Novel orally active growth hormone secretagogues

Article abstract of DOI:10.1021/jm980197u

A novel class of growth hormone-releasing compounds with a molecular weight in the range from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone secretagogues with oral bioavailability. By a rational approach we were able t

Full text of DOI:10.1021/jm980197u

J . Med. Chem. 1998, 41, 3705-3714
3705
Novel Or a lly Active Gr ow th Hor m on e Secr eta gogu es
Thomas K. Hansen,*,† Michael Ankersen,† Birgit S. Hansen,‡ Kirsten Raun,§ Karin K. Nielsen,∇ J esper Lau,†
Bernd Peschke,† Behrend F. Lundt,† Henning Thøgersen,† Nils L. J ohansen,† Kjeld Madsen,† and
Peter H. Andersen†
Departments of Medicinal Chemistry Research, Assay and Cell Technology, Growth Hormone Pharmacology, and
Pharmacokinetics, Health Care Discovery and Preclinical Development, Novo Nordisk A/ S, Novo Nordisk Park,
DK2760 Ma˚løv, Denmark
Received March 30, 1998
A novel class of growth hormone-releasing compounds with a molecular weight in the range
from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone
secretagogues with oral bioavailability. By a rational approach we were able to reduce the
size of the lead compound ipamorelin (4) and simultaneously to reduce hydrogen-bonding
potential by incorporation of backbone isosters while retaining in vivo potency in swine. A rat
pituitary assay was used for screening of all compounds and to evaluate which compounds
should be tested further for in vivo potency in swine and oral bioavailability, fpo, in dogs. Most
of the tested compounds had fpo in the range of 10-55%. In vivo potency in swine after iv
dosing is reported, and ED50 was found to be 30 nmol/kg of body weight for the most potent
compound.
In tr od u ction
it seems unlikely that patients will accept daily injec-
tions. As most of these conditions are not life-threaten-
ing, it is of great importance to find safe, orally active
substances that offer the convenience of oral adminis-
tration and that have no or few adverse effects.
The first nonpeptide GHS, L-692,429 (1), was discov-
ered by scientists from Merck6 after a directed screening
program. This compound had low oral bioavailability,
but the same group later discovered orally active
compounds of which the prototype is MK-677 (2).7
The release of growth hormone (GH) from somatotro-
phs in the pituitary gland is known to be controlled via
two separate receptor systems triggered by somatostatin
and GHRH (growth hormone-releasing hormone). GH
release is probably influenced by a third, separate
mechanism as wellsa growth hormone secretagogue
(GHS) pathway (also known as the GHRP pathway).
The mechanism of action, for the compounds now known
as GHSs, implies binding to one or more receptor(s)
different from those used by somatostatin or GHRH.
Recently, Howard et al. disclosed the isolation of a cDNA
encoding a putative GHS receptor1 that binds GHSs
with high affinity, but so far there are no reports on an
endogenous ligand.
The field of GHSs originates from Momany and
Bowers work2 on enkephalines in the 1970s which led
to potent and specific compounds such as the heptapep-
tide GHRP-1 (3) and hexapeptide GHRP-6. A substan-
tial level of worldwide interest3 has now been reached.
This process has been driven not only by an increase in
the understanding of the varied effects of GH itself and
the potential clinical applications but also by the
discovery of novel small molecules, which mimic the
action of Momany and Bowers’ original peptides.
At present, a number of studies4 suggest benefits of
using GH in elderly patients with osteoporosis, wasting
conditions, and recovery, i.e., following major surgery.
And there is reason to believe that orally administered
GHS therapy could substitute direct GH replacement
by parenteral routes and at the same time produce a
GH concentration profile that mimics the natural
pulsatile pattern better.5 Considering the nature of the
conditions that may be treated with GHS in the future,
Resu lts
Our program took a different route and was an
attempt directly to reduce the size of GHRP-1 (3) in a
rational, stepwise manner with the aim of retaining
activity and achieving oral bioavailability (Scheme 1).
The initial part of our program was based on classical
peptide chemistry and has been reported elsewhere.8
Major achievements during this program were the
elimination of the central Ala-Trp sequence from
GHRP-1 leading to the very potent clinical candidate
ipamorelin (4) (NNC 26-0161) and the substitution of
the N-terminal Aib-His residues with 3-(aminomethyl)-
benzoic acid leading to 5 (NNC 26-0235). This was
achieved without loss of in vitro or in vivo potency.
Removal of the C-terminal Lys residue from 5 gave us
the first compound (6) with oral bioavailability (fpo
)
20% in rats), but with a more than 100-fold loss of
activity in the rat pituitary in vitro assay (EC50 from
2.0 nM for 5 to 265 nM for 6). Yet, compound 6 served
as a lead compound at later stages of our program where
our task was to improve the potency without losing oral
bioavailability.
Starting from 6, our strategy was to maintain or
reduce the number of potential hydrogen-bonding sites
and to keep the molecular weight as low as possible in
order to achieve high oral bioavailability. This strategy
was based on the suggestions provided by other groups,9
† Department of Medicinal Chemistry Research.
‡ Department of Assay and Cell Technology.
§ Department of Growth Hormone Pharmacology.
∇ Department of Pharmacokinetics.
S0022-2623(98)00197-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/14/1998

3706 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hansen et al.
Sch em e 1
thereby maximize the chance of absorption across the
intestinal membrane. Previous results in our peptide
program indicated that shielding of the N-terminal
nitrogen atom by the bulk of two methyl groups was
particularly advantageous. Furthermore, 3-(aminom-
ethyl)benzoic acid10 constitutes a very rigid mimic of an
Aib-His dipeptide unit, leading us to believe that
substitution of the m-phenylene with an allyl unit was
attractive in order to achieve more flexibility. These
considerations led to the synthesis and incorporation of
5-amino-5-methyl-2-hexenoic acid as an N-terminal
dipeptide mimic in a series of novel modified tripeptides,
7a -s. Indeed, when this substitution was carried out
on 6 along with a simple modification in the C-terminal,
we obtained compounds that were generally very much
improved with respect to in vitro potency. The first
compound in this series (7a ), was 15 times more potent
than 6. After the discovery of 7a , a series of close
analogues was prepared and screened for oral bioavail-
ability in beagle dogs and for GH-releasing properties
in swine after iv administration.
Ch em istr y
The synthesis of compounds 7a -s (Scheme 2, Table
1) was based on commercially available Boc-protected
amino acids (with exception of the N-terminal) which
were N-methylated in THF with methyl iodide in the
presence of sodium hydride. This procedure11 allows
virtually racemization-free N-methylation without con-
comitant esterification of the carboxylic acid. Attach-
ment of a C-terminal amine proceeded as HOBt-
mediated couplings in almost quantitative yield. All
peptide couplings on secondary amines were performed
as activated esters with 7-azahydroxybenzotriazole12
(HOAt) and EDAC as activating agents. In general,
couplings were relatively slow13 and required prolonged
reaction times to go to completion.
Yields in all steps were good, except for the final
deprotection. In all cases studied (7a -s) the molecules
showed pronounced tendency to cleave between the Nal
and Phe residues14 during treatment with TFA. This
cleavage was clearly induced by the anhydrous and very
acidic conditions. Minimizing this undesired cleavage
required that the Boc-protected precursors were exposed
to TFA for a very short time. Already after 7 min in
50% TFA in methylene chloride at room temperature
we observed substantial cleavage between Nal and Phe
residues. Fast neutralization was essential. However,
once the compounds were purified, they showed good
stability even during prolonged storage in aqueous
solution at pH 4-5.
(2E)-5-(tert-Butoxycarbonylamino)-5-methylhex-2-eno-
ic acid (18) was used as a general N-terminal and was
unknown in the literature. The acid was prepared in
four reaction steps from the known6 3-(tert-butoxycar-
bonylamino)-3-methylbutanoic acid (14) (Scheme 3).
