New highly potent dipeptidic growth hormone secretagogues with low molecular weight

Article abstract of DOI:10.1016/S0223-5234(00)00160-4

Based on NN703, low molecular weight growth hormone secretagouges (GHSs) with a reduced number of hydrogen binding sites were designed by removal of the C-terminal amide group. The compounds were highly potent in combination with high efficacy in a rat pituitary cell assay, being characterized with EC₅₀ values down to 0.8 nM. Selected compounds were tested in in vivo animal models. The oral bioavailability in dogs was 16-44%. Also, the ED₅₀ values of the compounds were determined both in dog and swine. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00160-4

Eur. J. Med. Chem. 35 (2000) 599−618
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001604/FLA
599
Original article
New highly potent dipeptidic growth hormone secretagogues with low molecular
weight
Bernd Peschkea*, Michael Ankersena, Thomas Kruse Hansena, Birgit Sehested Hansenb,
Jesper Laua, Karin Kramer Nielsenc, Kirsten Raund
aHealth Care Chemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark
bGeneral Cell Biology, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark
cPharmacokinetics, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark
dPharmacological Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark
Received 11 October 1999; revised and accepted 12 January 2000
Abstract – Based on NN703, low molecular weight growth hormone secretagouges (GHSs) with a reduced number of hydrogen binding sites
were designed by removal of the C-terminal amide group. The compounds were highly potent in combination with high efficacy in a rat
pituitary cell assay, being characterized with EC50 values down to 0.8 nM. Selected compounds were tested in in vivo animal models. The
oral bioavailability in dogs was 16–44%. Also, the ED50 values of the compounds were determined both in dog and swine. © 2000 Éditions
scientifiques et médicales Elsevier SAS
growth hormone secretagogue / NN703 / peptidomimetic / oral bioavailability / GH release
1. Introduction
[10] or NN703 [11, 12], or of non-peptidic nature, such as
benzolactams [13, 14].
Growth hormone secretagouges (GHSs) [1, 2] have
been a fast evolving area of medicinal chemistry and
clinical research since Momany et al. discovered the
hexapeptides GHRP-6 [3, 4] and GHRP-2 [5] that were
able to release growth hormone from the pituitary gland.
These findings gave the possibility to develop small,
orally available drugs for growth hormone deficient
patients. Most current methods of treatment use recom-
binant human growth hormone (hGH). A major drawback
of this treatment is the way of administration. As a
peptide of 191 amino acids the usual administration is by
injection.
It is known that GHSs act via a different receptor [6, 7]
than the known endogenous growth hormone releasing
hormone (GHRH), but the exact mechanism of action is
not known. Despite these uncertainties, a number of
clinical candidates have been developed during the years,
both of peptidic nature, such as hexarelin [8] or ipamo-
relin [9], of peptidomimetic nature, such as MK-0677
Analogues of NN703 (figure 1) have been synthesized
where variations have been made at the N-terminal [15]
and at the C-terminal [16, 17]. In this paper, we present
the synthesis of new NN703 analogues (figure 1) with
further reduced molecular weight. The working hypoth-
esis was, that a reduction of molecular weight would
improve the oral activity.
2. Chemistry
The backbone of a peptide does not usually contribute
to the binding of the peptide to a receptor. In a first
attempt we therefore decided to remove the amide group
at the C-terminal. The commercially available amine 1,
was reacted with BOC-protected D-N-(2-naphthyl)alanine
[12, 18, 19] with 1-hydroxy-7-azabenzotriazole (HOAt)
[20],
N-(dimethylaminopropyl)-N′-ethylcarbodiimide
(EDAC) and ethyldiisopropylamine as coupling reagent
and deprotected with trifluoroacetic acid to give amine 2.
This was coupled under the same conditions as above to
* Correspondence and reprints: bpes@novo.dk

600
Earlier results [10, 12, 22] suggested that a polar group
at the C-terminal improved potency. We therefore de-
cided to attach different polar groups such as alcohol,
amides or sulfonamides at the aromatic ring. The
2-hydroxyethyl chain was attached to phenol 5, which
had been synthesized from acid 4, via alkylation with
ethyl bromoacetate. The reduction furnished the desired
aminoalcohol 6 (figure 3).
The synthesis of the chain-homologue 11 was per-
formed via phenol 10, as it is outlined in figure 4, from
the commercially available acid 7, which was transferred
into the corresponding methylamide 8. The amide 8 was
reduced with sodium borohydride/iodine and the result-
ing amine was BOC-protected to yield the benzyl ether 9.
The free phenol 10 was obtained by catalytic hydrogena-
tion. Subsequent alkylation of phenyl 10 with 3-bromo-
1-propanol followed by deprotection of the amino group
with trifluoroacetic acid furnished the aminoalcohol 11.
Figure 1. Structure of compound NN703.
BOC-protected (2E)-5-amino-5-methylhex-2-enoic acid
[12]. The subsequent deprotection had to be carried out
carefully at 0 °C with 50% trifluoroacetic acid in dichlo-
romethane (figure 2). Prolonged reaction time under those
acidic conditions usually cleaved the resulting compound
3 between the phenethyl and the naphthylalanine moieties
[21].
In order to prepare the analogues 15 and 16, the key
intermediate 14 had to be prepared (figure 5). The reac-
tion of the known diamine 12 [23] with one equivalent of
di-tert-butyl dicarbonate gave a mixture of di- and
mono-protected amines 13 and 14, which were easily
separated on silica gel. The anilino group of 14 was
Figure 2. a) i BOC-N-Me-D-(2-naphthyl)alanine, EDAC, HOAt, DIPEA; ii 50% TFA in CH2Cl2, 0 °C; b) i BOC-5-methylhex-2-enoic
acid, EDAC, HOAt, DIPEA; ii 50% TFA in CH2Cl2, 0 °C.

601
Figure 3. a) i H2NCH3, HOBT, EDAC; ii ethyl bromoacetate, K2CO3, KI, acetone; b) NaBH4/I2, THF.
reacted with FMOC-protected glycine using EDAC as
coupling reagent or with methanesulfonyl chloride using
triethylamine as base in dichloromethane at –78 °C. After
deprotection of the amino group with trifluoroacetic acid,
amines 15 and 16 were obtained.
The compounds 17–28 (figure 6) were synthesized
either from known building blocks such as N-methyl-N-
(2-(2-thienyl)ethyl)amine [24] or N-methyl-N-(3-
phenylpropyl)amine [25] or the building blocks described
in this paper with BOC-protected amino acids as de-
scribed for the synthesis of 3. The synthesis of those
amino acids are described in the literature [12, 15]. When
27 was synthesized, the FMOC group was removed
before the final BOC-deprotection step with triethanol-
amine as base [26].
synthesized with cyclic constraints in the C-terminal
(figure 7). One synthesis started with commercially avail-
able tetrahydroisoquinoline leading to compound 29. The
cyclopropane-derivative 33 was obtained as a diastereo-
meric mixture from racemic trans-2-phenylcyclo-
propylamine (30), afterwards this was formylated with
formic acid and acetic anhydride to give 31, which
was subsequently reduced with sodium borohydride/
iodine [27] to give 32. A standard peptide-coupling
deprotection sequence furnished the cyclopropyl deriva-
tive 33 (figure 8).
In order to achieve cyclization in the central part of the
GHSs, we decided to prepare an amide to amide bridge.
Since we believe that the activity of the GHSs is mainly
achieved by the aromatic moieties and the amine and not
by the amide bonds, a cyclization of this type should not
influence the binding, other than restrict the number of
possible conformations. The synthesis of the cyclic central
moiety started with (2R)-2-amino-3-(2-naphthyl)propionic
Aiming for higher potency we decided to prepare
conformationally constrained analogues of 3. A cyclic
constraint can be introduced either in the C-terminal part
of the molecule or in the central part. Two examples were
Figure 4. a) H2NCH3, HOBT, EDAC; b) i NaBH4/I2, THF; ii (BOC)2O, NaOH; c) H2 Pd/C; d) i 3-bromopropan-1-ol, K2CO3, CsCl,
DMF; ii TFA, CH2Cl2.

602
Figure 5. a) (BOC)2O, NaOH, THF, H2O; b) i FMOCHNCH2COOH, EDAC; ii HCl, EtOAc; c) i CH3SO2Cl, NEt3, CH2Cl2, –78 °C;
ii TFA, CH2Cl2.
acid, which was subjected to an esterification and subse-
quently N-alkylated [28] to give ester 34 (figure 9). Upon
deprotection of the amino group, a ring formation oc-
curred after neutralization yielding piperazinone 35. Pro-
tection of the secondary amine followed by alkylation
under strongly basic conditions (potassium hydroxide in
DMSO) furnished the intermediate 36. The GHSs 37 and
38 were prepared from 36 by coupling of the known
BOC-protected amino acids [12, 15] and subsequent
deprotection.
As has been shown in the benzolactam series of GHSs
[13], an (R)-hydroxypropyl group as substitutent on the
free amine can be advantageous in achieving highly
potent compounds [29]. We therefore synthesized one
example (39) of this type, by reductive alkylation of 3
with the silyl protected 2-hydroxypropanal [30]. After
removal of the protection group with tetrabutylammo-
nium fluoride (TBAF), the aminoalcohol 39 was isolated
(figure 10).
ability was also tested in swine, since 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 or dog [12].
4. Results and discussion
The in vitro data of table I show that several potent
GHSs have been found within this series of compounds.
In compound 3, which is structurally closest to the
clinical candidate NN703, the number of groups which
are able to form hydrogen bonds was decreased. This
could eventually improve the oral availability and result
in an improved oral activity. Compound 3 was found to
have an EC50 of 32 nM. Even though this is a loss of
potency of nearly one order of magnitude, we were
excited to see this result. Both the exchange of the
benzene moiety to a thiophene (17) and an additional
methyl group at the N-terminal (21) gave compounds
with higher potency. The combination of those exchanges
gave compound 18, the most potent compound in this
series. Also, an exchange of the naphthalene with a
biphenyl-moiety, as shown in compound 24, enhanced
the activity to a compound almost equipotent to NN703.
The elongation of the spacer for the benzene moiety with
one methylene group in compound 19 resulted in a
significant loss of activity. Similarly, the change of the
structure of the N-terminal from a propendiyl spacer to a
phenylene spacer, which is realized in compound 20, led
to a compound with only weak GHS activity. This change
3. Pharmacology
The primary test in our screening was a determination
of the EC50 values in a rat pituitary cell assay. The
efficacy of the compounds was compared to the maximal
stimulation obtained with GHRP-6 [9]. Compounds,
showing high potency and efficacy in the primary screen-
ing, were tested in vivo. An oral bioavailability of at least
20% in beagle dogs was a minimum requirement for
further development of the compounds. The GH release

