Novel octreotide dicarba-analogues with high affinity and different selectivity for somatostatin receptors

Article abstract of DOI:10.1021/jm1005868

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts_1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst₄, and derivative 8 exhibited high affinity and selectivity toward sst₅. Actually, compound 8 has similar sst₅ affinity (IC₅₀ 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS_-d25 micelles solution shows that all these analogues have the pharmacophore β-turn spanning Xaa ⁷-d-Trp⁸-Lys⁹-Yaa¹⁰ residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst₅ selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

Full text of DOI:10.1021/jm1005868

6188 J. Med. Chem. 2010, 53, 6188–6197
DOI: 10.1021/jm1005868
Novel Octreotide Dicarba-analogues with High Affinity and Different Selectivity
for Somatostatin Receptors
Alessandra Di Cianni,†,‡ Alfonso Carotenuto,*,§ Diego Brancaccio,§ Ettore Novellino,§ Jean Claude Reubi,
Karin Beetschen, Anna Maria Papini,†,‡ and Mauro Ginanneschi*,†,‡
†Laboratory of Peptides & Proteins, Chemistry & Biology, University of Firenze, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy,
‡Department of Chemistry “Ugo Schiff”, University of Firenze, Via della Lastruccia 5-13, I-50019, Sesto Fiorentino, Italy,
§Department of Pharmaceutical Chemistry and Toxicology, University of Napoli, Via Domenico Montesano 49, I-80131 Napoli, Italy, and
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Murtenstrasse 31,
CH-3010 Berne, Switzerland
Received May 17, 2010
A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7
and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a
pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward
sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-
activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the
receptors. The NMR study on the conformations of these compounds in SDS-d25 micelles solution
shows that all these analogues have the pharmacophore β-turn spanning Xaa7-D-Trp8-Lys9-Yaa10
residues. Notably, the correlation between conformation families and affinity data strongly indicates
that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-
SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded
conformational states.
Introduction
related to specific physiological activities.2 SRIF receptors are
strongly expressed in various types of malignant cells, parti-
cularly in some neuroendocrine or neuroendocrine-like tumors.
Over the last three decades, this has prompted researchers to
prepare a huge number of new cyclic and acyclic analogues,
which are more stable than SRIF in physiological conditions.
Among these, a largenumber of reduced-size cyclic analogues,
with or without the disulfide bridge, were synthesized and
tested for their affinity toward the ssts. Furthermore, their
pharmacological behavior was studied and several NMR
investigations on their affinity/conformations relationships
were carried out. J. E. Rivier’s group, at the Salk Institute of
LaJolla, carriedouta careful structure/affinity studyonSRIF
analogues, introducing non-natural amino acids in the se-
quence and preparing variably sized S-S bridged cyclopep-
tides. These authors related the structure/conformation of the
cyclopeptides to the sst1-4 specific affinity by means of NMR
studies.3-6
The cyclic tetradecapeptide somatostatin (H-Ala1-Gly2-
c[Cys3-Lys4-Asn5-Phe6-Phe7-Trp8-Lys9-Thr10-Phe11-Thr12-Ser13-
Cys14]-OH, SRIF-14a) was first isolated from mammalian
hypothalamus.1 This hormone is widely distributed in the human
body and is found in the gut, pancreas, nervous system, and in
some exocrine and endocrine glands. By interactions with a
family of five SRIF receptors (ssts), the native peptide exerts
a great number of regulatory effects, especially those related
to GH release. Different receptor subtypes mediate various
functions, but only sst2 and sst5 activities have been precisely
*To whom correspondence should be addressed. For A.C.: phone,
þ39-081-678626; fax, þ39-081-678644; E-mail, alfonso.carotenuto@
unina.it. For M.G.: phone, þ39-055-4573525; fax, þ39-055-4573531;
E-mail: mauro.ginanneschi@unifi.it.
a Abbreviations Bzl, benzyl; dh-DSA-C, dehydrodiaminosuberic acid,
C-terminus; dh-DSA-N, dehydrodiaminosuberic acid, N-terminus; DMF,
dimethyl formamide; DMSO-d6, hexadeuteriodimethylsulfoxide; DOTA,
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DQF-COSY,
double quantum filtered-correlation spectroscopy; EDT, 1,2-ethane-
dithiol; ESI-MS, electrospray ionization-mass spectrometry; Fmoc,
9-fluorenylmethoxycarbonyl; GH, growth hormone; Hag, L-2-allyl-Gly;
GPCR, G-protein coupled receptor; HATU, exafluorophosphate salt of
the O-(7-azabenzotriazol-yl)-tetramethyl uranium cation (this acronym
does not longer correspond to the true structure); LC, liquid chromato-
graphy; MD, molecular dynamics; MW, molecular weight; 1-Nal,
1-naphthyl-alanine; NMM, N-methyl morpholine; NOE, nuclear Over-
hauser effect; NOESY, nuclear Overhauser enhanced spectroscopy; RCM,
ring closing metathesis; rmsd, root-mean-square deviation; RP-HPLC,
reverse phase-high performance liquid chromatography; SDS, sodium
dodecyl sulfate; SPE, solid phase extraction; SPPS, solid phase peptide
synthesis; SRIF, somatostatin; sst, somatostatin receptor; TFA, trifluoro-
acetic acid; TOCSY, total correlated spectroscopy; TSP, [(2,2,3,3-tetra-
deuterio-3-(trimethylsilanyl)]propionic acid.
