Growth hormone secretagogues derived from NN703 with hydrazides as C- terminal

Article abstract of DOI:10.1016/S0223-5234(00)00146-X

A series of GH secretagogues based on modifications in the C-terminal of NN703 is reported. The C-terminal N-methyl amide of NN703 has been replaced with alkylated hydrazides in order to decrease the volume of distribution and identify GH secretagogues with shorter duration of action. Most of the prepared compounds show high potency in a rat pituitary assay. Subsequent to an initial in vivo screening in dogs, four compounds were selected for further pharmacological and pharmacokinetic evaluation. The four compounds showed oral bioavailability around 35% and equipotency in vitro compared to NN703. The relationship between lipophilicity and volume of distribution is discussed and it is speculated whether the lower volume of distribution is attributed to the observed higher in vivo potency and shorter plasma elimination half-life. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00146-X

Eur. J. Med. Chem. 35 (2000) 487−497
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S022352340000146X/FLA
487
Original article
Growth hormone secretagogues derived from NN703 with hydrazides
as c-terminal
Michael Ankersena*, Karin Kramer Nielsenb, Thomas Kruse Hansena, Kirsten Raunc,
Birgit Sehested Hansend
aHealth Care Chemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
bPreclinical Development, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
cHealth Care Pharmacology, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
dCell and Assay Biotechnology, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
Received 10 September 1999; revised 29 November 1999; accepted 1 December 1999
Abstract – A series of GH secretagogues based on modifications in the C-terminal of NN703 is reported. The C-terminal N-methyl amide
of NN703 has been replaced with alkylated hydrazides in order to decrease the volume of distribution and identify GH secretagogues with
shorter duration of action. Most of the prepared compounds show high potency in a rat pituitary assay. Subsequent to an initial in vivo
screening in dogs, four compounds were selected for further pharmacological and pharmacokinetic evaluation. The four compounds showed
oral bioavailability around 35% and equipotency in vitro compared to NN703. The relationship between lipophilicity and volume of
distribution is discussed and it is speculated whether the lower volume of distribution is attributed to the observed higher in vivo potency and
shorter plasma elimination half-life. © 2000 Éditions scientifiques et médicales Elsevier SAS
growth hormone releasing peptides / growth hormone secretagogue / NN703 / hydrazides / MK677 / GHRP / volume of distribution
/ half-life
1. Introduction
synthetic compounds has not been reported yet, whereas
a receptor (GHS 1A) with high affinity for the GH
secretagogues has been identified and cloned recently
[14, 15].
Since Momany and Bowers [1–3] discovered a series
of peptides that specifically released growth hormone
(GH) from the rat pituitary, a number of small peptides
and non-peptides with similar effect have been found [4].
These compounds (generally identified as GHS or GHRP)
have shown GH releasing capability in a number of
species, including man [5]. It is well established that their
mechanism of action differs from that of GHRH [6, 7].
Prominent members of the GH secretagogue family
(figure 1) are the peptides GHRP-1, GHRP-2, GHRP-6
[3], hexarelin [8] and ipamorelin [9], the non-peptides
L-692,429 (1) [10], MK677 (2) [11] and the acylated
dipeptide, NN703 (3) [12]. The GH secretagogues were
discovered via ‘reverse pharmacology’ [13] or more
precisely ‘reverse drug discovery’, referring to the fact
that a putative endogenous ligand corresponding to these
Compounds 2 and 3 are systematically available after
oral administration (i.e. oral bioavailable) and this is
clearly a point of utmost importance if a GH secretagogue
shall enter the market in competition with the existing
recombinant GH therapy [16]. Another issue of high
importance is based on a previously proposed link be-
tween the plasma elimination half-life and receptor-
desensitization and magnitude of GH-induced IGF-I re-
lease [17]. For instance, chronic dosing of 2 in humans
results in down regulation of the GH release but still
exhibits prolonged elevation of the IGF-I level [11]. This
has been associated with the relatively long plasma
elimination half-life of 2 (t
6 h). In this context it is
½
noteworthy that short acting compounds such as GHRP-2
have demonstrated efficacy with regard to growth veloc-
ity in children despite the absence of a sustained IGF-I
* Correspondence and reprints: miak@novo.dk

488
Figure 1. Some of the most prominent members of the GH secretagogue family.
elevation [18]. At present it is not clear exactly which
2. Chemistry
pharmacological profile is optimal.
The GH secretagogues were prepared by stepwise
peptide synthesis using Fmoc as the protecting group for
N-methyl-D-2-phenylalanine (N-Me-D-Phe-OH), N-me-
thyl-D-4-biphenylalanine (N-Me-D-Bip-OH), N-methyl-
D-thienylalanine (N-Me-D-Thi-OH) and N-methyl-D-
naphthylalanine (N-Me-D-2-Nal-OH) in the two first
coupling steps (figure 2). Fmoc-N-Me-D-Phe-OH or
Fmoc-N-Me-D-Thi-OH were readily coupled with vari-
ous substituted hydrazines [22] using the standard EDC/
HOBT coupling procedure. Removal of the FMOC pro-
tecting group with piperidine in dimethylformamide
(DMF) resulted in the corresponding hydrazide in good
yields. The resulting hydrazide was coupled to Fmoc-N-
Me-D-2-Nal-OH or Fmoc-N-Me-D-Bip-OH using the
same coupling procedure. Boc-protection was used in the
final coupling step introducing 5-amino-5-methylhex-2-
enoic acid. The final deprotection using trifluoroacetic
acid in methylene chloride was carried out with care (as
previously described) [20] affording the desired acylated
dipeptides 4–20.