603
Figure 6. Structures of compounds 17–28.

604
Figure 6. Continued.
was perfectly tolerated in NN703 [15]. Our attempts to
achieve higher activity by reduction of conformational
freedom by cyclization failed. The different types of
cyclization at the C-terminal, as represented by com-
pounds 29 and 33, led to compounds that showed
significantly lower GH release. Similar results were
obtained when the cyclization was performed in the
central part of the molecules. The piperazinone 37 proved
to be the most potent of the cyclized analogues in this
series, which is almost equipotent to our initial compound
3. A simple change of the N-terminal amino acid resulted
in compound 38 with much lower activity. An additional
hydroxyl group located at the C-terminal seems to be
beneficial when it is positioned properly. It is known from
similar compounds [12], that such as a hydroxyl group
can enhance the GH releasing activity. The enhanced
activity of 22, when compared to 3, could be due to an
additional binding interaction of the GHS with the
receptor. This hypothesis is supported by the fact that an
elongation with only one methylene group destroys this
effect completely, as is shown with compound 28. The
rather poor activity of 39 suggests that the binding site for
the hydroxyl group is not in the vicinity of the N-terminal,
as there is a binding site for a hydroxyl group in the
benzolactam GHSs [29]. The additional amine moiety in
27 on the other hand, did not have a great influence on the
activity. The diamine 27 is equipotent to 3, which may be
due to the position of the amino group. Since we expected
a decreased oral bioavailability for diamines compared to
monoamines, we did not try to optimize the activity of
this class of compounds. A comparison of this type of
GHSs with MK-0677 suggested that an addition of a
sulfonamide group in the ortho-position of the benzene-
ring could be beneficial for the activity as GHS. The
impact on activity of the sulfonamide group was, how-
ever, limited; a slight enhancement in potency of the
sulfonamide 23 compared to compound 3 was observed.
This effect is completely diminished when a more bulky
group is attached to the sulfonamide, as demonstrated in
compound 26.
Figure 7. Structure of compound 29.

605
Figure 8. a) HCOOH, Ac2O; b) NaBH4/I2.
The most promising compounds 17, 18, 22, 23 and 25
were chosen for in vivo experiments. The results are
shown in table II. We expected very good oral availability
since the molecules were designed to be of low molecular
mass and to bear only a very limited number of hydrogen-
bonding elements. The oral bioavailability in dogs of all
five compounds was relatively high. The growth hormone
release induced by the compounds in dogs, however, is
much lower than would be suggested from the in vitro
data, when compared to NN703. Alcohol 22, for example,
is equipotent to NN703 with a slightly higher efficacy in
the in vitro rat pituitary cell assay, and has the same oral
availability. The amount of GH, which is released in the
dog is significantly lower than that released by NN703 at
the same dose. One explanation could be that the com-
pounds had a short plasma elimination half-life of ap-
proximately 1 h and therefore a short action of duration
and an overall low exposure of the compounds. A second
hypothesis is that more than one receptor [6, 7] is
involved in GH release, and that various GHSs act
differently at these subtypes.
5. Conclusion
We have found dipeptides which are highly potent
GHSs. The compounds show equipotency with the start-
ing lead compound NN703 in the in vitro rat pituitary cell
assay. The most active compounds in this assay are those
with either a (2-thienyl)ethyl or a hydroxyethoxy substi-
tuted ethylphenyl group at the C-terminal. As predicted
from empirical rules [31], the oral bioavailability of those
compounds is high. The high activity as GHS of the
thiophene containing compound 18, when given orally to
swine, shows that dipeptides with even lower molecular
weights than NN703 can be efficient GHSs. In general,
however, the growth hormone release compared to NN703
was not as high as predicted from the efficacy in the in
vitro rat pituitary cell assay. The C-terminal amide bond
of NN703 seems to have a major influence on the plasma
elimination half-life of NNC703, since compounds lack-
ing this amide bond show significantly shorter half-lives.
The poor in vivo efficacy of the compounds is most likely
due to overall low exposure of the compounds in vivo.
Similar observations were made when the compounds
were tested in swine after p.o. administration. Only
compound 18 induced a growth hormone secretion com-
parable to NN703. All other compounds showed much
weaker activity at 50 nmol/kg than the reference com-
pound.
6. Experimental protocols
6.1. Pharmacology
The in vitro studies were performed as described in the
literature [9, 32].

606
Figure 9. a) i CH3OH, SOCl2; ii BOCHNCH2CHO, NaCNBH3, mole sieves; b) i TFA; ii NaHCO3; c) i NaOH, H2O, (BOC)2O, THF;
ii KOH, DMSO PhCH2CH2Br; d) i TFA, CH2Cl2; ii (2E)-5-tert-butoxycarbonylamino-5-methylhex-2-enoic acid, HOAT, EDAC,
DIPEA; iii TFA, CH2Cl2; e) i TFA, CH2Cl2; ii (2E)-4-(1-(tert-butoxycarbonylamino)cyclobutyl)but-2-enoic acid, HOAT, EDAC,
DIPEA; iii TFA, CH2Cl2.
The oral bioavailability of each compound was studied
in a single male beagle dog, except for NN703 which was
studied in two male and two female dogs. The dogs were
fasted overnight prior to dosing. Diet was withheld for at
least 3 h post-dosing. A one-week washout period sepa-
rated oral (p.o.) and intravenous (i.v.) dosing. The com-
pounds were administered in a vehicle of citrate/phosphate
buffer, pH 5.0. For p.o. administration the dogs received
a dose of 2.5 mg/kg of body weight via gavage. For i.v.
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 calcu-
lated as the total area under the plasma concentration

607
Figure 10. a) i (R)-(tert-butyldimethylsilyloxy)propanal, NaCNBH3, HOAc; ii TBAF, THF.
versus time curve following p.o. administration divided
by the area following i.v. administration, appropriately
corrected for dose.
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 the in vivo characterisation in conscious swine,
four female 30–40 kg Danish slaughter swine of the breed
Landrace Yorkshire cross were used for each GH secre-
tagogue. 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
haloghane anaesthesia at day 0. The test compounds were
administered as 50 nmol/kg i.v. 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%
6.2. Chemistry
Amino acids were purchased from Synthetech or
Bachem. The MS analyses were performed on a PE Sciex
API 100 LC/MS System using a Waterst 3 × 150 mm
3.5 m C-18 Symmetry column and positive ionspray with
a flow rate of 20 mL/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. NMR spectra were obtained at 400
MHz on a Bruker instrument.
Table I. Initial screening, in vitro rat-pituitary cell assay (n = 2).
Entry
NN703
3 ± 1
3
17
18
19
20
21
EC50 [nM]
32 ± 9
90 ± 5
23
7.5 ± 0.5
135 ± 2
24
0.8b ± 0.1
115b ± 20
25
175 ± 125
65 ± 12
26
143 ± 58
100 ± 19
27
3 ± 3
105 ± 25
28
a
Emax
110 ± 7
22
Entry
EC50 [nM]
5 ± 4
18b ± 9
115b ± 14
33
5 ± 4
105 ± 26
37
5b ± 3
150b ± 9
38
80 ± 1
80 ± 5
39
27 ± 14
145 ± 27
40 ± 10
88 ± 12
a
Emax
120 ± 22
29
Entry
EC50 [nM]
60b ± 26
75b ± 12
165 ± 20
80 ± 17
45 ± 5
80 ± 8
140 ± 140
90 ± 18
750 ± 50
110 ± 143
a
Emax
b
a [% of GHRP-6]; n = 3.

608
Table II. In vivo data of selected compounds.
Entry
NN703d
17
18
22
23
25
fp.o. [%]a
44
16
20
44
25
21
t
[h]b
3.2
10.5
2.40
64
0.9
0.47
0.36
23
1.1
1.25
0.79
10
0.9
3.58
2.75
14
0.3
1.85
3.81
22
1.9
3.45
1.24
4
½
Volume of distribution [l/kg]b
Total clearance [L/(h kg)]b
Maximal GH release in dog [ng/mL]b
Maximal GH release in swine [ng/mL]c
25e
2
30
10
13
15
a In beagle dogs; b in beagle dogs after i.v. administration at 0.5 mg/kg; c at 50 nmol/kg compound in swine after p.o. administration; d NN703
was tested in two male and two female beagle dogs (results are therefore listed as means of four), all other compounds were tested in one male
beagle dog; e extrapolation from a dose–response curve (Hill plot) using 1, 13, 30, 100, 300, 1 000 and 10 000 nmol/kg.
6.2.1. HPLC-methods
6.2.2. Protocols
Three different elution conditions were used.
Method A1:
6.2.2.1. (2R)-2-(Methylamino)-3-(2-
naphthyl)propionic acid N-methyl-N-phenethylamide 2
(2R)-2-(N-tert-Butoxycarbonyl-N-methylamino)-3-(2-
naphthyl)propionic acid (1.40 g, 4.3 mmol) was dissolved
in N,N-dimethylformamide (5 mL) and dichloromethane
(5 mL). Hydroxy-7-azabenzotriazole (0.59 g, 4.3 mmol)
was added as a solid. The solution was cooled to 0 °C.
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydro-
chloride (0.99 g, 5.2 mmol) was added. The solution was
stirred for 20 min at 0 °C. N-Methyl-N-phenethylamine
(1, 0.86 mL, 6.0 mmol) was added. The solution was
stirred for 16 h, while it was warming up to room
temperature. It was diluted with water (300 mL) and ethyl
acetate (150 mL). Ten percent sodium hydrogen sulfate
solution (80 mL) was added. The phases were separated.
The aqueous phase was extracted with ethyl acetate (4 ×
50 mL). The combined organic layers were washed with
saturated sodium hydrogen carbonate solution (200 mL)
and dried over magensium sulfate. The solvent was
removed in vacuo. The crude product was purified by
flash chromatography on silica (90 g), using ethyl acetate/
heptane 1:1 as eluent to give 1.89 g (100%) of N-methyl-
N-((1R)-1-(N-methyl-N-phenethylcarbamoyl)-2-(2-naph-
thyl)ethyl)carbamic acid tert-butylester. 1H-NMR
(CDCl3): δ 1.01, 1.09, 1.25 and 1.30 (all s, together 9 H);
2.60–3.85 (m, 12 H); 4.75, 5.03, 5.31 and 5.37 (all dd,
together 1 H); 7.00–7.85 (m, 12 H).
The RP-HPLC analysis was performed using UV
detections at 214, 254, 276 and 301 nm on a Vydac
218TP54 4.6 × 250 mm 5m C-18 silica column (The
Seperations Group, Hesperia), which was eluted at 1 mL/
min at 42 °C. 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 for 50 min.
Method B1:
The RP-HPLC analysis was performed using UV
detections at 214, 254, 276, and 301 nm on a Vydac
218TP54 4.6 × 250 mm 5m C-18 silica column (The
Seperations Group, Hesperia), which was eluted at 1 mL/
min at 42 °C. 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 for 50 min.
Method H8:
The RP-HPLC analysis was performed using UV
detections at 214 and 254 nm on a 218TP54 4.6 ×
150 mm C-18 silica column, which was eluted at 1 mL/
min at 42 °C. The column was equilibrated with 5%
acetonitrile, 85% water and 10% of a solution of 0.5%
trifluoroacetic acid in water and eluted by a linear
gradient from 5% acetonitrile, 85% water and 10% of a
solution of 0.5% trifluoroacetic acid to 90% acetonitrile
and 10% of a solution of 0.5% trifluoroacetic acid over
15 min.
N-Methyl-N-((1R)-1-(N-methyl-N-phenethylcarbamoyl)-
2-(2-naphthyl)ethyl)carbamic acid tert-butylester (1.84 g,
4.12 mmol) was dissolved in dichloromethane (6 mL). The
solution was cooled to 0 °C. Trifluoroacetic acid (6 mL)
was added. The solution was stirred for 10 min at 0 °C. The