Octreotide7 (compound 1, Figure 1), a cyclic octapeptide
analogue of somatostatin, containing a disulfide tether and
showing high affinity and selectivity for sst2, was the first ana-
logue to be used in clinical protocols. Following the enormous
growth in preparation and application of radiolabeled pep-
tides for tumor imaging and therapy, the somatostatin ana-
logues thus far obtained were designed mainly for the targeting
of malignant cells with γ- or β-emitting radionuclides.4,8,9 As a
matter of fact, octreotide derivatives [111In-DTPA]octreotide
(OctreoScan) and [90Y-DOTA-Tyr3]octreotide (OctreoTher)
are both quite successfully used in the clinical diagnosis and
therapy of neuroendocrine tumors, respectively.10 Neverthe-
less, the vulnerability of the S-S bridge to endogenous and
r
pubs.acs.org/jmc
Published on Web 07/28/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6189
Figure 1. Structure of octreotide (SMS201-995) (1) and of the first dicarba SRIF mimetic (2).12 (Note: numbering of the residues follows that
of the native SRIF).
Table 1. Peptide Sequences: General Formula: D-Phe2-c[dhDSA-N3-
Xaa7-D-Trp8-Lys9-Yaa10-dhDSA-C14]-Thr(ol)15-OH
peptide
Xaa7
Yaa10
double-bond geometry
2a
3a
4
Phe
Phe
Thr
Z
E
Z
Z
E
Z
Z
Tyr(Bzl)
Tyr(Bzl)
Thr
Phe
5
1-Nal
Phe
Phe
6
Tyr
Tyr
7
8
1-Nal
Tyr(Bzl)
a These compounds were previously reported.13
Figure 2. Second generation Grubbs catalyst.
exogenous oxidating and reducing agents, such as those emp-
loyed in the experimental conditions of labeling with the radio-
isotopes 99 mTc or 188Re,11 prompted us to synthesize dicarba-
analogues of similar ring size by the RCM reaction on two
allylglycines, substituting the relevant Cys3,14 residues in the
linear peptide. Compound 2, reported in Figure 1 as an example,
is the first octreotide dicarba-tethered analogue synthesized
by us and has the same amino acid sequence of the corres-
ponding, S-S bridged, molecule.12,13
The resulting unsaturated dicarba bridge proved to be
insensitive to the conditions used for 99 mTc or 188Re labeling
(unpublished results), and the molecules obtained were very
stable in human serum.12,13 Recently, the stability of these
compounds was exploited in the successful conjugation of
cytotoxic dichloroplatinumcomplexes to analogue2 aswell as
to thedouble-bond hydrogenated derivative.14 The same reac-
tion, attempted with the octreotide molecule, failed. When the
affinities of these analogues toward the five ssts were deter-
mined, we ascertained that some of them showed unexpected
specific affinity for the sst5 subtype, which led us to define a
novel pharmacophore model for this receptor.13
This paper reports the synthesis of new cyclooctapeptide
dicarba-analogues that have structures similar to those depicted
in Figure 1 but are designed to carry different aromatic residues
in positions 7 and/or 10 (Table 1). In the following, ssts subtypes
affinities found for the new compounds 4-8 are correlated with
the CdC bridge geometry and with the conformational beha-
vior in SDS-d25 micelles solution, investigated by NMR experi-
ments. Characteristic structure-affinity relationships of this
class of somatostatin analogues are widely discussed.
articles.12,13 Starting from H-L-Thr(tBu)-ol-2-chlorotrityl resin
(0.5 mmol/g) already containing [Thr(ol)15], the elongation of
the peptide sequence was stopped after the coupling of Hag3
residue, with the aim of removing any possible interference of
the aromatic ring of D-Phe in the correct orientation of the
allylglycine side chains. After the Fmoc-Hag3 coupling, the
resin loading (0.5 mmol/g) already met the requirements of
the pseudodilution effect, minimizing the risk of the formation
of intermolecular bonds. The linear heptapeptides were then
converted by RCM by the second generation Grubbs catalyst
(9) (Figure 2) to the corresponding cyclic analogues.
The D-Phe2 terminal residue was added only after ring-
closing, thus facilitating the cyclization step. Cleavage of the
crude peptides from the resin was obtained using the standard
cleavage mixture TFA/H2O/EDT/phenol (94:2:2:2, 3 h) for
compounds 5, 6, and 7 and with the new percentage mixture
(70:26:2:2, 2,30 h) for compounds 4 and 8 in order to over-
come the loss of the benzyl group of the Tyr(Bzl) residue by
hydrolysis, as described in our previous article.13 All com-
pounds obtained by RCM with 9 were prepurified by SPE.
The concentrated compound adsorbed on the SPE was
eluted with an increased percentage of CH3CN in H2O
(from 0% to 100%). The fractions enriched with each desired
compound were then purified by semipreparative RP-HPLC
and characterized by ESI-MS. For each peptide, with the
exception of 5 and 8, the HPLC chromatogram showed two
peaks with the same MW, corresponding to the geometric
isomers (Z/E ratio ≈ 90:10). In particular, the E structure of
the C-CdC-C tether of the sample eluted at lower Rt and
the Z structure one of the second, more intense peak, was
1
ascertained by H NMR inspection. The HPLC purity of
Results
each compound studied was >97%, and the isolated com-
pounds showed unique E or Z configuration, confirmed by
NMR analysis. No oligomer byproduct were observed.
