The arginine mimicking β-amino acid β3hPhe(3-H 2N-CH2) as S1 ligand in cyclotheonamide-based β-tryptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.09.050

β-Tryptase, a mast-cell specific serine protease with trypsin-like activity, has emerged in the last years as a promising novel therapeutic target in the field of allergic inflammation. Recently, we have developed a potent and selective β-tryptase inhibitor based on the natural product cyclotheonamide E4 by implementing a basic P3 residue that addresses the determinants of the extended substrate specificity of β-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now investigated β-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding β-homo amino acids derived from lysine or arginine, we demonstrate that this particular basic β-homo amino acid is a privileged S1 ligand for the development of β-tryptase inhibitors. Besides affinity, selectivity and reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma and serum.

Full text of DOI:10.1016/j.bmc.2011.09.050

Bioorganic & Medicinal Chemistry 19 (2011) 7236–7243
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The arginine mimicking b-amino acid b3hPhe(3-H2N-CH2) as S1 ligand
in cyclotheonamide-based b-tryptase inhibitors
Dennis Janke a, Christian P. Sommerhoff b, Norbert Schaschke a,
⇑
a Fakultät für Chemie, Universität Bielefeld, Universitätsstr. 25, D-33615 Bielefeld, Germany
b Abteilung für Klinische Chemie und Klinische Biochemie, Klinikum der Ludwig-Maximilians-Universität, Nußbaumstr. 20, D-80336 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Tryptase, a mast-cell specific serine protease with trypsin-like activity, has emerged in the last years as
a promising novel therapeutic target in the field of allergic inflammation. Recently, we have developed a
potent and selective b-tryptase inhibitor based on the natural product cyclotheonamide E4 by
implementing a basic P3 residue that addresses the determinants of the extended substrate specificity
of b-tryptase. To further improve the affinity/selectivity profile of this lead structure, we have now inves-
tigated b-homo-3-aminomethylphenylalanine as S1 ligand. In contrast to the corresponding b-homo
amino acids derived from lysine or arginine, we demonstrate that this particular basic b-homo amino acid
is a privileged S1 ligand for the development of b-tryptase inhibitors. Besides affinity, selectivity and
reduced basicity, these novel cyclotheonamide E4 analogs show excellent stability in human plasma
and serum.
Received 14 June 2011
Revised 9 September 2011
Accepted 10 September 2011
Available online 2 October 2011
Keywords:
Mast cells
Cyclic peptides
Serine proteases
Structure–activity relationship
Solid phase peptide synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
circulation are utilized clinically to confirm anaphylactic reac-
tions.7 Typically, in insect sting-induced systemic anaphylaxis the
Like other leukocytes, mast cells are derived from hematopoi-
etic progenitor cells of the bone marrow. In contrast to basophiles
that circulate as mature cells in the blood, mast cells reside in vir-
tually all peripheral tissues of the human body, in particular the
dermis, the lung, and the mucosa and submucosa of the intestine.
It is well accepted that mast cells play a pivotal role in the patho-
genesis of a variety of allergic and inflammatory diseases, most
prominent among them allergic asthma, psoriasis, and rheumatoid
arthritis.1–3 Beside by other mechanisms, mast cells are activated
level of b-tryptase reaches its maximum within 15–120 min after
the sting and subsequently declines with a half live of 1.5–2.5 h.8
The magnitude of b-tryptase levels observed in patients suffering
from insect sting reactions correlate with the clinical severity as
determined by the drop in mean arterial pressure.9
In addition to its well established role as clinical marker, b-tryp-
tase has emerged as promising new target for therapeutic interven-
tion in allergic asthma.10 The secreted protease has been implicated
in modulation of bronchial tone, airway inflammation and tissue
remodeling, the key features of asthma. Indeed b-tryptase degrades
and inactivates the neuropeptides vasoactive intestinal peptide
(VIP)11 and calcitonin gene-related peptide (CGRP)12 and thus via
its peptidolytic activity can modulate airway smooth muscle tone,
leading to bronchoconstriction. In addition, b-tryptase was shown
to induce the formation of edema,13,14 to recruit neutrophils and
eosinophils,15–17 and to act as a mitogen for airway smooth muscle
cells.
upon crosslinking of FceRI-bound IgE with multivalent allergens
on the cells’ surface. The signaling cascade elicited eventually re-
sults in downstream events that trigger the release of a set of pre-
formed mediators, which are stored in cytoplasmic granules, into
the surrounding tissue by degranulation.4
The mediators released by degranulation include, for example,
histamine and heparin proteoglycan as well as b-tryptase, a tryp-
sin-like serine protease that is abundantly and virtually exclusively
expressed in mast cells.1 The amount of catalytically active b-tryp-
tase accounts for up to 20% of the entire protein of a mast cell and
thus is higher than those of proteases like elastase and cathepsin G
found in other granulocytes.5 Therefore b-tryptase is used as a
valuable marker for the activation and localization of mast cells.6
In particular, increased levels of b-tryptase in the patient’s
By implementing information derived from substrate specificity
screening18 onto the cyclotheonamide E4 scaffold we have recently
developed
Scheme 1).19 For this, the natural product cyclotheonamide E4
was modified at two key positions: the -amino function of (S)-
2,3-diamino propionic acid was used as anchoring point for -ami-
a potent and selective b-tryptase inhibitor (1,
a
e
no hexanoic acid as optimized basic P3 residue. Based on modeling
studies performed by Harris et al. to elucidate the structural
features responsible for the extended substrate specificity of
⇑
Corresponding author.
E-mail address: norbert.schaschke@uni-bielefeld.de (N. Schaschke).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.050

D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
7237
OH
O
2. Results and discussion
2.1. Chemistry
S1' pocket
S3 pocket
NH2
2.1.1. Synthesis of Fmoc-b3hPhe(3-BocHN-CH2)-OH (4)
HN
HN
N
To incorporate b-homo-3-aminomethylphenylalanine as S1
ligand onto the cyclotheonamide E4 scaffold we have focused on
a solid phase-assisted assembly based on the Fmoc/tBu protecting
group scheme. First, it was necessary to establish a synthetic
approach towards the Fmoc amino acid building block
Fmoc-b3hPhe(3-BocHN-CH2)-OH (4). For this, we took partially
advantage from a synthetic approach towards Ac-DL-Phe(3-H2N-
CH2)-OMe that we have established previously.20 Thus, as starting
material for the synthesis H-Phe(3-CN)-OH (5) was selected. Upon
conversion into Pht-Phe(3-CN)-OMe (7) by standard procedures
(Scheme 3), the nitrile function was reduced smoothly using
hydrogen in the presence of 10% Pd-C, and the resulting amino
function was Boc-protected yielding Pht-Phe(3-Boc-HN-CH2)-
OMe (9). After mild hydrazinolysis of the Pht-group by hydrazine
acetate, saponification of the methyl ester, and Fmoc-protection
of the free amino function Fmoc-Phe(3-Boc-HN-CH2)-OH (12)
was obtained over seven steps in 18% yield. Subsequently, applying
the Seebach protocol21 the protected amino acid 12 was converted
into the corresponding b-homo amino acid 4. Briefly, 12 was
smoothly transferred into the diazoketone 13 which was then sub-
jected to Arndt–Eistert homologation yielding the Fmoc amino acid
building block 4.
