The influence of conformational restriction in the C-terminus of growth hormone secretagogues on their potency

Article abstract of DOI:10.1016/S0223-5234(02)01370-3

In order to obtain more potent growth hormone secretagogues, a comparison of ipamorelin and NN703 suggested the addition of a polar group at the C-terminus of NN703. A study was conducted using constrained amines for this purpose. Here, substituted 4-piperidinylamino- and 4-dimethylaminopiperidino-substitutents were found to give the most active compounds. A replacement of the 4-dimethylaminopiperidino-substituent with 4-hydroxypiperidino resulted in a series of compounds, which showed in vitro activity with EC₅₀ values in the low nanomolar range, and favourable kinetic properties, such as 40% oral bioavailability. The most promising compound was also tested in a swine in vivo model, resulting in a growth hormone level with a C_max of over 40 ng mL^-1.

Full text of DOI:10.1016/S0223-5234(02)01370-3

Eur. J. Med. Chem. 37 (2002) 487–501
www.elsevier.com/locate/ejmech
Original article
The influence of conformational restriction in the C-terminus of
growth hormone secretagogues on their potency
Bernd Peschke a,*, Michael Ankersen a, Michael Bauer b, Thomas Kruse Hansen a,
Birgit Sehested Hansen c, Karin Kramer Nielsen d, Kirsten Raun b, Lutz Richter a,
Lisbet Westergaard e
a Disco6ery Chemistry, No6o Nordisk A/S, No6o Nordisk Park, 2760 Ma˚lø6, Denmark
b Pharmacological Research, No6o Nordisk A/S, No6o Nordisk Park, 2760 Ma˚lø6, Denmark
c General Cell Biology, No6o Nordisk A/S, No6o Nordisk Park, 2760 Ma˚lø6, Denmark
d Pharmacokinetics, No6o Nordisk A/S, No6o Nordisk Park, 2760 Ma˚lø6, Denmark
e Drug Metabolism, No6o Nordisk A/S, No6o Nordisk Park, 2760 Ma˚lø6, Denmark
Received 5 October 2001; received in revised form 8 March 2002; accepted 11 March 2002
Abstract
In order to obtain more potent growth hormone secretagogues, a comparison of ipamorelin and NN703 suggested the addition
of a polar group at the C-terminus of NN703. A study was conducted using constrained amines for this purpose. Here, substituted
4-piperidinylamino- and 4-dimethylaminopiperidino-substitutents were found to give the most active compounds. A replacement
of the 4-dimethylaminopiperidino-substituent with 4-hydroxypiperidino resulted in a series of compounds, which showed in vitro
activity with EC50 values in the low nanomolar range, and favourable kinetic properties, such as 40% oral bioavailability. The
most promising compound was also tested in a swine in vivo model, resulting in a growth hormone level with a Cmax of over 40
ng mL−1. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
´
Keywords: Growth hormone secretagogue; NN703; Peptidomimetic; Oral bioavailability; GH release
identified in the late nineties a number of orally active
1. Introduction
growth hormone secretagogues [3,4] have been tested in
clinical trials, for indications such as GH deficiency,
catabolic states and sleep enhancement [5].
Growth hormone secretagogues (GHSs) are com-
pounds that are able to release growth hormone (GH)
through the Ghrelin Pathway. This pathway was
known through the eighties as the GHRP Pathway,
reflecting the identification of GHRP-6 and related
analogues. Through the last decade of the 20th century
it was called the GHS Pathway, referring to the identifi-
cation of small molecule analogues of the GHRP’s, and
finally after the identification of Ghrelin in 1999 [1], this
system is now referred to as the GHrelin Pathway.
Ghrelin is a 28-amino acid octanoylated peptide and is
released from the stomach. It has a high affinity to the
G-protein coupled GHS-1A receptor [2], and is able to
release GH in vitro as well as in vivo. Despite the fact
that Ghrelin and its corresponding receptor, were only
A number of different strategies have been used in
the identification of orally active GHSs. One successful
strategy has comprised the application of pepti-
domimetic methods on the pentapeptide ipamorelin [6]
[7]. The orally active NN703 [7,8] was developed by
systematic reduction of molecular weight and removal
of the hydrogen-donating amide bonds [9]. Extensive
work has been done to improve NN703 by N-terminus
modifications [10], C-terminus modifications [11,12], or
libraries with changes in the core of the tripeptide [12].
A recent publication suggests, that the C-terminal
amide is necessary to obtain a reasonable in vivo
activity [13] (Fig. 1).
A comparison of the starting lead compound—
ipamorelin—and NN703 shows, that an additional
group with hydrogen-bonding capability at the C-ter-
* Correspondence and reprints
E-mail address: bpes@novonordisk.com (B. Peschke).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(02)01370-3

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B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
minus, such as an amine, could be beneficial for the in
vivo activity. In the original approach [8] this addi-
tional pharmacophore was sacrificed in order to obtain
good oral bioavailability. In this paper we want to
show our attempts to identify the structural demands as
well as an optimal positioning of this additional phar-
macophoric group.
trifluoroacetic acid as reagent for the deprotection of
the BOC-groups.
3. Pharmacology
The in vitro-screening of the compounds was per-
formed in rat pituitary cell assay [6,22], where the
GH-releasing ability of the compounds were tested. The
EC50 value was determined for each compound and is
given as a mean value of at least two separate experi-
ments. The efficacy was given as a fraction of the
maximal stimulation obtained with GHRP-6 [6] as re-
ference compound. The pharmacokinetic properties of
compounds with sufficient potency in vitro were subse-
quently investigated in vivo in beagle dogs with special
focus on oral bioavailability. Finally acute GH release
was measured for compounds with promising pharma-
cokinetics in vivo in a single dose swine experiment.
2. Chemistry
In our first approach, we used the working-hypothe-
sis, that tertiary amines at the C-terminus would be a
sufficient pharmacophoric group, if a basic hydrogen-
bond acceptor was needed. Tertiary amines are not too
polar to inhibit oral absorption of the compound.
Therefore, we chose commercially available 1-
methylpiperazine or 4-dimelthylaminopiperidine as con-
formationally of constrained amines for building blocks
for the C-terminus. In order to have a greater variety
constrained amines, we synthesised amines 4, 13 and 14
starting from the BOC-protected amino acids
isonipecotic acid (1), prolin (5) or nipecotic acid ethyl
ester (6). In order to synthesise diamine 4, acid 1 was
reacted to amide 2, which then was reduced with
sodium borohydride–iodine to give the BOC-protected
amine 3. After removal of the protection group diamine
4 was isolated. For the synthesis of diamines 13 and 14,
a different strategy was chosen. Starting with the com-
mercially available compounds 5 and 6, the BOC-pro-
tected esters 7 and 8 were prepared. Reduction to the
aldehydes 9 and 10 and reductive amination led to the
BOC-protected amines 11 and 12, which then upon
deprotection gave the desired diamines 13 and 14 (Fig.
2).
4. Results and discussion
All the tripeptides 15–41 showed high or moderate
activity as GHSs. The activity was, however, dependent
on the positioning of the polar group at the C-termi-
nus. Compounds 15–18 with a piperazine at the C-ter-
minus showed moderate potency in the range of
39–310 nM. Steric bulk at the N-terminal amino acid
resulted in more active compounds. When, however, an
amine was placed one atom further away from the core
of the tripeptide, as realised in compounds 19–24,
much more potent growth hormone secretagogues were
obtained with EC50 between 2 and 12 nM. Thus, the
extension can be done on both sides of the six-mem-
bered ring. It is noteworthy, that the heavily shielded
primary amine in compounds 19–21 apparently had the
same effect as a secondary amine in compounds 22–24.
The EC50-values for these compounds were determined
to be between 2.5 and12 nM. A further extension of the
spacer between the peptide core and the amine as polar
group with one carbon atom resulted in the GHS 25
with slightly reduced potency. For the investigation of a
The building blocks for the core N-methylated
D-
amino acids with aromatic side chains [8,14,15] were
used. At the N-terminus, (2E)-5-aminoalk-2-enoic acids
[8,10] or 3-aminomethylbenzoic acid [7,16–20] were
used as dipeptidomimetics.
The tripeptides 15–41 were constructed via a BOC-
strategy in solution as described in the literature [8,12]
using EDAC and HOAt [21] as coupling reagents and
Fig. 1.

