NG-Acyl-argininamides as NPY Y1 receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity

Article abstract of DOI:10.1016/j.bmc.2010.07.028

N^G-Acylated argininamides, covering a broad range of lipophilicity (calculated log D values: -1.8-12.5), were synthesized and investigated for NPY Y₁ receptor (Y₁R) antagonism, Y ₁R affinity and stability in buffer (N^G-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K_i (binding) and K_b values (Y₁R antagonism) varied from low nM to one-digit μM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90 min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism.

Full text of DOI:10.1016/j.bmc.2010.07.028

Bioorganic & Medicinal Chemistry 18 (2010) 6292–6304
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
NG-Acyl-argininamides as NPY Y1 receptor antagonists: Influence of
structurally diverse acyl substituents on stability and affinity
Stefan Weiss a, Max Keller b, Günther Bernhardt b, Armin Buschauer b, Burkhard König a,
*
a Institut für Organische Chemie, Universität Regensburg, D-93040 Regensburg, Germany
b Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
NG-Acylated argininamides, covering a broad range of lipophilicity (calculated log D values: ꢀ1.8–12.5),
were synthesized and investigated for NPY Y1 receptor (Y1R) antagonism, Y1R affinity and stability in buf-
fer (NG-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The Ki
Article history:
Received 16 February 2010
Revised 3 July 2010
Accepted 10 July 2010
Available online 16 July 2010
(binding) and Kb values (Y1R antagonism) varied from low nM to one-digit lM. Most of the compounds
proved to be sufficiently stable at pH 7.4 over 90 min to determine reliable pharmacological data in vitro.
Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, prob-
ably, due to an intramolecular cleavage mechanism.
Keywords:
Acyl guanidines
NPY
Ó 2010 Elsevier Ltd. All rights reserved.
BIBP 3226
GPCR
1. Introduction
Arginine derivatives such as the Y1R antagonist BIBP 322616 (1a,
Fig. 1) have been proven as valuable pharmacological tools regard-
G Protein-coupled receptors (GPCRs) represent a major class of
biological targets in drug discovery. Focusing on neuropeptide Y
(NPY) and histamine receptors as models of aminergic and peptider-
gic GPCRs, respectively, we are particularly interested in bioisosteric
approaches to develop special receptor subtype-selective tools, for
example, bivalent ligands, prodrugs, radiolabeled and fluorescent
compounds.1–6 Strongly basic groups such as guanidines (including
Arg residues) are essential structural features of numerous GPCR li-
gands, but are unfavourable with respect to bioavailability and brain
penetration. This is also true for many high affinity NPY and hista-
mine receptor agonists and antagonists.7,8 Therefore, special effort
was put into the search for bioisosteric replacements of strongly ba-
sic functional groups. Recently, we reported on the exploitation of
guanidine–acylguanidine bioisosterism with respect to histamine
H2, H3 and H4 receptor ligands5,9–12 and arginine-type NPY Y1 recep-
tor antagonists.1,3,4,6,7,13,14
NPY is a highly conserved peptide which plays an important
role as a neurotransmitter in the central and peripheral nervous
system.15 In humans, four receptor subtypes, referred to as NPY
Y1, Y2, Y4 and Y5 receptors, mediate the biological effects of NPY.
For instance, in the periphery NPY Y1 receptor (Y1R) stimulation
causes an increase in blood pressure. In the central nervous system
(CNS) Y1R activation elicits anxiolytic and sedative effects and is in-
volved in the stimulation of food intake.
less of properties being far from drug-like. Interestingly, the reduc-
tion of the basicity of the Arg-derived Y1R antagonist 1a by
introducing electron-withdrawing NG-substituents such as acyl
groups turned out to be a promising general route to guanidine
derivatives with increased potency, to fluorescent ligands and
radioactive tracers.3–7,14 Even bulky fluorophores attached to the
guanidine via spacers of different size were tolerated; a moderate
decrease in affinity did not compromise suitability of the com-
pounds as pharmacological tools. However, depending on the
chemical nature of the linker cleavage of the acylguanidine group
may occur as demonstrated for a model compound.17
In continuation of our studies on the structure–activity relation-
ships of argininamide-type Y1R antagonists we synthesized a series
of NG-acylated analogues of 1a in order to explore the impact of
structurally diverse substituents on stability and pharmacological
activity in vitro (Y1R binding and antagonism). As the small library
of Y1R antagonists was synthesized to cover a wide range of distri-
bution coefficients (log D) the BIBP 3226 skeleton was substituted
with hydrophobic alkyl chains or sugars and amines to alter the
polarity of the parent compound (Fig. 1).
2. Results and discussion
2.1. Synthesis of the NG-acylated argininamides
The NG-acylated argininamides were efficiently prepared
according to the general synthetic route shown in Scheme 1.
* Corresponding author. Tel.: +49 941 943 4576; fax: +49 941 943 1717.
E-mail address: Burkhard.koenig@chemie.uni-regensburg.de (B. König).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.028

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6293
H2N
N
R
NH
OH
O
H
N
Pyr =
N
H
O
no. R
logD
0.7 (1.4)
no. R
logD
1.8 (1.4)
OH
1a
H
(BIBP 3226)
N
N
1l
O
N
N
1b
12.5
7
NH2
11
O
O
N
N
1m
1.7
O
1c
1d
9.4
13
(R)
OH OH
O
O
O
OH
1n
0.9 (1.0)
0.7
OH
Pyr
O
(R)
(S)
7.6 (5.3)
7.2
(R)
O
O
O
O
Pyr
1e
1f
1o
N
OH
5
5
H
O
O
H
N
O
6.0 (3.1)
O
5
N
H
Pyr
NH2
1p
-0.3 (1.3)
-0.3 (1.4)
O
O
N
H
3
O
O
O
NH
NH2
Bn
1g
1h
4.6 (3.2)
3.9
N
O
1q
N
H
H
O
O
O
O
O
O
H
N
OMe
5
1r
-0.6
-1.3
N
NH
H
O
OAc OAc OAc
OAc OAc
O
1i
1j
3.5 (2.9)
3.5
NH2
O
1s
N
H
F
O
O
O
O
O
H
N
NH2
NH
NH
O
1t
-1.4
N
H
N
H
O
O
O
O
H
N
1u
-1.8 (1.3)
NH2
1k
2.9
N
H
N
OMe
5
H
O
Figure 1. Structures of NG-acylated derivatives of the Y1R antagonist BIBP 3226 (1a) ranked according to calculated log D values (ACD-labs software version 12, pH 7.4);
experimental log D values from HPLC measurements18 in parentheses.
N-Boc-S-methylisothiourea (3) was acylated with the respective
carboxylic acids yielding the isothiourea derived guanidinylation
reagents 4b–4g, 4i, 4k–4q, 4s–4u. Guanidinylation of D-ornithina-
of amines 19 and 21 with succinic anhydride (Scheme 2). The sub-
eric acid derivatives 27 and 28 were prepared through amidation
of octandioic acid mono-benzyl ester with amines 24 and 21,
respectively, followed by benzyl ester cleavage (Scheme 3). Octa-
ndioic acid derivative 30 was obtained via amidation of non-pro-
tected suberic acid with 3-aminopropanoic acid benzyl ester
(Scheme 3). The synthesis of carboxylic acid 36 started from pyr-
ene-1-carboxylic acid (31), which was amidated with mono-Boc-
protected ethylenediamine (21) in moderate yield (Scheme 4).
The Boc group was removed with hydrochloric acid yielding amine
33, which was coupled with 3,6,9-trioxaundecandioic acid mono-
benzyl ester (34) to give compound 35. Hydrogenolysis of 35 re-
sulted in acid 36 (Scheme 4).
mide 5 and subsequent removal of protecting groups gave the
envisaged NG-acylated argininamides. Amine 5 is available from
D
-ornithine in six steps in 32% overall yield.4
Synthetic strategies for the preparation of carboxylic acids 7, 8,
11, 14, 17, 20, 22, 27, 28, 30 and 36 are depicted in Schemes 2–4.
Acids 7 and 8, containing a triazole entity, were prepared through
a Cu(I)-catalysed ‘click-reaction’ between 4-azidobutanoic acid and
tBu-protected propargyl alcohol or Boc-protected propargyl amine
(Scheme 2). Carboxylic acid 14 was prepared via guanidinylation of
6-aminohexanoic acid methyl ester with N,N0-diBoc-S-meth-
ylisothiourea followed by ester hydrolysis (Scheme 2). For the syn-
thesis of succinic acid derivative 17 ethylenediamine was onefold
guanidinylated with N,N0-diBoc-S-methylisothiourea yielding
amine 16, which was treated with succinic anhydride (Scheme
2). Acid precursors 20 and 22 were obtained through the treatment
D-Gluconic acid derivative 1i was synthesized from penta-acet-
ylated -gluconic acid. Unfortunately, deprotection of the hydroxyl
D
groups failed. The base-labile acylguanidine moiety prevents basic
selective cleavage of the acetyl protecting groups. Enzymatic
deprotection using a lipase preparation (Novozym 435) was also

6294
S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
Boc
unsuccessful. To circumvent such problems, sugar derivative 1n,
N
O
NH
NH2
NBoc
NH2
a
b
containing a galactose entity, was prepared. The synthesis of the
respective carboxylic acid 11 is shown in Scheme 2. Two acetal
protecting groups were introduced in the first step and the remain-
ing primary hydroxyl function was esterified with succinic
anhydride. The acetal groups are compatible with the general
synthetic strategy outlined in Scheme 1 and can be easily removed
in the final deprotection step with TFA/DCM.
MeS
N
H
R
MeS
MeS
HO
R
O
2
3
4b-4g, 4i, 4k-4q, 4s-4u
Ph
Ph
NH
NH
O
O
Ph
NH2
Ph
HN
HN
HN
HN
HN
BIBP 3226 derivatives 1j and 1r were prepared through 4-fluor-
obenzoylation and propionylation of amines 1s and 1u, respec-
tively (Scheme 5).
c, d
O
R
4b-4g,
O
O
4i,
4k-4q,
4s-4u
t-Bu
2.2. Stability and Y1R antagonistic activity
O
OH
5
1b-1g, 1i, 1k-1q, 1s-1u
As the acylguanidine moiety of NG-acylated argininamide-type
Y1R antagonists may be considered as the most probable cleavage
site the stability of the presented BIBP 3226 (1a) derivatives was
investigated with respect to the formation of 1a at physiological
pH of 7.4. A release of 1a under the conditions of the pharmacolog-
Scheme 1. General route for the synthesis of NG-acylated BIBP 3226 derivatives
1b–1g, 1i, 1k–1q and 1s–1u. Reagents and conditions: (a) Boc2O, NaOH, tBuOH,
90%; (b) DIPEA, EDC, HOBT, DCM, (1s: TBTU, DIPEA, DMF), 31–95%; (c) HgCl2, NEt3,
DMF, 39–78%; (d) TFA/DCM 1:1, quantitative yield.
R
O
HO
a
N
N
N3
R
HO
O
N
6
7 R = OtBu
8 R = NHBoc
O
O
CH2OH
CH2OH
OH
O
O
O
HO
O
O
O
O
c
b
O
OH
O
OH
OH
O
O
O
O
9
10
11
Cl-
NH3
H
N
H
N
+
d
NBoc
e
O
O
NHBoc
NBoc
NHBoc
NBoc
4
4
4
OMe
OMe
OH
MeS
NHBoc
12
13
14
NBoc
NHBoc
OH
NBoc
NHBoc
NH2
g
H
N
f
HN
O
O
N
H
NBoc
O
NH2
MeS
NHBoc
O
NH2
15
16
17
O
Boc
NH2
HN
OH
Boc
NH
H
h, i, j
k
N
NH
O
N
Boc
O
N
Boc
O
O
H2N
H2N
H2N
18
19
20
O
O
H
H
l
O
N
Boc
N
HO
N
H
Boc
O
O
O
21
22
Scheme 2. Synthesis of carboxylic acids 7, 8, 11, 14, 17, 20 and 22. Reagents and conditions: (a) CuSO4 (5 mol %), Na-ascorbate (10 mol %), MeOH, H2O, 67–86%; (b) acetone,
iodine, 71%; (c) succinic anhydride, DMAP, NEt3, 73%; (d) HgCl2, NEt3, DMF, 84%; (e) THF, NaOH, 71%; (f) CHCl3, 16 h, 97%; (g) THF, NEt3, 49%; (h) CF3COOEt, DCM; (i) Boc2O,
DCM; (j) K2CO3, MeOH, H2O, 47% overall; (k) succinic anhydride, THF, NEt3, 77%; (l) DCM, 99%.