O
O
O
O
NH
N
H
H
N
H
O
H2N
N
NH
1
S2 pocket
S1 pocket
Scheme 1. Chemical structure of the cyclotheonamide E4-based b-tryptase
inhibitor 1. The proposed binding mode is indicated.
b-tryptase,18 it is supposed that this S3 ligand interacts with the
negatively charged residues Glu-217 of the cognate as well as
Asp-60B’ of the adjacent subunit of the b-tryptase tetramer. In
addition, the S1 ligand present in the natural product, (S)-3-ami-
no-6-guanidino-2-oxo-hexanoic acid, was substituted by the
structurally related b-homoarginine to shift the binding from a
covalent into a fully reversible mode.
In a previous study we have utilized 3-aminomethyl-phenyl-
alanine as S1 ligand in bivalent b-tryptase inhibitors20 and
shown that Ac-DL-Phe(3-H2N-CH2)-OMe is a moderately potent
2.1.2. Synthesis of the cyclotheonamide E4-based b-tryptase
inhibitors 2 and 3 containing b-homo-3-aminomethyl-
phenylalanine as S1 ligand
and selective inhibitor of b-tryptase (Ki (b-tryptase) 14
Ki (trypsin) 400 M). Taking advantage of these findings for the
lM vs
l
design and synthesis of cyclotheonamide E4-based b-tryptase
inhibitors we have now investigated whether b-homo-3-
aminomethylphenylalanine as S1 ligand in cyclotheonamide
E4-based inhibitors increases selectivity for b-tryptase among
trypsin-like serine proteases. Therefore, we have synthesized
and assessed the inhibitory profile of inhibitor 2 (Scheme 2)
which does not address the proposed determinants of b-trypta-
ses’ extended substrate specificity. To further improve the
inhibitory profile of our lead compound 1, the cyclotheonamide
E4-based inhibitor 3 (Scheme 2) containing b-homo-3-amino-
methyl-phenylalanine at P1 position was synthesized and its
affinity and selectivity properties against a representative panel
of trypsin-like serine proteases determined. In addition, the sta-
bility of both inhibitors against proteolytic degradation in human
plasma and serum was determined.
The synthesis of inhibitors 2 and 3 was performed by a combi-
nation of solid phase and solution phase chemistry following a
strategy that we have utilized previously to establish an optimal
basic S3 ligand (Scheme 4).19 Briefly, after immobilization of H-
Dap(Fmoc)-OAl through its
a-amino function on a 2-chlorotrityl
resin the linear pentapeptide was built stepwise according to the
Fmoc/tBu protocol for solid phase peptide synthesis using opti-
mized excesses of each Fmoc-amino acid building block for the
coupling step. After stepwise deprotection of the termini of the re-
sin-bound and side chain protected linear pentapeptide 15, cycli-
zation was performed smoothly on-resin without appreciable
oligomerization using PyBOP essentially according to a procedure
described by the group of Albericio.22 Then the fully protected
cyclotheonamide E4 scaffold 16 was cleaved from the resin under
mild acidic conditions, thereby liberating the a-amino function of
(S)-2,3-diamino propionic acid for further functionalization in
solution. Eventually 16 was reacted either with acetic anhydride
or with Boc-protected
e-aminohexanoic acid in the presence of
OH
TBTU and subjected to the final deprotection step yielding the
inhibitors 2 and 3 based on the initial resin loading with 29% and
32%, respectively, which were fully characterized by RP-HPLC, 1H
NMR and HRMS.
O
HN
HN
O
2: R= -CO-CH3
3: R= -CO-(CH2)5-NH2
2.2. Biological activity
O
R
O
NH
N
H
H
N
2.2.1. Inhibitory profile of cyclotheonamide E4 analogs 2 and 3
The equilibrium dissociation constants Ki for inhibition of
b-tryptase by the inhibitors 2 and 3 reported in Table 1 were
determined essentially as described previously.19,23 In addition,
to assess the selectivity of the inhibitors for b-tryptase among
the trypsin-like serine proteases, Ki values for trypsin were
determined. The comparison of the different S1 ligands including
b-homolysine (17), b-homoarginine (18) and b-homo-3-amino-
methylphenylalanine (2) on the affinity/selectivity profile of
N
O
NH2
Scheme 2. Chemical structures of the cyclotheonamide E4-based b-tryptase
inhibitors 2 and 3 containing b-homo-3-aminomethylphenylalanine as S1 ligand.

7238
D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
NHBoc
NHBoc
CN
OR
R
O
v
ix
ii
OMe
R2
FmocHN
N
R1
H2N
O
O
O
O
OH
O
4
10: R1 = NH2 , R2 = OMe
11: R1 = NH2 , R2 = OH
5: R = H
7: R = CN
i
iii
iv
vi
6: R = Me
8: R = CH2 -NH2
vii
viii
9: R = CH2 -NHBoc
12: R1 = NHFmoc, R2 = OH
13: R1 = NHFmoc, R2 = CHN2
Scheme 3. Synthesis of Fmoc-b3hPhe(3-BocHN-CH2)-OH (4). Reagents and conditions: (i) MeOH, SOCl2, 89%; (ii) N-ethoxycarbonylphthalimide, Na2CO3, dioxane/H2O (1:1, v/
v), 87%; (iii) 10% Pd-C/H2, AcOH, 52%; (iv) (Boc)2O, NaHCO3, dioxane/H2O (1:1, v/v), 96%; (v) H2N-NH2 Â AcOH, MeOH, 76%; (vi) NaOH, dioxane/H2O (2:1, v/v), 82%; (vii) Fmoc-
OSu, NaHCO3, dioxane/H2O (2:1, v/v), 76%; (viii) (1) Et3N, ClCO2Et, THF, (2) CH2N2, Et2O, 86%; (ix) C6H5COOAg, dioxane/H2O (5:3, v/v), 91%.
HN
ii, iii, iv, v
i
OAl
Cl
FmocHN
Cl
O
OtBu
O
14
OtBu
HN
HN
O
vi, vii, viii, ix
H
N
HN
H
N
H
N
OAl
O
O
N
N
H
O
O
NH
O
O
NH2
O
O
Fmoc
H
N
N
15
NHBoc
16
OH
O
NHBoc
HN
HN
O
2: R = -CO-CH3
3: R = -CO-(CH2)5-NH2
O
R
O
NH
x, xi
N
H
H
N
N
O
NH2
Scheme 4. Synthesis of the cyclotheonamide E4 analogs 2 and 3 containing b-homo-3-aminomethylphenylalanine as S1 ligand. Reagents and conditions: (i) (1) H-
Dap(Fmoc)-OAl  HCl (2 equiv), DIPEA, CH2Cl2, (2) MeOH/CH2Cl2, DIPEA; (ii) (1) piperidine/DMF (1:4 v/v), (2) Fmoc-vTyr(tBu)-OH/TBTU/HOBt/DIPEA (1:1:1:1, 2  1 equiv),
DMF; (iii) (1) piperidine/DMF (1:4 v/v), (2) Fmoc-D
-allo-Ile-OH/TBTU/HOBt/DIPEA (1:1:1:1, 4 equiv), DMF; (iv) (1) piperidine/DMF (1:4 v/v), (2) Fmoc-b3hPhe(3-BocHN-CH2)-
OH (4)/TBTU/HOBt/DIPEA (1:1:1:1, 2 Â 1 equiv), DMF; (v) (1) piperidine/DMF (1:4 v/v), (2) Fmoc-Pro-OH/TBTU/HOBt/DIPEA (1:1:1:1, 4 equiv), DMF; (vi) Pd(PPh3)4
(3 Â 0.4 equiv), PhSiH3 (3 Â 24 equiv), CH2Cl2; (vii) piperidine/DMF (1:4 v/v); (viii) PyBOP/HOBt/DIPEA (1:1:2, 3 equiv), DMF; (ix) TFA/TIS/CH2Cl2 (1:1:98 v/v/v), over eight
steps 96%; (x) BocHN(CH2)5COOH/TBTU/HOBt/DIPEA (1:1:1:1, 2 equiv), DMF or Ac2O/pyridine (1:1, 10 equiv), CH2Cl2; (xi) TFA/H2O, (95:5 v/v), over two steps: 2: 30%, 3: 33%.