NN703 is a highly specific compound, has a long
plasma elimination half-life (t
4 h) similar to that of 2
½
and a rather high volume of distribution (Vz 8 L/kg) in
beagles. Consequently, we see down-regulation of GH-
release with NN703, as is the case with 2 [12]. This led
us to search for compounds in the NN703 series with
similar good in vitro and in vivo potencies, good oral
bioavailability but a significantly shorter half-life. Since
the half-life is related to the volume of distribution (Vz =
CL/λz; t = ln 2/λz), and the volume of distribution is
½
believed to be related to the fat/water partition, we
speculated whether a decrease in lipophilicity, provided
that clearance is unchanged or decreased, would decrease
such volume of distribution. Since NN703 is a rather
lipophilic compound (clogP 4.2) we hypothesised that a
moderate increase in hydrophilicity could lead to the
desired change in pharmacokinetic parameters. This was
clearly a narrow balance as we had optimized the oral
bioavailability of the NN703 series by applying ‘the rule
of five’ [19], and we realized that major changes in size
and polarity could easily be detrimental to the oral
bioavailability. Therefore, we chose to replace the
C-terminal amide with various hydrazides, because a
hydrazide would maintain some of the amide character,
which was important according to our SAR [20], and it
would add an extra charge group to the molecule and
supposedly increase polarity. This transformation was
also synthetically easy and would not lead to a major
increase in molecular weight. According to our SAR the
C-terminal was also the region of NN703 that was most
tolerant with regard to addition of extra steric bulk [20].
3. Results and discussion
Due to the assumed influence of pharmacokinetic
parameters on GH down-regulation and IGF-I prolonga-
tion [17], GH secretagogues derived from 3 with shorter
duration of action were required. Therefore, the role of
the C-terminal amide of 3 with respect to potency and
pharmacokinetics was investigated by replacing the
C-terminal amide with various hydrazides. The introduc-
tion of a basic moiety was hoped only to influence the in
vitro and in vivo potency and oral bioavailability to a
minor extent, but to have a major impact on the pharma-
cokinetic profile. The volume of distribution is related to
clearance and plasma elimination half-life and is believed
to be related to the lipophilicity of the compound. Often
Those considerations led to the present series of
analogues of 3 with small hydrazides in the C-terminal
combined with isosteric modifications in the naphthyl and
the phenyl moieties [21].

489
Figure 2. The first two coupling steps of the synthesis.
compounds which are relatively more polar would tend
not to distribute to fatty tissues and exhibit a lower
volume of distribution [23] and consequently to a shorter
half-life, provided the clearance remain relatively unaf-
fected. Thus, a series of various hydrazide analogues of 3
was prepared and tested in vitro and a selection of these
was further pharmacologically and pharmacokinetically
evaluated.
Growth hormone in vitro was measured in rat pituitary
cells as described previously [9]. In this assay and in this
series of experiments, the reference compound 3 had a
potency (EC50) of 8.3 ± 1.3 nM (mean ± SEM, n = 4) and
an efficacy (Emax) of 100% of that measured for GHRP-6.
In this assay GHRP-6 had a potency at 2.7 ± 0.8 nM; 1
was 169 ± 53 nM and 2 was 1.8 ± 0.5 nM.
Table I shows the in vitro potency (EC50) and efficacy
(Emax) of a series of hydrazide analogues of 3. A number
of derivatives are equipotent to 3, such as 4, 5, 8, 12, 14
and 18 (EC50s below 10 nM), while others are slightly
less potent, such as 7, 9, 10, 11, 13 and 17 (EC50s above
40 nM). With respect to efficacy, 4, 10, 11 and 17 are
more efficacious in vitro than the other compounds (Emax
> 100%).
The series contains simple methylated hydrazides (5, 8,
14, 16, 18 and 20), hydrazides holding an additional
carbonyl (4, 6, 7 and 11) and hydrazides holding bulky
groups (9, 10, 11, 12, 13, 15, 17 and 19). In general, all
compounds are full agonists (i.e. Emax > 80%), and
despite the introduction of a basic moiety, an additional
carbonyl or a bulky group in the C-terminal, no major
changes in in vitro potency were observed. This may
suggest the presence of a space between the C-terminal
and the receptor, which is insensitive to the presence of
such different groups as amines, carbonyls and lipophilic
groups. Such additional free space may be used for
further enhancement of the interaction of the compounds
and the receptor.
shorter half-life than 3, we screened a selection of the
compounds in dogs after oral administration.
3.1. In vivo pharmacology and pharmacokinetics in dogs
Our setup used fasted dogs that were dosed by gavage
as well as by intravenous administration in a hind leg vein
[12]. We have previously shown that at a dose of
2.5 mg/kg, compound 3 released 12.2 ± 9.9 ng/mL GH in
dogs after oral administration (mean ± SEM, four dogs)
[12]. To get a fast overview of the most interesting
compounds, we screened several compounds (table II) in
four dogs after oral administration of 1.0 mg/kg. Our
selection criterion for further pharmacological and phar-
macokinetic studies in this initial screening was a mean
GH release above 10 ng/mL. As shown in table II the
compounds 5, 8, 14, 16, 17, 18 and 20 met this criterion.
We selected 5, 8, 14 and 18 for further pharmacological
and pharmacokinetic evaluation. These four compounds
are structurally different to 3, either they contain a
trimethylhydrazine (e.g. 5 and 18) or N,N-dimethyl-
hydrazine (e.g. 8 and 14) instead of methylamine, or that
the phenylalanine has been replaced with a thienylalanine
in combination with di- or trimethylhydrazine (e.g. 14
and 18). The four compounds were tested in dogs after
oral administration of a dose between 2.0 and 2.8 mg/kg
and an i.v. administration at 0.5 mg/kg and compared
with data for 3. The results are shown in table II. All four
compounds had slightly lower clearance (however, only 5
was significantly different to 3) and while 8, 14 and 18
had a significantly lower half-life, 5 showed a longer
half-life, although not significantly different to 3.
The volume of distribution (Vz) was derived from the
half-life and the clearance (i.e. Vz = CL/λz; t½ = ln 2/λz)
and the Vz was lower for all four compounds, although
only 18 was significantly lower than 3. All four com-
pounds had an oral bioavailability similar to 3, and all
four compounds showed a better GH release after oral
administration than 3, although none of them was signifi-
cantly higher.
Since our main objective was to identify in vivo active
GH secretagogues with good oral bioavailability and

490
Table I. Potency (EC50) and efficacy (Emax) of a series of GH secretagogues with modifications in the C terminal. Mean values of at least
two separate experiments.