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6295
R
R
O
O
O
O
O
O
a
b
BnO
OH
BnO
N
H
HO
N
H
R
6
6
6
H2N
23
24 R = COOMe 25 R = COOMe
27 R = COOMe
28 R = NHBoc
21 R = NHBoc
26 R = NHBoc
O
O
O
H
c
N
OBn
HO
OH
HO
O
6
O
30
O
H2N
OBn
29
Scheme 3. Synthesis of suberic acid derivatives 27, 28 and 30. Reagents and conditions: (a) DCM, EDC, HOBt, DIPEA, 62%; (b) Pd/C, H2, 90–94%; (c) DCM, EDC, HOBt, DIPEA,
44%.
H
N
H
N
Pyr
OH
a
21
b
Pyr
Pyr
OR
NHBoc
NH3+ Cl-
O
O
O
31
32
33
O
O
35 R = Bn
36 R = H
H
N
c
Pyr
O
33
d
N
O
O
H
3
O
O
HO
OBn
3
34
Pyr =
Scheme 4. Synthesis of the pyrene-1-carboxamide derivative 36. Reagents and conditions: (a) DCM, EDC, HOBt, DIPEA, 52%; (b) MeOH, HCl, 83%; (c) DMF, EDC, HOBt, DIPEA,
59%; (d) Pd/C, MeOH, quantitative.
resulting in the N-alkyl succinimide derivative 39. The lability of
a
a
O
1s
1j
1u
1r
the acylguanidines bearing nucleophiles in position 5 or 6 of the
acyl side chain is in accordance with the recently reported reactiv-
ity of 5-aminopentanoyl substituted guanidine.17
O
O
O
F
N O
N O
All compounds proved to be stable at acidic pH (0.1 % aqueous
trifluoroacetic acid, pH 2–3). Under these conditions the acylguani-
dine group is almost quantitatively protonated due to pKa values in
the range of 7–8. Obviously, this changes the susceptibility against
hydrolysis and prevents intramolecular attacks of nucleophiles at
the carbonyl group, respectively.
O
O
37
38
Scheme 5. Synthesis of the NG-acylated BIBP 3226 derivatives 1j and 1r. Reagents
and conditions: (a) NEt3, DMF, 65% (1j), 21% (1r).
ical assays has to be taken into account as 1a is a highly potent Y1R
antagonist (Ki = 1.3 nM) and could pretend a higher potency of the
investigated compounds. Therefore the stability of the NG-acylated
argininamides was investigated at pH 7.4 on the time scale of the
assays. Formation of 1a was not observed for the strongly hydro-
phobic compounds 1c, 1d and 1e, which are devoid of hetero atoms
in their NG-acyl substituents. The hydrophobic ligands 1f and 1g,
both containing an amide group in the acyl substituent, showed
a very minor decomposition over 90 min (<0.5%). Instability was
more pronounced (decomposition up to 2.6% over 90 min) for the
more polar compounds (1h, 1i, 1k–1q) bearing amide, ester, tria-
zole, sugar, amine, carboxylic or guanidine functions in the acyl
residue.
Exceptionally high instabilities were found for argininamides
with succinyl attached to the guanidine (1j, 1r–1u). Compounds
1r–1u quantitatively decomposed to 1a over 90 min (Table 1). This
process was exemplarily explored by RP-HPLC–MS using the
hydrophobic compound 1j, which allows the detection of the acyl
substituent upon cleavage. As becomes obvious from Figure 2 com-
pound 1j is cleaved via an intramolecular attack of the succinic
amide nitrogen at the carbonyl group attached to the guanidine
Except for the highly unstable compounds 1j and 1r–1u the NG-
acylated argininamides were characterised in terms of Y1R antago-
nism (Kb values) and Y1R affinity (Ki values) using a Fura-2 assay on
human erythroleukemia (HEL) cells19 and a radioligand binding as-
say on SK-N-MC human neuroblastoma cells,4 respectively. Kb and
Ki values are summarised in Table 1. Except for 1h (Ki = 0.9 nM) all
NG-acylated argininamides showed a reduced Y1R affinity com-
pared to the parent compound 1a (Ki = 1.3 nM, Table 1). The de-
crease in affinity was most pronounced (up to three orders of
magnitude) for the most hydrophobic compounds (1b–1e, Table
1), which have limited solubility and might interact with the cell
membrane.
Direct attachment of pyrene-1-carboxylic acid to the guanidine-
N (compound 1e) resulted in complete loss of Y1R affinity. By con-
trast, high Y1R affinity can be retained, if the pyrene moiety is at-
tached to the guanidine group through a linker (compounds 1d
and 1f, Fig. 1, Table 1).
The Y1R affinity of the polar sugar and guanidine substituted
compounds 1n and 1q is about 35 times lower compared to the
affinity of 1a. The argininamide 1a may be considered a mimic of
the C-terminal dipeptide in NPY (. . .-Arg35-Tyr36-NH2). It may be

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S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
Table 1
log D values, stability, Y1R antagonistic activity (Kb) and Y1R binding affinity (Ki) of
BIBP 3226 derivatives 1b–1u
No.
log Da
% Decomposition
(pH 7.4, 20 °C)
after 20/90 min
Y1R Antagonism
Kb [nM]
Y1R Affinity
b
Kic [nM]
1ad
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
0.7 (1.4)
12.5
9.4
7.6 (5.3)
7.2
6.0 (3.1)
4.6 (3.2)
3.9
3.5 (2.9)
3.5
2.9
1.8 (1.4)
1.7
0.9 (1.0)
0.7
—
n.d.
0/0
0/0
1.5 0.2
140 10
1.3 0.2
1500 120
460 19
270 70
Inactive
59 16
170
8
n.d.e
n.d.e
n.d.e
0/0
0/<0.5
0/<0.5
0/0.8
0.9/2.6
30/69
0/0.7
0.9/2.1
0.6/1.7
<0.5/1.3
<0.5/1.4
<0.5/1.4
<0.5/1.6
64/100
51/100
61/100
40/100
1.10 0.04
40
9
0.06 0.01f
0.9 0.1f
110 38
n.d.g
83
8
n.d.g
170 18
0.40 0.03
260 82
3.0 0.5
6.7 0.1
14
4
510 190
450 52
230 64
41
5
73 11
130 10
51 18
n.d.g
ꢀ0.3 (1.3)
ꢀ0.3 (1.4)
ꢀ0.6
18
8
n.d.g
n.d.g
n.d.g
n.d.g
1s
1t
1u
ꢀ1.3
n.d.g
ꢀ1.4
n.d.g
ꢀ1.8 (1.3)
n.d.g
a
Calculated with ACD-labs software version 12, pH 7.4; in brackets: experi-
mental log D determined with HPLC measurements.
b
Kb values for inhibition of NPY (10 nM) induced calcium mobilisation in HEL
cells (Fura-2 assay); all mean values SEM from two or three (1k, 1n, 1o, 1p, 1u)
independent experiments.
c
Ki values determined from the displacement of 1.5 nM [3H]-UR-MK1144 on SK-
N-MC cells; all mean values SEM from two or three (1c, 1f, 1g, 1k, 1l, 1p) inde-
pendent experiments.
d
BIBP 3226.
e
Due to their fluorescent properties pyrene ligands are not compatible with the
Figure 2. Exploration of the decomposition mechanism of BIBP 3226 derivative 1j
incubated in an aqueous buffer (pH 7.4) at 20 °C. LC–MS analysis was performed
after an incubation period of 20 and 90 min. Compound 1j is decomposed to BIBP
3226 (1a) and the succinimide derivative 39 through an intramolecular attack of
the succinic amide nitrogen at the guanidine linked carbonyl group. Chromato-
grams were acquired by single ion monitoring analysis.
Fura-2 assay.
Keller et al.1
f
g
Not determined due to the high instability.
speculated that the introduction of a second guanidine moiety (as
provided by compound 1q) could enhance binding affinity through
mimicking the second arginine residue (Arg33).20 However, the Ki
value of compound 1q was only 51 nM—perhaps due to an inap-
propriate distance between the two guanidine groups.
lar loop regions7 and, depending on chemical nature and size of the
acyl residue, an interaction with membrane lipids cannot be ruled
out.
The hydroxy substituted Y1R antagonist 1l (Ki = 3.0 nM), proved
to be as potent as 1a. It is conceivable that the hydroxy group mim-
ics Thr32 or Tyr27 of the natural ligand NPY. Both amino acids are
very important for the binding of NPY to the Y1R, as identified by
alanine scan.21 The amine analogue of 1l, compound 1m, binds
with similar affinity to the Y1R (Ki = 6.7 nM).
As partial decomposition of compounds 1i, 1k and 1o–1q occurs
(up to 2.6% over 90 min), the determined Ki values of these five
compounds have to be considered with reservation, as the affinity
of the cleavage product 1a (Ki = 1.3 nM) and the assay periods
(from preparation of solutions to read out: 60–90 min) have to
be taken into account.
With respect to the prediction of the affinity of new compounds,
three issues have to be taken into account: substitution with bulky
groups directly at the BIBP 3226 backbone results in a complete
loss of affinity that can be compensated with a spacer of adequate
length. Derivatives with a small aromatic or heteroaromatic substi-
tuent (1g, 1l, 1m) show slightly higher binding affinity compared
to the more flexible derivatives (1g vs 1k, 1o, 1p; 1m vs 1q). Rela-
tively small hydrophobic substituents, as in 1h (Ki = 0.9 nM) and
the radioligand [3H]-UR-MK114 (Ki = 1.3 nM),4 result in similar
and even higher affinity compared to BIBP 3226. Interactions of
the acyl substituent with the Y1R are difficult to predict, because
these residues are presumably oriented toward flexible extracellu-
3. Conclusion
The stability of the NG-acylated argininamides strongly depends
on the nature of the acyl residue. It becomes obvious from the high
instability of the succinyl derivatives (1j, 1r–1u) that cleavage is fa-
voured when the residues harbour nucleophilic functional groups
capable of an intramolecular attack on the acyl carbonyl. This is
also conceivable for argininamides decomposing to a minor extent
and bearing NG-acyl residues containing amide, ester, triazole, su-
gar, amine, carboxylic or guanidine functions (1h, 1i, 1k–1q). This
has to be taken into account in pharmacological investigations, in
particular, when cleavage products are highly bioactive as in the
case of 1a (Ki = 1.3 nM). However, despite these limitations broad
structural variation of NG-acyl substituents was tolerated (affinities
in the nM range). Most of the investigated compounds proved to be
sufficiently stable at pH 7.4 to determine reliable in vitro pharma-
cological data. In conclusion, the NG-acylation of the stongly basic
guanidine group in argininamides is a successful bioisosteric ap-
proach to more drug-like properties provided that the outlined sta-
bility considerations are taken care of. This is not restricted to the
NPY field, as guanidine groups are crucial structural features of
many different biologically active compounds including GPCR
ligands.

