5, France, CBS, UMR 5048, Faculte´ de
Pharmacie, 34093 Montpellier Ce´dex 5, France, Europeptides, Zentaris Group, Bt. Aristote, 9 Avenue du Marais,
95108 Argenteuil Ce´dex, France, Department of Experimental, Environmental Medicine and Biotechnology,
School of Medicine, University of Milano-Bicocca, Via Cadore 48, 20052 Monza (MI), Italy, and Division of Pharmacology,
Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Via P. Giuria, 13, 10125 Turin, Italy
Received J uly 5, 2002
New growth hormone secretagogue (GHS) analogues were synthesized and evaluated for growth
hormone releasing activity. This series derived from EP-51389 is based on a gem-diamino
structure. Compounds that exhibited higher in vivo GH-releasing potency than hexarelin in
rat (subcutaneous administration) were then tested per os in beagle dogs and for their binding
affinity to human pituitary GHS receptors and to hGHS-R 1a. Compound 7 (J MV 1843, H-Aib-
(D)-Trp-(D)-gTrp-formyl) showed high potency in these tests and was selected for clinical studies.1
In tr od u ction
Growth hormone (GH) is an important endocrine
regulator of growth and anabolic processes.2 The clinical
use of recombinant human GH has demonstrated ben-
efits in the therapy of GH-deficient children,3 and it
reverses some of the effects of aging in the elderly.4 In
recent years, the GH-releasing peptides (GHRPs) and
peptidomimetics have received considerable attention
as potential alternatives to the expensive recombinant
human GH. During their studies on enkephalin ana-
F igu r e 1. Sequences of GHRP analogues.
logues Bowers and co-workers discovered a series of
peptides able to stimulate GH release from rat pituitary
and opened a new avenue of research.5 This new family
of peptides6 including GHRP-6, GHRP-1, GHRP-2, and
hexarelin promotes the release of GH in man (Figure
1). This GH release mechanism was found to be different
from the endogeneous growth hormone releasing hor-
mone (GHRH)7 and to be mediated through a G-protein-
coupled receptor (GHS-R 1a).8 The natural ligand of
this receptor has been isolated and characterized very
recently from rat stomach9 and further identified in
humans.10 It is a 28 amino acid peptide in which serine
3 is n-octanoylated with the following sequence GSS-
(octanoyl)FLSPEHQRVQQRKESKKPPAKLQPR. Sev-
eral classes of small non-peptide secretagogues (ben-
zolactam biphenyl tetrazoles,11 camphor derivatives,12
and 4-spiropiperidines13) have been described (Figure
2) that are able to release GH from the pituitary.
F igu r e 2. Structures of non-peptide GH secretagogues.
Starting from the GHRP-6 sequence, various peptide
molecules have been reported as potent GHRPs (Figure
3). Starting from hexarelin, we earlier described a
tripeptide EP-51389, Aib-(D)-2-Me-Trp-(D)-2-Me-Trp-
NH2,14 that is more potent than hexarelin in stimulating
GH secretion in an infant rat model when injected
subcutaneously, and it is orally active in dogs and
horses. This 2-Me-Trp (Mrp) containing tripeptide was
also proved to be subcutaneously more potent than
hexarelin in an infant rat model and orally active in
dogs and man.15,16 In this paper, we report the identi-
fication of a new series of potent pseudotripeptide
analogues that act as GH secretagogues.
* To whom correspondence should be addressed. Phone: 33 467 548
651. Fax: 33 467 548 654. E-mail: fehrentz@colombes.pharma.univ-
montp1.fr.
† Universite´ Montpellier I et II.
‡ CBS.
Our experience with bombesin analogues has shown
that in this peptide, replacement of the C-terminus
carboxamide residue Leu-NH2 (or Met-NH2) with the
§ Zentaris Group, Bt. Aristote.
| University of Milano-Bicocca.
University of Turin.
10.1021/jm020985q CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/25/2003

1192 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Guerlavais et al.
and the C-terminal formyl group was replaced by an
acetyl moiety. Methyl groups were also introduced on
the amide bond between the two Trp residues or on the
R carbon of the Trp residues. All attempts to introduce
the methyl group between Trp and Aib residues were
unsuccessful. N-methylation between both tryptophan
residues was performed following Scheme 2. H-(D)-
N(Me)Trp-amide 9 was obtained in three steps from 8
by the Fukuyama methylation method.20 The coupling
step to obtain the pseudodipeptide amide was then
performed with HBTU,21 and after coupling, the indole
groups were protected as previously described to yield
10. Compound 10 was then allowed to react with BTIB.
At this stage we observed an instability of the gem-
diamino compound 11 that led to a partial cleavage of
the dipeptide at the N-methyl bond to yield 12 (Scheme
2). The gem-diamino dipeptide 11 was acylated with
acetic anhydride to yield 13, and compound 19 N(Me)-
Aib-(D)-Trp-(D)-N(Me)-gTrp-acetyl was obtained as de-
scribed for 7. Compounds incorporating a methyl group
at the C-terminus gem-diamino position were synthe-
sized according to Scheme 3 by the Fukuyama proce-
dure. The dipeptide amide 4 reacted with BTIB to yield
the C-terminus gem-diamino compound in which amino
group was trapped by o-nitrobenzenesulfonyl chloride
to yield 14. After N-methylation and obtention of 15,
the C-terminus amino group was deprotected in the
presence of thioanisole and immediately acylated with
acetic anhydride. After deprotection of 16, Boc-N(Me)-
Aib was introduced with DCC as a coupling reagent and
the Boc group was removed to yield a mixture of
diastereoisomers 17 and 18 corresponding to epimer-
ization of the C-terminus gem-tryptophan residue that
occurred during the synthesis. These diastereoisomers
were separated by RP preparative HPLC and tested. A
methyl group was also incorporated in the R position of
the tryptophan residues. Preparation of the (R)-(Me)-
Trp residue was performed following the Zhang and Fill
procedure22 in five steps and with an enantiomeric
excess of 92%.
F igu r e 3. Structure of peptide or pseudo-peptide GH secre-
tagogue containing an Aib moiety or an Aib substitute.
Sch em e 1. Synthetic Pathway for J MV 1843 and
Analoguesa
a
Reagents and conditions: (a) IBCF, NMM, DME, 0 °C; (b)
NH4OH; (c) H2, Pd/C, EtOH, HCl; (d) BOP, NMM, DMF, Boc-(D)-
Trp-OH; (e) Boc2O, DMAP cat., anhydrous CH3CN; (f) BTIB,
pyridine, DMF/H2O; (g) 2,4,5-trichlorophenylformate, DIEA, DMF;
(h) TFA/anisole/thioanisole (8:1:1), 0 °C; (i) BOP, NMM, DMF, Boc-
Aib-OH; (j) TFA/anisole/thioanisole (8:1:1), 0 °C; (k) RP preparative
HPLC.
formyl gem C-terminus residue gLeu-For (or gMet-For)
leads to very potent agonists (unpublished results of this
laboratory). This strategy was also explored in the GHS
analogues presented in this paper.
Ch em istr y
The preparation of 7 (J MV 1843, H-Aib-(D)-Trp-(D)-
gTrp-formyl) and all parent analogues has been carried
out in six steps in 10-20% overall yield from the
commercially available N-Z-(D or L)-tryptophan, N-Boc-
(D or L)-tryptophan, and N-Boc-aminoisobutyric acid as
described in Scheme 1.
Resu lts a n d Discu ssion
Because the aim of our search was the discovery of a
new orally active drug, the first biological test of our
compounds was performed in vivo. This was done by
the sc route in infant rats at a dose of 300 µg/kg. This
dose was selected on the basis of previous experiments
that showed it elicited a near-maximal response with a
large number of active GH secretagogues. Compounds
that exhibited in this model a better biological activity
(GH release) than hexarelin used as control were then
tested for their activity in beagle dogs when they were
given orally at a dose of 1 mg/kg. Only some of our
compounds were tested for their ability to displace
radiolabeled ghrelin from human pituitary membranes
in order to determinate their IC50.
In Vivo Biologica l Eva lu a tion in Ra ts. Com-
pounds were administered by the sc route in infant rats
at a dose of 300 µg/kg, and results are gathered in Table
1. Surprisingly, in the strict analogue of EP-51389
(compound 23), no activity could be detected. Replace-
ment of Mrp in EP-51389 by (D)-Trp led to the recovery
of potency. The importance of the configuration of each
residue was also studied. The presence of two tryp-
Dipeptide 3 was synthesized by standard procedures
and then treated with di-tert-butyl dicarbonate (Boc2O)
in the presence of a catalytic amount of 4-(dimethylami-
no)pyridine (DMAP) to protect the two indole functions.
Compound 4 was submitted to bis-trifluoroacetoxyiodo-
benzene (BTIB)17 treatment in a mixture of DMF and
water in the presence of pyridine to afford the pseudo-
dipeptide, which was immediately acylated by 2,4,5-
trichlorophenylformate (2,4,5-TCPF)18 to yield 5. All
attempts to perform the Hoffman transposition without
protection of the indole groups led to poor yields.
Removing Boc protecting groups and coupling with Boc-
Aib-OH in the presence of BOP19 and NMM in DMF
produced the N-protected desired compound 6. Com-
pound 7 was then obtained by treatment with a mixture
of TFA/anisole/thioanisole (8:1:1) at 0 °C and purified
by reversed-phase HPLC preparative chromatography.
A structure-activity study of compound 7 was per-
formed. The N-terminal amino group was methylated,

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1193
Sch em e 2. N-Methylation between Both Trp Residuesa
a
Reagents and conditions: (a) o-NBSCI, DMF, Et3N; (b) MeI, K2CO3, DMF; (c) PhSH, K2CO3, DMF, 84% over three steps, no purification
needed; (d) Boc-(D)-TrpOH, NMM, HBTU, DMF, 56%; (e) Boc2O, DMAP, ACN, 89%; (f) BTIB, Py, DMF; (g) Ac2O, DMF, DIEA, 35-45%
over last two steps.
Sch em e 3. N-Methylation at the gem-Diamino C-Terminus Aminea
a
Reagents and conditions: (a) BTIB, Py, DMF; (b) o-NBSCI, DMF, Et3N, 47% over two steps; (c) MeI, K2CO3, DMF, 60%; (d) PhSH,
K2CO3, DMF; (e) Ac2O, DMF, DIEA, 85% over last two steps.
tophan residues of D configuration (compound 7) was
required to recover the activity (see compounds 20-22).
Compound 7 exerted a higher activity than the parent
molecule and was considered to be our new lead
compound. It can be seen that MK-0677 was found to
be less potent than hexarelin in the in vivo rat test.
To increase lipophilicity, enzyme stability, and per-
haps the bioavailability of compound 7 and analogues,
we decided to methylate amide bonds or the N-terminus
amino function. These modifications led us to the second
group of molecules, which are gathered in Table 1.
Introduction of one or two methyl groups on the amino
function of Aib resulted in less potent compounds as
shown by the results obtained with compounds 25 and
26. Replacement of the formyl with an acetyl group also
decreased the potency of compound 27, while concomi-
tant introduction of a methyl group and an acetyl moiety
led to compound 28, which exhibited activity similar to
that of our lead compound 7. A second methyl group on
the Aib residue resulted in a total loss of activity
(compound 29). Introduction of a methyl group between
Trp residues led to compound 30, which is less potent
than its analogue 27, but the concomitant methylation
of the N-terminal part yielded one of the most potent
compounds, 19. Alkylation of the R carbon of the first
Trp residue with a methyl group yielded an inactive
compound (31), while the same alkylation on the R
carbon of the second Trp residue led to the analogue
32, which is almost as potent as hexarelin. Replacement
in this analogue of the formyl by an acetyl group and
methylation of the N-terminus primary amine yielded
compound 33 exhibiting the same activity as hexarelin,
confirming that the concomitant introduction of an
acetyl group at the C terminus and of a methyl group
at the N terminus was favorable for biological activity.