609
solvent was removed in vacuo at 20 °C. The residue was
dissolved in dichloromethane (100 mL) and the solvent
was removed in vacuo. This latter procedure was repeated
twice. The crude product was purified by flash chroma-
tography on silica (70 g), using dichloromethane/
methanol/25% aqueous ammonia (100:10:1) as eluent, to
give 350 mg (25%) of amine 2. 1H-NMR (CDCl3): δ 1.72
(br, 1 H); 2.12, 2.30, 2.44 and 2.87 (all s, together 6 H);
2.58, 2.76, 2.91, 2.98, 3.09, 3.25, 3.50, 3.61 and 3.73 (all
m, together 7 H); 6.90–7.85 (m, 12 H).
(100 mL) was added. A saturated aqueous solution of
sodium hydrogen carbonate (70 mL) was added. Solid
sodium hydrogen carbonate was added until pH 7 was
obtained. The phases were separated. The aqueous phase
was extracted with dichloromethane (3 × 70 mL). The
combined organic layers were dried over magnesium
sulfate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(90 g), using dichloromethane/methanol/25% aqueous
ammonia 100:10:1 as eluent, to give 1.08 g (75%) of
amine 3 at 20 °C. HPLC: Rt 33.92 min; purity: 95%
(254 nm; A1). Rt 35.67 min; purity: 88% (214 nm; B1).
NMR (CDCl3, selected values, free base): δ 1.04, 1.05,
1.11 and 1.12 (all s, together 6 H); 5.78 and 5.87 (both dd,
together 1 H); 6.14 and 6.23 (both d, together 1 H); 6.78
and 6.87 (both dt, together 1 H). MS: calc. for [M + H]+:
472; found 472.1.
6.2.2.2. (2R)-2-(N-((2E)-5-Amino-5-
methylhex-2-enoyl)-N-methylamino)-3-(2-
naphthyl)propionic acid N-methyl-N-phenethylamide 3
(2E)-5-(tert-Butoxycarbonylamino)-5-methylhex-3-enoic
acid (913 mg, 3.75 mmol) was dissolved in dichloromethane
(10 mL). Hydroxy-7-azabenzotriazole (511 mg, 3.75 mmol)
was added as a solid. The solution was cooled to 0 °C.
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (719 mg, 3.75 mmol) was added. The solution
was stirred for 10 min at 0 °C. Amine 2 (1.30 g,
3.75 mmol) was dissolved in dichloromethane (10 mL)
and N,N-dimethylformamide (10 mL) and added to the
reaction mixture. Ethyldiisopropylamine (0.64 mL,
3.75 mmol) was added. The reaction mixture was stirred
for 16 h while it was warming to room temperature. The
solution was diluted with ethyl acetate (150 mL). Ten
percent aqueous sodium hydrogen sulfate solution
(50 mL) was added. The phases were separated, and the
aqueous phase was extracted with ethyl acetate
(4 × 50 mL). The combined organic layers were washed
with saturated sodium hydrogen carbonate solution
(200 mL) and dried over magnesium sulfate. The solvent
was removed in vacuo. The crude product was purified by
flash chromatography on silica (90 g), using ethyl acetate/
heptane 1:1 as eluent, to give 1.76 g (82%) of (3E)-1,1-
dimethyl-4-(N-methyl-N-((1R)-1-(N-methyl-N-phenethyl-
carbamoyl)-2-(2-naphthyl)ethyl)carbamoyl)but-3-enyl-
6.2.2.3. Ethyl 2-(2-((N-methyl-
carbamoyl)methyl)phenoxy)acetate 5
(2-Hydroxyphenyl)acetic acid (4, 9.89 g, 63.7 mmol)
and 1-hydroxybenzotriazole hydrate (8.61 g, 63.7 mmol)
were dissolved in N,N-dimethylformamide (50 mL) and
dichloromethane (200 mL). The solution was cooled to
0 °C. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (8.67 g, 63.7 mmol) was added. The reac-
tion mixture was stirred for 30 min at 0 °C. An 8.0 M
solution of methyl amine (39 mL, 318 mmol) was added.
The reaction mixture was stirred for 16 h while it was
slowly warming up to room temperature. It was diluted
with ethyl acetate (600 mL) and washed with a 10%
aqueous solution of sodium hydrogen sulfate (2 ×
300 mL). The combined aqueous phases were extracted
with ethyl acetate. The combined organic layers were
washed with saturated sodium hydrogen carbonate
solution (300 mL) and dried over magnesium sulfate.
The solvent was removed in vacuo. The crude product
was purified by flash chromatography on silica (180 g),
using ethyl acetate/heptane (1:1) as eluent to give 4.90 g
(47%) of 2-(2-hydroxyphenyl)-N-methylacetamide. M.p.:
105–106 °C (ethyl acetate/heptane). 1H-NMR (CDCl3): δ
2.82 (d, 3 H); 3.56 (s, 2 H); 6.20 (br, 1 H); 6.83 (m, 1 H);
7.00 (m, 2 H); 7.18 (m, 1 H); 9.85 (s, 1 H).
1
carbamic acid tert-butylester. H-NMR (CDCl3): δ 1.14,
1.17, 1.23 and 1.26 (all s, together 6 H); 1.38 and 1.41
(both s, together 9H); 2.40–3.10, 3.30–3.60 and 3.92 (all
m, together 8 H); 2.78, 2.89 and 3.03 (all s, together 6 H);
4.28 and 4.40 (both br, together 1 H); 5.78 and 5.85 (both
dd, together 1 H); 6.15 and 6.23 (both d, together 1 H);
6.70 and 6.80 (both m, together 1 H); 7.00–7.85 (m,
12 H).
(3E)-1,1-Dimethyl-4-(N-methyl-N-((1R)-1-(N-methyl-
N-phenethylcarbamoyl)-2-(2-naphthyl)ethyl)carbamoyl)-
but-3-enylcarbamic acid tert-butylester (528 mg,
0.85 mmol) was dissolved in dichloromethane (10 mL).
Trifluoroacetic acid (10 mL) was added. The solution was
stirred at room temperature for 50 min. Dichloromethane
Potassium carbonate (2.81 g, 20.34 mmol) was given
to a solution of 2-(2-hydroxyphenyl)-N-methylacetamide
(3.36 g, 20.34 mmol) in acetone (150 mL). Ethyl bromo-
acetate (2.13 mL, 19.32 mmol) and potassium iodide
(166 mg, 1.02 mmol) were added successively. The reac-
tion mixture was heated to reflux for 6 h. It was left at
room temperature for 16 h. The solid was filtered off. The
solvent was removed in vacuo. The crude product was
purified by flash chromatography on silica (80 g), using