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6297
MeOH. Then ascorbic acid (65 mg, 0.33 mmol) dissolved in 1 mL
H2O and copper sulfate pentahydrate (8 mg, 0.03 mmol) dissolved
in 1 mL H2O were added and the reaction mixture was heated to
reflux overnight. Next day MeOH was evaporated completely and
dichloromethane (30 mL) and satd aqueous NaHSO4 solution
(20 mL) were added. The organic layer was collected and then di-
luted NaOH (40 mL, 1 mol/L) was added. The aqueous layer was
collected and acidified with NaHSO4 solution until pH <2. Dichloro-
methane (40 mL) was added and the acid was extracted. The or-
ganic phase was dried over MgSO4, the solvent was evaporated
and a white solid was obtained (674 mg, 86%), mp 81 °C. 1H NMR
(300 MHz, CDCl3): 1.26 (s, 9H), 2.15–2.25 (m, 2H), 2.39 (t,
J = 6.88, 2H), 4.42 (t, J = 6.94, 2H), 4.58 (s, 2H), 7.55 (s, 1H), 10.31
(br s, 1H). 13C NMR (75 MHz, CDCl3): 25.3, 27.5, 30.5, 49.3, 56.2,
74.1, 122.5, 146.8, 176.4. C11H19N3O3: MS (CI, NH3): m/z (%) 242
(100, MH+).
4. Experimental
4.1. General experimental conditions
Unless otherwise noted, solvents (analytical grade) were pur-
chased from commercial suppliers and used without further purifi-
cation. Ethyl acetate (EA), petrol ether (PE, 60–70 °C), methanol
and dichloromethane were obtained in technical grade and distiled
before application. Acetonitrile (MeCN) for HPLC was obtained from
Merck (Darmstadt, Germany). Pentacosa-10,12-diynoic acid (Sig-
ma–Aldrich Chemie GmbH, Munich, Germany), 4-(pyren-1-yl)buta-
noic acid (Sigma–Aldrich Chemie GmbH, Munich, Germany),
hexadecanoic acid (Riedel-de Haen, Seelze, Germany), suberic acid
(Fluka, Sigma–Aldrich Chemie GmbH, Munich, Germany) and
D
-(+)-galactose (Merck, Darmstadt, Germany) were purchased. Pre-
parative HPLC was performed with a system from Knauer (Berlin,
Germany) consisting of two K-1800 pumps and a K-2001 detector.
4.2.2. 4-{4-[(tert-Butoxycarbonylamino)methyl]-1H-1,2,3-tria-
zol-1-yl}butanoic acid (8)
A Nucleodur 100-5 C18 (250 ꢁ 21 mm, 5
l
m; Macherey-Nagel,
m; Knauer,
Germany) and a Eurospher-100 C18 (250 ꢁ 32 mm, 5
l
4-Azidobutanoic acid (0.89 g, 6.90 mmol) was mixed with Boc-
protected propargylamine (1.07 g, 6.90 mmol) in MeOH (10 mL).
NaOH (1 mol/L) was added until the pH value was between 6
and 8. Then ascorbic acid (65 mg, 0.33 mmol) dissolved in 1 mL
H2O and copper sulfate pentahydrate (8 mg, 0.03 mmol) dissolved
in 1 mL H2O were added and the reaction mixture was heated to
reflux overnight. Next day MeOH was evaporated under reduced
pressure and the residue was diluted with 80 mL EA and 80 mL
of aqueous NaHSO4 solution (5%w). The organic layer was sepa-
rated, dried over MgSO4 and the solvent was evaporated. The crude
material was recrystallised from EA yielding a white crystalline so-
lid (1.31 g, 67%), mp 117 °C. 1H NMR (300 MHz, CD3OD): 1.43 (s,
9H), 2.10–2.20 (m, 2H), 2.25–2.35 (m, 2H), 4.29 (s, 2H), 4.44 (t,
J = 6.89, 2H), 7.82 (s, 1H). 13C NMR (75 MHz, CD3OD): 26.7, 29.0,
31.5, 36.9, 50.6, 80.5, 124.2, 147.3, 158.3, 176.1. C12H20N4O4: MS
(LC–MS-II): m/z (%) [tR = 5.1 min]: 285 (35, MH+), 569 (100).
Germany) served as RP-columns at flow rates of 20 and 38 mL/min,
respectively. MixturesofMeCNanddilutedaqueousTFA(0.1%)were
used as mobile phase. 1H NMR spectra were recorded at 300 MHz on
a Bruker Avance 300 spectrometer or at 600 MHz on a Bruker Avance
III 600 with cryogenic probehead (Bruker, Karlsruhe, Germany). 13
C
NMR spectra were recorded at 75 MHz on a Bruker Avance 300 spec-
trometer. All chemical shifts values are reported in ppm. UV/Vis
spectrawererecordedwitha VarianCaryBIO50UV/Vis/NIRspectro-
photometer (Varian Inc., CA, USA). Mass spectra: Finnigan SSQ 710A
(EI), Finnigan MAT 95 (CI), Finnigan MAT TSQ 7000 (Thermo FINNI-
GAN, USA) (ES/LC–MS). LC-system for LC–MS: Agilent 1100 (Palo
Alto, USA). LC–MS method I (LC–MS-I): column: Phenomenex Luna
C18, 3.0
l
m, 100 ꢁ 2 mm HST (Phenomenex, Aschaffenburg, Ger-
many); flow: 0.30 mL/min; solvent A (water + 0.1% TFA), solvent B
(MeCN); gradient: 0 min [A/B 95:5], 1 min [A/B 95:5], 11 min [A/B
2:98], 18 min [A/B 2:98], 19 min [A/B 95:5], 24 min [A/B 95:5]. LC–
MS method II: column: Phenomenex Luna C18, 2.5
l
m, 50 ꢁ 2 mm
4.2.3. 6-(tert-Butoxycarbonylamino)-2,2-dimethyl-4,11-dioxo-
3-oxa-5,7,10-triaza-tetradec-5-en-14-oic acid (17)
HST (Phenomenex, Aschaffenburg, Germany); flow: 0.40 mL/min;
solvent A (water + 0.1% TFA), solvent B (MeCN); gradient: 0 min [A/
B 95:5], 8 min [A/B 2:98], 11 min [A/B 2:98], 12 min [A/B 95:5],
15 min [A/B 95:5]. Analytical HPLC (HPLC): Compounds 1c–1i, 1k–
Compound 16 (300 mg, 0.99 mmol) and succinic anhydride
(119 mg, 1.19 mmol) were dissolved in a mixture of THF (10 mL)
and NEt3 (150 mg, 1.49 mmol) and stirred overnight. Next day
water was added (10 mL) and the mixture was stirred for 1 h.
THF was evaporated completely and the residue was diluted with
water (20 mL). Aqueous NaHSO4 solution (5%w, 10 mL) was added
and the mixture was extracted with dichloromethane (2 ꢁ 50 mL).
The organic layer was dried over MgSO4 and the solvent was evap-
orated yielding a white solid (197 mg, 49%), mp 77–80 °C. 1H NMR
(300 MHz, CDCl3): 1.47 (s, 9H), 1.49 (s, 9H), 2.50–2.60 (m, 2H),
2.60–2.70 (m, 2H), 3.35–3.45 (m, 2H), 3.45–3.60 (m, 2H), 7.00–
8.40 (br s, 1H), 8.50 (br s, 1H), 8.67 (br s, 1H), 11.38 (br s, 1H).
13C NMR (75 MHz, CDCl3): 28.0, 28.2, 30.3, 30.5, 40.3, 41.9, 79.9,
83.9, 153.0, 157.6, 162.5, 172.9, 175.1. C17H30N4O7: MS (LC–MS-
I): m/z (%) [tR = 10.3 min]: 403 (100, MH+), 805 (10).
1q, 1t, 1u: Phenomenex Luna C18, 3.0
nex, Aschaffenburg, Germany); flow: 0.30 mL/min; solvent
(H2O + 0.0059%TFA), solvent B (MeCN); gradient: 0 min [A/B 95:5],
30 min [A/B 2:98]; 1j, 1r, 1s: Eurospher-100 C18, m,
lm, 150 ꢁ 2 mm (Phenome-
A
5
l
250 ꢁ 4.0 mm (Knauer, Berlin, Germany); flow: 0.80 mL/min; sol-
vent A (water + 0.05% TFA), solvent B (MeCN); gradient: 0 min [A/B
85:15], 28 min [A/B 45:55], 33 min [A/B 5:95], 40 min [A/B 5:95].
Melting points were determined with a Lambda Photometrics Opti-
melt MPA100 apparatus (Lambda photometrics, Harpenden, UK),
they are not corrected. Thin layer chromatography (TLC) was per-
formed on alumina plates coated with silica gel (Merck Silica Gel
60 F245, thickness 0.2 mm). Column chromatography (CC) was per-
formed with Merck Geduran SI 60 Silica Gel as the stationary phase.
The synthesis of 38, 1a and 1h was described elsewhere.1,4
Compounds 3,2 5,4 6,22 penta-acetyl gluconic acid,23 tBu-protected
propargyl alcohol,24 tBu-protected propargyl amine,25 10,26 11,27
14,28 16,29 19,30 21,31 23,32 31,33 374 were prepared according to
literature procedures.
4.2.4. 4-{2-[(2-tert-Butoxycarbonylaminoethyl)(tert-butoxy-
carbonyl)amino]ethylamino}-4-oxobutanoic acid (20)
Compound 19 (1.00 g, 3.30 mmol) was dissolved in 5 mL THF
and a solution of succinic anhydride (0.33 g, 3.30 mmol) in 5 mL
THF was added. Then NEt3 (0.50 g, 4.95 mmol) was added to the
mixture. The mixture was stirred overnight at ambient tempera-
ture. Next day THF was removed completely and the crude product
was dissolved in 20 mL dichloromethane. It was washed with
aqueous NaHSO4 soln (5%w, 30 mL), dried over MgSO4 and the
solvent was evaporated giving a white solid (1.03 g, 77%), mp
90 °C. 1H NMR (300 MHz, CDCl3): 1.40 (s, 9H), 1.43 (s, 9H), 2.48
(br s, 2H), 2.64 (br s, 2H), 3.10–3.40 (m, 8H), 4.95–5.25 (m, 1H),
4.2. Synthetic protocols and analytical data of 7, 8, 17, 20, 22,
25–28, 30 and 32–36
4.2.1. 4-[4-(tert-Butoxymethyl)-1H-1,2,3-triazol-1-yl]butanoic
acid (7)
4-Azidobutanoic acid (419 mg, 3.25 mmol) was mixed with
Boc-protected propargyl alcohol (364 mg, 3.25 mmol) in 5 mL