When a methyl group was introduced on the C-terminal
amino function of the gem-diamino residue simulta-
neously with a methyl group on the primary amine of
the Aib residue, a potent analogue (17) was obtained

1194 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Ta ble 1. Biological Activity in Infant Rat (sc Injection)a
Guerlavais et al.
compound
sequence
compound/hexarelin GH release
solvent
0.01
1.0
hexarelin
ghrelin
MK-0677
EP-51389
7
20
21
22
23
24
25
26
27
28
29
19
30
31
32
33
17
18
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
His-(D)-Mrp-Ala-Trp-(D)-Phe-Lys-NH2
0.65
0.31
0.89
1.31
0.08
0.07
0.28
0.08
0.12
0.64
0.69
0.77
1.38
0.15
1.45
0.65
0.12
0.91
1.00
1.21
0.20
0.95
0.46
0.82
0.53
0.14
0.11
0.09
0.13
0.09
0.09
0.09
0.69
0.93
0.24
0.10
0.11
0.16
1.34
0.85
0.16
0.17
0.15
0.16
1.40
0.20
H-Aib-(D)-Mrp-(D)-Mrp-NH2
H-Aib-(D)-Trp-(D)-gTrp-formyl
H-Aib-(L)-Trp-(L)-gTrp-formyl
H-Aib-(L)-Trp-(D)-gTrp-formyl
H-Aib-(D)-Trp-(L)-gTrp-formyl
H-Aib-(D)-Mrp-(D)-gMrp-formyl
H-Aib-(D)-Mrp-(L)-gMrp-formyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-formyl
N(Me)2-Aib-(D)-Trp-(D)-gTrp-formyl
H-Aib-(D)-Trp-(D)-gTrp-acetyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-acetyl
N(Me)2-Aib-(D)-Trp-(D)-gTrp-acetyl
N(Me)-Aib-(D)-Trp-(D)-N(Me)-gTrp-acetyl
H-Aib-(D)-Trp-(D)-N(Me)-gTrp-acetyl
H-Aib-(R)-(Me)Trp-(D)-gTrp-formyl
H-Aib-(D)-Trp-(R)-(Me)-gTrp-formyl
N(Me)-Aib-(D)-Trp-(R)-(Me)-gTrp-acetyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-N(Me)-acetyl
N(Me)-Aib-(D)-Trp-(L)-gTrp-N(Me)-acetyl
4-aminopiperidine-4-carbonyl-(D)-Trp-(D)-gTrp-formyl
4-aminopiperidine-4-carbonyl-(D)-Trp-(D)-gTrp-acetyl
isonipecotyl-(D)-Trp-(D)-gTrp-formyl
isonipecotyl-(D)-Trp-(D)-gTrp-acetyl
phenylsulfonyl-(D)-Trp-(D)-gTrp-formyl
isovaleryl-(D)-Trp-(D)-gTrp-acetyl
iovaleryl-(D)-Trp-(D)-gTrp-formyl
acetyl-(D)-Trp-(D)-gTrp-acetyl
N(Me)-(D)-Trp-(D)-gTrp-formyl, isomer A
N(Me)-(D)-Trp-(D)-gTrp-formyl, isomer B
methylsulfonyl-(D)-Trp-(D)-gTrp-formyl
H-Aib-(D)-Trp-(D)-gTrp-propionyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-propionyl
H-Aib-(D)-Trp-(D)-gTrp-isovaleryl
N(Me)-Aib-(D)-Trp-(D)-gTrp-isovaleryl
H-Aib-(D)-Trp-(D)-gTrp-phenylacetyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-phenylacetyl
H-Aib-(D)-Trp-(D)-gTrp-piperidine-2-carbonyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-piperidine-2-carbonyl
H-Aib-(D)-Trp-(D)-gTrp-3-indoleacetyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-3-indoleacetyl
H-Aib-(D)-Trp-(D)-gTrp-cyclohexane-3-propionyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-cyclohexane-3-propionyl
H-Aib-(D)-Trp-(D)-gTrp-C(O)NHCH2CH3
H-Aib-(D)-Trp-(D)-gTrp-C(O)O CH2CH3
a
Results are normalized as a ratio of GH release stimulated by 300 µg/kg of test compounds and GH release stimulated by 300 µg/kg
of hexarelin.
that exhibited an activity greater than that of hexarelin.
Its diastereoisomer 18 was found to be inactive.
inactive compounds, showing the importance of the Aib
residue or of a basic residue in the interaction with the
receptors.
A structure-activity relationship study was also
performed on the N-terminal part of the molecule. In
both compounds 7 and 28, we replaced the Aib residue
with a 4-aminopiperidine-4-carboxyl acid residue (com-
pounds 34, 35), isonipecotyl (compounds 36, 37), phe-
nylsulfonyl (compound 38), isovaleryl (compounds 39,
40), acetyl (compound 41), methyl (compounds 42, 43),
or methylsulfonyl group (compound 44). As shown in
Table 1, replacement of the Aib residue in compound 7
by the 4-aminopiperidine-4-carboxyl group (compounds
34) or the isonipecotyl moiety (compound 36) resulted
in less potent molecules than the parent compounds;
nevertheless, these compounds were almost as potent
as hexarelin. When, at the same time, the formyl group
was replaced by the acetyl group, half of the biological
activity was lost (compounds 35, 37). In all other
compounds, attempts to replace the Aib moiety led to
Modifications on the C-terminal part were then
performed. The formyl and acetyl groups were replaced
to change the steric hindrance and the lipophilicity of
the molecules. Propionyl, isovaleryl, phenylacetyl, pi-
peridine-2-carboxyl, 3-indoleacetyl, and cyclohexane-3-
propionyl groups were introduced for this purpose, and
the corresponding compounds were tested. Increase of
the lipophilicity of the C-terminal part of compound 7
produced a loss in biological activity as shown in Table
1 (compounds 27, 45, 46, and 49). The concomitant
introduction of a N(Me)-Aib with a propionyl group at
the C terminus led to an increase of potency and to a
compound as potent as hexarelin (compound 46). This
observation was not true in the case of isovaleryl
(compounds 47, 48) and phenylacetyl (compounds 49,
50) groups. The presence of a piperidine-2-carbonyl

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1195
Ta ble 2. Biological Activity in Beagle Dogs by Oral
Administration at a Dose of 1 mg/kg
an IC50 of 9.8 ( 1.5 nM in this model, since hexarelin
was found to be 2 times less potent and MK-0677 about
3 times less potent. Compounds 7, 19, 29, 51, 57, and
58 exhibited almost the same affinity as hexarelin.
Compounds 25, 28, and 48 were slighly less potent than
hexarelin, while compounds 27, 34, 36, 45, and 48 were
10-100 times less potent than hexarelin. Compounds
35, 37, and 46 were not able to displace 125I-Tyr4-ghrelin
from its binding sites. Some results were surprising.
Although compounds 57 and 58 showed the same in
vitro affinity data, compound 57 was able to elicit GH
secretion while its close analogue 58 did not. This
discrepancy could be explained by different metabolic
stability and brain penetration properties or by differ-
ences of compounds in functional activity at the recep-
tors. Compound 28, which was found to be potent in
releasing GH in the rat model, binds with a modest
affinity to the human GHS receptor on pituitary mem-
branes. Compounds 34 and 36, which were in vivo as
good as hexarelin, were revealed to be weak ligands for
the human pituitary GHS receptor. The correlation
between in vivo activity in rats and in vitro binding on
human pituitary GHS receptor was satisfactory for
other compounds.
compound
GH peak (µg/L)
GH AUC0f180 (µg min L-1
)
saline
MK-0677
7
28
51
19
57
0.4 ( 0.1
5.4 ( 2.4
5.5 ( 1.1
1.1 ( 0.5
0.5 ( 0.1
5.5 ( 2.0
0.4 ( 0.1
76.18 ( 1.28
544.55 ( 166.74
675.35 ( 94.47
85.85 ( 18.49
87.06 ( 15.36
262.43 ( 27.87
79.54 ( 1.7
group in this part of the molecule was well accepted by
the receptors (compound 51), leading to a potent com-
pound that is even more active than compound 7,
suggesting a possible interaction between the secondary
amine of the piperidyl group and a putative acid
function in the receptor subsite. In this compound,
introduction of a N(Me)-Aib led to a decrease in the
potency but resulted in compound 52 almost as potent
as hexarelin. Indolacetyl and cyclohexylpropionyl groups
produced inactive compounds. An interesting activity
was obtained by incorporation of a urea function on the
C-terminal gem-diamine, leading to compound 57 which
exhibited a strong in vivo activity in rat. This activity
was surprisingly lost when the urea function was
replaced by a carbamate moiety (compound 58), al-
though these two last compounds exhibited similar
binding affinities in the receptor assays.
Or a l Activity in Dog. The most potent compounds
were tested for their activity in beagle dogs when they
were given orally at a dose of 1 mg/kg. Results are
gathered in Table 2. In this test, hexarelin at a dose of
1 mg/kg by oral route was completely ineffective in
stimulating GH secretion in the dog. Two compounds 7
and 19 were found to be active when administered per
os in dogs, while compounds 28 and 51, which revealed
good in vivo activity in rats when injected sc, were
inactive in this model. Compound 7 had a more sus-
h GHS-R 1a R ecep t or s. Compounds were also
tested for their affinity to the cloned hGHS-R 1a
receptor. Binding affinities of human ghrelin and MK-
0677 obtained from this model were in accordance with
the literature.23 The binding affinity of Tyr-Ala-hexare-
lin was found to be lower than of human pituitary
membranes. Compounds 25, 28, and 19 exhibited a high
binding affinity to the cloned hGHS-R 1a receptor (22.7
( 7.4, 58 ( 20, and 29.5 ( 16.0 nM respectively). A
comparison of these values with the ones obtained for
compounds 7 and 27 (123 ( 25 and 228 ( 162 nM
respectively) clearly indicated that N-methylation of the
amino function of Aib was well accepted for the binding
to the cloned hGHS-R 1a receptor. This observation
was confirmed when comparing the IC50 obtained for
compounds 45 versus 46 and 47 versus 48. The obtained
results also showed the importance of the steric hin-
drance of the gem-diamino acylating group: the binding
affinity was reduced when the chain length was in-
creased (non-N-methylated compounds 7, 27, 45, and
47; N-methylated compounds 25, 28, 46 and 48). Di-
methylation of the Aib amino function led to less potent
compound 29, while Aib replacement by 4-aminopiperi-
dine-4-carboxyl or isonipecotyl moieties resulted in
reduced binding affinity (compounds 34, 36, and 37).
Compounds 51, 57, and 58 presented an affinity similar
to that of our lead compound 7.
tained GH response (peak, 5.5 ( 1.1 ng/mL; AUC0-180
,
675.35 ( 94.47; n ) 6) than compound 19 (peak, 5.5 (
2.0 ng/mL; AUC0-180, 262.43 ( 27.87; n ) 4), while other
compounds (28, 51, and 57) and the vehicle did not
induce a GH response. Compound 7 was found to be
more active than MK-0677, hexarelin, or ghrelin. This
result prompted us to choose this compound for further
studies.
Bin d in g to th e Hu m a n P itu ita r y GHS Recep tor s
a n d to th e Clon ed Hu m a n GHS-R 1a Recep tor s.
Compounds exhibiting good in vivo activity in rats (sc
injection) were further tested for their binding affinity
to human GHS receptors. This study was performed in
two distinct tests: (1) on autoptic human pituitary
tissue membranes and (2) on the cloned hGHS-R 1a
receptor transiently expressed in LLC PK-1 cells. Re-
sults are gathered in Table 3. In the first test, 125I-Tyr4-
ghrelin was used as radiolabeled ligand. It was dem-
onstrated to be a suitable ligand in this model as
described in the experimental part. 125I-His9-ghrelin was
used as a ligand in the second model in order to compare
the binding affinities of our compounds with literature
compounds.
Results obtained in these two models led to several
comments. The high observed binding affinities of
ghrelin and MK-0677 toward the cloned hGHS-R 1a
are in accordance with the literature because it was
discovered with the radiolabeled 35S-MK-0677 com-
pound. The same receptor was used for the discovery of
ghrelin. Affinity binding values of ghrelin and MK-0677
toward the human pituitary receptors were quite dif-
ferent. When comparing binding affinities of Tyr-Ala-
hexarelin in both models, it appears that pituitary
membranes also contain other receptor subtypes. These
observations are well correlated with results reported
in the literature. 35S-MK-0677 and 125I-His9-ghrelin
Hu m a n P itu ita r y GHS Recep tor s. The ability of
unlabeled ghrelin, hexarelin, MK-0677, and other com-
pounds to compete with 125I-Tyr4-ghrelin on human
pituitary membranes was measured. Ghrelin exhibited

1196 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Guerlavais et al.
Ta ble 3. Binding Affinity of Ghrelin, MK-0677, Hexarelin, Tyr-Ala-hexarelin, and Other Compounds to the Human Pituitary GHS
Receptors and to the Cloned Human GHS-R 1a Receptors
IC50 (nM) for
IC50 (nM) for
125I[His9]ghrelin
hGHS-R 1a
125I[Tyr4]ghrelin human
pituitary membranes
compound
sequence
Tyr-Ala-hexarelin
hexarelin
ghrelin
MK-0677
7
25
27
28
34
35
36
37
45
47
51
46
48
Tyr-Ala-His-(D)-Mrp-Ala-Trp-(D)-Phe-Lys-NH2
His-(D)-Mrp-Ala-Trp-(D)-Phe-Lys-NH2
12 ( 1
286 ( 70
ND
18.0 ( 2.7
9.8 ( 1.5
32.0 ( 1.7
22.9 ( 1.3
39.0 ( 3.4
3852 ( 290
74.0 ( 5.4
1791 ( 171
>10000
0.39 ( 0.10
0.76 ( 0.10
123 ( 25
22.7 ( 7.4
228 ( 162
58 ( 20
H-Aib-(D)-Trp-(D)-gTrp-formyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-formyl
H-Aib-(D)-Trp-(D)-gTrp-acetyl
N(Me)-Aib-((D)-Trp-(D)-gTrp-acetyl
4-aminopiperidine-4-carbonyl-(D)-Trp-(D)-gTrp-formyl
4-amino-piperidine-4-carbonyl-(D)-Trp-(D)-gTrp-acetyl
isonipecotyl-(D)-Trp-(D)-gTrp-formyl
isonipecotyl-(D)-Trp-(D)-gTrp-acetyl
H-Aib-(D)-Trp-(D)-gTrp-propionyl
337 ( 110
ND
391 ( 40
>10000
185 ( 27
59.8 ( 12
21.5 ( 1.6
>10000
213 ( 30
25.6 ( 1.8
20.1 ( 1.5
16.9 ( 2.8
16.2 ( 1.4
1.155 ( 235
807 ( 154
460 ( 11
535 ( 140
116 ( 35
167 ( 48
397 ( 141
29.5 ( 16.0
440 ( 80
162 ( 20
122 ( 1
H-Aib-(D)-Trp-(D)-gTrp-isovaleryl
H-Aib-(D)-Trp-(D)-gTrp-piperidine-2-carbonyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-propionyl
N(Me)-Aib-(D)-Trp-(D)-gTrp-isovaleryl
N(Me)-Aib-(D)-Trp-(D)-N(Me)-gTrp-acetyl
N(Me)2-Aib-(D)-Trp-(D)-gTrp-acetyl
H-Aib-(D)-Trp-(D)-gTrp-C(O)NHCH2CH3
H-Aib-(D)-Trp-(D)-gTrp-C(O)OCH2CH3
19
29
57
58
have Kd values of about 10-10 M in various tissues and
interact with few sites (about 10 fmol/mg of protein).24
On the other hand, 125I-Tyr4-ghrelin or 125I-Tyr-Ala-
hexarelin revealed a larger number of different binding
sites in various tissues (Bmax ) 1000-2000 fmol/mg of
protein) and had Kd values of about 10-9 M.25,26 Use of
a photoactivable hexarelin derivative allowed identifica-
tion of a 57 kDa protein that is different from hGHS-R
1a in pituitary membranes.27 In this context, discrep-
ancies observed for the binding affinities of our com-
pounds with the cloned hGHS-R 1a and with the
human pituitary GHS receptors also suggest the pres-
ence of receptor subtypes as already mentioned.28-32
Actually, it is not known which receptor(s) is (are)
involved in stimulation of GH release. It has been
clearly shown that MK-0677, ghrelin, and J MV 1843
were able to elicit in vivo stimulation of GH release, but
their specific target has still not been clearly identified.