Compound 14 was converted to 3-(tert-butoxycarbony-
lamino)-3-methylbutanol (15) by reduction with lithium
borohydride after in situ formation of an anhydride with
ethyl chloroformate. Alcohol 15 was oxidized under
Swern conditions to the aldehyde 16 which underwent
smooth two-carbon elongation by a Horner-Emmons
reaction with triethyl phosphonoacetate in THF. Fi-
although peptide drug absorption across membranes
still is a relatively poorly understood process. According
to this strategy, we consistently employed N-methylated
amino acids in order to reduce desolvation energy and

Novel Orally Active GH Secretagogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3707
Sch em e 2a
a
(a) HOBt, EDAC, R3NH2; (b) TFA; (c) AA, HOAt, EDAC.
Ta ble 1. Prepared Compounds
compd
R1
R2
R3
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
benzyloxy
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
4-fluorophenyl
phenyl
phenyl
2-thienyl
phenyl
4-iodophenyl
3,4-difluorophenyl
phenyl
methyl
(2S)-2-hydroxypropyl
(2-tetrahydrofuranyl)methyl
cyclopropylmethyl
methyl
methyl
methyl
1-naphthyl
2-naphthyl
3-benzothienyl
1-naphthyl
2-naphthyl
4-biphenylyl
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
4-biphenylyl
4-biphenylyl
2-naphthyl
methyl
(2-tetrahydrofuranyl)methyl
methyl
methyl
methyl
methyl
ethyl
phenyl
2-thienyl
4-fluorophenyl
2-thienyl
3,4-difluorophenyl
(2R)-2-hydroxypropyl
(2S)-2-hydroxypropyl
(2R)-2-hydroxypropyl
(2S)-2-hydroxypropyl
(2S)-2-hydroxypropyl
7s
nally saponification with lithium hydroxide gave 18 in
good overall yield.
administration, appropriately corrected for dose. The
second test was an in vivo test for GH release in swine
after iv dosing. A single dose per compound (50 nmol/
kg of body weight (BW)) was given iv. We favor a swine
model for GH release because this species has better
resemblance to humans with respect to physiology in
general and endocrinology in particular, than other
common laboratory animals such as rat and dog. On
the basis of our previous experience, measurements of
pGH release following a 50 nmol/kg BW iv dose allowed
us to make a crude selection of compounds for further
evaluation. Results from this test are tabulated as Cmax
(peak pGH levels at the given dose). Compounds which
simultaneously showed a relatively high fpo and Cmax
at the given dose were selected for further investigation.
Biology
For our screening strategy, the classical rat pituitary
cell assay was chosen as the primary assay in order to
narrow the focus to compounds with superior GH-
releasing properties. The more potent compounds within
a series were chosen for further evaluation. The next
step was divided into two parts. The first was a test
for oral bioavailability. Beagle dogs were chosen for this
purpose, due to their convenient temperament during
administration. The GH release was also measured, but
this was not the primary objective of the test. The oral
bioavailability was calculated as the total area under
the plasma concentration versus time curve following
po. administration divided by the area following iv
The screening data are collected in Table 2. All
compounds were active in the rat pituitary assay with

3708 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hansen et al.
Ta ble 3. Oral Bioavailability and GH Release in Beagle Dogsa
Sch em e 3a
dose
Cmax
fpo
Cmax for GH
(mg/kg BW)
(nmol/L)
(%)
(ng/mL)
7b
7g
0.5
2.5
10
nd
nd
21 ( 4
41 ( 16
1.8 ( 1.1
6.6 ( 5.1
42.5 ( 9.9*’**
260 ( 61
2318 ( 1266
0.5
2.5
10
99 ( 32
53 ( 23
55 ( 41
138 ( 64
2.7 ( 3.4
4.5 ( 3.3
12.0 ( 8.4
247 ( 106
3031 ( 917*,**
a
Results are shown as mean ( SEM (n ) 4). nd, not detectable;
*p < 0.05 vs 0.5 mg/kg BW; **p < 0.05 vs 2.5 mg/kg BW.
Ta ble 4. Potency and Efficacy of Different GH Secretagogues
in Swine after iv Administrationa
potency ED50
(nmol/kg BW)
Emax
(ng of pGH/mL)
compd
GHRP-6
ipamorelin
7b
3.9 ( 1.4
2.3 ( 0.03
31 ( 8*,**
129 ( 42*,**
74 ( 7
65 ( 0.2
96 ( 5*,**
86 ( 7**
7g
a
The values were calculated from dose-response curves based
on mean plasma GH concentrations obtained after iv administra-
tion of test compounds in a dose range of 10-10000 nmol/kg. Each
value is given as mean ( SEM, (n ) 6). *p < 0.05 vs GHRP-6; **p
< 0.05 vs ipamorelin.
a
(i) LiBH4; (ii) oxalyl chloride DMSO; (iii) Homer-Emmons;
(iv) LiOH.
The two compounds chosen for further biological study
were evaluated in a pharmacokinetic and pharmacody-
namic model using three ascending oral doses (0.5, 2.5,
and 10 mg/kg BW) and a single intravenous dose (0.5
mg/kg BW) in fasted beagle dogs (n ) 4). Table 3
summarizes the phamacokinetic parameters that were
calculated from these experiments. In particular both
compounds showed dose-dependent oral bioavailability.
Compound 7b showed an increase in fpo from 21% to
41% with increasing doses (the 0.5 mg/kg BW dose could
not be detected), whereas fpo for compound 7g increased
from 55% to 138%, eventually attributed to enterohe-
patic circulation or a saturation phenomenon of elimi-
nation pathways. Plasma elimination half-lives were
1.3 h for 7b and 1.1 h for 7g.
Ta ble 2. Results of in Vitro and in Vivo Screening of GH
Secretagoguesa
rat pit assay
swine,
fpo, oral
EC50
Emax (%
GH Cmax
bioavail,
compd
(nM) of GHRP-6) (ng of pGH/mL) beagle dog (%)
GHRP-6
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
1.7
18
8.9
16
1.8
5.0
24
8.0
35
21
2.0
3.0
20
100
110
130
100
105
125
115
90
26
9
14
25
17
24
11
0.4
12
30*
49
80
110
110
120
110
85
105
105
120
90
55**
15
0
An investigation of the dose dependency of GH release
for 7b,g in swine after iv dosing is summarized in Table
4 and compared to the values found for ipamorelin and
GHRP-6.
0.4
6
26
54
14
0
17
13
9.0
45
6.0
9
We also wanted to investigate the molecular site of
action of 7b,g and to compare it with that of GHRP-6.
For this purpose the effect of GHS and GHRH antago-
nists were tested, and the data are summarized in Table
5. We used the peptide (GHS) antagonist15 His-D-Trp-
D-Lys-Trp-D-Phe-Lys-NH2, the nonpeptide (GHS) an-
tagonist16 L-692,400, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-
substance P.17,18 Thus, GH release induced by all three
compounds was inhibited by GHS antagonists but not
by a GHRH receptor antagonist. For a comparison, the
GHRH(1-29)NH2-induced GH release was potently
inhibited by the GHRH antagonist19 [N-acetyl-Tyr1,D-
Arg2]-hGHRH(1-29), whereas the GHS antagonists had
no effect. The data clearly suggest a similar site of
action of 7b,g and GHRP-6.
24
56
5
10
0
1.5
5.0
110
100
7s
a
Dose-response curves of GH release from rat pituitary cells
in culture were constructed as described in the Experimental
Section, and the potency and efficacy were calculated from these.