6298
S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
7.00–7.30 (m, 1H), 9.31 (br s, 1H). 13C NMR (75 MHz, CDCl3): 20.4,
29.7, 30.6, 39.3, 39.6, 47.0, 47.8, 79.6, 80.7, 156.6, 156.7, 172.8,
175.7. C18H33N3O7: MS (ES): m/z (%) 204 (5), 245 (45), 304 (50),
348 (25), 404 (100, MH+), 426 (5), 808 (7), 825 (15).
moved by filtration over Celite, MeOH was evaporated and a white
solid was obtained (146 mg, 94%), mp 73 °C. 1H NMR (300 MHz,
CD3OD): 1.34 (m, 4H), 1.59 (m, 4H), 2.16 (t, J = 7.45, 2H,), 2.27 (t,
J = 7.39, 2H), 2.52 (t, J = 6.64, 2H), 3.41 (t, J = 6.64, 2H), 3.67 (s,
3H). 13C NMR (75 MHz, CD3OD): 26.0, 26.9, 29.90, 29.92, 34.8,
34.9, 36.4, 36.9, 52.2, 173.9, 176.4, 177.7. C12H21NO5: MS (CI,
NH3): m/z (%) 260 (100, MH+), 277 (41).
4.2.5. 3-(2-tert-Butoxycarbonylaminoethyl)aminocarbonyl-
propanoic acid (22)
Succinic anhydride (0.41 g, 4.06 mmol) and NEt3 (56
lL,
0.41 mmol) were added to solution of amine 21 (0.65 g,
a
4.2.9. 8-[2-(tert-Butoxycarbonylamino)ethylamino]-8-oxoocta-
noic acid (28)
4.06 mmol) in dichloromethane (3 mL). The mixture was heated
to 60 °C for 25 min (microwave synthesizer (Biotage Initiator 8)).
After removal of the solvent under reduced pressure the product
(insoluble in dichloromethane) was afforded as a white solid
(1.05 g, 99%). 1H NMR (300 MHz, DMSO-d6): 1.37 (s, 9H), 2.25–
2.32 (m, 2H), 2.37–2.44 (m, 2H), 2.91–2.99 (m, 2H), 3.00–3.08
(m, 2H), 6.78 (t, J = 5.45, 1H), 7.85 (t, J = 5.29, 1H), 12.08 (br s,
1H). 13C NMR (75 MHz, DMSO-d6): 28.1, 29.0, 29.9, 38.6, 39.5,
Compound 26 (172 mg, 0.42 mmol) was dissolved in 5 mL
MeOH. Pd/C (17 mg) was added and the mixture was stirred at
15 bar hydrogen pressure in an autoclave overnight. Pd/C was fil-
tered off using Celite and MeOH was evaporated. The product
was obtained as a white solid (120 mg, 90%), mp 95 °C. 1H NMR
(300 MHz, CDCl3): 1.15–1.25 (m, 4H), 1.31 (s, 9H), 1.40–1.55 (m,
4H), 2.00–2.10 (m, 2H), 2.10–2.20 (m, 2H), 3.00–3.10 (m, 2H),
3.10–3.17 (m, 2H), 4.05 (br s, 3H). 13C NMR (75 MHz, CDCl3):
24.5, 25.3, 28.1, 28.5, 28.6, 33.9, 36.1, 39.6, 39.7, 79.6, 157.1,
174.9, 176.5. C15H28N2O5: MS (ES): m/z (%) 217 (100), 261 (45),
317 (85, MH+), 339 (15), 633 (10), 650 (25).
77.5, 155.5, 171.0, 173.8.
C11H20N2O5: (LC–MS-II): m/z (%)
[tR = 4.63 min]: 261 (40, MH+), 278 (6, MNH4+), 521 (35, 2MH+),
538 (100, 2MNH4+).
4.2.6. Benzyl 8-(3-methoxy-3-oxopropylamino)-8-oxooctanoate
(25)
4.2.10. 8-[3-(Benzyloxy)-3-oxopropylamino]-8-oxooctanoic acid
(30)
Acid 23 (250 mg, 0.95 mmol), DIPEA (368 mg, 2.85 mmol), and
HOBTꢂH2O (142 mg, 1.05 mmol) were dissolved in ice-cold dichloro-
methane (20 mL) and EDC (147 mg, 0.95 mmol) was added under
nitrogen atmosphere. After 15 min amine 24 (98 mg, 0.95 mmol)
was added. The reaction mixture was stirred overnight. Next day
the reaction mixture was washed with aqueous NaHSO4 soln
(5%w, 20 mL) and satd aqueous NaHCO3 solution (20 mL). The sol-
vent was dried over MgSO4, filtered and evaporated. The crude prod-
uct was purified by column chromatography (PE/EA 3:1?EA Rf = 0.4
[EA]) yielding the product as a white solid (253 mg, 76%), mp 48 °C.
1H NMR (300 MHz, CDCl3): 1.31 (m, 4H), 1.60 (m, 4H), 2.10 (m, 2H),
2.30 (t, J = 7.48, 2H), 2.50 (m, 2H), 3.50 (dd, J1 = 6.07, J2 = 11.99, 2H),
Octanedioic acid (500 mg, 2.87 mmol), DIPEA (1.11 g,
8.61 mmol) and HOBTꢂH2O (427 mg, 3.16 mmol) were dissolved
in ice-cold dichloromethane (20 mL) and EDC (490 mg, 3.16 mmol)
was added under nitrogen atmosphere. After 15 min 3-aminoprop-
anoic acid benzyl ester (617 mg, 2.87 mmol) was added and the ice
bath was removed. The mixture was stirred overnight. Next day an
aqueous NaHSO4 solution (5%w, 20 mL) was added and the crude
product was extracted with dichloromethane (2 ꢁ 20 mL), the or-
ganic phase was dried over MgSO4, filtered and evaporated. The
crude material was purified by column chromatography,
(EA?EA/MeOH 9:1). The obtained material is a 2:1 mixture of
the desired product (420 mg, 44%) and the appropriate octanedioic
acid diamide. The mixture was used in the next synthesis step
without further purification.
3.68 (s, 3H), 5.10 (s, 2H), 6.00–6.10 (br s, 1H), 7.30–7.38 (m, 5H). 13
C
NMR (75 MHz, CDCl3): 24.0, 25.4, 28.75, 28.80, 33.9, 34.2, 34.7, 36.6,
51.8, 66.1, 128.2, 128.6, 136.1, 173.0, 173.2, 173.6. C19H27NO5: MS
(CI, NH3): m/z (%) 350 (100, MH+).
4.2.11. tert-Butyl 2-(pyrene-1-carbonylamino)ethylcarbamate (32)
Pyrene-1-carboxylic acid (246 mg, 1.00 mmol), DIPEA (387 mg,
3.00 mmol) and HOBTꢂH2O (149 mg, 1.10 mmol) were dissolved
in ice-cold dichloromethane (20 mL). EDC (171 mg, 1.10 mmol)
was added under nitrogen atmosphere. After 15 min Boc-protected
ethylenediamine (160 mg, 1.0 mmol) was added and the ice bath
was removed. The mixture was stirred overnight. Next day the
reaction mixture was diluted with dichloromethane (40 mL),
washed with aqueous NaHSO4 solution (5%w, 40 mL) and satd
aquoeus NaHCO3 solution (1 ꢁ 40 mL). The organic layer was dried
over MgSO4 and evaporated. The crude compound was recrystal-
lised from EA yielding the product as a yellow solid (213 mg,
55%), mp >190 °C. 1H NMR (300 MHz, DMSO-d6): 1.41 (s, 9H),
3.20–3.30 (m, 2H), 3.40–3.50 (m, 2H), 7.00 (t, J = 5.57, 1H), 8.05–
8.30 (m, 5H), 8.30–8.40 (m, 3H), 8.50–8.54 (m, 1H), 8.71 (t,
J = 5.37, 1H). 13C NMR (75 MHz, DMSO-d6): 28.2, 39.58, 39.61,
77.6, 123.5, 123.7, 124.2, 124.7, 125.3, 125.5, 125.7, 126.5, 127.1,
127.9, 128.2, 130.1, 130.6, 131.5, 131.8, 155.7, 168.9. C24H24N2O3:
MS (EI): m/z (%) 201 (58), 229 (100), 245 (57), 258 (15), 389 (22,
MH+).
4.2.7. Benzyl 8-[2-(tert-butoxycarbonylamino)ethylamino]-8-
oxooctanoate (26)
Compound 23 (264 mg, 1.00 mmol) was dissolved in 10 mL of
dichloromethane. HOBTꢂH2O (149 mg, 1.10 mmol) and DIPEA
(387 mg, 3.00 mmol) were added under nitrogen atmosphere.
The mixture was stirred and cooled in an ice bath. EDC (171 mg,
1.10 mmol) was added and after 15 min amine 21 (160 mg,
1.00 mmol) was added. The mixture was stirred overnight. Next
day DCM was added (10 mL), the organic phase was washed once
with aqueous NaHSO4 (5%w, 20 mL) and satd aqueous NaHCO3 soln
(20 mL), was dried over MgSO4 and the organic layer was evapo-
rated. The crude product was purified by column chromatography
(PE/EA 1:1?EA Rf = 0.3 [EA]). A white, wax-like solid (251 mg,
62%), mp 60 °C was obtained. 1H NMR (300 MHz, CDCl3): 1.25–
1.25 (m, 4H), 1.43 (s, 9H), 1.55–1.70 (m, 4H), 2.10–2.17 (m, 2H),
2.30–2.37 (m, 2H), 3.20–3.30 (m, 2H), 3.30–3.40 (m, 2H), 4.97 (br
s, 1H), 5.10 (s, 2H), 6.20 (br s, 1H), 7.30–7.40 (m, 5H). 13C NMR
(75 MHz, CDCl3): 24.7, 25.4, 28.4, 28.7, 28.8, 34.1, 36.5, 40.3,
40.7, 66.1, 79.6, 128.2, 128.6, 136.1, 157.0, 173.6, 173.8.
C22H34N2O5: MS (ES): m/z (%) 407 (100, MH+).
4.2.12. 2-(Pyren-1-carbonylamino)ethanaminium chloride (33)
Compound 32 (549 mg, 1.41 mmol) was suspended in a mixture
of 20 mL MeOH and 5 mL HCl (37%). It was stirred 30 min at 70 °C.
The solvent was evaporated completely and a yellow solid was ob-
tained (445 mg, 97%), mp >190 °C. 1H NMR (300 MHz, DMSO-d6):
3.05–3.20 (m, 2H), 3.65–3.75 (m, 2H), 8.05–8.15 (m, 1H), 8.15–
4.2.8. 8-(3-Methoxy-3-oxopropylamino)-8-oxooctanoic acid (27)
Compound 25 (210 mg, 0.60 mmol) was dissolved in MeOH,
placed into an autoclave and Pd/C (25 mg) was added after flushing
the autoclave with N2. The reaction mixture was stirred overnight
at room temperature and 12 bar hydrogen pressure. Pd/C was re-

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6299
8.30 (m, 3H), 8.30–8.35 (m, 4H), 8.35–8.50 (br s, 3H), 8.62 (m, 1H),
9.02 (t, J = 5.33, 1H). 13C NMR (75 MHz, DMSO-d6): 37.3, 38.4,
123.5, 123.7, 124.2, 124.7, 125.6, 125.7, 125.8, 126.5, 127.1,
128.0, 128.3, 130.0, 130.6, 130.8, 131.7, 169.2. C19H16N2O: MS
(CI, NH3): m/z (%) 289 (100, MH+).
in a flask under nitrogen atmosphere with 10 mL of cold DMF. After
15 min compound 3 (1 mmol) was added and the mixture was stir-
red overnight at room temperature. Next day the solvent was re-
moved completely and the crude material was purified with
column chromatography.
4.2.13. 3-Oxo-1-phenyl-2,5,8,11-tetraoxatridecan-13-oic acid (34)
3,6,9-Trioxaundecanedioic acid (5.55 g, 25 mmol), benzyl alco-
hol (2.70 mg, 25 mmol) and toluene sulfonic acid (43 mg,
4.3.1. tert-Butyl (pentacosa-10,12-diynamido)methylthiomethy-
lenecarbamate (4b)
Column chromatography (PE/EA 9:1, Rf = 0.3). The product is a
white solid (489 mg, 89%), mp 37 °C. 1H NMR (300 MHz, CDCl3):
0.75–0.85 (t, J = 7.24, 3H), 1.10–1.70 (m, 41H), 2.17 (t, J = 6.92,
4H), 2.30–2.45 (m, 5H), 12.00–12.45 (m, 1H). 13C NMR (75 MHz,
CDCl3): 14.13, 14.45, 19.18, 19.20, 22.69, 24.50, 27.99, 28.29,
28.36, 28.74, 28.86, 28.91, 29.06, 29.11, 29.35, 29.49, 29.62,
29.64, 29.65, 31.92, 37.38, 65.25, 65.34, 77.35, 77.54, 81.17,
160.97, 171.32, 171.49. C32H54N2O3S: MS (LC–MS-I): m/z (%)
[tR = 15.3 min]: 547 (100, MH+).
0.25 mmol) were combined in
a 250 mL flask and toluene
(70 mL) was added. The flask was connected to a Dean–Stark appa-
ratus. The mixture was heated to reflux and the reaction was com-
pleted after 1 h. satd aqueous NaHCO3 (50 mL) solution was added
and the phases were separated. The aqueous layer was collected
and extracted with EA (3 ꢁ 100 mL). The organic layer was dried
over MgSO4 and evaporated. The product is a viscous oil (1.87 g,
24%). 1H NMR (300 MHz, CD3OD): 3.60–3.75 (m, 8H), 4.10 (s,
2H), 4.20 (s, 2H), 5.17 (s, 2H), 7.30–7.40 (m, 5H). 13C NMR
(75 MHz, CD3OD): 66.7, 68.5, 68.6, 70.3, 70.5, 70.7, 70.9, 128.44,
128.47, 128.52, 128.63, 128.65, 135.3, 170.5, 173.2. C15H20O7: MS
(CI, NH3): m/z (%) 242 (6), 286 (28), 313 (2, MH+), 330 (100).
4.3.2. tert-Butyl (hexadecanamido)methylthiomethylenecarba-
mate (4c)
Column chromatography (PE/EA 9:1 Rf = 0.5). The product is a
white solid (342 mg, 80%), mp 62 °C. 1H NMR (300 MHz, CDCl3):
0.82 (t, J = 6.68, 3H), 1.18–1.28 (m, 24H), 1.46 (s, 9H), 1.60 (m,
2H), 2.33 (s, 3H), 2.37 (m, 2H), 12.40 (br s, 1H). 13C NMR
(75 MHz, CDCl3): 14.1, 14.5, 28.0, 22.7, 29.1–29.7, 31.9, 80.2.
4.2.14. Benzyl 1,6-dioxo-1-(pyren-1-yl)-8,11,14-trioxa-2,5-diaza-
hexadecan-16-oate (35)
Compound 34 (462 mg, 1.48 mmol), DIPEA (476 mg,
3.69 mmol) and HOBTꢂH2O (200 mg, 1.48 mmol) were dissolved
in DMF (10 mL) and EDC (229 mg, 1.48 mmol) was added (ice
bath). After 15 min compound 33 (400 mg, 1.23 mmol) was added
and the mixture was stirred overnight. Next day DMF was removed
completely, the residue was dissolved in dichloromethane (20 mL)
and washed with aqueous NaHSO4 solution (5%w, 20 mL), satd
aqueous NaHCO3 solution (1 ꢁ 20 mL) and brine (1 ꢁ 20 mL). The
crude product was purified with column chromatography (PE/EA
1:1?EA/MeOH 4:1 Rf = 0.3 [EA/MeOH 9:1]). The product is a yel-
low solid (420 mg, 59%), mp >190 °C. 1H NMR (300 MHz, CDCl3):
3.45–3.80 (m, 12H), 3.97 (s, 2H), 4.02 (s, 2H), 5.07 (s, 2H), 7.20–
7.40 (m, 6H), 7.64 (t, J = 5.57, 1H), 7.95–8.10 (m, 6H), 8.14–8.19
(m, 2H), 8.55–8.60 (m, 1H). 13C NMR (75 MHz, CDCl3): 38.8, 40.9,
66.6, 68.4, 70.1, 70.27, 70.32, 70.7, 70.9, 124.3, 124.4, 124.6,
124.69, 124.7, 125.65, 125.72, 126.3, 127.1, 128.39, 128.47,
128.51, 128.55, 128.62, 130.7, 130.95, 131.12, 132.4, 135.2, 170.1,
170.4, 171.4. C34H34N2O7: MS (ES): m/z (%) 583 (100, MH+), 605
C23H44N2O3S: MS (CI, NH3): m/z (%) 329 (15), 429 (100, MH+).
4.3.3. tert-Butyl-[4-(pyren-1-yl)butanamido]methylthiomethyl-
enecarbamate (4d)
Column chromatography (PE/EA 9:1?PE/EA 3:1, Rf = 0.4 [PE/EA
3:1]). The product was obtained as a white solid (299 mg, 65%), mp
>190 °C (decomp.). 1H NMR (300 MHz, CDCl3): 1.53 (s, 9H), 2.18–
2.32 (m, 2H), 2.32–2.43 (m, 3H), 2.45–2.70 (m, 2H), 3.41 (t,
J = 7.46, 2H), 7.80–7.90 (m, 1H), 7.95–8.05 (m, 3H), 8.05–8.20 (m,
4H), 8.25–8.35 (m, 1H), 12.15–12.55 (m, 1H). 13C NMR (75 MHz,
CDCl3): 14.6, 26.1, 28.1, 32.5, 36.7, 81.3, 123.3, 124.86, 124.97,
125.01, 125.10, 125.88, 126.76, 127.32, 127.43, 127.47, 127.53,
128.74, 130.02, 130.03, 131.43, 135.26, 150.98, 161.04, 171.24.
C27H28N2O3S: MS (LC–MS-I): m/z (%) [tR = 15.5 min]: 461 (100,
MH+).
4.3.4. tert-Butyl-(pyren-1-carbonylamino)methylthiomethylen-
ecarbamate (4e)
(60), 621 (10). UV (MeOH): k (
e
) 233 (65 ꢁ 103), 242 (89 ꢁ 103),
265 (34 ꢁ 103), 275 (51 ꢁ 103), 326 (31 ꢁ 103), 340 (41 ꢁ 103).
Fluorescence (MeOH): k (type) 383 (monomer), 401 (monomer)
Column chromatography (PE/EA 9:1, Rf = 0.1). The product was
obtained as a bright yellow solid (190 mg, 45%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, CDCl3): 1.54–1.63 (m, 9H), 2.53–2.67
(m, 3H), 7.95–8.40 (m, 7H), 8.90–9.00 (m, 1H), 9.40–9.50 (m, 1H),
12.60–13.40 (m, 1H). 13C NMR (75 MHz, CDCl3): 15.1, 28.1, 83.6,
124.03, 124.36, 124.99, 125.55, 126.07, 126.21, 127.27, 127.38,
129.09, 129.54, 129.67, 130.42, 130.47, 131.05, 131.25, 134.25,
151.25, 171.15, 178.73. C24H22N2O3S: MS (LC–MS-I): m/z (%)
[tR = 16.3 min]: 419 (100, MH+), 837 (10).
4.2.15. 1,6-Dioxo-1-(pyren-1-yl)-8,11,14-trioxa-2,5-diazahexad-
ecan-16-oic acid (36)
Compound 35 (203 mg, 0.35 mmol) was dissolved in 5 mL of
MeOH. Pd/C (10 mg) was added and the mixture was stirred in
an autoclave at 1 bar hydrogen pressure for 4 h. After that time
Pd/C was filtered off with Celite and the solvent was removed.
The product is a yellow solid (172 mg, 100%), mp >190 °C. 1H
NMR (300 MHz, CD3OD): 3.50–3.58 (m, 4H), 3.58–3.65 (m, 4H),
3.65–3.75 (m, 4H), 4.01 (s, 2H), 4.03 (s, 2H), 8.00–8.30 (m, 8H),
8.45–8.55 (m, 1H). 13C NMR (75 MHz, DMSO-d6): 38.1, 39.2, 67.5,
69.4, 69.6, 69.7, 69.9, 70.2, 123.5, 123.7, 124.3, 124.7, 125.2,
125.5, 125.7, 126.5, 127.1, 127.7, 128.0, 128.2, 130.1, 130.6,
131.5, 131.8, 169.0, 169.6, 171.6. C27H28N2O7: MS (ES): m/z (%)
493 (100, MH+), 510 (10), 515 (50).
4.3.5. tert-Butyl 5,15,20-trioxo-20-(pyren-1-yl)-7,10,13-trioxa-
2-thia-4,16,19-triazaicosan-3-ylidenecarbamate (4f)
Column chromatography (EA?EA/MeOH 9:1, Rf = 0.3 [EA/
MeOH 9:1]). The product was obtained as a light yellow solid
(485 mg, 73%), mp >190 °C (decomp.). 1H NMR (300 MHz, CDCl3):
1.49 (s, 9H), 2.20–2.40 (m, 3H), 3.50–3.75 (m, 10H), 3.75–3.85
(m, 2H), 3.90–4.05 (m, 4H), 7.10–7.90 (m, 2H), 7.95–8.15 (m, 6H),
8.15–8.25 (m, 2H), 8.55–8.65 (m, 1H), 11.80–12.90 (m, 1H). 13C
NMR (75 MHz, CDCl3): 14.4, 28.0, 39.0, 40.9, 70.4, 70.5, 70.6,
70.8, 70.9, 71.5, 77.2, 124.4, 124.6, 124.7, 125.7, 125.8, 126.3,
127.1, 128.6, 128.7, 130.7, 131.2, 132.5, 165.9, 169.1, 170.4.
4.3. General procedure for the preparation of compounds4b–4g,
4i, 4k–4q, 4t, 4u
The appropriate carboxylic acid (1 mmol), HOBTꢂH2O
C
34H40N4O8S: MS (ES): m/z (%) 565 (50), 665 (100, MH+),
(1.2 mmol), EDC (1.2 mmol) and DIPEA (2 mmol) were combined
687 (15).