1.2. Meth od s. Pups were acutely challenged with solvent
(DMSO, final dilution 1:300), hexarelin, or new peptides (300
µg/kg, sc) and killed by decapitation 15 min later. Trunk blood
was collected and centrifuged immediately. Plasma samples
were stored at -20 °C until assayed for the determination of
plasma GH concentrations.
1.3. Gr ow th Hor m on e Assa y. GH concentrations in
plasma were measured by radioimmunoassay (RIA) using
materials kindly provided by the National Institute of Diabe-
tes, Digestive and Kidney Diseases (NIDDK) of the National
Institutes of Health. Values are expressed in terms of NIDDK-
rat-GHRP-2 standard (potency 2 IU/mg) as ng/mL plasma. The
minimum detectable value of rat GH was about 1.0 ng/mL,
and intra-assay variability was about 6%.
2. In Vivo Exp er im en ts in th e Dog. 2.1. An im a ls.
Experiments were performed in beagle dogs, a species that
behaves like humans for many aspects of GH regulation and
presents a GH secretory profile similar to that observed in
humans.
Four to six young (age, 2-3 years; weight, 11-15 kg; male
and female) well-trained beagle dogs were used in this study.
Animals were fed normal dry food (Diete Standar, Charles
River, Calco, Italy) once a day at 16.00 h with water available
ad libitum. Animals were on a 12-h light and 12-h dark
regimen, with lights on at 07.00 h. Before the study, the body
weights of all dogs were stable and they had no observable
disease. All the experimental procedures were in accordance
with the protocol previously authorized by the Committee on
Animal Care and Use of the University of Milan.
2.2. Acu te Com p ou n d s Testin g. After one baseline blood
sample (-20 min) had been taken from the cephalic vein, dogs
were administered, through an oral-gastric cannula in a
randomized sequence, hexarelin or other compounds (1 mg/
kg, per os) at time 0 and then samples were drawn at 15, 30,
60, 90, 120, and 180 min. All blood samples were collected in
chilled tubes containing 1.2 mg of EDTA/mL of blood (Sigma
Tau, Milan, Italy) and centrifuged. The plasma was then frozen
at -20 °C until assayed.
2.3. GH RIA. Highly purified canine GH, used for iodination
and as a standard, and monkey anticanine GH antiserum were
obtained through the courtesy of Dr. A. F. Parlow (Pituitary
Hormones and Antisera Center, Torrance, CA). The sensitivity
of the assay was 0.4 ng/mL. The intra-assay coefficients of
variation were 3.8% and 4.1% at concentrations of 12.5 and
3.1 ng/mL, respectively. To avoid possible interassay variation,
all samples of a given experiment were assayed in a single
RIA.
Con clu sion
We have synthesized a new class of GH secretagogues
based on a gem-diaminotryptophan residue. Among
these compounds, 7, 17, 19, 28, 51, and 57 showed in
vivo potencies higher than that of hexarelin in the rat
(sc injection). Only two compounds (7 and 28) were
found to be active per os in beagle dogs at 1 mg/kg.
Compound 7 was found to exhibit high oral activity and
specificity in human volunteers,1 and additional clinical
studies are in progress.
Exp er im en ta l Section
1. In Vivo Exp er im en ts in th e Ra t. 1.1. An im a ls. Male
10-day-old Sprague-Dawley rats weighing about 25 g (Charles
River, Calco, Italy) were used. Rats pups were received on the
fifth day after birth and were housed in our facilities under
controlled conditions (22 ( 2 °C, 65% humidity, and artificial
light from 06.00 to 20.00 h). A standard dry diet and water
were available ad libitum to the dams. One hour before the
experiments, pups were separated from their respective dams
and were divided randomly into groups of eight each. All the
experiments were performed in accordance with the Italian
Guidelines for the Use of Animals in Medical Research.

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1197
2.4. Sta tistica l An a lysis. GH was evaluated either in
absolute mean values (ng/mL) ( SEM or as area under the
GH response curve (AUC0-180 min; ng/mL × h) calculated by
the trapezoid method. Since no difference in analytical levels
between male and female dogs were observed, data were
pooled.
3. In Vitr o Deter m in a tion of th e Affin ities on Hu m a n
P itu ita r y Mem br a n es. 3.1. Tissu e P r ep a r a tion . Pituitary
glands were obtained at autopsy from seven subjects (four
males and three females ranging from 39 to 64 years, median
age 52 years) who died of trauma or neoplams and were
submitted to autopsy for diagnostic purposes in the Depart-
ment of Pathology, University of Turin. Tissue was removed
24-28 h after death with the approval of our hospital Ethical
Committee and immediately stored at -35 °C for 1-2 months
until processed for membrane preparation and binding studies.
At histopathological examination, pituitary glands were all
preserved from both an architectural and cytological point of
view.
fuged at 10000g for 20 min at 4 °C. The membrane pellet was
resuspended in a minimal volume of HB, aliquoted, and stored
at -80 °C until use. Membrane protein concentrations were
determined by the Bradford method using the Bio-Rad protein
assay kit.
4.2. Recep tor Bin d in g Stu d ies. Isolated plasma mem-
branes from LLC PK-1 cells (10 µg of protein) were incubated
in HB for 60 min at 25 °C (steady-state conditions) with 60
pM 125I-His9-ghrelin (Amersham) in the presence or absence
of competing compounds. Nonspecific binding was defined
using an excess (1 µM) of ghrelin. The binding reaction was
stopped by addition of 4 mL of ice-cold HB followed by rapid
filtration over Whatman GF/C filters presoaked with 0.5%
polyethyleneimine to prevent excessive binding of radioligand
to the filters. Filters were rinsed three times with 3 mL of
ice-cold wash buffer (50 mM Tris (pH 7.3), 10 mM MgCl2, 2.5
mM EDTA, and 0.015% (w/v) X-100 Triton), and the radioac-
tivity bound to membranes was measured in a γ counter.
5. Ch em istr y. Ascending TLC was performed on precoated
plates of silica gel 60 F254 (Merck). Peptide derivatives were
located with charring reagent or ninhydrine. Column chro-
matography was performed with silica gel Kieselguhr Merck
G, 0.05-0.2 mm. HPLC purifications were run on a Waters
4000 preparative apparatus on a C18 Deltapak column (100
mm × 40 mm, 15 µm, 100 Å), with UV detection at 214 nm,
at a flow rate of 50 mL/min of a mixture of solutions A (water
with 0.1% TFA) and B (acetonitrile with 0.1% TFA in gradient
mode). Analytical HPLC chromatograms were performed on
a Beckman Gold apparatus composed of the 126 solvent
module, the 168 detector, and the 32 Karat software. Runs
were performed on a Deltapak Waters C18 column (150 mm
× 3.9 mm, 5 µm) at a flow rate of 1 mL/min from solution A to
solution B in a 50 min gradient (conditions A) or on a
Symmetry Shield C18 column (50 mm × 4.6 mm, 3.5 µm) at a
flow rate of 1 mL/min from solution A to solution B in a 15
min gradient (conditions B). Mass spectrum analyses were
recorded on a Platform II (Micromass, Manchester, U.K.)
quadrupole mass spectrometer fitted with an electrospray
interface. The (L)- and (D)-amino acids and derivatives were
from Senn Chemicals, Neosystem, and Advanced Chemtech.
All reagents were of analytical grade.
3.2. Bin d in g Stu d ies. GHS binding assay with tissue
membranes (30 000 g fraction) was performed as previously
described25 using 125I-Tyr4-human ghrelin as the radioligand.
125I-Tyr4-ghrelin (specific activity: 2000 Ci/mmol) was radio-
iodinated as previously described33 and proved to be a reliable
probe for labeling ghrelin receptors in human hypothalamus
and pituitary gland.33,34 Pituitary membranes (corresponding
to 100 µg of membrane protein) were incubated in triplicate
at 20 °C for 120 min with constant shaking with approximately
0.5 nmol/L 125I-Tyr4-ghrelin in a final volume of 0.5 mL of assay
buffer (50 mmol/L Tris, 2.5 mmol/L EGTA, 0.003% bacitracin,
and 0.1% BSA, titrated with HCl to pH 7.4) in the absence
and in the presence of increasing concentrations (from 1
nmol/L to 10 µmol/L) of various unlabeled competitors. Parallel
incubations where 2 µmol/L unlabeled human ghrelin was also
present were used to determine nonspecific binding, which was
subtracted from total binding to yield specific binding values.
In all assays, the binding reaction was terminated by adding
ice-cold assay buffer followed by filtration through Whatman
GF/B filters. The radioactivity remaining bound to the filters
was measured by a Packard γ counter. The concentration of
competitor required to inhibit radiotracer binding by 50% (IC50
)
was calculated by iterative nonlinear curve-fitting with the
Prism 3 program (GraphPad Software, Inc., San Diego, CA).
Values are espressed as mean ( SE of three to four separate
experiments.
4. In Vitr o Deter m in a tion of th e Affin ities on th e
Clon ed Hu m a n GHS-R 1a . 4.1. Tr a n sien t Tr a n sfection
of LLC P K-1 Cells a n d Mem br a n e P r ep a r a tion . LLC PK-1
cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FCS (v/v), glutamine (2 mM),
and antibiotics (50 units/mL penicillin and 50 µg/mL strepto-
mycin). The growth of cells was performed in a humidified
incubator at 37 °C under an atmosphere of 5% CO2 in air.
The following abbreviations were used: BOP (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
DME, ethylene glycol dimethyl ether; DMF, dimethylforma-
mide; NMM, N-methylmorpholine; BTIB, bis(trifluoroacetoxy)-
iodobenzene; 2,4,5-TCP, 2,4,5-trichlorophenylformate; HBTU,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate. Other abbreviations used were those recom-
mended by IUPAC-IUB Commission (Eur. J . Biochem. 1984,
138, 9-37).
All final compounds were purified by reversed-phase HPLC.
The purity was assessed by analytical reversed-phase C18
HPLC, and the structure was confirmed by MS (electrospray)
1
and H NMR. In some compounds, the presence of two isomers
LLC PK-1 cells were transiently transfected using the
electroporation method (Easyject Optima apparatus, Equibio).
To this aim, 10 × 106 LLC PK-1 cells were washed twice in
serum-free growing medium by centrifugation (50 g for 10 min)
and transiently transfected with 0.5 µg of the pcDNA3 vector
containing the cDNA for the human GHS-R 1a using the
Optimix Kit according to the manufacturer’s protocol (Equibio).
Electroporation was carried out at room temperature according
to the following parameters: single pulse, 250 V, 1500 µF, and
infinite internal resistance (under these conditions, the pulse
time was 22 ms). Transfected cells were immediately with-
drawn and plated in 10 cm culture dishes containing complete
growth medium without phenol red. After an overnight
incubation, cells were washed with 10 mL of the same culture
medium. Approximately 48 h post-transfection, cells were
washed three times with phosphate-buffered saline, pH 6.95,
and once with 10 mL of homogeneisation buffer (HB) contain-
ing 50 mM Tris (pH 7.3), 5 mM MgCl2, 2.5 mM EDTA, and 30
µg/mL bacitracin, scrapped from the plate in 500 µL of HB,
and frozen in liquid N2. After thawing at 37 °C, cells were
subjected to another cycle of freeze-thawing and then centri-
could be detected by 1H NMR (for example, because of the
presence of cis- and trans-formylamide conformations). In the
NMR description, these isomers were called A and B. Com-
pounds (D)- and (L)-Mrp, EP-51389, hexarelin, Tyr-Ala-hexare-
lin, and MK-0677 were supplied by Europeptides (Argenteuil,
France).
Boc-N(Me)-Aib-OH. Boc-Aib-OH (5 g, 24.6 mmol) was
dissolved in THF (200 mL). Then, NaH (1.8 g, 78 mmol) and
ICH3 (12.3 mL, 203 mmol) were successively added at 0 °C.
After 24 h, the solvent was removed in vacuo. The mixture
was diluted in ether (150 mL) and water (150 mL). The
aqueous layer was acidified at pH 1. Then the residue was
dissolved in ethyl acetate (100 mL) and washed with aqueous
sodium thiosulfate (200 mL, 1 M), aqueous potassium hydro-
gen sulfate (200 mL, 1 M), and saturated aqueous sodium
chloride (200 mL). The organic layer was dried over sodium
sulfate and filtered and the solvent removed in vacuo to afford
Boc-N(Me)-Aib-OH as a white foam in 81% yield. 1H NMR (200
MHz, CDCl3): δ 1.45 (s, 9H, Boc), 1.48 (s, 6H, Aib), 2.93 (s,
3H, NMe). MS (ES), m/z: 118.45 [M + H - Boc]+, 162.20 [M

1198 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Guerlavais et al.