Results are showed as means. To obtain GH Cmax values four swine
were dosed with 50 nmol/kg BW of the test compounds; results
shown as mean. To obtain the fpo values four dogs were dosed
orally with 2.5 mg/kg BW of the test compounds and iv with 0.5
mg/kg BW; results shown as mean. *Exatrapolated from a Hill
plot using 5, 25, 270 nmol/kg BW. **Exatrapolated from a Hill
plot using 2, 240, 560 nmol/kg BW.
a factor of 30 between the most and least potent
compound. We chose the most potent of the compounds
and tested them for oral bioavailability and GH release.
We found two compounds, 7b,g, which at the same time
were systemically available following oral administra-
tion and were among the more potent with respect to
GH release.
Discu ssion
The antagonist data summarized in Table 5 indicate
that 7b,g and GHRP-6 mediate their effects through the
same receptor, which is distinct from the GHRH recep-

Novel Orally Active GH Secretagogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3709
Ta ble 5. Effect of Different GHS and GHRH Antagonists on the Stimulatory Effect of 7b (50 nM), 7g (50 nM), and GHRP-6 (10 nM)
on the GH Release from Rat Pituitary Cells in Vitro
IC50 (µM)
D-Lys3-GHRP-6
L-692,400
D-Arg1D-Phe5D-Trp7,9,Leu11 (21),
N-acetyl-Tyr1-D-Arg2(1-29)GHRH-NH2
(22)
compd
7b
7g
GHRP-6
(19)
(20)
substance P antagonist
8 ( 4
9 ( 4
8 ( 2
11 ( 3
14 ( 5
10 ( 3
0.14 ( 0.08
0.049 ( 0.02
0.038 ( 0.015
>10
>10
>10
a
Results are shown as mean ( SEM (n ) 4). No significant differences were observed (p > 0.05) between the compounds.
tor. Clearly, we are dealing with true peptide mimetics
of Momany and Bowers’ original peptides.
mum of 4.15 for MlogP and maximum of 5.0 for ClogP.
In our case, none of the “rules” could differentiate
compounds. Poor absorption is predicted if the molec-
ular weight is higher than 500. However, all our
molecules are above 500 with the heaviest molecule
being 7l (MW: 656), and this compound has an oral
bioavailability of 26%. The calculated log P values
varied between 2.72 for 7e and 4.60 for 7l using ClogP
calculations and between 1.64 for 7e and 3.37 for 7d
using MlogP. This is well within the limits of the “rules
of five”. Furthermore, all compounds had good solubility
in water, leading us to believe that the oral bioavail-
ability in this series is determined by very subtle
interactions during passage of the intestine wall as well
as quite unpredictable levels of first-pass elimination.
The two compounds that were chosen for further
investigation were each representatives for two sub-
classes within our series: 7g for those that had simple
aliphatic C-termini and 7b for those with a 2-hydrox-
ypropyl group in the C-terminal. There was a tendency
for the former ones to have higher oral bioavailability
on the average and for the latter ones to be more active
in the in vivo model. The unpredictability of the oral
bioavailability and the importance of the C-terminal
domain were highlighted by the difference in bioavail-
ability between the S-isomer 7b (25%) and the corre-
sponding R-isomer 7o (0%).
With regard to in vivo potency we observed high
potency in those cases where R2 (Table 2) was either
thienyl or 4-fluorophenyl instead of phenyl, leading us
to believe that these side chains produced a better fit
to the receptor. Also the use of 4-biphenylyl as R1
instead of 2-naphthyl was an acceptable substitution in
contrast to 1-naphthyl or 3-benzothienyl. However,
substitutions that had proven beneficial on the potency
parameter were not always additive, and their influence
on the oral bioavailability was unpredictable. One such
example was compound 7r which contained a substitu-
tion pattern that we prior to synthesis predicted optimal
for high in vivo potency based on previous SAR. Indeed,
this compound was the most potent we observed in this
series (Cmax ) 56 ng/mL) but turned out to have no oral
bioavailability at all.
The series of compounds presented above were all
prepared by classical peptide methods and were readily
applicable for scaleup. In the C-terminal domain we
used D-Phe (or isosters) coupled to commercially avail-
able amines. Simple amines caused no problems during
peptide couplings. We encountered less clean reactions
and slightly lower yields when the amine was 1-amino-
2-propanol, probably due to esterification of the hydroxy
group as a side reaction during acylation of the hindered
amines. (S)-1-Amino-2-propanol was introduced as a
C-terminal because there were reports in the literature20
indicating that the N- and C-termini might be situated
closely together in the active conformation and that
alkylation of the N-terminal with 2-hydroxypropyl was
advantageous.21 The employment of 5-amino-5-meth-
ylhex-2-enoic acid as a general N-terminal highlights
the use of this building block as a potentially useful
dipeptide isoster that seems particularly advantageous22
as a substitute for Aib-containing peptides. Compared
to 3-(aminomethyl)benzoic acid, it is clear that 5-amino-
5-methylhex-2-enoic acid resembles a peptide backbone
more closely in terms of flexibility and steric bulk. Our
choice to make consistent use of 5-amino-5-methylhex-
2-enoic acid as an N-terminal residue, D-2-Nal (or
isosters) as the core, and D-Phe (or isosters) as the
C-terminal inherently allows for relatively limited
structural variation, and focus of this study was indeed
on the importance of the C-terminal domain. This is
reflected in the fact that all compounds are active in
the lower nanomolar range in the rat pituitary assay.
Our experience is that the rat pituitary assay is useful
as a primary, crude test to select compounds with good
GH-releasing properties and reasonably predictive for
in vivo potency. We prefer that compounds show
potency in the range below 20 nM before we initiate
further investigation. Due to limited structural varia-
tions, relatively few compounds were rejected on the
basis of the rat pituitary assay.
The oral bioavailability in the dog turned out to be a
most difficult parameter to rationalize. Despite the
similarity within the series, oral bioavailability varied
considerably. Within the series presented we were
unable to correlate oral bioavailability to any physico-
chemical or structural parameter. All compounds have
approximately the same molecular weight and closely
related structure. Scientists from Pfizer23 have pre-
sented a “rule of five” for prediction of oral absorption.
According to their experience compounds have a poor
chance of being orally bioavailable if the molecular
weight exceeds 500 and if there are more than 5 H-bond
donors or more than 10 H-bond acceptors. Furthermore,
calculated log P values should not exceed certain values
that are dependent on the method of calculation: maxi-
From a comparison of 7b,g with the original leads
GHRP-6 and ipamorelin (Table 4), it is clear that
potency has been lost with regard to the GH-releasing
effect. GHRP-6 is approximately 8 times more potent
than 7b. However, the much reduced size of our
compounds and the fact that the compounds are orally
bioavailable are improvements that we believe will
make up for the loss in potency.
In conclusion, we have identified a novel class of GH
secretagogues for which oral bioavailability is the rule
rather than the exception. This class of compounds has
been developed directly from hexapeptide leads via

3710 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hansen et al.
218TP54 4.6-mm × 250-mm 5-µm C-18 silica column (The
Separations Group, Hesperia), which was eluted at 1 mL/min
at 42 °C. Two different elution conditions were used. Method
A1: The column was equilibrated with 5% acetonitrile in a
buffer consisting of 0.1 M ammonium sulfate, which was
adjusted to pH 2.5 with 4 M sulfuric acid. After injection the
sample was eluted by a gradient of 5-60% acetonitrile in the
same buffer during 50 min. Method B1: The column was
equilibrated with 5% acetonitrile/0.1% TFA/water and eluted
by a gradient of 5% acetonitrile/0.1% TFA/water to 60%
acetonitrile/0.1% TFA/water during 50 min.
