Structure-activity relationship studies optimizing the antiproliferative activity of novel cyclic somatostatin analogues containing a restrained cyclic β-amino acid

Article abstract of DOI:10.1021/jm049500j

The cyclic somatostatin analogue cyclo[Pro¹-Phe ²-D-Trp³-Lys⁴-Thr⁵-Phe⁶] (L-363,301) displays high biological activity in inhibiting the release of growth hormone, insulin, and glucagon. According to the sequence of L-363,301, we synthesized a number of cyclic hexa- and pentapeptides containing nonnatural α- and β-amino acids. The N-fluorenylmethoxycarbonyl protected cyclic β-amino acid [1S, 2S, 5R]-2-amino-3,5-dimethyl-2-cyclohex-3-enecarboxylic acid (cβAA), for the replacement of the Phe⁶-Pro¹ moiety of L-363,301, was synthesized in two steps by an enantioselective multicomponent reaction using (-)-8-phenylmenthol as a chiral auxiliary. The resulting peptide cyclo[cβAA¹-Tyr²-D-Trp ³-Nle⁴-Thr(Trt)⁵] (Trt = triphenylmethyl) shows high antiproliferative effects in an in vitro assay with A431 cancer cells. The same peptide without the Trt group does not reveal any biological activity, whereas L-363,301 and closely related hexapeptides show only minor activity. By comparison of the solution structure of cyclo[cβAA¹-Tyr ²-D-Trp³-Nle⁴-Thr(Trt)⁵] with the structure of L-363,301, a nearly perfect match of the βII′-turn region with D-Trp in the i + 1 position was observed. The cyclic β-amino acid cβAA is likely needed for the bioactive conformation of the peptide.

Full text of DOI:10.1021/jm049500j

2916
J. Med. Chem. 2005, 48, 2916-2926
Structure-Activity Relationship Studies Optimizing the Antiproliferative
Activity of Novel Cyclic Somatostatin Analogues Containing a Restrained
Cyclic â-Amino Acid†
Martin Sukopp,‡ Richard Schwab,§ Luciana Marinelli, Eric Biron,‡ Markus Heller,‡ Edit Va´rkondi,§ AÄ kos Pap,§
Ettore Novellino, Gyo¨rgy Ke´ri,§ and Horst Kessler*,‡
Lehrstuhl fu¨r Organische Chemie und Biochemie II, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4,
85747 Garching, Germany, Cooperative Research Center, Semmelweis University, Rippl-Ronai u. 37, and Peptide Biochemistry
Research Group of Hung. Acad. Sci., H-1062 Budapest, Hungary, and Dipartimento di Chimica Farmaceutica e Tossicologica,
Universita` “Federico II” di Napoli, Via Domenico Montesano, 49 Napoli, Italy
Received June 24, 2004
The cyclic somatostatin analogue cyclo[Pro1-Phe2-D-Trp3-Lys4-Thr5-Phe6] (L-363,301) displays
high biological activity in inhibiting the release of growth hormone, insulin, and glucagon.
According to the sequence of L-363,301, we synthesized a number of cyclic hexa- and
pentapeptides containing nonnatural R- and â-amino acids. The N- fluorenylmethoxycarbonyl
protected cyclic â-amino acid [1S, 2S, 5R]-2-amino-3,5-dimethyl-2-cyclohex-3-enecarboxylic acid
(câAA), for the replacement of the Phe6-Pro1 moiety of L-363,301, was synthesized in two steps
by an enantioselective multicomponent reaction using (-)-8-phenylmenthol as a chiral auxiliary.
The resulting peptide cyclo[câAA1-Tyr2-D-Trp3-Nle4-Thr(Trt)5] (Trt ) triphenylmethyl) shows
high antiproliferative effects in an in vitro assay with A431 cancer cells. The same peptide
without the Trt group does not reveal any biological activity, whereas L-363,301 and closely
related hexapeptides show only minor activity. By comparison of the solution structure of cyclo-
[câAA1-Tyr2-D-Trp3-Nle4-Thr(Trt)5] with the structure of L-363,301, a nearly perfect match of
the âII′-turn region with D-Trp in the i + 1 position was observed. The cyclic â-amino acid
câAA is likely needed for the bioactive conformation of the peptide.
Introduction
residues D-Trp-Lys and an opposite âVI-turn about the
Phe-Pro bridging region with a cis amide bond.16 In the
model, the side chains of D-Trp and Lys are in close
proximity and the Phe6 side chain prefers the trans
rotamer.15-17 In general, the side chains of D-Trp, Lys
and one further aromatic residue in the right spatial
arrangement are important pharmacophoric groups for
most of the somatostatin analogues,18,19 whereas the
backbone contributes little to receptor binding in this
case.17,20,21 The latter is also valid for nonpeptidic
somatostatin analogues which are highly selective for
different receptor subtypes.22
Somatostatin is a cyclic tetradecapeptide hormone
with a disulfide bridge, which was isolated from bovine
hypothalami in 1973.1 First known for its ability to
inhibit the release of growth hormone, somatostatin also
suppresses the release of many other bioactive mol-
ecules (e.g. glucagon, insulin, gastrin, and secretin)2-4
and mediates tumor cell growth inhibition.5,6 The
biological effects of somatostatin are elicited through a
family of G-protein-coupled receptors (GPCR), of which
five different forms have been characterized (SSTR1-
SSTR5).7
Another interesting somatostatin analogue is TT-232
(D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) containing seven
amino acids, a five-residue ring structure, and a cystein
bridge, which was developed by Ke´ri and co-workers.23
Also in this somatostatin analogue the biologically
important D-Trp-Lys moiety in combination with one
aromatic residue (D-Phe or Tyr) is preserved. TT-232
demonstrated potent and tumor-selective antiprolifera-
tive activity without an antisecretory action. Several
lines of evidence suggest that TT-232 does not cause its
effects through somatostatin receptors but by interac-
tion with cytoplasmic and nuclear targets after inter-
nalization.24,25 Another proof is that the binding affini-
ties of TT-232 to all cloned somatostatin receptors are
very low compared to that of somatostatin.26
Starting from somatostatin, a number of smaller
peptidic and nonpeptidic analogues have been synthe-
sized in order to increase receptor subtype selectivity
and metabolic stability.8,9 Among them is the cyclic
hexapeptide L-363,301 with the sequence cyclo[Pro1-
Phe2-D-Trp3-Lys4-Thr5-Phe6], which was developed by
Veber and co-workers.10-13 This somatostatin analogue
shows higher biological activity in inhibiting the release
of growth hormone, insulin, and glucagon as well as
higher selectivity for the receptor subtype SSTR2.
However, its clinical development has been halted due
to side effects.14,15 NMR studies of L-363,301 exhibited
a backbone structure which adopts a âII′-turn about
† This paper is dedicated to the memory of Prof. Murray Goodman,
who passed away on June 1, 2004.
Our objective was to develop somatostatin or L-363,301
analogues which are further reduced in ring size and
bear mostly hydrophobic, nonnatural residues which in
general are used for better pharmacological efficacy and
* To whom correspondence should be addressed. Telephone: +49-
89-289 13300. Fax: +49-89-289 13210. E-mail: Kessler@ch.tum.de.
‡ Technische Universita¨t Mu¨nchen.
§ Semmelweis University.
Universita` “Federico II” di Napoli.
10.1021/jm049500j CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/19/2005

Somatostatin Analogue Antiproliferative Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2917
Figure 1. Somatostatin analogues based on L-363,301: Hexa- and pentapeptides with Thr(Trt) residue and/or â-amino acid
with IC50 values of A431 cancer cell assay.
ADME profiles. Herein, we report the results of our
efforts, which led to cyclic pentapeptides with high
antiproliferative activity in a cellular assay with A431
human epidermoid carcinoma cells. To explain this
finding, we carried out structure-activity relationship
(SAR) studies by NMR/modeling techniques.
series of reaction steps include an aldol reaction of two
molecules of aldehyde, the condensation of the amide
with the aldol reaction product, isomerization of double
bonds, and a Diels-Alder reaction with an acrylate as
dienophile in the end (see Scheme 1). By the final
cycloaddition reaction the 1-(acylamino)-1,3-butadiene
intermediate is removed from the equilibrium in the
reaction mixture. The reaction works best using acetic
anhydride as water scavenger, camphor sulfonic acid
as catalyst, and 2,6-di-tert-butyl-4-methylphenol (BHT)
for inhibition of acrylate polymerization. Sterically less
hindered aldehydes such as propionaldehyde (4a) and
3-phenylpropionaldehyde (4b) were used in combination
with a number of amides, e.g. acetamide (5a) and
benzylcarbamate (Z-amide) (5b). The reaction did not
proceed with Fmoc-amide and trifluoroacetamide, but
any type of acrylate that we checked yielded the desired
product. The reaction was carried out in a pressure
reactor at 140-160 °C for 16 h with DMF as solvent,
which can be easily removed after the reaction. The
products were then isolated by flash chromatography
and characterized by NMR and high-resolution mass
spectroscopy (HRMS).