Entry
R1
R2
R3
EC50 (nM)
Emax (%)
3 (NN703)
4
5 (NNC 26-1089)
6
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
phenylene
phenylene
phenylene
phenylene
phenylene
phenylene
phenylene
–NHCH3
8
8
9
17
28
7
100
125
85
90
95
–NHNHCOCH3
–NCH3N(CH3)2
–NHNCH3COCH3
–NCH3NHCOCH3
–NHN(CH3)2
7
8 (NNC 26-1136)
9
90
90
24
10
11
2-naphthalene
2-naphthalene
phenylene
phenylene
24
22
110
105
12
13
2-naphthalene
2-naphthalene
phenylene
phenylene
9
80
85
35
14 (NNC 26-1137)
15
2-naphthalene
4-biphenylene
2-thienyl
phenylene
–NHN(CH3)2
8
11
95
100
16
17
4-biphenylene
2-naphthalene
phenylene
2-thienyl
–NCH3N(CH3)2
10
24
95
110
18 (NNC 26-1167)
19
2-naphthalene
4-biphenylene
2-thienyl
phenylene
–NCH3N(CH3)2
–NCH3N(CH3)2
9
15
85
100
20
4-biphenylene
2-thienyl
13
80

491
Table II. GH release (ng/mL) after the initial screening in dogs (n = 4) after oral administration of 1.0 mg/kg of 4–20, oral bioavailability
(Fpo), volume of distribution (Vz), half-life (t ), clearance (CL) and GH release after oral administration of 2.0–2.8 mg/kg of 3, 5, 8, 14 and
½
18, respectively. Values are mean ± SEM.
Entry
Screening (1.0 mg/kg) Fpo (%)
t
(h)
CL (l/h/kg) Vz (l/kg)
GH release (Cmax) (ng/mL)
½
3 (NN703)
4
5 (NNC 26-1089)
6
33 ± 6
4.1 ± 0.4
6.6 ± 1.5
1.4 ± 0.2
8.2 ± 1.6
5.6 ± 0.7
12.2 ± 5.0 (2.5 mg/kg)
< 10
> 10
> 10
< 10
> 10
< 10
< 10
< 10
> 10
< 10
> 10
> 10
> 10
< 10
> 10
32 ± 10
35 ± 3
0.6 ± 0.1*
18.5 ± 4.9 (2.5 mg/kg)
21.1 ± 6.8 (2.0 mg/kg)
7
8 (NNC 26-1136)
10
11
12
14 (NNC 26-1137)
15
16
17
2.7 ± 0.3*
2.5 ± 0.2*
1.9 ± 0.2*
0.8 ± 0.3
0.8 ± 0.3
0.8 ± 0.2
3.5 ± 1.5
28 ± 4
33 ± 5
3.0 ± 1.3*
23.2 ± 9.3 (2.0 mg/kg)
18 (NNC 26-1167)
19
20
2.26 ± 0.7* 21.0 ± 0.9 (2.8 mg/kg)
* P > 0.05 using student′s t-test.
In total, all four compounds showed lower volume of
distribution. The lower Vz was in all cases associated
with lower clearance and shorter half-life, except for 5,
which surprisingly showed a longer half-life, but this was
counteracted by a significantly lower clearance. The
generally lower Vz is, however, in accord with our
hypothesis that the introduction of a basic moiety into 3
would give a significantly lower volume of distribution
and that this decrease would be reflected by a shorter
half-life, given that the clearance is unchanged or de-
creased.
Meanwhile, one would expect 8 and 14 to be less
lipophilic than 5 and 18 since these two compounds have
an additional proton donor at the hydrazide, and accord-
ing to the hypothesis this should lead to a lower Vz. This
is only the case for 8 compared to 5 and is not the case for
14 compared to 18, although no significant differences
are observed. However, if the two trimethylhydrazides 5
and 18 are compared with respect to Vz, the introduction
of a thienyl group seems to significantly decrease the Vz,
whereas in the two dimethylhydrazides 8 compared to 14
no significant difference was observed. And compared to
3, only the thienyl containing compounds (14 and 18) are
significantly different, suggesting that the lower Vz may
be attributed to the thienyl group rather than the hy-
drazides.
Meanwhile, our main purpose of the study was to
identify compounds with a shorter half-life, and as shown
in table II, the compounds 8, 14 and 18 all showed
significantly shorter half-lives than 3, while 5 was not
significantly different to 3 but significantly higher than
the other hydrazides.
Overall, this could indicate that the substitution of a
proton donor (as in the secondary amide in 3) with a
positive charge alone (i.e. 5) is not enough to obtain a
shorter half-life, but that the combination of a positive
charge with a proton donor (i.e. 8 and 14) or combined
with a thienyl group (i.e. 18) in our series is necessary.
The obvious reference compound in this study would
be the thienyl analogue of NN703. This compound has
previously been prepared and studied in a different
set-up, but the compound did not meet the criterion for
further biological evaluation (due to low GH release after
oral administration) and therefore no data for comparison
is accessible [20].
The in vivo potency of all four hydrazides was in-
creased, although not significantly, and it may be specu-
lated whether this is a reflection of the lower volume of
distribution as well. It is likely that in this case, where the
clearance is only slightly affected, the lower volume of
distribution is directly translated into a higher amount of
substance in systemic circulation and this subsequently
results in a higher in vivo potency. Also it is interesting to
note that despite the introduction of an additional positive
charge, the oral bioavailability was unchanged. The in
vitro specificity of 5 and 18 were evaluated in more than
With respect to clearance, only 5 was significantly
different to 3, while no significant difference was ob-
served between 8, 14 and 18 to 3 and no significant
difference was observed between the four hydrazides.

492
100 receptor and enzyme assays and no significant
binding at 10 µM was observed.
column was equilibrated with a mixture composed of 5%
of solvent system I and 95% of solvent system II. After
injection of the sample a gradient of 20–80% of solvent
system I in solvent system II was run over 30 min. The
gradient was then extended to 100% of solvent system I
over 5 min followed by isocratic elution with 100% of
this system for 5 min.
4. Conclusion
We have described a series of GH secretagogues
derived from 3 (NN703) with hydrazides in the
C-terminal. Based on the GH release from a rat pituitary
assay and some initial in vivo screening, we selected the
hydrazide analogues 5, 8, 14 and 18 for further in vivo
characterization. In beagle dogs, all four compounds
showed improved potency after oral administration of
2.0–2.8 mg/kg, with respect to GH release and similar
oral bioavailability compared to 3. Meanwhile, the vol-
ume of distribution of all four compounds and the
half-life of all but 5 was significantly lower than 3, and
we speculate whether the decreased volume of distribu-
tion may explain the relatively higher in vivo potency.