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
489
Fig. 2. (a) HN(CH3)2, HOAt, EDAC; (b) NaBH4/I2; (c) HCl/EtOAc; (d) EtOH, HOBt, EDAC, DMAP; (e) (BOC)2O, NaOH; (f) DIBAL; (g)
HN(CH3)2, NaB(OAc)3H, HOAc.
served after a slight change in the N-terminal amino
acid, giving rise to compound 36. This sharp change in
the activity was dependent on the other building blocks
in the tripeptides as demonstrated by the comparison of
geometric change of the spacer, the amines 26 and 27,
with a substituent attached at the 3-position of the
piperidine were synthesised. The number of atoms be-
tween the peptide core and the C-terminal amine in
these compounds is the same as in the very active GHSs
19–24. The compounds were, however, less potent with
EC50 of 25 and 18 nM. Similar results were found with
the five-membered ring spacer in compound 28 or 29.
From these data it was concluded that C-termini as in
19–24 had the best geometrical constitution. From
these results with the 4-dimethylaminopiperidine used
in 23 and 24 it was decided to use 4-hydroxypiperidine
at the C-terminus instead. This was expected to im-
prove the oral bioavailability of the compounds, and
the strategy seemed to be very successful: The alcohol
30 showed with an EC50 of 3 nM even better potency
than the corresponding dimethylamine 23. Similarly,
the alcohol 41 had the same activity as the dimethy-
lamine 24. The thorough change and permutation of
the two core and the N-terminal amino acids resulted in
a number of highly active GHSs. The replacement of
the phenyl group with a 2-thienyl-group resulted in the
highly active compounds 31–34. Also the 4-fluoro-subs-
titution of the phenyl group was acceptable in a highly
active GHS, as it was demonstrated with compound 35.
Here, however, a sharp drop in the activity was ob-
the -(2-naphthyl)alanine containing compound 40 and
D
its close analogue 30. Compounds 37 and 38 with a
biphenylylalanine in the core were, however, equipotent
to each other despite the change in the N-terminal
amino acid. A change from the gem-dimethyl-feature at
the N-terminus to a cyclobutyl-moiety was tolerated
with both naphthyl and biphenylyl residues as it was
demonstrated with the compounds 39 and 41. Most of
the amino alcohols 30–41 were full GHSs, showing
efficacies comparable to that of the reference com-
pound GHRP-6. In this series compounds 33, 34, 36,
38, and 40 with a 5-amino-3,5-dimethylhex-2-enoyl
moiety as N-terminus were, however, only partial ago-
nists with efficacies between 46 and 75% compared with
the GH-release obtained by GHRP-6. The same effect
of this particular N-terminus could be observed for the
diamines. Also here the corresponding compounds 16
and 20 were rather ineffective GHSs despite their high
potency. The efficacy was independent of the tested
C-termini,
except
for
the
(S)-2-((dimethyl-
amino)methyl)pyrrolidin-1-yl-moiety which reduced the
efficacy in compound 28 to 75% compared with the

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B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
Table 1 (Continued)
Table 1
In vitro screening of growth hormone secrstagogues 15–41
a
% of maximal stimulation of GH-release obtained by GHRP-6.
bioavailability might be accomplished by substitution
of the amine with a hydroxyl group. This hypothesis
was explored with the compounds 30 and 39. Even
though the result of amino alcohol 30 was rather
disappointing, the amino alcohol 39 was orally ab-
sorbed very readily (Table 2).
In order to study the influence of the ring-restriction
at the C-terminus on the pharmacodynamic and
corresponding compounds with different C-termini 15,
19, 22 or 23, which efficacies lie in a range from 100 to
120% (Table 1).
Table 2
Pharmacokinetic screening results in beagle dogs
Entry Vz (l kg−1
)
CL b (ml min−1 t1/2 (min)
fpo (%)
a
c
d
The pharmacokinetic properties of the most promis-
ing compounds from the in vitro-screening were tested
in a beagle dog model. In contrast to compounds 19,
23, and 30, compounds 22 and 39 showed surprisingly
long plasma half lives. Poor oral bioavailability was
expected when the polar group at the C-terminus was
an amine-moiety, we did not expect good oral
bioavailability, because two amine groups would result
in a doubly charged species. Indeed, we found, that the
diamines 19, 22, and 23 were not orally absorbed and
further screening of diamines was omitted. Instead
amino alcohols were used because better oral
kg−1
)
19
22
23
30
39
42
0.71
0.40
0.37
0.85
8.35
6.57
57
5
8.4
53.4
7.8
0
0
0
0
40
1
32
35
27
13
16.8
180
354
a Volume of distribution (i.v.).
b Clearance (i.v.).
c Plasma half life (i.v.).
d Oral bioavailability.

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
491
pharmacokinetic properties of the compounds, we syn-
thesised the open-chain analogue of compound 30, the
aminoalcohol 42. In both molecules, a hydroxyl group
is placed on a three-carbon spacer at the C-terminal
amide bond. As it can be seen from Table 1, aminoal-
cohol 42 was similarly potent as compound 30, while
higher efficacy was observed for compound 42. The
compounds were, however, very different with respect
to pharmacokinetics. The volume of distribution for
compound 42 was with 6.57 l kg−1 much higher than
for compound 30 0.85 l kg−1) and accordingly the
plasma half life was almost one order of magnitude
higher for compound 42. The oral bioavailability of the
open-chain analogue 42 was very poor and similar to
that of the ring-restricted compound 30.
group proved to be successful. Substituted 4-piperidiny-
lamino-substitutents and 4-dimethylaminoperidino-sub-
stitutents were the most suitable C-termini, resulting in
the most active compounds in vitro (compounds 19–
24). None of those compounds were orally bioavailable,
probably as a result of the C-terminal amino group.
The change to a hydroxyl group, which was accom-
plished by the incorporation of a 4-hydroxypiperidino-
moiety gave, however, in one instance the orally
absorbed compound 39, which also showed very
promising in vivo efficacy in a swine model.
6. Experimental
6.1. Pharmacology
The pharmacokinetic data in combination with the
results form the functional in vitro-screening, clearly
suggested to test compound 39 in vivo for GH release.
A swine model was chosen, because the resemblance to
humans with respect to physiology in general and en-
docrinology in particular is better than for other com-
mon laboratory animals such as rat or dog [6–8]. A
dose-dependent GH secretion was observed, when com-
pound 39 was administered orally. As it is shown in
Fig. 3, at a dose of 5000 nmol kg−1 the maximal
concentration of GH, Cmax, was found to be over 40 ng
6.1.1. In 6itro studies
The in vitro studies were performed as described in
the literature [6,22].
6.1.2. Pharmacokinetic studies in dogs
The oral bioavailability of each compound was stu-
died 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 postdosing. A 1-week
washout period separated oral (p.o.) and intravenous
(i.v.) dosing. The compounds 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−1
of body weight via gavage. For i.v. administration the
dogs received a dose of 0.5 mg kg−1 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. Analyti-
cal C8 columns and disposable C3 extraction columns
were used. The oral bioavailability was calculated as
the total area under the plasma concentration versus
time curve following p.o. administration divided by the
area following i.v. administration, appropriately cor-
rected for dose.
mL−1
.
5. Conclusion
The strategy to determine the best position of an
additional amine-pharmacophore of compounds similar
to NN703, and a subsequent change to a hydroxyl
6.1.3. Oral dose–response to compound 39 in pigs
6.1.3.1. Animals. Female Danish slaughter pigs were
ordered with a weight range between 35 and 40 kg life
weight at time of delivery. The pigs had at least 2 weeks
to acclimatise to their new environment before they
entered the experiment.
6.1.3.2. Surgery. Pigs were fasted for 24 h before
Fig. 3. Growth hormone response to increasing doses of compound
39. Pigs were dosed p.o. at time 0. Data are mean9SEM.
surgery, but had free access to water. On the day of

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B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
surgery the animals were anaesthetised with a mixture
of: Zoletil, Xylazin, Ketaminol and Methadon. One mL
per 15 kg BW of this mixture was given i.m. to induce
anaesthesia within 5 min after injection. Atropin (0.05
mg kg−1) i.m. was given with the above mixture. Once
the animal had lost consciousness an ear vein catheter
was inserted to gain venous access. A standard dose of
antibiotics (Novocilin) was given i.v. at that time. The
flank and the neck of the animal was then carefully
clipped and shaved outside the operating theatre. The
shaved areas were thoroughly disinfected with chlorhex-
idin and iodine solution and all surgical procedures
were performed under strictly aseptic conditions. The
animal was positioned on the operating table and an
endotracheal tube was inserted. Total relaxation of the
pig was induced by administration of a 5–10 mL
Propofol (5 mg kg−1) (Rapinovet) bolus. After intuba-
tion anaesthesia was maintained by 1–1.5% isoflurane
in 2 l min−1 100% oxygen. The pigs were kept on
spontaneous respiration. All vital data were noted on
the anaesthesia protocol. The pigs were also connected
to a saline drip infusion (500 mL h−1) to stabilise their
cardio–vascular system. A laparotomy was performed
with the pig in left recumbency. The stomach was gently
retracted and a purse–string suture was placed in the
muscular part of the stomach wall 5–6 cm from the
pyloric end. A hole was made with a diatherimic needle
and a silicone catheter was inserted into the gastric
cavity. The catheter was held in place by two silicone
cuffs, one inside and one outside the stomach wall. The
wound was closed using standard techniques and the
catheter was exteriorised at the back of the animal. The
pig was then placed in dorsal recumbency and a para-
median skin incision was made over the jugular furrow.
The jugular vein and carotid artery were dissected and
fitted on 20 cm with a silicone catheter. The catheters
were tunnelled to the back of the neck and the wound
was closed using standard techniques.
10 min, 4 °C). Plasma was frozen at −20 °C until
analysis. The pigs were dosed with increasing doses
(1500, 5000, 15 000 nmol kg−1 BW) of compound 39
with a 48 h wash-out period between doses. The plasma
samples were analysed for growth hormone by radio-
immuno assay, fully validated for porcine plasma.
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 Waters® 3×150
mm 3.5 m C-18 Symmetry column and positive ion-
spray with a flow rate of 20 mL min−1. The column
was eluted with a linear gradient of 5–90% acetonitrile,
85–0% water and 10% trifluoroacetic acid (0.1%)–wa-
ter in 15 min at a flow rate of 1 mL min−1. NMR
spectra were obtained at 400 MHz on a Bruker
instrument.
6.2.1. HPLC-methods
Two different elution conditions were used.
6.2.1.1. Method A1. The RP–HPLC analysis was per-
formed using UV detections at 214, 254, 276, and 301
nm on a Vydac 218TP54 4.6×250 mm 5 m C-18 silica
column (The Seperations Group, Hesperia), which was
eluted at 1 mL min−1 at 42 °C. The column was
equilibrated with 5% acetonitrile in a buffer consisting
of 0.1 M ammonium sulphate, which was adjusted to
pH 2.5 with 4 M sulphuric acid. After injection the
sample was eluted by a gradient of 5–60% acetonitrile
in the same buffer during 50 min.
6.2.1.2. Method B1. The RP–HPLC analysis was per-
formed using UV detections at 214, 254, 276, and 301
nm on a Vydac 218TP54 4.6×250 mm 5 m C-18 silica
column (The Seperations Group, Hesperia), which was
eluted at 1 mL min−1 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 during 50
min.
6.1.3.3. Post surgical care. All animals received a stan-
dard 4 days course of antibiotics (Novocilin). Analgesia
(Fenadyne, 0.5 mL per 10 kg BW, i.v.) was provided for
3 days after surgery. All blood sampling catheters are
flushed daily with 10 mL of saline for the first 3 days
and thereafter very third.
6.2.2. Protocols
6.1.3.4. Experimental procedures. The pigs were dosed
orally at 09:00 h after an overnight fast. Compound 39,
as an acetate salt, was freshly dissolved in sterile water
to the needed concentration and dosed as 20 mL bolus.
The stomach catheters were flushed with 150 mL water
for injection. Blood samples (3.5 mL) were taken at
−30, −15, 0, 5, 15, 30, 45, 60, 90, 120, 180 and 240
min relative to the dosing of the animals. The blood was
transferred to tubes containing EDTA, placed on ice
and centrifuged at least once every 60 min (3000 rpm,
6.2.2.1. 4-(Dimethylcarbamoyl)piperidine-1-carboxylic
acid
tert-butyl
ester
(2).
1-(tert-Butoxycar-
bonyl)piperidine-4-carboxylic acid (1) (8.0 g, 35 mmol)
was dissolved in dichloromethane (70 mL) and N,N-
dimethylformamide (35 mL). 1-Hydroxy-7-azabenzotri-
azole (4.75 g, 35 mmol) was added. The solution was
cooled to 0 °C. N-(3-dimethylaminopropyl)-N%-ethyl-
carbodiimide hydrochloride (6.69 g, 35 mmol) was
added. The reaction mixture was stirred for 20 min at