Peptide Synthesis and Purification. The synthesis of dicarba-
analogues followed the procedure described in our previous

6190 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Table 2. Receptor Affinities of the Somatostatin Analogues
Di Cianni et al.
IC50 (nM)a
sst3
no.
sst1
sst2
sst4
sst5
SRIF-28
2.3 ( 0.4 (7)
>1000 (2)
>1000 (2)
25 ( 1 (3)
>1000 (3)
1000 (3)
3.0 ( 0.2 (7)
44 ( 1 (2)
>1000 (2)
3.6 ( 0.5 (7)
>1000 (2)
892 ( 245 (2)
25 ( 4 (3)
>1000 (3)
1000 (3)
1.6 ( 0.3 (7)
412 ( 68 (2)
>1000 (2)
346 ( 23 (3)
249 ( 51 (3)
1000 (3)
2.4 ( 0.2 (6)
28 ( 2 (2)
29 ( 1 (2)
12.3 ( 0.3 (3)
16.5 ( 4.5 (3)
418 ( 56 (3)
161 ( 27 (3)
4.9 ( 1.0 (4)
2b
3b
4
46 ( 3 (3)
5
9.6 ( 0.9 (3)
355.5 ( 45.5 (3)
87 ( 18 (3)
101 ( 9 (3)
6
7
>1000 (3)
57.5 ( 12.5 (3)
>1000 (3)
92.5 ( 0.5 (3)
>1000 (3)
>1000 (3)
8
a The number of independent repetitions to obtain the mean values ( SEM are indicated between brackets. SRIF-28 is used as internal control.
b Corresponds to data published previously.13
Binding Affinity to sst1-5 Receptors. All compounds were
tested for their ability to bind to the five human sst1-5
receptors subtypes in complete displacement experiments
using the universal somatostatin radioligand [125I]-[Leu8,
D-Trp22,Tyr25]-somatostatin-28. SRIF-28 was run in parallel
as control. IC50 values were calculated after quantification of
the data using a computer-assisted image processing system.
Binding data indicate that all compounds show sub-μM
binding affinities toward the sst5 (Table 2). Compounds 2
and 3 have already been described13 and are reported for
comparison. While peptide 3 was a potent and selective sst5
ligand, its Z-isomer, peptide 4, exhibited a pan-somatostatin
affinity, apart from sst4. In fact, the analogue 4 doubled the
affinity toward sst5 but completely lost the selectivity of 3.
Peptide 5 is the 1-Nal7 analogue of 2 (Table 1). This
peptide exhibited a low nanomolar sst2,5 affinity. Actually,
it is the most potent sst2 ligand among the dicarba-analogues
prepared to date. The double-bond isomer analogues 6 (E)
and 7 (Z), in which the phenolic group of Tyr10 replaces the
Tyr(Bzl) residue of 3 and 4, respectively, did not show any
significant affinity toward sst1-5 subtypes apart from a slight
affinity of 7 to sst2.
(3JCHdCH=15.1 Hz) between the two olefinic protons of the
bridge and NOE contacts between the same olefinic and the
Hβs of residue 14 (3). A qualitative analysis of short- and
medium-range NOEs, 3JNH-HR coupling constants, and tem-
perature coefficients for exchanging NH was used to charac-
terize the secondary structure of 3. Spectra analysis pointed
to the presence of a β-turn about residues 7-10. Interest-
ingly, the upfield shift observed for Hγs of Lys9 (δ = 0.52,
0.43 ppm) has been used for decades as diagnostic for
biological activity.20 NOE-derived constraints obtained for
3 were used as the input data for a simulated annealing
structure calculation (Table S9, Supporting Information).
The backbone arrangement of 3 was well-defined, possessing
an average root-mean-square deviation (rmsd) of the heavy
atoms equal to 0.15 A. No violation higher than 0.1 A was
observed again, indicating conformational stability (Table S7,
Supporting Information). Main backbone features were a
type II0 β-turn spanning residues D-Trp8-Lys9, followed by a
short 310-helix along residues Tyr(Bzl)10-dhDsa-C14-Thr-
(ol)15 (Figure 3). The turn structure is stabilized by hydrogen
bonds between Phe7-CO and Tyr(Bzl)10-NH. The helical
structure is stabilized by H-bonds between D-Trp8-CO and
dhDsa-C14-NH and between Lys9-CO and Thr(ol)15-NH.
These bonds are typical of 310-helix structure (i, i þ 3). The
side chains of dhDsa-N3, D-Trp8, Lys9, Tyr(Bzl)10, and
dhDsa-C14 showed well-defined χ1 values (i.e., trans, trans,
gauche-, gauche-, and gaucheþ orientations, respectively).
These orientations allowed a close spatial proximity between
D-Trp8/Lys9 side chains; moreover, the tyrosyl group of the
residue 10 points toward the Lys9 side chain. In contrast,
D-Phe2 and Phe7 side chain showed almost free rotation about
the χ1 torsion angle. Also, the Bzl group of residue 10 was
highly flexible.