610
ethyl acetate/dichloromethane (1:1) as eluent, to give
5.00 g (98%) of ether 5. H-NMR (CDCl3): δ 1.33 (t, 3
H); 2.74 (d, 3 H); 3.61 (s, 2 H); 4.30 (q, 2 H); 4.70 (s, 2
H); 6.68 (br, 1 H); 6.76 (d, 1 H); 6.98 (t, 1 H); 7.24 (t, 1
H); 7.32 (d, 1 H).
and dried over magnesium sulfate. The solvent was
removed in vacuo to give 10.24 g (65%) of amide 8.
1H-NMR (DMSO-d6): δ 2.57 and 2.58 (both s, together 3
H); 3.44 (s, 2 H); 5.60 and 5.61 (both s, together 2 H);
6.89 (t, 1 H); 7.03 (d, 1 H); 7.19 (m, 2 H); 7.30 (m, 1 H);
7.38 (m, 2 H); 7.45 (m, 2 H); 7.69 (br, 1 H).
1
6.2.2.4. 2-(2-(2-(Methylamino)ethyl)phenoxy)ethanol 6
At 0 °C, a solution of ethyl ether 5 (5.00 g, 19.9 mmol)
in tetrahydrofuran (75 mL) was added dropwise to a
suspension of sodium borohydride (2.26 g, 59.7 mmol) in
tetrahydrofuran (75 mL). A solution of iodine (5.05 g,
19.9 mmol) in tetrahydrofuran (150 mL) was added drop-
wise. The solution was warmed to room temperature and
heated to reflux for 16 h. It was cooled to 0 °C. Methanol
(150 mL) was added dropwise. The solvent was removed
in vacuo. The solid residue was dissolved in 20% aqueous
sodium hydroxide solution/tert-butyl methyl ether
(150 mL/150 mL). The phases were separated. The aque-
ous phase was extracted with tert-butyl methyl ether (3 ×
150 mL). The combined organic layers were dried over
magnesium sulfate. The solvent was removed in vacuo.
The crude product was purified by flash chromatography
on silica (80 g), using dichloromethane/methanol/10%
aqueous ammonia (first: 100:10:1, then 70:30:3) as elu-
ent, to give 1.12 g (29%) of alcohol 6. 1H-NMR (CDCl3):
δ 2.40 (s, 3 H); 2.82 (m, 2 H); 2.92 (m, 2 H); 3.05 (br, 2
H); 3.94 (m, 2 H); 4.10 (m, 2 H); 6.87 (d, 1 H); 6.92 (t,
1 H); 7.17 (m, 2 H).
6.2.2.6. N-(2-(2-Benzyloxyphenyl)-
ethyl)-N-methylcarbamic acid tert-butyl ester 9
At 0 °C, a solution of amide 8 (9.39 g, 36.8 mmol) in
tetrahydrofuran (150 mL) was added dropwise to a sus-
pension of sodium borohydride (1.67 g, 44.12 mmol) in
tetrahydrofuran (100 mL). After the addition was fin-
ished, a solution of iodine (4.67 g, 18.39 mmol) in
tetrahydrofuran (200 mL) was added dropwise. The so-
lution was warmed to reflux for 16 h. It was cooled to
0 °C. Methanol (200 mL) was added dropwise. The
solvent was removed in vacuo. The residue was dissolved
in a 20% aqueous solution of sodium hydroxide (200 mL)
and tert-butyl methyl ether (200 mL). The phases were
separated. The aqueous phase was extracted with tert-
butyl methyl ether (3 × 75 mL). The combined organic
layers were dried over magnesium sulfate. The solvent
was removed in vacuo. The crude product was purified
by flash chromatography on silica (400 g), using
dichloromethane/methanol/25% aqueous ammonia as
eluent (100:10:1), to give 4.73 g (38%) of N-(2-
(2-benzyloxyphenyl)ethyl)-N-methylamine.
(CDCl3): δ 2.40 (s, 3 H); 2.70 (br, 1 H); 2.87 (m, 4 H);
1H-NMR
6.2.2.5. 2-(2-Benzyloxyphenyl)-N-methylacetamide 8
2-(2-Benzyloxyphenyl)acetic acid (7, 15.0 g, 62 mmol)
was dissolved in dichloromethane (270 mL) and N,N-
dimethylformamide (70 mL). 1-Hydroxybenzotriazole
(8.37 g, 62 mmol) was added. The solution was cooled to
0 °C. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (11.89 g, 62 mmol) was added. The reac-
tion mixture was stirred for 20 min. An 8.0 M solution of
methylamine in ethanol (38.8 mL, 310 mmol) was added.
The solution was stirred for 16 h while it was warming up
to room temperature. It was diluted with ethyl acetate
(500 mL) and washed with a 10% aqueous solution of
sodium hydrogen sulfate solution (500 mL). The aqueous
phase was extracted with ethyl acetate (2 × 300 mL). The
combined organic layers were washed with a saturated
aqueous solution of sodium hydrogen carbonate (400 mL)
and dried over magnesium sulfate. The solvent was
removed in vacuo. The remaining crystals were washed
with a mixture of ethyl acetate/heptane 1:4 (100 mL).
They were dried in vacuo. They were dissolved in ethyl
acetate. The solution was washed with a saturated aque-
ous solution of sodium hydrogen carbonate (2 × 500 mL)
5.07 (s, 2 H); 6.89 (m, 2 H); 7.18 (m, 2 H); 7.35 (m, 5 H).
A solution of di-tert-butyl dicarbonate (3.80 g,
17.4 mmol) in tetrahydrofuran (8.7 mL) was added drop-
wise to a solution of N-(2-(2-benzyloxyphenyl)ethyl)-N-
methylamine (3.82 g, 15.8 mmol) in tetrahydrofuran
(8.7 mL) and a 1 N aqueous sodium hydroxide solution
(17.4 mL, 17.4 mmol). The reaction mixture was stirred
for 16 h at room temperature. It was diluted with ethyl
acetate (200 mL) and water (200 mL). The phases were
separated. The aqueous phase was extracted with ethyl
acetate (200 mL). The combined organic layers were
washed with a saturated aqueous solution of sodium
hydrogen carbonate (400 mL) and dried over magnesium
sulfate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(225 g), using ethyl acetate/heptane 1:4 as eluent, to give
1
4.73 g (88%) of carbamate 9. H-NMR (CDCl3): δ 1.32
and 1.42 (both br, together 9 H); 2.74 and 2.86 (both br,
together 5 H); 3.43 (t, 2 H); 5.08 (s, 2 H); 6.89 (m, 2 H);
7.17 (br, 2 H); 7,40 (m, 5 H).

611
6.2.2.7. N-(2-(2-Hydroxyphenyl)-
ethyl)-N-methylcarbamic acid tert-butyl ester 10
combined and dried over magnesium sulfate. The solvent
was removed in vacuo to give 227 mg (57%) of crude
aminoalcohol 11. The crude product was used in the next
Carbamate 9 (4.66 g, 13.65 mmol) was dissolved in
ethanol (35.6 mL) and was hydrogenated at room pres-
sure in the presence of 10% palladium on activated
carbon for 16 h. The reaction mixture was filtered through
a plug of celite. The celite was washed with ethyl acetate
(50 mL). The liquid phases were collected. The solvents
were removed in vacuo. The crude product was purified
by flash chromatography on silica (300 g), using ethyl
acetate/heptane (1:2) as eluent, to give 2.84 g (83%) of
1
step without further purification. H-NMR (CDCl3): δ
1.19 (s, 1 H); 2.03 (m, 2 H); 2.25 (br, 1 H); 2.39 (s, 3 H);
2.83 (m, 4 H); 3.87 (m, 2 H); 4.10 (m, 2 H); 5.90 (m, 2
H); 7.15 (m, 2 H).
6.2.2.9. {2-[2-(N-(tert-Butoxycarbonyl)-
N-methylamino)ethyl]-phenyl}carbamic acid
tert-butyl ester 13 and N-(2-(2-amino-
1
phenyl)ethyl)-N-methylcarbamic acid tert-butyl ester 14
A solution of di-tertbutoxy dicarboxylate (7.78 g,
36 mmol) in tetrahydrofuran (18 mL) was added to a
solution of amine 12 (4.87 g, 32 mmol) [23] in tetrahy-
drofuran (18 mL) and a 1 N aqueous sodium hydroxide
solution (36 mL, 36 mmol). The reaction mixture was
stirred for 16 h at room temperature. It was diluted with
ethyl acetate (400 mL) and water (300 mL). The phases
were separated. The aqueous phase was extracted with
ethyl acetate (4 × 100 mL). The combined organic layers
were washed with a saturated aqueous solution of sodium
hydrogen carbonate (300 mL) and dried over magnesium
sulfate. The solvent was removed in vacuo. Flash chro-
matography on silica (60 g), using ethyl acetate/heptane
1:2 gave 4.95 g (44%) of dicarbamate 13 and 3.08 g
(38%) of monocarbamate 14.
phenol 10. H-NMR (CDCl3): δ 1.45 (s, 9 H); 2.86 (t, 2
H); 2.90 (s, 3 H); 3.34 (br, 2 H); 6.81 (t, 1 H); 6.87 (br,
1 H), 7.03 (d, 1 H), 7.12 (t, 1 H).
6.2.2.8. 3-[2-(2-Methylamino-
ethyl)phenoxy]propan-1-ol 11
Phenol 10 (702 mg, 2.79 mmol) was dissolved in
N,N-dimethylformamide (6 mL). Potassium carbonate
(1.93 g, 13.97 mmol) and caesium chloride (24 mg,
0.14 mmol) were added. 3-Bromo-1-propanol (0.28 mL,
3.07 mmol) was added. The reaction mixture was stirred
at 80 °C for 16 h. It was cooled to room temperature and
diluted with ethyl acetate (75 mL) and water (75 mL).
The phases were separated. The aqueous phase was
extracted with ethyl acetate (2 × 50 mL). The combined
organic layers were washed with a 10% aqueous solution
of sodium hydrogen sulfate solution (70 mL) and a
saturated aqueous solution of sodium hydrogen carbonate
(70 mL). They were dried over magnesium sulfate. The
solvent was removed in vacuo. The crude product was
purified by flash chromatography on silica (80 g), using
ethyl acetate/heptane (1:1) as eluent, to give 606 mg
(70%) of {2-[2-(3-hydroxypropoxy)phenyl]ethyl}-N-
13: 1H-NMR (CDCl3): δ 1.45 (br, 9 H); 1.54 (s, 9 H);
2.78 (t, 2 H); 2.88 (s, 3 H); 3.30 (br, 2 H); 6.98 (br, 1 H);
7.08 (br, 1 H); 7.20 (t, 1 H); 7.60–8.10 (br, 2 H).
1
14: H-NMR (CDCl3): δ 1.47 (s, 9 H); 2.75 (t, 2 H);
2.90 (s, 3 H); 3.35 (br, 2 H); 3.71 (br, 1 H); 4.23 (br, 1 H);
6.68 (m, 2 H); 7.00 (d, 1 H); 7.05 (t, 1 H).
MS: calc. for [M + H]+: 152; found: 151.2.
1
methylcarbamic acid tert-butyl ester. H-NMR (CDCl3):
δ 1.39 (br, 9 H); 2.06 (m, 2 H); 2.82 (br, 5 H); 3.45 (br,
2 H); 3.90 (br, 2 H); 4.14 (t, 2 H); 6.85 (m, 2 H); 7.09 (br,
1 H); 7.17 (t, 1 H).
6.2.2.10. {[2-(2-(Methylamino)ethyl)phenylcarbamoyl]-
methyl}carbamic acid 9H-((fluoren-9-yl)methyl) ester 15
2-(((9-Fluorenyl)methoxycarbonyl)amino)acetic acid
(2.49 g, 8.36 mmol) was suspended in dichloromethane
(40 mL). The suspension was cooled to 0 °C. N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (802 mg, 4.19 mmol) was added. The reaction mix-
ture was stirred for 30 min at 0 °C. A solution of amine 14
(698 mg, 2.79 mmol) in dichloromethane (15 mL) was
added. The reaction mixture was stirred for 16 h while it
was warming up to room temperature. It was diluted with
dichloromethane (100 mL) and washed with brine
(100 mL). The aqueous solution was extracted with
dichloromethane (2 × 30 mL). The combined organic
layers were dried over magnesium sulfate. The solvent
was removed in vacuo. The crude product was purified by
flash chromatography on silica (80 g), using ethyl acetate/
{2-[2-(3-Hydroxypropoxy)phenyl]ethyl}-N-methyl-
carbamic acid tert-butyl ester (0.587 g, 1.90 mmol) was
dissolved in dichloromethane (5 mL). The solution was
cooled to 0 °C. Trifluoroacetic acid (5 mL) was added.
The reaction mixture was stirred for 30 min at 0 °C.
Dichloromethane (50 mL) was added. A saturated aque-
ous solution of sodium hydrogen carbonate (50 mL) was
added dropwise. Solid sodium hydrogen carbonate was
added, until pH 7 was obtained. The phases were sepa-
rated. The aqueous solution was extracted with dichlo-
romethane (3 × 70 mL). The aqueous phase was made
basic to pH 14 with a 20% aqueous sodium hydroxide
solution. It was extracted with tert-butyl methyl ether
(3 × 100 mL). The tert-butyl methyl ether extracts were

