Modifications at Arg and Ile Give Neurotensin(8-13) Derivatives with High Stability and Retained NTS1 Receptor Affinity

Article abstract of DOI:10.1021/acsmedchemlett.9b00122

Due to its expression in various malignant tumors, the neurotensin receptor 1 (NTS₁R) has been suggested and explored as a target for tumor diagnosis and therapy. Animal model-based investigations of various radiolabeled NTS₁R ligands derived from the hexapeptide neurotensin(8-13) (NT(8-13)), e.g. ⁶⁸Ga- and ¹⁸F-labeled compounds for PET diagnostics, give rise to optimize such radiotracers for clinical use. As NT(8-13) is rapidly degraded in vivo; structural modifications are required in terms of increased metabolic stability. In this study, the stabilization of the peptide backbone of NT(8-13) against enzymatic degradation was systematically explored by performing an N-methyl scan, replacing Ile¹² by tert-butylglycine¹² (Tle¹²) and N-terminal acylation. N-Methylation of either arginine, Arg⁸, or Arg⁹, combined with the Ile¹²/Tle¹² exchange, proved to be most favorable with respect to NTS₁R affinity (K_i < 2 nM) and stability in human plasma (t_1/2 > 48 h), a valuable result regarding the development of radiopharmaceuticals derived from NT(8-13).

Full text of DOI:10.1021/acsmedchemlett.9b00122

Subscriber access provided by UNIVERSITY OF TECHNOLOGY SYDNEY
Letter
Modifications at Arg and Ile give neurotensin(8-13) derivatives
with high stability and retained NTS1 receptor affinity
Lisa Schindler, Günther Bernhardt, and Max Keller
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00122 • Publication Date (Web): 10 May 2019
Downloaded from http://pubs.acs.org on May 11, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Modifications at Arg and Ile give neurotensin(8-13) derivatives with
high stability and retained NTS1 receptor affinity
Lisa Schindler,† Günther Bernhardt,† and Max Keller*,†
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitätsstr. 31, D-93053
Regensburg, Germany
KEYWORDS: peptide, neurotensin, NT(8-13), neurotensin receptor, plasma stability
ABSTRACT: Due to its expression in various malignant tumors, the neurotensin receptor 1 (NTS1R) has been suggested and explored
as a target for tumor diagnosis and therapy. Animal model-based investigations of various radiolabeled NTS1R ligands derived from
the hexapeptide neurotensin(8-13) (NT(8-13)), e.g. 68Ga- and 18F-labeled compounds for PET diagnostics, give rise to optimize such
radiotracers for clinical use. As NT(8-13) is rapidly degraded in vivo, structural modifications are required in terms of increased
metabolic stability. In this study, the stabilization of the peptide backbone of NT(8-13) against enzymatic degradation was
systematically explored by performing an N-methyl scan, replacing Ile12 by tert-butylglycine12 (Tle12) and N-terminal acylation. N-
methylation of either arginine, Arg8 or Arg9, combined with the Ile12/Tle12 exchange proved to be most favorable with respect to
NTS1R affinity (Ki < 2 nM) and stability in human plasma (t1/2 > 48 h), a valuable result regarding the development of
radiopharmaceuticals derived from NT(8-13).
Introduction. The neuromodulator neurotensin (NT), a 13
amino acid peptide (Figure 1), is found in the central nervous
system (CNS), mediating e.g. analgesic effects, as well as in the
periphery (primarily in the gastrointestinal tract).1-2 The
carboxy-terminal hexapeptide of NT (NT(8-13) (1), Figure 1),
is biologically equi-active to NT.3 The physiological effects of
NT are mediated by three cell-surface receptors: the NT
receptors 1 and 2 (NTS1R, NTS2R), both G-protein coupled
receptors,4 and the NTS3R, which belongs to the Vps10p-
domain receptor family.2, 5 The NTS1R has increasingly gained
interest as a target for tumor diagnosis and therapy, as it was
reported to be (over)expressed in a variety of malignancies,
The predominant approaches to stabilize the backbone of 1 are
N-methylation of Arg8 or Arg9, N-terminal acylation and the
exchange of Ile12 by tert-butylglycine (Tle).10-15,
17, 26-38
However, for some interesting analogs of 1, such as N-
methylated derivatives, investigations on the stability are
lacking.33, 39. It is worth mentioning that described derivatives
of 1, containing Tle12 instead of Ile12, include additional
29, 31-32, 38, 40
structural modifications throughout,10-12,
i.e.