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
493
0 °C. A 5.6 M solution of dimethylamine in ethanol (37
mL, 209 mmol) was added. The reaction mixture was
stirred for 3 days, while it was warming up to room
temperature (r.t.). It was diluted with ethyl acetate (400
mL) and washed with a 10% aqueous solution of
sodium hydrogen sulphate (400 mL). The aqueous
phase was extracted with ethyl acetate (2×200 mL).
The combined organic layers were washed with a satu-
rated aqueous solution of sodium hydrogen carbonate
(300 mL) and dried over magnesium sulphate. The
solvent was removed in vacuo. The crude product was
purified by flash chromatography on silica (300 g),
using dichloromethane–methanol 20:1 as eluent, to give
4.56 g of amide 2.
(24.38 g, 113 mmol) was dissolved in dichloromethane
(60 mL). Ethanol (7.9 mL, 135 mmol) and 1-hydroxy-
benzotriazole (15.25 g, 113 mmol) and 4-dimethy-
laminopyridine (1.52 g, 12.5 mmol) were added. The
solution was cooled to 0 °C. N-(3-dimethy-
laminopropyl)-N%-ethylcarbodiimide
hydrochloride
(23.88 g, 113 mmol) was added. The reaction mixture
was stirred for 16 h, while it was warming up to r.t..
Ethyl acetate (400 mL) was added. It was washed with
a 10% aqueous solution of sodium hydrogen sulphate
(300 mL). The aqueous phase was extracted with ethyl
acetate (3×200 mL). The combined organic layers
were washed with a saturated aqueous solution of
sodium hydrogen carbonate (300 mL) and dried over
magnesium sulphate. The solvent was removed in
vacuo. The crude product was purified by flash chro-
matography on silica (400 g), using ethyl acetate (1:4)
as eluent, to give 17.11 g of ester 7.
1H-NMR (CDCl3): l 1.47 (s, 9H); 1.70 (m, 4H);
2.60–2.90 (m, 3H); 2.96 (s, 3H); 3.08 (s, 3H); 4.17 (m,
2H).
6.2.2.2.
4-((Dimethylamino)methyl)piperidine-1-car-
1H-NMR (CDCl3): l 1.28 (m, 3H); 1.43 and 1.46
(both s, together 9H); 2.95 (m, 3H); 2.22 (m, 1H); 3.50
(m, 2H); 4.18 and 4.30 (m and dd, together 3H).
boxylic acid tert-butyl ester (3). At 0 °C a solution of
amide 2 (4.56 g, 18 mmol) in tetrahyrofuran (80 mL)
was added dropwise to a suspension of sodium borohy-
dride (1.61 g, 43 mmol) in tetrahydrofuran (80 mL).
The reaction mixture was stirred for 20 min at 0 °C. A
solution of iodine 4.51 g, 18 mmol) in tetrahydrofuran
(80 mL) was added dropwise at 0 °C. The reaction
mixture was heated to reflux for 16 h. It was cooled to
4 °C. Methanol (200 mL) was added dropwise. The
solvent was removed in vacuo. The residue was dis-
solved in a 20% aqueous solution of sodium hydroxide
(200 mL) and tert-butyl methyl ether (150 mL). The
phases were separated. The aqueous phase was ex-
tracted with tert-butyl methyl ether (3×100 mL). The
combined organic layers were dried over magnesium
sulphate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(100 g), using dichloromethane–methanol–25%
aqueous ammonia (100:10:1) as eluent, to give 4.07 g of
amine 3.
6.2.2.5. (3R)-Piperidine-1,3-dicarboxylic acid 1-tert-bu-
tyl ester 3-ethyl ester (8). (R)-Ethyl nipetcotate tartrate
(10.0 g, 32.5 mmol) were suspended in tetrahydrofuran
(90 mL). An 1 N solution of sodium hydroxide in water
(98 mL, 98 mmol) was added. A solution of di-tert-bu-
tyl dicarbonate (7.10 g, 32.5 mmol) in tetrahydrofuran
(90 mL) was added. The reaction mixture was stirred
for 16 h at r.t. Ethyl acetate (400 mL) was added. The
reaction mixture was washed with a 10% aqueous solu-
tion of sodium hydrogen sulphate (400 mL). The
aqueous solution was extracted with ethyl acetate (2×
200 mL). The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogen
carbonate (200 mL) and dried over magnesium sul-
phate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica
(90 g), using ethyl acetate–heptane 1:4 as eluent, to give
4.13 g of ester 8.
1H-NMR (CDCl3): l 1.22 (m, 2H); 1.44 (s, 9H); 1.85
(d, 2H); 2.09 (m, 1H); 2.61 (s, 6H); 2.65 (m, 2H); 2.78
(t, 2H); 4.05 (d, 2H).
1H-NMR (CDCl3): l 1.27 (t, 3H); 1.48 (s, 9H); 1.54
(m, 1H); 1.62 (m, 1H); 1.73 (m, 2H); 2.05 (m, 1H); 2.45
(m, 1H); 2.81 (m, 1H); 2.98 (br, 1H); 3.93 (m, 1H); 4.14
(q, 1H).
6.2.2.3. N,N-Dimethyl-N-((piperidin-4-yl)methyl)amine
(4). A 3 M solution of hydrogen chloride in ethyl
acetate (120 mL, 360 mmol) was added to a solution of
amine 3 (2.0 g, 14 mmol) in ethyl acetate (50 mL). The
reaction mixture was stirred for 30 min at r.t. The
solvent was removed in vacuo to give 2.3 g of the crude
dihydrochloride salt of diamine 4, which was used
without purification for the next step.
6.2.2.6. N-t-Butyloxycarbonyl-(S)-prolinal (9). At −
78 °C, a 1.2 M solution of diisobutylaluminum hydride
(31.7 mL, 38 mmol) in toluene was added dropwise to
a solution of ester 7 (4.02 g, 16.5 mmol) in diethyl ether
(15 mL). The reaction mixture was stirred for 3 h at
−78 °C. Water (9.9 mL) was added dropwise. The
reaction mixture was warmed to r.t. The mixture was
filtered through a plug of celite. The celite was washed
with tert-butyl methyl ether (3×100 mL). The com-
bined organic layers were dried over magnesium sul-
phate. The solvent was removed in vacuo, to give 2.34
1H-NMR (CDCl3, selected values): l 1.48 (m, 2H);
1.92 (s, 6H); 3.22 (d, 2H).
6.2.2.4. (2S)-Pyrrolidine-1,2-dicarboxylic acid 1-tert-bu-
tyl ester 2-ethyl ester (7). N-tert-Butoxycarbonylprolin