cyclotheonamide E4-based b-tryptase inhibitors clearly reveals
that b-homo-3-aminomethylphenylalanine is by far the most po-
tent one of the series (Table 1). Thus, the affinity of inhibitor 2 to
inhibit the activity of b-tryptase is more than 600-fold higher than
that of inhibitor 17 and about 70-fold higher than that of inhibitor
18. Furthermore, inhibitors 17 and 18 do not discriminate between
b-tryptase and trypsin, whereas compound 2 inhibits the activity
of b-tryptase more than two orders of magnitude more effectively
than that of trypsin. Substituting the acetyl group (inhibitor 2) by a
6-aminohexanoyl group (inhibitor 3) increases the affinity by a fac-
tor of ꢀ25 (2: Ki 0.6
lM vs 3: Ki 0.025 lM). These findings suggest
that the basic S3 ligand exploits interactions with the acidic resi-
dues Glu-217 of the cognate as well as Asp-60B’ of the adjacent
subunit of the b-tryptase tetramer that have been proposed to be
responsible for the extended substrate specificity of b-tryptase.18
More importantly, the selectivity ratio Ki(trypsin)/Ki(b-tryptase)

D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
7239
Table 1
Affinity/selectivity profiles of the cyclotheonamide E4-based b-tryptase inhibitors 2 and 3
OH
O
HN
O
HN
N
O
R2
O
NH
N
H
N
H
O
R1
Inhibitor
R1
R2
Ki (
l
M)a
Selectivityb
b-Tryptase
Trypsin
100
O
O
NH2
2
0.60
170
0.8
CH3
CH3
NH
NH2
18
40
30
N
H
O
NH2
NH2
380
230
50
0.6
17
3
CH3
O
O
0.025
2000
NH2
NH2
NH
NH2
1
0.007
10
1400
N
H
a
The Ki values of inhibitors 1, 17 and 18 were taken from Ref. 19.
Ki(trypsin)/Ki(b-tryptase).
b
Table 2
Expanded affinity/selectivity profiles of the cyclotheonamide E4-based b-tryptase inhibitors 2 and 3
Inhibitor
Ki (lM)
b-Tryptase
Trypsin
Thrombin
Factor Xa
Tissue kallikrein
Plasma kallikrein
2
3
0.60
0.025
100
50
680
330
310
360
1800
1300
3300
2000
is improved from 170 to 2000. A comparison of inhibitor 3 with our
previous lead compound 1 shows that inhibitor 3 exhibits virtually
the same affinity/selectivity profile as inhibitor 1 despite a consid-
erably reduced basicity.
The affinity of inhibitor 3 and even inhibitor 2 towards b-tryp-
tase prompted us to extend our selectivity studies on a broader pa-
nel of trypsin-like serine proteases. Therefore, the affinities of both
inhibitors for thrombin, factor Xa, tissue kallikrein, and plasma kal-
likrein were determined.23,24 The results summarized in Table 2
clearly show that both inhibitors exhibit an excellent selectivity
profile for b-tryptase. Importantly, the introduction of e-amino-
hexanoic acid as basic P3 residue (inhibitor 3) increases the affinity
towards b-tryptase but not those for other relevant trypsin-like
proteases, resulting in a superior selectivity profile in comparison
to inhibitor 2.
2.2.2. Stability of the inhibitors 2 and 3 in human plasma and
serum
In order to utilize cyclotheonamide E4-based inhibitors as tools
for the analysis of b-tryptase activity in body fluids and tissues the
A
B
120
120
100
80
60
40
20
0
100
80
60
40
20
0
0
2
6
8
24
48
0
2
6
8
24
48
Time of incubation at 37 ºC [h]
Time of incubation at 37 ºC [h]
Figure 1. Stability of inhibitors 2 ( ) and 3 ( ) in (A) human plasma and (B) human serum.

7240
D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
stability of these peptidic compounds is an important issue that
has to be addressed. To investigate this aspect, both inhibitors 2
and 3 were incubated in undiluted human plasma and human ser-
um at 37 °C for up to 48 h, and the remaining amount of inhibitor
was subsequently determined by analytical RP-HPLC. The data
summarized in Figure 1 verify that both inhibitors are remarkable
stable against proteolytic degradation or other modification in hu-
man plasma and even in serum containing the various activated
trypsin-like serine proteases of the blood coagulation cascade.
Even after 48 hours incubation at 37 °C the recovery of the inhibi-
tors 2 and 3 is almost quantitatively in human plasma and only
slightly reduced in human serum (inhibitor 2: 95% and inhibitor
3: 81%).
Phenomenex (column I; type Jupiter
5 lm, C18, 300 Å,
250 Â 4.60 mm) and eluting (flow rate: 1 mL/min) with the gradi-
ent I: 0–3 min, isocratic A/B (100:0); 3–35 min, linear gradient
from A/B (100:0) to A/B (0:100) or using a column from Mache-
rey-Nagel (column II; type ET 125/4 Nucleosil 100-5 C18 PPN)
and eluting (flow rate: 1.5 mL/min) with the gradient II: 0–1 min,
isocratic A/B (100:0); 1–14 min, linear gradient from A/B (100:0)
to A/B (0:100) (eluent A: H2O/MeCN/TFA, 95:5:0.1 v/v/v, eluent
B: MeCN/H2O/TFA, 95:5:0.1 v/v/v). Preparative RP-HPLC was per-
formed with a HPLC system Type LaChrome from Merck Hitachi
using a column from Phenomenex (Type Jupiter 10 lm, C18,
300 Å, 250 Â 21.20 mm) and eluting (flow rate: 10 mL/min) with
the following gradient: 0–5 min, isocratic A/B (100:0); 5–50 min,
linear gradient from A/B (100:0) to A/B (50:50) (eluent A: H2O/
MeCN/TFA, 95:5:0.1 v/v/v, eluent B: MeCN/H2O/TFA, 95:5:0.1
v/v/v).
3. Conclusion
We have identified b-homo-3-aminomethylphenylalanine as a
privileged S1 ligand for the development of b-tryptase inhibitors
based on the cyclotheonamide E4 scaffold. In particular inhibitor
3 is a remarkable potent b-tryptase inhibitor that blocks the
proteolytic activity of this enzyme within a panel of five physiolog-
ical relevant trypsin-like serine proteases with exceptional high
selectivity. Even inhibitor 2, which lacks the basic group proposed
to address the determinants of b-tryptases’ extended substrate
specificity, is still reasonably potent and selective. Furthermore,
both inhibitors are remarkable stable even during prolonged
incubation in human plasma and serum. Thus, inhibitors 2 and 3
are interesting new lead compounds for the development of
b-tryptase inhibitors based on the cyclotheonamide E4 scaffold.