Compound 4. The analogue 4 is the geometric (Z) isomer of
3 as established by the coupling constant (3JCHdCH=8.1 Hz)
between the two olefinic protons of the bridge and the
relative strong NOE between the same olefinic Hγs. This
analogue shows spectral features similar to those found in 3
but with a greater tendency to conformational heterogeneity.
In fact, NOESY spectra of 4 showed, simultaneously, both
diagnostic connectivities consistent with folded structures
dRN(i, i þ 2) between HR-8/NH-10, HR-9/NH-14, HR-10/
NH-15, and dRN(i, i þ 3) between HR-9/NH-15 and NOE
contacts characteristic of extended regions strong dRN(i, i þ 1)
between HR-9/NH-10, HR-10/NH-14, and HR-14/NH-15
(Table S10, Supporting Information). The apparently con-
tradictory NOEs are indicative of the presence of at least
two conformations in solution. The impossibility of resum-
ing all the data in a single structure prompted us to consider
incompatible NOEs separately in different calculation cycles
˚
˚
Finally, analogue 8 shared Tyr(Bzl)10 residue with peptide
4 and 1-Nal7 residue with peptide 5. Like compound 4, it
showed affinity for all the receptor subtypes except sst4.
However, the significant enhancement of the sst5 affinity
(nearly 3-fold compared to compound 4) and the simul-
taneous reduction of affinity toward sst1-4 make compound
8 a strong and selective sst5 ligand. Indeed, compound 8 is the
most potent sst5 dicarba-analogue synthesized so far, show-
ing an affinity close to the value found for the reference com-
pound SRIF-28 (Table 2).
NMR Analysis. NMR analysis of the analogues 3-8 was
performed by means of 1D and 2D proton homonuclear
experiments. NMR experiments were recorded on a Varian
Inova-Unity 700 MHz spectrometer. Spectra were collected
in SDS-d25 (200 mM) micelles solution. All samples (about
2 mM) were kept at 308 K and at pH = 5. Complete 1H NMR
chemical shift assignments were effectively achieved for all
15
€
the analyzed molecules according to the Wuthrich procedure
via the usual systematic application of TOCSY16 and NOESY17
experiments with the support of the XEASY software package
(TablesS3-S8, Supporting Information).18 NMR-derived con-
straints obtained for all compounds, were used as the input data
for a simulated annealing structure calculation, as implemented
within the standard protocol of the DYANA program.19
Compound 3. The analogue 3 bears Tyr(Bzl) in position 10.
We have already analyzed this peptide in our previous work
in water/DMSO-d6 solution.13 The geometry of the double
bond was confirmed as trans (E) from the coupling constant

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6191
Figure 3. Stereoview of the lowest energy conformer of compound 3. Backbone is evidenced as a ribbon. Side chains of the 10 lowest energy
conformers are also shown as mesh surface. Surfaces are distinguished with different colors. N-Term, N-terminus; C-Term, C-terminus.
(Experimental Section). Hence, we obtained two families of
conformations. The first calculation cycle gave an ensemble
of structures (family I) showing a similar conformation to
compound 3, with a type II0 β-turn spanning residues D-Trp8-
Lys9, followed by a short 310-helix along residues Tyr(Bzl)10-
dhDsa-C14-Thr(ol)15 (Figure 4a). Moreover, side chain orien-
tations were the same as those described for 3. The main
difference was a better definition of the Phe7 side chain which
preferred the trans rotamer. For this set of structures, a
number of consistent violations were observed (Table S10,
Supporting Information). In a second MD cycle, the violated
upper limit constraints were upweighted for the contribution
to the target function. Thus, a second conformational family
(family II) was obtained which differed from the first mainly
in that C-terminal residues were in extended conformations
(Figure 4b). Furthermore, the side chain orientation of Tyr-
(Bzl)10 was trans. Hence, the tyrosyl nucleus was further
from the Lys9 side chain. This is in accordance with the
downfield shifts of the Hγ resonances of Lys9 compared to
the corresponding shifts of compound 3. Interestingly, the
complete ensemble of structures (helix and extended) ful-
residues were in extended conformations (Figure 5b). In both
the families, residue 7 showed a defined trans orientation
which forces 1-Nal7 naphthyl moiety close to D-Trp8 residue.
This orientation is in accordance with the intense upfield
shift observed for many D-Trp8 proton resonances.
Compounds 6 and 7. The analogues 6 and 7 differ from
compounds 3 and 4 in that the Tyr(Bzl)10 residue was
replaced by a Tyr (i.e., without the Bzl group). Following
the same arguments given for 3 and 4, an E configuration was
assigned to compound 6 and a Z configuration to the
compound 7 at the double bond.
Many potential diagnostic NOEs could not be observed in
the NOESY spectra of these analogues due to signal over-
lapping and this precluded structure calculations. For ins-
tance, HR protons of Lys9 and Thr-ol15 resonated at the same
chemical shift for both peptides. Actually, the NMR para-
meters of 6 (HR shifts, coupling constants, and temperature
coefficients) are very similar to those of compound 3, and
this was also true for 4 and 7. Therefore, it could be hypo-
thesized that 3D structures should be similar too.