494
B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
g of crude aldehyde 9, which was used for the next step
without further purification.
was stirred for 16 h at r.t. An 1 N aqueous solution of
sodium hydroxide (70 mL) and tert-butyl methyl ether
(70 mL) were added. The phases were separated. The
aqueous solution was extracted with tert-butyl methyl
ether (3×70 mL). The combined organic layers were
dried over magnesium sulphate. The solvent was re-
moved 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 866 mg of amine 12.
1H-NMR (CDCl3): l 1.10 (m, 1H); 1.45 (s, 9H), 1.45
(m, 1H); 1.64 (m, 2H); 1.85 (m, 1H); 2.10 (m, 2H); 2.20
(s, 6H); 2.50 (br, 1H); 2.79 (m, 1H); 3.95 (m, 2H).
1H-NMR (CDCl3): l 1.42 and 1.47 (both s, together
9H); 1.70–2.20 (m, 4H); 3.20–4.30 (m, 3H); 9.45 and
9.55 (both s, together 1H).
6.2.2.7. (3R)-3-Formylpiperidine-1-carboxylic acid tert-
butyl ester (10). A 1.2 M solution of diisobutylalu-
minum hydride in toluene (30.8 mL, 36.9 mmol) was
added at −78 °C to a solution of ester 8 (4.13 g, 16.1
mmol) in diethyl ether (30 mL). The reaction mixture
was stirred for 2.5 h at −78 °C. Water (9.6 mL) was
added dropwise. The reaction mixture was warmed to
r.t. The precipitation was removed by filtration through
a plug of celite. The celite was washed with tert-butyl
methyl ether (3×100 mL). The liquids were combined
and dried over magnesium sulphate. The solvent was
removed in vacuo, to give 1.94 g of crude aldehyde 10,
which was used for the next step without further
purification.
6.2.2.10. N-Dimethyl-N-(((2S)-pyrrolidin-2-yl)methyl)-
amine (13). A 2.7 M solution of hydrogen chloride in
ethyl acetate (75 mL, 202 mmol) was given to a solu-
tion of amine 11 (1.29 g, 5.65 mmol) in ethyl acetate (30
mL). The reaction mixture was stirred for 30 min at r.t.
The solvent was removed in vacuo to give 1.36 g of the
crude dihydrochorlide salt of diamine 13, which was
used for the next step without further purification.
1H-NMR (CDCl3): l 1.90 (m, 2H); 2.17 (m, 1H);
2.40 (m, 1H); 2.90 (m, 2H); 3.14 (s, 6H); 3.55 (m, 2H);
4.35 (m, 1H).
1H-NMR (CDCl3): l 1.45 (s, 9H); 1.67 (m, 2H); 1.95
(m, 1H); 2.43 (m, 1H); 3.10 (m, 1H); 3.32 (dd, 1H); 3.52
(d, 1H); 3.66 (m, 1H); 3.95 (m, 1H); 9.69 (s, 1H).
6.2.2.8. (2S)-2-((Dimethylamino)methyl)pyrrolidine-1-
carboxylic acid tert-butyl ester (11). Crude aldehyde 9
(2.34 g, 11.7 mmol) was dissolved in dichloromethane
(90 mL). A 5.6 M solution of dimethylamine in ethanol
(4.19 mL, 23.5 mmol) was added. Mol sieves ( 0.4 nm,
10.0 g) was added. Sodium triacetoxyborohydride (7.47
g, 35.2 mmol) and glacial acetic acid (1.34 mL, 23.5
mmol) were added successively. The reaction mixture
was stirred for 3 days. It was filtered through a plug of
celite. The celite was washed with methanol (150 mL).
An 1 N aqueous solution of sodium hydroxide (150
mL) and tert-butyl methyl ether (150 mL) were added.
The phases were separated. The aqueous phase was
extracted with tert-butyl methyl ether (3×100 mL).
The combined organic layers were dried over magne-
sium sulphate. 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 1.29 g of
amine 11.
6.2.2.11.
N,N-Dimethyl-N-(((3R)-piperidin-3-yl)me-
thyl)amine (14). Amine 12 (1.25 g, 5.15 mmol) was
dissolved in ethyl acetate (30 mL). A 2.7 M solution of
hydrogen chloride in ethyl acetate (75 mL, 203 mmol)
was added. The reaction mixture was stirred for 45 min
at r.t. The solvent was removed in vacuo to give 976 mg
of the crude dihydrochloride salt of diamine 14, which
was used for the next step without further purification.
1H-NMR (CD3OD): l 1.42 (m, 1H); 1.86 (m, 1H);
2.00 (m, 2H); 2.38 (m, 1H); 2.85 (t, 1H); 2.95 (s, 6H);
2.98 (m, 1H); 3.16 (m, 2H); 3.42 (m, 1H); 3.53 (m, 1H).
6.2.2.12. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-methylpiperazin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2 -(2-naphthyl)ethyl)-
N-methylamide (15). Two hundred and ninety-eight mg
of compound 15 were prepared as described for com-
pound 26 using N-methylpiperazine, (2R)-2-(N-(tert-
butoxycarbonyl) - N - methylamino) - 3 - phenylpropionic
acid, (2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino-
3-(2-naphthyl))propionic acid, and (2E)-5-(butoxycar-
bonylamino)-5-methylhex-2-enoic acid.
1H-NMR (CDCl3): l 1.48 (s, 9H); 1.90 (m, 4H); 2.15
and 2.23 (AB, 2H); 2.26 (s, 6H); 3.31 (br, 2H); 3.85 (br,
1H).
6.2.2.9. (3S)-3-(Dimethylaminomethyl)piperidine-1-car-
boxylic acid tert-butyl ester (12). A solution of crude
aldehyde 10 (1.94 g, 9.1 mmol) in dichloromethane (80
mL) was prepared. A 5.6 M solution of dimethylamine
in ethanol (3.2 mL, 18.2 mmol) and molsieves were
added successively. Sodium triacetoxyborohydride (5.78
g, 27.3 mmol) was added to this mixture. Acetic acid
(1.04 mL, 18.2 mmol) was added. The reaction mixture
1H-NMR (CDCl3; selected values) l 1.24 (s, 6H);
1.65 (s, 3H); 2.35 (s, 3H); 2.80 (s, 3H); 5.68 (dd, 1H);
5.78 (dd, 1H); 6.18 (dd, 1H); 6.95(m, 1H); 7.15–7.80
(m, 12H).
HPLC: Tret=25.03 min (100%, 254 nm, A1); Tret
=
27.50 min (100%, 254 nm, B1).
MS (ES): m/z=598.4; calc. for ([M+H]+: 598.4).