4.1.1. H-Phe(3-CN)-OMe  HCl (6)
To MeOH (30 mL, 739.70 mmol) was added under stirring drop-
wise at À10 °C within 10 min thionyl chloride (2.11 mL,
29.07 mmol) followed by H-Phe(3-CN)-OH (4.93 g, 25.92 mmol)
as a solid. The resulting solution was stirred for 3.5 h at 45 °C
and then over night at r.t. The solvent was evaporated under re-
duced pressure and the title compound was isolated as colorless
solid by precipitation from MeOH/tert-butyl methyl ether; yield:
5.58 g (89%); TLC (n-butanol/AcOH/H2O/AcOEt, 3:1:1:5, v/v/v/v)
Rf 0.4; 1H NMR (500 MHz, DMSO-d6, 25 °C): d [ppm] = 3.25 (m,
2H, b-CH2), 3.69 (s, 3H, CH3), 4.36 (t, J = 6.6 Hz, 1H, a-CH), 7.55 (t,
J = 7.8 Hz, 1H, C5H Ar), 7.62 (d, J = 8.2 Hz, 1H, C6H Ar), 7.77 (d,
J = 7.5 Hz, 1H, C4H Ar), 7.79 (s, 1H, C2H Ar), 8.78 (s, 3H, NH3); 13C
NMR (125.7 MHz, DMSO-d6, 25 °C): d [ppm] = 35.4 (bC), 53.2 (aC
4. Experimental
4.1. Chemistry
and OCH3), 111.9 (C3 Ar), 119.2 (CN), 130.2, 131.6, 133.7, 135.0
(C2, C4, C5, C6 Ar), 137.0 (C1 Ar), 169.6 (C@O); ESI-MS: m/z 205.0
[M+H]+; calcd for C11H12N2O2: 204.1.
Solvents and reagents were of the highest purity commercially
available and were used without further purification. TBTU, HOBt,
Fmoc-Pro-OH, and Fmoc-OSu were purchased from Iris Biotech
(Marktredwitz, Germany), H-Phe(3-CN)-OH from Senn Chemicals
4.1.2. Pht-Phe(3-CN)-OMe (7)
H-Phe(3-CN)-OMe  HCl (6; 5.50 g, 22.85 mmol) was dissolved
in 1,4-dioxane (60 mL) and
a solution of Na2CO3 (2.42 g,
22.85 mmol) in H2O (40 mL) added. To the resulting solution at
r.t., N-ethoxycarbonylphthalimide (6.51 g, 29.71 mmol) was added
as solid under stirring and stirring was continued over night. The
solvent was evaporated and the resulting pale yellow solid was
distributed between AcOEt (100 mL) and H2O (50 mL). The organic
phase was washed with 5% aq KHSO4 (3 Â 50 mL), brine
(1 Â 50 mL), dried (Na2SO4), and the solvent evaporated. The crude
product was purified by flash chromatography (eluent: AcOEt/
petroleum ether 1:6; v/v). The title compound was obtained as col-
orless solid; yield: 6.64 g (87%); RP-HPLC (column I, gradient I): tR
25.5 min; TLC (AcOEt/petroleum ether 1:6; v/v): Rf 0.2; 1H NMR
(500 MHz, DMSO-d6, 25 °C): d [ppm] = 3.31–3.36 (m, 1H, b2-CH2),
3.57 (dd, J = 14.1, 4.9 Hz, 1H, b1-CH2), 3.70 (s, 3H, CH3), 5.35 (dd,
(Dielsdorf, Switzerland), Fmoc-D-allo-Ile-OH from Bachem (Buben-
dorf, Switzerland), DiazaldÒ, silver benzoate, and phenyl silane
from Sigma–Aldrich, triisopropylsilane (TIS), Boc2O, and hydrazine
hydrate from Fluka, and 2-chlorotrityl chloride resin from Nova-
biochem/Merck Chemicals Ltd (Nottingham, UK). Fmoc-vTyr
(tBu)-OH and H-Dap(Fmoc)-OAl were synthesized as described
previously19 and diazomethane using DiazaldÒ and the Diaz-
aldÒ-Distillation-Kit from Sigma–Aldrich according to a procedure
of de Boer.25 Manual peptide synthesis was carried out on a IKA KS
130 basic laboratory shaker (Staufen, Germany) using plastic syrin-
ges type Norm-Ject from Henke Sass Wolf (Tuttlingen, Germany)
equipped with PE frits (35 lm pore size) and PE stoppers from Ro-
land Vetter Laborbedarf (Ammerbuch, Germany). Precipitated pep-
tides were collected by centrifugation using a Labofuge I from
Heraeus Christ GmbH (Hanau, Germany). 1H and 13C NMR spectra
were recorded with a DRX 500 spectrometer from Bruker, ESI-MS
spectra with a Esquire 3000 from Bruker Daltonics, MALDI-MS
spectra with a Voyager-DE Biospectroscopy from PerSeptive Bio-
systems using 2,5-dihydroxy benzoic acid as matrix, and HRMS
(ESI) spectra with a APEX III FT-ICR mass spectrometer from Bruker
Daltonics. TLC was performed on silica gel 60 plates with fluores-
cence indicator F254 and flash chromatography using silica gel 60
(230–400 mesh) from VWR International GmbH (Darmstadt,
Germany), respectively. Spots were visualized with UV light
(k = 254 nm) using a lamp (type MinUVIS) from Desaga (Heidel-
berg, Germany). Analytical RP-HPLC was performed with a HPLC
system from Thermo Separation Products using a column from
J = 11.2, 4.9 Hz, 1H,
Ar), 7.50 (d, J = 7.5 Hz, 1H, C6H Phe(3-CN)-Ar), 7.61 (d, J = 7.5 Hz,
a-CH), 7.39 (t, J = 7.8 Hz, 1H, C5H Phe(3-CN)-
1H, C4H Phe(3-CN)-Ar), 7.71 (s, 1H, C2H Phe(3-CN)-Ar), 7.86 (m,
4H, CH Pht-Ar); 13C NMR (125.7 MHz, DMSO-d6, 25 °C):
d
[ppm] = 34.1 (bC), 52.6 (OCH3), 53.3 (aC), 111.7 (C3 Phe(3-CN)-
Ar), 119.1 (CN), 124.1, 130.0, 131.0, 133.1, 134.4, 135.6 (C Pht-Ar
and C Phe(3-CN)-Ar), 139.1 (C Pht-Ar), 167.3 (C@O Pht), 169.3
(C@O); ESI-MS: m/z 357.1 [M+Na]+; calcd for C19H14N2O4: 334.1.