Therefore, we have succeeded in our main goal in
identifying new GH secretagogues derived from NN703
with improved in vivo potency and shorter plasma
elimination half-lives.
The RP-analysis was performed using UV detection at
214, 254, 276 and 301 nm on a 218TP54 4.6 × 250 mm
5 µ C-18 silica column (The Seperations Group, Hespe-
ria), which was eluted at 1 mL/min at 42 °C. The column
was equilibrated with 5% acetonitrile in a buffer consist-
ing 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 for 50 min.
HPLC (method B1): the RP-HPLC analysis was per-
formed using UV detection at 214 nm and a Hibar
LiChrosorb RP-18 (5uM) 250-4 (Merck) column, which
was eluted at 1 mL/min. Two solvent systems were used:
solvent system I: 0.1% trifluoroacetic acid in acetonitrile.
Solvent system II: 0.1% trifluoroacetic acid in water. The
column was equilibrated with a mixture composed of 5%
solvent system I and 95% solvent system II. After
injection of the sample a gradient of 20–80% of solvent
system I in solvent system II was run over 30 min. The
gradient was then extended to 100% of solvent system I
over 5 min followed by isocratic elution with 100% of
this system for 5 min.
5. Experimental procedure
The four amino acids have been purchased from
Synthetech Inc. (PO Box 646, Albany, Oregon 97321) or
prepared as previously described [20]. All hydrazines
used were commercially available, except trimethylhy-
drazine and 1-(methylamino)piperidine, which were pre-
pared by formylation of unsym-dimethylhydrazine and
1-aminopiperidine, respectively, followed by reduction
with lithiumaluminumhydride according to a procedure
by Class et al. [22].
High performance liquid chromatography (HPLC) and
nuclear magnetic resonance (NMR, Bruker 400 MHz) or
liquid chromatography-mass spectrometry (LC-MS) con-
firms the structures of the compounds. NMR shifts (δ) are
given in parts per million (ppm) and only selected peaks
are given. M.p. is melting point and is given in °C. The
methanol/ammonia solution used is a 10% ammonia
solution in methanol.
The RP-analysis was performed using UV detection at
214, 254, 276 and 301 nm on a 218TP54 4.6 × 250 mm
5 µ C-18 silica column (The Seperations Group, Hespe-
ria), which was eluted at 1 mL/min at 42 °C. The column
was equilibrated with 5% (acetonitrile + 0.1% TFA) in an
aqueous solution of TFA in water (0.1%). After injection
the sample was eluted by a gradient of 5–60% (acetoni-
trile + 0.1% TFA) in the same aqueous buffer for 50 min.
HPLC (method H8): the HPLC analyses was per-
formed using a Waterst millenium system using a Watert
3 × 150 mm 3.5 µ C-18 Symmetry column. The column
was heated to 42 °C and eluted with a linear gradient of
5–90% acetonitrile, 85–0% water and 10% trifluoroacetic
acid (0.5%) in water in 15 min at a flow-rate of 1 mL/
min.
5.1. HPLC methods
The LC-MS analyses were performed on a PE Sciex
API 100 LC/MS System using a Waterst 3 × 150 mm
3.5 µ C-18 Symmetry column and positive ionspray with
a flow rate of 20 µL/min. The column was eluted with a
linear gradient of 5–90% acetonitrile, 85–0% water and
10% trifluoroacetic acid (0.1%)/water in 15 min at a flow
rate of 1 mL/min.
HPLC (method A1): the RP-HPLC analysis was per-
formed using UV detection at 214 nm and a Hibar
LiChrosorb RP-18 (5 uM) 250-4 (Merck) column, which
was eluted at 1 mL/min. Two solvent systems were used:
solvent system I: 0.1% trifluoroacetic acid in acetonitrile.
Solvent system II: 0.1% trifluoroacetic acid in water. The

493
5.2. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-2-(N′-acetylhydrazino)-
1-benzyl-2-oxoethyl]-N-methylcarbamoyl)-
2-(2-naphthyl)ethyl)-N-methylamide (4)
methanol/ammonia (9:1) to give 0.41 g of acetic acid
N′-((2R)-2-(methylamino)-3-phenylpropionyl)hydrazide
as an amorphous powder. Then (2R)-2-(N-tert-
butoxycarbonyl-N-methylamino)-3-(2-naphthyl)propio-
nic acid (0.63 g, 1.92 mmol) was dissolved in methylene
chloride (10 mL) and a mixture of 1-hydroxy-7-
azabenzotriazole (0.26 g, 1.92 mmol) and 1-ethyl-3-(3-
To a solution of tert-butyl carbazate (1.0 g, 7.6 mmol)
and pyridine (3.1 mL) in methylene chloride (5 mL) was
slowly added acetic acid anhydride (1.5 mL) and the
mixture was stirred overnight. The mixture was added to
methylene chloride (50 mL) and washed with water (2 ×
10 mL) and brine (10 mL) and dried (MgSO4), filtered
and concentrated in vacuo to give 0.95 g of
N′-acetylhydrazinecarboxylic acid tert-butyl ester as a
yellow oil. LC-MS: Rt = 5.39, m/z = 349.6 (m + 1);
1H-NMR (CDCl3) selected peaks: δ 1.5 (s, 9H,
(CH3)3C–O); 2.05 (s, 3H, CH3CO).
N′-Acetylhydrazinecarboxylic acid tert-butyl ester
(0.95 g, 5.45 mmol) was dissolved in methylene chloride
(10 mL) and trifluoroacetic acid (10 mL) was added and
the mixture was stirred at room temperature for 1 h. The
mixture was concentrated in vavuo and stripped three
times with methylene chloride to give 1.0 g of acetic acid
hydrazide. Then (2R)-2-(tert-butoxycarbonylmethyl-
amino)-3-phenyl propionic acid (0.76 g, 2.73 mmol) was
dissolved in a mixture of dimethyl formamide (3 mL) and
methylene chloride (6 mL) and a mixture of 1-hydroxy-
7-azabenzotriazole (0.45 g, 3.28 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide
hydrochloride
(0.37 g, 1.92 mmol) was added and stirred for 30 min. A
mixture of acetic acid N′-((2R)-2-(methylamino)-3-
phenylpropionyl)hydrazide (0.41 g, 1.74 mmol) and DIEA
(0.39 mL) was added and the mixture was stirred over-
night. Methylene chloride (50 mL) was added and the
mixture was washed with water (50 mL). The organic
layer was washed with aqueous sodium bicarbonate
(10 mL) and brine (50 mL) and dried (MgSO4), filtered,
concentrated to an oil, and chromatographed on silica
(40 g) with heptane:ethyl acetate (1:4) to give 0.59 g of
N-((1R)-1-(N-[(1R)-2-(N′-acetylhydrazino)-1-benzyl-2-
oxoethyl]-N-methylcarbamoyl)-2-(2-naphthyl)ethyl)-N-
methylcarbamic acid tert-butyl ester as an oil. LC-MS Rt
= 13.68 min, m/z = 547.2 (m + 1); HPLC: Rt = 13.53 min
(H8).