+ H - isobutene]+, 218.27 [M + H]+, 240.24 [M + Na]+, 435.05
[2M + H]+, 457.15 [2M + Na]+, 673.75 [3M + H]+.
(200 mL), aqueous potassium hydrogen sulfate (200 mL, 1 M),
and saturated aqueous sodium chloride (100 mL). The organic
layer was dried over sodium sulfate and filtered and the
solvent was removed in vacuo to afford 4.35 g (85%) of 3 as a
white foam. Rf ) 0.48 (chloroform/methanol/acetic acid, 85:
10:5). 1H NMR (200 MHz, DMSO-d6): δ 1.28 (s, 9H, Boc),
2.75-3.36 (m, 4H, 2 (CH2)â), 4.14 (m, 1H, CHR), 4.52 (m, 1H,
CHR), 6.83-7.84 (m, 14H, 2 indoles (10H), NH2, NH (urethane),
and NH (amide)), 10.82 (d, 1H, J ) 2 Hz, N1H), 10.85 (d, 1H,
J ) 2 Hz, N1H). MS (ES), m/z: 490 [M + H]+, 512 [M + Na]+,
979 [2M + H]+.
Boc-(D)-(NiBoc)-Tr p -(D)-(NiBoc)-Tr p -NH2 (4). 3 (3 g, 6.13
mmol) was dissolved in acetonitrile (25 mL). To this solution,
di-tert-butyl dicarbonate (3.4 g, 15.3 mmol) and 4-(dimeth-
ylamino)pyridine (150 mg, 1.2 mmol) were successively added.
After 1 h, the mixture was diluted with ethyl acetate (100 mL)
and washed with saturated aqueous sodium hydrogen carbon-
ate (200 mL), aqueous potassium hydrogen sulfate (200 mL,
1 M), and saturated aqueous sodium chloride (200 mL). The
organic layer was dried over sodium sulfate and filtered, and
the solvent was removed in vacuo. The residue was purified
by flash chromatography on silica gel, eluting with ethyl
acetate/hexane, 5:5, to afford 2.53 g (60%) of 4 as a white foam.
Rf ) 0.23 (ethyl acetate/hexane, 5:5). 1H NMR (200 MHz,
DMSO-d6): δ 1.25 (s, 9H, Boc), 1.58 (s, 9H, Boc), 1.61 (s, 9H,
Boc), 2.75-3.4 (m, 4H, 2 (CH2)â), 4.2 (m, 1H, CHR), 4.6 (m,
1H, CHR), 7.06-8 (m, 14H, 2 indoles (10H), NH (urethane),
NH and NH2 (amides)). MS (ES), m/z: 690 [M + H]+, 712 [M
+ Na]+, 1379 [2M + H]+, 1401 [2M + Na]+.
Boc-(D)-(NiBoc)-Tr p -(D)-(NiBoc)-gTr p -H (4′). 4 (3 g, 4.3
mmol) was dissolved in a mixture of DMF/water (18 mL:7 mL).
Then, pyridine (772 µL, 9.5 mmol) and bis(trifluoroacetoxy)-
iodobenzene (2.1 g, 4.7 mmol) were added. After 1 h, the
mixture was diluted with ethyl acetate (100 mL) and washed
with saturated aqueous sodium hydrogen carbonate (200 mL),
aqueous potassium hydrogen sulfate (200 mL, 1 M), and
aqueous saturated sodium chloride (200 mL). The organic layer
was dried over sodium sulfate and filtered, and the solvent
was removed in vacuo. 4′ was used immediately in the
acylation reaction. Rf ) 0.14 (ethyl acetate/hexane, 7:3). 1H
NMR (200 MHz, DMSO-d6): δ 1.29 (s, 9H, Boc), 1.61 (s, 18H,
2 Boc), 2.13 (s, 2H, NH2 (amine)), 3.1-2.8 (m, 4H, 2 (CH2)â),
4.2 (m, 1H, CHR), 4.85 (m, 1H, CHR), 6.9-8 (m, 12H, 2 indoles
(10H), NH (urethane), NH (amide)). MS (ES), m/z: 662 [M +
H]+, 684 [M + Na]+.
Boc-(D)-(NiBoc)-Tr p -(D)-(NiBoc)-gTr p -CHO (5). 4′ (2.84
g, 4.3 mmol) was dissolved in DMF (20 mL). Then, N,N-
diisopropylethylamine (815 µL, 4.7 mmol) and 2,4,5-trichlo-
rophenylformate (1.08 g, 4.7 mmol) were added. After 30 min,
the mixture was diluted with ethyl acetate (100 mL) and
washed with saturated aqueous sodium hydrogen carbonate
(200 mL), aqueous potassium hydrogen sulfate (200 mL, 1 M),
and saturated aqueous sodium chloride (200 mL). The organic
layer was dried over sodium sulfate and filtered, and the
solvent was removed in vacuo. The residue was purified by
flash chromatography on silica gel, eluting with ethyl acetate/
hexane (5:5) to afford 2.07 g of 5 (70% for both steps) as a white
foam. Rf ) 0.27 (ethyl acetate/hexane, 5:5). 1H NMR (200 MHz,
DMSO-d6): δ 1.28 (s, 9H, Boc), 1.6 (s, 9H, Boc), 1.61 (s, 9H,
Boc), 2.75-3.1 (m, 4H, 2 (CH2)â), 4.25 (m, 1H, (CH)RA and
(CH)RB), 5.39 (m, 0.4H, (CH)R′B), 5.72 (m, 0.6H, (CH)R′A),
6.95-8.55 (m, 14H, 2 indoles (10H), NH (urethane), 2 NH
(amides), CHO (formyl)). MS (ES), m/z: 690 [M + H]+, 712
[M + Na]+, 1379 [2M + H]+.
Boc-N(Me)-Aib-OBzl. Boc-N(Me)-Aib-OH (1 g, 4.6 mmol)
was dissolved in acetonitrile (50 mL). Then, DBU (690 µL, 4.63
mmol) and BzlBr (521 µL, 4.4 mmol) were successively added
to 60 °C. After 30 min, the solvent was removed in vacuo. The
residue was dissolved in ethyl acetate (100 mL) and washed
with saturated aqueous sodium hydrogen carbonate (200 mL),
aqueous potassium hydrogen sulfate (200 mL, 1 M), and
saturated aqueous sodium chloride (200 mL). The organic layer
was dried over sodium sulfate and filtered, and the solvent
was removed in vacuo to afford Boc-N(Me)-Aib-OBzl as an oil
in 85% yield. Rf ) 0.35 (ethyl acetate/hexane,1:9). 1H NMR
(200 MHz, CDCl3): δ 1.43 (s, 9H, Boc), 1.47 (s, 6H, Aib), 2.93
(s, 3H, NMe), 5.16 (s, 2H, CH2 (Bzl)), 7.38 (m, 5H, Bzl). MS
(ES), m/z: 208.16 [M + H - Boc]+, 252.12 [M + H -
isobutene]+, 308.33 [M + H]+, 330.27 [M + Na]+, 637.42 [2M
+ Na]+.
N(Me)-Aib-OBzl. Boc-N(Me)-Aib-OBzl (1.2 g, 3.9 mmol)
was dissolved in trifluoroacetic acid (20 mL) for 30 min at 0
°C. The solvent were removed in vacuo to give TFA, N(Me)-
Aib-OBzl as a white foam (yield: 98%). 1H NMR (200 MHz,
CDCl3): δ 1.64 (s, 6H, Aib), 2.69 (s, 3H, NMe), 5.26 (s, 2H,
CH2 (Bzl)), 7.39 (m, 5H, Bzl). MS (ES), m/z: 208.10 [M + H]+.
N,N-(d iMe)-Aib-OBzl. TFA, N(Me)-Aib-OBzl (1.47 g, 4.6
mmol) was dissolved in water (1.2 mL). Then, formic acid (868
µL, 23 mmol) and 37% formaldehyde (1.21 mL, 16.1 mmol)
were successively added, and the mixture was placed under
reflux. After 24 h, the mixture was diluted in ether (5 mL)
and water (5 mL). The aqueous layer was neutralized at pH
7. Then the residue was dissolved in ethyl acetate (10 mL)
and washed with saturated aqueous sodium chloride (20 mL).
The organic layer was dried over sodium sulfate and filtered,
and the solvent was removed in vacuo to afford N,N-(diMe)-
Aib-OBzl as an oil. Rf ) 0.41 (ethyl acetate/pyridine/acetic acid/
water, 40:20:5:10). 1H NMR (200 MHz, CDCl3): δ 1.34 (s, 6H,
Aib), 2.31 and 2.32 (2s, 6H, 2Me), 5.21 (s, 2H, CH2 (Bzl)), 7.39
(m, 5H, Bzl). MS (ES), m/z: 222.34 [M + H]+.
N,N-(d iMe)-Aib-OH. N,N-(diMe)-Aib-OBzl (4.6 mmol) was
dissolved in ethanol (100 mL), then palladium on activated
charcoal (10%) (0.1 equiv) was added to the stirred mixture.
The solution was bubbled under hydrogen for 24 h. When the
reaction went to completion, the palladium was filtered on
Celite. The solvent was removed in vacuo. N,N-(diMe)-Aib-
OH was precipitated in a solution of ether/hexane as a white
foam: 41% yield starting from TFA, N(Me)-Aib-OBzl. 1H NMR
(200 MHz, CDCl3): δ 1.42 (s, 6H, Aib), 2.7 (s, 6H, 2Me). MS
(ES), m/z: 132.44 [M + H]+.
Z-(D)-Tr p -NH2 (2). Z-(D)-Trp-OH, 1 (8.9 g, 26 mmol), was
dissolveld in DME (25 mL) and stored in an ice/water bath at
0 °C. NMM (3.5 mL, 31 mmol), IBCF (4.1 mL, 31 mmol), and
an ammonia solution 28% (8.9 mL, 130 mmol) were succes-
sively added. The mixture was diluted with water (100 mL),
and compound 2 was precipitated. It was filtered and dried in
vacuo to afford 8.58 g (98%) of a white foam. Rf ) 0.46
(chloroform/methanol/acetic acid, 180:10:5). 1H NMR (250
MHz, DMSO-d6): δ 2.9 (dd, 1H, Hâ, J ââ′ ) 14.5 Hz, J âR ) 9.8
Hz), 3.1 (dd, 1H, Hâ′, J â′â ) 14.5 Hz, J â′R ) 4.3 Hz), 4.2
(sextuplet, 1H, HR), 4.95 (s, 2H, CH2 (Z)), 6.9-7.4 (m, 11H),
7.5 (s, 1H, H2), 7.65 (d, 1H, J ) 7.7 Hz), 10.8 (s, 1H, N1H). MS
(ES), m/z: 338 [M + H]+, 360 [M + Na]+, 675 [2M + H]+, 697
[2M + Na]+.
Boc-(D)-Tr p -(D)-Tr p -NH2 (3). 2 (3 g, 8.9 mmol) was dis-
solved in DMF (100 mL). HCl, 36% (845 µL, 9.8 mmol), water
(2 mL), and palladium on activated charcoal (10%) (95 mg)
were added to the stirred mixture. The solution was bubbled
under hydrogen for 24 h. When the reaction went to comple-
tion, the palladium was filtered on Celite. The solvent was
removed in vacuo to afford HCl, H-(D)-Trp-NH2 as a colorless
oil. In 10 mL of DMF, HCl, H-(D)-Trp-NH2 (8.9 mmol), Boc-
(D)-Trp-OH (2.98 g, 9.8 mmol), NMM (2.26 mL, 20.3 mmol),
and BOP (4.33 g, 9. 8 mmol) were successively added. After 1
h, the mixture was diluted with ethyl acetate (100 mL) and
washed with saturated aqueous sodium hydrogen carbonate
Boc-Aib-(D)-Tr p -(D)-gTr p -CHO (6). 5 (1.98 g, 2.9 mmol)
was dissolved in a mixture of trifluoroacetic acid (16 mL),
anisole (2 mL), and thioanisole (2 mL) for 30 min at 0 °C. The
solvents were removed in vacuo, the residue was stirred in
ether, and the precipitated TFA salt was filtered. TFA, H-(D)-
Trp-(D)-gTrp-CHO (2.9 mmol), Boc-Aib-OH (700 mg, 2.9 mmol),
NMM (2.4 mL, 12.2 mmol), and BOP (1.53 g, 3.5 mmol) were
successively added in 10 mL of DMF. After 1 h, the mixture
was diluted with ethyl acetate (100 mL) and washed with
saturated aqueous sodium hydrogen carbonate (200 mL),

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1199
aqueous potassium hydrogen sulfate (200 mL, 1 M), and
saturated aqueous sodium chloride (200 mL). The organic layer
was dried over sodium sulfate and filtered, and the solvent
was removed in vacuo. The residue was purified by flash
chromatography on silica gel, eluting with ethyl acetate to
afford 1.16 g of 6 as a white foam (70%). Rf ) 0.26 (chloroform/
methanol/acetic acid, 180:10:5). 1H NMR (200 MHz, DMSO-
d6): δ 1.21 (s, 6H, 2 CH3(Aib)), 1.31 (s, 9H, Boc), 2.98-3.12
(m, 4H, 2 (CH2)â), 4.47 (m, 1H, (CH)RA and (CH)RB), 5.2 (m,
0.4H, (CH)R′B), 5.7 (m, 0.6H, (CH)R′A), 6.95-8.37 (m, 15H, 2
indoles (10H), 3 NH (amides), 1 NH (urethane), CHO (formyl)),
10.89 (m, 2H, 2 N1H (indoles)). MS (ES) m/z 575 [M + H]+,
597 [M + Na]+, 1149 [2M + H]+, 1171 [2M + Na]+.