Results and Discussion
Design and Synthesis of Novel Building Blocks
and Target Peptides. To compare the biological activ-
ity and structural features of L-363,301 (1a) with
L-363,301-analogues containing a â-amino acid instead
of the Phe-Pro moiety, we synthesized a number of
penta- and hexapeptides in which some residues were
replaced by homologous amino acids (Figure 1). It was
considered that incorporation of a â-amino acid and
nonnatural residues (e.g. norleucine) increases the
stability against proteases.27-29 Furthermore, to obtain
chemically stable pentapeptide analogues with a defined
conformation, the â-amino acid needed to fulfill the
following requirements: (1) small, rigid, and easily
accessible cyclic structure; (2) hydrophobic and chemical
resistant alkane backbone; (3) defined stereochemistry
with cis-configuration of amino and carboxylate sub-
stituent. A change in the biological activity should be
observed just like a previously published, bioactive
L-363,301-analogue with trans-configurated â-sugar
amino acid instead of the Phe-Pro moiety.30,31
The constitution and configuration of the câAA build-
ing block is controlled by several factors. The regiose-
lectivity of the Diels-Alder reaction yields an ortho
substituted cyclohexene â-amino acid and the endo
selectivity, which leads to an all-syn-substitution pat-
tern, causes the cis-configuration of carboxylate and
amide in positions 1 and 2 of câAA, respectively. The
primary structure of the expected R,â,ꢀ-substituted
The synthesis of câAA, which was used for incorpora-
tion into our somatostatin analogues, is based on a
multicomponent reaction published by Beller et al.32 The

2918 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sukopp et al.
Scheme 1. Synthesis of a Cyclic â-Amino Acid Building Block Using an Enantioselective Multicomponent Reaction
cyclohexene ring could be proven by TOCSY, COSY, and
HSQC NMR experiments. Measurements of JH,H cou-
For incorporation into peptides by solid-phase syn-
thesis, the amide or carbamate and the auxiliary had
to be removed from the products of the multicomponent
reaction. Therefore 7d was treated with 8% trifluo-
romethanesulfonic acid in anhydrous TFA for 2 h at 0
°C.35 After removal of acidic solvents by evaporation,
the free amino acid was protected with Fmoc-ONSu in
a 1:1 mixture of saturated NaHCO3 solution and THF
overnight. The resulting Fmoc-câAA (8) was then linked
to resin-bound L-tyrosine at the C-terminus and to both
enantiomers of Mosher’s acid36 at the N-terminus by
standard Fmoc solid-phase peptide synthesis (SPPS)37
to study the relative and absolute configuration of câAA
more in detail.
3
pling constants between HN-H2 (10.5 Hz) and H2-H1
(5.2 Hz) reveal a trans- and a cis-arrangement of the
corresponding protons to each other (Figure 2). For
control of the enantioselectivity of the Diels-Alder
reaction, easily accessible (-)-menthol and (-)-8-phe-
nylmenthol acrylates 6a,b (for synthesis see Experi-
mental Section) were used. It is well-known that in
cycloaddition reactions especially the application of (-)-
8-phenylmenthol esters favors the formation of nearly
only one diastereomer because the phenyl ring of the
chiral auxiliary blocks one side of the dienophile by
π-interaction.33,34 This is also reflected by high d.e.
(>90%) of the here-described multicomponent reaction
of cyclic â-amino acids, whereas the d.e. using (-)-
menthol is much lower. The two diastereomers of 7a-e
The ROESY spectra of 11a,b show very strong peaks,
respectively short distances of about 2.5 Å, between
H
5-H1, H5-H6proS, and HN-H6proR. Additionally the
3
with identical, characteristic JH,H coupling constants
amide proton of Tyr is in close proximity to H1 and H2
in about the same distance as that presented in Figure
(see above) could not be separated by chromatography
methods, but integration of well-separated 1H NMR
peaks (e.g. of H2) revealed the ratio of both compounds
for d.e. calculation.
6
2. Thereby, the methylene group CH2 of câAA points
in the same direction as the C5-CH3 group to bring the
three substituents of the cyclohexene ring in the steri-
cally less hindered equatorial position. Proof for the
absolute configuration is given by the differences in
chemical shifts (∆ ) δS - δR) of the S- and the R-Mosher
amides 11a,b. According to previous investigations
positive values have to be right of the H2-HN-CO-CF3
plane of the Mosher amide; negative values will be
found in the opposite direction.36 Especially the strong
high-field shifts of +110 and +75 Hz (measurements
at 500 MHz) of the C3-CH3 group and H4 confirm the
strong anisotropic shielding of the phenyl ring. Other
evidence for the absolute configuration is corresponding
ROE contacts of Tyr-HR and H6proR with the methoxy
group of the R-Mosher amide 11b, as well as one ROE
contact of Tyr-HR with the phenyl ring of the S-Mosher
amide 11a (Figure 3).
Figure 2. Conformational analysis of the câAA building block
by NMR studies. ROE contacts between protons of 11a,b are
represented by black lines.

Somatostatin Analogue Antiproliferative Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2919
peptides 3b,c, with IC50 values of 5 and 6 µM, respec-
tively. This behavior might indicate a different mode of
action that is not directly mediated by the somatostatin
receptors SSTR2 and SSTR5. Furthermore, the two
hexapeptides 2a,b that were designed to explore the
structural transition from L-363,301 (1a) to the most
active peptides 3b,c exhibit biological activities of 32
and 26 µM, respectively. It seems that already the
exchange of Lys4 to Nle4 and Phe2 to Thr2 (2a) has a
positive effect on the biological activity that can be
slightly improved by adding a Trt group to the Thr5
residue (2b). The greatest improvement in activity is
achieved by introduction of câAA1 (3b) instead of the
Phe6-Pro1 moiety, while shifting from a hexa- to an
extended pentapeptide. Additionally, the biological ac-
tivity of 3b,c is increased by approximately 50% com-
pared to a similar peptide with a trans-configurated
sugar amino acid instead of câAA (cis-configuration).30,31
Two further assumptions can be drawn from the
results of biological testing of compounds 3a-c. It seems
that the absence of trityl group in peptide 3a results in
the complete loss of antiproliferative activity (IC50 > 100
µM). Therefore, the trityl group might function as an
important pharmacophoric group. Another fact is that
the change of the Tyr2 configuration in peptides 3b,c
has only a minor influence on the activity of the
peptides. Possible reasons for these findings will be
discussed in the next paragraph dealing with the
structures of the tested peptides.
Figure 3. 1H chemical shift differences (∆δ ) δS - δR) and
essential ROE contacts for the Mosher amides of NH2-câAA-
Tyr-OH. ∆δ values are expressed in hertz (500 MHz). The 3D
structure of (R)-Mosher amide 11b shows the close proximity
of the OMe group to HR of Tyr. Arrows represent essential ROE
contacts.
The target L-363,301 analogues were successfully
synthesized via preparation of the linear peptide on solid
support and cyclization in solution (for example, see
Scheme 2). Synthesis commenced with TCP resin loaded
with side chain unprotected D- or L-Tyr. The Fmoc-Tyr
TCP resin 9 was allowed to react with 1.8 equiv of 8
using TBTU/HOBt/DIEA in NMP after deprotection of
the Fmoc group with 20% piperidine in NMP. The
peptide chain was then assembled by consecutive cleav-
age steps of Fmoc groups, and additions were done using
the same coupling reagents and 3 equiv of Fmoc amino
acid, respectively. Thereby the hydroxyl group of Thr
was protected by a Trt ether, and the D-Trp did not need
side chain protection. Since the Trt ether can be
removed easily under acidic conditions, the cleavage of
the linear peptide from the TCP resin was achieved
under very mild conditions with acetic acid in TFE and
DCM (2/1/6, v/v/v) after removal of the last Fmoc group
from the N-terminal residue. The resulting crude pep-
tide was cyclized in dilute DMF solution in the presence
of PyBOP, HOBt, and collidine. Racemization during
ring closure of about 10-20% of the C-terminal amino
acid could be observed due to sterical hindrance of the
Trt group. Target peptides with Thr(Trt) 3b,c were
purified by RP-HPLC not adding TFA to the mobile
phase in order to prevent cleavage of the Trt group. For
fully deprotected cyclic peptides 2a and 3a, nonpurified
intermediates 2b and 3b were treated with 2% TFA and
TIS in DCM for 1 h before final HPLC purification. All
target peptides exhibit a very low solubility in water
due to their hydrophobic residues, especially the ones
with a Thr(Trt) moiety.