To a solution of N-((1R)-1-(N-[(1R)-2-(N′-acetyl-
hydrazino)-1-benzyl-2-oxoethyl]-N-methylcarbamoyl)-2-
(2-naphthyl)ethyl)-N-methylcarbamic acid tert-butyl es-
ter (0.59 g, 1.08 mmol) in methylene chloride (5 mL) was
added trifluoroacetic acid (5 mL) at 0 °C and stirred for
90 min. The mixture was concentrated in vacuo and
stripped three times with methylene chloride. The ob-
tained oil was dissolved in methanol/ammonia (2 mL)
and added methylene chloride (20 mL) and silica gel
(5 g) and concentrated in vacuo. The obtained powder
was extracted by filtration with methylene chloride
(100 mL) and methylene chloride:methanol/ammonia
(9:1) and the combined extracts were concentrated
in vacuo to 0.41 g of crude product as a foam.
Then (2E)-5-(tert-butyloxycarbonylamino)-5-methylhex-
2-enoic acid (0.21 g, 0.89 mmol) was dissolved in me-
thylene chloride (10 mL) and a mixture of 1-hydroxy-7-
azabenzotriazole (0.13 g, 0.98 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide
hydrochloride
(0.62 g, 3.28 mmol) was added and stirred for 20 min.
Then a mixture of acetic acid hydrazide (1.0 g, 5.45 mmol)
and diisopropylethylamine (1.87 mL) was added and the
mixture was stirred overnight. Ethyl acetate (50 mL) was
added and the mixture was washed with water (50 mL).
The aqueous layer was extracted with ethyl acetate (3 ×
50 mL) and the combined organic layers were washed
with water (2 × 50 mL) and brine (50 mL) and dried
(MgSO4), filtered, concentrated to an oil, and chromato-
graphed on silica (40 g) with heptane:ethyl acetate (1:1)
to give 0.81 g of N-[(1R)-2-(N′-acetylhydrazino)-1-
benzyl-2-oxoethyl]-N-methylcarbamic acid tert-butyl es-
ter as a yellow oil. LC-MS Rt = 9.34, m/z = 336.4 (m +
1); HPLC: Rt = 10.17 min (H8); 1H-NMR (CDCl3)
selected peaks: δ 1.32 + 1.40 (2 s, 9H, (CH3)3C–O,
rotamere); 2.05 (s, 3H, COCH3); 2.78 (s, 3H, N-CH3).
N-[(1R)-2-(N′-Acetylhydrazino)-1-benzyl-2-oxoethyl]-
N-methylcarbamic acid tert-butyl ester (0.81 g,
2.42 mmol) was dissolved in methylene chloride (5 mL)
and trifluoroacetic acid (5 mL) was added and the mixture
was stirred for 30 min at room temperature. The mixture
was concentrated in vacuo and stripped three times with
methylene chloride and the remaining oil was chromato-
graphed on silica (40 g) with methylene chloride:
dimethylaminopropyl)-carbodiimide
hydrochloride
(0.17 g, 0.89 mmol) was added and stirred for 30 min. A
mixture of the above crude product (0.40 g, 0.89 mmol)
and diisopropylethylamine (0.20 mL) was added and the
mixture was stirred overnight. Methylene chloride
(50 mL) was added and the mixture was washed with
water (10 mL). The organic layer was washed with
aqueous sodium bicarbonate (10 mL) and brine (50 mL),
dried (MgSO4), filtered, concentrated to 0.42 g of a tan
foam. The foam was dissolved in methylene chloride
(5 mL), cooled to 0 °C and trifluoroacetic acid (5 mL)

494
was added and the mixture was stirred for 45 min. The
reaction mixture was quenched with methylene chloride
(50 mL) and water (10 mL) and titrated to pH 7 with
solid sodium bicarbonate. The organic layer was sepa-
rated and washed with water (10 mL), dried (MgSO4),
filtered and concentrated in vacuo. The obtained product
was dissolved in acetonitrile/water 1:20 (10 mL) and
applied to a C-18 Sep-Pak Classic© cartridge (2.0 g,
purchased from Waters™), which had been prewashed
with acetonitrile (100 mL) and water (100 mL). Then a
gradient of an eluent consisting of water/acetonitrile/
trifluoroacetic acid (10, 15, 20 and 25% acetonitrile in
water/0.1% trifluoroacetic acid) was run through the
Sep-Pak© (2 g). The relevant fractions were combined
and lyophilised to 0.19 g of the trifluoroacetic acid salt of
(2E)-5-amino-5-methylhex-2-enoic acid N-((1R)-1-
(N-[(1R)-2-(N′-acetylhydrazino)-1-benzyl-2-oxoethyl]-N-
methylcarbamoyl)-2-(2-naphthyl)ethyl)-N-methylamide
as a white amorphous powder. LC-MS: Rt = 9.21 min,
m/z = 572.4 (m + 1); HPLC: Rt = 26.45 min (A1), Rt =
28.40 (B1).
mixture of N,N,N′-trimethylhydrazine trifluoro acetate
(0.37 g, 1.96 mmol) and diisopropylethylamine (0.92 mL,
5.34 mmol) was added and the mixture was stirred for 2
days. Methylene chloride (50 mL) was added and the
mixture was washed with water (50 mL). The organic
layer was washed with saturated aqueous sodium bicar-
bonate (2 × 10 mL) and water (50 mL), dried (MgSO4),
filtered, concentrated in vacuo, and chromatographed on
silica (100 g) with heptane:ethyl acetate (1:1) to give
0.30 g
N-methyl-N-[(1R)-2-phenyl-1-(N,N′,N′-
trimethylhydrazinocarbonyl)ethyl]carbamic acid tert-butyl
ester as an amorphous powder. HPLC: Rt = 13.03 min
1
(H8); H-NMR (CDCl3) selected peaks: δ 1.23 + 1.35
(2 s, 9H, (CH3)3C–O, rotamere) 2.25 + 2.42 + 2.45 (3 s,
6H, N-N(CH3)2, rotamere).