Compound 8. The analogue 8 structure was rationalized
starting from the peptide sequence of the previous compounds
4 and 5. In fact, it bears both 1-Nal7 and Tyr(Bzl)10. For com-
pound 8, a Z configuration was established from the NOEs and
coupling constant (3JCHdCH=8.1 Hz) between the two olefinic
protons. NMR-based structure calculation (Table S12, Suppor-
ting Information) gave two conformational families, as it did for
compounds 4 and 5, the first (family I) showing a short 310-helix
along the Thr10-dhDsa-C14-Thr(ol)15 residues (Figure 6a), and
the second (family II) an extended conformation along the same
residues (Figure 6b). In both families, the D-Trp8, Lys9, and
Tyr(Bzl)10 side chains were spatially closed in accordance with
the increased upfield shift of the Hγ and Hβ resonances of Lys9.
Differently from compound 5, 1-Nal7 residue could not adopt
a trans conformation, probably due to steric hindrance with
Tyr(Bzl)10. In fact, 1-Nal7 side chain was preferentially in a
gauche- conformation.
˚
filled the NOE restraints, with no violations exceeding 0.5 A
(Table S10, Supporting Information).
Compound 5. Compound 5 maintains the same octreotide
scaffold except for position 7, which bears 1-Nal. Apart from
the dicarba bridge, it has the same peptide sequence of the
analogue NOC, formerly prepared and studied as DOTA-
conjugate by Maecke, Reubi, and co-workers.21 The cou-
pling constant (3JCHdCH =8.1 Hz) between the two olefinic
protons of the bridge and the relative strong NOE between
the same olefinic Hγs established (Z) configuration for
compound 5. Only one isomer is obtained from RCM.
NMR-based structure calculations gave two conforma-
tional families, like compound 4. Family I, obtained by a first
run of MD calculation, showed a type II0 β-turn spanning
residues D-Trp8-Lys9, followed by a short 310-helix along
residues Thr10-dhDsa-C14-Thr(ol)15 (Figure 5a). As found
with compound 4, a number of consistent violations were
observed (Table S11, Supporting Information). In a second
MD run, we obtained a second conformational family (II),
which differed from the first mainly because the C-terminal
Discussion
In our ongoing efforts to develop new somatostatin ligands
with improved stability and affinity toward sst receptors, we

6192 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Di Cianni et al.
Figure 4. Stereoview of the lowest energy conformer of compound 4: family I (a), family II (b). Backbone is evidenced as a ribbon. Side chains
of the 10 lowest energy conformers are also shown as mesh surface. Surfaces are distinguished with different colors. N-Term, N-terminus;
C-Term, C-terminus.
have rationally designed and analyzed a limited set of peptides
(Table 1). In these peptides, the labile disulfide bridge was
replaced by a dicarba-bridge, through the RCM reaction. As
can be seen from Table 2, variation of the residues 7 and 10
results in analogues having a low submicromolar potency and
a range of sst receptor subtype selectivities.
transition of the peptide.26 Actually, we succeeded in correlat-
ing the SDS-bound conformation of urotensin-II (U-II), a
peptidehormonestrictly correlated to somatostatin, to its bio-
logical activity.27
Some apparently contradictory NOEs were indicative of
the presence of at least two conformations in solution for
analogues 4, 5, and 8. To deal with this incongruence, we used
a practical approach. Incompatible NOEs were considered
separately in different calculation cycles. Hence, two families
of conformations were obtained which differed mainly in that
C-terminal residues were in 310-helix (family I) or extended
(family II) conformation. Eventually, the experimental re-
straints were fulfilled over the entire ensemble. It is noteworthy
thatthe NMR dataof the cognatemolecule octreotide, using a
single average conformation, reveal several important incon-
sistencies, including severe violations of mutually exclusive
backbone-to-backbone NOEs.28
On the basis of the NMR results, some general conformation-
affinity relationships concerning the binding to the sst recep-
tors can be outlined. Similar to most of the bioactive ana-
logues of SRIF reported so far,29 the structures of the
peptidomimetics presented here have a β-turn of type II0
spanning residues D-Trp8 and Lys9. The side chain of D-Trp8
is in the trans conformer, and the side chain of Lys9 is in the
Recently, we have investigated some octreotide analogues,
including compound 3, in a water/DMSO-d6 8:2 solution.13
Here, an NMR study was performed on the developed ana-
logues of octreotide in SDS micelles solution. The use of SDS
micelles to study the conformational properties of somato-
statin analogues is motivated on the basis of their interaction
with a membrane receptor. For peptides acting as ligands of
membrane receptors (such as GPCR), the use of membrane
mimetic media is suggested, hypothesizing a membrane-
assisted mechanism of interactions between the peptides and
their receptors.22 According to this model, the membrane
surface plays a key role in facilitating the transition of the
peptide from a random coil conformation adopted in the
extracellular environment to a conformation that is recogni-
zed by the receptor.23-25 The increase of the local concentration
of the peptide and the reduction of the rotational and transla-
tional freedom of the neuropeptide are membrane-mediated
events which are determinant steps for the conformational

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6193
Figure 5. Stereoview of the lowest energy conformer of compound 5: family I (a), family II (b). Backbone is evidenced as a ribbon. Side chains
of the ten lowest energy conformers are also shown as mesh surface. Surfaces are distinguished with different colors. N-Term, N-terminus;
C-Term, C-terminus.
gauche- conformer, bringing the two side chains adjacent to
each other in close proximity. Analogues with the Z config-
uration at the double bond can adopt both helical and
extended structures at the C-terminus, showing a conforma-
tional equilibrium (Figures 4-6). As a consequence of this
conformational behavior, Z analogues show greater potency
compared to the corresponding E isomers (4 vs 3 and 7 vs 6)
although it can go to the detriment of the selectivity, as in the
case of compound 4 compared to 3. It could be argued, from
the data of Table 2, that Z-geometry of the double bond is
a better mimic of the S-S bridge which, in turn, was hypo-
thesized to be directly involved in the interaction with the sst
receptors.30,31
Analogues 3 and 4 bear a Tyr(Bzl) residue in position 10.