612
heptane (1:1) as eluent, to give 1.41 g (96%) of ({2-[2-
(N-(tert-butoxycarbonyl)-N-methylamino)ethyl]phenyl-
carbamoyl}methyl)carbamic acid ((9-fluorenyl)methyl)
ester. 1H-NMR (CDCl3, selected values): δ 1.50 (br, 9 H);
2.80 (m, 2 H); 2.98 (br, 3 H); 3.22 (m, 2 H); 4.25 (m, 3
H); 4.40 (m, 2 H); 6.32 (br, 1 H); 9.20 (br, 1 H).
was purified by flash chromatography on silica (60 g),
using dichloromethane/methanol/25% aqueous ammonia
(100:10:1) as eluent to give 308 mg (53%) of amine 16.
1H-NMR (CDCl3) δ 2.51 (s, 3 H); 2.84 (m, 2 H); 2.94 (m,
2 H); 3.00 (s, 3 H); 5.70–6.70 (br, 1 H); 7.03 (m, 1 H);
7.12 (d, 1 H); 7.22 (t, 1 H); 7.53 (d, 1 H).
({2-[2-(N-(tert-Butoxycarbonyl)-N-methylamino)ethyl]-
phenylcarbamoyl}methyl)carbamic acid ((9-fluorenyl)-
methyl) ester (1.342 g, 2.53 mmol) was dissolved in 3.0
M hydrogen chloride in ethyl acetate (10 mL). The
reaction mixture was stirred for 2 h at room temperature.
Diethyl ether (40 mL) was added. The precipitation was
filtered off and dried in vacuo to give 857 mg (73%) of
crude amine 15 as hydrochloride, which was used for the
6.2.2.12. (2E)-5-Amino-5-methyl-
N-methyl-N-((1R)-1-(N-methyl-N-(2-(2-thienyl)-
ethyl)carbamoyl)-2-(2-naphthyl)ethyl)hex-2-enamide 17
Compound 17 (478 mg) (46% over 4 steps) was
synthesized analogously to compound 3, starting with
N-methyl-N-(2-(2-thienyl)ethyl)amine [24] instead of
amine 1. HPLC: Rt = 33.48 min; purity: 91% (254 nm;
A1). Rt = 35.13 min; purity: 86% (214 nm; B1). 1H-NMR
(CDCl3, selected values): δ 1.04, 1.05, 1.11 and 1.11 (all
s, together 6 H); 2.80, 2.90, 3.04 and 3.07 (all s, together
6 H); 5.83 and 5.88 (both dd, together 1 H); 6.14 and 6.25
(both d, together 1 H). MS: calc. for [M + H]+: 478;
found: 478.2. For biological testing, the title compound
was dissolved in 0.5 M acetic acid (25 mL) and lyo-
philized.
1
next step without purification. H-NMR (DMSO-d6, se-
lected values): δ 2.99 (br, 4 H); 9.05 (br, 2 H); 9.68 (br,
1 H).
6.2.2.11. N-(2-(2-(Methylamino)-
ethyl)phenyl)methanesulfonamide 16
A solution of amine 14 (723 mg, 2.9 mmol) and
triethylamine (0.48 mL, 3.5 mmol) in dichloromethane
(10 mL) was cooled to –78 °C. A solution of methane-
sulfonyl chloride (0.22 mL, 2.9 mmol) in dichloromethane
(2 mL) was added dropwise. The reaction mixture was
stirred for 16 h while it was warming up to room
temperature. It was diluted with ethyl acetate (50 mL)
and washed with 10% aqueous sodium hydrogen sulfate
solution (150 mL). The aqueous phase was extracted with
ethyl acetate (3 × 80 mL). The combined organic layers
were washed with saturated aqueous sodium hydrogen
carbonate solution (150 mL) and dried over magnesium
sulfate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(60 g), using ethyl acetate/heptane (1:1) as eluent, to give
870 mg (92%) of N-(2-(2-(methylsulfonylamino)phenyl)-
6.2.2.13. (2E)-5-Methyl-5-(methylamino)-
N-methyl-N-((1R)-1-(N-methyl-N-(2-(2-thienyl)-
ethyl)carbamoyl)-2-(2-naphthyl)ethyl)hex-2-enamide 18
Compound 18 (164 mg) (30% over 4 steps) was
synthesized analogously to compound 3 using N-methyl-
N-(2-(2-thienyl)ethyl)amine [24] instead of amine 1
and (2E)-5-(N-(tert-butoxycarbonyl)-N-methylamino)-5-
methylhex-2-enoic acid instead of (2E)-5-(tert-
butoxycarbonylamino)-5-methylhex-2-enoic acid. HPLC:
Rt = 33.27 min; purity: 89% (254 nm;A1). Rt = 35.28 min;
1
purity: 94% (214 nm; B1). H-NMR (CDCl3, selected
values): δ 1.00 and 1.08 (both s, together 6 H); 2.23 and
2.30 (both s, together 3 H); 2.80, 2.89, 3.04 and 3.07 (all
s, together 6 H); 5.85 (m, 1 H); 6.13 and 6.25 (both d,
together 1 H). MS: calc. for [M + H]+: 492; found: 492.0.
For biological testing, it was transformed into the acetate
by lyophilization from 0.5 N acetic acid (25 mL).
1
ethyl)-N-methylcarbamic acid tert-butyl ester. H-NMR
(CDCl3) δ 1.50 (s, 9 H); 2.87 (m, 2 H); 2.91 (s, 3 H); 3.02
(s, 3 H); 3.30 (br, 2 H); 7.05–7.30 (m, 3 H); 7.57 (br, 1 H);
8.65 (br, 1 H).
At 0 °C, trifluoroacetic acid (6 mL) was added to a
solution of N-(2-(2-(methylsulfonylamino)phenyl)ethyl)-
N-methylcarbamic acid tert-butyl ester (870 mg,
2.6 mmol) in dichloromethane (6 mL). The reaction mix-
ture was stirred for 50 min. Dichloromethane (25 mL)
was added. A saturated aqueous solution of sodium
hydrogen carbonate (35 mL) was added. Solid sodium
hydrogen carbonate was added, until pH 7 was obtained.
The phases were separated. The aqueous phase was
extracted with dichloromethane (3 × 50 mL). The com-
bined organic layers were dried over magnesium sulfate.
The solvent was removed in vacuo. The crude product
6.2.2.14. (2E)-5-Amino-5-methylhex-2-
enoic acid N-methyl-N-((1R)-1-(N-methyl-N-(3-
phenylpropyl)carbamoyl)-2-(2-naphthyl)ethyl)amide 19
Compound 19 (436 mg) (20% over 4 steps) was
prepared analogously to compound 3. N-Methyl-N-(3-
phenylpropyl)amine [25] was used instead of amine 1.
HPLC: Rt = 36.62 min; purity: 96% (254 nm; A1). Rt =
38.93 min; purity: 92% (214 nm; B1). 1H-NMR (CDCl3,
selected values): δ 1.10 and 1.11 (both s, together 6 H);
2.85, 3.13, 3.15, and 3.50 (all s, together 6 H); 5.89 and
5.97 (both dd, together 1 H); 6.23 and 6.24 (both d,
together 1 H). MS: calc. for [M + H]+: 486; found: 486.4.