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
495
6.2.2.13. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)1-{N-[(1R)1-benzyl-2-(4-methylpiperazin-1-yl)-2-
oxoethyl]-N-methylcarbamoyl}-2-(2-naphthyl)ethyl)-N-
methylamide (16). One hundred and thirty mg of com-
pound 16 were prepared as described for compound 26
using N-methylpiperazine, (2R)-2-(N-(tert-butoxycar-
bonyl)-N-methylamino)-3-phenylpropionic acid, (2R)-
2-(N-(tert-butoxycarbonyl)-N-methylamino-3-(2-nap-
hthyl))propionic acid, and (2E)-5-(butoxycarbonyl-
amino)-3,5-methylhex-2-enoic acid.
and seventy-eight mg of compound 19 were prepared as
described for compound 26 using 4-amino-2,2,6,6-te-
tramethylpiperidin, (2R)-2-(N-(tert-butoxycarbonyl)-
N-methylamino)-3-phenylpropionic acid, (2R)-2-(N-
(tert-butoxycarbonyl)-N-methylamino-3-(2-naphthyl))-
propionic acid, and (2E)-5-(butoxycarbonylamino)-5-
methylhex-2-enoic acid.
1H-NMR (CDCl3; selected values) l 1.25 (s, 6H);
1.40 (two s, 6H); 1.52 (two s, 6H); 2.92 (s, 3H); 3.02
(two s, 3H); 5.10 (dd, 1H); 5.50 (dd, 1H); 6.15 (d, 1H);
6.75 (m, 1H); 7.00–8.00 (m, 12H).
1H-NMR (CDCl3; selected values) l 1.18 (s, 6H);
1.68 (s, 3H); 1.95 (s, 3H); 2.30 (s, 3H); 2.85 (s, 3H); 3.40
(dd, 1H); 3.54–3.75 (m, 2H); 5.68–5.85 (m, 3H); 7.15–
7.80 (m, 12H).
HPLC: Tret=29.27 min (98%, 254 nm, A1); Tret
=
31.67 min (98%, 254 nm, B1).
MS (ES): m/z=654.8; calc. for ([M+H]+: 654.4).
HPLC: Tret=25.70 min (99%, 254 nm, A1); Tret
=
6.2.2.17. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
methyl-N-((1R)-1-{N-methyl-N-[(1R)-2-phenyl-1-((2,
2,6,6 - tetramethylpiperidin - 4 - yl)carbamoyl)ethyl]car-
bamoyl}-2-(2-naphthyl)ethyl)amide (20). Three hundred
and twenty-seven mg of compound 20 were prepared as
described for compound 26 using 4-amino-2,2,6,6-te-
tramethylpiperidin, (2R)-2-(N-(tert-butoxycarbonyl)-
N-methylamino)-3-phenylpropionic acid, (2R)-2-(N-
(tert-butoxycarbonyl)-N-methylamino-3-(2-naphthyl))-
propionic acid, and (2E)-5-(butoxycarbonylamino)-3,5-
methylhex-2-enoic acid.
28.27 min (93%, 254 nm, B1).
MS (ES): m/z=612.4; calc. for ([M+H]+: 612.4).
6.2.2.14. (2E)-4-(1-Aminocyclobutyl)but-2-enoic acid N-
((1R)1-{N-[(1R)1-benzyl-2-(4-methylpiperazin-1-yl)-2-
oxoethyl]-N-methylcarbamoyl}-2-(2-naphthyl)ethyl)-N-
methylamide (17). One hundred and eighty mg of com-
pound 17 were prepared as described for compound 26
using N-methylpiperazine, (2R)-2-(N-(tert-butoxycar-
bonyl)-N-methylamino)-3-phenylpropionic acid, (2R)-
2-(N-(tert-butoxycarbonyl)-N-methylamino-3-(2-nap-
hthyl))propionic acid, and (2E)-4-(1-(butoxycarbony-
lamino)cyclobutyl)but-2-enoic acid.
1H-NMR (CDCl3; selected values) l 3.92–4.30 (m,
1H); 5.05–5.88 (m, 3H); 7.00–7.80 (m, 12H).
HPLC: Tret=29.80 min (96%, 254 nm, A1); Tret
=
1H-NMR (CDCl3; selected peaks) l 1.62 (s, 3H); 2.35
(s, 3H); 2.80 (s, 3H); 5.70 (dd, 1H); 5.80 (dd, 1H); 6.22
(d, 1H); 6.98 (m, 1H); 7.15–7.80 (m, 12H).
32.43 min (93%, 254 nm, B1).
MS (ES): m/z=668.4; calc. for ([M+H]+: 668.4).
HPLC: Tret=25.88 min (99%, 254 nm, A1); Tret
=
6.2.2.18. 3-Aminomethyl-N-methyl-N-((1R)1-{N-me-
thyl-N-[(1R)-2-phenyl-1-((2,2,6,6-tetramethylpiperidin-
4-yl)carbamoyl)ethyl]carbamoyl}-2-(2-naphthyl)ethyl)-
benzamide (21). Three hundred and seventy two mg of
compound 21 were prepared as described for com-
pound 26 using 4-amino-2,2,6,6-tetramethylpiperidin,
(2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3-
phenylpropionic acid, (2R)-2-(N-(tert-butoxycarbonyl)-
N-methylamino-3-(2-naphthyl))propionic acid, and 3-
((tert-butoxycarbonylamino)methyl)benzoic acid.
1H-NMR (CDCl3; selected values) l 3.60–3.85 (m,
2H); 3.90–4.30 (m, 1H); 5.25–5.95 (m, 2H); 6.70–7.90
(m, 16H).
28.65 min (99%, 254 nm, B1).
MS (ES): m/z=610.4; calc. for ([M+H]+: 610.4).
6.2.2.15. 3-Aminomethyl-N-((1R)-1-{N-[(1R)-1-benzyl-
2-(4-methylpiperazin-1-yl)-2-oxoethyl]-N-methylcar-
bamoyl}-2-(2-naphthyl)ethyl)-N-methylbenzamide (18).
Three hundred and thirty mg of compound 18 was
prepared as described for example 26 using N-
methylpiperazine, (2R)-2-(N-(tert-butoxycarbonyl)-N-
methylamino)-3-phenylpropionic acid, (2R)-2-(N-(tert-
butoxycarbonyl)-N-methylamino-3-(2-naphthyl))pro-
pionic acid, and 3-((tert-butoxycarbonylamino)-me-
thyl)benzoic acid.
HPLC: Tret=29.27 min (93%, 254 nm, A1); Tret
=
1H-NMR (CDCl3; selected values) l 3.30 (m, 1H);
3.50 (dd, 1H); 3.75 (m, 1H); 3.95 (s, 2H); 5.78 (t, 1H);
3.88 (m, 1H); 7.00–7.80 (16H).
31.55 min (93%, 254 nm, B1).
MS (ES): m/z=662.4; calc. for ([M+H]+: 662.4).
HPLC: Tret=24.55 min (95%, 254 nm, A1); Tret
=
6.2.2.19. (2E)-5-Amino-5-methylhex-2-enoic acid N-
methyl-N-[(1R)-1-(N-methyl-N-{(1R)-1-[N-methyl-N-
(1-methylpiperidin-4-yl)carbamoyl]-2-phenylethyl}car-
bamoyl)-2-(2-naphthyl)ethyl]amide (22). Two hundred
and ten mg of compound 22 were prepared as described
for compound 26 using 1-methyl-4-(methylamino)-
piperidine, (2R)-2-(N-(tert-butoxycarbonyl)-N-methy-
lamino)-3-phenylpropionic acid, (2R)-2-(N-(tert-bu-
26.52 min (93%, 254 nm, B1).
MS (ES): m/z=606.4; calc. for ([M+H]+: 606.3).
6.2.2.16. (2E)-5-Amino-5-methylhex-2-enoic acid N-
methyl-N-((1R)-1-{N-methyl-N-[(1R)-2-phenyl-1-((2,
2,6,6 - tetramethylpiperidin - 4 - yl)carbamoyl)ethyl]car-
bamoyl}-2-(2-naphthyl)ethyl)amide (19). Three hundred

496
B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
toxycarbonyl) - N - methylamino - 3 - (2 - naphthyl))pro-
pionic acid, and (2E)-5-(butoxycarbonylamino)-5-
methylhex-2-enoic acid.
6.2.2.23. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-((3S)-3-(dimethylamino-
methyl)piperidin-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-
2-(2-naphthyl)ethyl)-N-methylamide (26). At 0 °C N-
(3-dimethylaminopropyl)-N%-ethylcarbodiimide hydro-
chloride (870 mg, 4.54 mmol) was added to a solution
(2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-3-
phenylpropionic acid (1.27 g, 4.54 mmol) and 1-hy-
droxy-7-azabenzotriazole (617 mg, 4.54 mmol) in
dichloromethane (20 mL) and N,N-dimethylformamide
(10 mL). The reaction mixture was stirred for 20 min at
0 °C. A solution of the crude dihydrochloride salt of
diamine 14 (976 mg, 4.54 mmol) in dichloromethane
(20 mL) and N,N-dimethylformamide (10 mL) and
ethyldiisopropylamine (3.9 ml, 22.7 mmol) were added
successively. The reaction mixture was stirred for 3
days, while it was warming up to r.t. Ethyl acetate (300
ml) was added. The solution was washed with a satu-
rated aqueous solution of sodium hydrogen carbonate
(300 ml). The aqueous phase was extracted with ethyl
acetate (2×200 ml). The combined organic layers were
dried over magnesium sulphate. The solvent was re-
moved 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 1.69 g of N-[(1R)-1-benzyl-
2-((3S)-3-(dimethylaminomethyl)piperidin-1-yl)-2-oxo-
ethyl]-N-methylcarbamic acid tert-butyl ester.
1H-NMR (CDCl3; selected values) l 5.50–6.08 (m,
2H); 6.20–6.70 (m, 2H); 7.10–7.85 (m, 12H).
6.2.2.20. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-(dimethylamino)piperi-
din-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-2-(2-naphth-
yl)ethyl)-N-methylamide (23). Two hundred and four-
teen mg of compound 23 were prepared as described
for compound 26 using 4-(dimethylamino)piperidine
hydrochloride salt, (2R)-2-(N-(tert-butoxycarbonyl)-N-
methylamino)-3-phenylpropionic acid, (2R)-2-(N-(tert-
butoxycarbonyl)-N-methylamino-3-(2-naphthyl))pro-
pionic acid, and (2E)-5-(butoxycarbonylamino)-5-
methylhex-2-enoic acid.
1H-NMR: (CDCl3; selected values) l 1.40 (s, 6H);
2.00 (s, 6H); 4.42–4.85 (2H); 5.45–5.90 (m, 2H); 6.28
(dd, 1H); 6.85(m, 1H); 7.10–7.85(m, 12H).
MS (ES): m/z=626.2; calc. for ([M+H]+: 626.4).
6.2.2.21. (2E)-4-(1-Aminocyclobutyl)but-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-(dimethylamino)piperi-
din-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-2-(2-nap-
hthyl)ethyl)-N-methylamide (24). Two hundred and
forty-nine mg of compound 24 were prepared as de-
scribed for compound 26, using 4-N,N-dimethylpiper-
azine,
(2R)-2-(N-(tert-butoxycarbonyl)-N-methyl-
1H-NMR (CDCl3, selected values): l 1.20, 1.24, 1.31,
and 1.32 (all s, together 9H); 2.12, 2.13, and 2.18 (all s,
together 6H); 2.81 (m, 3H); 4.97 and 5.30 (both m,
together 1H); 7.05–7.35 (m, 5H).
amino)-3-phenylpropionic acid, (2R)-2-(N-(tert-butoxy-
carbonyl) - N - methylamino - 3 - (2 - naphthyl))propionic
acid,
and
(2E)-4-(1-(butoxycarbonylamino)cyclo-
butyl)but-2-enoic acid.
At 0 °C, trifluoroacetic acid (25 ml) was added to a
solution of N-[(1R)-1-benzyl-2-((3S)-3-(dimethylamino-
methyl)piperidin-1-yl)-2-oxoethyl]- N -methylcarbamic
acid tert-butyl ester (1.69 g, 4.2 mmol) in di-
chloromethane (25 ml). The reaction mixture was
stirred for 30 min at 0 °C. The solvent was removed in
vacuo. The residue was dissolved in dichloromethane
(100 ml) and the solvent was removed in vacuo. The
latter procedure was repeated two times. 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 1.15 g of (2R)-1-
((3S)-3-((dimethylamino)methyl)piperidin-1-yl)-2-me-
thylamino-3-phenylpropan-1-one.
1H-NMR (CDCl3; selected peaks) l 1.90 (s, 3H); 2.38
(s, 3H); 2.45 and 2.47 (two s, 3H) 2.78 and 2.80 (two s,
3H); 6.32 (dd, 1H); 6.90 (m, 1H); 7.15–7.84 (m, 12H).
MS (ES): m/z=638.4; calc. for ([M+H]+: 638.4).
6.2.2.22. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R) - 1 - {N - [(1R) - 1 - benzyl - 2 - (4 - ((dimethylamino)-
methyl)piperidin1-yl)-2-oxoethyl]-N-methylcarbamoyl}-
2-(2-naphthyl)ethyl)-N-methylamide (25). Five mg of
compound 25 were prepared as described for com-
pound 26, using diamine 4, (2R)-2-(N-(tert-butoxycar-
bonyl)-N-methylamino)-3-phenylpropionic acid, (2R)-
2-(N-(tert-butoxycarbonyl)-N-methylamino)-3-(2-nap-
hthyl)propionic acid, and (2E)-5-(tert-butoxycarbony-
lamino)-5-methylhex-2-enoic acid.
1H-NMR (CDCl3, selected values): l 0.38, 1.11, 1.37,
and 1.65 (all m, together 4H); 2.11, 2.19, 2.25, and 2.31
(all s, together 9H); 4.37 and 4.53 (both m, together
1H); 7.10–7.35 (m, 5H).
1H-NMR (CDCl3, selected values): l 1.20 (s, 6H);
2.28, 2.32, 2.41, 2.49, 2.56, 2.57, 2.82, and 2.83 (all s,
together 12H); 5.58, 5.78, and, 5.92 (m, m, and dd,
together 2H); 6.16 and 6.19 (both d, together 1H); 7.00
(m, 1H).
At 0 °C N-(3-dimethylaminopropyl)-N%-ethylcar-
bodiimide hydrochloride (379 mg, 1.98 mmol) was
added to a solution of (2R)-2-(N-(tert-butoxycar-
bonyl)-N-methylamino)-3-(2-naphthyl)propionic acid
(651 mg, 1.98 mmol) and 1-hydroxy-7-azabenzotriazole
(269 mg, 1.98 mmol) in dichloromethane (10 ml) and
HPLC: Tret=39.23 min (94%, 254 nm, A1); Tret
=
41.55 min (95%, 254 nm, B1).
MS (ES): m/z=640.4; calc. for ([M+H]+: 640.4).