Syn t h esis: 3-H yd r oxy-1,1-d im et h ylp r op ylca r b a m ic
Acid ter t-Bu tyl Ester (15). At 0 °C, ethyl chloroformate (1.10
mL, 11.5 mmol) was added dropwise to a solution of 3-(tert-
butoxycarbonylamino)-3-methylbutanoic acid6 (14) (2.50 g, 11.5
mmol) and triethylamine (1.92 mL, 13.8 mmol) in tetrahydro-
furan (10 mL). The solution was stirred for 40 min at 0 °C.
The formed precipitate was filtered off and washed with
tetrahydrofuran (20 mL). The liquid was immediately cooled
to 0 °C. A 2 M solution of lithium borohydride in tetrahydro-
furan (14.4 mL, 28.8 mmol) was added dropwise. The solution
was stirred at 0 °C for 2 h and then warmed to room
temperature over a period of 4 h. The solution was again
cooled to 0 °C and methanol (5 mL) was added dropwise; 1 N
hydrochloric acid (100 mL) was added. The solution was
extracted with ethyl acetate (2 × 100, 3 × 50 mL). The
combined organic layers were washed with saturated sodium
hydrogen carbonate solution (100 mL) and dried over magne-
sium sulfate. The solvent was removed in vacuo. The crude
product was chromatographed on silica (110 g) with ethyl
acetate/heptane (1:2) as eluent to give 1.84 g (79%) of 15. 1H
NMR (CDCl3): δ 1.33 (s, 6H); 1.44 (s, 9H); 1.88 (t, 2H); 1.94
(br, 1H); 3.75 (q, 2H); 4.98 (br, 1H).
3-(ter t-Bu toxyca r bon yla m in o)-3-m eth ylbu ta n a l (16).
DMSO (1.22 mL, 17.2 mmol) was added to a solution of oxalyl
chloride (1.1 mL, 12.9 mmol) at -78 °C in dichloromethane
(15 mL). The mixture was stirred for 15 min at -78 °C. A
solution of 15 (1.75 g, 8.6 mmol) in dichloromethane (10 mL)
was added dropwise over a period of 15 min. The solution was
stirred at -78 °C for another 15 min. Triethylamine (6.0 mL,
43 mmol) was added. The solution was stirred at -78 °C for
5 min and then warmed to room temperature. The solution
was diluted with dichloromethane (100 mL) and extracted with
1 N hydrochloric acid (100 mL). The aqueous phase was
extracted with dichloromethane (50 mL). The combined
organic layers were washed with saturated sodium hydrogen
carbonate solution (100 mL) and dried over magnesium sulfate.
The solvent was removed in vacuo. The crude product was
purified by column chromatography on silica (140 g) with ethyl
acetate/heptane (1:3) to give 1.10 g (63%) of 16. 1H NMR
(CDCl3): δ 1.39 (s, 6H); 1.45 (s, 9H); 2.85 (d, 2H); 4.73 (br,
1H); 9.80 (t, 1H).
Eth yl (2E)-5-(ter t-Bu toxyca r bon yla m in o)-5-m eth ylh ex-
2-en oa te (17). Triethyl phosphonoacetate (1.96 mL, 9.8
mmol) was dissolved in tetrahydrofuran (30 mL). Potassium
tert-butoxide (1.10 g, 9.8 mmol) was added. The solution was
stirred for 40 min at room temperature. A solution of 16 (1.10
g, 5.5 mmol) in tetrahydrofuran (6 mL) was added. The
solution was stirred at room temperature for 75 min. It was
diluted with ethyl acetate (100 mL) and 1 N hydrochloric acid
(100 mL). The phases were separated. The aqueous phase
was extracted with ethyl acetate (2 × 50 mL). The combined
organic phases were washed with saturated sodium hydrogen
carbonate solution (60 mL) and dried over magnesium sulfate.
The solvent was removed in vacuo. The crude product was
purified by column chromatography on silica (90 g) with ethyl
acetate/hepatane (1:4) to give 1.27 g (79%) of 17. 1H NMR
(CDCl3): δ 1.30 (s, 6H); 1.30 (t, 3H); 1.46 (s, 9H); 2.62 (d, 2H);
4.27 (q, 2H); 4.42 (br, 1H); 5.88 (d, 1H); 6.94 (td, 1H).
F igu r e 1. Overlap of 6, 7b, and MK-677.
ipamorelin. The synthetic strategy has concentrated on
replacement of backbone amide bonds with amide
isosters with less hydrogen-bonding capacity. The final
outcome was compounds with one amide substituted to
a trans double bond and two amides substituted to
N-methylated amides. Also a reduction in size has been
achieved. Throughout, the optimization process has
been stepwise and guided by rational considerations.
Nevertheless, the resulting tripeptides bear structural
resemblance with MK-677. In both cases a shielded
amine and two aromatic fragments are present24 and
found to be important. To substantiate the similarity
of our molecules to MK-677, we have perfomed a
molecular modeling study on the overlap of 6, 7b, and
MK-677 based on the assumption that these molecules
bind to the same receptor. A pharmacophore model was
derived from Catalyst25 in HipHop mode, and refined
overlays of the three molecules were generated by
distance geometry calculations (DGEOM9526) based on
the Catalyst model and were followed by energy opti-
mizations by a SYBYL multifit27 approach. The result
is shown in Figure 1 and indicates that the molecules
readily overlap each other despite their differences. The
phenyl ring of the benzylserine moiety of MK-677 could
be overlapped with the outer ring in the naphthylala-
nines in 6 and 7b, while the phenyl rings in the
phenylalanines overlapped the phenyl ring in the spiropi-
peridine. Also the N-terminal amines overlapped readily
even with substantial difference in length and rigidity.
The relatively low potency of 6 compared to MK-677
may be due to a less optimal or missing pharmacophore
in the C-terminal. It is also noteworthy that the
hydroxy group in 7b is capable of reaching the same
place in space as the sulfonamide of MK-677, perhaps
serving as a mimic of this pharmacophore.
As a result of this investigation we have shown that
a direct stepwise method for size reduction of heptapep-
tides is viable and can result in orally active tripeptides.
Although the resulting substances are somewhat less
potent than the lead ipamorelin, we believe that the
potency is in a range that is satisfactory and allows for
further examination and development.
Exp er im en ta l Section
Amino acids were purchased from Synthetec. HRMS was
performed at Odense University using a Varian M311 ap-
paratus. PDMS spectra were run on a Bioion 1100 instrument
(Upsala, Sweeden). NMR spectra were obtained at 400 MHz
on a Bruker instrument.
(2E)-5-(ter t-Bu toxyca r bon yla m in o)-5-m eth ylh ex-2-en o-
ic Acid (18). Compound 17 (1.233 g, 4.54 mmol) was dissolved
in dioxane (20 mL). Lithium hydroxide (0.120 g, 5.00 mmol)
was added as a solid. Water (10 mL) was added, until a clear
solution was obtained. The solution was stirred for 16 h at
HP LC Meth od s. The RP-HPLC analysis was performed
using UV detection at 214, 254, 276, and 301 nm on a Vydac

Novel Orally Active GH Secretagogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3711
room temperature. The solution was diluted with water (70
mL) and extracted with tert-butyl methyl ether (2 × 100 mL).
The aqueous phase was acidified with 1 N sodium hydrogen
sulfate solution (pH ) 1) and was extracted with tert-
butylmethyl ether (3 × 70 mL). The organic phases were
combined and dried over magnesium sulfate. The solvent was
removed in vacuo to give 1.05 g (95%) of 18. The crude product
was used for further syntheses. An analytical sample was
obtained by recrystallization from heptane. 1H NMR (DMSO-
d6): δ 1.15 (s, 6H); 1.35 (s, 9H); 2.53 (d, 2H); 5.75 (d, 1H); 6.57
(br, 1H); 6.75 (td, 1H); 12.15 (s, 1H). Anal. (C12H21NO4) C,
H, N.