4.1.3. Pht-Phe(3-H2N-CH2)-OMe  HCl (8)
To a solution of Pht-Phe(3-CN)-OMe (7; 3.66 g, 10.95 mmol) in
AcOH (170 mL), aq HCl (2.2 mL, 10 M), and 10% Pd-C (3.60 g) were
added. The reduction was performed at r.t. under static hydrogen
pressure (4.8 bar) for 5 d. Subsequently, the catalyst was filtered
off and the solvent evaporated under reduced pressure. The

D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
7241
resulting material was dissolved in a small amount of AcOEt, and
the title compound was precipitated by addition of tert-butyl
methyl ether and petroleum ether as colorless solid; yield: 2.12 g
(52%); TLC (cyclohexane/CHCl3/AcOH; 45:45:10; v/v/v): Rf 0.1;
RP-HPLC (column I, gradient I): tR 18.5 min; 1H NMR (500 MHz,
DMSO-d6, 25 °C): d [ppm] = 3.31–3.35 (m, 1H, b2-CH2), 3.51 (dd,
J = 14.2, 4.8 Hz, 1H, b1-CH2), 3.70 (s, 3H, CH3), 3.86 (s, 2H, CH2-
4.1.6. H-Phe(3-Boc-HN-CH2)-OH (11)
H-Phe(3-Boc-HN-CH2)-OMe  HCl (10; 1.38 g, 4.00 mmol) was
dissolved in 1,4-dioxane/H2O (120 mL; 2:1, v/v), and 1 N NaOH
(7.60 mL, 7.60 mmol) was added at r.t. under stirring. After 3 h,
the solution was neutralized with 1 N HCl and the solvent evapo-
rated. The obtained solid was suspended in H2O (50 mL), and the
aqueous phase extracted with H2O-saturated 1-butanol
(5 Â 20 mL). The solvent was evaporated under reduced pressure
from the combined organic phases. The resulting material was sus-
pended in tert-butyl methyl ether (20 mL), petroleum ether
(20 mL) added, collected by filtration, washed with petroleum
ether and finally dried. The title compound was obtained as color-
less solid; yield: 969 mg (82%); TLC (CHCl3/MeOH/AcOH; 8:8:1; v/
v/v): Rf 0.5; RP-HPLC (column I, gradient I): tR 17.4 min; 1H NMR
(500 MHz, DMSO-d6, 25 °C): d [ppm] = 1.33 (s, 9H, Boc-CH3), 2.84
(dd, J = 14.2, 8.4 Hz, 1H, b2-CH2), 3.11 (dd, J = 14.3, 4.1 Hz, 1H, b1-
NH3), 5.27 (dd, J = 11.0, 4.8 Hz, 1H,
a-CH), 7.19–7.29 (m, 4H,
Phe(3-H2N-CH2)-Ar), 7.87 (s, 4H, CH Pht-Ar), 8.35 (s, 3H, NH3);
13C NMR (125.7 MHz, DMSO-d6, 25 °C): d [ppm] = 27.3, 34.3 (bC),
42.4, 49.2, 52.9 (OCH3), 53.3 (aC), 124.1, 127.7, 129.1, 130.0,
131.1, 134.6, 135.6, 137.8, 167.4 (C@O Pht), 169.5 (C@O); ESI-MS:
m/z 339.2 [M+H]+; calcd for C19H18N2O4: 338.1.
4.1.4. Pht-Phe(3-Boc-HN-CH2)-OMe (9)
Pht-Phe(3-H2N-CH2)-OMe  HCl (8; 2.66 g, 7.09 mmol) was dis-
solved in 1,4-dioxane/H2O (250 mL; 3:2, v/v) and a solution of
NaHCO3 (715 mg, 8.51 mmol) in H2O (30 mL) added. To the result-
ing solution Boc2O (2.01 g, 9.22 mmol) was added as solid at r.t.
under stirring and stirring was continued for 4.5 h. The solvent
was evaporated under reduced pressure and the obtained material
distributed between AcOEt (300 mL) and H2O (100 mL). The organ-
ic phase was washed with 5% aq KHSO4 (3 Â 50 mL), brine
(1 Â 50 mL), dried (Na2SO4), and the solvent evaporated. The title
compound was obtained as colorless foam; yield: 2.97 g (96%);
TLC (cyclohexane/CHCl3/AcOH; 45:45:10; v/v/v): Rf 0.5; RP-HPLC
(column I, gradient I): tR 28.0 min; 1H NMR (500 MHz, DMSO-d6,
25 °C): d [ppm] = 1.36 (s, 9H, Boc-CH3), 3.28–3.35 (m, 1H, b2-
CH2), 3.48 (dd, J = 14.1, 4.8 Hz, 1H, b1-CH2), 3.69 (s, 3H, CH3), 3.96
CH2), 3.54 (dd, J = 8.2, 4.4 Hz, 1H,
a-CH), 4.07 (s, 2H, CH2-NH),
7.08–7.11 (m, 3H, Phe(Boc-HN-CH2)-Ar), 7.23 (m, 1H, Phe(Boc-
HN-CH2)-Ar); 13C NMR (125.7 MHz, DMSO-d6, 25 °C):
[ppm] = 28.5 (Boc-CH3), 36.8 (bC), 43.7 (CH2-NH-Boc), 55.9 (
d
C),
a
79.2 (Boc-CCH3), 125.9, 128.3, 128.5, 129.1, 136.7, 140.2, 156.7
(C@O Boc), 171.7 (COOH); ESI-MS: m/z 317.2 [M+Na]+; calcd for
C15H22N2O4: 294.2.
4.1.7. Fmoc-Phe(3-Boc-HN-CH2)-OH (12)
H-Phe(3-Boc-HN-CH2)-OH (11; 904 mg, 3.07 mmol) was dis-
solved in a mixture of 1,4-dioxane (30 mL) and H2O (20 mL), and
the pH was adjusted to 8-9 by 5% aq NaHCO3. To the resulting solu-
tion Fmoc-OSu (1.00 g, 2.98 mmol) was added at r.t. as solid. After
stirring over night the solvent was removed under reduced pres-
sure. The obtained pale yellow solid was distributed between
AcOEt/H2O (150 mL; 2:1, v/v), and upon addition of KHSO4
(711 mg, 5.22 mmol), the free acid was extracted into the organic
phase. The organic phase was washed with 5% aq KHSO4
(3 Â 50 mL), brine (1 Â 50 mL), dried (Na2SO4), and the solvent
evaporated. The title compound was isolated as colorless solid by
precipitation from tert-butyl methyl ether/petroleum ether; yield:
1.20 g (76%); TLC (CHCl3/MeOH/AcOH; 8:8:1; v/v/v): Rf 0.8; RP-
HPLC (column I, gradient I): tR 29.4 min; 1H NMR (500 MHz,
DMSO-d6, 25 °C): d [ppm] = 1.39 (s, 9H, Boc-CH3), 2.87 (m, 1H,
b2-CH2), 3.06 (m, 1H, b1-CH2), 4.10 (d, J = 5.4 Hz, 2H, CH2-NH),
(d, J = 6.3 Hz, 2H, CH2-NH), 5.24 (dd, J = 11.2, 4.8 Hz, 1H,
a-CH),
6.99–7.14 (m, 4H, Phe(Boc-HN-CH2)-Ar), 7.24 (t, J = 6.0 Hz, 1H,
Boc-HN-CH2), 7.85 (s, 4H, CH Pht-Ar); 13CNMR (125.7 MHz,
DMSO-d6, 25 °C): d [ppm] = 28.7 (Boc-CH3), 34.4 (bC), 43.6 (CH2-
NH-Boc), 53.2 (aC and OCH3), 78.2 (Boc-CCH3), 124.0, 125.7,
127.4, 128.0, 128.7, 131.1, 135.5, 137.2, 140.7, 156.2 (C@O Boc),
167.4 (C@O Pht), 169.5 (C@O); ESI-MS: m/z 461.2 [M+Na]+; calcd
for C24H26N2O6: 438.2.