The side chain of Tyr(Bzl)10 was designed to replace Phe6,
Phe7, and Phe11 of SRIF14.32 Compound 3 (E-isomer) selec-
tively binds sst5 while its Z-isomer, 4, showed a pan-SRIF-
activity, apart from sst4. In compound 3, the β-turn motif is
followedbya short310-helix along residues Tyr(Bzl)10-dhDsa-
C14-Thr(ol)15. The side chain of Tyr(Bzl)10 is in the gauche-
conformer and is located in close proximityto the D-Trp8-Lys9
pair (Figure 3). This is in accordance with our recent results
which correlates sst5 selectivity to conformationally restricted
helical structure at the C-terminus.13 The conformational
properties of 3 in SDS micelles are similar to those observed
in water/DMSO solution (data not shown). Only N-terminal
residue D-Phe2 is more flexible in the SDS solution compared
to DMSO.
In addition to high-affinity binding to sst2,3,5 like octreotide
(1), compound 4 also exhibited a low nanomolar binding to
sst1, hence its affinity pattern resembles that of the hexa-cyclic
peptide SOM230 (pasireotide), which also bears a Tyr(bzl)10
residue.32 Because compound 4 fits 4/5 receptor binding sites,
it was expected to display a high degree of flexibility. In fact, a
dynamical equilibrium between extended and helical confor-
mations was observed. Moreover, Tyr(Bzl)10 side chain orien-
tation was different in the two conformations (Figure 4).
Notably, NMR28 and X-ray crystallography analyses have
already suggested an equilibrium between extended and folded
conformational states for the parent peptide 1.33 Further-
more, SOM230 exhibited similar backbone conformational
equilibrium in a theoretical MD study; the side chain of
Tyr(Bzl) of SOM230 underwent great flexibility which was
associated with low selectivity.34 Although sst2 is probably the
most abundantly expressed SRIF receptor in human cancer,35
recent literature data indicates that also sst1 and sst3-5 may
also be present in some human tumors.36 Hence, peptides with
an improved receptor binding profile are desirable in order to

6194 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Di Cianni et al.
Figure 6. Stereoview of the lowest energy conformer of compound 8: family I (a), family II (b). Backbone is evidenced as a ribbon. Side chains
of the 10 lowest energy conformers are also shown as mesh surface. Surfaces are distinguished with different colors. N-Term, N-terminus;
C-Term, C-terminus.
extend the spectrum of tumors accessible to diagnosis and
internal radiotherapy. As a matter of fact, SOM230 is being
investigated in clinical trials as a potential treatment for acro-
megaly, neuroendocrine tumors, and Cushing’s disease.37,38
Amongthe testedcompounds, peptide5 showed thehighest
affinity toward sst2 and also a good affinity toward sst5. The
sequence of this cyclopeptide is the same as the S-S bridged
NOC,21 whose DOTA derivative, DOTA-NOC, exhibited
high affinity toward sst2,3,5. The loss of sst3 affinity in 5 is
probably due to the absence of the D-Phe2-bonded DOTA
chelating group. Insertion of different arms at the N-terminus
may, in fact, have a dramatic effect, particularly on sst3 affi-
nity.39 Compound 5 is also closely related to the previously
described compound 2, sharing its configuration at the double
bond and the amino acid sequence but with Nal7 in replacing
Phe7.12 The activity profile of analogues 2 and 5 is similar,
showing an increase in the sst2 (∼4-fold) and sst5 affinity
(∼2-fold) of 5 compared to 2 (Table 2). Because 2 showed
similar conformational properties as 5 (data not shown), the
improved affinity toward sst2 and sst5 is probably attributable
to the 1-Nal7 aromatic side chain which, oriented in a trans con-
formation, adequately fits the binding pocket of both receptors.