successively added. After 1 h, the mixture was diluted with
ethyl acetate and washed with saturated aqueous sodium
hydrogen carbonate, aqueous potassium hydrogen sulfate (1
M), and saturated aqueous sodium chloride. The organic layer
was dried over sodium sulfate and filtered, and the solvent
was removed in vacuo. The residue was purified by flash
chromatography on silica gel, eluting with ethyl acetate/hexane
(9:1) to afford 2.92 g (5.80 mmol, 54%) of Boc-(D)-Trp-N(Me)-
(D)-Trp-NH2 as a white foam. Boc-(D)-Trp-N(Me)-(D)-Trp-NH2
(2.81 g, 5.60 mmol) was dissolved in acetonitrile (25 mL). To
this solution, di-tert-butyl dicarbonate (2.4 g, 11.2 mmol) and
4-(dimethylamino)pyridine (164 mg, 1.3 mmol) were succes-
sively added. After 3 h, the mixture was diluted with ethyl
acetate (100 mL) and washed with saturated aqueous sodium
hydrogen carbonate, aqueous potassium hydrogen sulfate, and
saturated aqueous sodium chloride. The organic layer was
dried over sodium sulfate and filtered, and the solvent was
removed in vacuo. The residue was purified by flash chroma-
tography on silica gel, eluting with ethyl acetate/hexane, 5:5,
to afford 3.51 g (5.0 mmol, 89%) of 10 as a white foam. Rf )
TF A, H-Aib-(D)-Tr p -(D)-gTr p -CHO (7). 6 (1 g, 1.7 mmol)
was dissolved in a mixture of trifluoroacetic acid (8 mL),
anisole (1 mL), and thioanisole (1 mL) for 30 min at 0 °C. The
solvents were removed in vacuo, the residue was stirred in
ether, and the precipitated TFA, H-Aib-(D)-Trp-(D)-gTrp-CHO
was filtered. 7 was purified by preparative HPLC and obtained
1
in 52% yield. 1H NMR (400 MHz, DMSO-d6) + correlation H-
1
1H: δ 1.21 (s, 3H, CH3 (Aib)), 1.43 (s, 3H, CH3 (Aib)), 2.97 (m,
2H, (CH2)â), 3.1 (m, 2H, (CH2)â′), 4.62 (m, 1H, (CH)RA and
(CH)RB), 5.32 (q, 0.4H, (CH)R′B), 5.71 (q, 0.6H, (CH)R′A), 7.3
(m, 4H, H5 and H6 (2 indoles)), 7.06-7.2 (4d, 2H, H2A and H2B
(2 indoles)), 7.3 (m, 2H, H4 or H7 (2 indoles)), 7.6-7.8 (4d, 2H,
0.44 (ethyl acetate/hexane, 7:3). H NMR (400 MHz, DMSO-
d6): δ 1.15-1.70 (m, 27H, 3 × Boc), 2.60-3.40 (m, 7H, 2 (CH2)â
+ N-CH3), 4.46 (m, 1/2H, CHR), 4.60 (m, 1/2H, CHR), 5.00 (m,
1/2H, CHR′), 5.20 (m, 1/2H, CHR′), 6.80-8.08 (m, 13H, 2 indoles
(10H), NH (urethane), 2 NH2 (amides)). MS (ES), m/z: 648.4
[M + H - 56]+, 704.3 [M + H]+, 726.7 [M + Na]+.
H
4A and H4B or H7A and H7B), 7.97 (s, 3H, NH2 (Aib) and CHO
(formyl)), 8.2 (d, 0.4H, NH1B (diamino)), 8.3 (m,1H, NHA and
NHB), 8.5 (d, 0.6H, NH1A (diamino)), 8.69 (d, 0.6H, NH2A
(diamino)), 8.96 (d, 0.4H, NH2B (diamino)), 10.8 (s, 0.6H, N1H1A
(indole)), 10.82 (s, 0.4H, N1H1B (indole)), 10.86 (s, 0.6H, N1H2A
(indole)), 10.91 (s, 0,4H, N1H2B (indole)). MS (ES), m/z: 475
[M + H]+, 949 [2M + H]+. HPLC tR: 16.26 min (conditions A).
Boc-(D)-(NiBoc)-Tr p -N(Me)-(D)-(NiBoc)-gTr p -C(O)CH 3
(13). Compound 13 was synthesized as described for compound
5 starting from 10 (500 mg, 0.71 mmol). The gem-diamino
compound was acetylated with acetic anhydride (88 µL, 0.92
mmol) in the presence of N,N-diisopropylethylamine (158 µL,
0.92 mmol). The usual workup was performed and the residue
was purified by flash chromatography on silica gel, eluting
with ethyl acetate/hexane, 5:5, to afford 340 mg (48%) of Boc-
(D)-(NiBoc)-Trp-N(Me)-(D)-(NiBoc)-gTrp-C(O)CH3 as a white
N(Me)-(D)-Tr p -NH2 (9). H-(D)-Trp-NH2 (2.49 g, 12.3 mmol)
was dissolved in DMF (25 mL). Et3N (2.6 mL, 18.84 mmol)
and o-NBSCl (2.85 g, 12.9 mmol) were successively added.
After 15 min, the mixture was diluted with ethyl acetate (300
mL) and washed with saturated aqueous sodium hydrogen
carbonate, aqueous potassium hydrogen sulfate (1 M), and
saturated aqueous sodium chloride. The organic layer was
dried over sodium sulfate and filtered and the solvent was
removed in vacuo to afford 4.30 g of o-NBS-(D)-Trp-NH2 as an
orange foam. An amount of 4.26 g of o-NBS-(D)-Trp-NH2 was
dissolved in DMF (25 mL). K2CO3 (2.20 g, 15.9 mmol) and MeI
(1.53 mL, 24.5 mmol) were successively added. After 1 h, the
mixture was diluted with ethyl acetate (300 mL) and washed
with saturated aqueous sodium hydrogen carbonate, aqueous
potassium hydrogen sulfate (1 M), and saturated aqueous
sodium chloride. The organic layer was dried over sodium
sulfate and filtered, and the solvent was removed in vacuo to
afford 4.10 g of o-NBS-N(Me)-(D)-Trp-NH2 as a yellow foam.
An amount of 4.10 g of o-NBS-N(Me)-(D)-Trp-NH2 was dis-
solved in DMF (30 mL). K2CO3 (3.39 g, 24.5 mmol) and PhSH
(1.51 mL, 14.7 mmol) were successively added. After 1 h, the
mixture was diluted with ethyl acetate (300 mL) and washed
with aqueous potassium hydrogen sulfate and the organic
layer was discarded. The aqueous layer was then treated with
NaOH pellets until pH 10 was attained and washed with 300
mL of ethyl acetate. The organic layer was washed with
saturated aqueous sodium chloride, dried over sodium sulfate,
and filtered and the solvent was removed in vacuo to afford
2.24 g (10.31 mmol, 84% over three steps) of N(Me)-(D)-Trp-
NH2 as a colorless oil. Rf ) 0.19 (ethyl acetate/pyridine/acetic
acid/water, 80:20:5:10). 1H NMR (400 MHz, DMSO-d6): δ 2.15
(s, 3H, N(CH3), 2.75 (dd, 1H, Hâ′, J â′â ) 14.7 Hz, J â′R ) 7.8
Hz), 2.95 (dd, 1H, Hâ, J ââ′ ) 14.4 Hz, J âR ) 5.7 Hz), 3.08 (dd,
1H, HR, J Râ′ ) 14.4 Hz, J Râ ) 5.7 Hz), 6.90-6.98 (m, 2H, 1H
indole and 1H amide), 7,02 (t, 1H, J ) 7.2 Hz, 1H indole), 7.10
(s, 1H, H indole), 7.22 (s, 1H, 1 NH amide), 7.25 (d, 1H, J )
8.0 Hz, H indole), 7.54 (d, 1H, J ) 7.8 Hz, 1H indole), 10.75
(s, 1H, N1H). MS (ES), m/z: 218.1 [M + H]+.
1
solid. H NMR (200 MHz, DMSO-d6): δ 1.20-1.71 (m, 27H, 3
× Boc), 1.79 (2s, 2.1H, acetyl), 1.81 (s, 0.9H, acetyl), 2.75-
3.15 (m, 7H, 2 (CH2)â + N-CH3), 4.61 (m, 0.7H, CHR), 5.05
(m, 0.3H, CHR), 5,91 (m, 0.3H, CHR′), 6.21 (m, 0.7H, CHR′),
6.90-8.1 (m, 11H, 2 indoles (10H), 1NH (urethane), 8.41 (d,
0.7H, 1 NH (amide)), 8.85 (d, 0.3H, 1 NH (amide)). MS (ES),
m/z: 717.8 [M + H]+, 739.8 [M + Na]+.
Boc-(D)-(NiBoc)-Tr p-(D)-(NiBoc)-gTr p-o-NBS (14). 14 was
synthesized starting from 4 (500 mg, 0.72 mmol) to generate
4′. The gem-diamino dipeptide was used without purification
and was reacted with o-NBSCl (290 mg, 1.08 mmol) in the
presence of NEt3 (354 µL, 2.5 mmol) in 10 mL of DCM for 1 h.
The mixture was diluted with ethyl acetate (100 mL) and
washed with saturated aqueous sodium hydrogen carbonate
(100 mL), aqueous potassium hydrogen sulfate (100 mL, 1 M),
and saturated aqueous sodium chloride (50 mL). The organic
layer was dried over sodium sulfate and filtered, and the
solvent was removed in vacuo. The residue was purified by
flash chromatography on silica gel, eluting with ethyl acetate/
hexane, 3:7, to afford 320 mg (52%) of 14 as a white solid. Rf
) 0.39 (ethyl acetate/hexane, 3:7). 1H NMR (400 MHz, DMSO-
d6): δ 1.27 (1s, 9H, Boc), 1.60 (1s, 18H, 2 × Boc), 2.73 (m, 2H,
CH2â), 3.03 (m, 2H, CH2â′), 4.14 (m, 1H, CHR), 5.44 (m, 1H,
CHR′), 6.93 (d, 1H, NH urethane), 7.19-7.73 (m, 12H, arom),
7.91 (d, 1H, arom), 8.03 (d, 1H, arom), 8.55 (d, 1H, NH amide),
8.70 (d, 1H, NH amide). MS (ES), m/z: 589.07 [M + H - (56
+ NO2 - C6H4 - SO2NH2)]+, 645.26 [M + H - (NO2 - C6H4 -
SO2NH2)]+, 847.5 [M + H]+, 869.7 [M + Na]+.
Boc-(D)-(NiBoc)-Tr p-(D)-(NiBoc)-gTr p-N(Me)-o-NBS (15).
An amount of 310 mg (0.37 mmol) of 14 was dissolved in dry
acetonitrile (2 mL), and the mixture was stored under an argon
atmosphere. Both 202 mg (1.58 mmol) of potassium carbonate
and 228 µL (3.7 mmol) of ICH3 were added to the solution at
room temperature. The reaction mixture was brought to 35
°C for 3 h and was monitored by TLC. After conversion was
complete, the mixture was diltuted with DCM (10 mL) and
water (10 mL). The aqueous layer was separated and extracted
with DCM (3 × 5 mL), and the combined organic layers were
washed with brine (10 mL), dried over sodium sulfate, and
Boc-(D)-(NiBoc)-Tr p -N(Me)-(D)-(NiBoc)-Tr p -NH2 (10).
N(Me)-(D)-Trp-NH2 (2.23 g, 10.3 mmol) and Boc-(D)-Trp-OH
(3.29 g, 10.8 mmol) were dissolved in 30 mL of DMF. NMM
(2.84 mL, 25.8 mmol) and HBTU (3.91 g, 10.3 mmol) were

1200 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Guerlavais et al.
concentrated to give a crude product that was purified by flash
chromatography on silica gel, eluting with ethyl acetate/
hexane, 3:7, to afford 200 mg (65%) of 15. Rf ) 0.41 (ethyl
acetate/hexane, 3:7). 1H NMR (400 MHz, DMSO-d6): δ 1.25
(1s, 9H, Boc), 1.59 (1s, 18H, 2 × Boc), 2.60-2.78 (m, 2H, CH2â),
2.86 (2s, 3H, N-Me), 3.00-3.29 (m, 2H, CH2â′), 4.21 (m, 1H,
CHR), 6.12 (m, 1H, CHR′), 6.98 (2d, 1H, NH urethane), 7.15-
8.10 (m, 14H, arom), 9.10 (2d, 1H, NH amide). MS (ES), m/z:
589.2 [M + H - (56 + NO2 - C6H4 - SO2NH - Me)]+, 645.4
[M + H - (NO2 - C6H4 - SO2NH - Me)]+, 861.0 [M + H]+,
883.6 [M + Na]+.
NH1A (diamino)), 8.4 (d, 0.6H, NH2A (diamino)), 8.75 (d, 0.4H,
NH2B (diamino)), 10.69 (s, 0.6H, N1H1A (indole)), 10.71 (s, 0.4H,
N1H1B (indole)), 10.80 (s, 0.6H, N1H2A (indole)), 10.92 (s, 0.4H,
N1H2B (indole)). MS (ES), m/z: 503.1 [M + H]+. HPLC tR: 21.08
min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -CHO (25). The titled com-
pound was synthesized from compound 5 but with coupling of
Boc-N(Me)-Aib-OH. After coupling and classical workup, it was
purified by flash chromatography on silica gel, eluting with
ethyl acetate/methanol (9:1) to afford 180 mg (53%) of Boc-
N(Me)-Aib-(D)-Trp-(D)-gTrp-CHO as a white foam. This com-
pound was deprotected as described earlier and precipitated
in ether to yield TFA, N(Me)-Aib-(D)-Trp-(D)-gTrp-CHO (25),
which was filtered. The product was purified by preparative
HPLC (39 mg, 21%). 1H NMR (200 MHz, DMSO-d6): δ 1.19
(s, 3H, CH3 (Aib)), 1.42 (s, 3H, CH3 (Aib)), 2.26 (s, 3H, NCH3),
3.12 (m, 4H, 2 (CH2)â), 4.66 (m, 1H, (CH)R), 5.32 and 5.7 (m,
1H, (CH)R), 6.9-7.8 (m, 10H, 2 indoles), 8 (m, 1H, CHO
(formyl)), 8.2-9 (m, 4H, 3 NH (amides) and NH (amine)), 10.87
(m, 2H, 2 N1H (indoles)). MS (ES), m/z: 489.29 [M + H]+.