613
For biological testing, the title compound was transferred
into its acetate salt by lyophilization from 0.5 M aqueous
acetic acid (50 mL).
pound was transferred into its acetate salt by lyophiliza-
tion with 0.5 M acetic acid (50 mL).
6.2.2.18. (2R)-2-(N-((2E)-5-Amino-5-methylhex-
6.2.2.15. (2R)-2-(N-(3-(1-Aminoethyl)-
2-enoyl)-N-methylamino)-N-methyl-3-(2-naphthyl)-N-
(2-(2-methylsulfonylaminophenyl)ethyl)propionamide 23
Compound 23 (82 mg) (12% over 4 steps) was pre-
pared analogously to compound 3. Amine 16 was used
instead of amine 1. HPLC: Rt = 32.08 min; purity: 94%
(254 nm; A1). Rt = 32.53 min; purity: 92% (214 nm; B1).
1H-NMR (CDCl3, selected values) δ 1.08 and 1.15 (both
s, together 1 H); 2.93 and 2.95 (both s, together 3 H); 2.99
and 3.05 (both s, together 3 H); 3.12 and 3.13 (both s,
together 3 H); 5.57 and 5.88 (t and dd, together 1 H); 6.18
and 6.30 (both d, together 1 H). MS: calc. for [M + H]+:
565; found: 565.0. For biological testing, the title com-
pound was transferred into its acetate salt by lyophiliza-
tion with 0.5 M acetic acid (40 mL).
benzoyl)-N-methylamino)-N-methyl-3-(2-
naphthyl)-N-(2-(2-thienyl)ethyl)propionamide 20
Compound 20 (293 mg) (50% over 4 steps) was
synthesized analogously to compound 3. N-Methyl-N-(2-
(2-thienyl)ethyl)amine was used instead of amine 1.
(3-1-(tert-Butoxycarbonylamino)ethyl)benzoic acid was
used instead of (2E)-5-(tert-butoxycarbonylamino)-5-
methylhex-2-enoic acid. HPLC: Rt = 34.30; purity: 99%
(254 nm; A1). Rt = 36.85; purity: 88% (214 nm; B1).
1H-NMR (CDCl3, selected peaks): δ 1.15 and 1.27 (both
d, together 3 H); 2.87 (s, 3 H); 3.00 and 3.03 (both s,
together 3 H); 5.90 and 6.00 (both dd, together 1 H). MS:
calc. for [M + H]+: 500; found: 500.0. For biological
testing, the compound was dissolved in 0.5 M acetic acid
(50 mL) and lyophilized.
6.2.2.19. (2E)-5-Amino-N-((1R)-2-
(biphenyl-4-yl)-1-(N-methyl-N-(2-(2-thienyl)ethyl)-
carbamoyl)ethyl)-5-methyl-N-methylhex-2-enamide 24
Compound 24 (152 mg) (39% over 4 steps) was
synthesized analogously to compound 3. (2R)-2-(N-(tert-
Butoxycarbonyl)-N-methylamino)-3-(biphenyl-4-yl)pro-
pionic acid was used instead of (2R)-2-(N-(tert-
buoxycarbonyl)-N-methylamino)-3-(2-naphthyl)propionic
acid. HPLC Rt = 37.87 min; purity: 96% (254 nm; A1). Rt
= 38.52 min; purity: 92% (214 nm; B1). 1H-NMR (CDCl3,
selected values): δ 1.09 and 1.22 (both s, together 6 H);
2.21 and 2.27 (both d, together 2 H); 2.85, 2.90, 3.07 and
3.08 (all s, together 6 H); 5.78 (m, 1 H); 6.20 and 6.26
(both d, together 1 H); 6.65–6.95, 7.09 and 7.20–7.60 (all
m, together 13 H). MS: calc. for [M + H]+: 504; found:
504.0. For biological testing, the title compound was
transferred into its acetate salt by lyophilization with 0.5
M acetic acid (40 mL).
6.2.2.16. (2E)-5-Methyl-N-methyl-5-
(methylamino)-N-((1R)-1-(N-methyl-N-phenethyl-
carbamoyl)-2-(2-naphthyl)ethyl)hex-2-enamide 21
Compound 21 (80 mg) (39% over 2 steps) was synthe-
sized analogously to compound 3. (2E)-5-(N-(tert-
butoxycarbonyl)-N-methylamino)-5-methylhex-2-enoic
acid was used instead of (2E)-5-(tert-butoxy-
carbonylamino)-5-methylhex-2-enoic acid. HPLC: Rt =
34.30 min; purity: 97% (254 nm; A1). Rt = 36.28 min;
1
purity: 99% (214 nm; B1). H-NMR (CDCl3, selected
values): δ 1.00 and 1.06 (both s, together 6 H); 2.25 and
2.31 (both s, together 3 H); 2.76, 2.87 and 3.05 (all s,
together 6 H); 5.77 and 5.85 (t and dd, together 1 H); 6.14
and 6.23 (both d, together 1 H); 6.78 (m, 1 H); 7.00–7.90
(m, 12 H). MS: calc. for [M + H]+: 486; found 486.0. For
biological testing, the title compound was transferred into
its acetate salt by lyophilization with 0.5 M acetic acid
(50 mL).
6.2.2.20. (2E)-N-((1R)-1-(N-(2-(2-(2-Hydroxyethoxy)-
phenyl)ethyl)-N-methylcarbamoyl)-2-(2-naphthyl)ethyl)-
N-methyl-5-methyl-5-(methylamino)hex-2-enamide 25
6.2.2.17. (2R)-2-(N-((2E)-5-Amino-5-methylhex-
2-enoyl)-N-methylamino)-N-(2-(2-(2-hydroxyethoxy)-
phenyl)ethyl)-N-methyl-3-(2-naphthyl)propionamide 22
Compound 22 (218 mg) (44% over 4 steps) was
synthesized analogously to compound 3. Amine 6 was
used instead of amine 1. HPLC: Rt = 32.75 min; purity:
96% (254 nm; A1). Rt = 33.82 min; purity: 96% (214 nm;
B1). 1H-NMR (CDCl3, selected values): δ 1.04 and 1.12
(both s, together 6 H); 2.93, 2.99, 3.02 and 3.07 (all s,
together 6 H); 5.68 and 5.87 (both dd, together 1 H); 6.05
and 6.25 (both d, together 1 H). MS: calc. for [M + H]+:
532; found: 532.2. For biological testing, the title com-
Compound 25 (118 mg) (24% over 4 steps) was
prepared analogously to compound 3. Amine 6 was used
instead of amine 1 and (2E)-5-(N-(tert-butoxycarbonyl)-
N-methylamino)-5-methylhex-2-enoic acid was used in-
stead of (2E)-5-(tert-butoxycarbonylamino)-5-methylhex-
2-enoic acid. HPLC: Rt = 33.47 min; purity: 92% (254 nm;
A1). Rt = 34.25 min; purity: 97% (214 nm; B1). 1H-NMR
(CDCl3, selected values): δ 1.02 and 1.10 (both s,
together 6 H); 2.27 and 2.32 (both s, together 3 H); 2.91,
2.98, 3.02 and 3.06 (all s, together 6 H); 5.65 and 5.86

614
(both dd, together 1 H); 6.10 and 6.25 (both d, together 1
H); 6.55–7.90 (m, 12 H). MS: calc. for [M + H]+: 546;
found: 546.0. For biological testing, the title compound
was transferred into its acetate salt by lyophilization with
0.5 M acetic acid (40 mL).
N-{(1R)-1-[N-(2-{2-[2-((fluoren-9-ylmethoxycarbonyl)-
amino)acetylamino]phenyl}ethyl)-N-methylcarbamoyl]-
2-(2-naphthyl)ethyl}-N-methylcarbamic acid tert-butyl
1
ester. H-NMR (CDCl3, selected values): δ (1.25 and
1.30, both br, together 9 H); 2.85, 3.03 and 3.04 (all s,
together 6 H); 5.08 and 5.45 (both t, together 1 H); 9.31
and 9.45 (both s, together 1 H).
N-{(1R)-1-[N-(2-{2-[2-((Fluoren-9-ylmethoxycarbonyl)-
amino)acetylamino]phenyl}ethyl)-N-methylcarbamoyl]-2-
(2-naphthyl)ethyl}-N-methylcarbamic acid tert-butyl es-
ter (1.18 g, 1.59 mmol) was dissolved in 3.0 M hydrogen
chloride in ethyl acetate (8 mL). The reaction mixture
was stirred for 2.25 h at room temperature. The solvent
was removed in vacuo. The residue was washed with
diethyl ether (3 × 20 mL) and dried in vacuo to give crude
[(2-{2-[N-methyl-N-((2R)-2-methylamino-3-(2-naphthyl)-
propionyl)amino]ethyl}phenylcarbamoyl)methyl]carbamic
acid (fluoren-9-yl)methyl ester as hydrochloride, which
was used for the next step without purification. 1H-NMR
(DMSO-d6, selected values): δ 4.41 and 4.67 (both m,
together 1 H).
(2E)-5-(tert-Butoxycarbonylamino)-5-methylhex-2-enoic
acid (200 mg, 0.82 mmol) and 1-hydroxy-7-azabenzo-
triazole (112 mg, 0.82 mmol) were dissolved in dichlo-
romethane (4 mL) and N,N-dimethylformamide (2 mL).
The solution was cooled to 0 °C. N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (157 mg, 0.82 mmol) was added. The reaction mix-
ture was stirred for 20 min at 0 °C. The hydrochloride of
6.2.2.21. (2E)-5-Amino-5-methylhex-
2-enoic acid N-((1R)-1-{N-[2-(2-(benzene-
sulfonylamino)phenyl)ethyl]-N-methyl-
carbamoyl}-2-(2-naphthyl)ethyl)-N-methylamide 26
Compound 26 (252 mg) (33% over 6 steps) was
prepared analogously to compound 3. The amine used
was N-[2-(2-(methylamino)ethyl)phenyl]benzenesulfon-
amide, which was prepared analogously to amine 16,
using benzensulfonyl chloride instead of methansulfonyl
chloride. HPLC Rt = 37.87 min; purity: 87% (254 nm;
A1). Rt = 40.23 min; purity: 91% (214 nm; B1). 1H-NMR
(CDCl3, selected values): δ 1.09 and 1.12 (both s,
together 6 H); 2.84 and 2.86 (both s, together 3 H); 3.15
and 3.20 (both s, together 3 H); 5.65 and 5.87 (t and dd,
together 1 H); 6.22 and 6.32 (both d, together 1 H). MS:
calc. for [M + H]+: 627; found: 627.2. For biological
testing, the title compound was transferred into its acetate
salt by lyophilization with 0.5 M acetic acid (40 mL).
6.2.2.22. 2-Amino-N-(2-(2-(N-((2R)-2-(N-((2E)-5-amino-
5-methylhex-2-enoyl)-N-methylamino)-3-(2-naphthyl)-
propionyl)-N-methylamino)ethyl)phenyl)acetamide 27
crude
[(2-{2-[N-methyl-N-((2R)-2-methylamino-3-(2-
naphthyl)propionyl)amino]ethyl}phenylcarbamoyl)methyl]-
carbamic acid (fluoren-9-yl)methyl ester (553 mg,
0.82 mmol) and ethyldiisopropylamine (0.28 mL,
1.64 mmol) were added successively. The reaction mix-
ture was stirred for 16 h, while it was warming up to
room temperature. It was diluted with ethyl acetate
(50 mL) and washed with 10% aqueous sodium hydrogen
sulfate solution (50 mL). The aqueous phase was ex-
tracted with ethyl acetate (2 × 20 mL). The combined
organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate (50 mL) and
dried over magnesium sulfate. The solvent was removed
in vacuo. The crude product was purified by flash
chromatography on silica (40 g), using ethyl acetate/
heptane (2:1) as eluent, to give 365 mg (58% over 2
steps) of (3E)-4-(N-((1R)-1-(N-(2-(2-((((9H-9-fluorenyl)-
methoxycarbonyl)amino)acetylamino)phenyl)ethyl)-N-
methylcarbmoyl)-2-(2-naphthyl)ethyl)-N-methylcarba-
moyl)-1,1-dimethylbut-3-enylcarbamic acid tert-butyl
ester. 1H-NMR (CDCl3, selected values): δ 5.51 and 5.94
(t and dd, together 1 H); 6.13 and 6.25 (both d, together
1 H); 6.25 (br, 1 H); 6.75 and 6.83 (both m, together 1 H).
(2R)-2-(N-(tert-Butoxycarbonyl)-N-methylamino)-3-
(2-naphthyl)propionic acid (590 mg, 1.79 mmol) and
1-hydroxy-7-azabenzotriazole (243 mg, 1.79 mmol) were
dissolved in dichloromethane (12 mL) and N,N-
dimethylformamide (6 mL). The solution was cooled to
0 °C. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (343 mg, 1.79 mmol) was added. The so-
lution was stirred for 25 min at 0 °C. The hydrochloride
of amine 15 (834 mg, 1.79 mmol) and ethyldiisopropyl-
amine (0.62 mL, 3.58 mmol) were added successively.
The reaction mixture was stirred for 16 h while it was
warming up to room temperature. It was diluted with
ethyl acetate (30 mL) and washed with 10% aqueous
sodium hydrogen sulfate solution (30 mL). The aqueous
phase was extracted with ethyl acetate (3 × 30 mL). The
combined organic layers were washed with a saturated
aqueous solution of sodium hydrogen carbonate (30 mL)
and dried over magnesium sulfate. The solvent was
removed in vacuo. The crude product was purified by
flash chromatography on silica (60 g), using ethyl acetate/
heptane (1:1) as eluent, to give 1.29 g (98%) of