Compounds 6 and 7 are analogues to 3 and 4, respectively,
with the Tyr(Bzl)10 residue replaced by a tyrosine. This change
renders compounds 6 and 7 strictly related to U-II. Analogue
6 showed a markedreduction of affinity toward sst5 compared
to the correlated compound 3. Analogously, compound 7
showed a marked reduction of affinity toward sst1,3,5 and a
2-fold reduction toward sst2 compared to 4. NMR data of the
analogue couples pointed to similar conformational behavior,
hence it can be argued that the Tyr10 phenol group is detri-
mental for binding to the sst receptors. This is in accordance
with the low affinity of U-II to the sst2A receptor.40 On the
other hand, residual affinity of compound 7 toward sst2 and
sst5 (Table 2) parallels the capability of U-II to activate these
two receptors at high doses.41
By combining 1-Nal7 and Tyr(Bzl)10 residue replacements,
we obtained compound 8 as a pure Z-isomer. Compound 8
showed the highest affinity toward sst5 (Table 2) with at least a
10-fold selectivity compared to the other ssts. Compound 8 is
closely related to compound 4, sharing its configuration at the
double bond and the amino acid sequence but with 1-Nal7
replacing Phe7. Actually, the activity profile of the two
analogues is similar (pan-SRIF-activity, apart from sst4) with
a decrease of the sst1-3 affinity (2- to 4-fold) and increase of
the sst5 activity (∼3-fold) of 8 compared to 4. The conforma-
tional behavior of 8 also resembles that of 4, in accordance to
the activity similarity (Figures 4 and 6). Because the helical-
extended conformational equilibrium is also observable in the
case of analogue 8, the affinity changes could be tentatively

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6195
˚
Table 3. Cγ-Cγ Distances (A) between Putative Pharmacophoric
RP-HPLC analyses were performed on the same instrument using a
flow rate of 4 mL/min with the same solvent system on a Pheno-
menex Juppiter C18 column (10 μm, 250 mmꢀ10 mm). Mass spectra
were registered on an ESI LCQ Advantage mass spectrometer
(Thermo-Finnigan). LC-ESI-MS analyses were performed on a
Phenomenex Jupiter C18 column (5 μm, 150 mm ꢀ 2.0 mm) using
a flow rate of 500 μL/min on a ThermoFinnigan Surveyor HPLC
system coupled to ESI-MS, using the solvent system: H2O (A),
MeCN (B), 1% TFA in H2O (C). Routine NMR spectra were
acquired on a Varian Inova 700 apparatus. TSP was purchased from
MSD Isotopes (Montreal, Canada). 2H2O was obtained from
Aldrich. SDS-d25 was obtained from Cambridge Isotope Laborato-
ries, Inc. (Andover, MA). SPPS was performed in Teflon reactor on
a manual synthesizer PLS 4 ꢀ 4 (AdvancedChemTech). Receptor
autoradiography was performed on 20 μm thick cryostat (Microm
HM 500, Walldorf, Germany).
Residuesa
b
compd
Ar2-Ar7
Ar2-Ar8
3
4
5
8
sst2/3/5
8.5 ( 0.8c
9.0 ( 1.2
9.5 ( 0.5
8.4 ( 1.0 5-11
14.3 ( 0.6 14.0 ( 0.5 14.3 ( 0.6 13.4 ( 0.5 11-15
Ar2-Lys9 14.8 ( 0.9 14.4 ( 1.0 13.9 ( 1.0 14.5 ( 1.0 12-15
Ar2-Ar10
Ar7-Ar8
8.1 ( 1.1
7.8 ( 0.8
7.8 ( 1.2
7.6 ( 0.3
9.8 ( 0.7
5.6 ( 0.2
8.9 ( 0.2
7.2 ( 0.1
13.4 ( 1.0
8.2 ( 0.5 7-9
9.7 ( 0.3 11.0 ( 0.6 9-11
6.8 ( 0.2
Ar7-Lys9 10.9 ( 0.6
Ar8-Lys9
Ar8-Ar10
Lys9-Ar10
5.5 ( 0.2
8.8 ( 0.2
7.2 ( 0.2
4.7 ( 0.3
5.2 ( 0.4
8.1 ( 0.1
5.9 ( 0.2
5
a Only the family I of peptides 4, 5, 8 were considered. b Pharmaco-
phore for the sst2, sst3, sst5 selective SRIF analogues.30 c Average distance
and standard deviation calculated from the ensemble of 10 structures.
Synthesis and Purification of Compounds 3-8. Peptides were
synthesized following the method reported in the preceding
paper.13 Briefly, the peptides were prepared using the general
Fmoc-SPPS strategy on preswelled H-L-Thr(tBu)-ol-2-chloro-
trityl resin. Couplings were performed by adding 2 equiv of
protected amino acid activated by HATU and 4 equiv of NMM
in DMF. Each coupling was monitored by the qualitative
ninhydrin (Kaiser) test.42 At the end of the linear peptides syn-
thesis, a microscale cleavage was performed. RP-HPLC analysis
of the crude products revealed the presence of the linear peptides
in approximately 95% purity, without traces of isomers due to
amino acid racemization. The cyclization was performed on-resin
by second generation Grubbs catalyst (0.5 mol equiv calculated
on the basis of 0.5 mmol/g of peptide). After swelling, NH2
terminal Fmoc-Hag was deprotected and coupled with Fmoc-
D-Phe, affording the on-resin peptides 4-8, which were depro-
tected and cleaved [5, 6, and 7 with TFA/H2O/EDT/phenol
(94:2:2:2, 3 h) while 4 and 8 with TFA/H2O/EDT/phenol
(70:26:2:2, 2.30 h)]. The aqueous solutions of the peptides 4-8
were prepurified by SPE and after subjected to the purification
by semipreparative RP-HPLC and subsequently characterized
by ESI-MS. Analytical RP-HPLC and ESI-MS analysis of the
crude compounds revealed two chromatographic peaks with the
same MW for compounds 4-7, corresponding to the geometric
isomers (Z/E ratio ≈ 90:10). Compounds were then purified by
semipreparative RP-HPLC, and the most abundant chromato-
graphic peaks were collected. For all the products, HPLC purity
was g97%. Further experimental data are reported in the Sup-
porting Information.
attributed to the orientation of the 1-Nal7 side chain which
was differently oriented in the two peptides. In particular, it
passed from a trans conformation observed in 4, to a gauche-
conformation in 8. Such gauche orientation of the naphthyl
group is likely still suitable (or preferred) for sst5 but not for
sst1-3 binding.
