Solid-phase synthesis of a pepticinnamin E library

Article abstract of DOI:10.1016/S0968-0896(03)00159-7

Pepticinnamin E is a naturally occurring bisubstrate inhibitor of farnesyltransferase. Based on the structure of the natural product, a compound library was synthesized by variation of eight structural parameters. Following three different routes, a total

Full text of DOI:10.1016/S0968-0896(03)00159-7

Bioorganic & Medicinal Chemistry 11 (2003) 2591–2615
Solid-Phase Synthesis of a Pepticinnamin E Library
Michael Thutewohl and Herbert Waldmann*
Max-Planck-Institut fur molekulare Physiologie, Abt. Chemische Biologie,
¨
Otto-Hahn-Str. 11, D-44227, Dortmund und Fachbereich 3, Organische Chemie, Universitat Dortmund, Germany
¨
Received 14 February 2003; accepted 7March 2003
Abstract—Pepticinnamin E is a naturally occurring bisubstrate inhibitor of farnesyltransferase. Based on the structure of the nat-
ural product, a compound library was synthesized by variation of eight structural parameters. Following three different routes, a
total of 51 analogues was synthesized on the polymeric support in 6–11-step parallel syntheses. Overall yields ranged from 3 to
63%, and the compounds were obtained with >90% purity.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
unknown.2 In this context, inhibitors which induce
apoptosis (i.e., programmed cell death), and which are
bisubstrate inhibitors of the PFT, are of particular
interest.2,4
Proteins with intrinsic GTPase-activity are involved in a
multitude of important biological processes like signal
transduction and vesicular trafficking. The biological
function often critically depends on the post-transla-
tional modification of these proteins with lipid residues.
A prime example are the Ras proteins which serve as
molecular switches in growth-factor mediated mitogenic
signalling events. The crucial biological role of the Ras
proteins is highlighted by the fact that mutations in the
Ras genes are found in approximately 30% of all
human tumours.1,2
To investigate these biological questions in a focussed
but flexible manner, a class of potential PFT inhibitors
are required, the structure of which can be varied
rapidly and efficiently by combinatorial solid-phase
synthesis. This approach should furnish inhibitors
which are competitive to the protein substrate, to the
farnesylpyrophosphate, or even to both substrates, as
well as substances which induce apoptosis in Ras-trans-
formed cells but not in the untransformed wild cells.
Oncogenic Ras only serves as a molecular switch for the
transduction of growth signals if the Ras is S-farnesy-
lated at the C-terminus and if located at the plasma
membrane. Thus, inhibitors of the enzyme protein far-
nesyltransferase (PFT), which catalyzes the transfer of a
farnesyl residue from farnesylpyrophosphate (FPP) to
the cysteine embedded in a C-terminal CAAX amino
acid sequence occurring in non-matured Ras (Scheme
1), are of particular interest as new antitumor thera-
peutic agents. However, despite the fact that several
PFT-inhibitors are in advanced clinical trials, some of
the most important and fundamental aspects of this
application of the signal transduction therapy3 remain
unclear. The crucial substrate of PFT, the farnesylation
of which is suppressed by these compounds, is still
In this article we report on the solid-phase synthesis of a
library of potential PFT-inhibitors that is based on the
structure of a naturally occurring bisubstrate inhibi-
tor.5a In the accompanying paper, the biological inves-
tigation of the compound collection and the delineation
of a structure–activity relationship are described.5b
Results and Discussion
Starting point for the synthesis of a library of potential
farnesyltransferase inhibitors was pepticinnamin E 1, a
naturally occurring bisubstrate inhibitor of the enzyme.6
The peptidic natural product is composed of five build-
ing blocks (Fig. 1). The central tripeptide embodies
three aromatic amino acids. Tyrosine 4 is D-configured
while DOPA-derivative 6 and phenylalanine 5 are
N-methylated. A lipophilic cinnamic acid derivative 2 is
*Corresponding author. Tel.: +49-231-133-2400; fax: +49-231-133-
2499; e-mail: herbert.waldmann@mpi-dortmund.mpg.de
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00159-7

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M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
Scheme 1. Farnesylation of Ras-proteins by protein farnesyltransfer-
ase (PFT).
Figure 2. Building blocks used for the solid phase synthesis.
variation of the structure of all five building blocks (Fig. 2).
In particular, the lipophilic N-terminal group, the
amino acid side chains and the polar C-terminus, as well
as the degree of N-methylation were varied. To this end,
it was planned to attach the first amino acid to a poly-
meric carrier via the aromatic hydroxyl group of a tyr-
osine (Scheme 2). Depending on the amino acid chosen
for resin attachment the subsequent chain elongation
should proceed in C-terminal-, N-terminal- or alternat-
ing C- and N-terminal direction (routes A, B and C, see
Scheme 2). Different immobilized tripeptide inter-
mediates 9, 10 and 11 formed thereby would then be
deprotected at their N-termini followed by attachment
of lipophilic carboxylic acids and liberation of the
C-terminus (step D, Scheme 2). Release of acylated tri-
peptides 13 from the polymeric carrier would give a set
of compounds analogous to chain-shortened farnesyl-
transferase inhibitor 7 (step E, Scheme 2). Coupling of
polymer-bound intermediate 12 to polar building blocks
would yield immobilized esters and amides 14. These
ultimately would be released to deliver analogues 15 of
pepticinnamin E mimicking all building blocks incor-
porated in the natural product (step E, Scheme 2). From
a preparative point of view the coupling of the sterically
demanding N-methylated amino acids which addition-
ally are prone to racemization and side reactions8 was
considered particularly challenging. Also, efficient acyl-
ation reactions with acrylic acid derivatives often is
Figure 1. Structure of the farnesyltransferase-inhibitor pepticinnamin
E and its components.
attached to the N-terminus of the tripeptide, and the
C-terminus is esterified with a diketopiperazine via the
side chain alcohol of a serine incorporated into this
heterocycle.
It is prudent to speculate that lipophilic structural unit 2
occupies the farnesylpyrophosphate binding site of far-
nesyltransferase, and that building blocks 3–6 imitate
the CAAX recognition sequence of the Ras protein
(compare Scheme 1).1,7 In this respect, it should be
noted that N-acylated tripeptide 7, an intermediate in
the synthesis of pepticinnamin E, displays an inhibitory
activity that is similar to the potency of the natural
product (Fig. 1).
In the total synthesis of pepticinnamin E7 first the core
tripeptide had to be built up followed by attachment of
the pentenylcinnamic acid and the diketopiperazine.
Drawing from this successfully realized strategy, a
library of pepticinnamin E analogues was developed by

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2593
Scheme 2. Planned synthesis of a pepticinnamin E library.
Scheme 3. (a) DEAD, PPh3, THF, 0 ꢁC, 1 h, rt, 12 h, loading-level:
0.40–0.53 mmol gꢀ1, 53–59%; (b) Pd(PPh3)4, PhSiH3 or NMA, THF,
24 h; (c) 25, HATU, HOAt, iPrNEt2, NMP; 48 h; (d) 26, PyAOP,
HOAt, iPrNEt2, NMP; 48 h; (e) piperidine/DMF 1/4, 10–40 min;
(f) 29, HATU, HOAt, iPrNEt2, NMP, 12–24 h; (g) 30, PyAOP,
DMAP, iPrNEt2, CH2Cl2/DMF 1/2, 12–17h; (h) 31, HOAt, DMAP,
iPrNEt2, CH2Cl2/DMF 2/1, 7–20 h; (i) Pd(PPh3)4, morpholine, THF,
4 h; (j) 33, PyAOP, iPrNEt2, NMP, 16 h (Y=NH); (h) Cs2CO3, KI,
32, DMF, 24 h, rt (X=Br) or 24–48 h, 50 ꢁC (X=Cl) (Y=O); (l) TFA/
H2O 95/5, 4–24 h; (m) 10% Pd/C, H2, MeOH, 8 h.
accompanied by unwanted side reactions and compli-
cations.9 As polymeric carrier, the Wang resin was cho-
sen which embodies an acid-labile linking group. The
base-labile Fmoc-urethane was employed for temporary
masking of the N-terminus, and the C-terminus of the
intermediates was protected as palladium0 (Pd0)-sensitive
allyl ester. These three types of blocking groups are
orthogonally stable to each other.
C-Terminal elongation of the peptide chain (route A)
and PyBroP resulted in incomplete conversion and sub-
stantial racemization (Scheme 3). The best results were
obtained with PyAOP resulting in 89% conversion and
7% epimerization of the activated amino acids. Double
coupling led to an increase of yield to 94% accom-
panied with 9% racemization. These findings are not
unexpected. It has been reported previously that effi-
cient coupling of sterically demanding N-methylated
amino acids requires particular coupling reagents and
conditions,8 in particular on the solid phase. Also, the
C-terminal chain elongation of peptides frequently is
accompanied by epimerization,12,13 and N-methylated
amino acids are also known to be prone to racemiza-
tion.14 In the light of these findings the results recorded
in the synthesis of solid-phase bound N-methylated tri-
peptide 18 can be regarded as fairly satisfying.
Orthogonally protected tyrosine building blocks 16 were
attached to the Wang resin by means of a Mitsunobu
reaction10 with loading loading levels of 0.4–0.53 mmol
g
ꢀ1 (54–59%; determined with the Fmoc method11).
For the synthesis of tripeptide 18 by C-terminal elonga-
tion the allyl ester was removed from D-8 by Pd0-cata-
lyzed allyl transfer to phenylsilane or N-methylaniline as
trapping nucleophiles. Attachment of N-methylated
building block 25 could then successfully be achieved by
employing HATU as highly reactive coupling reagent.
In contrast, further chain elongation with building
block 26 to give tripeptide 18 turned out to be problem-
atic. After cleavage of the allyl ester application of
numerous coupling reagents including HATU, TFFH

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M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
Table 1. Results for the synthesis of pepticinnamin E analogues via route A
Entry
Compd
Yield (yield/step) (%)
Purity
1
23/1
=a
14 (80)
>95
2
3
4
23/2
20/1
21
a
a
10 (77)
12 (76)
14 (78)
>95
>95
>95
–OH
–OH
=b
5
20/2
20/3
20/4
=c
–OH
–OH
–OH
13 (77)
13 (77)
18 (81)
>95
95
8
=d
=e
10
>95
12
13
14
20/5
20/6
23/4
=f
–OH
–OH
10 (75)
11 (76)
10 (77)
>95
>95
>95
Fm=g
=h
15
23/5h
5 (72)
>95
16
17
18
23/6
20/7
20/8
h
h
13 (78)
5 (69)
60
>95
>95
–OH
–OH
=I
13 (77)
19
20/9
=j
–OH
13 (77)
>95
(continued on next page)

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2595
Table 1 (continued)
Entry Compd
Yield (yield/step) (%)
Purity
20
23/7
=k
8 (76)
91
21
22
23/8
23/9
k
k
28 (87)
40 (90)
>95
>95
23
24
23/10
20/10
k
k
–OAll
–OH
31 (85)
36 (88)
>95
95
In the next step of the synthesis, the Fmoc-urethane was
cleaved under basic conditions and cinnamic and
hydrocinnamic acid derivatives 30 and 29 as well as
chloroformic acid esters 31 were attached to the N-ter-
minus. While the coupling reactions with 29 and 31
proceeded smoothly in the presence of HATU/HOAt
and DMAP/HOAt, respectively, efficient activation of
the unsaturated acids 30 could only be achieved with
PyAOP/DMAP. Subsequent liberation of the C-termi-
nus by Pd0-mediated allyl transfer to morpholine yiel-
ded resin-bound tripeptides 19, which were then
released from the solid support by repeated treatment
with trifluoroacetic acid/H2O 95/5. Under these condi-
tions, the use of additional scavenging reagents was not
necessary.15 After purification by HPLC, tripeptides 20,
which can be regarded as analogues to compound 7,
were obtained with >95% purity and acceptable overall
yields. Hydrogenation of the double bonds incorporated
into the N-acyl substituents yielded further analogues
with saturated carbon chains.
Furthermore, polymer-bound carboxylic acids 19 were
converted to amides 22 by activation with PyAOP.
Coupling to primary amino acids 33 proceeded effi-
ciently under these conditions without any epimeriza-
tion. In addition, conversion of the carboxylic acids to
the cesium salts followed by alkylation with alkyl
halides 32 yielded C-terminal esters. Compounds 23 (see
Table 1) were finally released from the resin under the
conditions described above.
N-Terminal elongation of the peptide chain (route C)
As an alternative, the C-terminal amino acid was
employed for attachment to the resin, and the peptide
chain was elongated in the N-terminal direction via
sequential Fmoc-deprotection and coupling steps
(Scheme 4). Monitoring the reactions with the ninhydrin
test15,16 and examination of tripeptides 34 by HPLC
revealed that the couplings proceeded with high yield and
without racemization. If histidine was attached to the
peptide, the couplings were carried out with diisopropyl
carbodiimide (DIC) and HOAt, since histidine derivatives
are prone to racemization under basic conditions.
Scheme 4. (a) Piperidine/DMF 1/4, 2ꢂ5 min or 2ꢂ20 min; (b) 27,
HATU, iPrNEt2, NMP, 2.5 h; (c) 27, HATU, iPrNEt2, NMP or DIC,
HOAt, DMF, 14 h; (d) 29, HATU, HOAt, iPrNEt2, NMP, 12–24 h;
(e) 30, PyAOP, DMAP, iPrNEt2, CH2Cl2/DMF 1/2, 12–24 h; (f) 31,
HOAt, DMAP, iPrNEt2, CH2Cl2/DMF 2/1, 7–10 h; (g) Pd(PPh3)4,
morpholine, THF, 4–16 h; (h) TFA/H2O 95/5, 4–5 h; (i) 10% Pd/C,
H2, dioxane, 8 h.

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M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
Table 2. Results for the synthesis of pepticinnamin E analogues via route C
Entry
1
Compd
R1
R2
*
R3
R4
H
Yield (per step) (%)
14 (78)
Purity
78
35/1
=i
HOPh–
d
Me
2
3
4
5
6
7
8
35/2
35/3
35/4
35/5
35/6
35/7
35/8
i
i
i
i
i
i
i
HOPh–
HOPh–
HOPh–
Ph–
Imid
Imid
Imid
d
d
l
l
l
l
d
H
H
H
H
H
H
OH
OH
H
H
H
18 (81)
21 (82)
31 (86)
18 (81)
24 (84)
24 (84)
24 (84)
>95
>95
>95
>95
95
Me
Me
85
>95
H
9
35/9
=h
Imid
d
d
H
H
H
H
58 (81)
31 (86)
>95
>95
10
35/10
=d
HOPh–
11
12
35/11
35/12
d
Imid
Imid
d
d
H
H
H
H
15 (79)
24 (84)
95
=e
>95
13
14
15
35/13
35/14
35/15
e
e
e
Imid
Imid
Imid
l
l
d
H
Me
Me
H
H
H
19 (81)
30 (86)
24 (84)
93
>95
>95
16
17
18
19
20
21
22
35/16
35/17
35/18
36
=l
Imid
Imid
Imid
Imid
Imid
Imid
Imid
d
d
d
d
d
d
d
H
H
H
H
H
H
H
H
H
3 (65)
13 (77)
38 (89)
30 (86)
34 (87)
21 (82)
10 (75)
E/Z-mixt. 1:1
=c
>95
=a
H
90
95
=b
=m
=n
=o
H
35/19
35/20
35/21
H
>95
Me
H
E/Z-mixt. 1.7/1
E/Z-mixt. 1/2.5
ortho
23
24
25
26
27
35/22
35/23
35/24
35/25
35/26
ortho o
meta o
meta o
para o
para o
Imid
Imid
Imid
Imid
Imid
l
l
d
l
d
H
H
H
H
H
H
H
H
H
H
13 (77)
21 (82)
28 (85)
11 (76)
10 (75)
89
91
E/Z-mixt. 1.7/1
E/Z-mixt. 1.6/1
>95

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2597
Scheme 6. (a) Pd(PPh3)4, NMA, THF; (b) 26, PyAOP, iPrNEt2, NMP,
8 h; (c) piperidine/DMF 1/4, 2ꢂ5 min; (d) Fmoc-D-Tyr(tBu)OH,
HATU, iPrNEt2, NMP, 14 h; (e) 29, HATU, HOAt, iPrNEt2, NMP,
12 h; (f) Pd(PPh3)4, morpholine, THF, 24 h. TFA/H2O 95/5, 3.5 h.
Scheme 5. (a) Piperidine/DMF 1/4, 215 min; (b) o-NO2C6H4SO2Cl,
lutidine, CH2Cl2, 11 h; (c) p-NO2PhSO3Me, MTBD, DMF, 1.5 h, (d)
DBU, 2-mercaptoethanol, DMF, 1 h; (e) Fmoc-l-His(Trt)OH, DIC,
HOAt, lutidine, DMF, 24 h; (f) Pd(PPh3)4, NMA, THF; 24 h, (g) 26,
PyAOP, iPrNEt2, NMP, 24 h; (h) 31, DMAP, HOAt, iPrNEt2, DMF/
CH2Cl2, 8 h; (i) 29c, HATU, HOAt, iPrNEt2, NMP, 8 h; (j)
Pd(PPh3)4, morpholine, THF, 24 h; (k) TFA/H2O 95/5, 24 h.
N- and C-terminal elongation of the peptide chain (route
B)
After deprotection of the N-termini, they were acylated
with cinnamic acids, hydrocinnamic acids and chloro-
formic acid esters, the C-terminal carboxylic acid was
liberated and N-acylated tripeptides 35 were released from
the polymeric carrier as described above. Hydrogenation
yielded compounds 36 embodying a saturated acyl chain.
The results of the synthesis are given in Table 2.
In the third approach, the central amino acid was
linked to the solid support and N-methylated according
to Miller et al.20 (Scheme 5). After removal of the
Fmoc group from tyrosine L-8 the 1-nitrobenzene-
sulfonic acid amide was formed and the N–H proton
was abstracted with a guanidine base. Treatment with
methyl-p-nitrobenzenesulfonate yielded desired inter-
mediate 37. After removal of the sulfonamide blocking
group a histidine building block was introduced under
weakly basic conditions without recordable epimeriza-
tion. Next the allyl ester was cleaved and the C-terminal
amino acid was introduced. In the presence of PyAOP as
coupling reagent, conversion was quantitative but two
diastereomeric compounds were formed due to epimer-
ization of the central amino acid upon activation. After
cleavage from the solid support, isomers 40a and 40b
could be separated by hplc. In addition, 39 was converted
to N-acylated tripeptides 41a and 41b according to the
methods described above, and isomers 42a and 42b were
released from the solid support under acidic conditions.
Surprisingly, in the products 35 with a para-pentenyl-,
phenyl- and benzyloxy-substituted cinnamic acid sub-
stituent (Table 2, entries, 16, 21, 22, 25 and 26) E/
Z-isomerization of the originally E-configured double
bond occurred. This was proven by LC–MS and NMR
analysis and comparison of the chemical shifts and
coupling constants with literature data17 for E- and
Z-configured cinnamic acid amides. Such isomerizations
of cinnamic acid derivatives have been observed before.
They occur under the influence of light,18 are favored
when electron-donating substituents are present at the
aromatic ring17 and are promoted by Brønstedt and
Lewis acids.19

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M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
Finally, peptides 45 were synthesized by reversal of the
order of the chain extension steps, that is by elongating
the peptide chain first in the C-terminal direction fol-
lowed by acylation of the N-terminus. To this end, L-8
was first converted to polymer-bound dipeptides 42
which then yielded access to tripeptide intermediates 44
(Scheme 6). Epimerization was not detected in any of the
coupling steps. Compounds 44 were then converted into
peptides 45 according to the methods described above.
brackets indicate rotamers. Mass spectra were recorded
on the following spectrometers: EI=Finnigan MAT
MS70, FAB=Finnigan MAT MS 70 (3-nitrobenzy-
lalcohol (NBA) as matrix), MALDI-TOF=Perseptive
Biosystems Voyager BioSpectrometry Workstation or
Voyager-DE Pro BioSpectrometryTM Workstation (2.5
dihydroxybenzoic acid (DHB) as matrix), ESI=Finni-
gan Thermoquest LCQ (ESI 1) or Waters ZMD 2000
(ESI 2). If not indicated otherwise, the ESI-mass spec-
trometers were coupled with analytical HPLC-systems:
ESI-1, coupled with HP Modell 1100/Hewlett Packard;
column: VP 50/10 Nucleosil C18PPN/ Macherey-Nagel,
100ꢂ5 mm, 5-mm particle size; detection: 210, 220, 254
and 305 nm; flow rate: 1 mL/min eluent gradient
(CH3CN/H2O/HCO2H): A: 45/55/0.1 to 90/10/0.1 in 30
min; B: 20/80/0.1 to 90/10/0.1 in 30 min. ESI 2, coupled
with Waters 2790 with 996 DAD-Detector, column:
RPC18 Synergy MAX, 50ꢂ2 mm, 3.5-mm particle size/
Phenomenex, detection: 254 nm; flow rate: 1.1 mL/min;
eluent-gradient: (CH3CN/H2O/HCO2H): 2.5/97.5/
0.05%, to 97.5/2.5/0.05% in 4 min. For analytical
HPLC was used HP Modell 1100/Hewlett Packard;
column: VP 50/10 Nucleosil C18PPN/Macherey-Nagel,
100ꢂ5 mm, 5-mm particle size, detection: 210, 220, 254
and 305 nm; flow rate: 1 mL/min; eluent gradients are
indicated for the appropriate compound. For preparative
HPLC a Pro Star 215/Varian with Varian detector model
340 was used, column: VP 250/21 Nucleosil C18PPN,
100ꢂ5 mm, 5 mm particle size/Macherey–Nagel, detec-
tion: 210 or 305 nm; flow rate: 10 mL/min; eluent-gra-
dient: (CH3CN/H2O/TFA): 20/80/0.05% to 80/20/
0.05%, 30 min; The used Wang resin (100–200 mesh, 1%
DVB) was supplied by Novabiochem and Advanced
ChemTech. The solid-phase synthesis of the compounds
6 were performed on an Argonaut Technologies Quest-
210-Synthesizer.
Conclusion
Employing four different synthesis routes, a collection
of 51 pepticinnamin E analogues was synthesized by
variation of up to eight structural parameters. The
syntheses proceeded in 6–11 steps on the polymeric
support and yielded the desired compounds in 3–63%
overall yield which corresponds to an average yield per
step of 65–91% (in the majority of the cases >80% are
reached). With a few exceptions, the compounds were
obtained in quantities more than sufficient for sub-
sequent biological evaluation and with >90% purity.
The library contains compounds with a structure that is
close to the natural product itself (e.g., 23/1) or which
embody at least the twice N-methylated backbone of
pepticinnamin E which may be prone to adopting a
b-turn conformation (see Table 1). In addition, ana-
logues were synthesized that carry only one N-methyl
group (Table 2) and which should adopt a different
conformation. Furthermore, several compounds
embody a histidine as N-terminal amino acid. Its imi-
dazole group may serve as a good ligand for the Zn2t in
the active site of farnesyltransferase.
The synthesized compounds were investigated in differ-
ent enzymatic farnesyltransferase assays and in cell-
assays with Ras-transformed cancer cell lines. The results
of these investigations and the delineation of a structure–
activity relationship are described in the accompanying
paper.5b
Compound 16 was synthesized by azeotropic esterifica-
tion21 of tyrosine, followed by standard Fmoc-protec-
tion under Schotten–Baumann conditions.
Compound 30i was synthesized as published before.6
Cinnamic acid derivatives 30a–h were synthesized by
standard knoevenagel-condensation starting from the
commercial aldehydes. Hydrogenation of 30a, 30b and
30f furnished the hydrocinnamic acids 29a–c.
Experimental
General
Amino acid allyl esters 28a and 28b were synthesized
by azeotropic esterification according to standard
procedures.21
All melting points were determined using a Buchi 530 or
a Buchi B 540 apparatus and are uncorrected. Optical
rotations ½aŠ were measured with a Perkin–Elmer
20
D
polarimeter 241 or 341. TLC was performed on Kie-
selgel 60F254 (Merck). Column chromatography is
referred to flash chromatography and was performed on
silica gel (230–400 mesh). IR spectra were recorded on a
Bruker IFS 88 Fourier-Transform-IR-spektrometer.
Elemental analyses were measured with an Elementar
CHN-Rapid Analysator. NMR spectra were recorded
with Bruker AC 250, Bruker AM 400, Varian Mercury
400 and Bruker DRX 500. The signal of the residual
protonated solvent (CDCl3 or CD3OD) was taken as
reference [1H: d, 7.24 (CHCl3) or 3.31 (CH3OH), 13C: d,
77.0 (CHCl3) or 49.0 (CH3OH)]. Values in square
3-(2-Naphthyl)-L-alanine allyl ester hydrotosylate (28b).
Yield 96%; brownish solid, Rf=0.28 [ethyl acetate/
cyclohexane 2/1, 2% NEt3 (v/v)]; HPLC (A2, CH3CN/
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC):
20
Rt=9.83 min; mp: 188 ꢁC; ½aŠ ꢀ4.9 (c 0.51, MeOH);
D
1H NMR (400 MHz, CD3OD): d 2.37(s, 3H), 3.38–
3.46 (d, 3J=6.8 Hz, 2H), 4.46 (t, 3J=6.8 Hz, 1H),
4.68–4.70 (m, 2H), 5.19–5.30 (m, 2H), 5.81-5.88 (m,
1H), 7.24 (d, 3J=8.4 Hz, 2H), 7.38–7.41 (m, 1H),
7.49–7.52 (m, 2H), 7.71–7.76 (m., 1H), 7.72 (d,
3J=8.2 Hz, 2H), 7.83–7.90 (m, 3H); 13C NMR
(100.6 MHz, CD3OD): d 21.30, 37.56, 55.11, 67.84,