615
At room temperature tris(2-aminoethyl)amine
(2.99 mL, 19.8 mmol) was added to a solution of (3E)-
4-(N-((1R)-1-(N-(2-(2-((((9-fluorenyl)methoxycarbonyl)-
amino)acetylamino)phenyl)ethyl)-N-methylcarbmoyl)-2-
(2-naphthyl)ethyl)-N-methylcarbamoyl)-1,1-dimethylbut-
3-enylcarbamic acid tert-butyl ester (346 mg, 0.40 mmol)
in dichloromethane (2.8 mL). The reaction mixture was
stirred for 1.2 h at room temperature. It was diluted with
dichloromethane (40 mL) and washed with brine (50 mL).
The aqueous phase was extracted with dichloromethane
(3 × 20 mL). The combined organic layers were washed
with a buffer of sodium dihyrogen phosphate and dipo-
tassium hydrogen phosphate (pH 6.4, 3 × 30 mL) and
successively with brine (20 mL). They were dried over
magnesium sulfate. The solvent was removed in vacuo.
The crude product was purified by flash chromatography
on silica (40 g), using dichloromethane/methanol/25%
aqueous ammonia (100:10:1) as eluent, to give 185 mg
(72%) of (3E)-4-(N-((1R)-1-(N-(2-(2-(2-aminoacetyl-
amino)phenyl)ethyl)-N-methylcarbamoyl)-2-(2-naphthyl)-
ethyl)-N-methylcarbamoyl)-1,1-dimethylbut-3-enylcarba-
mic acid tert-butyl ester. 1H-NMR (CDCl3, selected
values): δ 1.14, 1.15, 1.23, and 1.24 (all s, together 6 H);
1.39 and 1.40 (both s, together 9 H); 2.90, 2.94, 3.02 and
3.10 (all s, together 6 H); 5.64 and 5.90 (t and dd, together
1 H); 6.12 and 6.26 (both d, together 1 H); 6.63 and 6.82
(both m, together 1 H); 9.42 and 9.53 (both br, together
1 H).
6.2.2.23. (2R)-2-(N-((2E)-5-Amino-5-methylhex-
2-enoyl)-N-methylamino)-N-(2-(2-(3-hydroxypropoxy)-
phenyl)ethyl)-N-methyl-3-(2-naphthyl)propionamide 28
Compound 28 (64 mg) (17% over 4 steps) was pre-
pared analogously to compound 3. Amine 11 was used
instead of amine 1. HPLC: Rt = 32.57 min; purity: 95%
(254 nm; A1). Rt = 34.60 min; purity: 100% (254 nm;
1
B1). H-NMR (CDCl3, selected values): δ 0.98, 0.99,
1.10 and 1.11 (all s, together 6 H); 2.82, 2.85, 2.91 and
3.03 (all s, together 6 H); 5.47 and 5.84 (both dd, together
1 H); 5.95 and 6.19 (both d, together 1 H); 6.55 (m, 1 H).
MS: calc. for [M + H]+: 546; found: 546.0. For biological
testing, the title compound was transferred into its acetate
salt by lyophilization with 0.5 M actic acid (40 mL).
6.2.2.24. (2E)-5-Amino-5-methylhex-2-enoic
acid N-((1R)-2-(1,2,3,4-tetrahydroisoquinolin-2-yl)-
1-((2-naphthyl)methyl)-2-oxoethyl)-N-methylamide 29
Compound 29 (234 mg) (14% over 4 steps) was
prepared analogously to compound 3. Commercially
available 1,2,3,4-tetrahydroqunoline was used as amine
instead of amine 1. HPLC: Rt = 33.48 min; purity: 96%
(254 nm, A1). 1H-NMR (CDCl3, selected values): δ 1.08
and 1.10 (both s, together 6 H); 2.20 and 2.25 (both d,
together 2 H); 2.96 and 3.08 (both s, together 3 H); 5.98
(m, 1 H); 6.06 and 6.23 (both d, together 1 H); 6.70–7.80
(m, 12 H). MS: calc. for [M + H]+: 470; found: 470.0. For
biological testing, the title compound was transferred into
its acetate salt by lyophilization with 0.5 M acetic acid
(50 mL).
(3E)-4-(N-((1R)-1-(N-(2-(2-(2-Aminoacetylamino)phe-
nyl)ethyl)-N-methylcarbamoyl)-2-(2-naphthyl)ethyl)-N-
methylcarbamoyl)-1,1-dimethylbut-3-enylcarbamic acid
tert-butyl ester (175 mg, 0.27 mmol) was dissolved in
dichloromethane (2 mL). The solution was cooled to
0 °C. Trifluoroacetic acid (2 mL) was added. The reaction
mixture was stirred for 25 min at 0 °C. Dichloromethane
(20 mL) and ethanol (20 mL) were added successively.
The solvent was removed in vacuo without warming. The
residue was dissolved in dichlromethane (40 mL) and the
solvent was removed in vacuo. The last procedure was
repeated. The crude product was purified by flash chro-
matography on silica (15 g), using dichloromethane/
methanol/25% aqueous ammonia /(first: 100:10:1, then:
100:20:2) as eluent, to give 128 mg (87%) of compound
27. HPLC: Rt = 7.15 min; purity: 89% (214 nm; H8).
1H-NMR (CDCl3, selected values): δ 1.03 and 1.12 (both
s, together 6 H); 2.92, 2,94, 3.01 and 3.12 (all s, together
6 H); 5.62 and 5.90 (t and dd, together 1 H); 6.10 and 6.25
(both d, together 1 H); 6.70 and 6.89 (both m, together 1
H); 9.48 and 9.52 both br, together 1 H). For biological
testing, the title compound was transferred into its acetate
salt by lyophilization with 0.5 M acetic acid (40 mL).
6.2.2.25. N-((1R*,2S*)-2-
phenylcyclopropyl)formamide 31
Commercially available trans-2-phenylcyclopropyl-
amine hydrochloride (30, 5.00 g, 29.47 mmol) was dis-
solved in ethyl acetate (100 mL) and a 1 N aqueous
solution of sodium hydroxide (100 mL). The phases were
separated. The aqueous phase was extracted with ethyl
acetate (80 mL). The combined organic layers were dried
over magnesium sulfate. The solvent was removed. The
residue was dissolved in formic acid (34 mL). The
solution was cooled to 0 °C. Acetic anhydride (13 mL)
was added dropwise. The reaction mixture was stirred for
16 h while it was warming up to room temperature. It was
cooled to 0 °C. Water (13 mL) was added dropwise. The
solvents were removed in vacuo. The residue was dis-
solved in ethyl acetate (150 mL) and washed with brine
(2 × 100 mL). The organic phase was dried over magne-
sium sulfate. The solvent was removed to give 4.40 g
(93%) of crude 31. The crude product was used for the
next step without further purification. 1
H-NMR (CDCl3):
δ 1.30 (m, 2 H); 2.15 (m, 1 H); 2.90 (m, 1 H); 5.90 and

616
6.10 (both br, together 1 H); 7.00–7.40 (m, 5 H); 8.20 and
8.32 (s and d, together 1 H).
[28] (3.4 g, 23 mmol), sodium cyanoborohydride (1.9 g,
31 mmol) and molecular sieves (50 g, Fluka, 3 Å) were
added and the mixture was left overnight. The mixture
was filtered and the filtrate was added to water (200 mL)
and extracted with dichloromethane (3 × 100 mL). The
combined organic phases were dried (magnesium sul-
fate), and the solvent was removed in vacuo. The residue
was purified by flash chromatography on silica (80 g) to
6.2.2.26. N-Methyl-N-((1R*,2S*)-
2-phenylcyclopropyl)amine 32
A solution of crude 31 (3.00 g, 18.61 mmol) in tetrahy-
drofuran (41 mL) was added dropwise to a mixture of
sodium borohydride (0.84 g, 22.33 mmol) in tetrahydro-
furan (70 mL). A solution of iodine (2.36 g, 9.31 mmol)
in tetrahydrofuran (80 mL) was added dropwise. After the
addition was finished, the reaction mixture was heated to
reflux for 16 h. It was cooled to 0 °C. Methanol (160 mL)
was added dropwise. The solvents were removed in
vacuo. The residue was dissolved in a 20% aqueous
solution of sodium hydroxide (250 mL) and tert-butyl
methyl ether (150 mL). The phases were separated. The
aqueous phase was extracted with tert-butyl methyl ether
(2 × 100 mL). The combined organic layers were dried
over magnesium sulfate. The solvent was removed. The
crude product was purified by flash chromatography on
silica (80 g), using first ethyl acetate/heptane/triethylamine
(10:10:1) and then dichloromethane/methanol/25% aque-
ous ammonia (100:10:1) as eluent, to give 1.05 g (38%)
of 32. 1H-NMR (CDCl3): δ 0.95 (m, 1 H); 1.08 (m, 1 H);
1.77 (br, 1 H); 1.90 (m, 1 H); 2.33 (m, 1 H); 2.50 (s, 3 H);
7.00–7.30 (m, 5 H).
1
afford 3.55 g (41%) of ester 34. H-NMR: (CDCl3): δ
1.39 (s, 9H); 2.56 (m, 1H); 2.75 (m, 1H); 3.09 (m, 3H);
3.59 (m, 1H); 3.65 (s, 3H); 7.28–7.81 (7 arom. H).
6.2.2.29. (3R)-3-((2-Naphthyl)methyl)piperazin-2-one 35
Ester 34 (3.4 g, 9.1 mmol) was stirred for 1 h in a
mixture of trifluoroacetic acid (5 mL) and dichlo-
romethane (5 mL). The volatiles were removed in vacuo
and the residue was dissolved in a mixture of water
(40 mL) and methanol (100 mL). Sodium hydrogencar-
bonate (2.3 g) was added and the mixture was stirred
overnight. The solvent was removed in vacuo and the
residue was dissolved in water (40 mL) and extracted
with ethyl acetate (10 × 50 mL). The combined organic
phases were dried (magnesium sulfate) and the solvent
was removed in vacuo to afford 1.96 g (90%) of piper-
1
azinone 35. H-NMR: (CDCl3; selected peaks for major
6.2.2.27. (2E)-5-Amino-5-methylhex-2-enoic acid
rotamer): δ 2.95 (m, 1H); 3.05 (m, 2H); 3.24 (m, 1H);
N-methyl-N-((1R)-1-(N-methyl-N-((1R*,2S*)-2-phenyl-
cyclopropyl)carbamoyl)-2-(2-naphthyl)ethyl)amide 33
Compound 33 (193 mg) (23% over 4 steps) was
prepared as a mixture of diastereoisomeres analogously
to compound 3. The diasetereosiomeres were inseparable
on HPLC. Amine 32 was used instead of amine 1. HPLC:
Rt = 36.15 min; purity: 100% (254 nm; A1). Rt =
38.33 min; purity: 100% (214 nm; B1). 1H-NMR (CDCl3,
selected values): δ 0.80–1.40 (m, 2 H); 1.11 and 1.11
(both s, together 6 H); 2.22 (d, 2 H); 2.87, 2.95, 3.14 and
3.19 (all s, together 6 H); 5.85 and 6.25 (both m, together
2 H); 6.70–7.80 (m, together 12 H). MS: calc. for [M +
H]+: 484; found: 484.2. For biological testing, the title
compound was transferred into its acetate salt by lyo-
philization with 0.5 M acetic acid (50 mL).
3.39 (m, 1H); 3.59 (dd, 1H); 3.72 (dd, 1H).
6.2.2.30. (2R)-2-(2-Naphthyl)methyl-3-oxo-4-
phenethylpiperazine-1-carboxylic acid tert-butyl ester 36
Piperazinone 35 (1.9 g; 7.9 mmol) and di-tert-butyl
dicarbonate (2.1 g; 9.5 mmol) was suspended in a mix-
ture of tetrahydrofuran (20 mL) and aqueous sodium
hydroxide (1 M, 8 mL) and stirred overnight. The solvent
was removed in vacuo and water (30 mL) was added. The
aqueous phase was extracted with ethyl acetate (2 ×
50 mL). The combined organic phases were dried (mag-
nesium sulfate) and the solvent was removed in vacuo.
The residue was dissolved in a mixture of dimethylsulf-
oxide (15 mL) and potassium hydroxide (1.3 g). (2-
bromoethyl)benzene (2.2 g, 11 mmol) was added and the
mixture was stirred for 1 h. Water (30 mL) and dichlo-
romethane (60 mL) were added. The organic phase was
washed with water (5 × 10 mL) and the solvent was
removed in vacuo. The residue was chromatographed on
silica (60 g) using ethyl acetate/heptane 1:2 as eluent to
afford 1.25 g (36%) of piperazone 36. 1H-NMR (CDCl3;
selected peaks for major rotamer): δ 1.15 (s, 9H); 2.76 (t,
2H); 3.39 (t, 3H).
6.2.2.28. (2R)-2-((2-(tert-Butoxycarbonylamino)-
ethyl)amino)-3-(2-naphthyl)propionic acid methylester 34
(2R)-2-Amino-3-(2-naphthyl)propionic acid (5.0 g,
23 mmol) was added to methanol (150 mL) and thionyl
chloride (2.0 mL; 23 mmol) was added dropwise and the
mixture was stirred overnight and then refluxed for 2.5 h.
The solvent was removed in vacuo and the residue was
dissolved in a mixture of methanol (95 mL) and acetic
acid (5 mL). (2-Oxoethyl)carbamic acid tert-butyl ester