[Tle12]NT(8-13) (2, cf. Figure 1) has not been reported to date
to the best of the authors’ knowledge. Therefore, it is difficult
to estimate the impact of the Ile12/Tle12 exchange on the stability
of Tle12-containing derivatives of 1.
among
them
the
prognostically
poor
pancreatic
adenocarcinoma, Ewing’s sarcoma, breast cancer, and
colorectal carcinoma.6-9 Thus, radiolabeled NTS1R ligands
harbor the potential of being used as radiopharmaceuticals. The
majority of such compounds (e.g. 68Ga- and 18F-labeled for PET
diagnostics, 177Lu-labeled for radioendotherapy) has been
derived from the agonist 1.10-19 Noteworthily, also NTS1R
ligands derived from non-peptidic antagonists have been
explored as radiodiagnostics and radiotherapeutics.20-21
Recently reported data of a clinical trial on the treatment of
pancreatic adenocarcinoma in men by 177Lu-labeled NTS1R
antagonists give reason to develop clinical trial candidates with
improved properties.22 Therefore, peptidic NTS1R ligands, such
as radiolabeled derivatives of 1, should be considered for
clinical trials.
EC 3.4.24.16 EC 3.4.15.1
EC 3.4.24.11 EC 3.4.24.11
EC 3.4.24.15
pGlu1-Leu2-Tyr3-Glu4-Asn5-Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13
NT
iKi (NTS1R): 0.28 nMa
Ki (NTS1R): 0.14 nMb
1
NT(8-13),
Arg8-Arg9-Pro10-Tyr11-Tle12-Leu13
[Tle12]NT(8-13),
2
Tle = tert-butylglycine
Figure 1. Amino acid sequences of neurotensin, 1 (NT(8-13), in
blue) and 2, as well as major enzymatic cleavage sites (in red) of
1.3, 24-25 EC 3.4.24.15: metalloendopeptidase 24.15, EC 3.4.24.16:
metalloendopeptidase 24.16, EC 3.4.24.11: neutral endopeptidase
24.11, EC 3.4.15.1: angiotensin converting enzyme (ACE).24-25
aBarroso et al.41 bKeller et al.42
A major drawback of peptide 1 is its rapid degradation in vivo
by peptidases (see Figure 1).23-24 Enzymatic degradation of 1
occurs at three major sites: the Arg8-Arg9 bond, the Pro10-Tyr11
bond and the bond between Tyr11 and Ile12 (cf. Figure 1).24-25
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 13
Aiming at a systematic study on the stabilization of the NT(8-
methylation of Arg8 or Arg9 (7, 8) resulted in significantly
1
2
3
4
5
6
7
8
13) core structure, we synthesized compound 2, performed an
N-methyl scan of 1, combined N-methylation with the
Ile12/Tle12 exchange and, additionally, prepared N-terminally
acylated derivatives of 1. All compounds were studied with
respect to NTS1R binding and plasma stability.
higher plasma stabilities (t1/2 > 48 h) compared to 3 and 4. These
results confirmed that both, N-terminal (cleavage between Arg8
and Arg9) and C-terminal (cleavage between Tyr11 and Ile12)
degradation are highly relevant, and revealed that the former
occurs faster than the latter. As in case of N-terminal
methylation of 1 (peptide 3), N-terminal propionylation of 1
(peptide 11) resulted in a moderate increase in enzymatic
stability compared to 1 (t1/2 of 11 between 1 h and 2 h, cf. Table
1). The combination of N-terminal propionylation with an
Ile12/Tle12 exchange (compound 12) led to an excellent plasma
stability as also observed in case of combining N-terminal
methylation with an Ile12/Tle12 exchange (peptide 7) (Figure 3,
Table 1).