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2599
119.80, 126.91, 127.15, 127.38, 127.94, 128.63, 128.71,
129.60, 129.82, 132.29, 132.69, 134.21, 134.89, 141.72,
143.35, 169.71; MS (EI, 70 eV, m/z): 255 (M+);
HRMS (FAB): calcd for C16H18O2 [M+H]+
256.1338; found: 256.1361.
added. The aqueous phase was extracted with Et2O,
the combined organic phases were dried over MgSO4.
After concentration in vacuo chromatography on
silica gel [ethyl acetate/cyclohexane 1/2, 1% HOAc
(v/v) afforded 30g (140 mg, 65%) as yellowish solid;
Rf=0.40 (ethyl acetate/cyclohexane 1/1, 1% HOAc (v/
v)]; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/
0.1 in 30 min, 50 ꢁC): Rt=10.72 min; 1H NMR
The N-methylated amino acid building blocks 25 and
26 were synthesized in three steps starting from the
appropriate Boc-protected amino acids by N-methyla-
tion,22 alkylation of the carboxylic function by allyl-
bromide23 and standard Boc cleavage by HCl in
Et2O.
3
(400 MHz, CDCl3): d 1.01 (t, J=7.2 Hz, 3H), 1.55 (m,
2H), 2.24 (m, 2H), 6.27–6.33 (m, 1H), 6.40 (d,
3
3Jtrans=15.8 Hz, 1H), 6.48 (d, Jtrans=16.0, 1H), 7.37–
3
7.40 (m, 2H), 7.50 (s, 1H), 7.34 (d, J=7.6 Hz, 1H),
3
7.80 (d, Jtrans=16.0 Hz, 1H), 12.40 (s, 1H); 13C NMR
N,O-Dimethyl-L-tyrosine allyl ester hydrochloride (25).
Overall yield (three steps): 84%, white solid; Rf=0.25
[ethyl acetate/cyclohexane 3/1, 1% NEt3 (v/v)]; mp:
(100.6 MHz, CDCl3): d 14.03, 22.72, 35.38, 117.64,
126.24, 126.89, 128.44, 129.28, 129.37, 132.42, 134.47,
138.91, 147.39, 173.08; MS (ESI-1): 215.1 [M-H]ꢀ;
HRMS (FAB, m/z): calcd for C14H17O2 [M+H]+:
217.1229, found: 217.1256.
20
107 ꢁC; ½aŠ
+9.4 (c 0.5, MeOH); 1H NMR
D
(250 MHz, CDCl3): d 2.77 (s, 3H, NCH3), 3.28–3.37
(m, 1H), 3.53–3.61 (m, 1H), 3.76 (s, 3H), 4.05 (m,
1H), 4.57(d, 3J=5.9 Hz, 2H), 5.19–5.26 (m, 2H),
3-(4-(1-E-n-Pentenyl)phenyl)-E-acrylic acid (30h). The
synthesis was performed according to the procedure
described above for the synthesis of 30g starting from 4-
bromobenzaldehyde. Purification by cristallization from
cyclohexane afforded 30h [141 mg, 65%, as a yellowish
solid; Rf=0.40 (ethyl acetate/cyclohexane 1/1, 1%
HOAc (v/v)]; HPLC (A2, CH3CN/H2O/TFA, 45/55/
0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=11.58 min; mp:
3
5.66–5.82 (m, 1H), 6.82 (d, J=8.4 Hz, 2H), 7.20 (d,
J=8.4 Hz, 2H), 9.59 (bs, 1H), 10.53 (bs, 1H); 13C
NMR (100.6 MHz, CDCl3): d 32.10, 35.08, 55.22,
62.67, 66.91, 114.24, 119.92, 125.87, 130.44, 130.53,
159.05, 167.46; IR (KBr): 3476, 3003, 2913, 2839,
2698, 2449, 1745, 1653, 1605, 1575, 1465, 1453, 1397,
1252; MS (EI, 70 eV, m/z): 249 (2) [M+ꢀHCl], 164
(35) [M+ꢀHClꢀCO2ꢀC3H5], 128 (100) [M+ꢀNCH3ꢀ
OCH2ꢀOC3H5], 121 (23) [CH2C6H4OCH+3 ], 82 (5)
[2ꢂC4H+10], 41 (11) [C3H+5 ]; anal. calcd for
C14H30ClNO3 (285.77): C 58.84, H 7.05, N 4.90;
found: C 59.00, H 6.93, N 4.78.
175 ꢁC (decomp); H NMR (400 MHz, CDCl3): d 0.89
1
3
(t, J=7.2 Hz), 1.55 (m, 2H), 2.15 (m, 2H), 6.21–6.28
3
(m, 1H), 6.32 (d, Jtrans=16.0 Hz, 1H), 6.33 (d,
3
3Jtrans=16.0 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H), 7.40 (d,
3
3J=8.4 Hz, 2H), 7.60 (d, Jtrans=16.0 Hz, 1H); 13C
NMR (100.6 MHz, CDCl3): d 14.08, 23.43, 36.18,
118.31, 127.35, 129.38, 130.54, 133.49, 134.09, 141.36,
146.04, 170.41; MS (ESI-1): 215.1 [MꢀH]ꢀ; HRMS
(FAB): calcd for C14H17O2 [M+H]+: 217.1229, found:
217.1252.
N-Methyl-L-phenylalanine allyl ester hydrochloride (26).
Overall yield (three steps): 79%, white solid; Rf=0.27
[ethyl acetate/cyclohexane 3/1, 1% NEt3 (v/v)]; mp:
20
111–112 ꢁC; ½aŠ ꢀ17.2 (c 0.5, MeOH); 1H NMR
D
(400 MHz, CDCl3): d 2.78 (s, 3H), 3.32–3.42 (m, 1H),
3.62–3.69 (m, 1H), 4.09 (m, 1H), 4.55 (d, J=6.0 Hz,
3
General procedure for the pd0-catalysed cleavage of allyl
esters on the solid phase (GP P1)
2H), 5.15–5.21 (m, 2H), 5.61–5.77 (m, 1H), 7.21–7.34
(m, 5H), 9.81 (s, 1H), 10.61 (br. s, 1H); 13C NMR
(100.6 MHz, CDCl3): d 31.94, 35.85, 62.45, 66.85,
119.84, 127.63, 128.84, 129.31, 130.46, 134.13, 167.41;
IR (KBr): 3494, 2936, 2737, 2698, 2424, 1755, 1653,
1605, 1575, 1472 (CH), 1456 (CH), 1397 (CH3), 1264;
MS (FAB, m/z): 219.969 [M+H]+; anal calcd for
C13H18ClNO2 (255.74): C 61.05, H 7.09, N 5.48; found:
C 61.11, H 6.96, N 5.45.
Variant A. The resin (0.6 mmol) was washed under
argon atmosphere twice with 5 mL THF, 5 mL CH2Cl2
and the appropriate dry and degassed solvent. After
swelling in the solvent for some min 10–20 mol%
Pd(PPh3)4-catalyst and 10–30 equiv phenylsilane were
added and the mixture was shaken under argon and
under light exclusion. After filtration of the resin, it was
washed with 2ꢂ5 mL CH2Cl2, DMF/H2O 1/1 (v/v),
DMF, MeOH und CH2Cl2.
3-(3-(1-E-n-Pentenyl)phenyl)-E-acrylic acid (30g).
A
solution of 3-bromobenzaldehyde (117 mL, 1.0 mmol),
pent-1-enylboronic acid24 (137mg, 1.2 mmol), CsF
(365 mg, 2.4 mmol) and Pd(PPh3)4 (46 mg, 0.04
mmol) in 4 mL degassed DME was stirred at 100 ꢁC
for 14 h under argon atmosphere. After cooling to
room temperature water was added and the aqueous
phase was extracted with ethyl acetate. After drying
of the organic phases over MgSO4 the solvent was
evaporated in vacuo. To the residue was added a
solution of malonic acid (125 mg, 1.2 mmol) and
piperidine (9.9 mL, 0.1 mmol) in 1 mL pyridine and
the reaction mixture was stirred at 100 ꢁC for 6 h.
Then a mixture of ice and concentrated HCl were
Variant B. The resin (0.6 mmol) was washed under
argon atmosphere twice with 5 mL THF, 5 mL CH2Cl2
and dry and degassed NMA/THF [1/5 (v/v)]. After
swelling in 10 mL NMA/THF [1/5 (v/v)] for some min
10–20 mol% Pd(PPh3)4 catalyzator were added and the
mixture was shaken under argon and under light exclu-
sion. After filtration of the resin it was washed with 2ꢂ5
mL CH2Cl2, DMF/H2O 1/1 (v/v), DMF, MeOH und
CH2Cl2.
Variant C. The resin (0.6 mmol) was washed under
argon atmosphere twice with 5 mL THF, 5 mL CH2Cl2

2600
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
and morpholine/THF [1/9 (v/v)]. After swelling in 10
mL morpholine/THF mixture [1/5 (v/v)] for some min
10–20 mol% Pd(PPh3)4 catalyzator were added and the
mixture was shaken under argon and under light exclu-
sion. After filtration of the resin, it was washed with
2ꢂ5 mL CH2Cl2, DMF/H2O 1/1 (v/v), DMF, MeOH
und CH2Cl2.
v), 5ꢂ5 mL DMF, 5ꢂ5 mL MeOH und 8ꢂ5 mL
CH2Cl2 and dried in vacuo.
General procedure for the cleavage from the solid
support (GP P5)
Variant A. After washing the resin twice with 5 mL
CH2Cl2 a mixture of TFA and the appropriate scav-
enger reagent is added. After shaking for several hours,
the solid support is filtered off and washed with 5 mL
TFA, 5 mL CHCl3, 2ꢂ5 mL CH2Cl2, 5 mL MeOH and
5 mL toluene. The combined filtrates were evaporated
in vacuo. HPLC purification, followed by lyophilization
afforded the product.
General procedure for the base mediated cleavage of
Fmoc-protecting groups on the solid support (GP P2)
To the resin (0.5 mmol/g, 50 mmol) were added 3 mL
piperidine/DMF 1/4 (v/v) and the mixture was shaken
between 5 and 30 min. After filtration, the procedure
was repeated and the resin was filtered and washed with
4ꢂ5 mL DMF, 4ꢂ5 mL MeOH und 4ꢂ5 mL CH2Cl2.
Variant B. After washing the resin twice with 5 mL
CH2Cl2, the polymer-bound compound was reacted
several times for different periods of time with mixtures
of TFA/CH2Cl2/H2O 47/47/6 (v/v/v) and with TFA/
H2O 95/5 (v/v). The resin was filtered off, washed with
3ꢂ5 mL CH2Cl2, 2ꢂ5 mL EtOH and 5 mL toluene. The
combined filtrates were evaporated in vacuo. HPLC
purification, followed by lyophilization afforded the
product.
General procedure for the C-terminal coupling of amino
acid allyl esters on solid support (GP P3)
Variant A. To polymeric support (0.50 mmol/g, 0.50
mmol) swollen in NMP was added a solution of 3–6
equiv of HATU, 1–2 equiv of HOAt and 3–6 equiv of
DIEA in NMP. After shaking for 30–60 min the resin is
filtered, washed with 2ꢂ5 mL NMP and a solution of 3–
6 equiv of the amino component and 6–12 equiv of
DIEA in NMP were added. After shaking for several
hours the resin is filtered off, washed with 3ꢂ5 mL
DMF, 3ꢂ5 mL DMF/H2O 1/1 (v/v), 3ꢂ5 mL DMF,
3ꢂ5 mL MeOH und 4ꢂ5 mL CH2Cl2 and dried in
vacuo.
General procedure for the N-terminal coupling of acrylic
acid derivatives on solid support (GP P8)
After washing the polymer (0.5 mmol/g, 0.15 mmol)
with 2ꢂ5 mL THF, 2ꢂ5 mL CH2Cl2 and swelling in 3
mL DMF for 30 min, 10 equiv of DIEA were added. A
solution of 3–6 equiv of the acrylic acid, PyAOP and
HOAt in 6 mL of a mixture of dry DMF/CH2Cl2 1/1 (v/
v) was added. After shaking for 1 min, 0.5 equiv of
DMAP were added and the mixture was shaken for
several hours. The resin was filtered, washed with 3ꢂ5
mL DMF, 3ꢂ5 mL DMF/H2O 1/1, 3ꢂ5 mL DMF, 3ꢂ5
mL MeOH and 3ꢂ5 mL CH2Cl2 and dried in vacuo.
Variant B. To a solution of 3–6 equiv of PyAOP, 3–6
equiv of the amino-component and by choice 1–2 equiv
of HOAt in NMP, 12–15 equiv of DIEA were added
and the solution was immediately added to a pre-swol-
len (for 10 min) suspension of the polymer bound com-
pound (0.50 mmol/g, 0.50 mmol) in NMP. After
shaking for several hours, the resin is filtered off,
washed with 3ꢂ5 mL DMF, 3ꢂ5 mL DMF/H2O 1/1
(v/v), 3ꢂ5 mL DMF, 3ꢂ5 mL MeOH und 4ꢂ5 mL
CH2Cl2 and dried in vacuo.
General procedure for the N-terminal coupling of
saturated carboxylic acids on solid support (GP P9)
To a solution of 3–5 equiv carboxylic acid, 2.8–4.8 equiv
of HATU and by choice 1 equiv of HOAt in 10 mL
NMP were added 10–15 equiv of DIEA and the mixture
was shaken for some minutes. Afterwards, the pre-
activated mixture was added to the polymer-bound
compound (0.50 mmol/g, 55 mmol) in 5 mL NMP. After
shaking for several hours, the resin is filtered off,
washed with 3ꢂ5 mL DMF, 3ꢂ5 mL DMF/H2O 1/1 (v/
v), 3ꢂ5 mL DMF, 3ꢂ5 mL MeOH and 3ꢂ5 mL
CH2Cl2 and dried in vacuo.
General procedure for the N-terminal attachment of
Fmoc-amino acids GP P4
Variant A. To a solution of 3–5 equiv of the appropriate
Fmoc-amino acid, 2.8–4.8 equiv of HATU and by
choice 1 equiv of HOAt in NMP were added 10–16
equiv of DIEA and the mixture was shaken for some
minutes. This mixture was shaken then for several
hours, the resin was filtered off, washed with 3ꢂ5 mL
DMF, 3ꢂ5 mL DMF/H2O 1:1, 3ꢂ5 mL DMF, 3ꢂ5 mL
MeOH and 4ꢂ5 CH2Cl2 and dried in vacuo.
General procedure for the N-terminal coupling of
benzylchloroformiates on solid support (GP P10)
Variant B. To a solution of 4–6 equiv of the Fmoc-
amino acid, HOAt and by choice 2,6-lutidine in 15 mL
dry DMF were added 4–6 equiv of N,N0-diisopro-
pylcarbodiimide. After shaking the solution for 5 min-
utes this solution is added to a mixture of pre-swollen
polymer (0.50 mmol/g, 1.0 mmol) in 10 mL DMF. After
shaking for several hours, the resin is filtered off,
washed with 5ꢂ5 mL DMF, 5ꢂ5 mL DMF/H2O 1/1 (v/
To a solution of 3–6 equiv of HOAt, 0.1–1 equiv of
DMAP and 10–15 equiv of DIEA in 6 mL of a mixture
of dry CH2Cl2/DMF 1/1 (v/v), were added slowly 10–20
equiv of benzylchloroformiate at 0 ꢁC. After 10 min at
0 ꢁC, the mixture was added to the polymer-bound
compound (0.5 mmol/g, 0.15 mmol) in 3 mL dry
CH2Cl2 and the suspension was shaken for several

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2601
hours at room temperature. The polymer is filtered off,
washed with 3ꢂ5 mL DMF, 3ꢂ5 mL DMF/H2O 1/1,
3ꢂ5 mL DMF, 3ꢂ5 mL MeOH and 3ꢂ5 mL CH2Cl2
and dried in vacuo.
For the blocking of unreacted hydroxy functions of the
Wang linker, the resin is reacted with a solution of 1.5
mL pyridine and 1.5 mL acetic anhydride in 20 mL
CH2Cl2 for 30 min. The polymer is then filtered off,
washed with 3ꢂ10 mL CH2Cl2, 3ꢂ10 mL DMF, 3ꢂ10
mL MeOH and 2ꢂ10 mL CH2Cl2 and dried in vacuo.
General procedure for the electrophilic attachment (GP
P11):
Solid-phase bound N-9-fluorenylmethoxycarbonyl-D-tyro-
syl-N,O-dimethyl-L-tyrosine allyl ester (17). Compound
D-8 (2.00 g, 0.50 mmol/g, 1.04 mmol) was reacted with
Pd(PPh3)4 in 50 mL NMA/THF for 24 h according to
GP P1. Then the resin bound compound was pre-acti-
vated with HATU (1.54 g 4.05 mmol), HOAt (184 mg,
1.35 mmol) and DIEA (690 mL, 4.05 mmol) in 5 mL
NMP for 60 min and then coupled with 25 (1.16 g, 4.05
mmol) and DIEA (1.05 mL) in 10 mL NMP for 24 h. IR
(KBr): 3434, 3083, 3060, 3026, 2925, 2852, 1945, 1874,
1802, 1731, 1650, 1602, 1583, 1511, 1494, 1453, 1423,
1365, 1239, 1181; analysis after TFA-cleavage from a
resin sample: The starting material was completely
transformed into 17. Rf=0.40 [ethyl acetate/hexane 1/1,
1% HOAc (v/v)]; HPLC (A1): Rt=17.72 min, purity:
>95%; HRMS (FAB): calcd for C38H39N2O7
[M+H]+: 635.2757, found: 635.2893.
Variant A. To the polymer-bound compound (200 mg,
100 mmol) was added a solution of 10 equiv of Cs2CO3
in 0.5 mL H2O, 0.5 mL EtOH and 3 mL DMF and the
mixture was shaken for 5 h under exclusion of light.
After filtering off the resin and washing with DMF,
EtOH, CH2Cl2 and dry DMF, 1 mL dry DMF, variable
equiv of the halogenide and by choice 1–2 equiv of KI
(1–2 equiv) were added and the mixture was shaken for
several hours under exclusion of light. The resin is fil-
tered off then, washed with DMF (4ꢂ5 mL), DMF/H2O
1:1 (4ꢂ5 mL), DMF (4ꢂ5 mL), MeOH (2ꢂ5 mL) und
CH2Cl2 (6ꢂ5 mL) and dried in vacuo.
Variant B. After washing the polymer-bound compound
(200 mg, 100 mmol) with dry THF (2ꢂ5 mL), CH2Cl2
(2ꢂ5 mL) and DMF (2ꢂ5 mL), 1 mL dry DMF,
Cs2CO3 (10 equiv) and halogenide (10–20 equiv) were
added. The mixture was shaken for several hours under
exclusion of light and was worked up and dried
according to variant A.
Solid-phase bound N-9-Fluorenylmethoxycarbonyl-D-tyr-
osyl-N,O-dimethyl-L-tyrosyl-N-methyl-L-phenylalanine
allyl ester (18). According to GP P1 compound 17
(6.46 g, 0.50 mmol/g, 3.23 mmol) was reacted with
Pd(PPh3)4 in 50 mL NMA/THF 1/4 (v/v) for 24 h. Then
resin bound compound (2.16 g) was coupled with 26
(731 mg, 3.24 mmol), PyAOP (1.69 g, 3.24 mmol),
HOAt (147mg, 1.08 mmol) and DIEA (2.22 mL, 13.0
mmol) for 42 h in 20 mL NMP. Analysis after TFA-
cleavage from a resin sample: HPLC (A2, CH3CN/
H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min): Rt=8.48
min [11%, Fmoc-Tyr-Me(OMe)Tyr-OH], 17.28 min
(7%, 18, Epimer 1), 17.62 min (82%, 18, Epimer 2);
LC–MS (ESI-1, Grad. A): 595.2 [M+H]+, Rt=10.01
min [Fmoc-Tyr-Me(OMe)Tyr-OH]; 818.4 [M+Na]+,
Rt=20.45 min (18, Epimer 1); 818.4 [M+Na]+,
Rt=20.89 min (18, Epimer 2); 18: HRMS (FAB): calcd
for C48H49N3O8 [M+H]+ 796.3597, found: 796.3550.
General procedure for the nucleophilic attachment (GP
P12)
After washing of the polymer-bound compound (100
mg, 50 mmol) with a mixture of 8ꢂ2 mL MeOH/CH2Cl2
1/1, 1% HOAc, 3ꢂ5 mL THF, 3ꢂ5 mL CH2Cl2 and
2ꢂ5 mL NMP, a solution of 5–6 equiv of amino com-
ponent, 5–6 equiv of PyAOP and 1 equiv of HOAt in 6
mL NMP were added, followed by addition of 12–19
equiv of DIEA. The mixture was shaken for several
hours, then, the resin is filtered off, washed with 4ꢂ5
mL DMF, DMF/H2O 1/1, 4ꢂ5 mL DMF, 2ꢂ5 mL
MeOH und 5ꢂ5 mL CH2Cl2 and dried in vacuo.
Synthesis of polymer-bound compound 8
N-3-(2-(1-Z-n-Pentenyl)phenyl)-E-acryloyl-D-tyrosyl-
N,O-dimethyl-L-tyrosyl-N-methyl-L-phenylalanine-2,4-di-
oxo-1,2,3,4-tetrahydro-pyrimidine-5-ylmethylester (23/1).
According to GP P2, the polymer-bound compound 18
(260 mg, 0.50 mmol/g, 0.13 mmol) was reacted for 2ꢂ10
min with 5 mL piperidine/DMF, respectively. Then the
polymer bound compound was coupled with 30i (130
mg, 0.60 mmol), PyAOP (313 mg, 0.60 mmol), HOAt
(82 mg, 0.60 mmol), DMAP (18 mg, 0.15 mmol) and
DIEA (257 mL, 1.5 mmol) in 9 mL DMF/CH2Cl2 for 20
h according to GP P8. After cleavage of the allyl group
with Pd(PPh3)4 in 8 mL morpholine/THF mixture for 4
h according to GP P1 variant C the C-terminus was
alkylated by reaction with 32b (209 mg, 1.3 mmmol),
Cs2CO3 (424 mg, 1.3 mmol) and KI (43 mg, 0.26 mmol)
in 5 mL DMF according to GP P11 variant A for 24 h
at room temperature and for 24 h at 50 ꢁC. Then the
product is cleaved from the resin by treatment with
2ꢂ10 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 2ꢂ20
After washing Wang-resin (500 mg, 1.29 mmol/g, 0.65
mmol) with 2ꢂ5 mL THF and 2ꢂ5 mL CH2Cl2 drying
it in vacuo and swelling in 4 mL dry THF, a solution of
16 (865 mg, 1.95 mmol) and PPh3 (511 mg, 1.95 mmol)
in 4 mL THF was added. A solution of DEAD (303 mL,
1.95 mmol) was added slowly at 0 ꢁC. After 1 h at 0 ꢁC,
the mixture was shaken at room temperature for 24 h.
The resin was filtered off, washed with 2ꢂ5 mL THF,
2ꢂ5 CH2Cl2, 2ꢂ5 DMF, 2ꢂ5 mL DMF/H2O 1/1 (v/v),
2ꢂ5 DMF, 2ꢂ5 MeOH und 2ꢂ5 CH2Cl2 and dried in
vacuo; IR (KBr): 3530, 3430, 3083, 3060, 3026, 2923,
2848, 1944, 1873, 1802, 1734, 1602, 1507, 1493, 1452,
1182, 1068, 1028; loading: 0.53 mmol/g, 59% (according
to Fmoc-determination10); analysis after TFA-cleavage
from a resin sample: HPLC (A2, CH3CN/H2O/TFA, 45/
55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=11.52 min, purity
>95%, Rf=0.45 [ethyl acetate/hexane 1/1 (v/v)]). These
determined analytic results correspond to the data of 16.