Biological Results. Human A431 epidermoid carci-
noma cells were used for evaluating the antiproliferative
efficacy of peptides 1a, 2a,b, and 3a-3c. Thereby,
comparison of drug-treated cells with untreated cells
revealed no necrotic side effects after 6 h, indicated by
the unchanged proliferation of both cell cultures after
staining with methylene blue. However, cells treated
with different amounts of some of the peptides showed
significant reduced proliferation after 48 h compared to
the negative controls. Cycloheximide, used as a positive
control, exhibited the same behavior. The IC50 values
were generated from the growth (%) to concentration
(µM) graphs and are given in Figure 1.
Structural Analysis of L-363,301 Analogues. Struc-
turally relevant 1H and 13C chemical shifts of L-363,301
analogues 2a,b and 3a-c were assigned by TOCSY,
HMQC, and DQF-COSY experiments in DMSO-d6 at
3
300 K. Additionally, JH,H coupling constants of amide
protons and Hâ-protons were extracted from 1D spectra,
as well as temperature gradients of amide protons in
the range of 295-320 K. For structural comparison,
NMR data and 3D structures of 1a and two analogues
1b,c were taken from refs 12, 16, 18, and 19.
First structural similarities among the examined
peptides were revealed by comparison of chemical shifts,
coupling constants, and temperature gradients of amide
protons (Table 1). It is shown that the HN chemical
shifts of peptides 1a-c, which differ only in the methy-
lation of a D-Trp side chain or in the attachment of a
Lys side chain protecting group, are nearly identical.
This is reflected by a good match of secondary structures
published by Goodman et al.19 and our group.16 Espe-
cially, the âΙΙ′-turn of the D-Trp-Lys-Thr moiety is highly
conserved which is also expressed by an exact match of
HN coupling constants. A strong â-turn hydrogen bond
is indicated by a very low temperature gradient of the
Thr amide proton of approximately 0 ppb/K.38
Since chemical shifts and temperature gradients of
amide protons (Lys4/Nle4 and Thr5) as well as the
coupling constants of D-Trp3 and Lys4/Nle4 (highlighted
in italic characters in Table 1) exhibit strong consis-
tency, it can be assumed that also the secondary
structures, especially the â-turn region, of the listed
peptides are very much the same. Corresponding values
of amino acids flanking the â-turn region show larger
differences due to substitutions of residues and changes
in conformation, especially by introducing the câAA
residue.
Surprisingly, the reference peptide 1a (>50 µM) was
biologically less active in this assay than the two

2920 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sukopp et al.
Scheme 2. Synthesis of L-363,301 Analogues with Thr(Trt) Residuea
a (a) (i) 20% piperidine in NMP, (ii) 1.8 equiv of Fmoc-câAA-OH, TBTU, HOBt, DIEA, NMP, (iii) 20% piperidine in NMP, (iv) Fmoc-
Thr(Trt)-OH, TBTU, HOBt, DIEA, NMP, (v) 20% piperidine in NMP, (vi) Fmoc-Nle-OH, TBTU, HOBt, DIEA, NMP, (vii) 20% piperidine
in NMP, (viii) Fmoc-Trp-OH, TBTU, HOBt, DIEA, NMP, (ix) 20% piperidine in NMP; (b) (i) AcOH, TFE, DCM (2/1/6), RT, 1 h, (ii) 1.2
equiv of PyBOP, HOBt, collidine, DMF, RT, 16 h, (iii) purification by RP-HPLC (without TFA); (c) (i) 2% TFA in DCM, TIS, 1 h, (ii)
purification by RP-HPLC.
Table 1. Chemical Shifts, 3JH
Coupling Constants, and Temperature Gradients of Amide Protons of Cyclic Somatostatin
N,Ha
Analogues 1a-c, 2a,b, and 3a,ba
peptide
Phe2/Tyr2
D-Trp3
Lys4/Nle4
Thr5
câAA1/Phe6
chemical shift (ppm)
1a
1b
1c
2a
2b
3a
3b
3c
1a
1b
1c
2a
2b
3a
3b
3c
1a
1b
1c
2a
2b
3a
3b
3c
7.20
7.21
7.42
7.15
7.00
7.82
7.84
7.96
b
8.40
8.41
8.41
8.31
8.25
8.06
8.16
8.56
7.8
8.67
8.65
8.65
8.59
8.49
8.62
8.61
8.17
7.0
6.96
6.95
6.95
6.95
6.79
7.03
6.86
6.90
7.4
8.30
8.30
8.30
8.28
7.87
7.44
7.53
7.60
4.9
3JH
(Hz)
N,Ha
6.4
6.0
b
8.3
7.2
7.5
6.0
8.2
7.1
7.5
4.5
8.2
7.3
7.4
5.4
b
8.2
7.3
5.8
6.5
8.5
8.7
7.8
1.6
1.0
5.4
1.0
0.3
4.2
4.4
4.4
7.5
7.3
5.0
9.7
7.7
7.3
5.2
9.9
6.2
6.4
6.4
9.6
-∆δ/∆T (ppb/K)
5.1
5.4
0.3
2.8
4.8
5.0
0.2
2.8
6.0
5.4
-0.7
0.0
4.8
5.8
5.8
2.8
5.5
5.4
0.3
-0.2
5.3
8.5
6.7
-0.9
-1.0
0.0
9.2
4.9
13.1
9.2
3.6
1.2
a The values of peptides 1a-c were taken from refs 12,16,18,and 19. b No coupling constant available due to signal overlap.
Another important observation is that by removal of
the trityl group from the side chain of Thr (e.g. see 2a/b
and 3a/b) the order of chemical shifts, the coupling
constants, and probably also the backbone structure do
not change to a larger extent. In contrast to this, the
inversion of D-Tyr2 into L-Tyr2 in peptide 3c indicates
stronger conformational changes reflected by substan-
tial differences in chemical shifts and coupling constants
of the amide protons compared to 3b. Nevertheless, the
amide proton of Thr5 remains to be internally oriented
(e.g. by a hydrogen bond) indicated by a low-tempera-
ture gradient of 0 ppb/K.
For a detailed structural comparison with L-363,301,
the NMR structure of cyclo[câAA-Tyr-D-Trp-Nle-Thr-
(Trt)] (3b) was determined using a combined approach
of metric matrix distance geometry (DG) calculations
and molecular dynamics (MD) simulations based on 35
distance restraints obtained from ROESY experiments
with mixing times of 250 ms (0.8 mg of peptide in 0.5
mL of DMSO-d6 at 300 K) following standard strate-
gies.39 The prochiral methylene protons of câAA-H6 and
Tyr-Hâ could be stereospecifically assigned on the basis
of ROE values and 3JH,H coupling constants. The NMR
data and the results from DG calculations of 3b revealed
a rigid and well-defined structure of the backbone,
showing a slightly distorted âII′-turn with D-Trp3 in the
i + 1 position (Figure 4). The MD calculations were
performed first by using the experimental restraints
followed by a free dynamic simulation in an explicit
solvent box filled with DMSO.40 The rmsd over all
backbone atoms between the average structures from
restrained and free MD simulations is only 0.13 Å.
Small violation of distance restraints (<0.2 Å) during
MD calculations was observed for the Thr backbone
representing partial flexibility of this part of the cyclo-
peptide. Preferred conformations for the side chains of
Tyr2, D-Trp3, Nle4, and Thr5 were also obtained by
3
restrained DG in combination with JHR,Hâ coupling
constants. As shown in Figure 4, the bulky γ-methyl
group of Thr5 points in the same direction as the amide
proton of Thr and shields the latter from surrounding
DMSO. In combination with the internal â-turn hydro-
gen bond between HN-Thr5 and CO-Tyr2 which is
populated to a degree of 31% during restrained MD [the

Somatostatin Analogue Antiproliferative Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2921
Figure 4. Stereoview of cyclo[câAA-Tyr-D-Trp-Nle-Thr(Trt)] (3b) of the most representative structure in DMSO obtained by MD
simulations.
D-Trp in the i + 1 position) is also a key element for the
biological activity of 3b. Interestingly, câAA is able to
shorten and substitute the Phe-Pro moiety of L-363,-
301 while having the same structural effect of a âVI-
turn on the rest of the cyclopeptide. This means that
the D-amino acid can keep its strong conformational
influence on the cyclopeptide and the âII′-turn remains
in its original position. Thereby the cis-configuration of
amino and carboxylate substituents of câAA seems to
fit the conformational requirements for high activity
better than a â-amino acid building block with a trans-
configuration (2-fold increase of activity).