4.1.5. H-Phe(3-Boc-HN-CH2)-OMe  HCl (10)
To a stirred solution of Pht-Phe(3-Boc-HN-CH2)-OMe (9; 2.30 g,
5.25 mmol) in MeOH (160 mL), hydrazine monohydrate (2.09 mL,
43.05 mmol), and AcOH (2.46 mL, 43.05 mmol) were added. The
resulting reaction mixture was stirred at r.t. until the reaction
was completed as monitored by analytical RP-HPLC. Subsequently,
the solvent was removed under reduced pressure and H2O
(170 mL) added. The formed pale yellow precipitate was filtered
off and the pH of the filtrate was adjusted to 8-9 by NaHCO3. The
resulting aqueous phase was extracted with CHCl3 (3 Â 100 mL),
and the combined organic phases were washed with brine
(1 Â 50 mL), dried (Na2SO4), and the solvent evaporated under re-
duced pressure. The obtained material was dissolved in MeOH
(80 mL) and HCl in dioxane (1 M) was added to adjust the pH to
ꢀ4. Subsequently, the solvent was removed under reduced pres-
sure and title compound was isolated as colorless solid by precip-
itation from iPrOH/tert-butyl methyl ether; yield: 1.38 g (76%); TLC
(CHCl3/MeOH/AcOH; 8:8:1; v/v/v): Rf 0.9; RP-HPLC (column I, gra-
dient I): tR 18.7 min; 1H NMR (500 MHz, DMSO-d6, 25 °C): d
[ppm] = 1.39 (s, 9H, Boc-CH3), 3.07 (dd, J = 13.9, 7.7 Hz, 1H, b2-
CH2), 3.21 (dd, J = 13.7, 5.0 Hz, 1H, b1-CH2), 3.65 (s, 3H, CH3), 4.10
4.19 (m, 4H,
a-CH and Fmoc-CH-CH2), 7.08–7.41 (m, 9H,
Phe(Boc-HN-CH2)-Ar, Fmoc-Ar and Boc-NH), 7.66 (t, J = 8.8 Hz,
2H, Fmoc-Ar), 7.77 (d, J = 8.2 Hz, 1H,
a-CH-NH), 7.88 (d,
J = 7.2 Hz, 2H, Fmoc-Ar), 12.76 (s, 1H, COOH); 13C NMR
(125.7 MHz, DMSO-d6, 25 °C): d [ppm] = 14.6, 21.2, 23.0, 27.3,
28.7 (Boc-CH3), 36.9 (bC), 43.8 (CH2-NH-Boc), 47.0 (Fmoc), 56.0
(aC), 60.2, 66.1 (Fmoc), 78.2 (Boc-CCH3), 120.6 (Fmoc), 125.4,
125.7 (Fmoc), 127.5 (Fmoc), 127.9, 128.1 (Fmoc), 128.5, 138.4,
140.5, 141.1 (Fmoc), 144.2 (Fmoc), 156.3, 156.4, 173.9 (COOH);
ESI-MS: m/z 539.3 [M+Na]+; calcd for C30H32N2O6: 516.2.
4.1.8. Fmoc-Phe(3-Boc-HN-CH2)-CHN2 (13)
To a solution of Fmoc-Phe(3-Boc-HN-CH2)-OH (12; 795 mg,
1.54 mmol) in dry THF (15 mL) at À15 °C under argon was added
Et3N (214 lL, 1.54 mmol) and ClCO2Et (147 lL, 1.54 mmol). After
15 min, freshly distilled ethereal CH2N2 solution (5.70 mL,
1.54 mmol, c = 0.27 mol/L) was added to the resulting suspension
at À5 °C. Stirring was continued over night at r.t. The solvent was
evaporated under reduced pressure and the obtained material dis-
tributed between AcOEt (100 mL) and H2O (50 mL). The organic
phase was washed with 5% aq KHSO4 (3 Â 30 mL), 5% aq NaHCO3
(3 Â 30 mL), brine (1 Â 30 mL), dried (Na2SO4), and the solvent
evaporated. The title compound was isolated as yellowish solid
by precipitation from tert-butyl methyl ether/petroleum ether;
yield: 714 mg (86%); TLC (AcOEt/petroleum ether 1:2; v/v): Rf
(d, J = 6.0 Hz, 2H, CH2-NH), 4.18 (m, 1H,
Phe(Boc-HN-CH2)-Ar), 7.38 (t, J = 6.0 Hz, 1H, Boc-HN-CH2), 8.83
(s, 3H, NH3); 13C NMR (125.7 MHz, DMSO-d6, 25 °C):
[ppm] = 28.7 (Boc-CH3), 36.3 (bC), 43.7 (CH2-NH-Boc), 52.9
(OCH3), 53.7 ( C), 78.3 (Boc-CCH3), 126.4, 128.2, 128.4, 129.0,
a-CH), 7.06–7.29 (m, 4H,
d
a
135.0, 140.9, 156.2 (C@O Boc), 169.8 (C@O); ESI-MS: m/z 309.2
[M+H]+; calcd for C16H24N2O4: 308.2.

7242
D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
0.6; RP-HPLC (column II, gradient II): tR 12.2 min; 1H NMR
(500 MHz, DMSO-d6, 25 °C): d [ppm] = 1.39 (s, 9H, Boc-CH3), 2.75
(m, 1H, b2-CH2), 3.00 (dd, J = 13.8, 4.3 Hz, 1H, b1-CH2), 4.09 (d,
Subsequently, the resin was washed three times alternately with
DMF (5 mL) and isopropanol (5 mL). Fmoc cleavage was performed
prior to each coupling step by treating (2 Â 15 min) the resin-
bound peptide with piperidine/DMF (1:4, v/v, 5 mL). Subsequently,
the resin was washed three times alternately with DMF (5 mL), and
isopropanol (5 mL).
J = 5.9 Hz, 2H, CH2-NH), 4.16–4.30 (m, 4H,
a-CH and Fmoc-CH-
CH2), 6.04 (s, 1H, CH@N2), 7.07–7.43 (m, 9H, Phe(Boc-HN-CH2)-
Ar, Fmoc-Ar and Boc-NH), 7.64 (m, 2H, Fmoc-Ar), 7.85–7.89 (m,
3H,
a
-CH-NH and Fmoc-Ar); 13C NMR (125.7 MHz, DMSO-d6,
25 °C): d [ppm] = 27.2, 29.1 (Boc-CH3), 37.0 (bC), 44.2 (CH2-NH-
Boc), 47.5 (Fmoc), 66.4 (Fmoc), 78.6 (Boc-CCH3), 120.9 (Fmoc),
125.8, 126.0 (Fmoc), 127.9 (Fmoc), 128.3, 128.4 (Fmoc), 128.6,
128.9, 138.6, 140.9, 141.5 (Fmoc), 144.6 (Fmoc), 156.6; ESI-MS:
m/z 563.2 [M+Na]+; calcd for C31H32N4O5: 540.6.
4.1.12. c[–Pro–b3hPhe(3-BocHN-CH2)–
D-allo-Ile–vTyr(tBu)–NH-
CH2-CH(NH2)-CO–] (16)
Prior on-resin cyclization the termini of the resin-bound linear
pentapeptide 15 (0.20 mmol) were deprotected. For cleavage of
the C-terminal allyl ester the resin-bound linear pentapeptide 15
was treated (3 Â 15 min) under an argon atmosphere at r.t. with
4.1.9. Fmoc-b3hPhe(3-Boc-HN-CH2)-OH (4)
Pd(PPh3)4 (93 mg, 0.08 mmol, 0.4 equiv) and PhSiH3 (593 lL,
To a solution of Fmoc-Phe(3-Boc-HN-CH2)-CHN2 (13; 710 mg,
1.31 mmol) in 1,4-dioxane (15 mL) and H2O (8 mL) was added sil-
ver benzoate (23 mg, 0.10 mmol). It was stirred at 90 °C for 5 h. The
solvent was evaporated under reduced pressure and the obtained
grey solid distributed between AcOEt (100 mL) and 5% aq KHSO4
solution (50 mL). The organic phase was washed with 5% aq KHSO4
(2 Â 30 mL), brine (1 Â 30 mL), dried (Na2SO4), and the solvent
evaporated. The title compound was isolated as colorless solid by
precipitation from tert-butyl methyl ether/petroleum ether; yield:
633 mg (91%); TLC (CHCl3/MeOH/AcOH; 45:2:1; v/v/v): Rf 0.4; RP-
HPLC (column II, gradient II): tR 11.4 min; 1H NMR (500 MHz,
DMSO-d6, 25 °C): d [ppm] = 1.35 (s, 9H, Boc-CH3), 2.33 (m, 2H,
4.8 mmol, 24 equiv) in dry dichloromethane (5 mL). Upon each
treatment the resin was washed with dry dichloromethane
(8 Â 5 mL). Then, the N-terminal Fmoc-group was cleaved as de-
scribed above. Subsequently, PyBOP (312 mg, 0.6 mmol, 3 equiv),
HOBt (81 mg, 0.6 mmol, 3 equiv), and DIPEA (205 lL, 1.2 mmol,
6 equiv) in DMF (5 mL) were added to the resin-bound peptide.