2602
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
min and with 2ꢂ15 mL TFA/H2O 95/5 (v/v) for 2ꢂ30
min according to GP P5 variant B. Purification by
HPLC afforded 23/1 (13 mg, 13%, nine steps) as a white
solid; Rf=0.64 [ethyl acetate/EtOH 5/1 (v/v)]; HPLC
(A2, CH3CN/H2O/TFA, 20/80/0.1–60/40/0.1 in 60 min,
N-3-(2-(1-Z-n-Pentenyl)phenyl)-E-acryloyl-D-tyrosyl-
N,O-dimethyl-L-tyrosyl-N-methyl-L-phenylalanine (21/
1). According to GP P2, the polymer-bound compound
18 (260 mg, 0.50 mmol/g, 0.13 mmol) was reacted twice
for 20 min with 5 mL piperidine/DMF. Then the poly-
mer bound compound was coupled with 30i (112 mg,
520 mmol), PyAOP (313 mg, 600 mmol), HOAt (82 mg,
600 mmol), DMAP (18 mg, 150 mmol) and DIEA (205
mL, 1.2 mmol) in 9 mL DMF/CH2Cl2 for 20 h accord-
ing to GP P8. Cleavage of the allyl group with
Pd(PPh3)4 in 8 mL morpholine/THF mixture for 4 h
according to GP P1 furnishes the polymer bound com-
pound.
50 ꢁC): Rt=50.41 min, purity: >95%; mp: >250
20
D
(decomp); ½aŠ ꢀ50.2 (c 0.64, MeOH); 1H NMR
(400 MHz, CD3OD): d 0.79 (t, 3J=7.5 Hz, 3H), 1.34 (m,
2H), 1.93 (td, J=7.34 Hz, J=1.5 Hz, 2H), 2.14–2.23
3
3
(dd, 2J=15.0 Hz, 3J=6.0 Hz, 1H), 2.43–2.48 (dd,
3
2J=15.0 Hz, J=7.5 Hz, 1H), 2.56 [2.57] (s, 3H), 2.67–
2.70 (m, 2H), 2.87 [2.88] (s, 3H), 2.96–3.04 (m, 2H), 3.70
[3.73] (s, 3H), 4.61–4.65 [4.72–4.74] (m, 1H), 4.87–4.91
(m, 2H), 4.96–5.02 [5.07–5.11] (m, 1H), 5.36–5.39 (m,
1H), 5.59–5.63 (m, 1H), 5.73–5.78 (m, 1H), 6.48 (d,
N-3-(2-(1-Z-n-pentenyl)phenyl)-E-acryloyl-D-tyrosyl-
N,O-dimethyl-L-tyrosyl-N-methyl-L-phenylalanine (19/1).
The product is cleaved from the resin by treatment with
2ꢂ10 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 10 and
20 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 30
and 180 min according to GP P5 variant B. Purification
by HPLC afforded 21/1 (11 mg, 12%, eight steps) as a
white solid; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–
90/10/0.1 in 30 min, 50 ꢁC): Rt=11.96 min, purity:
3
3Jcis=10.6 Hz, 1H), 6.50 (d, Jtrans=15.8 Hz, 1H),
6.64–6.70 (m, 3H), 6.80–6.89 (m, 3H), 6.91–6.94 (m,
1H), 7.00–7.02 (m, 1H), 7.12–7.19 (m, 4H), 7.21–7.37
3
(m, 5H), 7.53–7.58 (m, 1H), 7.64 (d, Jtrans=15.8 Hz,
1H); LC–MS (ESI): 854.4 [M–H]ꢀ, 856.1 [M+H]+,
878.3 [M+Na]+; Rt=11.80 min; MS (MALDI-TOF):
878.93 [M+Na]+, 894.88 [M+K]+; HRMS (FAB):
calcd for C40H54N5O9 [M+H]+: 856.3922, found:
856.3941.
20
D
>95%; mp: 93–94 ꢁC; ½aŠ ꢀ63.6 (c 0.11, MeOH); H
1
NMR (400 MHz, CD3OD): 0.81–0.87[0.92–1.0] (t,
3J=7.4 Hz, 3H), 1.33–1.42 (m, 2H), 1.94–2.0 [2.03–2.09]
(m, 2H), 2.13–2.43 (m, 1H), 2.32 [2.41] (s, 3H), 2.43–
2.70 (m, 1H), 2.56 [2.57] (s, 3H), 2.67–2.70 (m, 2H),
2.96–3.04 (m, 2H), 3.70 [3.73] (s, 3H), 4.67–4.73 (m,
1H), 5.45–5.51 (m, 1H), 5.68–5.74 [5.78–5.86] (m, 1H),
5.92–6.08 (m, 1H), 6.40 (d, Jcis=11.3 Hz, 1H), 6.54 (d,
3Jtrans=15.6 Hz, 1H). 6.61–6.77 (m, 3H), 6.80–6.89 (m,
3H), 6.91–6.94 (m, 1H), 7.00–7.02 (m, 1H), 7.12–7.19
N-(3-(2-(1-Z-n-Pentenyl)phenyl)-E-acryloyl-D-tyrosyl-
N,O-dimethyl-L -tyrosyl-N-methyl-L -phenylalanine-2-
methyl-thiazole-5-yl methyl ester (23/2). According to
GP P2, the polymer-bound compound 18 (260 mg, 0.50
mmol/g, 0.13 mmol) was reacted for 2ꢂ20 min with 5
mL piperidine/DMF, respectively. Then the polymer-
bound compound was coupled with 30i (97mg, 450
mmol), PyAOP (235 mg, 450 mmol), HOAt (61 mg, 450
mmol), DMAP (18 mg, 150 mmol) and DIEA (257 mL,
1.5 mmol) in 9 mL DMF/CH2Cl2 for 17h according to
GP P8. After cleavage of the allyl group with Pd(PPh3)4
in 8 mL morpholine/THF mixture for 4 h according to
GP P1 variant C, the C-terminus was alkylated by
reaction with 32a (209 mg, 1.3 mmol), Cs2CO3 (977 mg,
3 mmol) and KI (25 mg, 150 mmol) in 5 mL DMF
according to GP P11 variant B for 24 h at 50 ꢁC. Then
the product is cleaved from the resin by treatment with
2ꢂ10 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ2
h according to GP P5 variant B. Purification by HPLC
afforded 23/2 (15 mg, 14%, nine steps) as a brownish
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–90/10/
0.1 in 30 min, 50 ꢁC): Rt=23.17min, purity: >95%;
3
3
(m, 7H), 7.21–7.37 (m, 2H), 7.72 (d, Jtrans=16.0 Hz,
1H); LC–MS (ESI-2): 730.1 [MꢀH]ꢀ, Rt=3.02 min; MS
(MALDI-TOF): 754.9 [M+Na]+; HRMS (FAB, m/z):
calcd for C44H50N3O7 [M+H]+ 732.3649, found
732.3664.
N-3-[2-(n-Pentyl)phenyl]propionyl-D-tyrosyl-N,O-dimethyl-
L-tyrosyl-N-methyl-L-phenylalanine (21). Compound 19/1
(230 mg, 0.50 mmol/g, 114 mmol) is treated with 10 mL
TFA/triisopropylsilane/H2O 95/5/5 (v/v) for 18 h. After
purification by HPLC, the residue was dissolved 5 mL
methanol, Pd (50 mg, 10% on charcoal) was added and
the mixture was stirred for 6 h under hydrogen atmo-
sphere. After filtration over Celite, washing with
methanol (2ꢂ5 mL) and evaporation of the solvents
furnished 21 (12 mg, 14%, nine steps) as a white solid;
HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in
20
D
mp: 87–89 ꢁC; ½aŠ ꢀ72.1 (c 0.59, MeOH); H NMR
1
3
(400 MHz, CD3OD): d 0.79 [0.80] (t, J=7.2 Hz, 3H),
1.35 (q, J=7.2 Hz, 2H), 1.92–1.96 (m, 2H), 2.35–2.40
30 min, 50 ꢁC): Rt=14.72 min, purity: >95%; mp: 119–
3
20
D
(m, 1H), 2.40 (s, 3H), 2.56–2.58 (m, 1H), 2.56 [2.58] (s,
3H), 2.64–2.67 (m, 1H), 2.66 (s, 1H), 2.77–2.79 (m, 2H),
3.70 [3.73] (s, 3H), 4.76–4.81 (m, 1H), 4.87–4.91 [4.99–
5.10] (m, 1H), 5.17(s, 2H) 5.40–5.44 (m, 1H), 5.75–5.83
122 ꢁC; ½aŠ
ꢀ27.8 (c 0.32, MeOH); 1H NMR
(400 MHz, CD3OD): d 0.87–0.91 (m, 3H), 1.28–1.39 (m,
4H), 1.53–1.56 (m, 2H), 2.11–2.21 (m, 3H), 2.36–2.46
(m, 1H), 2.58–2.70 (m, 4H), 2.80–2.94 (m, 3H), 2.82 (s,
3H), 3.43–3.48 (m, 2H), 3.68 [3.72] (s, 3H), 4.61–4.72
(m, 1H), 5.04–5.16 [5.36–5.44] (m, 1H), 5.63–5.74 (m,
1H), 6.58–6.67 (m, 4H), 6.75–6.80 (m, 4H), 7.06–7.12
(m, 5H), 7.22–7.27 (m, 4H); LC–MS (ESI-1, Grad. A):
736.2 [M+H]+, 758.5 [M+Na]+; Rt=15.80 min; MS
(MALDI-TOF): 758.7 [M+Na]+; HRMS (FAB):
calcd for C44H54N3O7 [M+H]+: 736.3962, found:
736.3937.
3
(m, 1H), 6.49–6.55 (m, 1H), 6.53 (d, Jtrans=15.8 Hz,
2H) 6.63–6.75 (m, 3H), 6.82–6.89 (m, 4H), 6.99–7.00 (d,
3J=8.4 Hz), 7.12–7.18 (m, 5H), 7.25–7.32 (m, 4H), 7.55
3
3
[7.60] (d, J=7.6 Hz, 1H), 7.66 [7.71] (d, Jtrans=15.8
Hz, 1H); LC–MS (ESI-2): 843.10 [M+H]+, Rt=3.25
min; MS (MALDI-TOF): 865.7[M+Na] +; HRMS
(FAB): calcd for C49H55N4O7S [M+H]+: 843.3791,
found: 843.3803.

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2603
N-3-(3-(1-E-Pentenyl)phenyl)-E-acryloyl-D-tyrosyl-N,O-
dimethyl - L - tyrosyl - N - methyl - L - phenylalanine (20/2).
According to GP P2, the polymer-bound compound 18
(240 mg, 0.50 mmol/g, 0.12 mmol) was reacted twice for 20
min with 5 mL piperidine/DMF. The polymer-bound
compound was then coupled with 30g (97mg, 450 mmol),
PyAOP (235 mg, 450 mmol), HOAt (61 mg, 450 mmol),
DMAP (18 mg, 150 mmol) and DIEA (257 mmol, 1.5
mmol) in 9 mL DMF/CH2Cl2 for 66 h according to GP P8.
Cleavage of the allyl group with Pd(PPh3)4 in 8 mL mor-
pholine/THF mixture for 4 h furnished polymer-bound.
HRMS (FAB): calcd for C44H52N3O7 [M+H]+:
734.3805, found: 734.3831.
N-3-[4-(n-Pentyl)phenyl)]propionyl-D-tyrosyl-N,O-dimethyl-
L-tyrosyl-N-methyl-L-phenylalanine (20/4). According to
GP P2, the polymer-bound compound 18 (260 mg, 0.50
mmol/g, 130 mmol) was reacted for 2ꢂ20 min with 5 mL
piperidine/DMF, respectively. Then the polymer-bound
compound was coupled with 29b (99 mg, 450 mmol),
HATU (171 mg, 450 mmol), HOAt (20 mg, 150 mmol)
und DIEA (257 mL, 1.5 mmol) in 8 mL NMP for 66 h
according to GP P9. After cleavage of the allyl group
with Pd(PPh3)4 in 8 mL morpholine/THF mixture for 4
h according to GP P1, the product is cleaved from the
resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47 /
47/6 (v/v/v) for 10 and 30 min and with 2ꢂ5 mL TFA/
H2O 95/5 (v/v) for 60 and 120 min according to GP P5
variant B. Purification by HPLC afforded 20/4 (16 mg,
17%, eight steps) as a white solid; HPLC (A2, CH3CN/
N-3-(3-(1-E-Pentenyl)phenyl)-E-acryloyl-D-tyrosyl-N,O-
dimethyl - L - tyrosyl - N - methyl - L - phenylalanine (19/2).
According to GP P1, the product is cleaved from the
resin by treatment with 2ꢂ10 mL TFA/CH2Cl2/H2O 47 /
47/6 (v/v/v) for 10 and 30 min and with 2ꢂ5 mL TFA/
H2O 95/5 (v/v) for 60 and 120 min according to GP P5
variant B. Purification by HPLC afforded 20/2 (12 mg,
13%, eight steps) as a white solid; HPLC (A2, HPLC (A2,
H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC)
20
D
CH3CN/H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC)
Rt=21.34 min, purity: >95%; mp: 101 ꢁC; ½aŠ ꢀ71.7
20
D
Rt=26.21 min, purity: >95%; mp: 127–129 ꢁC, ½aŠ
(c 0.54, MeOH); H NMR (400 MHz, CD3OD): d 0.85
(t, J=6.9 Hz, 3H), 1.28–1.35 (m, 4H), 1.53–1.57(m,
1
1
3
ꢀ64.1 (c 0.32, MeOH); H NMR (400 MHz, CDCl3): d
0.93 [0.94] (t, 3J=8.0 Hz, 3H), 1.26–1.34 (m, 2H), 1.43–1.50
(m, 2H), 2.25–2.57(m, 2H), 2.31 [2.43] s, 3H) 2.63–2.92 (m,
2H), 2.78 [2.87] (s, 3H), 3.04–3.14 (m, 1H), 3.22–3.34 (m,
1H), 3.73 [3.74] (s, 3H), 4.79–4.83 (m, 1H), 5.03–5.15 (m,
1H), 5.37–5.41 (m, 1H), 5.77–5.84 (m, 1H), 6.28–6.42 (m,
3H), 6.67–6.90 (m, 8H), 7.09–7.21 (m, 2H), 7.18–7.23 (m,
2H), 1.80–2.16 (m, 2H), 2.27–2.58 (m, 7H), 2.67–2.73
(m, 2H), 2.82–2.91 (m, 2H), 2.93 [2.95] (s, 3H), 3.00–
3.10 [3.13–3.25] (m, 1H), 3.33–3.41 (m, 1H), 3.68 [3.71]
(s, 3H), 4.54–4.62 (m, 1H), 4.84–4.94 (m, 1H), 5.02–5.11
3
(m, 1H), 6.52 (d, 3J=8.4 Hz, 2H), 6.60 [6.66] (d, J=8.4
Hz, 2H), 6.71–6.83 (m, 4H), 7.18 (d, 3J=7.8 Hz, 2H), 7.04
(d, 3J=8.0 Hz, 2H), 7.11–7.22 (m, 3H), 7.24–7.29 (m, 2H);
LC–MS (ESI-2): 734.16 [MꢀH]ꢀ; Rt=3.15 min; MS
(MALDI-TOF): 758.7 [M+H]+; HRMS (FAB): calcd
for C44H54N3O7 [M+H]+: 736.3962, found: 736.3942.
3
4H), 7.27–7.35 (m, 3H), 7.43 [7.56] (d, Jtrans=15.6 Hz,
1H); LC–MS (ESI 2): 730.07 [M-H]ꢀ, Rt=3.10 min, MS
(MALDI-TOF): 754.8 [M+Na]+; HRMS (FAB): calcd
for C44H50N3O7 [M+H]+: 732.3649, found 732.3627.
N-3-[4-(n-Pentyl)phenyl)]-E-acryloyl-D-tyrosyl-N,O-di-
methyl - L - tyrosyl - N - methyl - L - phenylalanine (20/3).
According to GP P2, the polymer-bound compound 18
(240 mg, 0.50 mmol/g, 0.12 mmol) was reacted for 2ꢂ20
min with 5 mL piperidine/DMF, respectively. Then the
polymer-bound compound was coupled with 30a (105
mg, 480 mmol), PyAOP (313 mg, 600 mmol), DMAP (18
mg, 150 mmol), HOAt (82 mg, 600 mg) und DIEA (257
mL, 1.5 mmol) in 9 mL DMF/CH2Cl2 for 66 h accord-
ing to GP P8. After cleavage of the allyl group with
Pd(PPh3)4 in 8 mL morpholine/THF mixture for 4 h
according to GP P1, the product is cleaved from the
resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47 /
47/6 (v/v/v) for 10 and 30 min and with 2ꢂ5 mL TFA/
H2O 95/5 (v/v) for 60 and 120 min according to GP P5
variant B. Purification by HPLC afforded 20/3 (12 mg,
13%, eight steps) as a white solid; HPLC (A2, CH3CN/
N-3-(2-Benzyloxyphenyl)-E-acryloyl-D-tyrosyl-N,O-dime-
thyl-L-tyrosyl-N-methyl-L-phenylalanine (20/5). Accord-
ing to GP P2, the polymer-bound compound 18 (260
mg, 0.50 mmol/g, 130 mmol) was reacted twice for 20
min with 5 mL piperidine/DMF, respectively. Then the
polymer-bound compound was coupled with 30c (114
mg, 450 mmol), PyAOP (235 mg, 450 mmol), DMAP
(18 mg, 150 mmol), HOAt (61 mg, 450 mmol) and DIEA
(257 mL, 1.5 mmol) for 66 h according to GP P8. After
cleavage of the allyl group with Pd(PPh3)4 in 8 mL
morpholine/THF mixture for 4 h according to GP P1,
the product is cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 10 and
30 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 60
and 120 min according to GP P5 variant B. Purification
by HPLC afforded 20/5 (10 mg, 10%, eight steps) as a
white solid; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–
H2O/TFA, 40/55/0.1–90/10/0.1 in 30 min, 50 ꢁC):
90/10/0.1 in 30 min, 50 ꢁC): Rt=10.03 min, purity:
20
20
D
Rt=14.33 min, purity: 95%; mp: 134 ꢁC; ½aŠ ꢀ49.6 (c
>95%; mp: 120 ꢁC; ½aŠ ꢀ88.0 (c 0.1, MeOH); 1H
D
0.22, MeOH); 1H NMR (400 MHz, CDCl3): d 0.85–0.88
(m, 3H), 1.26–1.34 (m, 6H), 1.55–1.61 (m, 2H), 1.80–
2.16 (m, 2H), 2.27–2.55 (m, 3H), 2.32 [2.43] (s, 3H)
2.63–2.92 (m, 1H), 3.05–3.14 (m, 1H), 3.22–3.33 (m,
1H), 3.73 [3.75] (s, 3H), 4.79–4.82 (m, 1H), 5.05–5.15
(m, 1H), 5.38–5.41 (m, 1H), 5.77–5.84 (m, 1H), 6.28
NMR (400 MHz, CD3OD): d 2.23–2.70 (m, 4H), 2.32
[2.58] (s, 3H), 2.79–3.00 (m, 4H), 3.21–3.28 (m, 1H),
3.70 [3.73] (s, 3H), 4.72–4.80 [4.85–4.96] (m, 1H), 5.08
(m, 2H), 5.05–5.19 (m, 1H), 5.50 (m, 1H), 5.94–5.98 (m,
3
1H), 6.62–6.84 (m, 7H), 6.88 (d, J=8.6 Hz, 1H), 6.92–
7.02 (m, 4H), 7.08–7.18 (m, 4H), 7.19–7.36 (m, 5H),
3
3
[6.35] (d, J=15.6 Hz, 1H), 6.60–6.91 (m, 8H), 7.09–
3
7.40–7.46 (m, 1H), 7.54–7.61 (d, Jtrans=15.4 Hz, 1H);
7.21 (m, 5H), 7.29–7.45 (m, 4H), 7.56 (d, J=15.6 Hz,
1H); LC–MS (ESI-2): 732.14 [MꢀH]ꢀ, Rt=3.18 min;
LC–MS (ESI-2): 768.1 [MꢀH]ꢀ; Rt=2.86 min; MS
(MALDI-TOF): 792.9 [M+Na]+; HRMS (FAB): calcd
for C46H48N3O8 [M+H]+: 770.3441, found: 770.3428.
MS (MALDI-TOF): 757.1 [M+Na]+, 773.1 [M+K]+;