Similarities in orientation are also found for the side
chains of D-Trp and Nle/Lys, shown in Figure 5, whereas
significant structural differences emerge for the back-
bone and side chains of Tyr/Phe2 and Thr5 by the
influence of câAA. Especially, the voluminous trityl
group of Thr5 occupies a huge space besides the Thr
residue (right side of Figure 5) which is not populated
Figure 5. Superimposition of the NMR solution structures
by the Phe6 side chain of L-363,301 in its most favored
of cyclo[câAA-Tyr-D-Trp-Nle-Thr(Trt)] (3b; black) and L-363,-
NMR conformation. Nevertheless, superimposition of
the Trt group of Thr5 in 3b and the side chain of Phe6
in L-363,301 could be obtained if the side chain of Phe6
would switch from the preferred trans to a gauche
conformation by induced fit into the target receptor.
From biological and structural results it can be assumed
that an aromatic residue in the region of the trityl group
next to Thr5 is essential for antiproliferative activity of
L-363,301 analogues. L-363,301 itself seems to show
much lower activity since the only aromatic moiety Phe6
that might fit the pharmacophoric requirements does
not prefer the right spatial arrangement. Compared to
3b, the lower biological activity of peptide 2b with only
a Phe-Pro moiety instead of câAA also indicates this
finding. Therefore câAA seems to be needed for the
correct spatial arrangement of the Thr(Trt) side chain.
In contrast to the aromatic residue next to the Thr5
residue, the Tyr/Phe2 residue opposite of Thr5 seems not
to have importance for the biological activity of 3b. This
can be suspected from the fact that the side chain
orientations of Tyr2 or Phe2 in Figure 5 differ substan-
tially in the CR-Câ vector. Previously Goodman et al.
have shown that conformational changes of Phe2 by side
chain methylation did not influence the biological aci-
tivity of L-363,301 toward the somatostatin receptor,
whereas methylation of the more sensitive Phe6 residue
mostly yielded inactive peptides.18 Here, we observe that
the switch from L-Tyr (peptide 3b) to D-Tyr (peptide 3c)
301 (1a; gray). Only heavy atoms and amide hydrogens are
displayed for clarity.
tolerance of the angle was set to 70°, while the tolerance
of the distance was set to 3.2 Å], this might be the
reason for an extremely low temperature gradient of
-∆δ/∆T ) -1.0 ppb/K of the Thr amide proton. Other
protons such as HN-câAA1 and HN-D-Trp3 are fully
surrounded by solvent molecules according to their
temperature gradient of 13.1 and 9.2 ppb/K, respec-
tively. For the Tyr side chain a strongly preferred
gauche+ conformation can be stated on the basis of a
distinct stereospecific assignment of Hâ protons. In
contrast to the latter, the side chains of D-Trp3 and Nle4
show higher conformational freedom during DG calcula-
tions, but a close, parallel orientation of both side chains
has to be preferred since the ꢀ- and γ-protons of the Nle
chain are shifted to higher field and due to a ROE peak
ꢀ
between H -Nle and H6Ar-D-Trp with a corresponding
distance of 4.3 Å. Conformation and absolute configu-
ration of the câAA residue is in accordance with the
ROE data of 3b, coupling constants of H2 (9.5 and 3.6
Hz), and the structure of Mosher amide 11.
Structural comparison of 3b with 1a (Figure 5)
reveals a good fit of the â-turn backbone of both peptides
with a rmsd of 0.21 Å (based on Ca atoms of Tyr, D-Trp,
Nle/Lys, and Thr). It seems that the â-turn motive that
appears in a number of somatostatin analogues (with

2922 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sukopp et al.
solvent and internal reference on a Bruker DMX500 spectrom-
eter. Chemical shifts (δ) are reported in parts per million
(ppm), and coupling constants (J values) are given in hertz
(Hz). The following abbreviations were used to explain the
multiplicities: s, single; d, doublet; t, triplet; q, quartet; dd,
doublet of doublets; dt, doublet of triplets; m, multiplet; b,
broad. Thin-layer chromatography was performed on silica gel
60 F254 plates from Merck. Flash chromatography was per-
formed on silica gel 60 (230-400 mesh ASTM) from Merck.
RP-HPLC analyses and separations were conducted on Am-
ersham Pharmacia Biotech instruments using Omnicrom YMC
columns (analytical, 4.6 mm × 250 mm, 5 µm C18, 1 mL/min;
semipreparative, 20 mm × 250 mm, 10 µm C18, 8 mL/min) with
different 30 min linear gradients from water (0.1% TFA) and
CH3CN (0.1% TFA) and detection at 220 nm. Mass spectra
(ESI) were performed on a LCQ Finnigan instrument and
high-resolution mass spectra (HRMS) on a Finnigan MAT-
8200 instrument.
Synthesis of Intermediates and â-Amino Acids. (a) (-)-
Menthyl Acrylate (6a). To a solution of (-)-menthol (7.81 g,
0.05 mol) in 50 mL of THF (dry) first acrylic acid chloride (4.85
mL, 0.06 mol) and then triethylamine (10.5 mL, 0.075 mol)
were added under argon atmosphere at 0 °C and stirred for 2
h at the same temperature. After acidification with 2 N
buffered acetate solution (pH 4) and evaporation of the THF
under reduced pressure, the aqueous mixture was extracted
with EtOAc (3 × 100 mL). The combined organic layers were
dried over MgSO4, and the solvent was evaporated under
reduced pressure. Distillation (high vacuum < 1 mbar) of the
raw product afforded 6a as a colorless liquid (6.4 g, 0.03 mol,
61%): 1H NMR (CDCl3) δ 6.38 (dd, 1H, J ) 1.5 and 17.4 Hz,
CH1H2-acrylate), 6.11 (dd, 1H, J ) 10.5 and 17.4 Hz, CH-
acrylate), 5.79 (dd, 1H, J ) 1.4 and 10.3 Hz, CH1H2-acrylate),
4.76 (m, 1H, C1H), 2.04/1.02 (2 × m, 2H, C6H2), 1.87 (m, 1H,
CH-(CH3)2), 1.70/1.09 (2 × m, 2H, C3H2), 1.70/0.89 (2 × m, 2H,
C4H2), 1.51 (m, 1H, C5H), 1.42 (m, 1H, C2H), 0.91 (d, 3H, J )
6.9 Hz, C5-CH3), 0.89/0.77 (2 × d, 6H, J ) 7.0 Hz, C-(CH3)2);
13C NMR (CDCl3) δ 165.8 (CO), 130.3 (CH2-acrylate), 129.2
(CH-acrylate), 74.6 (C1), 47.4 (C2), 41.0 (C6), 34.6 (C4), 31.6 (C5),
26.7 (C-(CH3)2), 23.8 (C3), 22.4 (C5-CH3), 20.9/16.7 (C-(CH3)2);
RP-HPLC tR ) 21.2 (50-100%).
(b) (-)-8-Phenylmenthyl Acrylate (6b). To a solution of
(-)-8-phenylmenthol (5.0 g, 21.6 mmol) in 40 mL of THF (dry)
first acrylic acid chloride (2.2 mL, 27.2 mmol) and then
triethylamine (5.3 mL, 38.3 mmol) were added under argon
atmosphere at 0 °C and stirred for 2 h at the same tempera-
ture. After acidification with 2 N buffered acetate solution (pH
4) and evaporation of the THF under reduced pressure, the
aqueous mixture was extracted with EtOAc (3 × 100 mL). The
combined organic layers were dried over MgSO4, and the
solvent was evaporated under reduced pressure. Flash chro-
matography (EtOAc/n-hexane, 1/4, v/v) of the raw product
afforded 6b as a colorless oil (4.5 g, 15.8 mmol, 73%): Rf )
0.40 (n-hexane); 1H NMR (CDCl3) δ 7.29-7.22 (m, 4H, Ar),
7.10 (t, 1H, J ) 6.7 Hz, Ar), 6.01 (dd, 1H, J ) 14.2 and 4.6 Hz,
CH1H2-acrylate), 5.59 (dd, 1H, J ) 10.5 and 20.1 Hz, CH-
acrylate), 5.57-5.56 (m, 1H, CH1H2-acrylate), 4.88 (m, 1H,
C1H), 2.08-2.02 (m, 1H, C2H), 1.93/0.99 (2 × m, 2H, C6H2),
1.68/1.11 (2 × m, 2H, C3H2), 1.64/0.88 (2 × m, 2H, C4H2), 1.49
(m, 1H, C5H), 1.31 (s, 3H, C-CH3), 1.23 (s, 3H, C-CH3), 0.87
(d, 3H, J ) 6.6 Hz, C5-CH3); 13C NMR (CDCl3) δ 165.4 (CO),
151.6 (Ar), 129.9 (CH2-acrylate), 128.9 (CH-acrylate), 128.0
(Ar), 125.4 (Ar), 125.0 (Ar), 74.6 (C1), 50.5 (C2), 41.6 (C6), 39.7
(Cq), 34.6 (C4), 31.3 (C5), 27.6 (C-CH3), 26.7 (C3), 25.4 (C-CH3),
21.6 (C5-CH3); RP-HPLC tR ) 23.6 (50-100%).