HPLC tR: 17.09 min (conditions A).
N(Me)2-Aib-(D)-Tr p -(D)-gTr p -CHO (26). 1H NMR (400
MHz, DMSO-d6): δ 1.2 (s, 3H, CH3 (Aib)), 1.39 (s, 3H, CH3
(Aib)), 2.29 (m, 3H, NCH3), 2.99-3.33 (m, 4H, 2 (CH2)â), 4.68
(m, 1H, (CH)R), 5.3 and 5.69 (m, 1H, (CH)R′), 6.97-7.72 (m,
10H, 2 indoles), 7.97 (2s, 1H, formyl), 8.2-9.47 (m, 3H, 3 NH
(amides)), 10.85 (m, 2H, 2 NH (indoles)). MS (ES), m/z: 503.45
[M + H]+. HPLC tR: 6.93 min (conditions B).
Boc-(D)-(NiBoc)-Tr p -(D)-(NiBoc)-gTr p -N(Me)-C(O)CH 3
(16A a n d 16B). 15 (200 mg, 0.23 mmol) was dissolved in DMF
(3 mL). K2CO3 (151 mg, 1.10 mmol) and PhSH (112 µL, 1.10
mmol) were successively added. After 4 h, the mixture was
diluted with ethyl acetate (50 mL) and washed with aqueous
potassium hydrogen sulfate (20 mL) and brine (20 mL). The
organic layer was dried over sodium sulfate and filtered, and
the solvent was removed in vacuo. The residue was dissolved
in DMF (3 mL). Acetic anhydride (140 µL, 1.48 mmol) and
DIEA (370 µL, 2.15 mmol) were added, and the reaction was
monitored by TLC. After completion, the mixture was diluted
with ethyl acetate (100 mL) and washed with saturated
aqueous sodium hydrogen carbonate (100 mL), aqueous potas-
sium hydrogen sulfate (100 mL, 1 M), and saturated aqueous
sodium chloride (50 mL). The organic layer was dried over
sodium sulfate and filtered, and the solvent was removed in
vacuo. The residue was purified by flash chromatography on
silica gel, eluting with ethyl acetate/hexane, 5:5, to afford 80
mg (48% for two steps) of 16A and 16B, which were not
H-Aib-(D)-Tr p -(D)-gTr p -C(O)CH3 (27). 27 was synthesized
from 4′, but the amine function was acetylated instead of
formylated. The synthetic pathway after acetylation was the
same as for compound 7. Compound 27 was purified by
1
separated at this step. H NMR (400 MHz, DMSO-d6): δ 1.15
(2s, 9H, Boc), 1.35 (2s, 9H, Boc), 1.46 (s, 9H, Boc), 1.85 (2s,
3H, acetyl), 2.50-2.80 (m, 5H, CH2â + N(Me)), 2.85-3.05 (m,
2H, CH2â′), 4.13 (m, 1H, CHR), 5.58 (m, 1H, CHR′), 6.80 (2d,
1H, NH urethane), 7.10-8.00 (m, 10H, arom), 8.40 (d, 1/2H,
NH amide), 8.75 (d, 1/2H, NH amide). MS (ES), m/z: 718.5
[M + H]+, 740.6 [M + Na]+.
1
preparative HPLC (80 mg, 31%). H NMR (200 MHz, DMSO-
d6): δ 1.22 (s, 3H, CH3 (Aib)), 1.44 (s, 3H, CH3 (Aib)), 1.8 (s,
3H, C(O)CH3), 3.06 (m, 4H, 2 (CH2)â), 4.6 (m, 1H, (CH)R), 5.6
(m, 1H, (CH)R), 6.9-7.8 (m, 10H, 2 indoles), 7.99 (s, 2H, NH2
(Aib)), 8.2-8.6 (m, 3H, 3 NH (amides)), 10.83 (s, 2H, 2 N1H
(indoles)). MS (ES), m/z: 489.32 [M + H]+. HPLC tR: 16.76
min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -C(O)CH3 (28). 28 was syn-
thesized as the previous compound but by coupling Boc-N(Me)-
Aib-OH instead of Boc-Aib-OH on the dipeptide. Compound
28 was purified by preparative HPLC (220 mg, 40%). 1H NMR
(200 MHz, DMSO-d6): δ 1.17 (s, 3H, CH3 (Aib)), 1.4 (s, 3H,
CH3 (Aib)), 1.78 (s, 3H, C(O)CH3), 2.23 (s, 3H, NCH3), 3.15
(m, 4H, 2 (CH2)â), 4.7 (m, 1H, (CH)R), 5.55 (m, 1H, (CH)R), 6.9-
7.9 (m, 10H, 2 indoles), 8.2-8.8 (s, 4H, NH (amine) and 3 NH
(amides)), 10.8 (s, 2H, 2 N1H (indoles)). MS (ES), m/z: 503.19
[M + H]+. HPLC tR: 16.78 min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -N(Me)-C(O)CH3 (17) a n d N-
(Me)-Aib-(D)-Tr p -(L)-gTr p -N(Me)-C(O)CH3 (18). 16A and
16B were deprotected as described for compound 7, and the
deprotected compounds were allowed to couple with Boc-
N(Me)-Aib-OH as described for compound 25. The final
compounds were deprotected and after a classical workup were
purified by preparative HPLC. Both compounds exhibited the
1
same H NMR and mass spectra, suggesting an epimerization
during the synthesis. The D configuration was attributed to
the active compound 17. 1H NMR (360 MHz, DMSO-d6): δ 1.20
and 1.45 (2s, 6H, 2CH3 (Aib)), 2.25 (s, 3H, N-Me(Aib)), 2.70 (s,
3H, N(Me), 2.80-3.25 (2m, 4H, CH2â + CH2â′), 4.75 (m, 1H,
CHR), 5.71 (m, 1H, CHR′), 6.90-7.80 (m, 10H, arom), 8.38 (d,
1H, NH amide), 8.70 (m, 1H, NH amine), 9.10 (d, 1H, NH
amide), 10.80 and 10.91 (2s, 2NiH). MS (ES), m/z: 517.5 [M +
H]+, 539.0 [M + Na]+. HPLC tR: 6.79 min (conditions B). 18,
HPLC tR: 7.20 min (conditions B).
N(Me)-Aib-(D)-Tr p -N(Me)-(D)-gTr p -a cetyl (19). 19 was
synthesized from TFA, H-(D)-Trp-N(Me)-(D)-gTrp-acetyl as
described for compound 25. 1H NMR (200 MHz, DMSO-d6): δ
1.32-1.53 (m, 6H, CH3 (Aib)), 1.83 (m, 3H, C(O)CH3), 2.32-
2.4 (m, 3H, N(CH3)), 2.6-3.2 (m, 7H, 2(CH2)â and N(CH3)),
4.94 (m, 1H, (CH)R), 5.27 (m, 1H, (CH)R), 6.01 (m, 1H, (CH)R′),
6.26 (m, 1H, (CH)R′), 6.59-8.72 (m, 13H, 10H (2 indoles), 2NH
(amides) and 1 NH amine), 10.64-10.94 (m, 2H, 2N1H). MS
(ES), m/z: 516.77 [M + H]+. HPLC tR: 11.59 min (conditions
A).
TF A, H-Aib-(D)-Mr p -(D)-gMr p -CHO (23). 1H NMR (400
MHz, DMSO-d6) + correlation 1H-1H: δ 1.19 (s, 2H, (CH3)1A
(Aib)), 1.23 (s, 1H, (CH3)1B (Aib)), 1.41 (s, 2H, (CH3)2A (Aib)),
1.44 (s, 2H, (CH3)2B (Aib)), 2.33-2.35 (4s, 6H, 2 CH3 (indoles)),
2.93 (m, 2H, (CH2)â), 3.02 (m, 2H, (CH2)â′), 4.65 (m, 0.6H,
(CH)RA), 4.71 (m, 0.4H, (CH)RB), 5.2 (m, 0.4H, (CH)R′B), 5.6
(m, 0.6H, (CH)R′A), 6.95 (m, 4H, H5 and H6 (2 indoles)), 7.19
(m, 2H, H4 or H7 (2 indoles)), 7.6 (m, 2H, H4 or H7 (2 indoles)),
7.9 (s, 1H, CHO (formyl)), 7.95 (s, 2H, NH2 (Aib)), 8.05 (d, 0.4H,
NH1B (diamino)), 8.3 (m,1H, NHA and NHB), 8.35 (m, 0.6H,
N(Me)2-Aib-(D)-Tr p -(D)-gTr p -C(O)CH3 (29). 1H NMR (400
MHz, DMSO-d6): δ 1.22 (s, 3H, CH3 (Aib)), 1.4 (s, 3H, CH3
(Aib)), 1.8 (s, 3H, acetyl), 2.28 (d, 3H, NCH3), 2.96-3.22 (m,
4H, 2 (CH2)â), 4.7 (m, 1H, (CH)R), 5.60 (m, 1H, (CH)R′), 6.98-
7.75 (m, 10H, 2 indoles), 8.2-9.47 (m, 3H, 3 NH (amides)),
10.84 (m, 2H, 2 NH (indoles)). MS (ES), m/z: 517.34 [M + H]+.
HPLC tR: 7.07 min (conditions B).
Aib-(D)-Tr p -N(Me)-(D)-gTr p -a cetyl (30). 30 was synthe-
sized from TFA, H-(D)-Trp-N(Me)-(D)-gTrp-acetyl as described
1
for compound 7. H NMR (200 MHz, DMSO-d6): δ 1.35-1.53
(m, 6H, CH3 (Aib)), 1.82 (m, 3H, C(O)CH3), 2.6-3.2 (m, 7H,
2(CH2)â and N(CH3)), 4.94 (m, 1H, (CH)R), 5.27 (m, 1H, (CH)R),
6.01 (m, 1H, (CH)R′), 6.26 (m, 1H, (CH)R′), 6.59-8.64 (m, 14H,
10H (2 indoles), 2 NH (amides) and 2 NH2 amine), 10.64-
10.92 (m, 2H, 2 N1H). MS (ES), m/z: 502.80 [M + H]+, 1004.76
[2M + H]+. HPLC tR: 11.99 min (conditions A).
H-Aib-(R)-(Me)Tr p -(D)-gTr p -CHO (31). (R)-(Me)-Trp was
synthesized as reported,18 and the titled compound was
prepared following the procedure described for compound 7.
MS (ES), m/z: 489.37 [M + H]+, 977.56 [2M + H]+. HPLC tR:
7.35 min (conditions B).
H-Aib-(D)-Tr p -(R)-(Me)-gTr p -CHO (32). 32 was prepared
following the procedure described for compound 7. MS (ES),
m/z: 489.24 [M + H]+, 511.45 [M + Na]+. HPLC tR: 7.38 min
(conditions B).

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1201
N(Me)-Aib-(D)-Tr p -(R)-(Me)-gTr p -C(O)CH 3 (33). 33 was
prepared following the procedure described for compound 28.
MS (ES), m/z: 517.34 [M + H]+. HPLC tR: 7.76 min (conditions
B).
10.81 (m, 2H, 2 N1H). MS (ES), m/z: 488.41 [M + H]+, 975.91
[2M + H]+. HPLC tR: 19.99 min (conditions A).
Isova ler yl-(D)-Tr p -(D)-gTr p -CHO (40). 40 was synthe-
sized as described for compound 7 starting from TFA, H-(D)-
Trp-(D)-gTrp-CHO (100 mg, 0.20 mmol), isovaleric acid (26 µL,
0.24 mmol), BOP (105 mg, 0.23 mmol), and NMM (50 µL, 0.45
mmol). Isovaleryl-(D)-Trp-(D)-gTrp-CHO (40 mg, 43%) was
purified by preparative HPLC. 1H NMR (200 MHz, DMSO-
d6): δ 0.71 and 0.77 (2d, 6H, 2 CH3), 1.86 (m, 3H, CH and
CH2), 3 (m, 4H, 2 (CH2)â), 4.6 (m, 1H, (CH)R), 5.3 and 5.7 (2m,
1H, (CH)R), 6.9-7.2 (m, 6H), 7.3 (m, 2H), 7.6 (m, 2H), 7.9 (m,
1H, CHO), 7.8-8.7 (m, 3H, 3 NH (amides)), 10.9 (m, 2H, 2
N1H). MS (ES), m/z: 474.39 [M + H]+, 496.39 [M + Na]+,
967.64 [2M + H]+, 989.17 [2M + Na]+. HPLC tR: 16.26 min
(conditions A).
4-Am in op ip er id in e-4-ca r b on yl-(D)-Tr p -(D)-gTr p -CH O
(34). 34 was synthesized by coupling of Boc-(N4Boc)Pip-OH
on TFA, H-(D)-Trp-(D)-gTrp-CHO as described earlier. Com-
pound 34 was purified by preparative HPLC (127 mg, 37%).