P r oced u r e for Com p ou n d 7a (Com p ou n d s 7b-s w er e
p r ep a r ed in th e sa m e w a y): N-Meth yl-N-((R)-1-(m eth -
ylca r ba m oyl)-2-p h en yleth yl)ca r ba m ic Acid ter t-Bu tyl
Ester (9a ). N-(tert-Butoxycarbonyl)-N-methyl-D-phenylala-
nine (8) (1.22 g, 4.4 mmol), 1-hydroxybenzotriazole hydrate
(0.59 g, 4.4 mmol), and EDAC (1-ethyl-3-(3-(dimethylamino)-
propyl)carbodiimid hydrochloride) (0.88 g, 4.6 mmol) were
dissolved in methylene chloride (25 mL) and stirred for 15 min.
Methylamine (0.51 g of a 40% solution in methanol, 6.6 mmol)
was added, and the mixture was stirred overnight. Methylene
chloride (80 mL) and water (100 mL) were added, and the
phases were separated. The organic phase was washed with
aqueous sodium hydrogen carbonate (20 mL, saturated),
aqueous sodium hydrogen sulfate (50 mL, 10%), and water (50
mL). The organic phase was dried (magnesium sulfate), and
the solvent was removed in vacuo to afford 1.27 g (99%) of 9a
as a colorless oil. 1H NMR (CDCl3): δ 1.25, 1.35 (two s, 9H);
2.73-2.94 (m, 7H); 3.30-3.50 (m, 1H); 4.68, 4.90 (two m, 1H);
5.90, 6.12 (two s (br); 1H); 7.12-7.25 (m, 5H) (mixture of
rotamers).
(R )-N -Me t h y l-2-(m e t h y la m in o )-3-p h e n y lp r o p io n -
a m id e (10a ). Compound 9a (1.39 g, 7.23 mmol) was dissolved
in a mixture of trifluoroacetic acid (10 mL) and methylene
chloride (10 mL) and stirred for 1 h. The volatiles were
removed in vacuo, and the residue was stirred with aqueous
sodium hydrogen carbonate (100 mL, saturated, pH adjusted
to 9.5 with sodium carbonate) and extracted with methylene
chloride (3 × 100 mL). The organic phase was dried (magne-
sium sulfate), and the solvent was removed in vacuo to afford
810 mg (89%) of 10a as a white solid. 1H NMR (CDCl3): δ 2.1
(s (br), 3H); 2.32 (s, 3H); 2.77 (dd, 1H); 2.81 (s, 3H); 2.82 (s,
3H); 3.21 (dd, 1H); 3.32 (dd, 1H); 7.12 (s (br), 1H); 7.20-7.34
(m, 5H).
N-Met h yl-N-((1R)-1-(N-m et h yl-N-((1R)-1-(m et h ylca r -
ba m oyl)-2-p h en yleth yl)ca r ba m oyl)-2-(2-n a p h th yl)eth yl)-
ca r ba m ic Acid ter t-Bu tyl Ester (11a ). (R)-(tert-Butoxycar-
bonyl)-N-methylamino-3-(2-naphthyl)propionic acid (548 mg,
1.66 mmol) was dissolved in methylene chloride (5 mL), and
1-hydroxy-7-azabenzotriazole (HOAt) (227 mg, 1.66 mmol,
dissolved in DMF (2 mL)) was added. 1-Ethyl-3-(3-(dimethy-
lamino)propyl)carbodiimide hydrochloride (351 mg, 1.83 mmol)
was added, and the solution was stirred for 15 min. Compound
10a (320 mg, 1.66 mmol) (dissolved in methylene chloride (4
mL)) and diisopropylethylamine (0.28 mL, 1.66 mmol) were
added, and the mixture was stirred overnight. Methylene
chloride (50 mL) was added, and the organic phase was washed
with water (100 mL), sodium hydrogen sulfate (50 mL, 5%),
and sodium hydrogen carbonate (50 mL, saturated). The
organic phase was dried (magnesium sulfate), and the solvent
was removed in vacuo. The residue was chromatographed
(silica, 2 × 45 cm) using ethyl acetate/methylene chloride (1:
1) as eluent to afford 604 mg (72%) of 11a . 1H NMR (CDCl3):
δ 1.05, 1.31, 1.56 (three s, 9H); 2.28-3.37 (m, 13H); 5.04, 5.17,
5.29, 5.48 (four dd, 2H); 7.05-7.79 (m, 12H); (mixture of
rotamers).
organic phase was separated and dried (magnesium sulfate)
to afford 420 mg (87%) of 12a . 1H NMR (CDCl3): δ 1.69 (s,
3H); 2.08 (d, 3H); 2.54 (s, 3H); 2.76 (dd, 1H); 2.92 (dd, 1H),
3.12 (dd, 1H), 3.31 (dd, 1H); 3.72 (dd, 1H), 4.95 (q (br), 1H);
5.50 (dd, 1H) (selected values, mixture of rotamers).
(3E)-1,1-Dim eth yl-4-(N-m eth yl-N-((1R)-1-(N-m eth yl-N-
((1R)-1-(m eth ylca r ba m oyl)-2-p h en yleth yl)ca r ba m oyl)-2-
(2-n a p h t h yl)et h yl)ca r ba m oyl)bu t -3-en ylca r ba m ic Acid
ter t-Bu tyl Ester (13a ). Compound 18 (200 mg, 0.82 mmol),
1-hydroxy-7-azabenzotriazole (112 mg, 0.82 mmol), and 1-ethyl-
3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (173
mg, 0.90 mmol) were dissolved in a mixture of methylene
chloride (10 mL) and DMF (1 mL) and stirred for 15 min.
Compound 12a (332 mg, 0.82 mmol) dissolved in methylene
chloride (5 mL) and diisopropylethylamine (0.14 mL) were
added, and the mixture was stirred overnight under a nitrogen
atmosphere. The mixture was diluted with methylene chloride
(50 mL) and washed with water (50 mL), sodium hydrogen
carbonate (30 mL, saturated), and sodium hydrogen sulfate
(30 mL, 5%). The phases were separated, and the organic
phase was dried with magnesium sulfate and evaporated in
vacuo. The residue was chromatographed (silica, 2 × 40 cm)
to afford 450 mg (88%) of 13a . 1H NMR (CDCl3): (selected
values) δ 1.20, 1.22, 1.24, 1.30, 1.41, 1.55 (six s, 15H); 4.30,
4.40 (two s (br), 1H); 5.08, 5.18, 5.32, 5.60, 5.87 (five dd, 2H);
6.05 (dd, 1H); 6.75 (m, 1H) (mixture of rotamers).