changes the conformation of the câAA-Tyr moiety
heavily (see chemical shifts, coupling constants, and
temperature gradient of amide protons), while keeping
the antiproliferative efficacy. An explanation for this
observation could be that only the conformation of the
pharmacologically less important câAA-Tyr part of the
â-turn changes but not the conformation of the essential
D-Trp-Nle-Thr(Trt) moiety. Structural investigations of
3c by NMR and modeling calculations confirm this (data
not shown). The finding that only two aromatic residues
(D-Trp and Thr(Trt) in the case of 3b) are required for
GPCR binding is in accordance with many examples in
the literature, e.g. nonpeptidic somatostatin antago-
nists, enkephalin analogues, and urotensin II ana-
logues.22,41,42 The fact that peptide 3b is much more
potent than L-363,301 in the cellular assay might point
to a biological mode of action that is not dominated by
the somatostatin receptors such as that assumed in the
case of TT-232.26,43 Another indication for this finding
is that the Lys residue, which is present in all active
somatostatin analogues, can be replaced by Nle without
loss of activity in the cellular assay.
Conclusion and Outlook
The peptide cyclo[câAA-Tyr-D-Trp-Nle-Thr(Trt)] (3b)
that was designed starting from the somatostatin
analogue L-363,301 (1a) showed very good antiprolif-
erative activity on A431 cells in a cellular assay without
necrotic side effects. NMR/modeling together with SAR
studies revealed that the structural feature of a âII′-
turn about the D-Trp-Lys/Nle moiety, which is found in
a number of somatostatin analogues, is also preserved
in 3b. The rigid conformation of 3b is mainly deter-
mined by the D-Trp residue and cyclic â-amino acid
(câAA) that was synthesized by an enantioselective
multicomponent reaction. This cyclic â-amino acid seems
to play an important role for the orientation of Thr5 side
chain of peptides bearing a biologically important Trt
group. The stereochemistry of the Tyr2 residue does not
seem to play an important role for biological activity
because both enantiomers can be used without major
differences in activity.
Compound 3b turns out to be an interesting lead in
the development of biologically active peptides featuring
nonnatural building blocks, reduced number of residues,
and increased hydrophobicity. Its defined structure-
activity relationship provides important information
about pharmacophoric groups and their orientation for
further development of antiproliferative drugs for cancer
treatment.
Experimental Section
General Methods. Trityl chloride polystyrol (TCP) resin
(0.94 mmol/g) was purchased from PepChem. Coupling re-
agents and amino acid derivatives were purchased from
Advanced ChemTech, Perseptive Biosystems GmbH, Neosys-
tem, and PurChem. All other reagents and solvents were
purchased from Merck-Schuchardt, Lancaster, Aldrich, and
Fluka and were used as received. The solvents ethyl acetate,
diethyl ether, methanol, and n-hexane were distilled before
use. Standard syringe techniques were applied for transferring
dry solvents. Reactions on solid support were performed in
filter columns (5 and 10 mL) from Abimed. Melting points (mp)
were determined on a Bu¨chi 510 apparatus and were uncor-
rected. 1H spectra were obtained in CDCl3 or DMSO-d6 as
General Procedure for Multicomponent Reaction
(7a-e). Aldehyde 4 (10 mmol), amide 5 (10 mmol), acrylate 6
(3 mmol), 1.5 mL of acetic anhydride (16 mmol), 8 mL of DMF,
0.05 g of camphorsulfonic acid, and 0.05 g of 2,6-di-tert-butyl-
4-methylphenol (BHT) are placed in a pressure reactor and
stirred for 16 h at 140-160 °C. Afterward, volatile compounds
are removed by evaporation under high vacuum. Flash chro-
matography (EtOAc/n-hexane) of the raw product affords 7 as
a colorless oil (approximately 30% yield).

Somatostatin Analogue Antiproliferative Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2923
(a) [1S, 2S, 5R]-2-(Acetylamino)-3,5-dimethylcyclohex-
3-enecarboxylic Acid (-)-Menthyl Ester (7a). Rf ) 0.30
(CDCl3, HSQC) δ 131.5 (C4), 129.1-124.8 (Ar), 74.5 (C1-
menthol), 67.0 (CH2-benzyl), 50.2 (C2-menthol), 49.5 (C2), 44.3
(C1), 41.0 (C6-menthol), 34.7 (C4-menthol), 31.6 (C5-menthol),
30.6 (C5), 29.5 (CH3-menthol), 28.3 (C6), 26.6 (C3-menthol), 23.7
(CH3-menthol), 22.2 (C5-CH3-menthol), 21.7 (C5-CH3), 20.9 (C3-
CH3); MS (ESI) m/z 518.3 [M + H]+, 540.4 [M + Na]+; HRMS
(EI) calcd for C21H35NO3 517.3192, found 517.3195; RP-HPLC
tR ) 27.7 (50-100%).
(e) [1S, 2S, 5R]-2-(Benzyloxy)carbonylamino-3,5-diben-
zylcyclohex-3-enecarboxylic Acid (-)-8-Phenylmenthyl
Ester (7e). Rf ) 0.25 (EtOAc/n-hexane, 1/10, v/v); 1H NMR
(CDCl3) δ 7.41-6.87 (m, 20H, Ar), 5.37 (s, 1H, C4H), 5.06/4.87
(2 × d, 2H, J ) 12.2 Hz, CH2-Z), 4.71 (m, 1H, C1H-menthol),
4.40 (d, 1H, J ) 10.6 Hz, NH), 4.06 (dd, 1H, J ) 4.6 and 10.6
Hz, C2H), 3.21 (s, 2H, C3-CH2), 2.60/2.51 (2 × dd, 2H, J ) 7.2
and 13.5, C5-CH2), 2.18 (m, 1H, C5H), 2.12 (m, 1H, C2H-
menthol), 1.81/1.13 (2 × m, 2H, C3H2-menthol), 1.73/0.97 (2 ×
m, 2H, C6H2-menthol), 1.70/ 0.94 (2 × m, 2H, C6H2), 1.64/0.90
(2 × m, 2H, C4H2-menthol), 1.46 (m, 1H, C1H), 1.42 (m, 1H,
C5H-menthol), 1.22/1.12 (2 × s, 6H, CH3-menthol), 0.80 (d, 3H,
J ) 7.2 Hz, C5-CH3-menthol); 13C NMR (CDCl3, HSQC) δ 130.8
(C4), 130.2-124.5 (Ar), 74.6 (C1-menthol), 67.2 (CH2-Z), 50.1
(C2-menthol), 48.5 (C2), 44.1 (C1), 42.8 (C5-CH2), 41.1 (C3-CH3),
41.0 (C6-menthol), 37.8 (C5), 34.8 (C4-menthol), 30.6 (C5-
menthol), 30.1 (CH3-menthol), 26.6 (C3-menthol), 26.0 (C6), 23.1
(CH3-menthol), 22.1 (C5-CH3-menthol); MS (ESI) m/z 670.3 [M
+ H]+, 692.4 [M + Na]+; HRMS (EI) calcd for C21H35NO3
669.3818, found 669.3819; RP-HPLC tR ) 19.9 (80-100%).