6300
S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
4.3.6. Benzyl 3-{8-[(Boc-amino)-methylthio-methyleneamino]-
8-oxooctanamido}propanoate (4g = 4o)
viscous oil. 1H NMR (300 MHz, CDCl3): 1.29 (d, J = 2.47, 6H), 1.40
(s, 3H), 1.42–1.50 (m, 12H), 2.34 (s, 3H), 2.60–2.85 (m, 4H), 3.90–
4.00 (m, 1H), 4.19 (dd, J1 = 1.55, J2 = 7.85, 2H), 4.24 (d, J = 4.96,
1H), 4.28 (dd, J1 = 2.50, J2 = 4.99, 1H), 4.57 (dd, J1 = 2.46, J2 = 7.88,
1H), 5.48 (d, J = 4.98, 1H), 12.05–12.49 (m, 1H). 13C NMR
(75 MHz, CDCl3): 14.5, 24.5, 24.9, 25.9, 26.0, 27.9, 28.3, 29.3,
31.8, 35.8, 63.5, 63.8, 65.9, 70.4, 70.7, 71.0, 81.3, 83.4, 96.3,
108.7, 109.6, 150.9, 169.8, 170.9, 171.8, 172.8, 184.3.
Column chromatography (PE/EA 1:1?PE/EA 3:7, Rf = 0.15[PE/
EA 1:1]). The product is a viscous oil (364 mg, 72%). 1H NMR
(300 MHz, CDCl3): 1.25–1.40 (m, 4H), 1.51 (s, 9H), 1.55–1.70 (m,
4H), 2.05–2.15 (m, 2H), 2.35–2.50 (m, 2H), 2.38 (s, 3H), 2.55–2.60
(m, 2H), 3.45–3.55 (m, 2H), 5.13 (s, 2H), 6.02 (s, 1H), 7.30–7.40
(m, 5H), 12.45 (br s, 1H). 13C NMR (75 MHz, CDCl3): 14.5, 25.4,
28.0, 28.7, 34.1, 34.8, 36.6, 66.6, 77.3, 128.3, 128.4, 128.7, 135.6,
172.6, 172.9. C25H37N3O6S: MS (ES): m/z (%) 408 (25), 508 (100,
MH+).
C23H36N2O10S: MS (ES): m/z (%) 533 (100, MH+).
4.3.12. tert-Butyl [17,17-dimethyl-13-(methylthio)-4,11,15-tri-
oxo-16-oxa-3,12,14-triazaoctadec-13-en-1-yl]carbamate (4p)
Column chromatography (PE/EA 1:1?PE/EA 3:7, Rf = 0.1 [PE/EA
1:1]). The product was obtained as a white solid (368 mg, 77%), mp
95 °C 1H NMR (300 MHz, CDCl3): 1.25–1.35 (m, 4H), 1.37 (s, 9H),
1.40–1.50 (m, 9H), 1.50–1.65 (m, 4H), 2.05–2.15 (m, 2H), 2.32 (s,
3H), 2.32–2.50 (m, 2H), 3.15–3.35 (m, 4H), 4.80–4.95 (br s, 1H),
6.00–6.20 (br s, 1H), 12.00–12.50 (m, 1H). 13C NMR (75 MHz,
CDCl3): 14.5, 24.3, 25.4, 28.0, 28.4, 28.6, 28.8, 36.5, 37.2, 40.3,
40.8, 79.7, 81.3, 157.0, 161.0, 171.3, 171.5, 173.7. C22H40N4O6S:
MS (ES): m/z (%) 389 (18), 489 (100, MH+).
4.3.7. (2R,3R,4S,5R)-6-[(Boc-amino)-methylthio-methylene-
amino]-6-oxohexane-1,2,3,4,5-pentayl pentaacetate (4i)
Column chromatography (PE/EA 3:1, Rf = 0.1). Compound 4i
(252 mg, 44%) was obtained as a viscous oil. 1H NMR (300 MHz,
CDCl3): 1.48 (s, 9H), 2.00–2.10 (m, 12H), 2.23 (s, 3H), 2.42 (s,
3H), 4.19 (dd, J1 = 5.84, J2 = 12.33, 1H), 4.34 (dd, J1 = 3.53,
J2 = 12.33, 1H), 5.05 (dt, J1 = 3.53, J2 = 5.84, 1H), 5.40 (d, J = 3.08,
1H), 5.52 (dd, J = 5.84, 1H), 5.90 (dd, J1 = 3.08, J2 = 5.84, 1H), 11.88
(br s, 1H). 13C NMR (75 MHz, CDCl3): 14.8, 20.5, 20.6, 20.7, 20.8,
21.1, 27.9, 61.5, 69.1, 69.3, 70.1, 74.5, 84.1, 150.5, 169.5, 169.7,
169.8, 170.2, 170.5, 174.5, 176.8. C23H34N2O13S: MS (ES): m/z (%)
579 (100, MH+).
4.3.13. tert-Butyl-16,16-dimethyl-5,14-dioxo-15-oxa-2-thia-
4,11,13-triazaheptadecane-3,12-diylidenedicarbamate (4q)
Column chromatography (PE/EA 3:1, Rf = 0.35) Compound 4q
(413 mg, 76%) was obtained as a white solid, mp 116–120 °C. 1H
NMR (300 MHz, CDCl3): 1.30–1.45 (m, 2H), 1.48 (s, 9H), 1.49 (s,
9H), 1.52 (s, 9H), 1.55–1.75 (m, 4H), 2.30–2.55 (m, 5H), 3.35–
3.45 (m, 2H), 8.30 (br s, 1H), 11.49 (br s, 1H), 12.16–12.48 (m,
1H). 13C NMR (75 MHz, CDCl3): 14.5, 24.2, 26.3, 28.0, 28.1, 28.3,
28.8, 37.1, 40.6, 79.2, 81.3, 83.1, 153.3, 156.1, 161.0, 163.6, 171.2,
171.3. C24H43N5O7S: MS (LC–MS-I): m/z (%) [tR = 14.2 min]: 546
(100, MH+), 1092 (10).
4.3.8. Methyl 3-{8-[boc-amino(methylthio)methyleneamino]-8-
oxooctanamido}propanoate (4k)
Column chromatography (PE/EA 1:1?EA, Rf = 0.4 [EA]). The
product is a colourless oil (337 mg, 78%). 1H NMR (300 MHz,
CDCl3): 1.33 (m, 4H), 1.51 (s, 9H), 1.63 (m, 4H), 2.14 (m, 2H),
2.35–2.50 (m, 5H), 2.53 (m, 2H), 3.47–3.54 (dd, J1 = 6.07,
J2 = 11.94, 2H), 3.70 (s, 3H), 6.04 (br s, 1H), 12.00–12.70 (br s,
1H). 13C NMR (75 MHz, CDCl3): 14.5, 24.3, 25.4, 28.0, 28.7, 28.8,
33.9, 34.7, 36.6, 37.3, 51.8, 77.2, 81.3, 171.3, 172.9, 173.3.
C
19H33N3O6S: MS (CI, NH3): m/z (%) 332 (14), 432 (100, MH+).
4.3.14. Di-tert-butyl-17,17-dimethyl-5,8,15-trioxo-16-oxa-2-thia-
4,9,12,14-tetraaza-octadecane-3,13-diylidenedicarbamate (4t)
Column chromatography (PE/EA 4:6, Rf = 0.2). The product was
obtained as a colourless, viscous oil (180 mg, 31%). 1H NMR
(300 MHz, CDCl3): 1.43–1.52 (m, 27H), 2.34 (s, 3H), 2.50–2.90 (m,
4H), 3.35–3.75 (m, 4H), 7.60–7.90 (m, 1H), 8.40–8.70 (m, 1H),
11.37 (br s, 1H), 11.75–12.50 (m, 1H). 13C NMR (75 MHz, CDCl3):
14.5, 27.99, 28.04, 28.25, 38.6, 38.8, 40.4, 41.6, 79.6, 83.2, 83.6,
153.0, 153.1, 156.9, 157.4, 162.8, 177.5. C24H42N6O8S: MS (LC–
MS-I): m/z (%) [tR = 12.9 min]: 575 (100, MH+), 1149 (15).
4.3.9. tert-Butyl-{4-[4-(tert-butoxymethyl)-1H-1,2,3-triazol-1-
yl]butanamido}methylthio-methylenecarbamate (4l)
Column chromatography (PE/EA 3:1?PE/EA 4:6, Rf = 0.4[PE/EA
4:6]). Compound 4l (254 mg, 62%) was obtained as a white solid,
mp 93 °C. 1H NMR (300 MHz, CDCl3): 1.26 (s, 9H), 1.49 (s, 9H),
2.15–2.30 (m, 2H), 2.37 (s, 3H), 2.40–2.60 (m, 2H), 4.40 (t,
J = 6.33, 2H), 4.57 (s, 2H), 7.52 (s, 1H), 12.10–12.50 (m, 1H). 13C
NMR (75 MHz, CDCl3): 14.6, 25.2, 27.6, 28.0, 35.3, 49.1, 56.5,
73.8, 81.5, 83.6, 122.1, 147.0, 171.0, 171.4. C18H31N5O4S: MS (CI):
m/z (%) 314 (18), 414 (100, MH+).
4.3.15. tert-Butyl 14-boc-amino-12-boc-5,8-dioxo-2-thia-4,9,12-
triazatetradecan-3-ylidenecarbamate (4u)
4.3.10. tert-Butyl {4-[4-(Boc-aminomethyl)-1H-1,2,3-triazol-1-
yl]butanamido}methylthio-methylenecarbamate (4m)
Column chromatography (PE/EA 1:1?EA, Rf = 0.15 [PE/EA 1:1]).
The product was obtained as a colourless, viscous oil (386 mg,
67%). 1H NMR (300 MHz, CDCl3): 1.40 (s, 9H), 1.45 (s, 9H), 1.48
(s, 9H), 2.35 (s, 3H), 2.40–2.90 (m, 4H), 3.10–3.50 (m, 8H), 4.85–
5.10 (m, 1H), 6.50–7.00 (m, 1H), 11.70–12.60 (m, 1H). 13C NMR
(75 MHz, CDCl3): 14.5, 28.0, 28.2, 28.3, 28.4, 39.0, 39.6, 47.2,
47.9, 79.4, 80.5, 156.1, 156.7, 170.5, 171.2. C25H45N5O8S: MS (ES):
m/z (%) 576 (100, MH+).
Column chromatography (PE/EA 3:1?PE/EA 4:6, Rf = 0.3[PE/EA
4:6]). Compound 4m (341 mg, 75%) was obtained as a colourless,
viscous oil. 1H NMR (300 MHz, CDCl3): 1.34 (s, 9H), 1.41 (s, 9H),
2.10–2.25 (m, 2H), 2.29 (s, 3H), 2.30–2.50 (m, 2H), 4.25–4.40 (m,
4H), 5.20–5.40 (br s, 1H), 7.49 (s, 1H), 11.70–12.50 (m, 1H). 13C
NMR (75 MHz, CDCl3): 14.5, 24.9, 27.9, 28.3, 33.9, 37.6, 49.4,
79.5, 122.0, 145.6, 155.9, 160.7, 169.9, 184.5. C19H32N6O5S: MS
(LC–MS-II): m/z (%) [tR = 7.3 min]: 457 (100, MH+), 913 (40).
4.3.16. tert-Butyl 11-boc-amino-5,8-dioxo-2-thia-4,9-diazaund-
ecan-3-ylidenecarbamate (4s)
4.3.11. ((3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-
3aH-bis[1,3]dioxolo[4,5-b:40,50-d]pyran-5-yl)methyl 4-((boc-
amino)-methylthio-methyleneamino)-4-oxobutanoate (4n)
Column chromatography (PE/EA 9:1?PE/EA 3:1, Rf = 0.2 [PE/EA
9:1]). Compound 4n (396 mg, 74%) was obtained as a colourless,
DIPEA (0.37 g, 2.84 mmol) and TBTU (0.96 g, 3.0 mmol) were
added to a solution of 22 (0.74 g, 2.84 mmol) in DMF (5 mL). The
mixture was stirred under argon for 15 min prior to the addition
of 3 (0.595 g, 3.13 mmol) and DIPEA (0.74 g, 2.84 mmol). After stir-
ring over night glacial acetic acid (162 lL, 2.84 mmol) was added
and the solvent was removed under reduced pressure (1 mbar) at
a temperature of 45 °C. Purification with column chromatography
(dichloromethane/EA 3:1?1:2, Rf = 0.45[dichloromethane/EA
This compound was used for preparation of 1g and 1o.