Results and Discussion. Peptides 2, 3,34 4,33 5,33, 39 6,33 7, 8 and
933 were prepared by solid-phase peptide synthesis (SPPS)
according to the
9-fluorenylmethoxycarbonyl (Fmoc)
9
protecting group strategy using 1-hydroxybenzotriazole
(HOBt)/O-(1H-benzotriazol-1-yl)-N,N,N′,N′-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tetramethyluronium hexafluorophosphate (HBTU) and
diisopropylethylamine (DIPEA) for amide bond formation
(Scheme 1). Coupling of Fmoc-protected amino acids to the
secondary amino group of N-methylated amino acids turned out
to be the yield limiting factor in case of 5, 6 and 9 (overall
yields: 18%, 15% and 20%, respectively). The N-terminally
propionylated derivative 11 was obtained by treatment of the
respective resin-bound, side chain-protected, but N-terminally
deprotected precursor peptide with succinimidyl propionate
(10) followed by cleavage off the resin and side chain
deprotection. By contrast, the N-terminally propionylated
peptide 12 was prepared by solution phase treatment of 2 with
compound 10 (Scheme 1).
Scheme 1. Syntheses of the NT(8-13) derivatives 2-9, 11
and 12.a
I
II
2-8
H-Leu
Fmoc-aa or
Fmoc-N-Me-aa
chloro-
trityl
resin
III
I
II
Cl
H-N-Me-Leu
II
9
Fmoc-N-Me-Leu-OH
Fmoc-aa
I
IV
O
IV
11
2
12
H-Leu
Fmoc-aa
10
O
NTS1R binding data (Ki values) were determined for 1-9, 11 and
12 by competition binding with [3H]UR-MK30042 ([3H]13, for
structure see Figure S1, Supporting Information) at intact
hNTS1R expressing HT-29 colon carcinoma cells (Table 1).
The replacement of Ile12 by Tle12 in 1 (compound 2) resulted in
a minor decrease in NTS1R affinity (Ki values of 1 and 2: 0.33
vs. 1.17 nM, cf. Table 1). Regarding the N-methyl scan of 1
(peptides 3-6 and 9), methylation at Arg8 or Arg9 (3, 4) did not
affect NTS1R affinity (Ki < 0.5 nM, Table 1). By contrast, N-
methylation of Tyr11, Ile12 or Leu13 (5, 6, 9) led to a considerable
decrease in NTS1R affinity (Ki values: > 1,000 nM, 60 nM and
880 nM, respectively, cf. Table 1). As expected, the
combination of the N-methylation at Arg8 or Arg9 with the
replacement of Ile12 by Tle12 (peptides 7 and 8) resulted in
NTS1R affinities comparable to that of 2 (Table 1). The N-
terminally propionylated analogs of 1 and 2 (compounds 11 and
12) exhibited Ki values (NTS1R) of 1.0 and 18 nM, respectively.
N
O
O
10
H2N
HN
NH
·
nTFA
OH
(S)-1-methylpropyl
O
H
N
('Ile')
O
O
R4
N
O
R1
N
R2
N
N
R3
N
R5
COOH
O
A
HN
HN
tert-butyl
('Tle')
NH2
compd.
n
R1
R2
R3
R4
R5
A
2
3
4
3
3
3
3
3
3
3
3
2
2
H
H
H
H
H
H
H
H
H
H
H
H
tert-butyl
CH3
H
(S)-1-methylpropyl
(S)-1-methylpropyl
CH3
5
6
H
H
H
CH3
H
H
H
H
H
H
(S)-1-methylpropyl
(S)-1-methylpropyl
tert-butyl
Figure 2 illustrates a general decrease in NTS1R affinity caused
by the replacement of Ile12 by Tle12 in 1, 3, 4 and 11, giving 2,
7, 8 and 12, respectively, and a dependency of the extent of the
decrease in affinity on the primary structure of the peptides.