The resin was gently agitated at r.t. for 1 h. The progress of cycliza-
tion was monitored by using the chloranil test.27 Then, the resin
was washed three times alternately with DMF (5 mL) and isopro-
panol (5 mL). Eventually, at r.t., the cyclized resin-bound peptide
was treated (10 Â 5 min) with dichloromethane/TFA/TIS (98:1:1,
v/v/v, 5 mL). The cleavage solutions were collected in MeOH/pyri-
dine (45:5, v/v, 50 mL) and evaporated under reduced pressure.
The resulting material was distributed between AcOEt (200 mL)
and H2O (50 mL). The organic phase was washed with 5% aq NaH-
CO3 (3 Â 50 mL), brine (1 Â 50 mL), dried (Na2SO4), and the solvent
evaporated. The title compound was isolated as colorless solid by
precipitation from tert-butyl methyl ether/petroleum ether and
used for the next step without further purification; yield: 160 mg
(96%, based on resin loading); TLC (CHCl3/MeOH; 4:1; v/v): Rf
0.74; RP-HPLC (column II, gradient II) tR 9.7 min; MALDI-MS: m/z
854.8 [M+Na]+; calcd for C45H65N7O8: 831.5.
a-CH2), 2.64 (dd, J = 13.4, 6.4 Hz, 1H,
c2-CH2), 2.71 (dd, J = 13.3,
7.6 Hz, 1H, 1-CH2), 3.95 (m, 1H, b-CH), 4.05 (d, J = 5.9 Hz, 2H,
c
CH2-NH), 4.13 (t, J = 7.0 Hz, 1H, Fmoc-CH-CH2), 4.19 (d, J = 6.3 Hz,
2H, Fmoc-CH-CH2), 7.00–7.39 (m, 10H, Phe(Boc-HN-CH2)-Ar,
Fmoc-Ar, Boc-NH and b-CH-NH), 7.62 (d, J = 7.5 Hz, 2H, Fmoc-Ar),
7.85 (d, J = 7.5 Hz, 2H, Fmoc-Ar), 12.16 (s, 1H, COOH); 13C NMR
(125.7 MHz, DMSO-d6, 25 °C): d [ppm] = 29.1 (Boc-CH3), 39.5,
44.2 (CH2-NH-Boc), 47.6 (Fmoc), 50.5, 66.1 (Fmoc), 78.6 (Boc-
CCH3), 120.9 (Fmoc), 125.7, 126.0 (Fmoc), 127.9 (Fmoc), 128.4
(Fmoc), 128.7, 128.9, 139.3, 140.9, 141.5 (Fmoc), 144.7 (Fmoc),
156.2, 156.6, 173.2 (COOH); ESI-MS: m/z 553.1 [M+Na]+; calcd for
C31H34N2O6: 530.6.
4.1.13. c[–Pro–b3hPhe(3-H2N-CH2)–
D-allo-Ile–vTyr–NH-CH2-
CH(NHAc)-CO–] (2)
4.1.10. FmocHN-CH2-CH(NHÒ)-CO2Al (14)
To a stirred solution of the protected cyclic pentapeptide 16
To pre-swollen 2-chlorotrityl chloride resin (2.00 g, 1.08 mmol
Cl/g) was added a solution of HCl  H-Dap(Fmoc)-OAl (0.87 g,
2.16 mmol) and DIPEA (0.74 mL, 4.32 mmol) in dry dichloro-
methane (10 mL). The resin was gently agitated for 1.5 h at r.t.
Subsequently, the solution was sucked off and to the resin was
added a mixture of MeOH (2 mL), dichloromethane (8 mL), and DI-
PEA (0.37 mL) and agitation was continued for another 1 h at r.t.
The resin was washed three times alternately with DMF (10 mL)
and isopropanol (10 mL) then with tert-butyl methyl ether
(1 Â 10 mL), petroleum ether (1 Â 10 mL), and dried. Loading was
assessed by UV determination of the fulvene-piperidine adduct:26
0.36 mmol/g.
(34 mg, 0.041 mmol) in dichloromethane (5 mL) at r.t. was added
Ac2O (39 lL, 0.41 mmol) and pyridine (33 lL, 0.41 mmol) and stir-
ring was continued over night. The solvent was removed under re-
duced pressure and the resulting oil was dissolved in AcOEt
(100 mL). The organic phase was washed with 5% aq KHSO4
(3 Â 30 mL), 5% aq NaHCO3 (3 Â 30 mL), brine (1 Â 30 mL), dried
(Na2SO4), and the solvent evaporated. The acetylated compound
was isolated by precipitation from AcOEt/n-hexane. The obtained
material was treated at r.t. for 2 h with TFA/H2O (95:5, v/v;
3 mL). The peptide was isolated by adding the cleavage solution
dropwise into ice-cold tert-butyl methyl ether/n-hexane (2:1, v/v;
50 mL). The formed colorless precipitate was collected by centrifu-
gation, washed with petroleum ether (3Â), and dried. The crude
product was purified by preparative RP-HPLC and homogenous
fractions were pooled and lyophilized; yield: 10 mg (30%);
RP-HPLC (column II, gradient II) tR 6.1 min; 1H NMR (600 MHz,
4.1.11. Fmoc-Pro–b3hPhe(3-BocHN-CH2)–
NH-CH2-CH(NHÒ)-CO2Al (15)
D-allo-Ile–vTyr(tBu)–
Using FmocHN-CH2-CH(NHÒ)-CO2Al (14; 0.56 g, 0.20 mmol),
the linear pentapeptide was built up by coupling sequentially
DMSO-d6, 25 °C): d [ppm] = 0.55 (d, J = 6.7 Hz, 3H,
c
-CH3,
-allo-Ile), 0.66 (m, 1H,
-allo-Ile), 1.29 (m, 1H, b-CH,
-CH2, Pro), 1.89 (m,
2-CH2, b3hPhe(3-H2N-CH2)), 2.14 (m, 2H, b-CH2, Pro), 2.48
(m, 1H, d2-CH2, v-Tyr), 2.63 (m, 1H, b2-CH2, Dap), 2.67 (m, 1H,
1-CH2, b3hPhe(3-H2N-CH2)), 2.74 (m, 2H, -CH2, b3hPhe(3-H2N-
D
-allo-
Fmoc-vTyr(tBu)-OH (2 Â 1 equiv), Fmoc-D-allo-Ile (4 equiv), Fmoc-
Ile), 0.65 (d, J = 3.8 Hz, 3H, d-CH3,
D
c2-CH2,
b3hPhe(3-BocHN-CH2)-OH (4, 2 Â 1 equiv) and Fmoc-Pro-OH
(4 equiv). For each coupling step to a solution of equimolar
amounts of Fmoc amino acid, TBTU, and HOBt in DMF (5 mL)
was added at r.t. an equimolar amount of DIPEA. After 5 min of
preactivation, the solution was added to the resin-bound peptide
and the resin gently agitated for 1 h at r.t. The progress of coupling
was monitored by using the chloranil test27 and found to be
completed by applying the indicated excesses, respectively.