(f) [1S, 2S, 5R]-3,5-Dimethyl-2-[N-((9H-fluoren-9-yl-
methoxy)carbonyl)amino]cyclohex-3 -enecarboxylic Acid
(8), Fmoc-câAA-OH. A solution of 7d (0.2 g, 0.39 mmol) in
12 mL of TFA and 1 mL of trifluoromethanesulfonic acid was
stirred for 2 h at 0 °C. After evaporation of the solvent under
reduced pressure, the remaining oil was dissolved in saturated
NaHCO3 solution (15 mL) and THF (10 mL) and Fmoc-ONSu
(118 mg, 0.35 mmol) was added. The mixture was stirred for
10 h at room temperature and then adjusted to pH 2 by
addition of 2 N HCl solution and the THF removed by
evaporation under reduced pressure. The aqueous suspen-
sion was extracted with EtOAc (3 × 50 mL), the combined
organic layers dried over MgSO4, and the solvent evaporated
under reduced pressure. Flash chromatography (EtOAc/
n-hexane, 1/4, v/v, 1% AcOH) afforded 8 as white crystals
(98 mg, 0.25 mmol, 64%): mp 78-80 °C (dec); Rf ) 0.39
(EtOAc/n-hexane, 2/3, v/v, 1% AcOH); [R]25D -20° (2.0 mg/mL,
H2O) (without Fmoc); 1H NMR (CDCl3) δ 7.76 (t, 2 H, J ) 7.6
Hz, FmocAr), 7.68 (t, 2 H, J ) 7.6 Hz, FmocAr), 7.39 (t, 2 H, J
) 7.6 Hz, FmocAr), 7.31 (t, 2 H, J ) 7.6 Hz, FmocAr), 5.40 (s, 1
H, C4H), 4.68 (d, 1 H, J ) 10.6 Hz, HN), 4.52/4.38 (2 × m, 2
H, Fmoc-CH2), 4.51 (dd, 1 H, J ) 4.9 and 10.6 Hz, C2H), 4.21
(m, 1 H, Fmoc-CH), 2.74 (m, 1 H, C1H), 2.14 (m, 1 H, C5H),
1.97/1.16 (2 × m, 2 H, C6H2), 1.69 (s, 3 H, CH3), 1.03 (d, 3 H,
J ) 6.9 Hz, C5-CH3); 13C NMR (CDCl3) δ 172.8 (CO), 156.1
(COFmoc), 143.4 (Fmoc), 141.1 (Fmoc), 132.0 (C3), 131.8 (C4),
127.5 (FmocAr), 126.8 (FmocAr), 124.9 (FmocAr), 119.7 (FmocAr),
66.4 (Fmoc-CH2), 49.4 (C2), 47.2 (Fmoc-CH), 43.7 (C1), 30.3 (C5),
27.8 (C6), 21.1 (C5-CH3), 20.8 (CH3); MS (ESI) m/z 392.2 [M +
H]+, 414.2 [M + Na]+, 805.2 [2M + Na]+, 821.3 [2M + K]+,
1196.2 [3M + Na]+, 1212.1 [3M + K]+; HRMS (EI) calcd for
C24H25NO4 391.1784, found 391.1785; RP-HPLC tR ) 25.2 (10-
90%).
1
(EtOAc/n-hexane, 1/3, v/v); H NMR (CDCl3) δ 5.40 (d, 1H, J
) 10.2 Hz, NH), 5.38 (s, 1H, C4H), 4.81 (dd, 1H, J ) 5.0 and
10.2 Hz, C2H), 4.65 (m, 1H, C1H-menthol), 2.75 (m, 1H, C1H),
2.18 (m, 1H, C5H), 1.98 (s, 3H, CH3-acetyl), 1.97/1.15 (2 × m,
2H, C6H2), 1.85/0.98 (2 × m, 2H, C6H2-menthol), 1.84 (m, 1H,
C2-CH-menthol), 1.70 (s, 3H, C3-CH3), 1.67/0.87 (2 × m, 2H,
C4H2-menthol), 1.66/1.04 (2 × m, 2H, C3H2-menthol), 1.45 (m,
1H, C5H-menthol), 1.41 (m, 1H, C2H-menthol), 1.03 (d, 3H, J
) 7.2 Hz, C5-CH3), 0.90/ 0.74 (2 × d, 6H, J ) 6.9 Hz, CH3-
menthol), 0.90 (d, 3H, J ) 7.2 Hz, C5-CH3-menthol); 13C NMR
(CDCl3, HSQC) δ 132.2 (C4); 75.1 (C1-menthol); 47.6 (C2); 46.9
(C2-menthol); 44.6 (C1); 40.5 (C6-menthol); 34.5 (C4-menthol);
31.7 (C5-menthol); 30.9 (C5); 29.0 (C6); 26.7 (C2-CH-menthol);
23.9 (C3-menthol); 23.5 (CH3-acetyl); 22.3 (C5-CH3-menthol);
21.8 (C5-CH3); 21.1 (CH3-menthol); 21.0 (C3-CH3); 16.8 (CH3-
menthol); MS (ESI) m/z 350.2 [M + H]+, 372.2 [M + Na]+;
HRMS (EI) calcd for C21H35NO3 349.2617, found 349.2616; RP-
HPLC tR ) 17.8 (50-100%).
(b) [1S, 2S, 5R]-2-(Acetylamino)-3,5-dimethylcyclohex-
3-enecarboxylic Acid (-)-8-Phenylmenthyl Ester (7b). Rf
1
) 0.33 (EtOAc/n-hexane, 1/3, v/v); H NMR (CDCl3) δ 7.33-
7.22 (m, 4H, Ar), 7.07 (t, 1H, J ) 7 Hz, Ar), 5.29 (s, 1H, C4H),
5.26 (d, 1H, J ) 10.2 Hz, NH), 4.76 (m, 1H, C1H-menthol),
4.33 (dd, 1H, J ) 4.9 and 10.2 Hz, C2H), 2.12 (m, 1H, C2H-
menthol), 1.99 (m, 1H, C5H), 1.92 (s, 3H, CH3-acetyl), 1.80/
1.14 (2 × m, 2H, C3H2-menthol), 1.76/0.92 (2 × m, 2H, C6H2),
1.67/0.96 (2 × m, 2H, C6H2-menthol), 1.65/0.90 (2 × m, 2H,
C4H2-menthol), 1.62 (m, 1H, C1H), 1.60 (s, 3H, C3-CH3), 1.43
(m, 1H, C5H-menthol), 1.29/1.18 (2 × s, 6H, CH3-menthol), 0.99
(d, 3H, J ) 7.2 Hz, C5-CH3), 0.87 (d, 3H, J ) 7.2 Hz, C5-CH3-
menthol); 13C NMR (CDCl3, HSQC) δ 132.0 (C4), 128.1/125.7/
125.2 (Ar), 74.7 (C1-menthol), 50.3 (C2-menthol), 47.6 (C2), 44.0
(C1), 41.3 (C6-menthol), 34.8 (C4-menthol), 31.7 (C5-menthol),
30.7 (C5), 29.9 (CH3-menthol), 28.6 (C6), 26.8 (C3-menthol), 23.7
(CH3-menthol), 23.5 (CH3-acetyl), 22.1 (C5-CH3-menthol), 21.7
(C5-CH3), 20.9 (C3-CH3); MS (ESI) m/ z 426.2 [M + H]+, 448.3
[M + Na]+; HRMS (EI) calcd for C21H35NO3 425.2930, found
425.2932; RP-HPLC tR ) 19.8 (50-100%).
(c) [1S, 2S, 5R]-2-(Benzyloxy)carbonylamino-3,5-di-
methylcyclohex-3-enecarboxylic Acid (-)-Menthyl Ester
1
(7c). Rf ) 0.25 (EtOAc/n-hexane, 1/10, v/v); H NMR (CDCl3)
δ 7.41-7.23 (m, 5H, Ar), 5.35 (s, 1H, C4H), 5.17/4.96 (2 × d,
2H, J ) 12.2 Hz, CH2-benzyl), 4.78 (d, 1H, J ) 10.4 Hz, NH),
4.69 (m, 1H, C1H-menthol), 4.50 (dd, 1H, J ) 4.9 and 10.4 Hz,
C2H), 2.74 (m, 1H, C1H), 2.14 (m, 1H, C5H), 1.88/0.99 (2 × m,
2H, C6H2-menthol), 1.82 (m, 1H, C2-CH-menthol), 1.79/1.10 (2
× m, 2H, C6H2), 1.70 (s, 3H, C3-CH3), 1.66/1.03 (2 × m, 2H,
C3H2-menthol), 1.66/0.86 (2 × m, 2H, C4H2-menthol), 1.44 (m,
1H, C5H-menthol), 1.39 (m, 1H, C2H-menthol), 1.00 (d, 3H, J
) 7.0 Hz, C5-CH3), 0.88/ 0.74 (2 × d, 6H, J ) 6.9 Hz, CH3-
menthol), 0.86 (d, 3H, J ) 7.2 Hz, C5-CH3-menthol); 13C NMR
(CDCl3, HSQC) δ 132.0 (C4), 129.3-127.4 (Ar), 75.2 (C1-
menthol), 67.4 (CH2-benzyl), 50.0 (C2), 47.2 (C2-menthol), 45.0
(C1), 40.4 (C6-menthol), 34.5 (C4-menthol), 31.8 (C5-menthol),
30.9 (C5), 28.8 (C6), 26.6 (C2-CH-menthol), 23.7 (C3-menthol),
22.4 (C5-CH3-menthol), 21.8 (C5-CH3), 21.2 (CH3-menthol), 21.0
(C3-CH3), 16.7 (CH3-menthol); MS (ESI) m/z 442.2 [M + H]+,
464.4 [M + Na]+; HRMS (EI) calcd for C21H35NO3 441.2879,
found 441.2877; RP-HPLC tR ) 26.6 (50-100%).