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
497
N,N-dimethylformamide (5 ml). The reaction mixture
was stirred for 20 min at 0 °C. A solution of (2R)-1-
((3S)-3-((dimethylamino)methyl)piperidin-1-yl)-2- me-
thylamino-3-phenylpropan-1-one (600 mg, 1.98 mmol)
in dichloromethane (10 ml) and ethyldiisopropylamine
(0.51 ml, 2.97 mmol) were added successively. The
reaction mixture was stirred for 3 days, while it was
warming up to r.t. Ethyl acetate (100 ml) was added.
The solution was washed with a saturated aqueous
solution of sodium hydrogen carbonate (100 ml). The
aqueous phase was extracted with ethyl acetate (3×50
ml). The combined organic layers were dried over ma-
gnesium sulphate. The solvent was removed in vacuo.
The crude product was purified by flash chromatogra-
phy on silica (90 g), using dichloromethane–methanol–
25% aqueous ammonia (100:10:1) as eluent, to give 1.18
g of N-((1R)-1-{N-[(1R)-1-benzyl-2-((3S)-3-((dimethy-
lamino)methyl)piperidin-1-yl)-2-oxoethyl]-N-methyl-
carbamoyl} - 2 - (2 - naphthyl)ethyl) - N - methylcarbamic
acid tert-butyl ester.
propylamine (0.094 ml, 0.55 mmol) were added succes-
sively. The reaction mixture was stirred for 16 h, while
it was warming upto r.t. It was diluted with ethyl
acetate (70 ml) and washed with a saturated aqueous
solution of sodium hydrogen carbonate (70 ml). The
aqueous phase was extracted with ethyl acetate (3×50
ml). The combined organic layers were dried over mag-
nesium sulphate. The solvent was removed in vacuo.
The crude product was purified by flash chromatogra-
phy on silica (40 g), using dichloromethane–methanol–
25% aqueous ammonia (100:10:1) as eluent, to give 398
mg of {(3E)-4-[N-((1R)-1-{N-[(1R)-1-benzyl-2-((3S)-3-
((dimethylamino)methyl)piperidin-1-yl)-2-oxoethyl]-N-
methylcarbamoyl}-2-(2-naphthyl)ethyl)-N-methylcarba-
moyl]-1,1-dimethylbut-3-enyl}carbamic acid tert-butyl
ester.
1H-NMR (CDCl3, selected values): l 1.44 (s, 9H);
5.58, 5.75, and 5.86 (all m, 2H); 6.09 and 6.17 (both d,
together 1H); 6.84 (m, 1H); 7.10–7.80 (m, 12H).
At 0 °C, trifluoroacetic acid (7 ml) was added to a
solution of {(3E)-4-[N-((1R)-1-{N-[(1R)-1-benzyl-2-
((3S)-3-((dimethylamino)methyl)piperidin-1-yl)-2-oxo-
ethyl]-N-methylcarbamoyl}-2-(2-naphthyl)ethyl)-N-me-
thylcarbamoyl]-1,1-dimethylbut-3-enyl}carbamic acid
tert-butyl ester (398 mg, 0.54 mmol) in dichloro-
methane (7 ml). The reaction mixture was stirred for 40
min at 0 °C. The solvent was removed in vacuo. The
residue was dissolved in dichloromethane (20 ml) and
the solvent was removed in vacuo. The latter procedure
was repeated two times. 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 150 mg of compound 26.
1H-NMR (CDCl3, selected values): l 1.08, 1.12, 1.14,
and 1.15 (all s, together 6H), 5.46, 5.59, 5.75, and 5.94
(all m, together 2H); 6.15 (m, 1H); 6.93 (m, 1H).
1H-NMR (CDCl3, selected values): l 0.45 and 0.71
(both m, together 1H); 1.03, 1.05, 1.15, 1.20, 1.28, 1.36,
and 1.42 (all s, together 9H); 2.12, 2.15, 2.21, 2.26, 2.29,
2.85 (all s, together 6H); 5.05, 5.44, 5.58, 5.71, 5.85, and
6.00 (all s, together 2H); 7.10–7.80 (m, 12H).
At 0 °C, trifluoroacetic acid (20 ml) was added to a
solution of N-((1R)-1-{N-[(1R)-1-benzyl-2-((3S)-3-
((dimethylamino)methyl)piperidin-1-yl)-2-oxoethyl]-N-
methylcarbamoyl}-2-(2-naphthyl)ethyl)-N-methylcar-
bamic acid tert-butyl ester (1.18 g, 1.92 mmol) in
dichloromethane (20 ml). The reaction mixture was
stirred for 50 min at 0 °C. The solvent was removed in
vacuo. The residue was dissolved in dichloromethane
(80 ml) and the solvent was removed in vacuo. The
latter procedure was repeated two times. 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 788 mg of (2R)-
N-[(1R)-1-benzyl-2-((3S)-3-((dimethylamino)methyl)-
piperidin-1-yl)-2-oxoethyl]-N-methyl-2-(methylamino)-
3-(2-naphthyl)propionamide.
HPLC: Tret=27.55 min (72%, 254 nm, A1); Tret
=
30.23 min (70%, 254 nm, B1).
MS (ES): m/z=640.4; calc. for ([M+H]+: 640.4).
For biological testing, the title compound was trans-
ferred into its acetate salt, by lyophilisation of a solu-
tion of 150 mg of compound 26 in 0.5 M acetic acid (40
mL).
1H-NMR (CDCl3, selected values): l 2.01 and 2.25
(both s, together 9H); 3.72 (m, 2H); 3.95 and 4.27 (both
m, together 1H); 5.77, 5.86, and 6.03 (t, m, and dd,
together 1H); 7.10 and 7.85 (m, 12H).
6.2.2.24. (2E)-4-(1-Aminocyclobutyl)but-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-((3S)-3-(dimethylamino-
methyl)piperidin-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-
2-(2-naphthyl)ethyl)-N-methylamide (27). One hundred
and eighty mg of compound 27 were prepared as
described for compound 26, using diamine 14, (2R)-2-
(N-(tert-butoxycarbonyl)-N-methylamino)-3-phenyl-
propionic acid, (2R)-2-(N-(tert-butoxycarbonyl)-N-
methylamino)-3-(2-naphthyl)propionic acid, and (2E)-
4-(1-(tert-butoxycarbonylamino)cyclobutyl)but-2-enoic
acid.
At 0 °C, N-(3-dimethylaminopropyl)-N%-ethylcar-
bodiimide hydrochloride (105 mg, 0.55 mol) was added
to a solution of (2E)-5-(tert-butoxycarbonylamino)-5-
methylhex-2-enoic (136 mg, 0.55 mmol) and 1-hydroxy-
7-azabenzotriazole (74 mg, 0.55 mmol) in dichloro-
methane (5 ml). The reaction mixture was stirred for 20
min at 0 °C. A solution of (2R)-N-[(1R)-1-benzyl-2-
((3S)-3-((dimethylamino)methyl)piperidin-1-yl)-2-oxo-
ethyl]-N-methyl-2-(methylamino)-3-(2-naphthyl)propio-
namide (281 mg, 0.55 mmol) in dichloromethane (5 ml)
and N,N-dimethylformamide (5 ml) and ethyldiiso-