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6301
3:1]) yielded the product as a white solid (1.17 g, 95%). 1H NMR
(300 MHz, DMSO-d6): 1.37 (s, 9H), 1.42 (s, 9H), 2.27 (2.25) (s,
3H), 2.30–2.38 (m, 2H), 2.53–2.59 (m, 2H), 2.09–2.99 (m, 2H),
2.99–3.10 (m, 2H), 6.75–6.90 (m, 1H), 7.86–7.99 (m, 1H), 11.24
(10.98) (s, 1H). C18H32N4O6S: (LC–MS-II): m/z (%) [tR = 6.87 min]:
433 (100, MH+).
3.35–3.45 (m, 2H), 4.05–4.20 (m, 2H), 4.25–4.40 (m, 1H), 5.12 (s,
1H), 6.67 (d, J = 8.48, 2H), 7.00 (d, J = 8.47, 2H), 7.10–7.30 (m,
10H), 7.90–8.00 (m, 1H), 8.00–8.10 (m, 1H), 8.15 (br s, 2H), 8.20–
8.35 (m, 4H), 8.35–8.65 (m, 5H), 8.80–8.95 (m, 1H), 9.31 (br s,
1H), 11.32 (br s, 1H).
C47H45N5O4: MS (LC–MS-I): m/z (%)
[tR = 10.2 min]: 744 (100, MH+). HPLC: 100% (ELSD, tR = 19.1 min).
UV (MeCN): k (
e
) 264 (20 ꢁ 103), 275 (31 ꢁ 103), 312 (10 ꢁ 103),
4.4. General procedure for the preparation of compounds 1b–1g,
1i, 1k–1q and 1s–1u
326 (17 ꢁ 103), 342 (23 ꢁ 103). Fluorescence (kex. = 340 nm,
MeCN): 397 nm (monomer), 419 nm (monomer).
The appropriate S-methylisothiourea derivative 4 (0.2 mmol),
compound 5 (0.2 mmol) and HgCl2 (0.2 mmol) were dissolved sepa-
rately in small amounts of DMF (1–2 mL). The solutions of the com-
pounds 4 and 5 were combined in a small flask under nitrogen
atmosphere. NEt3 (2 mmol) was added under stirring, then theHgCl2
solution was added and the mixture was stirred overnight at room
temperature. Next day DMF was removed completely and the resi-
due was dissolved in DCM. The precipitate of mercury salts was fil-
tered off and the organic phase was concentrated. The crude
products were purified by column chromatography (PE/EA mix-
tures). The Boc- and tBu-protected target molecules were deprotec-
ted to the corresponding target compounds 1b–1g, 1i, 1k–1q and
1s–1u without further characterisation: The corresponding pro-
tected compound was dissolved in a dichloromethane/TFA 1:1 mix-
ture (5 mL) and stirred for 2 h. After that time the solvent was
removed completely under reduced pressure and the oily residue
was repeatedly dissolved in dichloromethane and the solvent evap-
orated to remove TFA. Except for 1b and 1s (1b: purified by column
chromatography, 1s: no purification after deprotection) the depro-
tected compounds were purified with preparative HPLC. The given
yield corresponds to the coupling step in the first part of this proce-
dure, the deprotection step was quantitative.
4.4.4. (R)-1-[4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]-2-(pyrene-1-carbonyl)guanidinium 2,2,2-
trifluoroacetate (1e)
The product is a yellow solid (106 mg, 68%), mp >190 °C
(decomp.). 1H NMR (300 MHz, CD3CN): 1.50–1.90 (m, 4H), 3.20–
3.40 (m, 2H), 4.10–4.30 (m, 2H), 4.35–4.45 (m, 1H), 5.05 (s, 1H),
6.72 (d, J = 8.51, 2H), 7.04 (d, J = 8.47, 2H), 7.15–7.40 (m, 13H),
8.05–8.40 (m, 8H), 8.50–8.60 (m, 1H), 9.25–10.30 (m, 2H), 13.05
(s, 1H). C44H39N5O4: MS (LC–MS-I): m/z (%) [tR = 9.7 min]: 702
(100, MH+). HPLC: 99% (ELSD, tR = 17.8 min). UV (MeCN): k (e)
242 (45 ꢁ 103), 277 (33 ꢁ 103), 348 (28 ꢁ 103). Fluorescence
(kex. = 340 nm, MeCN): 406 nm (very broad).
4.4.5. (R)-23-(4-Hydroxybenzylcarbamoyl)-1,6,16,25-tetraoxo-
26,26-diphenyl-1-(pyren-1-yl)-8,11,14-trioxa-2,5,17,19,24-pen-
taazahexacosan-18-iminium 2,2,2-trifluoroacetate (1f)
The product is a white solid (52 mg, 46%), mp >190 °C (decomp.).
1H NMR (300 MHz, DMSO-d6): 1.30–1.80 (m, 4H), 3.10–3.80 (m,
14H), 3.94 (s, 2H), 4.05–4.20 (m, 2H), 4.19 (s, 2H), 4.28–4.38 (m,
1H), 5.12 (s, 1H), 6.67 (d, J = 8.45, 2H), 7.00 (d, J = 8.45, 2H), 7.15–
7.30 (m, 10H), 7.98 (t, J = 5.65, 1H), 8.08–8.45 (m, 9H), 8.45–8.55
(m, 2H), 8.55–8.85 (m, 3H), 8.95–9.05 (m, 1H), 9.32 (br s, 1H),
10.61 (br s, 1H). C54H57N7O9: MS (ES): m/z (%) 474 (88), 948 (100,
4.4.1. (R)-1-[4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]-2-pentacosa-10,12-diynoylguanidinium
2,2,2-trifluoroacetate (1b)
MH+). HPLC: 99% (ELSD, tR = 16.3 min). UV (H2O): k (e) 267
(26 ꢁ 103), 277 (35 ꢁ 103), 330 (24 ꢁ 103), 343 (30 ꢁ 103). Fluores-
cence (kex. = 340 nm, H2O): k (type) 401 (monomer).
The product is a light yellow solid (170 mg, 72%), mp >190 °C
(decomp). 1H NMR (300 MHz, DMSO-d6): 0.85 (t, J = 6.51, 3H),
1.00–1.80 (m, 36H), 2.26 (t, J = 6.67, 4H), 2.41 (t, J = 7.19, 2H),
3.15–3.30 (m, 2H), 4.00–4.25 (m, 2H), 4.30–4.40 (m, 1H), 5.12 (s,
1H), 6.67 (d, J = 8.43, 2H), 7.00 (d, J = 8.41, 2H), 7.15–7.35 (m,
10H), 8.00–9.70 (m, 6H), 11.50–11.80 (m, 1H). 13C NMR (75 MHz,
DMSO-d6): 13.8, 18.2, 22.0, 23.73, 27.57, 27.59, 28.05, 28.06,
28.15, 28.27, 28.45, 28.60, 28.75, 28.83, 28.90, 31.19, 36.02,
52.15, 55.80, 65.23, 77.84, 77.90, 114.87, 126.47, 128.04, 128.07,
128.29, 128.38, 128.40, 129.00, 140.15, 140.34, 152.71, 156.15,
4.4.6. (R)-2-{8-[3-(Benzyloxy)-3-oxopropylamino]-8-oxoocta-
noyl}-1-[4-(2,2-diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]guanidinium 2,2,2-trifluoroacetate (1g)
The product is a white solid (58 mg, 78%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.00–1.80 (m, 12H), 1.95–
3.50 (m, 10H), 4.05–4.20 (m, 2H), 4.25–4.40 (m, 1H), 5.08 (s, 2H),
5.12 (s, 1H), 6.67 (d, J = 8.36, 2H), 7.00 (d, J = 8.40, 2H), 7.20–7.40
(m, 15H), 7.85–7.95 (m, 1H), 8.30–8.60 (m, 4H), 8.70–8.90 (br s,
1H), 9.31 (br s, 1H), 11.20 (br s, 1H). C45H54N6O7: MS (ES): m/z
(%) 791 (100, MH+), 1582 (1). HPLC: 100% (ELSD, tR = 16.2 min).
170.86, 170.91, 175.17.
C52H71N5O4: MS (LC–MS-I): m/z (%)
[tR = 12.3 min]: 830 (100, MH+).
4.4.7. (4R,12R,13S,14R,15R)-12,13,14,15-Tetraacetoxy-4-(2,2-diph-
enylacetamido)-1-(4-hydroxyphenyl)-3,11,18-trioxo-17-oxa-2,8,-
10-triazanonadecan-9-iminium 2,2,2-trifluoroacetate (1i)
The product is a white solid (197 mg, 66%), mp >190 °C
(decomp.). 1H NMR (300 MHz, DMSO-d6): 1.30–1.70 (m, 4H), 1.90–
2.20 (m, 15H), 3.20–3.30 (m, 2H), 4.00–4.40 (m, 5H), 5.00–5.10 (br
s, 1H), 5.12 (s, 1H), 5.24 (br s, 1H), 5.35–5.45 (m, 1H), 5.45–5.55
(m, 1H), 6.67 (d, J = 8.45, 2H), 7.00 (d, J = 8.45, 2H), 7.15–7.35 (m,
10H), 8.36 (t, J = 5.58, 1H), 8.47 (d, J = 8.14, 1H), 8.50–8.90 (m, 2H),
9.30 (br s, 1H), 11.80 (br s, 1H). C43H51N5O14: MS (ES): m/z (%) 862
(100, MH+), 884 (60). HPLC: 100% (ELSD, tR = 16.2 min).
4.4.2. (R)-1-[4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]-2-hexadecanoylguanidinium 2,2,2-tri-
fluoracetate (1c)
The product is
a white solid (48 mg, 53%), mp >190 °C
(decomp.). 1H NMR (300 MHz, CDCl3): 0.88 (t, 3H), 1.20–1.35 (m,
26H), 1.40–1.70 (m, 4H), 2.42 (t, 2H), 2.70–3.10 (m, 2H), 3.90–
4.10 (m, 1H), 4.15–4.35 (m, 2H), 4.99 (s, 1H), 5.80–6.50 (br s,
1H), 6.60 (d, 2H), 6.90 (d, 2H), 7.15–7.30 (m, 10H), 7.40–7.90 (m,
3H), 8.87 (br s, 1H), 9.45 (br s, 1H), 12.13 (br s, 1H), C43H61N5O4:
MS (ES): m/z (%) 712 (100, MH+). HPLC: 100% (ELSD, tR = 23.8 min).
4.4.3. (R)-1-[4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]-2-[4-(pyren-1-yl)butanoyl]guanidinium
2,2,2-trifluoroacetate (1d)
4.4.8. (R)-21-(4-Hydroxybenzylcarbamoyl)-3,7,14,23-tetraoxo-
24,24-diphenyl-2-oxa-6,15,17,22-tetraazatetracosan-16-iminium
2,2,2-trifluoroacetate (1k)
The product is
a
white solid (82 mg, 74%), mp >190 °C
The product is a white solid (149 mg, 64%), mp >190 °C
(decomp.). 1H NMR (300 MHz, CDCl3): 1.10–1.70 (m, 12H), 2.16
(t, J = 7.28, 2H), 2.35 (t, J = 6.51, 2H), 2.54 (t, J = 5.66, 2H), 2.95 (br
(decomp.). 1H NMR (300 MHz, DMSO-d6): 1.30–1.80 (m, 4H),
2.00–2.15 (m, 2H), 2.55–2.65 (t, J = 6.98, 2H), 3.15–3.25 (m, 2H),