1H NMR (200 MHz, DMSO-d6): δ 1.81 (m, 2H, CH2 (Pip)), 2.30
(m, 2H, CH2 (Pip)), 3.1 (m, 8H, 2 (CH2)â and 2 CH2 (Pip)), 4.68
(m, 1H, (CH)R), 5.30 and 5.73 (2m, 1H, (CH)R), 6.9-7.7 (m, 10H,
2 indoles), 7.98 (2s, 1H, CHO (formyl)), 8.2-9.2 (m, 6H, NH2
and NH (Pip) and 3 NH (amides)), 10.9 (m, 2H, 2 N1H
(indoles)).MS (ES), m/z: 516.37 [M + H]+, 538.27 [M + Na]+.
HPLC tR: 13.78 min (conditions A).
CH3C(O)-(D)-Tr p -(D)-gTr p -C(O)CH3 (41). In 5 mL of
DMF, TFA, H-(D)-Trp-(D)-gTrp-C(O)CH3 (100 mg, 0.19 mmol),
DIEA (36 µL, 0.21 mmol), and acetic anhydride (28 µL, 0.21
mmol) were successively added. After 1 h, the mixture was
diluted with ethyl acetate (25 mL) and washed with saturated
aqueous sodium hydrogen carbonate (50 mL), aqueous potas-
sium hydrogen sulfate (50 mL, 1 M), and saturated aqueous
sodium chloride (50 mL). The organic layer was dried over
sodium sulfate and filtered, and the solvent was removed in
vacuo. The product 41 was purified by preparative HPLC (47
mg, 46%). 1H NMR (200 MHz, DMSO-d6): δ 1.77 (s, 3H, C(O)-
CH3), 1.79 (s, 3H, C(O)CH3), 2.81-3.12 (m, 4H, 2 (CH2)â), 4.5
(m, 1H, (CH)R), 5.63 (m, 1H, (CH)R), 6.98-7.15 (m, 6H), 7.25
(m, 2H), 7.62 (m, 2H), 8.0 (d, 1H, NH (amide)), 8.16 (m, 1H,
NH (amide)), 8.30 (d, 1H, NH (amide)), 10.81 (s, 1H, 1 N1H),
10.83 (s, 1H, 1 N1H). MS (ES), m/z: 446.75 [M + H]+, 467.86
[M + Na]+. HPLC tR: 11.99 min (conditions A).
4-Am in op ip er id in e-4-ca r bon yl-(D)-Tr p -(D)-gTr p -C(O)-
CH3 (35). 35 was purified by preparative HPLC (135 mg, 42%).
1H NMR (200 MHz, DMSO-d6): δ 1.79 (m, 2H, CH2 (Pip)), 1.81
(s, 3H, C(O)CH3), 2.3 (m, 2H, CH2 (Pip)), 3.1 (m, 8H, 2 (CH2)â
and 2 CH2 (Pip)), 4.7 (m, 1H, (CH)R), 5.6 (m, 1H, (CH)R′), 6.9-
7.8 (m, 10H, 2 indoles), 8.2-9 (m, 6H, NH2 and NH (Pip) and
3 NH (amides)), 10.85 (m, 2H, 2 N1H (indoles)). MS (ES), m/z:
530.39 [M + H]+, 552.41 [M + Na]+. HPLC tR: 14.02 min
(conditions A).
Ison ip ecotyl-(D)-Tr p -(D)-gTr p -CHO (36). 36 was synthe-
sized as described for compound 34 from TFA, H-(D)-Trp-(D)-
gTrp-CHO (250 mg, 0.33 mmol) and Fmoc-isonipecotic-OH
(144 mg, 0.32 mmol). The Fmoc group was removed in a
mixture of DMF (8 mL) and piperidine (2 mL) in 30 min. The
solvents were removed in vacuo, the residue was stirred in
ether, and the precipitated isonipecotyl-(D)-Trp-(D)-gTrp-CHO
was recovered by filtration. Compound 36 was purified by
N(Me)-(D)-Tr p -(D)-gTr p -CHO A a n d B (42, 43). Boc-
N(Me)-(D)-(NiBoc)Trp-(D)-(NiBoc)-gTrp-formyl (295 mg, 0.42
mmol) was dissolved in a mixture of trifluoroacetic acid (8 mL),
anisole (1 mL), and thioanisole (1 mL) for 1 h at 0°C. The
solvents were removed in vacuo, the residue was stirred in
ether, and the precipitated TFA, N(Me)-(D)-Trp-(D)-gTrp-formyl
was filtered. Analytical HPLC chromatograms showed the
presence of two equivalent peaks that possessed the same MS
spectrum and were attributed to two isomers. Compounds
TFA, N(Me)-(D)-Trp-(D)-gTrp-formyl A (52 mg, 24%) and TFA,
N(Me)-(D)-Trp-(D)-gTrp-formyl B (60 mg, 28%) were separated
by preparative HPLC.
1
preparative HPLC (81 mg, 33%). H NMR (200 MHz, DMSO-
d6): δ 1.65 (m, 4H, 2 CH2 (Iso)), 2.4 (m, 1H, CH (Iso)), 2.7-3.3
(m, 8H, 2 (CH2)â and 2 CH2 (Iso)), 4.6 (m, 1H, (CH)R), 5.3 and
5.7 (2m, 1H, (CH)R), 6.9-7.7 (m, 10H, 2 indoles), 7.97 (2s, 1H,
CHO (formyl)), 8-8.8 (m, 4H, NH (Iso) and 3 NH (amides)),
10.9 (m, 2H, 2 N1H (indoles). MS (ES), m/z: 501.36 [M + H]+.
HPLC tR: 15.34 min (conditions A).
Ison ip ecotyl-(D)-Tr p -(D)-gTr p -C(O)CH3 (37). 37 was syn-
thesized as described for compound 35 from TFA, H-(D)-Trp-
(D)-gTrp-C(O)CH3 (250 mg, 0.33 mmol) and Fmoc-isonipecotic-
OH (141 mg, 0.4 mmol). The Fmoc group was removed as
described for compound 36. The solvents were removed in
vacuo, the residue was stirred in ether, and the precipitated
isonipecotyl-(D)-Trp-(D)-gTrp-C(O)CH3 was filtered. Compound
1
Com p ou n d 42. H NMR (200 MHz, DMSO-d6): δ 2.1 (m,
3H, NCH3), 3.1 (m, 4H, 2 (CH2)â), 3.89 (m, 1H, CHR), 5.42 and
5.82 (m, 1H, CHR), 6.9-7.7 (m, 10H, 2 indoles), 8 (s, 1H, CHO),
8.3-9.3 (m, 3H, 2 NH (amides) and NH (amine)), 10.9 (m, 1H,
N1H), 11.05 (m, 1H, N1H). MS (ES), m/z: 404.5 [M + H]+.
HPLC tR: 113.30 min (conditions A).
1
37 was purified by preparative HPLC (65 mg, 26%). H NMR
(200 MHz, DMSO-d6): δ 1.66 (m, 4H, 2 CH2 (Iso)), 1.79 (s,
3H, C(O)CH3), 2.7-3.3 (m, 8H, 2 (CH2)â and 2 CH2 (Iso)), 4.54
(m, 1H, (CH)R), 5.59 (m, 1H, (CH)R), 6.9-7.7 (m, 10H, 2
indoles), 8-8.6 (m, 4H, NH (Iso) and 3 NH (amides)), 10.82
(m, 2H, 2 N1H (indoles)). MS (ES), m/z: 515.44 [M + H]+.
HPLC tR: 15.32 min (conditions A).
1
Com p ou n d 43. H NMR (200 MHz, DMSO-d6): δ 2.49 (m,
3H, NCH3), 2.8 (m, 2H, (CH2)â), 3.1 (m, 2H, (CH2)â), 3.9 (m,
1H, CHR), 5.2 and 5.65 (m, 1H, CHR), 6.8-7.6 (m, 10H, 2
indoles), 7.9 (s, 1H, CHO), 8.2-9.3 (m, 3H, 2 NH (amides) and
NH (amine)), 10.85 (m, 1H, N1H), 11 (m, 1H, N1H). MS (ES),
m/z: 404.44 [M + H]+. HPLC tR: 15.69 min (conditions A).
Me-SO2-(D)-Tr p -(D)-gTr p -CHO (44). 44 was synthesized
as described for compound 7 starting from H-(D)-Trp-(D)-gTrp-
CHO (100 mg, 0.14 mmol) and MeSO2Cl (12 µL, 0.154 mmol).
Compound Me-SO2-(D)-Trp-(D)-gTrp-CHO was purified by
P h -SO2-(D)-Tr p -(D)-gTr p -CHO (38). 38 was synthesized
as described for compound 7 starting from TFA, H-(D)-Trp-
(D)-gTrp-CHO (100 mg, 0.14 mmol) and PhSO2Cl (20 µL, 0.154
mmol). Compound Ph-SO2-(D)-Trp-(D)-gTrp-CHO (77 mg, 73%)
was purified by preparative HPLC. 1H NMR (200 MHz,
DMSO-d6): δ 2.85 (m, 4H, 2 (CH2)â), 4 (m, 1H, (CH)R), 5 and
5.5 (2m, 1H, (CH)R), 6.9-7.6 (m, 15H, 2 indoles (10H) and
benzyl (5H)), 7.9 (s, 1H, CHO), 8-8.7 (m, 3H, NH (sulfona-
mide) and 2 NH (amides)), 10.98 (m, 2H, 2 N1H). MS (ES),
m/z: 530.21 [M + H]+. HPLC tR: 20.19 min (conditions A).
Isova ler yl-(D)-Tr p -(D)-gTr p -C(O)CH3 (39). 39 was syn-
thesized as described for compound 7 starting from TFA, H-(D)-
Trp-(D)-gTrp-C(O)CH3 (100 mg, 0.19 mmol), isovaleric acid (25
µL, 0.23 mmol), BOP (101 mg, 0.23 mmol), and NMM (48 µL,
0.43 mmol). Isovaleryl-(D)-Trp-(D)-gTrp-C(O)CH3 (49 mg, 52%)
was purified by preparative HPLC. 1H NMR (200 MHz,
DMSO-d6): δ 0.71 and 0.77 (2d, 6H, 2 CH3), 1.78 (s, 3H, CH3),
1.9 (m, 3H, CH and CH2), 3.0 (m, 4H, 2 (CH2)â), 4.6 (m, 1H,
(CH)R), 5.6 (m, 1H, (CH)R), 6.9-7.2 (m, 6H), 7.3 (m, 2H), 7.6
(m, 2H), 8.1 (m, 1H, NH (amide)), 8.2 (m, 2H, 2 NH (amides)),
1
preparative HPLC (46 mg, 51%). H NMR (200 MHz, DMSO-
d6): δ 2.26 and 2.31 (2s, 3H, CH3), 3 (m, 4H, 2 (CH2)â), 4.05
(m, 1H, (CH)R), 5.3 and 5.7 (2m, 1H, (CH)R), 6.9-7.7 (m, 11H,
2 indoles (10H) and NH (sulfonamide)), 8 (s, 1H, CHO), 8.1-
8.9 (m, 2H, 2 NH (amides)), 10.98 (m, 2H, 2 N1H). MS (ES),
m/z: 468.23 [M + H]+, 490.31 [M + Na]+. HPLC tR: 117.59
min (conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -p r op ion yl (45). 45 was synthe-
sized as described for compound 7 starting from Boc-(D)-(Ni-
Boc)-Trp-(D)-(NiBoc)-gTrp-H (4′) (958 mg, 1.45 mmol), NMM
(351 µL, 3.16 mmol), propionic acid (130 µL, 1.74 mmol), and
BOP (770 mg, 1.74 mmol) to afford 250 mg (85%) of Boc-Aib-
(D)-Trp-(D)-gTrp-propionyl as a foam. Boc-Aib-(D)-Trp-(D)-gTrp-
propionyl (250 mg, 1.18 mmol) was dissolved in a mixture of

1202 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Guerlavais et al.
trifluoroacetic acid (8 mL), anisole (1 mL), and thioanisole (1
mL) for 30 min at 0 °C. The solvents were removed in vacuo,
the residue was stirred in ether, and the precipitated TFA,
H-Aib-(D)-Trp-(D)-gTrp-propionyl was filtered. Compound 45
was purified by preparative HPLC (80 mg, 31%). 1H NMR (200
MHz, DMSO-d6): δ 0.96 (t, 3H, CH3), 1.23 and 1.44 (2s, 6H, 2
CH3 (Aib)), 2.1 (q, 2H, CH2), 3 (m, 4H, 2 (CH2)â), 4.6 (m, 1H,
(CH)R), 5.6 (m, 1H, (CH)R), 6.9-7.2 (m, 6H), 7.3 (m, 2H), 7.7
(2d, 2H), 7.9 (s, 2H, NH2), 8.2 (d, 1H, NH (amide)), 8.3 (d, 1H,
NH (amide)), 8.5 (d, 1H, NH (amide)), 10.83 (m, 2H, 2 N1H).
MS (ES), m/z: 503.4 [M + H]+. HPLC tR: 16.26 min (conditions
A).