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-Meth yl-N-
((1R )-1-(N -m e t h y l-N -((1R )-1-(m e t h y lc a r b a m o y l)-2-
p h e n y le t h y l)c a r b a m o y l)-2-(2-n a p h t h y l)e t h y l)a m id e
(7a ). Compound 13a (403 mg, 0.63 mmol) was stirred in a
mixture of trifluoroacetic acid (4 mL) and methylene chloride
(4 mL) for 7 min. The mixture was poured onto a stirred
mixture of ice (10 g), sodium hydrogen carbonate (6 g), and
water (10 mL). Methylene chloride (50 mL) was added, and
the organic phase was dried and evaporated. The crude
product was chromatograped on silica (200 g) using a mixture
of methylene chloride, absolute ethanol, and ammonia (25%
in water) (80/18/2) to afford 140 mg (41%) of 7a as a white
solidified foam. 1H NMR (CDCl3): δ 1.05, 1.10, 1.15, 1.16 (four
s, 6H); 2.07 (s (br), 3H); 5.12, 5.32, 5.40, 5.60, 5.91 (five dd,
2H); 6.05, 6.14 (two d, 1H); 6.80 (m, 1H) (mixture of rotamers,
selected peaks). 1H NMR (DMSO at 160 °C): δ 1.08 (s, 6H);
2.17 (d, 2H); 2.59 (d, 3H); 2.76 (s, 3H); 2.87 (s, 3H); 2.92 (dd,
1H); 3.02 (dd, 1H); 3.21 (dd, 1H); 3.25 (dd, 1H); 5.11 (dd, 1H);
5.59 (dd, 1H); 6.10 (d, 1H); (dt, 1H); 7.13 (m, 1H), 7.18 (m,
2H); 7.19 (m, 2H); 7.19 (m, 2H); 7.31 (ab-pattern, 1H); 7.42
(m, 1H); 7.44 (m, 1H); (s, 1H); 7.74 (ab-pattern, 1H); 7.77 (m,
1H); 7.81 (m, 1H) (full spectrum, no rotamers). HPLC: tR
)
29.02 min (99% pure) (method A1). HPLC: tR ) 33.45 min
(method B1). ESMS: m/z ) 529 (100%) (M + H)+. EIMS: m/z
) 528 (5%, M+), 470 (9), 279 (30), 184 (79), 58 (100). HRMS:
calculated for C32H40N4O3, m/z ) 528.3100; found, m/z )
528.3070.
The compounds listed below were all prepared by the
general procedure above. The purity (determined by HPLC)
was generally in the 95-100% range. In general compounds
were colorless and noncrystalline.
(2E)-5-Amino-5-methylhex-2-enoic Acid N-((1R)-1-(((1R)-1-
((2S)-2-Hyd r oxyp r op ylca r ba m oyl)-2-p h en yleth yl)m eth -
ylca r ba m oyl)-2-(2-n a p h th yl)eth yl)-N-m eth yla m id e (7b).
Prepared according to the general procedure without protection
of the hydroxyl group. 1H NMR (CDCl3) (selected peaks,
mixture of rotamers): δ 3.90 (m, 1H); 5.55 (dd, 1H); 5.58 (d,
1H). HPLC: tR ) 29.03 min (method A1). HPLC: tR ) 32.60
min (method B1). PDMS: m/z ) 573.5 (100%) (M + H)+.
EIMS: m/z ) 572 (3%, M+), 514 (8), 279 (38), 184 (95), 58 (100).
HRMS: calculated for C34H44N4O5, m/z ) 572.3363; found, m/z
) 572.3382.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-m eth yl-N-
((1R)-1-(m eth yl((1R)-2-p h en yl-1-(((tetr a h yd r ofu r a n -2-yl)-
m eth yl)ca r ba m oyl)-2-p h en yleth yl)ca r ba m oyl)-2-(2-n a p h -
th yl)eth yla m id e (7c). 1H NMR (CDCl3) (selected peaks,
mixture of rotamers): δ 1.06, 1.09 (two d, 6H); 2.78 (d, 2H);
5.25-5.62 (m, 2H); 6.05 (m, 1H). HPLC: tR ) 33.65 min
(2R )-N -Me t h yl-2-(m e t h yla m in o)-N -((1R )-1-(m e t h yl-
c a r b a m o y l)-2-p h e n y le t h y l)-3-(2-n a p h t h y l)p r o p io n -
a m id e (12a ). Compound 11a (600 mg, 1.19 mmol) was stirred
in trifluoroacetic acid/methylene chloride (1:1, 5 mL) for 10
min, and the volatiles were removed in vacuo. The residue
was dissolved in methanol (2 mL) and mixed with sodium
hydrogen carbonate (10 mL) and ethyl acetate (15 mL). The

3712 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hansen et al.
(method A1). HPLC: tR ) 36.02 min (method B1). EIMS: m/z
) 598 (2%, M+), 540 (5), 439 (11), 184 (100), 58 (78). HRMS:
calculated for C36H46N4O4, m/z ) 598.3519; found, m/z )
598.3552.
(CDCl3) (selected peaks for major rotamer): δ 1.18 (s, 6H); 2.75
(d, 3H); 2.78 (s, 3H); 2.97 (s, 3H); 5.45 (dd, 1H); 5.75 (dd, 1H);
6.08 (d, J ) 17 Hz, 1H). ESMS: m/z 555.8 (M + H)+. HPLC:
tR ) 34.45 min (method A1). HPLC: tR ) 36.55 min (method
B1). HRMS: calculated for C34H42N4O3, m/z ) 554.3257;
found, m/z ) 554.3275.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-((1R)-1-(N-
((1R )-2-(4-Iod op h e n yl)-1-(m e t h ylca r b a m oyl)e t h yl)-N -
m e t h y lc a r b a m o y l)-2-(2-n a p h t h y l)e t h y l)-N -m e t h y l-
a m id e (7l). 1H NMR (CDCl3) (selected peaks for major
rotamer): δ 1.15 (s, 6H); 2.09 (d, 3H); 2.69 (s, 3H); 2.70 (s,
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-((1R)-1-(((1R)-
1-((Cyclop r op ylm eth yl)ca r ba m oyl)-2-p h en yleth yl)m eth -
ylca r ba m oyl)-2-(2-n a p h th yl)eth yl)-N-m eth yla m id e (7d ).
1H NMR (CDCl3) (selected peaks, mixture of rotamers):
δ
0.08-0.20 (m, 2H); 1.05; 1.15 (two s, 6H); 6.02, 6.05 (two d,
1H). HPLC: tR ) 35.7 min (method A1). HPLC: tR ) 37.28
min (method B1). EIMS: m/z ) 568 (8%, M+), 510 (10), 439
(12), 279 (51), 184 (90), 58 (100). HRMS: calculated for
3H); 5.24 (dd, 1H); 5.90 (dd, 1H); 6.18 (d, 1H). HPLC: tR
)
C
35H44N4O3, m/z ) 568.3413; found, m/z ) 568.3442.
35.25 min (method A1). HPLC: tR ) 37.55 min (method B1).
PDMS: m/z ) 655.7 (M + H)+. HRMS: calculated for
(2E)-5-Am in o-5-m et h ylh ex-2-en oic Acid N-((1R)-2-
C
32H39N4O3I, m/z ) 654.2067; found, m/z ) 654.2101.
ben zyloxy)-1-(N-m eth yl-N-((1R)-1-(m eth ylca r ba m oyl)-2-
p h en yleth yl)ca r ba m oyl)eth yl)-N-m eth yla m id e (7e). 1H
NMR (CDCl3) (selected data for major rotamer): δ 1.27 (s, 3H);
1.28 (s, 3H); 2.84 (d, 3H); 2.95 (s, 3H); 3.08 (s, 3H); 4.32 (d,
1H); 4.40 (d, 1H); 5.12 (dd, 1H); 6.34 (d, J ) 18 Hz, 1H).
ESMS: m/z 509.7 (M + H)+. HPLC: tR ) 23.45 min (method
A1). HPLC: tR ) 31.71 min (method B1). EIMS: m/z ) 508
(1%, M+), 260 (64), 259 (55), 164 (43), 58 (100). HRMS:
calculated for C29H40N4O4, m/z ) 508.3050; found, m/z )
508.3055.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-Meth yl-N-
((1R )-1-(N -m e t h y l-N -((1R )-1-(m e t h y lc a r b a m o y l)-2-
p h en yleth yl)ca r ba m oyl)-2-(1-n a p h th yl)eth yl)a m id e (7f).