2604
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
1
N-9-Fluorenylmethoxycarbonyl-D-tyrosyl-N,O-dimethyl-
L-tyrosyl-N-methyl-L-phenylalanine (20/6). According to
GP P1 variant B, polymer-bound compound 18 (230
mg, 0.50 mmol/g, 115 mmol) was reacted with Pd(PPh3)4
in 10 mL NMA/THF 1/4 (v/v) for 16 h. Then the pro-
duct was cleaved from the resin for 19 h according to
GP P5 variant A with 10 mL TFA/triisopropylsilane/
H2O 95/5/5 (v/v). Purification by HPLC afforded 20/6
(10 mg, 11%, six steps) as a white solid; HPLC (A2,
ꢀ10.0 (c 0.29, MeOH); H NMR (400 MHz, CDCl3): d
2.28–2.63 (m, 5H), 2.74–2.99 (m, 6H), 3.00–3.02 (m,
1H), 3.66 [3.67] (s, 3H), 3.92–3.95 (m, 1H), 4.50–5.55
(m, 1H), 5.05–5.11 (m, 2H), 5.44–5.46 (m, 1H), 6.61–
6.80 (m, 10H), 6.92–7.11 (m, 3H), 7.32–7.39 (m, 2H),
8.09–8.18 (m, 2H); LC–MS (ESI-2): 735.00 [M+Na]+;
Rt=2.58 min; MS (MALDI-TOF): 735.7 [M+Na]+.
In a third fraction was isolated N-4-nitro-benzyloxy-
carbonyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-N-methyl-L-
phenylalanyl-L-methionine sulfoxide (23/5) (5.2 mg, 5%)
as a yellowish solid; LC–MS (ESI-1, Grad. B): 882.3
[M+Na]+; Rt=18.27min, purity: >95%.
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
20
50 ꢁC): Rt=9.87min, purity: >95%; mp: 223 ꢁC; ½aŠ
D
1
ꢀ18.9 (c 0.26, MeOH); H NMR (400 MHz, CDCl3): d
2.18–2.70 (m, 4H), 2.31 [2.50] (s, 3H) 2.81 [2.89] (s,
3H), 3.02–3.09 (m, 1H), 3.21–3.37(m, 1H), 3.75 [3.76]
(s, 3H), 4.15–4.18 (m, 1H), 4.25–4.55 (m, 2H), 4.63–
4.69 (m, 1H), 4.95–4.97[5.17–5.21] (m, 1H), 5.48–5.50
[5.54–5.62] (m, 1H), 6.67(d, 3J=8.4 Hz, 2H), 6.72–6.81
(m, 3H), 6.84–6.92 (m, 3H), 7.12–7.14 (m, 1H), 7.18–
N-4-Nitro-benzyloxycarbonyl-D-tyrosyl-N,O-dimethyl-L-
tyrosyl-N-methyl-L-phenylalanyl-piperidine-4-carboxylic
amide (23/6). According to GP P2, the polymer-bound
compound 18 (260 mg, 0.50 mmol/g, 0.13 mmol) was
reacted for 2ꢂ20 min with 5 mL piperidine/DMF,
respectively. Then the polymer-bound compound was
coupled with 31b (129 mg, 600 mmol), DMAP (18 mg,
150 mmol), HOAt (82 mg, 600 mmol) und DIEA (205
mL, 1.2 mmol) in 9 mL DMF/CH2Cl2 for 17h accord-
ing to GP P10. After cleavage of the allyl group with
Pd(PPh3)4 in 8 mL morpholine/THF mixture for 4 h
according to GP P1 variant C the compound was reac-
ted with 4-piperidine-amide (77 mg, 600 mmol), PyAOP
(313 mg, 600 mmol), HOAt (20 mg, 150 mmol) and
DIEA (257 mL, 1.5 mmol) in 5 mL NMP for 16 h
according to GP P12. Then the product was cleaved
from the resin by treatment with 2ꢂ10 mL TFA/
CH2Cl2/H2O 47/47/6 (v/v/v) for 10 and 30 min and with
2ꢂ5 mL TFA/H2O 95/5 (v/v) for 60 and 120 min
according to GP P5 variant B. Purification by HPLC
afforded 23/6 (11 mg, 13%, nine steps) as yellow solid as
an unseparable 3/2 compound mixture; HPLC (A2,
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
50 ꢁC): Rt=6.34 min (isomer 1); Rt=6.63 min (isomer
3
7.22 (m, 2H), 7.23–7.31 (m, 4H), 7.39 (t, J=6.8 Hz,
3
2H), 7.50–7.54 (m, 2H), 7.75 (d, J=7.0 Hz, 2H); LC–
MS (ESI-1, Grad. A): 756.1 [M+H]+, 778.2
[M+Na]+, 754.1 [MꢀH]ꢀ, Rt=13.27min; MS
(MALDI-TOF): 778.8 [M+Na]+, 794.8 [M+K]+.
N-4-Nitro-benzyloxycarbonyl-D-tyrosyl-N,O-dimethyl-L-
tyrosyl-N-methyl-L -phenylalanyl-L -methionine (23/4).
According to GP P2, the polymer-bound compound 18
(260 mg, 0.50 mmol/g, 0.13 mmol) was reacted for 2ꢂ20
min with 5 mL piperidine/DMF, respectively. Then the
polymer bound compound was coupled with 31b (129
mg, 600 mmol), DMAP (18 mg, 150 mmol), HOAt (82
mg, 600 mmol) and DIEA (205 mL, 1.2 mmol) in 9 mL
DMF/CH2Cl2 for 17h according to GP P10. After
cleavage of the allyl group with Pd(PPh3)4 in 8 mL
morpholine/THF mixture for 4 h according to GP P1
variant C the compound was reacted with tbutyl-
methionine hydrochloride (145 mg, 600 mmol), PyAOP
(313 mg, 600 mmol), HOAt (20 mg, 150 mmol) und
DIEA (257 mL, 1.5 mmol) for 16 h in 5 mL NMP
according to GP P12. Then the product is cleaved
from the resin by treatment with 2ꢂ10 mL TFA/
CH2Cl2/H2O 47/47/6 (v/v/v) for 10 and 30 min and
with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 60 and 120 min
according to GP P5 variant B. Purification by HPLC
afforded 23/4 (11 mg, 10%, nine steps) as a yellowish
solid; HPLC (A2, HPLC (A2, CH3CN/H2O/TFA, 45/
ꢁ
1
2); mp: 105 C; H NMR (400 MHz, CDCl3): d 1.71–
1.78 (m, 2H), 2.17–2.24 (m, 2H), 2.36–2.76 (m, 8H),
2.80–3.14 (m, 5H), 3.37–3.39 (m, 2H), 3.62–3.69 (m,
2H), 3.75 [3.76] (s, 3H), 4.36–4.46 [4.61–4.68] [4.74–4.83]
(m, 1H), 4.98–5.28 (m, 2H), 5.42–5.68 (m, 2H), 6.68–
6.82 (m, 5H), 6.88–6.95 (m, 3H), 7.12–7.24 (m, 5H),
7.33–7.46 (m, 2H), 8.18 (d, J=8.8 Hz, 2H); LC–MS
(ESI-2): 845.10 [M+Na]+, Rt=2.36 min; MS
(MALDI-TOF): 845.8 [M+Na]+.
3
55/0.1-90/10/0.1 in 30 min, 50 ꢁC) Rt=6.99 min, pur-
20
ity: >95%; mp: 122 ꢁC; ½aŠ ꢀ23.4 (c 0.30, MeOH);
D
1H NMR (400 MHz, CDCl3): d 1.82–2.18 (m, 2H),
2.19 [2.24] (s, 3H), 2.31–2.72 (m, 6H), 2.80–3.03 (m,
6H), 3.30–3.32 (m, 2H), 3.70 [3.72] (s, 3H), 4.36-4.46
[4.61–4.68] (m, 1H), 4.74–4.83 (m, 1H) 4.98–5.28 (m,
2H), 5.42–5.68 (m, 2H), 6.68–6.82 (m, 5H), 6.88–6.95
(m, 3H), 7.12–7.24 (m, 5H), 7.33–7.46 (m, 2H), 8.18
N-Benzyloxycarbonyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanine (20/8). According to GP P2,
the polymer-bound compound 18 (260 mg, 0.50 mmol/
g, 130 mmol) was reacted for 2ꢂ20 min with 5 mL
piperidine/DMF, respectively. Then the polymer-bound
compound was coupled with 31a (85 mL, 600 mmol),
DMAP (18 mg, 150 mmol), HOAt (82 mg, 600 mmol)
and DIEA (205 mL, 1.2 mmol) for 20 h in 9 mL DMF/
CH2Cl2 according to GP P10. After cleavage of the allyl
group with Pd(PPh3)4 in 8 mL morpholine/THF mix-
ture for 4 h according to GP P1, the product is cleaved
from the resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/
H2O 47/47/6 (v/v/v) for 10 and 20 min and with 2ꢂ5
mL TFA/H2O 95/5 (v/v) for 30 and 180 min according
to GP P5 variant B. Purification by HPLC afforded 20/
3
(d, J=8.8 Hz, 2H); LC–MS (ESI-1, Grad. B) 866.2
[M+Na]+; Rt=23.30 min; MS (MALDI-TOF): 867.1
[M+Na]+.
In a second fraction, N-4-nitro-benzyloxycarbonyl-D-
tyrosyl-N,O-dimethyl-L-tyrosyl-N-methyl-L-phenylalanine
(20/7) was isolated (4 mg, 5%) as a white solid; HPLC
(A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
20
50 ꢁC) Rt=6.66 min, purity: >95%; mp: 115 ꢁC; ½aŠ
D

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2605
8 (11 mg, 12%, eight steps) as a white solid; HPLC (A2,
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
(400 MHz, CDCl3): d 2.29–2.62 (m, 5H), 2.34 [2.59] (s,
3H), 2.56–2.83 (m, 3H), 2.79 [2.87] (s, 3H), 3.04–3.12
(m, 1H), 3.27–3.35 (m, 1H), 3.71 [3.73] (s, 3H), 4.60 (d,
3J=5.8 Hz, 2H), 4.82–5.08 (m, 2H), 5.20–5.33 (m, 2H),
20
D
50 ꢁC): Rt=6.87min; mp: 112 ꢁC; ½aŠ ꢀ127.1 (c 0.24,
1
MeOH); H NMR (400 MHz, CDCl3): d 2.17–2.45 (m,
3
2H), 2.27[2.42] (s, 3H) 2.46–2.91 (m, 2H), 2.76 [2.86] (s,
3H), 2.98–3.09 (m, 1H), 3.21–3.37(m, 1H), 3.72 [3.75]
(s, 3H), 4.40–4.42 [4.59–4.63] (m, 1H), 4.86–5.05 (m,
2H), 5.11–5.14 (m, 1H), 5.41–5.44 [5.62–5.66] (m, 1H),
(m, 8H), 7.12–7.22 (m, 4H), 7.28–7.32 (m, 6H); LC–MS
(ESI-2): 666.07[M ꢀH]ꢀ, Rt=2.60 min, purity: >95%;
MS (MALDI-TOF): 691.0 [M+Na]+; HRMS (FAB):
calcd for C38H42N3O8 [M+H]+: 668.2972, found:
668.2987.
5.50 (t, J=7.5 Hz, 1H), 5.73–5.91 (m, 1H), 6.13–6.22
(m, 1H), 6.58–6.64 (m, 3H), 6.72–6.75 (m, 2H), 6.79–
6.83 (m, 2H), 6.93–6.95 (m, 1H), 7.10–7.19 (m, 6H),
7.21–7.27 (m, 4H); MS (MALDI-TOF): 728.7
[M+Na]+, 744.7 [M+K]+; LC–MS (ESI-1, Grad. A):
728.4 [M+Na]+, Rt=13.41 min; FAB (3-NBA): 706.4
[M+H]+, 728.3 [M+Na]+; HRMS (FAB) calcd for
C42H48N3O7 [M+H]+: 706.3492, found: 706.3398.
N-3-Phenylpropionyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanine (20/10). Resin-bound com-
pound 22/10 (132 mg, 0.50 mmol/g, 66 mmol) was reac-
ted with Pd(PPh3)4 in 10 mL morpholine/THF mixture
for 5 h according to GP P1 variant C furnishing poly-
mer-bound N-3-phenylpropionyl-D-tyrosyl-N,O-dimethyl-
L-tyrosyl-N-methyl-L-phenylalanine (19/10). Cleavage
from the solid support by 10 mL TFA/triisopropyl-
silane/H2O 90/5/5 (v/v/v) for 12 h and then with 10 mL
TFA/H2O 95/5 (v/v) for 12 h, followed by purification
by HPLC affording 20/10 (23 mg, 53%) as a light
brown solid; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–
N-3-Phenyl-E-acryloyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanine (20/9). According to GP P2,
the polymer-bound compound 18 (260 mg, 0.50 mmol/
g, 130 mmol) was reacted twice for 20 min with 5 mL
piperidine/DMF, respectively. Then the polymer-bound
compound was coupled with 30f (77 mg, 0.52 mmol),
PyAOP (313 mg, 600 mmol), DMAP (18 mg, 150 mmol),
HOAt (82 mg, 600 mmol) and DIEA (205 mL, 1.2 mmol)
for 20 h in 9 mL DMF/CH2Cl2 according to GP P8.
After cleavage of the allyl group with Pd(PPh3)4 in 8 mL
morpholine/THF mixture for 4 h according to GP P1,
the product is cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 10 and
20 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 30
and 180 min according to GP P5 variant B. Purification
by HPLC afforded 20/9 (11 mg, 13%, eight steps) as a
white solid; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–
100/0/0.1 in 30 min): Rt=8.73 min, purity: 95%; mp:
20
D
126 ꢁC; ½aŠ
ꢀ105.1 (c 1.0, MeOH); 1H NMR
(400 MHz, CDCl3): d 2.16–2.53 (m, 4H), 2.24 [2.32] (s,
3H), 2.56–2.83 (m, 4H), 2.65 [2.76] (s, 3H), 2.93–3.03
(m, 1H), 3.12–3.29 (m, 1H), 3.63 (s, 3H), 4.17–4.28 (m,
1H), 4.63–4.72 [4.73–4.84] (m, 1H), 4.97 [5.30] (t,
3J=7.3 Hz, 1H), 6.48–6.82 (m, 8H), 6.91–7.26 (m, 10H);
13C NMR (100.6 MHz, CDCl3): d 29.67[29.94], 30.31,
31.28, 31.39, 33.94, 34.92, 36.81, 37.80, 50.10 [50.54],
53.78, 55.23 [55.26], 55.92 [60.88], 113.78 [113.85],
115.57 [15.67], 126.33 [126.38], 126.70 [127.12], 128.18
[128.26], 128.33 [128.54], 128.58 [128.64], 128.81
[128.98], 129.00 [130.06], 130.16 [130.40], 130.81, 136.78,
137.26, 140.06 [140.20], 155.41 [155.59], 158.31 [158.43],
168.99, 169.84, 171.71, 172.50; LC–MS (ESI-1, Grad.
A): 664.3 [MꢀH]ꢀ, Rt=8.18 min; MS (MALDI-TOF):
687.70 [M+Na]+, 703.33 [M+K]+; HRMS (FAB)
calcd for C39H44N3O7 [M+H]+: 666.3179, found:
666.3164.
90/10/0.1 in 30 min, 50 ꢁC): Rt=6.38 min, purity:
20
>95%; mp: 114–116 ꢁC; ½aŠ ꢀ10.0 (c 0.12, MeOH);
D
1H NMR (400 MHz, CDCl3): d 2.29–2.58 (m, 2H), 2.29
[2.42] (s, 3H) 2.68–2.91 (m, 2H), 2.79 [2.88] (s, 3H),
3.04–3.14 (m, 1H), 3.22–3.34 (m, 1H), 3.73 [3.75] (s,
3H), 4.13–4.15 (m, 1H), 4.81–4.84 [5.03–5.08] (m, 1H),
3
5.12–5.16 [5.37–5.41] (m, 1H), 6.31 [6.39] (d, J=15.6
Hz, 1H), 6.66–6.92 (m, 8H), 7.09–7.11 (m, 5H), 7.19–
7.21 (m, 3H), 7.29–7.35 (m, 1H), 7.37–7.47 (m, 1H), 7.58
3
(d, J=15.6 Hz, 1H); LC–MS (ESI-2): 662.1 [MꢀH]ꢀ,
+
Rt=2.55 min; MS (MALDI-TOF): 686.7[M+Na]
;
HRMS (FAB): calcd for C39H42N3O7 [M+H]+:
664.3023, found: 664.2990.
N-3-Phenylpropionyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanineallyl ester (23/10). According
to GP P2, the polymer-bound compound 18 (960 mg,
0.50 mmol/g, 0.48 mmol) was reacted twice for 20 min
with 5 mL piperidine/DMF. The polymer-bound com-
pound was then coupled with 29c (264 mg, 1.75 mmol),
HATU (627mg, 1.65 mmol), HOAt (75 mg, 0.55 mmol)
and DIEA (941 mL, 5.5 mmol) in 15 mL NMP accord-
ing to GP P9 for 4.5 h furnishing the polymer bound
N-3-Phenylpropionyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanyl-L-methionine methyl ester (23/
7). Compound 19/10 (126 mg, 0.50 mmol/g, 63 mmol)
was reacted twice with 33c (66 mg, 380 mmol), PyAOP
(197mg, 378 mmol), HOAt (8.6 mg, 63 mmol) and DIEA
(108 mL, 630 mmol) in 3 mL NMP for 8 h according to
GP P12, followed by cleavage from the resin according
to GP P5 variant B for 12 h with 10 mL TFA/triiso-
propylsilane/H2O 90/5/5 (v/v/v). Purification by HPLC
afforded 23/7 (4 mg, 8%, nine steps) as light brown
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–65/35/
intermediate
N-3-phenylpropionyl-d-tyrosyl-N,O-di-
methyl-l-tyrosyl-N-methyl-l-phenylalanine allyl ester
(22/10). A sample of the intermediate (168 mg, 0.52
mmol/g, 87 mmol) was treated with 10 mL TFA/triiso-
propylsilane/H2O 90/5/5 (v/v/v). Purification by HPLC
afforded 23/10 (19 mg, 31%, seven steps) as a white
solid; HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–100/0/
0.1 in 30 min, 50 ꢁC): Rt=14.65 min, purity: >95%;
0.1 in 60 min, 50 ꢁC): Rt=31.98 min, purity: 91%; mp:
20
D
80 ꢁC; ½aŠ ꢀ63.6 (c 0.11, MeOH); 1H NMR (400 MHz,
CDCl3): d 0.71–2.22 (m, 11H) 1.23 (s, 3H), 2.23–2.68
(m, 8H), 2.84 (s, 3H), 2.93 (m, 1H), 3.58 (s, 3H), 3.70 (s,
3H), 4.48–4.59 (m, 1H), 5.05–5.14 (m, 1H), 5.15–5.23
[5.39–5.47] (m, 1H), 6.05–6.13 [6.29–6.34] (m, 1H), 6.44–
6.62 (m, 3H), 6.64–6.83 (m, 5H), 7.08–7.20 (m, 10H);
20
D
mp: 77–80 ꢁC; ½aŠ ꢀ122.1 (c 0.33, MeOH); H NMR
1