To
a
solution of N-methyl-N-[(1R)-2-phenyl-1-
(N,N′,N′-trimethylhydrazinocarbonyl)ethyl]carbamic acid
tert-butyl ester (0.3 g, 0.89 mmol) in methylene chloride
(1 mL) was added trifluoroacetic acid (1 mL) and the
mixture was stirred for 30 min. The mixture was concen-
trated in vacuo, stripped three times with methylene
chloride and suspended in methylene chloride:methanol/
ammonia (1:1) (2 mL) and trifluoroammonium acetate
precipitated. Then diethyl ether (10 mL) was added and
the mixture was filtered and the filtrate was concentrated
in vacuo to give 0.31 g of (2R)-2-methylamino-3-
phenylpropionic acid trimethylhydrazide as an oil.
Then (2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-3-
(2-naphthyl)propionic acid (0.35 g, 1.07 mmol) was dis-
solved in methylene chloride (20 mL) and a mixture of
1-hydroxy-7-azabenzotriazole (0.15 g, 1.07 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (0.21 g, 1.07 mmol) was added and stirred for
30 min. Then a mixture of (2R)-2-methylamino-3-phenyl-
propionic acid trimethylhydrazide (0.21 g, 0.89 mmol)
and diisopropylethylamine (0.20 mL) was added and the
mixture was stirred overnight. Methylene chloride
(30 mL) was added and the mixture was washed with
water (10 mL). The organic layer was washed with
aqueous sodium bicarbonate (10 mL) and brine (50 mL),
dried (MgSO4), filtered, concentrated to an oil, and
chromatographed on silica (40 g) with heptane:ethyl
acetate (3:7) to give 0.43 g of N-methyl-N-((1R)-1-(N-
methyl-N-[(1R)-2-phenyl-1-(N,N′,N′-trimethylhydrazino-
carbonyl)ethyl]carbamoyl)-2-(2-naphthyl)ethyl)carbamic
acid tert-butyl ester as an oil.1H-NMR (CDCl3) selected
peaks: δ 0.92 + 1.22 (2 s, 9H, (CH3)3C–O, rotamere) 5.0
+ 5.3 (2 t, 1H, CH-CH2C6H5, rotamere) 6.3 + 6.45 (2 t,
1H, CH-CH2C10H7, rotamere).
5.3. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-(N-methyl-N-[(1R)-2-phenyl-
1-(N,N′,N′′-trimethylhydrazinocarbonyl)ethyl]-
carbamoyl)-2-(2-naphthyl)ethyl)amide (5)
To a solution of tert-butyl carbazate (1.0 g, 7.56 mmol)
in anhydrous tetrahydrofuran (40 mL) at 0 °C was added
sodium hydride (60% dispersion in oil, 2.73 g, 68 mmol)
and methyl iodide (11.3 mL, 181 mmol) and the mixture
was stirred for 3 days. Tetrahydrofuran (100 mL) was
added and the suspension was filtrated and the filtrate was
concentrated in vacuo. The obtained product was dis-
solved in ethyl acetate and chromatographed on silica gel
(40 g) with heptane:ethyl acetate (1:1) and concentrated
to give 0.53 g (40%) of N,N′,N′-trimethylhydrazine-
1
carboxylic acid tert-butyl ester as a thin oil. H-NMR
(CDCl3): δ 1.48 (s, 9H, (CH3)3C–O) 2.6 (s, 6H, 2
N–CH3) 2.9 (s, 3H, N–CH3).
To a solution of N,N′,N′-trimethylhydrazinecarboxylic
acid tert-butyl ester (0.52 g, 2.99 mmol) in methylene
chloride (4 mL) was added trifluoroacetic acid (4 mL)
and the mixture was stirred for 60 min. The mixture was
concentrated in vacuo and stripped three times with
methylene chloride to give 0.61 g of N,N,N′-
trimethylhydrazine trifluoroacetate as a thin oil. Then
(2R)-2-(tert-butoxycarbonylmethylamino)-3-phenyl pro-
pionic acid (1.0 g, 3.58 mmol) was dissolved in methyl-
ene chloride (5 mL) and 1-ethyl-3-(3-dimethyl-
To a solution of N-methyl-N-((1R)-1-(N-methyl-N-
[(1R)-2-phenyl-1-(N,N′,N′-trimethylhydrazinocarbonyl)
aminopropyl)carbodiimide
hydrochloride
(0.34 g,
1.79 mmol) was added and stirred for 30 min. Then a
ethyl]carbamoyl)-2-(2-naphthyl)ethyl)carbamic acid tert
-

495
butyl ester (0.43 g, 0.79 mmol) in methylene chloride
(2 mL) was added trifluoroacetic acid (2 mL) at 0 °C and
stirred for 30 min. The mixture was concentrated in
vacuo and stripped three times with methylene chloride.
The obtained oil was dissolved in methanol/ammonia
(2 mL) and added methylene chloride (20 mL) and silica
gel (5 g) and concentrated in vacuo. The obtained powder
was extracted by filtration with methylene chloride
(50 mL) and methylene chloride:methanol/ammonia (9:1)
and the combined extracts were concentrated in vacuo to
0.36 g of crude product as a foam. Then (2E)-5-(tert-
The compounds listed below were all prepared by the
general procedure above. The purity (determined by
HPLC) was generally in the 95–100% range.