6302
S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
s, 1H), 3.15 (br s, 1H), 3.50 (m, 2H), 3.69 (s, 3H), 4.00–4.35 (m, 3H),
4.98 (s, 1H), 6.48 (t, J = 5.40, 1H), 6.70 (d, J = 7.86, 2H), 6.94 (d,
J = 7.82, 2H), 7.15–7.30 (m, 10H), 7.40–7.70 (m, 3H), 8.89 (br s,
1H), 9.70 (br s, 1H), 12.19 (br s, 1H). C39H50N6O7: MS (ES): m/z
(%) 715 (100, MH+), 737 (5). HPLC: 100% (ELSD, tR = 14.1 min).
4.4.13. (R)-4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-
iminio-3,11,18-trioxo-2,8,10,19-tetraazahenicosan-21-amin-
ium 2,2,2-trifluoroacetate (1p)
The product is a white solid (125 mg, 70%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.10–1.70 (m, 12H), 2.00–
2.15 (m, 2H), 2.35–2.45 (m, 2H), 2.75–2.90 (m, 2H), 3.15–3.30
(m, 4H), 4.10–4.20 (m, 2H), 4.25–4.40 (m, 1H), 5.12 (s, 1H), 6.67
(d, J = 8.36, 2H), 7.00 (d, J = 8.36, 2H), 7.15–7.30 (m, 10H), 7.78
(br s, 3H), 8.00 (t, J = 5.52, 1H), 8.38 (t, J = 5.72, 1H), 8.51 (d,
J = 8.02, 1H), 8.65–8.80 (br s, 2H), 9.12 (br s, 1H), 9.32 (br s, 1H),
11.73 (br s, 1H). C37H49N7O5: MS (ES): m/z (%) 336 (100), 672
(25, MH+). HPLC: 97% (ELSD, tR = 11.2 min).
4.4.9. (R)-1-[4-(2,2-Diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]-2-{4-[4-(hydroxymethyl)-1H-1,2,3-tria-
zol-1-yl]butanoyl}guanidinium 2,2,2-trifluoroacetate (1l)
The product is a white solid (90 mg, 66%), mp >190 °C (decomp.).
1H NMR (300 MHz, DMSO-d6): 1.25–1.80 (m, 4H), 2.00–2.15 (m,
2H), 2.40–2.50 (m, 2H), 3.15–3.30 (m, 2H), 4.00–4.50 (m, 5H),
4.51 (s, 2H), 5.12 (s, 1H), 6.67 (d, J = 8.38, 2H), 7.00 (d, J = 8.38,
2H), 7.15–7.35 (m, 10H), 7.98 (s, 1H), 8.37 (t, J = 5.69, 1H), 8.40–
8.70 (m, 3H), 8.80–10.50 (m, 2H), 11.48 (br s, 1H). C34H40N8O5:
MS (ES): m/z (%) 641 (100, MH+). HPLC: 99% (ELSD, tR = 12.6 min).
4.4.14. (R)-18-Amino-4-(2,2-diphenylacetamido)-1-(4-hydroxy-
phenyl)-3,11-dioxo-2,8,10,17-tetraazaoctadecane-9,18-diimin-
ium 2,2,2-trifluoroacetate (1q)
The product is a white solid (93 mg, 71%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, CD3CN): 1.25–1.40 (m, 2H), 1.45–1.85
(m, 8H), 2.40–2.48 (t, J = 7.27, 2H), 3.00–3.10 (dd, J1 = 6.68,
J2 = 12.84, 2H), 3.10–3.30 (m, 2H), 4.10–4.25 (m, 2H), 4.30–4.40
(m, 1H), 5.02 (s, 1H), 6.40 (br s, 4H), 6.71 (d, J = 8.50, 2H), 6.90–
7.40 (m, 5H), 7.03 (d, J = 8.42, 2H), 7.20–7.35 (m, 10H), 9.45 (br s,
1H), 9.85 (br s, 1H), 12.73 (br s, 1H). 13C NMR (75 MHz, CD3CN):
23.2, 23.4, 25.0, 27.4, 28.6, 35.9, 40.1, 40.8, 41.8, 52.4, 57.0,
114.8, 126.67, 126.73, 128.14, 128.17, 128.34, 128.40, 129.6,
154.1, 155.7, 171.9, 176.3. C34H44N8O4: MS (LC–MS-I): m/z (%)
[tR = 7.6 min]: 315 (95), 335 (100), 629 (85, MH+), 675 (30), 743
(5). HPLC: 100% (ELSD, tR = 11.2 min).
4.4.10. (R)-2-{4-[4-(Ammoniomethyl)-1H-1,2,3-triazol-1-yl]but-
anoyl}-1-[4-(2,2-diphenylacetamido)-5-(4-hydroxybenzyl-
amino)-5-oxopentyl]guanidinium 2,2,2-trifluoroacetate (1m)
The product is a white solid (139 mg, 73%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.30–1.80 (m, 4H), 2.09
(p, J = 7.20, 2H), 2.45–2.50 (m, 2H), 3.15–3.30 (m, 2H), 4.05–4.20
(m, 4H), 4.25–4.40 (m, 1H), 4.45 (t, J = 7.08, 2H), 5.12 (s, 1H),
6.67 (d, J = 8.45, 2H), 7.00 (d, J = 8.45, 2H), 7.15–7.35 (m, 10H),
8.15 (s, 1H), 8.20–8.35 (br s, 2H), 8.37 (t, J = 5.79, 1H), 8.50 (d,
J = 8.02, 1H), 8.55–9.10 (m, 2H), 9.33 (br s, 1H), 12.11 (s, 1H).
C34H41N9O4: MS (LC–MS-I): m/z (%) [tR = 7.5 min]: 320 (100), 341
(35), 640 (50, MH+). HPLC: 100% (ELSD, tR = 10.7 min).
4.4.15. (R)-4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-
iminio-3,11,14-trioxo-2,8,10,15-tetraazaheptadecan-17-amin-
ium 2,2,2-trifluoroacetate (1s)
4.4.11. (13R)-13-(4-Hydroxybenzylcarbamoyl)-3,6,15-trioxo-
16,16-diphenyl-1-((2R,3R,4S,5R)-3,4,5,6-tetrahydroxytetra-
hydro-2H-pyran-2-yl)-2-oxa-7,9,14-triazahexadecan-8-
iminium 2,2,2-trifluoroacetate (1n)
The product is a white solid (35 mg, 76%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.30–1.80 (m, 4H), 2.55–
2.80 (m, 4H), 3.20–3.90 (m, 5H), 3.90–4.80 (m, 8H), 4.85–5.00
(m, 2H), 5.12 (s, 1H), 5.20–6.50 (m, 1H), 6.67 (d, J = 8.41, 2H),
7.00 (d, J = 8.41, 2H), 7.15–7.35 (m, 10H), 8.38 (t, J = 5.63, 1H),
8.50 (d, J = 8.06, 1H), 8.50–8.70 (br s, 2H), 8.86 (br s, 1H), 9.30 (br
s, 1H), 11.49 (br s, 1H). C37H45N5O11: MS (ES): m/z (%) 736 (100,
MH+), 758 (20). HPLC: 100% (ELSD, tR = 12.1 min).
The product is a white solid (0.74 g, 68%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.32–1.60 (m, 3H), 1.60–
1.76 (m, 1H), 2.46 (t, J = 6.71, 2H), 2.66 (t, J = 6.48, 2H), 2.80–2.88
(m, 2H), 3.18–3.31 (m, 4H), 4.11 (dd, J1 = 15.02, J2 = 5.76, 1H),
4.18 (dd, J1 = 15.05, J2 = 5.93, 1H), 4.28–4.38 (m, 1H), 5.12 (s, 1H),
6.67 (d, J = 8.53, 2H), 7.00 (d, J = 8.54, 2H), 7.18–7.32 (m, 10H),
7.76 (br s, 3H), 8.14 (t, J = 5.62, 1H), 8.38 (t, J = 5.74, 1H), 8.50 (d,
J = 8.06, 1H), 8.71 (br s, 2H), 9.16 (t, non-res., 1H), 9.31 (br s, 1H),
11.88 (s, 1H). C33H41N7O5: MS (LC–MS-I): m/z (%) [tR = 7.44 min]:
308.5 (100), 616 (27, MH+). HPLC: 96% (220 nm, tR = 16.5 min).
4.4.16. (R)-1-Amino-16-(4-hydroxybenzylcarbamoyl)-6,9,18-tri-
oxo-19,19-diphenyl-2,5,10,12,17-pentaazanonadecane-1,11-dii-
minium 2,2,2-trifluoroacetate (1t)
4.4.12. (R)-21-Carboxy-4-(2,2-diphenylacetamido)-1-(4-hydr-
oxyphenyl)-3,11,18-trioxo-2,8,10,19-tetraazahenicosan-9-imin-
ium 2,2,2-trifluoroacetate (1o)
The product is a white solid (35 mg, 49%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, CD3CN): 1.40–1.80 (m, 4H), 2.49 (t,
J = 6.59, 2H), 2.66 (t, J = 6.55, 2H), 3.10–3.30 (m, 6H), 4.10–4.25
(m, 2H), 4.25–4.40 (m, 1H), 5.02 (s, 1H), 6.10–7.50 (m, 10H), 6.70
(d, J = 8.50, 2H), 7.03 (d, J = 8.43, 2H), 7.20–7.35 (m, 10H), 9.39
(br s, 1H), 9.79 (br s, 1H), 12.82 (br s, 1H). C34H43N9O5: MS (LC–
MS-I): m/z (%) [tR = 7.5 min]: 329 (100), 658 (20, MH+). HPLC:
99% (ELSD, tR = 10.7 min).
t
1.) Removal of the benzyl-group: The Boc- and Bu-protected
precursor of compound 1o (35 mg, 0.04 mmol) was dis-
solved in 2-propanol (5 mL). Pd/C (10 mg) was added and
the mixture was stirred at 15 bar hydrogen pressure in an
autoclave. After 2 h TLC control confirmed complete conver-
sion. Pd/C was filtered off over Celite and the solvent was
evaporated. The product was obtained quantitative.
2.) The N-Boc and O-tBu protected compound was deprotected
as described in Section 4.4.
4.4.17. (R)-N1-[4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-
9-iminio-3,11,14-trioxo-2,8,10,15-tetraazaheptadecan-17-yl]-
ethane-1,2-diaminium 2,2,2-trifluoroacetate (1u)
The product is a white solid (62 mg, 39%), mp >190 °C (de-
comp.). 1H NMR (300 MHz, DMSO-d6): 1.30–1.80 (m, 4H), 2.62–
2.70 (t, J = 6.29, 2H), 3.00–3.40 (m, 12H), 4.00–4.20 (m, 2H),
4.25–4.40 (m, 1H), 5.12 (s, 1H), 6.70 (d, J = 8.45, 2H), 7.00 (d,
J = 8.45, 2H), 7.15–7.35 (m, 10H), 7.90–9.40 (m, 12H), 12.03 (br s,
1H). C35H46N8O5: MS (ES): m/z (%) 330 (100), 659 (20, MH+). HPLC:
99% (ELSD, tR = 9.1 min).
The product is a white solid (30 mg, 78%), mp >190 °C (decomp.).
1H NMR (300 MHz, DMSO-d6): 1.10–1.80 (m, 12H), 2.03 (t,
J = 7.28, 2H), 2.30–2.45 (m, 4H), 3.15–3.30 (m, 4H), 4.05–4.25 (m,
2H), 4.25–4.40 (m, 1H), 5.12 (s, 1H), 6.67 (d, J = 8.41, 2H), 7.00 (d,
J = 8.38, 2H), 7.15–7.35 (m, 10H), 7.86 (t, 1H), 8.30–8.70 (m, 4H),
8.80–9.00 (m, 1H), 9.20–9.40 (br s, 1H), 11.30–11.40 (br s, 1H).
C
38H48N6O7: MS (ES): m/z (%) 351 (11), 701 (100, MH+). HPLC:
99% (ELSD, tR = 13.7 min).