2606
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
LC–MS (ESI): 833.3 [M+Na]+, Rt=12.54 min; MS
(MALDI-TOF): 833.8 [M+Na]+, 849.8 [M+K]+;
HRMS (FAB): calcd for C45H54N3O9S [M+H]+:
812.3581, found: 812.3396.
(m, 1H); LC–MS (ESI-1, Grad. A): 724.0 [M+H]+,
Rt=7.45 min; MS (MALDI-TOF): 746.8 [M+Na]+,
762.8
[M+K]+;
HRMS
(FAB):
calcd
for
C41H45NaN3O9 [M+H]+ 746.3053, found: 724.3063.
N-3-Phenylpropionyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl-L-phenylalanine-methoxycarbonylmethylester
(23/8). Compound 19/10 (80 mg, 0.50 mmol/g, 40
mmol) was reacted first with Cs2CO3 (293 mg, 0.9 mmol)
and then with methyl-bromoacetate (166 mL, 1.8 mmol)
in 1 mL DMF for 44 h according to GP P11 variant A.
Afterwards, cleavage from the resin according to GP P5
variant B for 12 h with 10 mL TFA/triisopropylsilan/
H2O 90/5/5 (v/v/v) and purification by HPLC afforded
23/8 (11 mg, 38%, nine steps) as white solid; HPLC
(A2, CH3CN/H2O/TFA, 45/55/0.1–100/0/0.1 in 30 min,
N-Benzyloxycarbonyl-D-tyrosyl-N-methyl-L-phenylalanyl-
L-tyrosine (35/1). Starting from l-8 (300 mg, 0.40 mmol/
g, 120 mmol) the appropriate tripeptide was built up by
sequenzial N-terminal deblocking by treatment with
2ꢂ5 mL piperidine/DMF for 2ꢂ5 min according to GP
P2 and attachment of FmocMePheOH (169 mg, 420
mmol) for 2.5 h and of Fmoc-d-Tyr(tBu)OH (193 mg,
420 mmol) for 14 h by coupling with HATU (148 mg,
390 mmol) in the presence of DIEA (134 mL, 780 mmol)
in 5 mL NMP according to GP P4 variant A furnishing
34e. Then, after N-terminal deblocking according to GP
P2 and reaction with 31a (73 mL, 520 mmol), DMAP (16
mg, 130 mmol), HOAt (71 mg, 520 mmol) and DIEA
(178 mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 according
to GP P10 for 10 h followed by the liberation of the
C-terminus according GP P1 variant C for 16 h. The
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5
h, according to GP P5 variant B affording 92/21 (11 mg,
14%, eight steps) as a white solid; HPLC (A2, CH3CN/
50 ꢁC): Rt=12.17min, purity: >95%; Rf=0.31 [ethyl
20
D
acetate/hexane 2/1, 1% HOAc (v/v)]; mp: 110 ꢁC; ½aŠ
ꢀ119.4 (c 0.32, MeOH); 1H NMR (400 MHz, CDCl3): d
2.00–2.11 (m, 1H), 2.29–2.55 (m, 5H), 2.66 (s, 3H),
2.70–2.79 (dd, 2J=15.8 Hz, 3J=8.0 Hz, 2H), 2.85 [2.98]
(s, 3H), 3.01–3.10 (m, 1H), 3.27–3.37 (m, 1H), 3.70
[3.71] (s, 3H), 3.77 [3.72] (s, 3H), 4.44 [4.61] (d, 2J=15.8
Hz, 1H), 4.67(d, 2J=15.8 Hz, 1H), 4.77–4.82 (m, 2H),
3
5.11–5.15 (m, 1H), 5.46–5.50 (t, J=7.7 Hz, 1H), 6.26–
3
6.31 (m, 1H), 6.56–6.64 (m, 3H), 6.71–6.74 (d, J=8.3
3
Hz, 2H), 6.78–6.83 (d, J=8.5 Hz, 2H), 6.97–6.98 (d,
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC):
20
D
+23.6 (c 0.11, MeOH); H NMR (400 MHz, CD3OD):
3J=6.0 Hz, 1H), 7.08–7.20 (m, 5H), 7.23–7.26 (m, 4H),
7.33–7.42 (m, 1H); 13C NMR (100.6 MHz, CDCl3): d
29.88, 30.38, 31.43 [31.51], 33.44, 34.59, 37.26 [37.42],
37.98, 49.99 [50.19], 52.43 [52.48], 53.61, 54.89 [55.38],
59.64 [60.36], 61.15 [61.48], 113.74, [113.80], 115.33,
[115.38], 126.26 [126.30], 126.84 [126.97], 127.24, 128.17
[128.24], 128.40 [128.52], 128.63 [128.73], 128.82
[128.98], 130.18 [130.32], 136.63, 135.34, 140.31, 140.34,
155.03, 158.46, 167.83, 169.55, 169.75, 171.45, 171.88;
LC–MS (ESI-1, Grad. A): 736.2 [MꢀH]ꢀ, 760.5
[M+Na]+, Rt=12.23 min; MS (MALDI-TOF): 738.85
[M+H]+, 759.17 [M+Na]+, 775.45 [M+K]+; HR-MS
(FAB): calcd for C42H48N3O9 [M+H]+: 738.3391,
found: 738.3405.
Rt=15.74 min, purity: 78%; mp: 116–117 ꢁC; ½aŠ
1
d 2.28–2.40 (m, 1H), 2.47(s, 3H), 2.60–2.69 (m, 1H),
2.75–2.87 (m, 2H), 3.05–3.16 (m, 2H), 4.46–4.50 (m,
2
1H), 4.56–4.60 (m, 1H), 4.95 (d, J=12.5 Hz, 1H), 5.03
(d, 2J=12.5 Hz, 1H), 5.39–5.42 (t, 3J=5.5 Hz, 1H), 6.56
3
3
(d, J=8.6 Hz, 2H), 6.62 (d, J=8.4 Hz, 2H), 6.75 (d,
3J=8.4 Hz, 2H), 6.93 (d, 3J=8.4 Hz, 2H), 7.11–7.13 (m,
3H), 7.17–7.25 (m, 7H); LC–MS (ESI-2): 638.04
[MꢀH]ꢀ, Rt=2.22 min; MS (MALDI-TOF): 662.6
[M+Na]+, 678.6 [M+K]+; HR-MS (FAB, m/z): calcd
for C36H3N3NaO8 [M+Na]+ 662.2478, found:
662.2502.
N-Benzyloxycarbonyl-D-tyrosyl-L-phenylalanyl-L-tyrosine
(35/2). Starting from L-8 (300 mg, 0.40 mmol/g, 120
mmol) the appropriate tripeptide was built up by
sequenzial N-terminal deblocking by treatment with
2ꢂ5 mL piperidine/DMF for 2ꢂ5 min according to GP
P2 and attachment of FmocPheOH (163 mg, 420 mmol)
for 2.5 h and of Fmoc-d-Tyr(tBu)OH (193 mg, 420
mmol) for 14 h by coupling with HATU (148 mg, 390
mmol) in the presence of DIEA (134 mL, 780 mmol) in 5
mL NMP according to GP P4 variant A, furnishing 34f.
Then, after N-terminal deblocking according to GP P2
and reaction with 31a (73 mL, 520 mmol), DMAP (16
mg, 130 mmol), HOAt (71 mg, 520 mmol) and DIEA
(178 mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 according
to GP P10 for 10 h followed by the liberation of the
C-terminus according GP P1 variant C for 16 h. The
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5
h, according to GP P5 variant B affording 35/2 (14 mg,
18%, eight steps) as a white solid; HPLC (A2, CH3CN/
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC)
N-3-Phenylpropionyl-D-tyrosyl-N,O-dimethyl-L-tyrosyl-
N-methyl - L - phenylalanyl - carboxymethylester (23/9).
Compound 19/10 (144 mg, 0.50 mmol/g, 72 mmol) was
reacted first with Cs2CO3 (450 mg, 1.5 mmol) and then
with 32d (488 mL, 3.0 mmol) in 6 mL DMF for 44 h
according to GP P11 variant A. Afterwards, the product
was cleaved from the resin according to GP P5 variant
B for 12 h by treatment with 10 mL TFA/triisopro-
pylsilane/H2O 90/5/5 (v/v/v) and 10 mL TFA/H2O 95/5
(v/v). Purification by HPLC afforded 23/9 (10 mg, 35%,
nine steps) as a white solid; HPLC (A2, CH3CN/H2O/
TFA, 45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=9.11
20
D
min, purity: >95%; mp: 95–97 ꢁC; ½aŠ ꢀ93.1 (c 0.39,
1
MeOH); H NMR (400 MHz, CDCl3): d 1.93–1.98 (t,
3J=7.8 Hz, 1H), 2.21–2.43 (m, 4H), 2.40 [2.50] (s, 3H),
2.61–2.74 (m, 3H), 2.71 [2.86] (s, 3H), 2.92–3.05 (m,
1H), 3.18–3.29 (m, 1H), 3.64 [3.65] (s, 3H), 4.41–4.67
3
(m, 2H), 4.78–4.95 (m, 2H), 5.39–5.44 (t, J=7.7 Hz,
3
1H), 6.52–6.63 (m, 3H), 6.68 (d, J=8.5 Hz, 2H), 6.74
3
3
(d, J=8.3 Hz, 2H), 6.76 (m, 1H), 7.01 (d, J=7.2 Hz,
2H), 7.04–7.13 (m, 3H), 7.14–7.23 (m, 4H), 7.25–7.32

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2607
20
Rt=14.54 min, purity: >95%; mp: 165 ꢁC; ½aŠ +1.0 (c
to GP P10 for 10 h followed by the liberation of the
C-terminus according GP P1 variant C for 16 h. The
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5
h, according to GP P5 variant B affording 35/4 (24 mg,
31%, eight steps) as a white solid; HPLC (A2, CH3CN/
D
0.35, MeOH); 1H NMR (400 MHz, CD3OD): d 2.52
2
3
2
(dd, J=13.5 Hz, J=8.8 Hz, 1H), 2.72 (dd, J=14.1
Hz, 3J=8.8 Hz, 2H), 2.86 (dd, 2J=14.1 Hz, 3J=8.4 Hz,
1H), 2.95–3.06 (m, 2H), 4.15–4.18 (m, 1H), 4.50–4.56
2
2
(m, 2H), 4.90 (d, J=12.5 Hz, 1H), 5.01 (d, J=12.5
3
Hz, 1H), 6.57(d, 3J=8.4 Hz, 2H), 6.62 (d, J=8.4 Hz,
2H), 6.81 (d, J=8.2 Hz, 2H), 7.02 (d, J=8.6 Hz, 2H),
7.10–7.16 (m, 4H), 7.19–7.25 (m, 5H); LC–MS (ESI-2):
624.48 [MꢀH]ꢀ; Rt=3.88 min; MS (MALDI-TOF):
648.5 [M+Na]+, 664.5 [M+K]+; HRMS (FAB): calcd
for C35H36N3O8 [M+H]+ 626.2502, found: 626.2523.
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC):
3
3
20
D
Rt=11.20 min, purity: >95%; mp: 126 ꢁC; ½aŠ ꢀ12.7
1
(c 0.45, MeOH); H NMR (400 MHz, CD3OD): d 2.57
(dd, J=13.7Hz, J=9.6 Hz, 1H), 2.71 (dd, J=14.1
2
3
2
3
Hz, J=8.6 Hz, 1H), 2.82–2.87(m, 2H), 2.91–3.07(m,
3
3
2H), 4.19 (dd, J=9.4 Hz, J=4.9 Hz, 1H), 4.50 (dd,
3J=8.0 Hz, 3J=5.5 Hz, 2H), 4.88 (d, 2J=12.5 Hz, 1H),
N-Benzyloxycarbonyl-D-tyrosyl-L-tyrosyl-L-tyrosine (35/3).
Starting from L-8 (300 mg, 0.40 mmol/g, 120 mmol) the
appropriate tripeptide was built up by sequenzial
N-terminal deblocking by treatment with 2ꢂ5 mL
piperidine/DMF for 2ꢂ5 min according to GP P2 and
attachment of Fmoc-d-Tyr(tBu)OH (193 mg, 420 mmol)
for 2.5 h and of Fmoc-d-Tyr(tBu)OH (193 mg, 420
mmol) for 14 h by coupling with HATU (148 mg, 390
mmol) in the presence of DIEA (134 mL, 780 mmol) in 5
mL NMP according to GP P4 variant A, furnishing
34g. Then, after N-terminal deblocking according to GP
P2 and reaction with 31a (73 mL, 520 mmol), DMAP (16
mg, 130 mmol), HOAt (71 mg, 520 mmol) and DIEA
(178 mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 according
to GP P10 for 10 h followed by the liberation of the
C-terminus according GP P1 variant C for 16 h. The
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5
h, according to GP P5 variant B, affording 35/3 (16 mg,
21%, eight steps) as a white solid; HPLC (A2, CH3CN/
2
4.98 (d, J=12.5 Hz, 1H), 6.58–6.63 (m, 6H), 6.90–6.95
(m, 6H), 7.15–7.26 (m, 5H); LC–MS (ESI-2): 640.00
[MꢀH]ꢀ, Rt=1.90 min; MS (MALDI-TOF): 664.5
[M+Na]+; HRMS (FAB): calcd for C35H36N3O9
[M+H]+: 642.2452, found: 642.2462.
N-Benzyloxycarbonyl-L-phenylalanyl-L-phenylalanyl-L-
tyrosine (35/5). Starting from L-8 (300 mg, 0.40 mmol/
g, 120 mmol) the appropriate tripeptide was built up by
sequenzial N-terminal deblocking by treatment with
2ꢂ5 mL piperidine/DMF for 2ꢂ5 min according to GP
P2 and attachment of FmocPheOH (163 mg, 420 mmol)
for 2.5 h and of FmocPheOH (163 mg, 420 mmol) for 14
h by coupling with HATU (148 mg, 390 mmol) in the
presence of DIEA (134 mL, 780 mmol) in 5 mL NMP
according to GP P4 variant A furnishing 34i. Then,
after N-terminal deblocking according to GP P2 and
reaction with 31a (73 mL, 520 mmol), DMAP (16 mg,
130 mmol), HOAt (71 mg, 520 mmol) and DIEA (178
mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 according to
GP P10 for 10 h followed by the liberation of the
C-terminus according GP P1 variant C for 16 h. The
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v/v) for 30 and
60 min and with 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5
h, according to GP P5 variant B affording 35/5 (13 mg,
18%, eight steps) as a white solid; HPLC (A2, CH3CN/
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC):
20
D
Rt=11.33 min, purity: >95%; mp: 142–144 ꢁC; ½aŠ
1
+1.1 (c 0.54, MeOH); H NMR (400 MHz, CD3OD): d
2.53 (dd, 2J=13.9 Hz, 3J=8.6 Hz, 1H), 2.65 (dd,
2J=14.1 Hz, J=8.6 Hz, 1H), 2.77 (dd, J=14.1 Hz,
3
2
3J=5.8 Hz, 2H), 2.87(m, 2H), 3.03 (dd, 3J=8.6 Hz,
3
1H) 4.17(dd, 3J=8.9 Hz, J=5.7Hz, 1H), 4.47–4.53
(m, 2H), 4.89 (d, J=12.5 Hz, 1H), 5.01 (d, J=12.5
3
H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC)
2
2
20
D
Hz, 1H), 6.58–6.65 (m, 6H), 6.82 (d, J=8.4 Hz, 2H),
Rt=14.54 min, purity: >95%; mp: 201–202 ꢁC; ½aŠ
6.87(d, 3J=8.4 Hz, 2H), 6.96 (d, 3J=8.4 Hz, 2H), 7.20–
7.23 (m, 5H); LC–MS (ESI 2): 640.77 [MꢀH]ꢀ;
Rt=1.88 min; MS (MALDI-TOF): 664.8 [M+Na]+,
680.8 [M+K]+; HRMS (FAB): calcd for C35H36N3O9
[M+H]+ 642.2452, found: 642.2417.
ꢀ20.0 (c 0.39, MeOH); 1H NMR (400 MHz, CD3OD): d
2.52 (m, 1H), 2.78–2.94 (m, 3H), 2.98–3.08 (m, 2H), 4.26
(dd, J=9.8 Hz, J=4.9 Hz, 1H), 4.50 (dd, J=7.8 Hz,
3J=5.5 Hz, 1H), 4.58 (dd, 2J=8.4 Hz, 3J=5.5 Hz, 1H),
2
3
2
2
2
4.90 (d, J=12.5 Hz, 1H), 4.94 (d, J=12.5 Hz, 1H),
6.63 (d, 3J=8.6 Hz, 2H), 6.96 (d, 3J=8.6 Hz, 2H), 7.08–
7.22 (m, 15H); LC–MS (ESI 2): 608.47 [MꢀH]ꢀ;
Rt=2.43 min; MS (MALDI-TOF): 632.9 [M+Na]+,
648.8 [M+Na]+; HRMS (FAB): calcd for C35H36N3O7
[M+H]+ 610.2553, found: 610.2554.
N-Benzyloxycarbonyl-L-tyrosyl-L-tyrosyl-L-tyrosine (35/4).
Starting from L-8 (400 mg, 0.30 mmol/g, 120 mmol) the
appropriate tripeptide was built up by sequenzial
N-terminal deblocking by treatment with 2ꢂ5 mL
piperidine/DMF for 2ꢂ5 min according to GP P2 and
attachment of Fmoc-l-Tyr(tBu)OH (193 mg, 420 mmol)
for 2.5 h and of Fmoc-l-Tyr(tBu)OH (193 mg, 420
mmol) for 14 h by coupling with HATU (148 mg, 390
mmol) in the presence of DIEA (134 mL, 780 mmol) in 5
mL NMP according to GP P4 variant A, furnishing
34h. Then, after N-terminal deblocking according to GP
P2 and reaction with 31a (73 mL, 520 mmol), DMAP (16
mg, 130 mmol), HOAt (71 mg, 520 mmol) and DIEA
(178 mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 according
General procedure for the synthesis of polymer bound
tripeptides 34a–d (GP P13)
Starting from L-8 (2.1 g, 0.40 mmol/g, 840 mmol) the
appropriate tripeptide was built up by sequenzial N-
terminal deblocking by treatment with 2ꢂ10 mL piper-
idine/DMF for 2ꢂ5 min according to GP P2 and cou-
pling of FmocMePheOH or FmocPheOH (1.08 g, 2.67
mmol), HATU (958 mg, 2.52 mmol) in the presence of

2608
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
DIEA (913 mL, 5.34 mmol) in 20 mL NMP according to
GP P4 variant A for 2.5 h and of Fmoc-d-His(Trt)OH
or Fmoc-l-His(Trt)OH (193 mg, 420 mmol), DIC (520
mL, 3.36 mmol) and HOAt (457mg, 3.36 mmol) for 14 h
in 25 mL DMF for 14 h according to GP P4 variant B.
CD3OD): d 2.88–2.98 (m, 3H), 3.06–3.14 (m, 3H), 4.36–
3 3 3
4.40 (t, J=6.8 Hz, 1H), 4.58 (dd, J=5.3 Hz, J=8.4
3
3
Hz, 1H), 4.63 (dd, J=5.3 Hz, J=9.0 Hz, 1H), 5.04 (s,
2H), 6.68 (d, 3J=8.6 Hz, 2H), 7.06 (d, 3J=8.6 Hz, 2H),
7.16–7.27 (m, 5H), 7.31–7.34 (m, 5H), 8.63 (s, 1H); LC–
MS (ESI-2): 600.08 [M+H]+, 622.05 [M+Na]+;
Rt=1.45 min; MS (MALDI-TOF): 600.4 [M+H]+,
622.4 [M+Na]+; HRMS (FAB, m/z): calcd for
C32H34N5O7 [M+H]+ 600.2458, found: 600.2476.
Polymer-bound Nꢀ-9-Fluorenylmethoxycarbonyl-(NIm-
triphenylmethyl) - D - histidyl - L - phenylalanyl - L - tyrosine
allyl ester (34a). Reaction of L-8 with FmocPheOH
and Fmoc-d-His(Trt)OH according to GP P13 furn-
ished 34a; ninhydrine-test:15 negative.
Nꢀ-Benzyloxycarbonyl-L -histidyl -N-methyl-L -phenyl-
alanyl-L-tyrosine hydrotrifluoroacetate (35/7). Starting
from tripeptide 34d reaction according to GP P14 fur-
nishes 35/7 (28 mg, 24%, eight steps) as a white solid;
HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–70/30/0.1 in
Polymer-bound Nꢀ-9-fluorenylmethoxycarbonyl-(NIm-tri-
phenylmethyl)-L-histidyl-L-phenylalanyl-L-tyrosine allyl
ester (34b). Reaction of L-8 with FmocPheOH and
Fmoc-l-His(Trt)OH according to GP P13 furnished
34b; ninhydrine-test:15 negative.
30 min, 50 ꢁC): Rt=10.82 min, purity: >85; mp: 127 ꢁC;
20
D
½aŠ ꢀ64.2 (c 0.88, MeOH); 1H NMR (400 MHz,
CD3OD): d 2.44–2.59 (m, 2H), 2.74–2.97 (m, 5H), 3.13–
3.28 (m, 2H), 4.51–4.61 (m, 1H), 4.67–4.71 (m, 2H),
Analysis of samples of 34a and 34b after cleavage from
the resin: LC–MS (ESI-1, Grad. A): 742.3 [M+H]+,
Rt=1.95 min (Fmoc-His-Phe-Tyr-OH).
3
4.97–5.00 (m, 1H), 5.06–5.09 (m, 1H), 6.67 (d, J=8.6
3
Hz, 2H), 6.92 [6.97] (d, J=8.6 Hz, 2H), 7.13–7.20 (m,
6H), 7.26–7.34 (m, 5H), 8.68 [8.73] (s, 1H); LC–MS
Polymer-bound Nꢀ-9-fluorenylmethoxycarbonyl-(NIm-tri-
phenylmethyl)-D-histidyl-N-methyl-L-phenylalanyl-L-tyro-
sine allyl ester (34c). Reaction of L-8 with
FmocMePheOH and Fmoc-d-His(Trt)OH according to
GP P13 furnished 34c; ninhydrine-test:15 negative.
(LC–ESI 2): 614.08 [M+H]+, 636.07[M+Na]
Rt=1.49 min, MS (MALDI-TOF): 614.7[M+H]
;
,
+
+
636.7[M+Na] +; HRMS (FAB): calcd for C33H36N5O7
[M+H]+: 614.2615, found: 614.2637.
Nꢀ-Benzyloxycarbonyl-D-histidyl-N-methyl-L-phenylalanyl-
L-tyrosine hydrotrifluoroacetate (35/8). Starting from
tripeptide 34c reaction according to GP P14 furnishes
35/8 (28 mg, 24%, eight steps) as a white solid. HPLC
Polymer-bound Nꢀ-9-fluorenylmethoxycarbonyl-(NIm-tri-
phenylmethyl)-L-histidyl-N-methyl-L-phenylalanyl-L-tyro-
sine allyl ester (34d). Reaction of L-8 with
FmocMePheOH and Fmoc- l-His(Trt)OH according to
GP P13 furnished 34d; ninhydrine-test:15 negative.
(A2, CH3CN/H2O/TFA, 20/80/0.1–70/30/0.1 in 30 min,
50 ꢁC) Rt=9.74 min, purity: >95%; mp: 128 ꢁC; ½aŠ
20
D
ꢀ52.2 (c 0.51, MeOH); 1H NMR (400 MHz, CD3OD): d
Analysis of samples of 34c and 34d after cleavage from
the resin: LC–MS (ESI-1, Grad. A): 742.3 [M+H]+,
Rt=1.95 min (Fmoc-His-MePhe-Tyr-OH).
2.48–2.51 (m, 2H), 2.66 (s, 3H), 2.73–2.83 (m, 2H), 3.09
(dd, J=14.1 Hz, J=4.50 Hz, 1H), 3.18 (dd, J=14.7
2
3
2
3
2
Hz, J=5.09, 1H), 4.58–4.61 (m, 2H), 4.93 (d, J=12.5
2
3
Hz, 1H), 5.01 (d, J=12.3 Hz, 1H), 5.36 (dd, J=11.15
3
3
Hz, J=5.1 Hz, 1H), 6.56 [6.61] (d, J=8.6 Hz, 2H),
6.90–7.01 (m, 1H), 6.93 [6.99] (d, 3J=8.6 Hz, 2H), 7.07–
7.17 (m, 5H), 7.18–7.32 (m, 5H), 8.55 [8.58] (s, 1H); LC–
General procedure for the synthesis of the tripeptides 35/
6–35/8 (GP P14)
+
According to GP P2, the polymer-bound tripeptide 34
(400 mg, 0.40 mmol/g, 160 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 31a (113 mL, 800 mmol),
DMAP (24 mg, 200 mmol), HOAt (109 mg, 800 mmol)
and DIEA (274 mL, 1.6 mmol) in DMF/CH2Cl2 2/1
(v/v) for 12 h. After the liberation of the C-terminus
with Pd(PPh3)4 in 5 mL morpholine/THF for 16 h
according to GP P1 variant C, the product was cleaved
from the resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/
H2O 47/47/6 (v/v) for 2ꢂ45 min and 2ꢂ5 mL TFA/H2O
95/5 (v/v) for 2ꢂ1.5 h According to GP P5, variant B
followed by HPLC purification affording the appro-
priate product.
MS (LC-ESI 2): 614.08 [M+H]+, 636.07[M+Na]
,
Rt=1.42 min; MS (MALDI-TOF): 614.9 [M+H]+,
636.9 [M+Na]+; HRMS (FAB): calcd for C33H36N5O7
[M+H]+: 614.2615, found: 614.2650.
Nꢀ-4-Nitro-benzyloxycarbonyl-D-histidyl-L-phenylalanyl-
L-tyrosine hydrotrifluoroacetate (35/9). According to GP
P2 the polymer-bound tripeptide 34a (300 mg, 0.40
mmol/g, 120 mmol) was N-terminally deblocked by
piperidine/DMF treatment for 2ꢂ5 min, followed by
coupling with 31b (112 mg, 520 mmol), DMAP (16 mg,
130 mmol), HOAt (71 mg, 520 mmol) and DIEA (178
mL, 1.04 mmol) in 7.5 mL DMF/CH2Cl2 1/2 (v/v) for 20
h. After the liberation of the C-terminus with Pd(PPh3)4
in 5 mL morpholine/THF for 16 h according to GP P1
variant C, the product was cleaved from the resin by
treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6
(v/v) for 2ꢂ45 min and 2ꢂ5 mL TFA/H2O 95/5 (v/v)
for 2ꢂ1.5 h According to GP P5 variant B followed by
HPLC purification affording 35/9 (45 mg, 58%, eight
steps) as a light yellow solid; HPLC (A2, CH3CN/H2O/
TFA, 20/80/0.1–90/10/0.1 in 40 min, 50 ꢁC) Rt=8.96
Nꢀ-Benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tyro-
sine hydrotrifluoroacetate (35/6). Starting from tripep-
tide 34b reaction according to GP P14 furnishes 35/6
(28 mg, 24%, eight steps) as a slightly yellow solid;
HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–70/30/0.1 in
30 min, 50 ꢁC): Rt=10.24 min, purity: 95%; mp: 129 ꢁC;
20
D
½aŠ
ꢀ7.3 (c 0.56, MeOH); 1H NMR (400 MHz,