498
B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
1H-NMR (CDCl3, selected values): l 0.40 and 0.74
bonylamino-5-methylhex-2-enoic acid as starting
(both m, together 2H); 3.73 and 4.22 (both m, together
2H); 5.57, 5.77, and 5.91 (all m, together 2H); 6.15 and
6.24 (both d, together 1H); 6.85 and 6.96 (both m,
together 1H); 7.22, 7.92, and 7.74 (all m, together 12H).
materials.
1H-NMR (CD3OD, two sets of signals, selected val-
ues for the major rotamere): l 1.38 (s, 6H); 2.52 (s 3H);
2.99 (s, 3H); 5.87 (m, 1H); 6.38 (d, 1H).
HPLC: Tret=28.03 min (90%, 254 nm, A1); Tret
=
HPLC: Tret=29.88 min (95%, 254 nm, A1); Tret=
29.92 min (89%, 254 nm, B1).
31.41 min (92%, 254 nm, B1).
MS (ES): m/z=652.4; calc. for ([M+H]+: 652.4).
MS (ES): m/z=599.4; calc. for ([M+H]+: 599.4).
6.2.2.25. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-((2S)-2-((dimethylamino)-
methyl)pyrrolidin-1-yl)-2-oxoethyl]-N-methylcarbamo-
6.2.2.28. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(2R)-2-(4-hydroxypiperidin-1-yl)-2-oxo-1-
((2-thienyl)methyl)ethyl]-N-methylcarbamoyl}-2-(2-nap-
hthyl)ethyl)-N-methylamide (31). Two hundred and sev-
enteen mg of compound 31 were prepared as described
for compound 26 using 4-hydroxypiperidine, (2R)-2-
(N-tert-butoxycarbonyl-N-methylamino)-3-(2-thienyl)-
yl}-2-(2-naphthyl)ethyl)-N-methylamide
(28).
Two
hundred and eighty five mg of compound 28 were
prepared as described for compound 26, using diamine
13, (2R)-2-(N-(tert-butoxycarbonyl)-N-methylamino)-
3-phenylpropionic acid (2R)-2-(N-(tert-butoxycar-
bonyl)-N-methylamino)-3-(2-naphthyl)propionic acid,
and (2E)-5-(tert-butoxycarbonylamino)-5-methylhex-2-
enoic acid.
propionic
acid,
(2R)-2-(N-tert-butoxycarbonyl-N-
methylamino)-3-(2-naphthyl)propionic acid, and (2E)-
5-tert-butoxycarbonylamino-5-methylhex-2-enoic acid
as starting materials.
1H-NMR (CDCl3, selected values): l 0.55 (m, 1H);
1.11, 1.12, and 1.17 (all s, together 6H); 2.25 (s, 6H);
2.45 (s, 3H); 2.85 (s, 3H); 4.02 (m, 1H); 5.48 (dd, 1H);
5.80 and 5.93 (m, and dd, together 1H); 6.10 and 6.18
(both d, together 1H); 6.87 and 7.00 (both m, together
1H); 7.10–7.90 (m, 12H).
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 1.20 (s, 6H); 2.60 and 2.65 (both s, together 3H);
2.85 (s, 3H); 5.60 (m, 1H); 5.95 (m, 1H); 6.25 (d, 1H);
6.85 (m, 2H); 7.00 (m, 1H); 7.10–7.90 (m, 8H).
HPLC: Tret=29.07 min (96%, 254 nm, A1); Tret
=
30.81 min (91%, 254 nm, B1).
HPLC: Tret=27.97 min (96%, 254 nm, A1); Tret
=
MS (ES): m/z=605.4; calc. for ([M+H]+: 605.3).
27.80 min (95%, 254 nm, B1).
MS (ES): m/z=626.4; calc. for ([M+H]+: 626.4).
6.2.2.29. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-2-(biphenyl-4-yl)-1-{N-[(2R)-2-(4-hydroxypiperi-
din-1-yl)-2-oxo-1-((2-thienyl)methyl)ethyl]-N-methyl-
carbamoyl}ethyl)-N-methylamide (32). Two hundred
and seventy nine mg of compound 32 were prepared as
described for compound 26 using 4-hydroxypiperidine,
(2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-3-(2-
thienyl)propionic acid and (2R)-2-(N-tert-butoxycar-
bonyl-N-methylamino)-3-(biphenyl-4-yl)propionic acid
and (2E)-5-tert-butoxycarbonylamino-5-methylhex-2-
enoic acid as starting materials.
6.2.2.26. N-((1R)-1-{N-[(1R)-1-Benzyl-2-((2S)-2-((di-
methylamino)methyl)pyrrolidin-1-yl)-2-oxoethyl]-N-me-
thylcarbamoyl}-2-(2-naphthyl)ethyl)-N-methyl-3-((me-
thylamino)methyl)benzamide (29). Four hundred and
thirty-six mg of compound 29 were prepared as de-
scribed for compound 26, using diamine 13, (2R)-2-(N-
(tert - butoxycarbonyl) - N - methylamino) - 3 - phenylpro-
pionic acid (2R)-2-(N-(tert-butoxycarbonyl)-N-methy-
lamino)-3-(2-naphthyl)propionic acid, and 3-(N-(tert-
butoxycarbonyl)-N-methylamino)benzoic acid.
1H-NMR (CDCl3, selected values): l 0.87 (m, 1H);
1.22 (m, 1H); 1.45 (m, 1H); 1.67 (m, 1); 4.09 (m, 1H);
5.53 and 5.90 (dd and m, together 2H); 6.80–7.90 (m,
16H).
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 1.20 (s, 6H); 2.60 and 2.62 (both s, together 3H);
2.85 and 2.87 (both s, together 3H); 5.65 (m, 1H); 5.85
(m, 1H); 6.22 and 6.23 (both dd, together 1H); 6.75–
7.65 (m, 13H).
HPLC: Tret=28.43 min (89%, 254 nm, A1); Tret
=
30.63 min (85%, 254 nm, B1).
HPLC: Tret=32.20 min (97%, 254 nm, A1); Tret
=
MS (ES): m/z=648.4; calc. for ([M+H]+: 648.4).
34.09 min (95%, 254 nm. B1).
MS (ES): m/z=631.2; calc. for ([M+H]+: 631.3).
6.2.2.27. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-hydroxypiperidin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2-(2-naphthyl)ethyl)-
N-methylamide (30). One hundred and fifty-six mg of
compound 30 was prepared as described for compound
26 using 4-hydroxypiperidine, (2R)-2-(N-tert-butoxy-
6.2.2.30. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)-1-{N-[(2R)-2-(4-hydroxypiperidin-1-yl)-2-oxo-1-
((2-thienyl)methyl)ethyl]-N-methylcarbamoyl}-2-(2-nap-
hthyl)ethyl)-N-methylamide (33). Two hundred and
eight mg of compound 33 were prepared as described
for compound 26 using 4-hydroxypiperidine, (2R)-2-
(N-tert-butoxycarbonyl-N-methylamino)-3-(2-thienyl)-
carbonyl-N-methylamino)-3-phenylpropionic
acid,
(2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-3-(2-
naphthyl)propionic acid, and (2E)-5-tert-butoxycar-