5.4. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-2-(N′-acetyl-N′-methylhydrazino)-
1-benzyl-2-oxoethyl]-N-methylcarbamoyl)-
2-(2-naphthyl)ethyl)-N-methylamide (6)
LC-MS: Rt = 9.255 min, m/z = 586.4 (m + 1); HPLC:
Rt = 31.47 min (A1), Rt = 33.30 (B1); 1H-NMR (DMSO)
selected peaks: 5.3 (dd, 1H, C=CH–CO) 5.7 (t, 1H,
CH–CH2C6H5) 6.15 (d, 1H, CH2–CH=C) 6.4 (m, 1H,
CH–CH2C10H7).
butyloxycarbonylamino)-5-methylhex-2-enoic
acid
(0.12 g, 0.48 mmol) was dissolved in methylene chloride
(5 mL) and a mixture of 1-hydroxy-7-azabenzotriazole
(0.065 g, 0.48 mmol) and 1-ethyl-3-(3-dimethyl-
aminopropyl)-carbodiimide hydrochloride (0.092 g,
0.48 mmol) was added and stirred for 30 min. Then a
mixture of the obtained crude product (0.18 g, 0.40 mmol)
and diisopropylethylamine (0.09 mL, 0.53 mmol) was
added and the mixture was stirred overnight. Methylene
chloride (50 mL) was added and the mixture was washed
with water (10 mL). The organic layer was washed with
aqueous sodium bicarbonate (10 mL) and brine (50 mL),
dried (MgSO4), filtered, concentrated to 0.14 g of a
colourless oil. The oil was dissolved in methylene chlo-
ride (2 mL), cooled to 0 °C and trifluoroacetic acid
(2 mL) was added and the mixture was stirred for 30 min.
The reaction mixture was quenched with methylene
chloride (50 mL) and water (10 mL) and titrated to pH
7 with solid sodium bicarbonate. The organic layer was
separated and washed with water (10 mL), dried (MgSO4),
filtered and concentrated in vacuo. The obtained product
was dissolved in acetonitrile/water 1:20 (10 mL) and
applied to a C-18 Sep-Pak Classic© cartridge (2.0 g,
purchased from Waters™), which had been prewashed
with acetonitrile (100 mL) and water (100 mL). Then a
gradient of an eluent consisting of water/acetonitrile/
trifluoroacetic acid (10, 15, 20 and 25% acetonitrile in
water/trifluoroacetic acid) was run through the Sep-Pak©.
The relevant fractions were combined and lyophilised to
0.072 g of the trifluoroacetic acid salt of (2E)-5-amino-
5-methylhex-2-enoic acid N-methyl-N-((1R)-1-(N-methyl-
N-[(1R)-2-phenyl-1-(N,N′,N′-
5.5. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-2-(N′-acetyl-N-methylhydrazino)-
1-benzyl-2-oxoethyl]-N-methylcarbamoyl)-
2-(2-naphthyl)ethyl)-N-methylamide (7)
LC-MS: Rt = 8.74 min, m/z = 586.4 (m + 1); HPLC: Rt
= 29.53 (A1); Rt = 31.35 (B1); 1H-NMR (DMSO)
selected peaks: δ 1.05 (s, 6H, C–(CH3)2); 1.77 (s, 3H,
COCH3); 6.2 (d, 1H, C=CH–CO); 6.3 (m, 1H,
CH2–CH=C).
5.6. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-1-(N′,N′-dimethylhydrazino-
carbonyl)-2-phenylethyl]-N-methylcarbamoyl)-
2-(2-naphthyl)ethyl)-N-methylamide (8)
HPLC: Rt = 30.42 min (A1), Rt = 30.38 (B1), Rt =
8.40 min (H8); LC-MS: Rt = 8.99 min, m/z = 558.
5.7. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-{N-methyl-N-[(1R)-2-phenyl-
1-((piperidin-1-yl)carbamoyl)ethyl]carbamoyl}-
2-(2-naphthyl)ethyl)amide (9)
HPLC: Rt = 34.78 min (A1), Rt = 33.74 min (B1);
LC-MS: Rt = 9.81 min, m/z = 598.4 (m + 1).
trimethylhydrazinocarbonyl)ethyl]carbamoyl)-2-(2-
naphthyl)ethyl)amide as a white amorphous powder.
LC-MS: Rt = 9.96 min, m/z = 572.4 (m + 1). HPLC: Rt
5.8. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-(N-methyl-N-[(1R)-1-(N-
methyl-N-(piperidin-1-yl)carbamoyl)-2-phenylethyl]-
carbamoyl)-2-(2-naphthyl)ethyl)amide (10)
1
= 35.23 min (A1), Rt = 37.45 (B1); H-NMR (DMSO)
selected peaks: δ 5.1 (t, 1H, CH-CH2C6H5) 6.1 (t, 1H,
CH2–CH=C) 6.2 (d, 1H, C=CH–CO) 6.4 (t, 1H,
CH–CH2C10H7); anal (C34H45N5O3 x 2 C2H4O2 x ½
H2O) Calc: C 65.12; H 7.77; N 9.99; Found: C 64.54;
H7.73; N 9.97.
HPLC: Rt = 38.85 min (A1), Rt = 40.19 min (B1), Rt
= 11.42 min (H8); LC-MS: Rt = 12.11 min, m/z = 612.4
(m + 1).

496
5.9. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-1-benzyl-2-oxo-2-(3-oxopyrazolidin-
1-yl)ethyl]-N-methylcarbamoyl)-
5.15. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-(N-methyl-N-[(1R)-1-(N-
methyl-N-(piperidin-1-yl)carbamoyl)-2-(2-thienyl)ethyl]-
carbamoyl)-2-(2-naphthyl)ethyl)amide (17)
2-(2-naphthyl)ethyl)-N-methylamide (11)
HPLC: Rt = 39.47 min (A1), Rt = 41.43 min (B1), Rt
= 11.30 min (H8); LC-MS: Rt = 12.17 min, m/z = 618.4
(m + 1).
HPLC: Rt = 29.21 min (A1), Rt = 8.43 min (H8);
LC-MS: Rt = 9.24 min, m/z = 584.4 (m + 1).
5.10. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-{N-methyl-N-[(1R)-2-phenyl-
1-((pyrrolidin-1-yl)carbamoyl)ethyl]carbamoyl}-
2-(2-naphthyl)ethyl)amide (12)
5.16. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-(N-methyl-N-[(1R)-2-(2-thienyl)-1-
(N,N′,N′-trimethylhydrazinocarbonyl)-
ethyl]carbamoyl)-2-(2-naphthyl)ethyl)amide (18)
HPLC: Rt = 33.85 min (A1), Rt = 35.38 min (B1);
LC-MS: Rt = 10.02 min, m/z = 578.2 (m + 1).