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2609
20
min, purity: >95%; mp: 124 ꢁC; ½aŠ +3.9 (c 1.00,
and 180 min according to GP P5 variant B followed by
HPLC purification affording 35/11 (14 mg, 15%, eight
steps) as a white solid; HPLC (A2, CH3CN/H2O/TFA,
45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=16.19 min,
D
MeOH); 1H NMR (400 MHz, CD3OD): d 2.64–2.81 (m,
3
3H), 2.88–3.04 (m, 3H), 4.27(dd, 3J=6.1 Hz, J=7.8
3
Hz, 1H), 4.47(dd, 3J=5.1 Hz, J=8.41 Hz, 1H), 4.53
20
(dd, J=4.7Hz, 3J=9.8 Hz, 1H), 5.02 (s, 2H), 6.53 (d,
purity: 95%; mp: 149 ꢁC; ½aŠ ꢀ5.3 (c 0.53, MeOH); H
3
1
D
3
3
3J=8.4 Hz, 2H), 6.83 (s, 1H), 6.90 (d, J=8.6 Hz, 2H),
NMR (400 MHz, CD3OD): d 0.90 (t, J=7.0 Hz, 3H),
1.28–1.36 (m, 4H), 1.62 (quint, J=7.6 Hz, 2H), 2.62 (t,
3
3
7.03–7.09 (m, 5H), 7.39 (d, J=8.4 Hz, 2H), 8.05 (d,
3J=8.80 Hz, 2H), 8.51 (s, 1H); MS (MALDI-TOF):
645.6 [M+H]+, 667.6 [M+Na]+; HRMS (FAB):, calcd
for C32H33N6O9 [M+H]+: 645.2309, found: 645.2295.
3J=7.6 Hz, 2H), 2.81 (dd, 2J=14.1 Hz, 3J=9.8 Hz, 1H),
2.90–2.97(m, 2H), 3.08–3.16 (m, 3H), 4.58 (dd, 3J=5.28
3
3
Hz, J=8.6 Hz, 1H), 4.65 (dd, J=4.7Hz, 3J=9.2 Hz,
1H), 4.71 (m, 1H), 6.55 (d, 3Jtrans=15.6 Hz, 1H), 6.67(d,
3J=8.6 Hz, 2H), 6.95 (s, 1H), 7.04 (d, J=8.4 Hz, 2H),
7.15–7.26 (m, 8H), 7.45 (d, J=8.2 Hz, 1H), 7.50 (d,
3
N-3-[(4-(n-Pentyl)phenyl]-E-acryloyl-D-tyrosyl-L-phenyl-
alanyl-L-tyrosine (35/10). Starting from L-8 (400 mg,
0.30 mmol/g, 120 mmol), the appropriate tripeptide was
built up by sequenzial N-terminal deblocking by treat-
ment with 2ꢂ5 mL piperidine/DMF for 2ꢂ5 min
according to GP P2 and attachment of FmocPheOH
(163 mg, 420 mmol) for 2.5 h and of Fmoc-d-Tyr(t-
Bu)OH (193 mg, 420 mmol) for 14 h by coupling with
HATU (148 mg, 390 mmol) in the presence of DIEA
(134 mL, 780 mmol) in 5 mL NMP according to GP P4
variant A furnishing 34f. Then, after N-terminal
deblocking according to GP P2 and reaction with 30a
(85 mg mL, 390 mmol) and PyAOP (203 mg, 390 mmol)
in the presence of DIEA (223 mL, 1.3 mmol) in 7.5 mL
DMF/CH2Cl2 for 18 h according to GP P8, followed by
the liberation of the C-terminus according GP P1 var-
iant C for 16 h. The product was cleaved from the resin
by treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6
(v/v/v) for 30 and 60 min and with 2ꢂ5 mL TFA/H2O
95/5 (v/v) for 2ꢂ1.5 h, according to GP P5 variant B
affording 35/10 (26 mg, 31%, eight steps) as a white
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–90/10/
0.1 in 40 min, 50 ꢁC): Rt=22.25 min, purity: >95%;
3
3Jtrans=15.6 Hz, 1H), 8.64 (s, 1H); LC–MS (ESI-1, Grad.
A): 666.3 [M+H]+, 688.3 [M+Na]+, 664.4 [MꢀH]ꢀ;
Rt=17.78 min; MS (MALDI-TOF): 666.7 [M+H]+,
688.7[M+Na] +, 704.6 [M+K]+; HRMS (FAB): calcd
for C38H44N5O6 [M+H]+: 666.3292, found: 666.3317.
Nꢀ-3-[4-(n-Pentyl)phenyl]propionyl-D-histidyl-L-phenyl-
alanyl-L-tyrosine hydrotrifluoroacetate (35/12). Accord-
ing to GP P2, the polymer-bound tripeptide 34a (300
mg, 0.40 mmol/g, 120 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 29b (106 mg, 480 mmol),
HATU (175 mg, 460 mmol), HOAt (27mg, 200 mmol)
and DIEA (342 mL, 2.0 mmol) in 5 mL NMP for 24 h.
After the liberation of the C-terminus with Pd(PPh3)4 in
5 mL morpholine/THF for 16 h according to GP P1
variant C the product was cleaved from the resin by
treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6
(v/v) for 10 and 20 min and 2ꢂ5 mL TFA/H2O 95/5
(v/v) for 30 and 180 min according to GP P5 variant B
followed by HPLC purification affording 92/34 (23 mg,
24%, eight steps) as a light yellow solid; HPLC (A2,
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
20
D
mp: 131–132 ꢁC; ½aŠ ꢀ1.4 (c 0.58, MeOH); H NMR
1
3
(400 MHz, CD3OD): d 0.82 (t, J=7.5 Hz, 3H), 1.22–
1.29 (m, 4H), 1.52–1.56 (m, 2H), 2.53 (t, J=7.4 Hz,
50 ꢁC): Rt=16.69 min, purity: 95%; mp: 130–131 ꢁC;
3
20
D
2
3
2H), 2.61 (dd, J=13.9 Hz, J=8.0 Hz, 1H), 2.71 (dd,
3
½aŠ
+7.6 (MeOH, c 1.0); 1H NMR (400 MHz,
2J=14.1 Hz, J=8.8 Hz, 1H), 2.79 (dd, J=14.1 Hz,
CD3OD): d 0.87(t, 3J=6.8 Hz, 3H, CH3), 1.15–1.37(m,
4H), 1.55 (quint, J=7.6 Hz, 2H), 2.41–2.55 (m, 2H),
2
3J=6.5 Hz, 1H), 2.88 (dd, 2J=13.9 Hz, 3J=8.4 Hz,
3
2
3
3
1H), 2.96 (dd, J=14.1 Hz, J=5.1 Hz, 1H), 3.04 (dd,
3
2.54 (t, J=7.6 Hz, 2H), 2.79 (m, 4H), 2.90–3.02 (m,
2J=14.1 Hz, J=5.1 Hz, 1H), 4.49–4.55 (m, 3H), 6.48 (d,
2H), 3.10–3.16 (m, 2H), 4.55–4.64 (m, 3H), 6.68 (d,
3J=8.4 Hz, 2H), 6.88 (s, 1H), 7.08–7.11 (m, 6H), 7.14–
7.22 (m, 5H), 8.64 (s, 1H); LC–MS (ESI-1, Grad. A):
668.4 [M+H]+, 690.3 [M+Na]+, 666.4 [MꢀH]ꢀ;
Rt=17.27 min; MS (MALDI-TOF): 668.8 [M+H]+,
690.8 [M+Na]+; HRMS (FAB, m/z): calcd for
C38H46N5O6 [M+H]+: 668.3448, found: 668.3418.
3Jtrans=15.6 Hz, 1H), 6.57(d, 3J=8.6 Hz, 2H), 6.60 (d,
3J=8.6 Hz, 2H), 6.82 (d, 3J=8.8 Hz, 2H), 6.97(d, J=8.6
3
Hz, 2H), 7 .03 (d,3J=8.4 Hz, 2H), 7.09–7.13 (m, 5H), 7.36
(d, 3J=8.2 Hz, 2H), 7.56 (d, 3Jtrans=15.6 Hz, 1H); LC–MS
(ESI-2): 690.84 [MꢀH]ꢀ, Rt=2.63 min; MS (MALDI-
TOF): 714.6 [M+Na]+; HR-MS (FAB, m/z): calcd for
C41H46N3O7 [M+H]+: 632.3336, found: 692.3332.
General procedure for the synthesis of tripeptides 35/13–
35/15 (GP P15)
Nꢀ-3-[4-(n-Pentyl)phenyl]-E-acryloyl-D-histidyl-L-phenyl-
alanyl-L-tyrosine hydrotrifluoroacetate (35/11). Accord-
ing to GP P2 the polymer bound tripeptide 34a (300 mg,
0.40 mmol/g, 120 mmol) was N-terminally deblocked by
piperidine/DMF treatment for 2ꢂ5 min, followed by
coupling with 30a (105 mg, 480 mmol), PyAOP (250 mg,
480 mmol), DMAP (15 mg, 120 mmol) and DIEA (103
mL, 600 mmol) in 3 mL DMF/CH2Cl2 for 24 h. After the
liberation of the C-terminus with Pd(PPh3)4 in 5 mL
morpholine/THF for 16 h according to GP P1 variant C
the product was cleaved from the resin by treatment
with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v) for 10
and 20 min and 2ꢂ5 mL TFA/H2O 95/5 (v/v) for 30 min
According to GP P2, the polymer-bound tripeptide 34
(400 mg, 0.40 mmol/g, 160 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 29b (110 mg, 500 mmol),
HATU (175 mg, 460 mmol), HOAt (27mg, 200 mmol)
und DIEA (342 mL, 2.0 mmol) in 5 mL NMP fur 12 h.
After the liberation of the C-terminus with Pd(PPh3)4 in
5 mL morpholine/THF for 16 h according to GP P1
variant C the product was cleaved from the resin by
treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/
v) for 2ꢂ45 min and 2ꢂ5 mL TFA/H2O 95/5 (v/v) for

2610
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2ꢂ1.5 h according to GP P5 variant B followed by
PyAOP (250 mg, 480 mmol), DMAP (15 mg, 120 mmol),
DIEA (205 mL, 1.2 mmol) in 7.5 mL DMF/CH2Cl2 for
24 h According to GP P8. After the liberation of the
C-terminus with Pd(PPh3)4 in 5 mL morpholine/THF
for 16 h according to GP P1 variant C the product was
cleaved from the resin by treatment with 2ꢂ5 mL TFA/
CH2Cl2/H2O 47/47/6 (v/v) for 10 and 20 min and 2ꢂ5
mL TFA/H2O 95/5 (v/v) for 30 min and 180 min
according to GP P5 variant B followed by HPLC pur-
ification affording 35/16 (3 mg, 3%, eight steps) as a
light brown solid as a mixture of E- und Z-isomer
(E/Z=1:1, according to 1H NMR); HPLC (A2,
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
50 ꢁC): Rt=16.19 min; HPLC (A2, CH3CN/H2O/TFA,
HPLC purification affording the appropriate product.
Nꢀ-3-[4-(n-Pentyl)phenyl]propionyl-L-histidyl-L-phenyl-
alanyl-L-tyrosine hydrotrifluoroacetate (35/13). Starting
from tripeptide 34b, reaction according to GP P15
furnished 35/13 (24 mg, 19%, eight steps) as a white
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–70/30/
0.1 in 30 min, 50 ꢁC): Rt=20.04 min, purity: 93%;
20
D
mp: >210 ꢁC (decomp.); ½aŠ ꢀ13.2 (c 0.19, MeOH);
1H NMR (400 MHz, CD3OD): d 0.87(t, 3J=6.8 Hz,
3H), 1.25–1.36 (m, 4H), 1.52–1.59 (quint, J=7.6 Hz,
3
3
3
2H), 2.44 (t, J=8.4 Hz, 2H), 2.54 (t, J=7.6 Hz, 2H),
3
2.79 (t, J=7.6 Hz, 2H), 2.81–2.94 (m, 3H), 3.00–3.13
3
(m, 3H), 4.56 (dd, J=5.1 Hz, J=8.2 Hz, 1H), 4.56–
4.62 (m, 2H), 6.67(d, 3J=8.6 Hz, 2H), 6.93 (s, 1H), 7.04
20/80/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=13.47min
3
20
D
+6.3 (c 0.24, MeOH); H NMR (400 MHz, CD3OD)
(isomer 1), Rt=15.41 min (isomer 2); mp: 165 ꢁC; ½aŠ
3
1
(d, J=8.6 Hz, 2H), 7.04–7.08 (m, 4H), 7.17–7.25 (m,
+
5H), 8.58 (s, 1H); LC–MS (ESI 2): 668.17[M+H]
,
Z-isomer: d 0.96 (m, 3H), 1.27–1.30 (m, 2H), 1.49–
1.52 (m, 2H), 2.72–2.78 (m, 6H), 4.56–4.72 (m, 3H),
690.15 [M+Na]+, Rt=1.94 min; MS (MALDI-TOF):
668.8 [M+H]+, 690.8 [M+Na]+; HRMS (FAB): calcd
for C38H46N5O6: 668.3448, found: 668.3433.
3
5.93 (d, Jcis=12.7Hz, 1H), 6.30–6.39 (m, 2H), 6.65–
6.68 [6.74–6.77] (m, 2H), 6.89 (m, 1H), 6.99 (d,
3J=8.2 Hz, 1H), 7.15–7.27 (m, 7H), 7.39 (t, 3J=8.8
Nꢀ-3-[4-(n-Pentyl)phenyl]propionyl-L-histidy-N-methyl-
L-phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/14).
Starting from tripeptide 34d, reaction according to GP
P15 furnished 35/14 (38 mg, 30%, eight steps) as a white
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–90/10/
0.1 in 40 min, 50 ꢁC) Rt=18.36 min, purity: >95%; mp:
120 ꢁC; ½aŠ2D0, ꢀ12.3 (c, 1.0, MeOH); 1H NMR
(400 MHz, CD3OD): d, 0.87(m, 3H), 1.29 (m, 4H), 1.54
(m, 2H), 2.33–2.45 (m, 4H), 2.52 (s, 3H), 2.65–2.71 (m,
2H), 2.79–2.92 (m, 4H), 3.12–3.23 (m, 2H), 4.55–4.67
(m, 1H), 4.94–5.00 (m, 1H), 5.27–5.39 (m, 1H), 6.67 (m,
2H), 6.94–6.96 (m, 3H), 7.02–7.20 (m, 9H), 8.71 (s, 1H);
LC–MS (ESI-2):, 682.18 [M+H]+, 704.16 [M+Na]+,
Rt=1.99 min; MS (FAB): 682.3 [M+H]+, 704.3
[M+Na]+; HRMS (FAB): calcd for C39H48N5O6
[M+H]+: 682.3605, found: 682.3625.
Hz, 2H), 7.47 (d, J=8.8 Hz, 1H), 8.54 (s, 1H, His);
E-isomer: d 0.96 (m, 3H), 1.27–1.30 (m, 2H), 1.49–
1.52 (m, 2H), 2.72–2.78 (m, 6H), 4.56–4.72 (m, 3H),
3
3
6.30–6.39 (m, 2H), 6.56 (d, Jtrans=15.9 Hz, 1H),
3
6.74–6.77 (m, 2H), 6.93 (m, 1H), 7.04 (d, J=8.2 Hz,
3
1H), 7.15–7.27 (m, 6H), 7.39 (t, J=8.8 Hz, 2H), 7.47
3
3
(d, J=8.8 Hz, 1H), 7.47 (d, J=16.0 Hz, 1H), 8.56
(s, 1H); LC–MS (ESI-1, Grad. A): 664.3 [M+H]+,
686.3 [M+Na]+; Rt=16.06 min (isomer 1), Rt=17.67
min (isomer 2); MS (MALDI-TOF): 664.9 [M+H]+,
686.9 [M+Na]+; HR-MS (FAB, m/z): calcd for
C38H42N5O6 [M+H]+ 664.3135, found: 664.3096.
Nꢀ-3-[3-(1-E-n-Pentenyl)phenyl)]-E-acryloyl-D-histidyl-
L-phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/17).
According to GP P2, the polymer-bound tripeptide 34a
(150 mg, 0.40 mmol/g, 60 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 30g (39 mg, 180 mmol),
PyAOP (94 mg, 180 mmol), DMAP (7mg, 60 mmol),
HOAt (8 mg, 60 mmol), DIEA (103 mL, 600 mmol) in 3 mL
DMF/CH2Cl2 for 24 h according to GP P8. After the lib-
eration of the C-terminus with Pd(PPh3)4 in 5 mL mor-
pholine/THF for 16 h according to GP P1 variant C the
product was cleaved from the resin by treatment with 2ꢂ5
mL TFA/CH2Cl2/H2O 47/47/6 (v/v) for 10 and 20 min and
2ꢂ5 mL TFA/H2O 95/5 (v/v) for 30 min and 180 min
according to GP P5 variant B followed by HPLC purifica-
tion affording 35/17 (6 mg, 13%, eight steps) as a white
solid. HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1
in 30 min, 50 ꢁC); Rt=15.85 min, purity: >95%; mp:
Nꢀ-3-[4-(n-Pentyl)phenyl]propionyl-D-histidyl-N-methyl-
L-phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/15).
Starting from tripeptide 34c, reaction according to GP
P15 furnished 35/15 (27mg, 21%, eight steps) as a
slightly yellow solid. HPLC (A2, CH3CN/H2O/TFA,
20/80/0.1–70/10/0.1 in 30 min, 50 ꢁC) Rt=18.95 min,
20
purity: >95%; mp: 127 ꢁC; ½aŠ ꢀ20.0 (c 0.75, MeOH);
D
1H NMR (400 MHz, CD3OD): d 0.86 (t, 3J=7.0 Hz, 3H),
1.29–1.31 (m, 4H), 1.54 (quint, 3J=7.4 Hz, 2H), 2.32–2.39
(m, 1H), 2.46–2.54 (m, 3H), 2.60 (dd, 2J=15.3 Hz, 3J=6.3
Hz, 2H), 2.70 (s, 3H), 2.78 (t, 3J=7.2 Hz, 2H), 2.86–2.92
(m, 4H), 4.56 (dd, 3J=5.1 Hz, 3J=8.2 Hz, 1H), 4.56–4.62
(m, 2H), 6.67(d, 3J=8.6 Hz, 2H), 6.93 (s, 1H), 7.04 (d,
3J=8.6 Hz, 2H), 7.04–7.08 (m, 4H), 7.17–7.25 (m, 5H),
8.58 (s, 1H); LC–MS (ESI-2): 682.18 [M+H]+, 7 04.16
[M+Na]+, Rt=1.92 min; HRMS (FAB): calcd for
C39H47N5O6 [M+H]+ 682.3605, found: 682.3615.
20
D
144 ꢁC; ½aŠ ꢀ4.2 (MeOH, c 0.33); H NMR (400MHz,
1
CD3OD): d 0.97(t, 3J=7.2 Hz, 3H), 1.49–1.55 (m, 2H),
2
3
2.17–2.23 (m, 2H), 2.81 (dd, J=14.1 Hz, J=10.0, 1H),
2.91–2.97(m, 2H), 3.09–3.19 (m, 3H), 4.48–4.52 (m, 1H),
4.58–4.66 (m, 2H), 6.29–6.34 (m, 1H), 6.41 (d, 3Jtrans=16.0
Hz, 1H), 6.60 (d, J=15.6 Hz, 1H), 6.67(d, 3J=8.6 Hz,
Nꢀ-3-[4-(1-E-n-Pentenyl)phenyl)]-E/Z-acryloyl-D-histidyl-
L-phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/16).
According to GP P2, the polymer-bound tripeptide 34a
(300 mg, 0.40 mmol/g, 120 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 30h (60 mg, 270 mmol),
3
3
2H), 6.95 (s, 1H), 7.04 (d, J=8.4 Hz, 2H), 7 .16–7 .24 (m,
4H), 7.29–7.33 (m, 1H), 7.37 (m, 2H), 7.50–7.54 (m, 2H),
7.73 (d, 3J=15.8 Hz, 1H), 8.40 (s, 1H); LC–MS (LC–ESI
1): 664.3 [M+H]+, 686.3 [M+Na]+, Rt=16.06 min; MS