617
6.2.2.31. (3R)-4-((2E)-5-Amino-5-methylhex-2-enoyl)-
3-((2-naphthyl)methyl)-1-phenethylpiperazin-2-one 37
Piperazone 36 (1.2 g; 2.7 mmol) was dissolved in a
mixture of trifluoroacetic acid (5 mL) and dichloro-
methane (5 mL) and stirred for 15 min. Dichloromethane
(30 mL) and a saturated aqueous solution of sodium
hydrogencarbonate was added to pH 8. The mixture was
extracted with dichloromethane (3 × 10 mL) and the
combined aqueous phases were dried (magnesium sul-
fate) and the solvent was removed in vacuo. Part of the
residue (400 mg 1.2 mmol) was added to a mixture of
(2E)-5-(tert-butoxycarbonylamino)-5-methylhex-2-enoic
acid (282 mg; 1.2 mmol); 1-hydroxy-7-azabenzotriazole
(158 mg; 1.2 mmol), EDAC (245 mg; 1.3 mmol) and
ethyldiisopropylamine (150 mg; 1.2 mmol) and stirred
overnight. Dichloromethane (50 mL) was added and the
mixture was washed with 10% solution of aqueous
sodium hydrogensulfate (50 mL); a saturated aqueous
solution of sodium hydrogencarbonate (50 mL) and water
(50 mL). The organic phase was dried (magnesium sul-
fate) and the solvent removed in vacuo. The residue was
purified by flash chromatography on silica (20 g) and the
residue was dissolved in a mixture of trifluoroacetic acid
(2 mL) and dichloromethane (2 mL) and stirred for 5 min.
Dichloromethane and a saturated aqueous solution of
sodium hydrogencarbonate was added until pH 8 was
obtained. The mixture was extracted with dichloromethane
(2 × 10 mL). The organic phase was dried (magnesium
sulfate) and the solvent was removed in vacuo to afford
5.30 mmol) and mol. sieves (3 Å, 5.0 g) were added
successively. (2R)-2-(tert-Butyldimethylsilyloxy)propanal
[30] (500 mg, 2.66 mmol) was dissolved in methanol
(3 mL) and added to the reaction mixture. Sodium cy-
anoborohydride (95 mg, 1.51 mmol) was added as a
solid. The reaction mixture was stirred for 3 h at room
temperature. Another portion of sodium cyanoborohy-
dride (95 mg, 1.51 mmol) was added. The reaction mix-
ture was stirred 16 h at room temperature. The mol.
sieves was filtered off through a plug of celite, which was
washed with methanol (30 mL). The solvent was re-
moved in vacuo. The residue was dissolved in water/1 N
sodium hydroxide solution (50 mL/50 mL). The solution
was extracted with diethyl ether (3 × 50 mL). The
combined organic layers were dried over magnesium
sulfate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(60 g) using ethyl acetate/heptane/triethylamine (10:10:1)
as eluent to give 160 mg (65%) of (2R)-2-(N-((2E)-
5-((2R)-2-(tert-butoxydimethylsilyloxy)-propylamino)-
5-methylhex-2-enoyl)-N-methylamino)-N-methyl-3-(2-
1
naphthyl)-N-phenethylpropionamide. H-NMR (CDCl3,
selected values): δ = 5.80 and 5.86 (t and dd, together 1
H); 6.14 and 6.23 (both d, together 1 H); 6.85 (m, 1 H).
(2R)-2-(N-((2E)-5-((2R)-2-(tert-Butoxydimethylsilyl-
oxy)-propylamino)-5-methylhex-2-enoyl)-N-methylamino)-
N-methyl-3-(2-naphthyl)-N-phenethylpropionamide (135 mg,
0.21 mmol) was dissolved in tetrahydrofuran (2 mL). A
1.1 M solution of tetrabutylammonium fluoride in tet-
rahydrofuran (0.42 mL, 0.46 mmol) was added. The re-
action mixture was stirred for 1 h at room temperature.
The solution was diluted with ethyl acetate (50 mL). It
was extracted with saturated sodium hydrogen carbonate
solution (3 × 20 mL). The combined organic layers were
dried over magnesium sulfate. The solvents were re-
moved in vacuo. The residue was purified on silica (20 g),
using dichloromethane/methanol/25% aqueous ammonia
(100:10:1) as eluent to give 24 mg of the crude product.
The residue was purified by HPLC-chromatography on a
25 × 250 mm 10m C18 silica column at 40 °C with a
gradient of 30.0–43.5% acetonitrile in a 0.1 M ammo-
nium sulfate buffer, which was adjusted to pH 2.5 with
4 M sulfuric acid. The peptide containing fractions were
collected, diluted with 3 volumes of water and applied to
a Sep-Pakt C18 cartridge which was equilibrated with
0.1% trifluoroacetic acid. The peptide was eluted from
the Sep-Pakt cartridge with 70% acetonitrile in a 0.1%
trifluoroacetic acid solution in water. The product was
lyophilized to give 10.7 mg (10%) of compound 39 as
trifluoroacetate. HPLC: Rt = 35.13; purity: 88% (254 nm;
A1). Rt = 37.08; purity: 77% (214 nm; B1). MS: calc. for
[M + H]+: 530; found: 530.8.
1
310 mg (66% over 3 steps) of compound 37. H-NMR:
(CDCl3; selected peaks for major rotamer): δ 0.99 (s,
6H); 4.51 (dd, 1H); 5.61 (d, 1H); 6.56 (m, 1H). HPLC: Rt
= 32.47 min, purity: 99% (214 nm; A1). MS: calc. for [M
+ H]+: 470; found: 470.5.
6.2.2.32. (3R)-4-(4-(1-Aminocyclobutyl)but-2-enoyl)-
3-((2-naphthyl)methyl)-1-phenethylpiperazin-2-one 38
Compound 38 (250 mg) (46% over 3 steps) was
prepared analogously to compound 37. (2E)-4-(1-(tert-
Butoxycarbonylamino)cyclobutyl)but-2-enoic acid was
used instead of (2E)-5-(tert-butoxycarbonylamino)-5-
methylhex-2-enoic aicd. HPLC: Rt = 33.45 min; purity:
1
97% (214 nm; A1). H-NMR (CDCl3, selected peaks): δ
3.90 (m, 1 H); 4.51 (d, 1 H); 4.63 (dd, 1 H); 5.65 (d, 1 H);
6.48 (td, 1 H); 7.00–7.90 (m, 12 H). MS: calc. for [M +
H]+: 482; found: 481.9.
6.2.2.33. (2R)-2-(N-((2E)-5-((2R)-2-Hydroxy-
propylamino)-5-methylhex-2-enoyl)-N-methylamino)-
N-methyl-3-(2-naphthyl)-N-phenethylpropionamide 39
Compound 3 (179 mg, 0.38 mmol) was dissolved in
methanol (10 mL). Glacial acetic acid (0.30 mL,

618
[12] Hansen T.K., Ankersen M., Hansen B.S., Raun K., Nielsen K.K.,
Acknowledgements
Lau J. et al., J. Med. Chem. 41 (1998) 3705–3714.
[13] Devita R.J., Wyvratt M.J., Drugs Future 21 (1996) 273–281.
We thank J. Gredal Bundgaard, H. Christensen, K.
Frandsen, N. Birkebæk Hammerum, N. Hansen, A. Heer-
wagen, S. Kold, M. Munk, J. Møller, and V. Rode for
their commitment and enthusiastic work which made
these results possible.
[14] Hansen T.K., Thøgersen H., Hansen B.S., Bioorg. Med. Chem. Lett.
7 (1997) 2951–2954.
[15] Peschke B., Ankersen M., Hansen B.S., Hansen T.K., Johansen
N.L., Lau J. et al., Eur. J. Med. Chem. 34 (1999) 363–380.
[16] Peschke B., Hansen B.S., Bioorg. Med. Chem. Lett. 9 (1999)
1295–1298.
[17] Ankersen M., Hansen B.S., Hansen T.K., Lau J., Peschke B.,
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