On the basis of the results reported above, we updated the
previously proposed pharmacophore model for sst5-selective
analogues.13 The model involves the classical four side chains
of the sst2/3/5 pharmacophore,30 namely those of residues D-Phe2,
Phe7 (Nal7), D-Trp8, and Lys9, plus the Tyr(Bzl)10 side chain.
The distances between the Cγ atoms of these side chains,
observed in the potent sst5 ligands 3-5, 8arereportedinTable3.
The Cγ-Cγ distances found by Melacini et al. for the sst2/3/5
-
selective SRIF analogues are also reported in the same table.30
It can be observed that the distances found in our derivatives
agree with the sst2/3/5 pharmacophore.
Conclusions
A limited set of compounds of biostable SRIF analogues
with dicarba bridge replacing the disulfidebridge of the parent
octreotide (1) were prepared. Compounds were obtained by
on-resin RCM by second generation Grubbs catalyst. All the
analogues were tested for their affinity toward the sst1-5
receptor subtypes. Among the synthesized compounds, deri-
vative4 exhibited a pan-somatostatin activity(except sst4) and
derivative 8 exhibited high affinity and selectivity toward sst5.
Actually, compound 8 had a similar sst5 affinity (IC50 4.9 nM)
to SRIF-28 and octreotide. Conformation-affinity relation-
ships confirmed that helical propensity correlates with the
peptide sst5-affinity, while a pan-SRIF activity is obtained by
conformational equilibria. Both pan- and selective-SRIF ana-
logues are potentially useful for the diagnosis and internal
radiotherapy of tumors.
NMR Spectroscopy. The samples for NMR spectroscopy
were prepared by dissolving the appropriate amount of peptide
in 0.55 mL of 1H2O (pH 5), 0.05 mL of 2H2O to obtain a con-
centration 1-2 mM of peptides and 200 mM of SDS-d25. TSP
was used as internal chemical shift standard. The water signal
was suppressed by gradient echo.43 NMR experiments were
recorded on a Varian Inova-Unity 700 MHz at 308.1 K. Com-
plete 1H NMR chemical shift assignments were effectively
achieved for all the analyzed peptides (Tables S3-S8, Support-
ing Information) according to the Wuthrich procedure15 via the
€
usual systematic application of TOCSY16 and NOESY17 experi-
ments recorded in the phase-sensitive mode using the method
from States.44
Experimental Section
General Procedures. Fmoc protected amino acids were purchased
from Calbiochem-Novabiochem (Laufelfingen. Switzerland).
Second generation Grubbs catalyst was obtained from Aldrich.
Fmoc-Hag, Fmoc-O-benzyl-L-tyrosine and H-L-Thr(tBu)-ol-2-
chlorotrityl resin were purchased from Iris Biotech (Marktredwitz,
Germany). HATU was obtained from Chempep (Miami, FL).
Peptide grade DMF was from Scharlau (Barcelona, Spain). All
the other solvents and reagents used for SPPS were of analytical qua-
lity and used without further purification. Analytical RP-HPLCs
were performed on a Waters instrument equipped with a UV detec-
tor on a Phenomenex Jupiter C18 column (5 μm, 250 mmꢀ4.6 mm)
using a flow rate of 1 mL/min, with the following solvent system:
0.1% TFA in H2O (A), 0.1% TFA in MeCN (B)). Semipreparative
Typical data block sizes were 2048 addresses in t2 and 512
equidistant t1 values. Before Fourier transformation, the time
domain data matrices were multiplied by shifted sin2 functions
in both dimensions. A mixing time of 70 ms were used for the
TOCSY experiments. NOESY experiments were run with mix-
ing times of 100 and 200 ms. The qualitative and quantitative
analyses of TOCSY and NOESY spectra were obtained with the
support of the XEASY software package.18
Structural Determinations and Computational Modeling. The
NOE-based distance restraints were obtained from NOESY spec-
tra collected with the mixing time of 100 ms. The NOE cross peaks
were integrated with the XEASY program and were converted

6196 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Di Cianni et al.
into upper distance bounds using the CALIBA program incor-
porated into the program package DYANA.19 Only NOE deri-
ved constraints (Tables S9-S12, Supporting Information) were
considered in the annealing procedures. In a first calculation run,
all the upper distance bounds were used, generating an ensemble
of 100 structures with the simulated annealing standard proto-
col of the program DYANA. For peptides 4, 5, and 8, a number
of consistent (i.e., in all calculated structures) violated upper
Supporting Information Available: Analytical data of the
synthesized peptides and NMR data of the analyzed peptides.
Atomic coordinates of the lowest energy conformer of com-
pounds 3, 4, 5, and 8. This material is available free of charge via
the Internet at http://pubs.acs.org.
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limit constraints (>0.1 A) were observed (Tables S9-S12,
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