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2611
(MALDI-TOF): 664.7[M+H] +, 686.7[M+Na] +, 7 02.7
[M+K]+; HRMS (FAB): calcd for C38H41NaN5O6
[M+Na]+: 686.2955, found: 686.2938.
(m, 3H), 6.68 (d, 3J=8.6 Hz, 2H), 6.85 (s, 1H), 7.04–7.09
(m, 7H), 7.15–7.22 (m, 4H), 8.60 (s, 1H); LC–MS (ESI-1,
Grad. A): 668.3 [M+H]+, 690.3 [M+Na]+, 666.5
[MꢀH]ꢀ, Rt=16.33 min; MS (MALDI-TOF): 668.5
[M+H]+, 690.5 [M+Na]+; HR-MS (FAB, m/z): calcd
for C38H46N5O6 [M+H]+: 668.3448, found: 668.3421.
Nꢀ-3-[2-(1-Z-n-Pentenyl)phenyl]-E-acryloyl-D-histidyl-L-
phenylalanyl - L - tyrosin - hydrotrifluoroacetate (35/18).
According to GP P2, the polymer-bound tripeptide 34a
(300 mg, 0.40 mmol/g, 120 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 30i (104 mg, 480 mmol),
PyAOP (250 mg, 480 mmol), DMAP (15 mg, 120 mmol),
DIEA (205 mL, 1.2 mmol) in 3 mL DMF/CH2Cl2 for 24
h according to GP P8. Liberation of the C-terminus
with Pd(PPh3)4 in 5 mL morpholine/THF for 16 h
according to GP P1 variant C furnished the polymer
bound compound Nꢀ-3-[2-(1-Z-n-pentenyl)phenyl]-E-
acryloyl-D-histidyl-L-phenylalanyl-L-tyrosine-hydrotri-
fluoroacetate. The product was cleaved from the resin by
treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6
(v/v) for 10 and 20 min and 2ꢂ5 mL TFA/H2O 95/5
(v/v) for and 180 min according to GP P5 variant B
followed by HPLC purification affording 35/18 (35 mg,
38%, eight steps) as a yellowish solid. HPLC (A2,
Nꢀ-3-(4-Biphenyl)-E-acryloyl-D-histidyl-L-phenylalanyl-
L-tyrosine-hydrotrifluoracetate (35/19). According to
GP P2, the polymer-bound tripeptide 34a (300 mg, 0.40
mmol/g, 120 mmol) was N-terminally deblocked by
piperidine/DMF treatment for 2ꢂ5 min, followed by
coupling with 29a (109 mg, 480 mmol), HATU (186 mg,
480 mmol), HOAt (16 mg, 120 mmol) und DIEA (205
mL, 1.2 mmol) in 5 mL NMP fur 24 h. After the libera-
tion of the C-terminus with Pd(PPh3)4 in 5 mL mor-
pholine/THF for 16 h according to GP P1 variant C the
product was cleaved from the resin by treatment with
2ꢂ5 mL TFA/CH2Cl2/H2O 47/47/6 (v/v) for 10 and 20
min and 2ꢂ5 mL TFA/H2O 95/5 (v/v) for and 180 min
according to GP P5 variant B followed by HPLC pur-
ification affording 35/19 (32 mg, 34%, eight steps) as a
yellowish solid. HPLC (A2, CH3CN/H2O/TFA, 45/55/
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
20
50 ꢁC): Rt=14.90 min, purity: 90%; mp: 134 ꢁC; ½aŠ
0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=13.00 min, purity:
20
D
>95%; mp: 118–119 ꢁC; ½aŠ +8.9 (c 1.00; MeOH); 1H
D
1
+7.0 (c 1.00, MeOH); H NMR (400 MHz, CD3OD): d
NMR (400 MHz, CD3OD): d 2.41–2.45 (m, 2H), 2.63–
2.93 (m, 6H), 2.99–3.04 (m, 2H), 4.29 (t, J=7.0 Hz,
3
3
0.76 (t, J=7.2 Hz, 3H, CH2CH3), 1.28–1.33 (m, 2H),
1.89–1.95 (m, 2H), 2.72–2.78 (dd, 2J=13.9 Hz, 3J=10.0
Hz, 1H), 2.86–2.95 (m, 2H), 2.99–3.10 (m, 3H), 4.48–
4.52 (m, 1H), 4.58–4.66 (m, 2H), 5.63–5.80 (m, 1H), 6.46
1H), 4.35 (dd, J=5.9 Hz, J=8.6 Hz, 1H), 4.66 (dd,
3
3
3
3J=5.5 Hz, J=8.8 Hz, 1H), 6.58 (d, J=8.6 Hz, 2H),
6.78 (m, 1H), 6.94 (d, J=8.4 Hz, 2H), 7.04–7.15 (m,
7H), 7.16–7.21 (m, 2H), 7.27–7.40 (m, 2H), 7.39 (d,
3J=7.8 Hz, 2H), 7.45 (d, J=7.8 Hz, 2H), 8.49 (s, 1H).
3
3
3
3
(d, Jcis=11.1 Hz, 1H), 6.51 (d, Jtrans=15.6 Hz, 1H),
3
6.61 (d, J=8.6 Hz, 2H), 6.87(s, 1H), 7.00 (d, 3J=8.6
Hz, 2H), 7.08–7.16 (m, 6H), 7.21–7.27 (m, 2H), 7.59 (d,
3
LC–MS (ESI-1, Grad. A): 674.3 [M+H]+; Rt=13.63
min; MS (MALDI-TOF): 674.9 [M+H]+, 696.9
[M+Na]+, 712.9 [M+K]+; HRMS (FAB): calcd for
C39H40N5O6 [M+H]+ 674.2979, found: 674.2947.
3J=7.0 Hz, 1H), 7.73 (d, Jtrans=15.8 Hz, 1H), 8.40 (s,
3
1H); LC–MS (ESI-1 Grad. A): 662.4 [MꢀH]ꢀ, 664.3
[M+H]+, 686.3 [M+Na]+; Rt=15.53 min; MS
(MALDI-TOF): 664.9 [M+H]+, 686.9 [M+Na]+,
702.9 [M+K]+; HRMS (FAB): calcd for C38H42N5O6
[M+H]+: 664.3135, found: 664.3150.
Nꢀ-3-(4-Biphenyl)-E/Z-acryloyl-D-histidyl-N-methyl-L-
phenylalanyl-L-tyrosine hydrotrifluoracetate (35/20).
According to GP P2, the polymer-bound tripeptide 34a
(400 mg, 0.40 mmol/g, 160 mmol) was N-terminally
deblocked by piperidine/DMF treatment for 2ꢂ5 min,
followed by coupling with 30b (179 mg, 800 mmol),
PyAOP (417mg, 800 mmol), HOAt (109 mg, 800 mmol),
DMAP (25 mg, 200 mmol), DIEA (343 mL, 2.0 mmol) in
Nꢀ-3-[2-(n-Pentyl)phenyl]propionyl-D-histidyl-L-phenyl-
alanyl-L-tyrosin hydrotrifluoroacetate (36). Starting
from 34a (300 mg, 0.40 mmol/g, 120 mmol) polymer-
bound compound Nꢀ-3-[2-(1-Z-n-pentenyl)phenyl]-
E-acryloyl-D-histidyl-L-phenylalanyl-L-tyrosine-hydrotri-
fluoroacetate was synthesized according to the proce-
dure described for 35/18. After cleavage from the resin
the product by treatment with 2ꢂ5 mL TFA/CH2Cl2/
H2O 47/47/6 (v/v) for 10 and 20 min and 2ꢂ5 mL TFA/
H2O 95/5 (v/v) for 30 and 180 min according to GP P5
variant B the cleaving solutions were concentrated in
vacuo. The residue was dissolved in 5 mL dioxane, Pd (50
mg, 10% on charcoal) was added and the suspension was
stirred for 8 h under an hydrogen atmosphere. After fil-
tration over Celite, the filtrate was concentrated. Purifica-
tion by HPLC and lyophilisation afforded 36 (28 mg,
30%, eight steps) as a white solid; HPLC (A2, CH3CN/
7mL DMF/CH Cl2 2/1 (v/v) for 18 h according to GP
2
P8. After the liberation of the C-terminus with
Pd(PPh3)4 in 5 mL morpholine/THF for 16 h according
to GP P1 variant C, the product was cleaved from the
resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47 /
47/6 (v/v) for 2ꢂ45 min and 2ꢂ5 mL TFA/H2O 95/5 (v/
v) for 2ꢂ1.5 h according to GP P5 variant B followed
by HPLC purification affording 35/20 (27mg, 21%) as
a white solid as a mixture of E- and Z-isomer (E/
Z=1.6/1, according to 1H NMR). HPLC (A2, CH3CN/
H2O/TFA, 20/80/0.1–70/30/0.1 in 30 min, 50 ꢁC):
Rt=15.20 min (E-isomer), Rt=16.34 min (Z-isomer);
20
D
H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min, 50ꢁC):
mp: 161 ꢁC; ½aŠ ꢀ43.9 (c 0.59, MeOH); 1H NMR
20
D
Rt=14.70 min, purity: >95%; mp: 120–121 ꢁC; ½aŠ
(400 MHz, CD3OD): Z-isomer: d 2.29–2.43 [2.59–2.70]
(m, 1H), 2.42 [2.58] (s, 3H), 2.81–3.05 (m, 4H), 3.06–
3.28 (m, 2H), 4.50–4.68 (m, 1H), 5.01–5.20 (m, 1H), 5.31–
5.50 (m, 1H), 4.68–4.72 (m, 1H), 5.86 [5.96] [6.01] (d,
1
+7.2 (c 1.00, MeOH); H NMR (400 MHz, CD3OD): d
0.89 (t, 3J=7.2 Hz, 3H), 1.31–1.35 (m, 4H), 1.52–1.58 (m,
2H), 2.43–2.49 (m, 2H), 2.60 (t, 3J=7.8 Hz, 2H), 2.73–2.89
(m, 4H), 2.92–3.03 (m, 2H), 3.10–3.15 (m, 2H), 4.55–4.64
3
3Jcis=12.5 Hz, 1H), 6.58 (d, J=8.4 Hz, 2H), 6.65–6.69

2612
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
[6.78–6.82] [6.88–6.93] (m, 1H), 6.97–7.01 (m, 1H), 7.09–
7.25 (m, 9H), 7.32–7.36 (m, 1H), 7.41–7.45 (m, 3H),
7.52–7.56 (m, 1H), 7.59–7.70 (m, 7H), 8.65 [8.70] [8.73]
(s, 1H); E-isomer: d 2.29–2.43 [2.59–2.70] (m, 1H), 2.42
[2.58] (s, 3H), 2.81–3.05 (m, 4H), 3.06–3.28 (m, 2H),
4.50–4.68 (m, 1H), 5.01–5.20 (m, 1H), 5.31–5.50 (m,
6H), 4.43–4.58 (m, 3H), 5.04 (s, 2H), 5.90 (d, 3Jcis=12.5
Hz, 1H), 6.60 (d, J=8.4 Hz, 2H), 6.71–6.75 (m, 1H),
3
6.79–6.82 (m, 1H), 6.93–6.97 (m, 3H), 7.10–7.12 (m,
6H), 7.25–7.33 (m, 3H), 7.36–7.42 (m, 3H), 7.53 (d,
3J=8.8 Hz, 1H), 8.27(s, 1H); E-isomer: d 2.74–3.10 (m,
6H), 4.43–4.58 (m, 3H), 5.14 (s, 2H), 6.54 (d,
3
3
1H), 4.68–4.72 (m, 1H), 6.46 [6.56] (d, Jtrans=15.8 Hz,
3Jtrans=15.8 Hz, 1H), 6.60 (d, J=8.4 Hz, 2H), 6.71–
1H), 6.58 (d, 3J=8.4 Hz, 2H), 6.65–6.69 [6.78–6.82]
[6.88–6.93] (m, 1H), 6.97–7.01 (m, 1H), 7.09–7.25 (m,
8H), 7.32–7.36 (m, 1H), 7.41–7.45 (m, 3H), 7.52–7.56
(m, 1H), 7.59–7.70 (m, 8H), 8.65 [8.70] [8.73] (s, 1H);
LC–MS (ESI-1, Grad. B): 686.2 [M+H]+, 708.3
[M+Na]+, Rt=14.22 min (isomer 1), Rt=15.31 (iso-
mer 2); MS (MALDI-TOF): 686.1 [M+H]+; HRMS
(FAB): calcd for C40H40N5O6 [M+H]+: 686.2979,
found: 686.3008.
6.75 (m, 1H), 6.79–6.82 (m, 1H), 6.93–6.97 (m, 3H),
7.10–7.12 (m, 6H), 7.25–7.33 (m, 3H), 7.36–7.42 (m,
3
3H), 7.53 (d, J=8.8 Hz, 1H), 8.31 (s, 1H); LC–MS
(ESI-1, Grad. B): 702.3 [M+H]+, 724.3 [M+Na]+,
Rt=17.77 min (isomer 1), 702.3 [M+H]+, 724.3
[M+Na]+, Rt=18.73 min (isomer 2).
Nꢀ-3-[3-(Benzyloxy)phenyl]-E-acryloyl-L-histidyl-L-phe-
nylalanyl-L-tyrosin-hydrotrifluoroacetate (35/23). Reac-
tion with 30d starting from tripeptide 34b according to
GP P16 furnished 35/23 (28 mg, 21%, eight steps) as a
white solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–
General procedure for the synthesis of the compounds
35/21–35/26 (GP P16)
70/30/0.1 in 30 min, 50 ꢁC): Rt=16.72 min, purity: 91%;
20
D
According to GP P2, the polymer-bound tripeptide 34a
or 34b (400 mg, 0.40 mmol/g, 160 mmol) was N-termin-
ally deblocked by piperidine/DMF treatment for 2ꢂ5
min, followed by coupling with 30c, 30d, or 30e (122
mg, 480 mmol), PyAOP (250 mg, 480 mmol), DMAP (20
mg, 160 mmol) and DIEA (274 mL, 1.6 mmol) in 7mL
DMF/CH2Cl2 2/1 (v/v) for 18 h. After the liberation of
the C-terminus with Pd(PPh3)4 in 5 mL morpholine/
THF for 16 h according to GP P1 variant C the product
was cleaved from the resin by treatment with 2ꢂ5 mL
TFA/CH2Cl2/H2O 47/47/6 (v/v) for 2ꢂ45 min and 2ꢂ5
mL TFA/H2O 95/5 (v/v) for 2ꢂ1.5 h according to GP
P5 variant B followed by HPLC purification affording
the appropriate product.
mp: 147 ꢁC; ½aŠ ꢀ23.9 (c 0.90, MeOH); 1H NMR
(400 MHz, CD3OD): d 2.80–3.21 (m, 3H) 3.07–3.15 (m,
3H), 4.56 (dd, J=5.3 Hz, J=8.4 Hz, 1H), 4.61 (dd,
3
3
3
3J=5.3, J=9.2 Hz, 1H), 4.71–4.73 (m, 1H), 5.07 (s,
3
3
2H), 6.52 (d, J=15.6 Hz, 1H), 6.65 (d, J=8.6 Hz,
2H), 6.98–7.04 (m, 3H), 7.06–7.20 (m, 8H), 7.24–7.37
3
(m, 4H), 7.38–7.42 (m, 2H), 7.45 (d, J=15.8 Hz, 1H),
8.62 (s, 1H); LC-MS (ESI-1, Grad. B): 700.4 [M-H]ꢀ,
702.3 [M+H]+, 724.3 [M+Na]+, Rt=20.76 min; MS
(MALDI-TOF): 703.66 [M+H]+, 725.80 [M+Na]+;
HRMS (FAB): calcd for C40H40N5O7 [M+H]+:
702.2928, found: 702.2945.
Nꢀ-3-[3-(benzyloxy)phenyl]-E/Z-acryloyl-D-histidyl-L-
phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/24).
Reaction with 30d starting from tripeptide 34a accord-
ing to GP P16 furnished 35/24 (37mg, 28%) as a white
solid as a mixture of E- und Z-isomer (E/Z=1.7/1,
according to 1H NMR); HPLC (A2, CH3CN/H2O/
TFA, 20/80/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=14.76
Nꢀ-3-[2-(Benzyloxy)phenyl]-E-acryloyl-L-histidyl-L-phenyl-
alanyl-L-tyrosine hydrotrifluoroacetate (35/21). Reaction
with 30c starting from tripeptide 34b according to GP
P16 furnished 35/21 (16 mg, 13%, eight steps) as a white
solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/0.1–70/30/
0.1 in 30 min, 50 ꢁC): Rt=15.61 min, purity: 89%; mp:
min (Z-isomer), Rt=15.72 min (E-isomer); mp: 144 ꢁC;
20
D
20
D
118 ꢁC; ½aŠ ꢀ3.7( c 0.43, MeOH); 1H NMR (400 MHz,
½aŠ
ꢀ3.5 (c 0.48, MeOH); 1H NMR (400 MHz,
CD3OD): d 2.80 (d, 3J=9.8 Hz, 1H), 2.89–2.95 (m, 2H),
CD3OD) Z-isomer: d 2.69–2.88 (m, 3H), 2.94–3.09 (m,
3H), 4.50–4.59 (m, 3H), 4.98 (s, 2H), 5.78 (d, J=12.7
3
3
3.07–3.15 (m, 3H), 4.58 (d, J=5.3 Hz, 1H), 4.64 (d,
3J=4.9 Hz, 1H), 4.68–4.72 (m, 1H), 5.09 (s, 2H), 6.56
(d, 3Jtrans=15.8 Hz, 1H), 6.66 (d, 3J=8.4 Hz, 2H), 6.92-
7.05 (m, 2H), 7.02–7.17 (m, 8H), 7.27–7.37 (m, 5H), 7.42
Hz, 1H), 6.59–6.61 (m, 2H), 6.81–6.89 (m, 2H), 6.92–
7.01 (m, 4H), 7.05–7.17 (m, 5H), 7.20–7.45 (m, 7H), 8.52
(s, 1H); E-isomer: d=2.69–2.88 (m, 3H), 2.94–3.09 (m,
3
3
3
(d, J=7.2 Hz, 2H), 7.56 (d, Jtrans=15.8 Hz, 1H), 8.63
(s, 1H); LC–MS (ESI-1, Grad. B): 700.4 [MꢀH]ꢀ, 702.3
[M+H]+, Rt=19.21 min; MS (Maldi-TOF): 703.09
[M+H]+, 725.59 [M+Na]+; HRMS (FAB): calcd for
C40H40N5O7, [M+H]+; 702.2928, found: 702.2937.
3H), 4.50–4.59 (m, 3H), 5.05 (s, 2H), 6.41 (d, J=15.6
Hz, 1H), 6.59–6.61 (m, 2H), 6.81–6.89 (m, 2H), 6.92–
7.01 (m, 4H), 7.05–7.17 (m, 5H), 7.20–7.45 (m, 7H), 8.54
(s, 1H); LC–MS (ESI-1, Grad. B): 702.3 [M+H]+,
724.3 [M+Na]+, Rt=14.07min (isomer 1), 072.3
[M+H]+, 724.3 [M+Na]+, Rt=15.02 min (isomer 2);
MS (MALDI-TOF): 704.2 [M+H]+, 725.6 [M+Na]+.
Nꢀ-3-[2-(benzyloxy)phenyl]-E/Z-acryloyl-D-histidyl-L-
phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/22).
Reaction with 30c starting from tripeptide 34a accord-
ing to GP P16 furnished 35/22 (13 mg, 10%, eight steps)
as a white solid as a mixture of E- and Z-isomer
(E/Z=1/2.5, according to 1H NMR); HPLC (A2,
CH3CN/H2O/TFA, 20/80/0.1–90/10/0.1 in 40 min,
50 ꢁC): Rt=14.12 min (Z-isomer), Rt=14.50 min (E-
Nꢀ-3-[4-(benzyloxy)phenyl]-E/Z-acryloyl-L-histidyl-L-
phenylalanyl-L-tyrosine hydrotrifluoroacetate (35/25).
Reaction with 30e starting from tripeptide 34b accord-
ing to GP P16 furnished 35/25 (14 mg, 11%, eight steps)
as a white solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/
0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=15.84 min (Z-iso-
20
D
20
D
(c 0.55, MeOH); H NMR (400 MHz, CD3OD) Z-iso-
isomer); mp: 127 ꢁC; ½aŠ ꢀ15.3 (c 0.50, MeOH); H
mer), Rt=16.78 min (E-isomer); mp: 145 ꢁC; ½aŠ ꢀ22.2
1
1
NMR (400 MHz, CD3OD): Z-isomer: d 2.74–3.10 (m,