(d) [1S, 2S, 5R]-2-(Benzyloxy)carbonylamino-3,5-di-
methylcyclohex-3-enecarboxylic Acid (-)-8-Phenylmen-
thyl Ester (7d). Rf ) 0.32 (EtOAc/n-hexane, 1/10, v/v); 1H
NMR (CDCl3) δ 7.34-7.23 (m, 9H, Ar), 7.07 (t, 1H, J ) 7 Hz,
CH-Ar), 5.24 (s, 1H, C4H), 5.13/4.91 (2 × d, 2H, J ) 12.2 Hz,
CH2-benzyl), 4.80 (m, 1H, C1H-menthol), 4.49 (d, 1H, J ) 10.6
Hz, NH), 4.00 (dd, 1H, J ) 4.9 and 10.6 Hz, C2H), 2.15 (m,
1H, C2H-menthol), 1.97 (m, 1H, C5H), 1.80/1.15 (2 × m, 2H,
C3H2-menthol), 1.77/1.01 (2 × m, 2H, C6H2-menthol), 1.75/0.84
(2 × m, 2H, C6H2), 1.67/0.92 (2 × m, 2H, C4H2-menthol), 1.67
(m, 1H, C1H), 1.63 (s, 3H, C3-CH3), 1.42 (m, 1H, C5H-menthol),
1.31/1.19 (2 × s, 6H, CH3-menthol), 0.96 (d, 3H, J ) 7.2 Hz,
C5-CH3), 0.83 (d, 3H, J ) 7.2 Hz, C5-CH3-menthol); 13C NMR
Solid-Phase Peptide Synthesis and Purification. Pep-
tide synthesis was carried out using TCP resin following
standard Fmoc strategy.37 Side chain unprotected Fmoc-Tyr-
OH (97 mg, 0.24 mmol) was attached to the TCP resin (0.20
g) with DIEA (100 µL) in anhydrous DCM (2 mL) at room
temperature for 1 h, followed by addition of MeOH (0.2 mL)
for 15 min for quenching, yielding 0.24 g of Fmoc-Tyr-TCP-
resin (substitution level 0.43 mmol/(g of resin)). For Fmoc
deprotection the resin was treated with 20% piperidine in NMP
(v/v) and washed with NMP. The coupling of 1.8 equiv of Fmoc-
câAA-OH (8; 60 mg, 15 mmol) was achieved using TBTU (49
mg, 15 mmol), HOBt (24 mg, 16 mmol), DIEA (72 µL), in NMP
(2 mL) for 2 h at room temperature. For attachment of (S)-/

2924 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sukopp et al.
(R)-Mosher acid to the unprotected N-terminus of the câAA-
Tyr dipeptide, 60 mg of NH2-câAA-Tyr-TCP-resin (approxi-
mately 27 µmol) was treated with 5.0 equiv of (R)- or (S)-
Mosher acid chloride (34 mg, 0.14 mmol) and collidine (40 µL)
in dry DMF for 1 h at room temperature. The Mosher amides
11a,b were cleaved from resin by treatment with 20% HFIP
in DCM (v/v) for 15 min and purified by preparative RP-HPLC
(>98% purity).
Cyclo[câAA-Tyr-D-Trp-Nle-Thr(Trt)] (3b): 98% purity (de-
termined by HPLC and 1H NMR); RP-HPLC tR1 ) 29.18 (10-
90%); MS (ESI) m/z 979.5 [M + Na]+, 715.4 [M - Trt + H]+.
Cyclo[câAA-D-Tyr-D-Trp-Nle-Thr(Trt)] (3c): 95% purity (de-
termined by HPLC and 1H NMR); RP-HPLC tR1 ) 28.82 (10-
90%), MS (ESI) m/z 979.4 [M + Na]+, 715.4 [M - Trt + H]+.
Biological Assay. Human A431 epidermoid carcinoma cells
were cultures in DMEM (Dulbecco’s Mod Eagle Medium)
supplemented with 10% FCS (fetal calf serum), 200 mM
L-glutamine, 10 000 U/mL penicillin, and 10 mg/mL strepto-
mycin (Gibco Life Sci) at 37 °C and 5% CO2. Cells were seeded
in 96-well plates and incubated for 16 h before serial dilutions
of compounds were added. Cells were treated for 6 and 48 h.
Cells used for 6 and 48 h treatments were seeded at 4 × 104
and 1 × 104 per well, respectively. Antiproliferative efficacy
of the compounds was analyzed with methylene blue test.
All compounds were dissolved in DMSO and diluted in cell
culture medium for the proliferation test in final concentra-
tions of 50, 10, 2, 0.4, and 0.08 µM and tested in duplicate.
Cycloheximide, a well-established inducer of apoptosis, was
used as positive control.
Antiproliferative effect was first expressed as a percentage
of the optical density (OD) of treated (T) and negative control
(D) wells after both 6 and 48 h (T/C × 100). Because new
protein synthesis is required for apoptosis in immortalized cell
lines, compounds that induce programmed cell death will show
significantly less antiproliferative activity after 6 h than after
48 h. In the optimal case an apoptosis-inducing compound will
cause 100% viability after 6 h and 0% after 48 h. Therefore,
analyzing T/C48h versus T/C6h will correlate with the apoptosis-
inducing “specific” of a compound. The cutoff limit for “effec-
(a) (S)-Mosher Amide of H2N-câAA-Tyr-OH (11a). 1H
NMR (DMSO-d6) δ 9.13 (bs, 1H, OH), 8.08 (d, 1H, J ) 8.0 Hz,
HN-Tyr), 7.49 (d, 1H, J ) 9.7 Hz, HN-câAA), 7.36-7.46 (m,
5H, MosherAr), 6.93/6.62 (2 × d, 4H, J ) 8.4 Hz, TyrAr), 5.38
(s, 1H, C4H), 4.64 (dd, 1H, J ) 4.7 and 9.7 Hz, C2H), 4.39 (m,
1H, HR), 3.13 (s, 3H, MeO), 2.88/2.72 (2 × dd, 2H, 2J ) 14 Hz,
3J ) 5.4 and 7.5 Hz, Hâ), 2.71 (m, 1H, C1H), 2.09 (m, 1H, C5H),
1.61 (s, 3H, CH3), 1.49/1.34 (2 × m, 2H, C6H2), 0.96 (d, 3H, J
) 7.0 Hz, C5-CH3); 13C NMR (DMSO-d6) δ 172.8 (CO-Tyr),
171.9 (CO-câAA), 164.9 (CO-Mosher), 156.1 (Cq-Tyr), 132.0
(C3), 131.8 (C4), 131.6 (Cq-Mosher), 130.4 (TyrAr), 129.7 (Mosher-
Ar), 128.9 (MosherAr), 128.6 (MosherAr), 127.6 (Cq-Tyr), 124.3
(CF3), 115.1 (TyrAr), 84.2 (C-CF3), 54.7 (MeO), 53.7 (CR), 47.9
(C2), 43.9 (C1), 37.3 (Câ), 30.6 (C5), 28.7 (C6), 21.3 (CH3), 20.8
(C3-CH3); MS (ESI) m/z 549.3 [M + H]+, 571.4 [M + Na]+,
1119.3 [2M + Na]+; RP-HPLC tR ) 22.4 (10-90%).
1
(b) (R)-Mosher Amide of H2N-câAA-Tyr-OH (11b). H
NMR (DMSO-d6) δ 9.14 (s, 1H, OH), 7.96 (d, 1H, J ) 7.8 Hz,
HN-Tyr), 7.55 (d, 1H, J ) 9.5 Hz, NH-câAA), 7.52/7.39 (2 ×
m, 5H, MosherAr), 6.95/6.63 (2 × d, 4H, J ) 8.4 Hz, TyrAr),
5.23 (s, 1H, C4H), 4.64 (dd, 1H, J ) 4.3 and 9.3 Hz, C2H), 4.28
(m, 1H, HR), 3.48 (s, 3H, MeO), 2.91/2.73 (2 × dd, 2H, 2J )
13.8 Hz, 3J ) 5.8 and 7.3 Hz, Hâ), 2.62 (m, 1H, C1H), 2.02 (m,
1H, C5H), 1.50/1.38 (2 × m, 2H, C6H2), 1.39 (s, 3H, CH3), 0.86
(d, 3H, J ) 7.1 Hz, C5-CH3); 13C NMR (DMSO-d6) δ 172.8 (CO-
Tyr), 172.2 (CO-câAA), 165.4 (CO-Mosher), 156.2 (Cq-Tyr),
134.3 (Cq-Mosher), 131.8 (C3), 131.6 (C4), 130.4 (TyrAr), 129.5
(MosherAr), 128.4 (MosherAr), 127.8 (Cq-Tyr), 127.1 (MosherAr),
124.3 (CF3), 115.1 (TyrAr), 83.6 (C-CF3), 55.6 (MeO), 54.0 (CR),
48.2 (C2), 44.1 (C1), 37.0 (Câ), 30.6 (C5), 28.3 (C6), 21.0 (CH3),
21.0 (C3-CH3); MS (ESI) m/z 549.3 [M + H]+, 571.4 [M + Na]+,
1119.3 [2M + Na]+; RP-HPLC tR ) 22.1 (10-90%).
tive” compounds was set for differences expressed as (T/C6h
-
T/C48h) > 80%. IC50 values were generated from IC50 graphs.