S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
6303
4.5. (R)-1-(4-Fluorophenyl)-16-(4-hydroxybenzylcarbamoyl)-
1,6,9,18-tetraoxo-19,19-diphenyl-2,5,10,12,17-pentaazanon-
adecan-11-iminium 2,2,2-trifluoroacetate (1j)
60
and 90 min a 125
MeCN, H2O and 1% aq TFA (for compounds 1c–1j: 5:1:4, 125
compounds 1k–1u: 1:1:1.33, 125 L). One hundred microlitres of
the resulting solution (pH ꢃ2) were analysed with analytical HPLC
on a RP-column (Eurospher-100 C18, 250 ꢁ 4 mm, 5 m; Knauer,
l
L of a 0.25 mM solution were added to 240
l
L of buffer). After 20
l
L aliquot was taken and diluted with a mixture of
L; for
l
l
Compound 1s (24 mg, 28.4
were dissolved in DMF (200 L) followed by the addition of active
ester 37 (4.5 mg, 19 mol). The reaction was stopped by addition
of 10% aq TFA (corresponding to 60 mol of TFA) after an incuba-
tion period of 3.5 h at rt. Purification with preparative HPLC (col-
umn: Eurospher-100 C18 (250 ꢁ 32 mm, 5 m), Knauer, Berlin,
lmol) and NEt3 (21 lL, 152 lmol)
l
l
l
Berlin, Germany) using a system from Thermo Separation Products
(composed of a SN400 controller, a P4000 pump, a degasser (Degas-
sex DG-4400, phenomenex), an AS3000 autosampler and a Spectra
Focus UV–Vis detector). Mixtures of 0.05% aq TFA (A) and acetoni-
trile (B) were used as mobile phase. The flow rate was set to
0.80 mL/min and the column temperature to 30 °C. Due to the wide
range of log D values of the compounds two different gradients were
used for HPLC analysis (compounds 1c–1j: 0 min [A/B 75:25],
28 min [A/B 80:20], 30 min [A/B 5:95], 38 min [A/B 5:95]; com-
pounds 1k–1u: 0 min [A/B 85:15], 28 min [A/B 55:45], 32 min [A/B
5:95], 40 min [A/B 5:95]). A five-point calibration curve (0.3, 1.5, 4,
l
l
Germany) and lyophilisation afforded the product as white fluffy
solid (10.55 mg, 65%), mp >190 °C (decomp.). 1H NMR (300 MHz,
DMSO-d6): 1.31–1.60 (m, 3H), 1.60–1.73 (m, 1H), 2.45 (t, J = 6.60,
2H), 2.64 (t, J = 6.38, 2H), 3.16–3.34 (m, 6H), 4.10 (dd, J1 = 14.80,
J2 = 5.66, 1H), 4.18 (dd, J1 = 15.06, J2 = 5.76, 1H), 4.28–4.38 (m,
1H), 5.12 (s, 1H), 6.67 (d, J = 8.52, 2H), 7.00 (d, J = 8.52, 2H), 7.18–
7.33 (m, 12H), 7.86–7.93 (m, 2H), 8.11 (t, J = 5.51, 1H), 8.38 (t,
J = 5.78, 1H), 8.47–8.62 (m, 4H), 8.87 (t, non-res., 1H), 9.30 (br s,
1H), 11.44 (s, 1H). C40H44FN7O6: (LC–MS-I): m/z (%) [tR = 8.69 min]:
738 (100, MH+). HPLC: 98% (220 nm, tR = 23.7 min).
10 and 25 lM) of BIBP 3226 (1a) was acquired for quantification of
the decomposition product 1a.
To study the decomposition products of 1j with LC–MS, 1j was
incubated in 10 mM HEPES buffer as described above, but at a con-
4.6. (R)-4-(2,2-Diphenylacetamido)-1-(4-hydroxyphenyl)-9-
iminio-3,11,14-trioxo-N-(2-propionamidoethyl)-2,8,10,15-
tetraazaheptadecan-17-aminium 2,2,2-trifluoroacetate (1r)
centration of 30
0.05% aq TFA (20:80) to 470
125 L aliquot was taken and diluted with a mixture of MeCN,
H2O and 1% aq TFA (1:1:1.33, 125 L). Ten microlitres of the result-
l
M (30
l
L of a 0.5 mM solution of 1j in MeCN/
l
L of buffer). After 20 and 90 min a
l
l
Compound 1u (35 mg, 35
were dissolved in DMF (200
ester 38 (2.4 mg, 14 mol). The reaction was stopped by addition
of 10% aq TFA (corresponding to 80 mol of TFA) after an incuba-
tion period of 3 h at rt. Purification with preparative HPLC (col-
umn: Eurospher-100 C18 (250 ꢁ 32 mm, 5 m), Knauer, Berlin,
l
mol) and NEt3 (23
l
L, 186
l
mol)
ing solution were injected into the LC–MS system and analysed with
LC–MS method II (cf. Section 4.1) (source type: ESI (capillary tem-
perature: 250 °C, spray voltage:4.0 kV, sheath and auxiliarygas: on).
l
L) followed by the addition of active
l
l
4.9. Radioligand competition binding assay
l
Germany) and lyophilisation afforded the product as white fluffy
solid (2.71 mg, 21%), mp >190 °C (decomp.). 1H NMR (600 MHz,
DMSO-d6): 0.99 (t, J = 7.59, 3H), 1.35–1.47 (m, 2H), 1.48–1.56 (m,
1H), 1.62–1.69 (m, 1H), 2.11 (q, J = 7.58, 2H), 2.45 (t, J = 6.73, 2H),
2.49–2.52 (m, 4H) 2.65 (t, J = 6.53, 2H), 2.95–3.01 (m, 2H), 3.19–
3.24 (m, 2H), 3.27–3.33 (m, 2H), 4.10 (dd, J1 = 14.76, J2 = 5.66,
1H), 4.17 (dd, J1 = 14.83, J2 = 6.03, 1H), 4.30–4.35 (m, 1H), 5.11 (s,
1H), 6.66 (d, J = 8.52, 2H), 6.99 (d, J = 8.53, 2H), 7.19–7.31 (m,
10H), 8.00 (t, J = 5.64, 1H), 8.17 (t, J = 5.73, 1H), 8.35 (t, J = 5.82,
1H), 8.44 (br s, 2H), 8.47 (d, J = 8.15, 1H), 8.59 (br s, 2H), 8.93 (t,
non-res., 1H), 9.28 (br s, 1H), 11.56 (s, 1H). C38H50N8O6: (LC–MS-
I): m/z (%) [tR = 7.54 min]: 358 (100), 715 (18, MH+). HPLC: 97%
(220 nm, tR = 17.3 min).
Radioligand competition experiments at SK-N-MC neuroblas-
toma cells using the radioligand [3H]UR-MK114 (1.5 nM) were per-
formed as described elsewhere.4
4.10. Fura-2 assay on HEL cells
The Fura assay was performed with HEL cells as previously de-
scribed using a Perkin–Elmer LS50 B spectrofluorimeter (Perkin–
Elmer, Überlingen, Germany).19
4.11. Data processing
Data from radioligand competition experiments were analyzed
by four parameter sigmoidal fits (SigmaPlot 9.0, Systat Software).
IC50 values from radioligand competition studies were converted
to Ki values according to the Cheng–Prusoff equation34 using the
respective KD value of the radioligand. Three data points (between
20% and 80% inhibition, from fura-2 assays) served for the calcula-
tion of IC50 values after logit-log transformation. IC50 values were
converted to Kb values according to the Cheng–Prusoff equation33
using an EC50 value of 1.8 nM for pNPY (mean value from four inde-
pendently determined concentration–effect curves on HEL cells).
4.7. Experimental determination of log D values with HPLC
The log D values of compounds 1a, 1d, 1f, 1g, 1i, 1l, 1n, 1p, 1q
and 1u were determined by RP-HPLC.
(3.0
a flow of 0.30 mL/min was used. An ammonium acetate buffer
(10 mM, pH 7.4) was used as solvent A. Solvent B was acetonitrile.
The gradient was: 0 min [A/B 95:5], 30 min [A/B 2:98]. A volume of
A Luna C18 column
m, 150 ꢁ 2 mm, Phenomenex, Aschaffenburg, Germany) with
l
2 lL of a solution of the analytes, dissolved in DMSO (1 mM), was
injected. Column temperature was 25 °C. The compounds were de-
tected with a diode array detector. The log D value was calibrated
to the retention time (tR) by running a mixture of six reference
compounds and plotting the tR versus log D (Supplementary data).
Acknowledgement
This work was supported by the Graduate Training Program
(Graduiertenkolleg) GRK 760, ‘Medicinal Chemistry: Molecular
Recognition—Ligand-Receptor Interactions’, of the Deutsche
Forschungsgemeinschaft.
4.8. Investigation of stability
Decomposition of the NG-acylated argininamides to 1a was
investigated at neutral pH in the buffer used for radioligand binding4
Supplementary data
(10 mM HEPES, pH 7.4, filtered: 0.45
addition of 15 L of a 1 mM solution of the compounds in DMSO to
285 L of buffer to give a final concentration of 50 M (in case of 1c
lm). Incubation was started by
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.028.
l
l
l

6304
S. Weiss et al. / Bioorg. Med. Chem. 18 (2010) 6292–6304
16. Rudolf, K.; Eberlein, W.; Engel, W.; Wieland, H. A.; Willim, K. D.; Entzeroth, M.;
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