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2613
mer: d 2.80–2.91 (m, 2H), 2.97–3.17 (m, 4H), 4.54–4.78 (m,
were added 15 mL dry DMF and after swelling for some
min, DBU (1.05 mL, 7mmol) and 2-mercaptoethanol
(981 mL, 14 mmol). The suspension was shaken for 1 h,
whereby the solution becomes yellow-green. After fil-
tering the resin was washed with 3ꢂ5 mL DMF, 3ꢂ5
mL DMF/H2O 1/1 (v/v), 3ꢂ5 mL DMF, 3ꢂ5 mL
MeOH and 4ꢂ5 mL CH2Cl2 and dried in vacuo. Then
the deprotected, polymer-bound compound (1.20 g, 0.50
mmol/g, 600 mmol) was reacted with Fmoc-l-His(Trt)OH
(2.08 g, 3.36 mmol), N,N-diisopropylcarbodiimide (520
mL, 3.36 mmol), HOAt (457mg, 3.36 mmol) and lutidine
(390 mL, 3.36 mmol) in 15 mL DMF for 24 h according
GP P4 variant B furnishing 38. Ninhydrine test:15 nega-
tive; analysis after cleavage from a polymer sample:
HPLC (A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in
30 min, 50 ꢁC): Rt=5.13 min; LC–MS (ESI-1, Grad. A):
595.4 [M+H]+, 617.3 [M+Na]+, Rt=4.22 min.
3
3H), 5.04 (s, 2H), 5.79 (d, Jcis=12.5 Hz, 1H), 6.64–6.67
(m, 3H), 6.86 (d, 3J=8.8 Hz, 1H), 6.98–7.04 (m, 5H), 7.07–
7.18 (m, 2H), 7.26–7.51 (m, 9H), 8.60 (s, 1H). E-isomer: d
2.80–2.91 (m, 2H), 2.97–3.17 (m, 4H), 4.54–4.78 (m, 3H),
3
5.09 (s, 2H), 6.41 (d, Jtrans=15.8 Hz, 1H), 6.64–6.67(m,
3H), 6.86 (d, 3J=8.8 Hz, 1H), 6.98–7.04 (m, 5H), 7.07–7.18
(m, 2H), 7.26–7.51 (m, 9H), 8.62 (s, 1H); LC–MS (ESI-1,
Grad. B): 702.3 [M+H]+, 724.3 [M+Na]+, Rt=15.22
min (isomer 1); 702.3 [M+H]+, 724.3 [M+Na]+,
Rt=16.16 min (isomer 2); MS (MALDI-TOF): 703.9
[M+H]+, 725.5 [M+Na]+; HRMS (FAB): calcd for
C40H40N5O7 [M+H]+ 702.2928, found: 702.2859.
Nꢀ-3-[4-(Benzyloxy)phenyl]-E-acryloyl-D-histidyl-N-L-
phenylalanyl-L-tyrosin hydrotrifluoroacetate (35/26).
Reaction with 30e starting from tripeptide 34a accord-
ing to GP P16 furnished 35/26 (27mg, 19%, eight steps)
as a white solid; HPLC (A2, CH3CN/H2O/TFA, 20/80/
Nꢀ-9-Fluorenylmethoxycarbonyl-L-histidyl-N-methyl-L/
D-tyrosine-N-methyl-L-phenylalanine allyl ester hydrotri-
fluoracetate (40). According to GP P1 variant B, poly-
mer-bound compound 38 (1.20 g, 0.50 mmol/g, 600
mmol) was reacted with Pd(PPh3)4 in 15 mL THF/NMA
4/1 (v/v), then it was coupled with 26 (569 mg, 2.52
mmol), PyAOP (1.32 g, 2.52 mmol), HOAt (114 mg, 840
mmol) and DIEA (1.29 mL, 7.56 mmol) in 15 mL NMP
according to GP P3 variant B furnishing the polymer-
bound product 39.
0.1–90/10/0.1 in 40 min, 50 ꢁC): Rt=13.48 min, purity:
20
>95%; mp: 127 ꢁC; ½aŠ +5.5 (c 0.33, MeOH); 1H
D
NMR (400 MHz, CD3OD): d 2.74 (d, 2J=13.9 Hz,
3J=9.8 Hz, 1H), 2.83–2.90 (m, 2H), 3.01–3.09 (m, 3H),
4.51 (dd, 3J=5.3 Hz, 3J=8.3 Hz, 1H), 4.69 (dd, 3J=4.7
3
3
3
Hz, J=9.6 Hz, 1H), 4.65 (dd, J=6.5 Hz, J=7.4 Hz,
3
1H), 4.83 (s, 2H), 6.52 (d, Jtrans=15.8 Hz, 1H), 6.55–
6.63 (m, 3H), 6.68–6.77 (m, 1H), 6.87 (s, 1H), 6.93–6.99
3
(m, 2H), 7.07–7.15 (m, 11H), 7.31 (d, J=7.6 Hz, 1H),
3
7.88 (d, Jtrans=15.8 Hz, 1H), 8.51 (s, 1H); LC–MS
(ESI-2): 702.38 [M+H]+, Rt=1.70 min; MS (MALDI-
Treatment of 39 twice with TFA/H2O/EMS 90/5/5 for
2ꢂ2.5 h and HPLC-purification furnished two isomers
40a and 40b of the tripeptide Nꢀ-9-fluorenylmethoxy-
carbonyl-L-histidyl-N-methyl-L/D-tyrosine-N-methyl-L-
phenylalaninallyl ester-hydrotrifluoroacetate.
TOF): 702.6 [M+H]+, 724.6 [M+Na]+.
Solid-phase bound N-methyl,N-(o-nitro-benzolsulfonyl)-
L-tyrosine allyl ester (37). According to GP P2, poly-
mer-bound compound L-8 (2.80 g, 0.50 mmol/g, 1.4
mmol) was reacted twice with 10 mL piperidine/DMF
for 15 min, then it was coupled with o-nitrobenzene-
sulfonic acid chloride (1.24 g, 5.6 mmol) and 2,6-luti-
dine (813 mL, 7.0 mmol) in 10 mL CH2Cl2. After
shaking the suspension for 11 h under argon atmo-
sphere, the resin was filtered off and washed with 3ꢂ5
mL CH2Cl2, 3ꢂ5 mL DMF/H2O 1/1 (v/v), 3ꢂ5 mL
DMF, 3ꢂ5 mL MeOH und 5ꢂ5 mL CH2Cl2 and finally
with 8ꢂ5 mL dry THF, 4ꢂ5 mL dry CH2Cl2 and 2ꢂ5
mL dry DMF under argon. To the polymer bound
compound were added 5 mL dry DMF, MTBD (603
mL, 4.2 mmol) and afterwards a solution of methyl-4-
nitrobenzenesulfonate (1.22 g, 5.6 mmol) 15 mL DMF.
After shaking for 1.5 h, the resin is filtered off, washed
with 2ꢂ10 mL DMF, 2ꢂ10 mL MeOH and 3ꢂ10 mL
CH2Cl2 and dried in vacuo. Analysis of a sample of the
polymer: The analytical data confirms the complete
reaction of the starting material. HPLC (A2, CH3CN/
H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min): Rt=8.67min,
purity: >95%; MS [EI, 70 eV, m/z (%)]: 420 (7) [M+],
40a. 38 mg, 29%, seven steps, brownish solid; HPLC
(A2, CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
50 ꢁC): Rt=6.35 min, purity: >95%; mp: 115–116 ꢁC,
20
D
½aŠ ꢀ100.7( c 1.0, MeOH), 1H NMR (400 MHz,
CD3OD): d 2.34–2.54 (m, 2H), 2.40 [2.54] (s, 3H), 2.67–
3.05 (m, 3H), 2.53 [2.74] (s, 3H), 3.11–3.18 (m, 1H),
4.01–4.08 [4.22–4.30] (m, 1H), 4.37–4.59 (m, 4H), 4.61–
4.68 (m, 1H), 4.80 (m, 2H), 5.03–5.34 (m, 3H), 5.78–5.85
(m, 1H), 6.55 (d, 3J=8.4 Hz, 2H), 6.80 (m, 2H), 6.90 (m,
1H), 7.04–7.06 (m, 2H), 7.10–7.19 (m, 5H), 7.22–7.28
3
3
(m, 2H), 7.58 (d, J=7.6 Hz, 2H), 7.67 (d, J=7.4 Hz,
2H), 8.56 (s, 1H); LC-MS (ESI 1, Grad. B): 756.2
[M+H]+, 778.4 [M+Na]+, Rt=12.09 min.
40b. 31 mg, 23%, brownish solid; HPLC (A2,
CH3CN/H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min,
50 ꢁC): Rt=7.99 min, purity: 90%; mp: 113–114 ꢁC;
20
D
½aŠ ꢀ192.3 (c 0.20, MeOH); 1H NMR (400 MHz,
335
(14)
[M+ꢀC3H5ꢀCO2],
149
107(100)
(49),
CD3OD): d 2.38–3.23 (m, 12H), 4.09–4.20 (m, 1H),
4.37–4.59 (m, 2H), 4.49–4.70 (m, 2H), 4.72–4.85 (m,
2H), 5.14–5.25 (m, 2H), 5.50 (m, 1H), 5.79–5.89 (m,
[M+ꢀCO2ꢀSO2C6H4NO2ꢀC3H5ꢀOH],
[C7H7O+], 77 (4), [C6H+5 ]; HRMS (EI, 70 eV, m/z): calcd
for C19H20N2O7S [M+]: 420.0991, found: 420.0999.
3
1H), 6.62 (d, J=8.4 Hz, 2H), 6.80–6.83 (m, 2H), 7.00–
7.01 [7.02–7.04] (m, 1H), 7.09–7.21 (m, 5H), 7.27–7.31
Polymer-bound Nꢀ-9-Fluorenylmethoxycarbonyl-L-histi-
dyl-N-methyl-L-tyrosine-allyl ester (38). To polymer-
bound compound 37 (2.70 g, 0.50 mmol/g, 1.35 mmol)
(m, 2H), 7.37–7.40 (t, J=7.4 Hz, 2H), 7.57 (d, J=7.4
Hz, 2H), 7.78 (d, 3J=7.6 Hz, 2H), 8.67 (s, 1H); LC–ESI
(ESI-1, Grad. B): 756.3 [M+H]+, Rt=14.02 min;
3
3

2614
M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
HRMS (FAB): calcd for C44H45N5O7 [M+H]+
756.3397, found: 756.3410.
mmol) according to GP P3 in 5 mL NMP for 8 h
affording dipeptides 43.
Nꢀ-Benzyloxycarbonyl-L-histidyl-N-methyl-L/D-tyrosyl-N-
General procedure for the synthesis of dipeptides of type
44 (GP P18)
methyl-L-phenylalanine
hydrotrifluoroacetate
(42b).
According to GP P2, polymer-bound compound 39
(336 mg, 0.52 mmol/g, 175 mmol) was reacted twice with
6 mL piperidine/DMF for 15 min, then it was coupled
with 18a (191 mL, 1.12 mmol), DMAP (14 mg, 112
mmol), HOAt (152 mg, 1.12 mmol) and DIEA (383 mL,
2.24 mmol) in 9 mL DMF/CH2Cl2 for 8 h according to
GP P10. After deblocking of the carboxylic function
with Pd(PPh3)4 in 10 mL morpholine/THF for 24 h
according to GP P1 variant C the product is cleaved
from the resin by treatment with TFA/H2O/TES 90/5/5
(v/v/v) for 24 h according to GP P5 variant A affording
42b (21 mg, 16%) as a brown solid as a diastereomeric
mixture (7:3); HPLC (A2, CH3CN/H2O/TFA, 20/80/
According to GP P2, polymer-bound compounds 43
were N-terminally deblocked with 2ꢂ5 mL piperidine/
DMF for 2ꢂ3 min, followed by coupling with Fmoc-d-
Tyr(tBu)OH (223 mg, 486 mmol) by reaction with
HATU (170 mg, 440 mmol), HOAt (22 mg, 162 mmol)
and DIEA (277 mL, 162 mmol) in 5 mL NMP for 14 h
according to GP P4 variant A affording the polymer
bound compounds 44.
Polymer bound N-9-Fluorenylmethoxycarbonyl-(O-tert-
butyl)-D-tyrosyl-L-tyrosyl-L-phenylalanine allyl ester
(44a). Reaction of L-8 (400 mg, 0.41 mmol/g, 162
mmol) with 28a (185 mg, 490 mmol) according to GP
P17furnished the dipeptide 43a which was reacted with
Fmoc-d-Tyr(tBu)OH according to GP P18 affording
44a; Ninhydrine test: negative; TFA-cleavage from a
polymer sample furnished the deprotected compound
Fmoc-Tyr-Tyr-Ph-OH: HPLC (A2, CH3CN/H2O/TFA,
45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=16.81 min;
LC–MS (ESI-1, Grad. A): 754.1 [M+H]+, Rt=13.58
min; C45H43N3O8 (753.84).
0.1–90/10/0.1 in 40 min, 50 ꢁC): Rt=11.33 min (isomer
20
D
1), Rt=11.83 min (Isomer 2); mp: 109 ꢁC; ½ꢀŠ ꢀ65.3 (c
1
1.00, MeOH); H NMR (400 MHz, CD3OD): d 2.34–
3.22 (m, 12H), 4.58–4.78 (m, 2H), 5.08–5.14 (m, 1H),
5.41–5.49 (m, 2H), 6.56–6.71 (m, 3H), 6.91 (m, 1H),
6.98–7.03 (m, 1H), 7.08–7.35 (m, 10H), 8.63–8.67 (m,
1H); LC-MS (ESI-1, Grad A): 628.2 [M+H]+, 650.2
[M+Na]+, Rt=2.43 (isomer 1), Rt=2.48 (isomer 2);
MS (MALDI-TOF): 628.62 [M+H]+, 650.61
[M+Na]+; HRMS (FAB): calcd for C34H38N5O7
[M+H]+ 628.2771, found: 628.2761.
Polymer-bound N-9-Fluorenylmethoxycarbonyl-(O-tert-
butyl)-D-tyrosyl-L-tyrosyl-L-3-(2-naphthyl)alanine allyl
ester (44b). Reaction of L-8 (400 mg, 0.41 mmol/g, 162
mmol) with 28b (209 mg, 490 mmol) according to GP
P17furnished the dipeptide 43b which was reacted with
Fmoc-d-Tyr(tBu)OH according to GP P18 affording
44b. Ninhydrine Test:15 negative; TFA-cleavage from a
polymer sample furnished the deprotected compound
Fmoc-Tyr-Tyr-Naph-OH: HPLC (A2, CH3CN/H2O/
TFA, 45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=13.96
min; MS (MALDI-TOF): 827.0 [M+Na]+, 842.9
Nꢀ-3-Phenylpropionyl-L-histidyl-N-methyl-L/D-tyrosyl-
N-methyl-L-phenylalanin-hydrotrifluoroacetate
(42a).
According to GP P2, polymer-bound compound 39
(336 mg, 0.52 mmol/g, 175 mmol) was reacted twice with
6 mL piperidine/DMF for 15 min, then it was coupled
with 29c (135 mg, 896 mmol), HATU (319 mg, 840
mmol), HOAt (38 mg, 280 mmol) and DIEA (479 mL) in
9 mL NMP for 8 h according to GP P9. After deblock-
ing of the carboxylic function with Pd(PPh3)4 in 10 mL
morpholine/THF for 24 h according to GP P1 variant C
the product is cleaved from the resin by treatment with
TFA/H2O/TES 90/5/5 (v/v/v) for 24 h according to GP
P5 variant A affording 42a (66 mg, 50%) as a brown
solid as a diastereomeric mixture (7/3); HPLC (A2,
CH3CN/H2O/TFA, 20/80/0.1–90/10/0.1 in 50 min,
50 ꢁC) Rt=17.88 min (isomer 1), Rt=19.14 min (isomer
[M+K]+; Fmoc-Tyr-Tyr-Naph-OH:
(803.91).
C49H45N3O8
General procedure for the synthesis of tripeptides of the
type N-3-[(4-(n-pentyl)phenyl]propionyl-D-Tyr-Tyr-X-
OH (GP P19)
20
D
2); mp: 122 ꢁC; ½aŠ ꢀ71.8 (c 0.32, MeOH); H NMR
According to GP P2, the polymer-bound tripeptide 44
(400 mg, 0.41 mmol/g, 162 mmol) was reacted twice with
5 mL piperidine/DMF for 5 min, the compound is then
reacted with 29b (117mg, 530 mmol), HATU (188 mg,
480 mmol), HOAt (22 mg, 162 mmol) and DIEA (277
mL, 1.62 mmol) in 5 mL NMP for 12 h according to GP
P9. After deblocking of the carboxylic function with
Pd(PPh3)4 in 10 mL morpholine/THF for 24 h accord-
ing to GP P1 variant C the product is cleaved from the
resin by treatment with 2ꢂ5 mL TFA/CH2Cl2/H2O 47 /
47/6 (v/v/v) for 2ꢂ15 min and with 2ꢂ5 mL TFA/H2O
95/5 (v/v) for 2ꢂ1.5 h according to GP P5 variant B.
1
(400 MHz, CD3OD): d 2.29–3.23 (m, 16H), 4.58–4.64
(m, 1H), 5.08–5.14 (m, 1H), 5.28–5.48 (m, 1H), 6.56–
6.75 (m, 3H), 6.85–6.93 (m, 1H), 7.01–7.04 (m, 1H),
7.13–7.38 (m, 10H), 8.61 [8.64] (m, 1H). LC–MS (ESI-1,
Grad A): 626.2 [M+H]+, 648.3 [M+Na]+; 2.50 min;
HRMS (FAB): calcd for C35H41N5O6 [M+H]+
626.2979, found: 626.2958.
General procedure for the synthesis of dipeptides of type
43 (GP P17)
According to GP P1 variant B, polymer-bound com-
pound L-8 (400 mg, 0.41 mmol/g, 162 mmol) was C-
terminally deblocked with Pd(PPh3)4 in 5 mL NMA/
THF followed by coupling with 3 equiv of 28a or 28b,
PyAOP (256 mg, 490 mmol) and DIEA (528 mL, 3.10
N-3-[(4-(n-pentyl)phenyl]propionyl-D-tyrosyl-L-tyrosyl-L-
phenylalanine (45a). Starting from tripeptide 44a reac-
tion according to GP P19 furnishes 45a (16 mg, 14%,
eight steps) as a white solid; HPLC (A2, CH3CN/H2O/

M. Thutewohl, H. Waldmann / Bioorg. Med. Chem. 11 (2003) 2591–2615
2615
TFA, 45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC): Rt=8.25
4. (a) Schlitzer, M.; Bohm, M.; Sattler, I.; Dahse, H.-M.
Bioorg. Med. Chem. 2000, 8, 1991. (b) Manne, V.; Yan, N.;
Carboni, J. M.; Tuomari, A. V.; Ricca, C. S.; Gullo Brown, J.;
Andahazy, M. L.; Schmidt, R. J.; Patel, D.; Zahler, R.;
Weinmann, R.; Der, C. J.; Cox, A. D.; Hunt, J. T.; Gordon,
E. M.; Barbacid, M.; Seizinger, B. R. Oncogene 1995, 10, 1763.
5. (a) Part of this work was published in preliminary form:
Thutewohl, M.; Kissau, L.; Popkirova, B.; Karaguni, I.-M.;
Nowak, T.; Bate, M.; Kuhlmann, J.; Muller, O.; Waldmann,
H. Angew. Chem., Int. Ed. 2002, 41, 3616. (b) Accompanying
article in this issue. doi: 10.1016/S0968-0896(03)00160-3.
6. Shiomi, K.; Yang, H.; Inokoshi, J.; Van der Pyl, D.; Naka-
gawa, A.; Takeshima, H.; Omura, S. J. Antibiot. 1993, 46, 229.
7. (a) Hinterding, K.; Hagenbuch, P.; Retey, J.; Waldmann,
H. Angew. Chem., Int. Ed. 1998, 37, 1236. Hinterding, K.;
Hagenbuch, P.; Retey, J.; Waldmann, H. Angew. Chem. 1998,
110, 1298. (b) Hinterding, K.; Hagenbuch, P.; Retey, J.;
Waldmann, H. Chem. Eur. J. 1999, 5, 227.
8. (a) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997,
97, 2243. (b) Albericio, F.; Carpino, L. A. Meth. Enzym. 1997,
289, 104.
9. Bernasconi, S.; Comini, A.; Corbella, A.; Gariboldi, P.;
Sisti, M. Synthesis 1980, 385.
10. Cabrele, C.; Langer, M.; Beck-Sickinger, A. G. J. Org.
Chem. 1999, 64, 4353.
11. Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Chang, C. D. Int. J. Pep. Prot. Res. 1979,
13, 35.
20
min, purity: >95%; mp: 214 ꢁC; ½aŠ ꢀ1.1 (c 0.47,
D
MeOH); 1H NMR (400 MHz, CD3OD): d 0.74 (t,
3J=6.8 Hz, 3H), 1.16–1.18 (m, 4H), 1.42–1.47(m, 2H),
2.26–2.32 (m, 2H), 2.39–2.43 (m, 2H), 2.51–2.61 (m,
2H), 2.65–2.72 (m, 4H), 2.86–2.91 (m, 1H), 3.03–3.07
(m, 1H), 4.26–4.33 (m, 2H), 4.51–4.54 (m, 1H), 6.51–
3
3
6.55 (m, 4H), 6.61 (d, J=8.4 Hz), 6.70 (d, J=8.4 Hz,
2H), 6.92–7.15 (m, 9H); LC–MS (ESI-1, Grad. A):
694.2 [M+H]+, 716.4 [M+Na]+; Rt=11.78 min; MS
(MALDI-TOF): 694.9 [M+H]+, 716.8 [M+Na]+;
HRMS (FAB): calcd for C41H48N3O7 [M+H]+
694.3492, found: 694.3522.
N-3-[(4-(n-Pentyl)phenyl]propionyl-D-tyrosyl-L-tyrosyl-L-
3-(2-naphthyl)alanine (45b). Starting from tripeptide 44b
reaction according to GP P19 furnishes 45b (26 mg,
22%, eight steps) as a white solid; HPLC (A2, CH3CN/
H2O/TFA, 45/55/0.1–90/10/0.1 in 30 min, 50 ꢁC)
20
D
Rt=9.74 min, purity: >95%; mp: 102 ꢁC; ½aŠ +2.0 (c
1
1.0, MeOH); H NMR (400 MHz, CD3MeOD): d 0.78
(t, J=6.94 Hz, 3H), 1.20–122 (m, 4H), 1.46–1.49 (m,
2H), 2.24–2.29 (m, 2H), 2.45 (t, J=7.8 Hz, 2H), 2.48–
2.62 (m, 2H), 2.63–2.75 (m, 4H), 3.06–3.11 (dd, 2J=13.9
Hz, 3J=7.8 Hz, 1H), 3.23–3.28 (m, 1H), 4.29 (t, 3J=7.0
3
3
3
3
Hz, 1H), 4.35 (dd, J=5.9 Hz, J=7.8 Hz, 1H), 4.66
3
12. Angell, Y. M.; Garcıa-Echeverrıa, C.; Rich, D. H. Tetra-
hedron Lett. 1994, 35, 5981.
3
(dd, J=5.5 Hz, J=7.6 Hz, 1H), 6.52–6.57 (m, 4H),
3
3
6.64 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.4 Hz, 2H), 6.94
(d, 3J=8.2 Hz), 6.98 (d, 3J=8.2 Hz, 2H), 7.18 (dd,
3J=8.4 Hz, 4J=1.8 Hz, 1H), 7.30–7.34 (m, 2H), 7.52 (s,
1H), 7.61–7.68 (m, 3H); LC–MS (ESI 1, Grad. A): 744.2
[M+H]+, 766.4 [M+Na]+, Rt=14.45 min; MS
(MALDI-TOF): 766.5 [M+Na]+; HRMS (FAB): calcd
for C45H50N3O7 [M+H]+: 744.3649, found: 744.3660.
13. (a) Henkel, B.; Zhang, L. S.; Bayer, E. J. Liebigs Ann.
Chem. 1997, 2161. (b) Leger, R.; Yen, R.; She, M. W.; Lee,
V. J.; Hecker, S. J. Tetrahedron Lett. 1998, 39, 4171. (c)
Thierit, N.; Guibe, F.; Albericio, F. Org. Lett. 2000, 2, 1815.
(d) Johanssen, A.; Akerblom, E.; Ersmark, K.; Lindeberg, G.;
Hallberg, A. J. Comb. Chem. 2000, 2, 496.
14. Davies, J. S.; Mohammed, A. K. J. Chem. Soc., Perkin
Trans. 1 1981, 2982.
15. Guy, C. A.; Fields, G. B. Meth. Enzym. 1997, 289, 67 .
16. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
Anal. Biochem. 1970, 34, 595.
17. Lewis, F. D.; Elbert, J. E.; Upthagrove, A. L.; Hale, P. D.
J. Am. Chem. Soc. 1988, 110, 5191.
Acknowledgements
18. Lewis, D.; Oxman, J. D.; Gibson, L. L.; Hampsch, H. L.;
Quillen, S. L. J. Am. Chem. Soc. 1986, 108, 3005.
19. Brettle, R.; Mosedale, A. J. J. Chem. Soc., Perkin Trans. 1
1988, 2185.
20. (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997,
119, 2301. (b) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc.
1998, 120, 2690.
This work was supported by the Fonds der chemischen
Industrie and AstraZeneca (Case Award to M.T.).
References and Notes
21. Waldmann, H.; Kunz, H. J. Liebigs Ann. Chem. 1983, 17 12.
22. Cheung, S. T.; Benoiton, N. L. Can. J. Chem. 1977, 55, 906.
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24. Miyaura, N.; Suginome, H.; Suzuki, A. Tetrahedron 1983,
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1. (a) Waldmann, H.; Thutewohl, M. Top. Curr. Chem. 2001,
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