D-allo-Ile), 0.88 (m, 1H, c1-CH2,
D
D-allo-Ile), 1.84 (s, 3H, CH3, Ac), 1.87 (m, 2H,
c
1H,
a
a
c
CH2)), 2.88 (dd, J = 13.8, 4.2 Hz, 1H, d1-CH2, v-Tyr), 3.41 (m, 1H,
d2-CH2, Pro), 3.63 (m, 1H, d1-CH2, Pro), 4.01 (m, 2H, CH2-NH3,
b3hPhe(3-H2N-CH2)), 4.04 (m, 1H, b1-CH2, Dap), 4.17 (m, 1H,

D. Janke et al. / Bioorg. Med. Chem. 19 (2011) 7236–7243
7243
a
-CH,
b3hPhe(3-H2N-CH2)), 4.46 (m, 1H,
v-Tyr), 5.93 (dd, J = 15.3, 1.7 Hz, 1H,
D
-allo-Ile), 4.37 (m, 1H,
a
-CH, Pro), 4.39 (m, 1H, b-CH,
-CH, Dap), 4.50 (m, 1H, -CH,
-CH, v-Tyr), 6.63 (d,
degradation stopped by acidification with an equal volume of
0.1% TFA. After centrifugation at 8000 Â g for 10 min, the superna-
tants were injected onto a C18 column (LiChrospher 100 RP18,
125 Â 4 mm, Merck, Darmstadt, Germany) and eluted (eluent A:
H2O/TFA, 99.9:0.1 v/v, eluent B: MeCN/TFA, 99.9:0.1 v/v) with a lin-
ear gradient from A/B (100:0) to A/B (0:100) over 200 min using a
Sykam HPLC system (Fürstenfeldbruck, Germany). The residual
inhibitors were quantified at 206 nm by comparison to standard
curves obtained with the native compounds under identical
conditions.
a
c
a
J = 8.4 Hz, 2H, Ar, v-Tyr), 6.65 (m, 1H, b-CH, v-Tyr), 7.05 (d,
J = 8.2 Hz, 2H, Ar, v-Tyr), 7.21–7.36 (2 m, 4H, Ar, b3hPhe(3-H2N-
CH2)), 7.65 (d, J = 9.3 Hz, 1H, NH, D-allo-Ile), 8.06 (m, 1H, a-NH,
Dap), 8.07 (m, 1H, NH, v-Tyr), 8.10 (br s, 3H, CH2-NH3, b3hPhe(3-
H2N-CH2)), 8.27 (d, J = 9.6 Hz, 1H, NH, b3hPhe(3-H2N-CH2)), 8.64
(d, J = 10.5 Hz, 1H, b-NH, Dap), 9.13 (s, 1H, OH, v-Tyr); HRMS
(ESI) calcd for C38H51N7O7+H+ ([M+H]+); m/z: 718.39227, found:
718.39239.
Acknowledgments
4.1.14. c[–Pro–b3hPhe(3-H2N-CH2)–
D-allo-Ile–vTyr–NH-CH2-
CH(NHCO-(CH2)5-NH2)-CO–] (3)
The study was supported by the Deutsche Forschungsgemein-
schaft (Heisenberg Fellowship for N.S., grant SCHA 1012/1-3, and
project SO 249/1-1 of the priority program ‘Mast cells – promoters
of health and modulator of diseases’).
To a stirred solution of the protected cyclic pentapeptide 16
(40 mg, 0.048 mmol) in DMF (4 mL) at r.t. was added upon 5 min
preactivation a solution of BocHN-(CH2)5-COOH (22 mg, 0.096
mmol), TBTU (31 mg, 0.096 mmol), HOBt (13 mg, 0.096 mmol),
and DIPEA (17
lL, 0.096 mmol) in DMF (1 mL) and stirring was con-
References and notes
tinued over night. The solvent was removed under reduced pressure
and the resulting oil was dissolved in AcOEt (100 mL). The organic
phase was washed with 5% aq KHSO4 (3 Â 50 mL), 5% aq NaHCO3
(7 Â 50 mL), brine (1 Â 50 mL), dried (Na2SO4), the solvent evapo-
rated and further processed as described for inhibitor 2; yield:
16 mg (33%); RP-HPLC (column II, gradient II) tR 5.8 min; 1H NMR
(600 MHz, DMSO-d6, 25 °C): d [ppm] = 0.55 (d, J = 6.7 Hz, 3H,
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c
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(m, 3H, b-CH,
d-CH2, -Ahx), 1.86 (m, 2H,
b3hPhe(3-H2N-CH2)), 2.01–2.21 (m, 4H, b-CH2, Pro;
2.48 (m, 1H, d2-CH2, v-Tyr), 2.62–2.68 (m, 2H, b2-CH2, Dap;
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D
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D-allo-Ile), 0.67 (m,
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D
c
D
D
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c
-CH2, b3hPhe(3-H2N-
e
e-Ahx), 2.88 (dd, J = 13.7, 4.1 Hz, 1H, d1-CH2, v-Tyr),
3.41 (m, 1H, d2-CH2, Pro), 3.63 (m, 1H, d1-CH2, Pro), 4.00 (m, 2H,
CH2-NH3, b3hPhe(3-H2N-CH2)), 4.02 (m, 1H, b1-CH2, Dap), 4.17 (m,
1H,
b3hPhe(3-H2N-CH2)), 4.47 (m, 1H,
v-Tyr), 5.94 (d, J = 13.7 Hz, 1H, -CH, v-Tyr), 6.63 (d, J = 8.2 Hz, 2H,
a-CH,
D
-allo-Ile), 4.37 (m, 1H,
a-CH, Pro), 4.41 (m, 1H, b-CH,
a
-CH, Dap), 4.51 (m, 1H, -CH,
c
a
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Ar, v-Tyr), 6.66 (m, 1H, b-CH, v-Tyr), 7.05 (d, J = 8.2 Hz, 2H, Ar,
v-Tyr), 7.20–7.35 (2 m, 4H, Ar, b3hPhe(3-H2N-CH2)), 7.63 (d,
J = 9.3 Hz, 1H, NH,
1H,
D-allo-Ile), 7.70 (br s, 3H, e-NH3, e-Ahx), 8.07 (m,
a
-NH, Dap), 8.08 (m, 1H, NH, v-Tyr), 8.19 (br s, 3H, CH2-NH3,
b3hPhe(3-H2N-CH2)), 8.26 (d, J = 9.5 Hz, 1H, NH, b3hPhe(3-H2N-
CH2)), 8.65 (d, J = 10.5 Hz, 1H, b-NH, Dap), 9.13 (s, 1H, OH, v-Tyr);
HRMS (ESI) calcd for C42H60N8O7+H+ ([M+H]+); m/z: 789.46577,
found: 789.46685.
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and plasma
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To determine the stability of the inhibitors 2 and 3 in biological
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At various time points, samples were removed and any ongoing