2 CH2 (Iso) and 2 (CH2)â), 4.66 (m, 1H, (CH)R), 5.64 (m, 1H,
(CH)R′), 6.98-7.18 (m, 6H), 7.3 (2d, 2H), 7.7 (2d, 2H), 8.34 and
8.64 (2m, 2H and 3H, 3 NH (amides) and 2 NH (amines)), 10.85
(m, 2H, 2 N1H). MS (ES), m/z: 571.91 [M + H]+. HPLC tR:
9.11 min (conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -3-in d olyla cetyl (53). 53 was syn-
thesized from 4′ as described for compound 45, by substituting
propionic acid for 3-indolylacetic acid. 1H NMR (200 MHz,
DMSO-d6): δ 1.18 and 1.42 (2s, 6H, 2 CH3 (Aib)), 3 (m, 4H, 2
(CH2)â), 3.53 (s, 2H, CH2), 4.6 (m, 1H, (CH)R), 5.7 (m, 1H,
(CH)R), 6.9-7.8 (m, 15H), 7.9 (s, 2H, NH2), 8.3 (m, 2H, 2 NH
(amides)), 8.6 (d, 1H, NH (amide)), 10.8 (m, 1H, N1H), 10.85
(s, 2H, 2 N1H). MS (ES), m/z: 604.51 [M + H]+. HPLC tR: 19.50
min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -p r op ion yl (46). 46 was puri-
1
fied by preparative HPLC (92 mg, 50%). H NMR (200 MHz,
DMSO-d6): δ 0.96 (t, 3H, CH3), 1.19 and 1.42 (2s, 6H, 2 CH3
(Aib)), 2.07 (q, 2H, CH2), 2.58 (s, 3H, N(CH3)), 3 (m, 4H, 2
(CH2)â), 4.65 (m, 1H, (CH)R), 5.6 (m, 1H, (CH)R), 6.9-7.2 (m,
6H), 7.3 (m, 2H), 7.7 (2d, 2H), 8.2 (d, 1H, NH (amide)), 8.3 (d,
1H, NH (amide)), 8.55 (d, 1H, NH (amide)), 8.65 (m, 1H, NH
(amine)), 10.83 (m, 2H, 2 N1H). MS (ES), m/z: 517.38 [M +
H]+, 539.45 [M + Na]+. HPLC tR: 12.12 min (conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -isova ler yl (47). 47 was synthe-
sized from 4′ as described for 45, by substituting propionic acid
with isovaleric acid. 1H NMR (200 MHz, DMSO-d6): δ 0.83
(m, 6H, 2 CH3), 1.22 and 1.43 (2s, 6H, 2 CH3 (Aib)), 1.9 (m,
3H, CH and CH2), 3 (m, 4H, 2 (CH2)â), 4.6 (m, 1H, (CH)R), 5.6
(m, 1H, (CH)R), 6.9-7.2 (m, 6H), 7.3 (m, 2H), 7.7 (2d, 2H), 7.9
(s, 2H, NH2), 8.2 (d, 1H, NH (amide)), 8.3 (d, 1H, NH (amide)),
8.6 (d, 1H, NH (amide)), 10.82 (m, 2H, 2 N1H). MS (ES), m/z:
531.48 [M + H]+. HPLC tR: 18.41 min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -3-in d olyla cetyl (54). 54 was
synthesized from TFA, H-(D)-Trp-(D)-gTrp-3-indolylacetyl as
described for compound 46. 1H NMR (200 MHz, DMSO-d6): δ
1.06 and 1.39 (2s, 6H, 2 CH3 (Aib)), 2.23 (m, 3H, NCH3), 2.92-
3.2 (m, 4H, 2 (CH2)â), 3.53 (s, 2H, CH2), 4.67 (m, 1H, (CH)R),
5.68 (m, 1H, (CH)R), 6.59-7.15 (m, 9H), 7.31-7.35 (m, 3H),
7.43 (d, 1H), 7.63 and 7.72 (2d, 2H), 8.32 (m, 2H, NH (amine)
and NH (amide)), 8.6 (m, 2H, 2 NH (amides)), 8.6 (d, 1H, NH
(amide)), 10.81 (d, 1H, N1H), 10.85 (s, 2H, 2 N1H). MS (ES),
m/z: 618 [M + H]+, 640 [M + Na]+. HPLC tR: 19.20 min
(conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -cycloh exyl-3-p r op ion yl (55). 55
was synthesized from 4′ as described for compound 45, by
substituting propionic acid for cyclohexyl-3-propionic acid. 1H
NMR (200 MHz, DMSO-d6): δ 0.83 (m, 2H, CH2), 1.22 and
1.44 (2s, 6H, 2 CH3 (Aib)), 1-1.7 (m, 11H, 5 CH2 and CH), 2
(t, 2H, CH2), 3 (m, 4H, 2 (CH2)â), 4.6 (m, 1H, (CH)R), 5.6 (m,
1H, (CH)R), 6.9-7.2 (m, 6H), 7.3 (m, 2H), 7.7 (2d, 2H), 7.9 (s,
2H, NH2), 8.1 (d, 1H, NH (amide)), 8.3 (m, 1H, NH (amide)),
8.5 (d, 1H, NH (amide)), 10.82 (m, 2H, 2 N1H). MS (ES), m/z:
585.65 [M + H]+. HPLC tR: 23.47 min (conditions A).
N(Me)-Aib-(D)-Tr p-(D)-gTr p-cycloh exyl-3-pr opion yl (56).
56 was synthesized from TFA, H-(D)-Trp-(D)-gTrp-cyclohexyl-
3-propionyl as described for compound 46. 1H NMR (200 MHz,
DMSO-d6): δ 0.8 (m, 2H, CH2), 1.09 (m, 4H, 2 CH2), 1.19 (s,
3H, CH3 (Aib)), 1.3-1.4 (m, 2H, CH2), 1.42 (s, 3H, CH3 (Aib)),
1.61-1.7 (m, 5H, 2 CH2 and CH), 2.06 (m, 2H, CH2), 2.25 (s,
3H, NCH3), 3-3.2 (m, 4H, 2 (CH2)â), 4.66 (m, 1H, (CH)R), 5.62
(m, 1H, (CH)R), 7-7.16 (m, 6H), 7.31-7.36 (m, 2H), 7.64 and
7.73 (2d, 2H), 8.2 (d, 1H, NH (amine)), 8.3 (d, 1H, NH (amide)),
8.5 (d, H, NH (amide)), 8.67 (d, 1H, NH (amide)), 10.83 (m,
2H, 2 N1H). MS (ES), m/z: 599 [M + H]+. HPLC tR: 12.58
min (conditions B).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -isova ler yl (48). 48 was syn-
thesized from TFA, H-(D)-Trp-(D)-gTrp-isovaleryl as described
1
for the previous title compound. H NMR (200 MHz, DMSO-
d6): δ 0.83 (m, 6H, 2 CH3), 1.19 and 1.41 (2s, 6H, 2 CH3 (Aib)),
1.94 (m, 3H, CH and CH2), 2.25 (m, 3H, NCH3), 2.99-3.21 (m,
4H, 2 (CH2)â), 4.67 (m, 1H, (CH)R), 5.64 (m, 1H, (CH)R), 7-7.07
(m, 4H), 7.15-7.18 (m, 2H), 7.3-7.36 (m, 2H), 7.66 and 7.73
(2d, 2H), 8.21 (d, 1H, NH (amide)), 8.3 (d, 1H, NH (amide)),
8.58 (d, 1H, NH (amide)), 8.6 (m, 1H, NH (amine)), 10.83 (m,
2H, 2 N1H). MS (ES), m/z: 545 [M + H]+. HPLC tR: 18.34
min (conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -p h en yla cetyl (49). 49 was syn-
thesized from 4′ as described for 45, by substituting propionic
acid with phenylacetic acid. 1H NMR (200 MHz, DMSO-d6):
δ 1.22 and 1.42 (2s, 6H, 2 CH3 (Aib)), 3 (m, 4H, 2 (CH2)â), 3.43
(s, 2H, CH2), 4.6 (m, 1H, (CH)R), 5.6 (m, 1H, (CH)R), 6.9-7.4
(m, 13H), 7.7 (2d, 2H), 7.9 (s, 2H, NH2), 8.3 (d, 1H, NH
(amide)), 8.5 (d, 1H, NH (amide)), 8.7 (d, 1H, NH (amide)),
10.83 (m, 2H, 2 N1H). MS (ES), m/z: 565.40 [M + H]+. HPLC
tR: 119.56 min (conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -p h en yla cetyl (50). 50 was
synthesized from TFA, H-(D)-Trp-(D)-gTrp-phenylacetyl as
described for compound 46. 1H NMR (200 MHz, DMSO-d6): δ
1.19 and 1.4 (2s, 6H, 2 CH3 (Aib)), 2.24 (m, 3H, NCH3), 2.91-
3.2 (m, 4H, 2 (CH2)â), 4.66 (m, 1H, (CH)R), 5.63 (m, 1H, (CH)R),
6.99-7.37 (m, 13H), 7.66 and 7.74 (2d, 2H), 8.29 (d, 1H, NH
(amide)), 8.52 (d, 1H, NH (amide)), 8.6 (m, 2H, NH (amide)
and NH (amine)), 10.88 (m, 2H, 2 N1H). MS (ES), m/z: 565.40
[M + H]+. HPLC tR: 19.23 min (conditions A).
H-Aib-(D)-Tr p -(D)-gTr p -C(O)-NHCH2CH3 (57). 57 was
synthesized by coupling Boc-(D)-(NiBoc)-Trp-(D)-(NiBoc)-gTrp-H
(4) with ethyl isocyanate, under usual deprotection conditions,
1
and coupling to Aib residue as described for compound 7. H
NMR (400 MHz, DMSO-d6): δ 0.95 (t, 3H, -C(O)-NHCH2CH3),
1.05 and 1.10 (2s, 6H, 2 CH3 (Aib)), 2.88-3.18 (m, 6H, 2(CH2â
)
and -C(O)-NHCH2CH3), 4.55 (m, 1H, (CH)R1), 5.45 (m, 1H,
(CH)R2), 6.08 (t, 1H, -C(O)-NHCH2CH3), 6.25 (d, 1H, -NH-
C(O)-NHCH2CH3), 6.90-7.60 (10H, 2 indoles), 8.00 (sl, 1H,
NH amide), 8.35 (d, 1H, NH amide), 10.75 (s, 1H, 1N1H), 10.80
(s, 1H, 1 N1H). MS (ES), m/z: 430.1 [M + H - CH3CH2NH-
(CO)NH2]+, 518.4 [M + H]+, 540.3 [M + Na]+. HPLC tR: 7.25
min (conditions B).
H-Aib-(D)-Tr p -(D)-gTr p -ison ip ecotyl (51). 51 was syn-
thesized from 4′ as described for compound 45, by substituting
H-Aib-(D)-Tr p -(D)-gTr p -C(O)-OCH2CH3 (58). 58 was syn-
thesized by coupling gem-diamino Boc-(D)-(NiBoc)-Trp-(D)-(Ni-
Boc)-gTrp-H (4) with ethyl chloroformate (ClC(O)OCH2CH3)
in the presence of Et3N and under the usual deprotection
conditions and coupling to Aib as described previously. 1H
NMR (360 MHz, DMSO-d6): δ 1.12 (t, 3H, -C(O)-OCH2CH3),
1
propionic acid for Fmoc-isonipecotic acid. H NMR (200 MHz,
DMSO-d6): δ 1.22 and 1.43 (2s, 6H, 2 CH3 (Aib)), 1.7 (m, 4H,
2 CH2 (Iso)), 2.4 (m, 1H, CH (Iso)), 2.8-3.3 (m, 8H, 2 CH2 (Iso)
and 2 (CH2)â), 4.6 (m, 1H, (CH)R), 5.6 (m, 1H, (CH)R′), 6.9-7.2
(m, 6H), 7.3 (2d, 2H), 7.7 (2d, 2H), 7.9 (s, 2H, NH2), 8.3 (m,
2H, 2 NH (amides)), 8.7 (d, 1H, NH (amide)), 10.82 (m, 2H, 2
N1H). MS (ES), m/z: 558.44 [M + H]+. HPLC tR: 13.51 min
(conditions A).
N(Me)-Aib-(D)-Tr p -(D)-gTr p -ison ip ecotyl (52). 52 was
synthesized from TFA, H-(D)-Trp-(D)-gTrp-isonipecotyl as de-
scribed for compound 46. 1H NMR (200 MHz, DMSO-d6): δ
1.20 and 1.41 (2s, 6H, 2 CH3 (Aib)), 1.69 (m, 4H, 2 CH2 (Iso)),
2.25 (m, 3H, NCH3), 2.4 (m, 1H, CH (Iso)), 2.85-3.34 (m, 8H,
1.21 and 1.41 (2s, 6H, 2 CH3 (Aib)), 2.99 (d, 1H, Hâ1, J â1â′1
)
13.5 Hz), 3.04 (m, 2H, CH2â2), 3.15 (dd, 1H, Hâ′1, J â′1 ) 14.5
â1
Hz, J â′1R1 ) 3.5 Hz), 3.95 (q, 2H, -C(O)-OCH2CH3), 4.63 (m,
1H, (CH)R1), 5.45 (m, 1H, (CH)R2), 6.95-7.15 (m, 4H, H indole),
7.12 (s, 1H, H indole), 7.18 (s, 1H, H indole), 7.30-7.40 (m,
2H, H indole), 7.55 (m, 1H, -NH-C(O)-OCH2CH3), 7.65 (d,
1H, H indole), 7.71 (d, 1H, H indole), 8.25 (d, 1H, NH amide),
8.60 (d, 1H, NH amide), 10.80 (s, 1H, 1N1H), 10.82 (s, 1H, 1

Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1203
N1H). MS (ES), m/z: 430.3 [M + H - CH3CH2O(CO)NH2]+,
519.1 [M + H]+, 541.6 [M + Na]+. HPLC tR: 7.22 min
(conditions B).
1999; pp 19-24.
(16) Deghenghi, R.; Boutignon, F.; Muccioli, G.; Ghigo, E.; Locatelli,
V. Impervious peptides as growth hormone secretagogues. In
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Ack n ow led gm en t. The pcDNA3 vector containing
the cDNA for the human GHS-R 1a has been kindly
provided by Andrew D. Howard from Merck Research
Laboratories, Rahway, New J ersey. This work is a
Eureka Project No. 1923.
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