HPLC: Rt = 34.80 min (A1), Rt = 34.04 min (B1);
LC-MS: Rt = 9.46 min, m/z = 610.4 (m + 1).
5.17. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-2-(biphenyl-4-yl)-1-{N-methyl-N-
[(1R)-2-phenyl-1-((pyrrolidin-1-yl)carbamoyl)-
ethyl]carbamoyl}ethyl)-N-methylamide (19)
5.11. (2E)-5-Amino-5-methylhex-2-enoic acid
N-methyl-N-((1R)-1-{N-methyl-N-[(1R)-2-phenyl-
1-((pyrrol-1-yl)carbamoyl)ethyl]carbamoyl}-
2-(2-naphthyl)ethyl)amide (13)
HPLC: Rt = 34.80 min (A1), Rt = 34.04 min (B1);
LC-MS: Rt = 9.46 min, m/z = 610.4 (m + 1).
HPLC: Rt = 35.94 min (A1), Rt = 37.55 min (B1);
LC-MS: Rt = 10.14 min, m/z = 580.4 (m + 1).
5.18. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-2-(biphenyl-4-yl)-1-{N-methyl-N-[(1R)-
2-(2-thienyl)-1-(N,N′,N′-trimethylhydrazinocarbonyl)
ethyl]carbamoyl}ethyl)-N-methylamide (20)
5.12. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-1-(N-[(1R)-1-(N′,N′-dimethylhydrazino-
carbonyl)-2-(2-thienyl)ethyl]-N-methylcarbamoyl)-
2-(2-naphthyl)ethyl)-N-methylamide (14)
HPLC: Rt = 36.92 min (A1), Rt = 39.91 min (B1);
LC-MS: Rt = 5.47 min, m/z = 604.2 (m + 1).
HPLC: Rt = 30.03 min (A1), Rt = 29.88 min (B1);
LC-MS: Rt = 8.97 min, m/z = 564.4 (m + 1).
5.19. Pharmacokinetic
and pharmacodynamic studies in dogs
5.13. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-2-(biphenyl-4-yl)-1-(N-methyl-N-[(1R)
-2-phenyl-1-((piperidin-1-yl)carbamoyl)ethyl]-
carbamoyl)ethyl)-N-methylamide (15)
Oral bioavailability studies were conducted in male
and female beagle dogs. The dogs were fasted overnight
prior to dosing. Diet was withheld for at least 3 h post
dosing. A 1 week wash-out period separated p.o. and i.v.
dosing. The compounds were administered in a vehicle of
citrate/phosphate buffer, pH 5.0 by stomach tubing. For
i.v. administration the dogs received a dose of 0.5 mg
compound/kg as a bolus in a hind leg vein. EDTA blood
samples were drawn from a front leg vein at intervals up
to 23 h after dosing. Blood samples were placed on
ice-water bath immediately after sampling. Plasma was
separated by centrifugation and stored frozen pending
analysis for canine GH and the compounds.
HPLC: Rt = 37.03 min (A1), Rt = 35.93 min (B1), Rt
= 10.45 min (H8); LC-MS: Rt = 10.24 min, m/z = 624.4
(m + 1).
5.14. (2E)-5-Amino-5-methylhex-2-enoic acid
N-((1R)-2-(biphenyl-4-yl)-1-(N-methyl-N-[(1R)-
2-phenyl-1-(N,N′,N′-trimethylhydrazinocarbonyl)-
ethyl]carbamoyl)ethyl)-N-methylamide (16)
The oral bioavailability (f) was calculated as the total
area under the plasma concentration versus time curve
following p.o. administration divided by the area follow-
ing i.v. administration, appropriately corrected for dose.
HPLC: Rt = 37.23 min (A1), Rt = 38.88 min (B1), Rt
= 10.88 min (H8); LC-MS: Rt = 11.14 min, m/z = 598.4
(m + 1).

497
[7] Bercu B.B., Walker R.F., Growth Hormone Secretagogues, Springer
Verlag, NY, 1996.
The AUC (0-tn) is the area under the plasma concentra-
tion curve from time zero to the last measurable concen-
tration estimated by the trapezoidal rule. λz is the terminal
elimination rate constant, calculated by log–linear regres-
sion. The systemic clearance, CL, is:
[8] Deghenghi R., Cananzi M.M., Torsello A., Battisti C., Muller E.E.,
Locatelli V., Life Sci. 54 (1994) 1321.
[9] Raun K., Hansen B.S., Johansen N.L., Thogersen H., Madsen K.,
Ankersen M., Andersen P.H., Eur. J. Endocrinol. 139 (1998) 552.
CL = doseiv/AUCiv
and the volume of distribution during the terminal phase,
Vz, is:
[10] Smith R.G., Cheng K., Schoen W.R., Pong S.S., Hickey G., Jacks T.
et al., Science 260 (1993) 1640.
[11] Patchett A.A., Nargund R.P., Tata J.R., Chen M.H., Barakat K.J.,
Johnston D.B.R. et al., Proc. Natl. Acad. Sci. USA 92 (1995) 7001.
Vz = doseiv/(AUCiv · λz)
[12] Hansen B.S., Raun K., Nielsen K.K., Johansen P.B., Hansen T.H.,
Peschke B. et al., Eur. J. Endocrinol. 141 (1999) 180–189.
[13] Conn P.M., Bowers C.Y., Science 273 (1996) 923.
Acknowledgements
[14] Howard A.D., Feighner S.D., Cully D.F., Arena J.P., Liberator P.A.,
Rosenblum C.I. et al., Science 273 (1996) 974.
We would like to thank Peter Andersen (synthesis)
Anette Heerwagen (in vitro pharmacology) and Edward
Kristensen (in vivo pharmacology and pharmacokinetics)
for excellent technical assistance.
[15] Ong H., McNicoll N., Escher E., Collu R., Deghenghi R., Locatelli
V. et al., Endocrinology 139 (1998) 432.
[16] Bengtsson B.A., Johannsson G., GH & IGF Research 8 (1998)
27–35.
[17] Yang L.H., Morriello G., Patchett A.A., Leung K., Jacks T., Cheng
K. et al., J. Med. Chem. 41 (1998) 2439–2441.
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