1H NMR (CDCl3) (selected peaks for major rotamer): δ 1.22
(s, 3H); 1.23 (s, 3H); 2.82 (d, 3H); 2.92 (s, 3H); 3.08 (s, 3H);
5.22 (dd, 1H); 5.92 (dd, 1H); 6.12 (d, 1H). ESMS: m/z 529.2
(M + H)+. HPLC: tR ) 30.90 min (method A1). HRMS:
calculated for C32H40N4O3, m/z ) 528.3100; found, m/z )
528.3101.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid ((1R)-1-(((1R)-
2-(4-F lu or op h en yl)-1-(m et h ylca r b a m oyl)et h yl)m et h yl-
ca r ba m oyl)-2-(2-n a p h th yl)eth yl)m eth yla m id e (7g). 1H
NMR (CDCl3) (selected peaks for major rotamer): δ 1.15 (s,
6H); 2.14 (d, 3H); 2.73 (s, 3H); 3.09 (s, 3H); 5.23 (dd, 1H); 5.90
(dd, 1H); 6.12 (dd, 1H). PDMS: m/z 547.4 (M + H)+. HPLC:
tR ) 32.05 min (method A1). HPLC: tR ) 34.18 min (method
B1). EIMS: m/z ) 546 (10%, M+), 488 (19), 279 (48), 184 (81),
58 (100). HRMS: calculated for C32H39N4FO3, m/z ) 546.3006;
found, m/z ) 546.3026.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid ((1R)-2-(Ben -
zo[b]th ioph en e-3-yl)-1-(m eth yl((1R)-1-(m eth ylcar bam oyl)-
2-p h en yleth yl)ca r ba m oyl)eth yl)m eth yla m id e (7h ). 1H
NMR (CDCl3) (selected peaks for major rotamer): δ 1.25 (s,
6H); 2.47 (s, 3H); 2.75 (d, 3H); 2.96 (s, 3H); 4.98 (dd, 1H); 5.89
(dd, 1H); 6.10 (d, 1H). PDMS: m/z 535.7 (M + H)+. HPLC: tR
) 30.87 min (method A1). HPLC: tR ) 33.03 min (method
B1). EIMS: m/z ) 534 (11%, M+), 476 (17), 320 (32), 231 (40),
190 (36), 187 (42), 58 (100). HRMS: calculated for C30H38N4O3S,
m/z ) 534.2665; found, m/z ) 534.2619.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid Meth yl((1R)-
1-(m e t h y l((1R )-2-p h e n y l-1-(((t e t r a h y d r o fu r a n -2-y l)-
m eth yl)car bam oyl)eth yl)car bam oyl)-2-(1-n aph th yl)eth yl)-
a m id e (7i). 1H NMR (CDCl3) (selected peaks for major
rotamer): δ 1.25 (s, 6H); 2.25 (d, 2H); 2.08 (s, 3H); 2.89 (d,
3H); 3.18 (s, 3H); 5.90 (dd, 1H); 6.65 (d, 1H). PDMS: m/z 599.8
(M + H)+. HPLC: tR ) 33.50 (method A1). HPLC: tR ) 35.83
min (method B1). HRMS: calculated for C36H46N4O4, m/z )
598.3519; found, m/z ) 598.3512.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-Meth yl-N-
((1R )-1-(N -m e t h y l-N -((1R )-1-(m e t h y lc a r b a m o y l)-2-
(th iop h en e-2-yl)eth yl)ca r ba m oyl)-2-(2-n a p h th yl)eth yl)-
a m id e (7j). 1H NMR (CDCl3) (selected peaks for major
rotamer): δ 2.31 (d, 3H); 2.63 (s, 3H); 2.91 (s, 3H); 5.18 (dd,
1H); 5.55 (dd, 1H); 6.19 (d, 1H). HPLC: tR ) 30.3 min (method
A1). HPLC: tR ) 32.93 min (method B1). PDMS: m/z 534.8
(M + H)+. HRMS: calculated for C30H38N4O3S, m/z ) 534.2665;
found, m/z ) 534.2619.
5-(Meth yla m in o)h ex-2-en oic Acid ((1R)-1-(((1R)-2-(3,4-
Diflu or op h en yl)-1-(m et h ylca r b a m oyl)et h yl)m et h ylca r -
ba m oyl)-2-(2-n a p h th yl)eth yl)m eth yla m id e (7m ). 1H NMR
(CDCl3) (selected peaks for major rotamer): δ 1.22 (s, 6H); 2.10
(d, 3H); 2.71 (s, 3H); 2.85 (s, 3H); 5.22 (dd, 1H); 5.86 (dd, 1H);
6.17 (d, 1H). HPLC: tR ) 33.18 min (method A1). HPLC: tR
) 35.47 min (method B1). PDMS: m/z 566.0 (M + H)+.
5-(Meth ylam in o)h ex-2-en oic Acid ((1R)-1-(((1R)-2-P h en -
yl-1-(eth ylca r ba m oyl)eth yl)m eth ylca r ba m oyl)-2-(2-n a p h -
th yl)eth yl)m eth yla m id e (7n ). 1H NMR (CDCl3) (selected
peaks for major rotamer): δ 0.61 (t, 3H); 1.05 (s, 6H); 1.98 (s,
3H); 2.24 (s, 3H); 2.97 (d, 3H); 5.57 (dd, 1H); 5.85 (dd, 1H);
6.07 (d, 1H). HPLC: tR ) 33.0 min (method A1). HPLC: tR
)
35.27 min (method B1). PDMS: m/z 544.0 (M + H)+. HRMS:
calculated for C33H42N4O3, m/z ) 542.3257; found, m/z )
542.3296.
5-Am in o-5-m eth ylh ex-2-en oic Acid ((1R)-1-(((1R)-1-
((2S)-2-H yd r oxyp r op ylca r b a m oyl)-2-(2-t h ien yl)et h yl)-
m eth ylcar bam oyl)-2-(2-n aph th yl)eth yl)m eth ylam ide (7o).
1H NMR (CDCl3) (selected peaks for major rotamer): δ 1.10
(d, 3H); 1.14 (s, 3H); 1.15 (s, 3H); 2.18 (d, 2H); 2.95 (s, 3H);
3.05 (s, 3H); 5.28 (dd, 1H); 5.72 (dd, 1H); 6.07 (d, 1H). HPLC:
tR ) 30.4 min (method A1). HPLC: tR ) 32.68 min (method
B1). PDMS: m/z 580.0 (M + H)+. HRMS: calculated for
C
34H44N4O5, m/z ) 572.3363; found, m/z ) 572.3387.
5-Am in o-5-m et h ylh ex-2-en oic Acid ((1R)-1-(((1R)-1-
((2R)-2-Hyd r oxyp r op ylca r ba m oyl)-2-p h en yleth yl)m eth -
ylca r ba m oyl)-2-(2-n a p h th yl)eth yl)m eth yla m id e (7p ). 1H
NMR (CDCl3) (selected peaks for major rotamer): δ 1.09 (d,
3H); 1.19 (s, 6H); 2.97 (s, 3H); 2.99 (s, 3H); 5.15 (dd, 1H); 5.53
(dd, 1H); 6.12 (d, 1H). HPLC: rt) 31.8 min (method A1).
HPLC: tR ) 33.28 min (method B1). PDMS: m/z 573.7 (M +
H)+.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-((1R)-2-
(Bip h en yl-4-yl)-1-(N-((1R)-2-(4-flu or op h en yl)-1-((2R)-2-
h yd r oxyp r op ylca r b a m oyl)et h yl)-N-m et h ylca r b a m oyl)-
eth yl)-N-m eth yla m id e (7q). 1H NMR (CDCl3) (selected
peaks for major rotamer): δ 1.08 (d, 3H); 1.30 (s, 3H); 1.37 (s,
3H); 2.91 (s, 3H); 3.04 (s, 3H); 5.15 (dd, 1H); 5.57 (dd, 1H);
6.38 (d, 1H); 6.70 (m, 1H). HPLC: tR ) 35.08 min (method
A1). HPLC: tR ) 37.18 min (method B1).