B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
499
propionic acid and (2R)-2-(N-tert-butoxycarbonyl-N-
methylamino)-3-(2-naphthyl)propionic acid and (2E)-5-
tert-butoxycarbonylamino-3,5-dimethylhex-2-enoic acid
as starting materials.
idin-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-2-(2-nap-
hthyl)ethyl)-N-methylamide (36). One hundred and
thirty-five mg of compound 36 were prepared as de-
scribed for compound 26 using 4-hydroxypiperidine,
(2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-3-(4-
fluorophenyl)propionic acid and (2R)-2-(N-tert-bu-
toxycarbonyl-N-methylamino)-3-(2-naphthyl)propionic
acid and (2E)-5-tert-butoxycarbonylamino-3,5-dime-
thylhex-2-enoic acid as starting materials.
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 1.20 (s, 6H); 1.95 and 1.97 (both s, together 3H);
2.55 and 2.60 (both s, together 3H); 3.87 (s, 3H); 5.60
(m, 1H); 5.80 (s, 1H); 5.95 (m, 1H); 6.70–7,90 (m,
10H).
HPLC: Tret=29.76 min (96%, 254 nm, A1); Tret
=
1H-NMR (CD3OD, trifluoroacetate salt, two sets of
signals, selected values): l 1.25, 1.30, 1.35, and 1.40 (all
s, together 6H); 2.50, 2.95, 3.00, 3.30 (all s, together
9H); 5.70 (m, 1H); 5.80 (m, 1H); 5.95 (m, 1H); 6.90–
7.90 (m, 12H).
31.62 min (95%, 254 nm., B1).
MS (ES): m/z=619.4; calc. for ([M+H]+: 619.3).
6.2.2.31. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)-2-(biphenyl-4-yl)-1-{N-[(1R)-2-(4-hydroxypiperi-
din-1-yl)-2-oxo-1-((2-thienyl)methyl)ethyl]-N-methyl-
carbamoyl}ethyl)-N-methylamide (34). Two hundred
and twenty-three mg of compound 34 were prepared as
described for compound 26 using 4-hydroxypiperidine,
(2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-3-(2-
thienyl)propionic acid and (2R)-2-(N-tert-butoxycar-
bonyl-N-methylamino)-3-(biphenyl-4-yl) propionic acid
and (2E)-5-tert-butoxycarbonylamino-3,5-dimethylhex-
2-enoic acid as starting materials.
HPLC: Tret=30.98 min (86%, 254 nm, A1); Tret
=
32.38 min (100%, 254 nm, B1).
MS (ES): m/z=631.4; calc. for ([M+H]+: 631.4).
6.2.2.34. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-hydroxypiperidin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2-(biphenyl-4-yl)ethyl)-
N-methylamide (37). Compound 37 (1.20 g) were pre-
pared as described for compound 26 using 4-hydroxyp-
iperidine,
(2R)-2-(N-tert-butoxycarbonyl-N-methyl-
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 1.15 (s, 6H); 1.92 and 1.95 (both s, together 3H);
2.50 and 2.55 (both s, together 3H); 2.87 and 2.90 (both
s, together 3H); 5.70 (m, 1H); 5.75 (s, 1H); 5.85 (m,
1H); 6.80–7.60 (m, 12H).
amino)-3-phenylpropionic acid and (2R)-2-(N-tert-bu-
toxycarbonyl-N-methylamino)-3-(biphenyl-4-yl)pro-
pionic acid and (2E)-5-tert-Butoxycarbonylamino-5-
methylhex-2-enoic acid as starting materials.
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 0.75 and 0.95 (both m, together 1H); 1.05 and
1.25 (both s, together 6H); 2.30 and 2.20 (m, 2H); 2.35,
2.40, 2.85 and 2.88 (all s, together 6H); 2.70 (m, 1H);
2.75–3.35 (m, 8H); 3.45 and 3.65 (both m, together
1H); 3.85 and 4.00 (both m, together 1H); 5.70 (m, 1H);
5.80 (m, 1H); 6.15 (m, 1H); 7.00 (m, 1H); 7.10–7.65 (m,
14H).
HPLC: Tret=32.89 min (100%, 254 nm, A1); Tret
=
34.82 min (98%, 254 nm, B1).
MS (ES): m/z=645.4; calc. for ([M+H]+: 645.3).
6.2.2.32. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-(4-fluorobenzyl)-2-(4-hydroxypiper-
idin-1-yl)-2-oxoethyl]-N-methylcarbamoyl}-2-(2-nap-
hthyl)ethyl)-N-methylamide (35). One hundred and
eighty mg of compound 35 were prepared as described
for compound 26 using 4-hydroxypiperidine, (2R)-2-
(N-tert-butoxycarbonyl-N-methylamino)-3-(4-fluoro-
phenyl)propionic acid and (2R)-2-(N-tert-butoxycar-
HPLC: Tret=32.65 min (93%, 254 nm, A1); Tret
=
34.02 min (92%, 254 nm, B1).
MS (ES): m/z=625.4; calc. for ([M+H]+: 625.4).
6.2.2.35. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-hydroxypiperidin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2-(biphenyl-4-yl)ethyl)-
N-methylamide (38). Two hundred and twenty five mg
of compound 38 were prepared as described for com-
pound 26, using 4-hydroxypiperidine, (2R)-2-(N-tert-
butoxycarbonyl - N - methylamino) - 3 - phenylpropionic
acid, (2R)-2-(N-tert-butoxycarbonyl-N-methylamino)-
3-(biphenyl-4-yl)propionic acid and (2E)-5-tert-butoxy-
carbonylamino-3,5-dimethylhex-2-enoic acid as starting
materials.
bonyl-N-methylamino)-3-(2-naphthyl)propionic
acid
and (2E)-5-tert-butoxycarbonylamino-5-methylhex-2-
enoic acid as starting materials.
1H-NMR (CD3OD, trifluoroacetate salt, two sets of
signals, selected values): l 1.00 (m, 1H); 1.22, 1.24, and
1.35 (all s, together 6H); 2.70, 2.90, and 2.97 (all s,
together 6H); 5.65 and 5.75 (both dd, together 1H);
5.90 (m, 1H); 6.40 and 6.65 (both m, together 2H);
6.85–7.90 (m, 11H).
HPLC: Tret=30.27 min (99%, 254 nm, A1); Tret
=
31.60 min (99%, 254 nm, B1).
1H-NMR (CDCl3, two sets of signals, selected val-
ues): l 0.70 and 0.95 (both m, together 1H); 1.00–1.80
(m, 4H); 2.30 (m, 4H); 2.35, 2.40, 2.85 and 2.87 (all s,
together 6H); 2.60–4.10 (m, 6H); 5.72 (m, 1H); 5.80 (m,
1H); 6.15 (dd, 1H); 7.00 (m, 1H); 7.15–7.5 (m, 10H).
MS (ES): m/z=617.4; calc. for ([M+H]+: 617.3).
6.2.2.33. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-(4-fluorobenzyl)-2-(4-hydroxypiper-

500
B. Peschke et al. / European Journal of Medicinal Chemistry 37 (2002) 487–501
HPLC: Tret=33.29 min (99%, 254 nm, A1); Tret
=
1H-NMR (CD3OD, two sets of signals, selected val-
36.40 min (97%, 254 nm, B1).
ues of the major rotamer) l 1.48 (s, 6H); 2.44 (s, 3H);
2.94 (s, 3H); 5.78 (m, 1H); 5.94 (d, 1H).
MS (ES): m/z=639.4; calc. for (M+H]+: 639.4).
HPLC: Tret=30.82 min (95%, 254 nm, A1); Tret
=
6.2.2.36. (2E)-4-(1-Aminocyclobutyl)but-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-hydroxypiperidin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2-(biphenyl-4-yl)ethyl)-
N-methylamide (39). Compound 39 (1.4 g) were pre-
pared as described for compound 26, but using 4-hy-
droxypiperidine, (2R)-(N-tert-butoxycarbonyl-N-me-
thylamino)-3-phenylpropionic acid, (2R)-2-(N-tert-bu-
toxycarbonyl-N-methylamino)-3-(biphenyl-4-yl)pro-
pionic acid, and (2E)-4-(1-(tert-butoxycarbonyl-
amino)cyclobutyl)but-2-enoic acid as starting materials.
32.40 min (88%, 254 nm, B1).
MS (ES): m/z=611.4; calc. for ([M+H]+: 611.4).
6.2.2.39. (2E)-5-Amino-5-methylhex-2-enoic acid N-
((1R)-1-(((1R)-1-((2S)-2-hydroxypropylcarbamoyl)-2-
phenylethyl)-methylcarbamoyl)-2-(2-naphthyl)ethyl)-N-
methylamide (42). One hundred and five mg of com-
pound 42 were prepared as described for compound 26,
using 3-aminopropanol, (2R)-2-(N-tert-butoxycar-
bonyl-N-methylamino)-3-phenylpropionic acid, (2R)-2-
(N - tert - butoxycarbonyl - N - methylamino) - 3 - (2-nap-
hthyl)propionic acid, and (2E)-5-tert-butoxycarbony-
lamino-5-methylhex-2-enoic acid as starting materials.
1H-NMR (CDCl3, selected values) l 3.90 (m, 1H);
5.55 (dd, 1H); 5.58 (d, 1H).
HPLC: Tret=33.58 min (99%, 254 nm, A1); Tret
=
34.95 min (99%, 254 nm, B1).
MS (ES): m/z=637.4; calc. for ([M+H]+: 637.4).
Compound 39 was transferred into its acetate salt by
lyophilisation of a solution of compound 39 in an 0.5
M aqueous solution of acetic acid.
HPLC: Tret=29.03 min (87%, 254 nm, A1); Tret
=
1H-NMR (DMSO-d6, selected values): l 2.37, 2.40,
2.75, and 2.80 (all s, together 6H); 5.60 (m, 2H); 6.30
(m, 1H); 6.70 (m, 1H); 7.10–7.70 (m, 14H).
C39H48N4O4·C2H4O2·H2O (636.84·60.05·18.02) Calc.
for: C, 68.88; H, 7.61; N, 7.84. Found: C, 68.86; H,
7.48; N, 7.81%.
32.25 min (81%, 254 nm, B1).
MS (ES): m/z=573.5; calc. for ([M+H]+: 573.4).
Acknowledgements
We thank P. Andersen, S. Andersen, J.G.
Bundgaard, H. Christensen, N.B. Hammerum, N.
Hansen, A. Heerwagen, K. Holmberg, J. Møller, and
V. Rode for their commitment and enthusiastic work.
6.2.2.37. (2E)-5-Amino-3,5-dimethylhex-2-enoic acid N-
((1R)-1-{N-[(1R)-1-benzyl-2-(4-hydroxypiperidin-1-yl)-
2-oxoethyl]-N-methylcarbamoyl}-2-(2-naphthyl)ethyl)-
N-methylamide (40). One hundred and fifty-two mg of
compound 40 were prepared as described for com-
pound 26, using 4-hydroxypiperidine, (2R)-2-(N-tert-
butoxycarbonyl - N - methylamino) - 3 - phenylpropionic
acid and (2R)-2-(N-tert-butoxycarbonyl-N-methyl-
amino)-3-(2-naphthyl)propionic acid and (2E)-5-tert-
butoxycarbonylamino-3,5-dimethylhex-2-enoic acid as
starting materials.
References
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as starting materials.

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