NMR Spectroscopy of Peptides. The spectra were re-
corded at 300 K on a Bruker DMX spectrometer operating at
500 MHz. The samples were prepared in DMSO-d6 at a
concentration of 1.5-3 mM. DMSO-d6 was used as an internal
standard (2.49 ppm for 1H and 39.5 ppm for 13C). JH,H coupling
constants and amide hydrogen temperature coefficients were
measured from 1D experiments carried out at 295, 300, 305,
310, and 315 K. The data were processed with XWINNMR 3.5
software from Bruker. Homo- and heteronuclear experiments
DQF-COSY, TOCSY, ROESY, and HSQC were performed
Elongation of the peptide chain for synthesis of cyclic
peptides 2a,b and 3a-c was done using 3.0 equiv of Fmoc-
Pro-OH, Fmoc-Phe-OH, Fmoc-Thr(Trt)-OH, Fmoc-Nle-OH, and
Fmoc-D-Trp-OH each and proportionally higher amounts of
TBTU, HOBt, and DIEA, respectively. Before cleavage of the
partly protected linear peptides from its resin support, the
Fmoc protection was removed. After washings with DCM, the
peptides were cleaved from resin with a mixture of AcOH,
TFE, and DCM (2/1/6, v/v/v) for 1 h at room temperature. After
evaporation of the solvents, the crude linear peptides were
cyclized without further purification. For cyclization, the linear
fragments were dissolved in DMF to a concentration of 4 mM,
treated with 3 equiv of HOBt, 5 equiv of collidine, and 1.2 equiv
of PyBOP and stirred 16 h at room temperature until comple-
tion. The solvent was almost completely removed in vacuo, and
the products were then precipitated with ether and dried.
Preparative HPLC purification (mobile phase H2O/ACN, no
TFA) afforded Trt protected peptides 2b and 3b,c. For
complete deprotection, the peptides 2b and 3b were dissolved
at 0 °C in TFA/TIS/DMC (2/1/97, v/v/v) and the mixture was
stirred for 1 h in the cold. After evaporation of the solvent the
products were precipitated with ether and dried. Preparative
HPLC purification afforded fully deprotected peptides 2a and
3a.
1
with a spectral width of 12 ppm for H and 150 ppm for 13C.
In all of the experiments, spectra were recorded with 1024
increments in t1 and 4096 complex data points in t2. For
ROESY, 32 transients were averaged for each t1 value; for
COSY, 13C-HSQC, and TOCSY, 16 transients. A mixing time
of 70 or 250 ms was used for TOCSY (spin lock field, 10 kHz;
mixing sequence, MLEV-17) and ROESY spectra, respectively.
Water signal suppression was achieved by WATERGATE (W5)
techniques.44 Sequential assignment and quantitative infor-
mation on interproton distances were obtained from ROESY
spectra (250 ms mixing time; spin lock field, 5.00 kHz). The
volume integrals of the individually assigned cross-peaks were
converted into distance constraints using the isolated spin pair
approximation45 and taking the offset effect into account. The
ROESY cross-peak volumes were calibrated against the dis-
tance (1.78 Å) between the prochiral H6 methylene protons of
câAA.
Upper and lower distance limits were set to plus and minus
10% of the calculated distances, respectively. For nondiaste-
reotopically assigned methyl groups, 0.9 Å were added to the
upper bounds as pseudoatom corrections, respectively.
Molecular Modeling. The structural NMR refinement
protocol included distance geometry, energy minimizations,
and molecular dynamics (MD) simulations. A modified version
of the distance geometry (DG) program DISGEO46,47 was used
to generate structures consistent with 35 distance constraints
derived from ROEs. The DG procedure started with the
embedding of 100 structures using random metrization. The
structure with the smallest total error was placed in a cubic
box with a box length of 40 Å and soaked with DMSO.40 The
Cyclo[Pro-Tyr-D-Trp-Nle-Thr-Phe] (2a): 98% purity; RP-
HPLC tR1 ) 17.38 (20-80%) tR2 ) 17.41 (10-100%); MS (ESI)
m/z 808.5 [M + H]+, 830.6 [M + Na]+.
Cyclo[Pro-Tyr-D-Trp-Nle-Thr(Trt)-Phe] (2b): 97% purity;
RP-HPLC tR1 ) 11.69 (60-100%), tR2 ) 26.01 (10-100%); MS
(ESI) m/z 1072.6 [M + Na]+, 808.6 [M - Trt + H]+.
Cyclo[câAA-Tyr-D-Trp-Nle-Thr] (3a): 97% purity (deter-
mined by HPLC and 1H NMR); RP-HPLC tR1 ) 18.27 (10-
90%); MS (ESI) m/z 715.4 [M + H]+, 737.5 [M + Na]+.

Somatostatin Analogue Antiproliferative Activity
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2925
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MD calculations were carried out employing the module
DISCOVER of the INSIGHT II 2001 program (Biosym/MSI
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included with a force constant of 10 kcal/(mol Å2). The
calculations were done with the explicit-image model of
periodic boundary conditions. After energy minimization using
the steepest descent and conjugate gradient, the system was
heated gradually starting from 10 K and increasing to 50, 100,
150, 200, 250, and 300 K in 1 ps steps, each by direct scaling
of velocities. Configurations were saved every 100 fs for
another 150 ps. The averaged structure of the restrained MD
was minimized by using the method of steepest descent.
Distance violations were calculated by r-3 -1/3 averaging. The
MD calculation was continued without restraints for another
150 ps with an unchanged sampling rate.
Acknowledgment. The authors gratefully acknowl-
edge technical assistance from M. Kranawitter, B.
Cordes, and M. Wolff. We would like to thank J. Krause
for the EI-HRMS analyses of the intermediates. E.B.
thanks the Fonds FQRNT of Quebec and the Founda-
tion Alexander von Humboldt for Postdoctoral Fellow-
ships. Financial support was provided by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen
Industrie, as well as grants from the Hungarian Min-
istry of Education KKK-01/2001, NKFP-21/2002, and
OTK 49478.
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Appendix
Abbreviations. All optically active amino acids
are of the L variety unless otherwise stated. Other
abbreviations are the following: Bzl, benzyl; câAA, cyclic
â-amino acid; CDCl3, deuterated chloroform; COSY,
correlation spectroscopy; DG, distance geometry; DIEA,
diisopropylethylamine; DMF, dimethylformamide;
DMSO-d6, fully deuterated dimethyl sulfoxide; FACS,
fluorescence activated cell sorter; Fmoc, fluorenyl-
methoxycarbonyl; Fmoc-ONSu, (9-fluorenylmethoxycar-
bonyloxy) succinimide; HOBt, 1-hydroxybenzotriazole;
HSQC, heteronuclear single quantum coherence; MD,
molecular dynamics; NMP, N-methylpyrrolidinone; Py-
BOP, benzotriazol-1-yloxytrispyrrolidinophosphonium
hexafluorophosphate; rmsd, root-mean-square devia-
tion; ROESY, rotating frame nuclear Overhauser spec-
troscopy; RP-HPLC, reverse-phase high-performance
liquid chromatography; TBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate; TCP,
trityl chloride polystyrol; TFA, trifluoroacetic acid; TFE,
2,2,2-trifluoroethanol; TIS, triisopropylsilane; TOCSY,
total correlation spectroscopy; Trt, triphenylmethyl; Z,
benzyloxycarbonyl.
Supporting Information Available: Distance restraints
and their violations from the 150 ps restrained MD simulation
of peptide 3b, proton and carbon chemical shifts of peptide
3b and temperature coefficients for the amide protons, and
3JH,H coupling constants of amide and side chain protons of
peptide 3b. This material is available free of charge via
Internet at http://pubs.acs.org.
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