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid N-((1R)-2-
(Bip h en yl-4-yl)-1-(N-((1R)-1-((2S)-2-h yd r oxyp r op ylca r -
bam oyl)-2-(2-th ien yl)eth yl)-N-m eth ylcar bam oyl)eth yl)-N-
m eth yla m id e (7r ). 1H NMR (CDCl3) (selected peaks for
major rotamer): δ 1.07 (d, 3H); 1.36 (s, 3H); 1.39 (s, 3H); 2.81
(s, 3H); 3.05 (s, 3H); 5.21 (dd, 1H); 5.55 (dd, 1H); 6.36 (d, 1H);
6.69 (m, 1H). HPLC: tR ) 33.92 min (method A1). HPLC: tR
) 35.93 min (method B1).
5-Am in o-5-m eth ylh ex-2-en oic Acid ((1R)-1-(((1R)-1-
((2S )-2-H y d r o x y p r o p y lc a r b a m o y l)-2-(3,4-d i flu o r o -
p h en yl)et h yl)m et h ylca r b a m oyl)-2-(2-n a p h t h yl)et h yl)-
m eth yla m id e (7s). 1H NMR (CDCl3) (selected peaks for
major rotamer): δ 1.01 (t, 3H); 1.10 (s, 6H); 2.74 (s, 3H); 3.04
(s, 3H); 5.08 (dd, 1H); 5.56 (dd, 1H); 6.07 (d, 1H). HPLC: tR
)
32.9 min (method A1). PDMS: m/z 610.3 (M + H)+.
(2E)-5-Am in o-5-m eth ylh ex-2-en oic Acid ((1R)-2-(Bi-
p h e n y l-4-y l)-1-(m e t h y l((1R )-1-(m e t h y lc a r b a m o y l)-2-
ph en yleth yl)car bam oyl)eth yl)m eth ylam ide (7k). 1H NMR
Biologica l Met h od s: P h a r m a cok in et ics, Or a l Bio-
a va ila bility. Oral bioavailability studies were conducted in
male and female beagle dogs. The dogs were fasted overnight

Novel Orally Active GH Secretagogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3713
Nargund, R.; Griffin, P. R.; DeMartino, J . A.; Gupta, S. K.;
Schaeffer, J . M.; Smith, R. G.; Van der Ploeg, L. H. T. A Receptor
in Pituitary and Hypothalamus That Functions in Growth
Hormone Release. Science 1996, 273, 974.
prior to dosing. Diet was withheld for at least 3 h postdosing.
A 1-week washout period separated peroral (po) and intrave-
nous (iv) dosing. The compounds were administered in a
vehicle of citrate/phosphate buffer, pH 5.0. For po administra-
tion the dogs received a dose of 2.5 mg/kg of body weight via
gavage (three doses for 7b,g). For iv administration the dogs
received a dose of 0.5 mg/kg of body weight as a bolus in a
hind leg vein. EDTA blood samples were drawn from a front
leg vein at intervals up to 6 h after dosing. Blood samples
were placed on an ice-water bath immediately after sampling.
Plasma was separated by centrifugation and stored frozen
pending analysis. An HPLC assay with UV detection and
solid-phase extraction was developed for each compound.
Analytical C8 columns and disposable C3 extraction columns
were used. The oral bioavailability was calculated as the total
area under the plasma concentration versus time curve
following po administration divided by the area following iv
administration, appropriately corrected for dose (eq 1):
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On the in vitro and in vivo activity of a new synthetic hexapep-
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hormone. Endocrinology 1984, 114, 1537-1545. (c) Bowers, C.
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R. P.; Patchett, A. A. Peptidomimetic regulation of growth
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catabolic response to surgically produced acute stress and on
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peptide. J Clin. Endocrinol. Metab. 1994, 79, 940-942.
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Fisher, M. H.; Cheng, K.; Chan, W. W. S.; Butler, B.; Smith, R.
G.; Ball, R. G. A novel 3-substituted Benzazepinone Growth
Hormone Secretagogue L-692,429. J . Med. Chem. 1994, 37, 897-
906.
(7) Patchett, A. A.; Nargund, R. P.; Tata, J . R.; Chen, M.-H.;
Barakat, K. J .; J ohnsyon, D. B. R.; Cheng, K.; Chan, W. W.-S.;
Butler, B.; Hickey, G.; J acks, T.; Schleim, K.; Pong, S.-S.;
Chaung, L.-Y. P.; Chen, H. Y.; Frazier, E.; Leung, K. H.; Chiu,
S.-H. L.; Smith, R. G. Design and biological-activities of L-163,191
(MK-0677) - a potent, orally active growth-hormone secreta-
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Lundt, B. F.; Thøgersen, H.; Andersen, P. H. A new series of
highly potent growth hormone-releasing peptides derived from
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Mechanisms of Peptide and protein absorption. Adv. Drug Deliv.
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C.-M.; Qian, Y.; Hamilton, A. D.; Sebti, S. M. Potent Inhibition
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AUCpo‚doseiv
AUCiv‚dosepo
f )
(1)
In Vivo Ch a r a cter iza tion in Con sciou s Sw in e. For the
initial screening, four female 30-40-kg Danish slaughter swine
of the breed Landrace Yorkshire cross were used for each GH
secretagogue. The swine were housed at least 1 week prior
to experiments. Prior to experiments it was tested that the
swine used had similar basal GH levels. Indwelling jugular
catheters were inserted and fixed under general halothane
anesthesia at day 0. The test compounds were administered
as 50 nmol/kg iv bolus injections with at least 48-h intervals
between compounds. Each group of swine was used to test a
maximum of five different compounds. The test compounds
were dissolved in phosphate/citrate buffer diluted in saline
containing 0.5% porcine serum albumin. Blood samples were
drawn from the jugularis catheter at frequent intervals from
1 h prior to stimulation until 3 h poststimulation. For a more
detailed characterization of 7b,g, the same setup was used
except six animals were used for each compound. The two
compounds were administered as single iv injections in six
increasing doses with 72-h intervals between doses (10, 30,
100, 300, 1000, and 10000 nmol/kg). Plasma was analyzed
for porcine GH (pGH) by ELISA. The basal GH level, for the
individual swine, was calculated as the average of the three
GH values obtained prior to stimulation. Peak hormone levels
(Cmax) adjusted for basal level, obtained following administra-
tion of test compounds, were used to characterize the hormone
response of individual swine. In single-dose experiments Cmax
was used directly. In multidose experiments dose-response
curves were constructed using Cmax GH plasma concentrations.
Fitting to the Hill equation or hyperbolic Michaelis-Menten
equation was performed by nonlinear regression using the
Prism software (GraphPad). Using the efficacy (Emax values)
of the individual compounds, the potency (ED50 values) was
calculated as the dose inducing half-maximal stimulation, i.e.,
the increase in plasma GH from basal. The results were tested
for normal probability distribution by Shapiro-Wilk test. All
statistical comparisons were performed using Prism and SAS
software. When appropriate, the data are given as means (
SEM.
Ack n ow led gm en t. We are thankful to Anette Heer-
wagen, Lotte G. Sørensen, Anne G. Christiansen, Nille
B. Hammerum, Karina Frandsen, Anette Carlsen,
Kirsten Holmberg, Lene von Voss, Tine R. Ankersen,
and Peter Andersen for technical assistance.
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lamino acids by N-methylation. Can. J . Chem. 1977, 55, 6-910.
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(13) Humphrey, J . M.; Chamberlin, A. R. Chemical synthesis of
Natural Product Peptides: Coupling Methods for the Incorpora-
tion of Noncoded Amino Acids into Peptides. Chem. Rev. 1997,
97, 2243.
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