This is in agreement with reported NTS1R binding data of
derivatives of 1 containing Tle12.10-11, 27, 31, 38, 40
H
CH3
H
7
CH3
H
H
H
8
CH3
H
H
H
tert-butyl
9
H
H
H
CH3 (S)-1-methylpropyl
11
12
CO-C2H5
CO-C2H5
H
H
H
H
H
(S)-1-methylpropyl
tert-butyl
H
H
H
In order to investigate the effect of N-methylation (3-9), the
Ile12/Tle12 exchange (2, 7, 8, 12) and N-terminal acylation (11,
12) on the stability of the peptides against enzymatic cleavage,
the stability of all compounds was investigated in human
plasma at 37 °C for up to 48 h (Figure 3, Table 1). Whereas N-
methylation of Arg8 or Arg9 in 1 (compounds 3 and 4)
significantly enhanced the peptide stability in plasma compared
to 1, methylation of Tyr11, Ile12 and Leu13 (5, 6, 9) did not lead
to higher plasma stabilities. Strikingly, peptide 2, which
differed from 1 only with respect to the replacement of Ile12 by
Tle12, proved to be as unstable as 1 (Figure 3, Table 1).
However, the combination of the Ile12/Tle12 exchange with N-
aReagents and conditions: (I) Fmoc strategy SPPS using
HBTU/HOBt and DIPEA, solvent: DMF/NMP (80:20 v/v), 35 °C,
2 × 1 h or 2 × 2 h, Fmoc-deprotection: 20% piperidine in
DMF/NMP (80:20 v/v), rt, 2 × 8-10 min; (II) (1) hexafluoro-2-
propanol (HFIP)/CH2Cl2 (1:3 v/v), rt, 2 × 20 min, (2) TFA/H2O
(95:5 v/v), rt, 3 h; (III) DIPEA, solvent: CH2Cl2, 35 °C, 14 h; (IV)
DIPEA, solvent: DMF/NMP (80:20 v/v), rt, 1 h; overall yields:
77% (2), 67% (3), 56% (4), 18% (5), 15% (6), 42% (7), 38% (8),
20% (9), 56% (11), 85% (12).
ACS Paragon Plus Environment

Page 3 of 13
ACS Medicinal Chemistry Letters
Figure 4 provides an overview of the major degradation
(Figure 4), being in agreement with reported data on the
fragments identified by LC-HRMS. The Arg8-Arg9, Pro10-Tyr11
and Tyr11-Ile12 bonds were identified as the major cleavage sites
metabolic stability of 1.24-25 As outlined above, the present study
suggests that cleavage of Arg8 in 1 occurs faster than its
1
2
3
4
5
6
7
8
9
Table 1. Peptide sequences and NTS1R affinities of 1-9, 11 and 12, as well as stabilities of 1-9, 11 and 12 in human plasma/PBS
(1:2 v/v) (37 °C).
% intact peptide in plasmab after the specified incubation times:
compd. sequence
Ki [nM] NTS1Ra
10 min 30 min
1 h
< 1
2 h
6 h
24 h
< 1
48 h
< 1
23.1
n.d.
0.33 [0.35, 0.31]
(lit. 0.14c)
1
2
3
4
5
6
7
8
9
Arg-Arg-Pro-Tyr-Ile-Leu
n.d.
n.d.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
± 0.2
10.8
n.d.
Arg-Arg-Pro-Tyr-Tle-Leu
1.17 [1.17, 1.17]
< 1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
> 99
> 99
n.d.
n.d.
> 99
< 1
n.d.
n.d.
n.d.
n.d.
< 1
n.d.
n.d.
n.d.
n.d.
± 0.5
92.1
88.2
79.7
± 0.1
70.8
± 0.1
0.223 ± 0.005
(lit. 0.29d)
N(Me)-Arg-Arg-Pro-Tyr-Ile-Leu
Arg-N(Me)-Arg-Pro-Tyr-Ile-Leu
Arg-Arg-Pro-N(Me)-Tyr-Ile-Leu
Arg-Arg-Pro-Tyr-N(Me)-Ile-Leu
N(Me)-Arg-Arg-Pro-Tyr-Tle-Leu
Arg-N(Me)-Arg-Pro-Tyr-Tle-Leu
Arg-Arg-Pro-Tyr-Ile-N(Me)-Leu
± 0.1
± 0.2
93.6
± 0.1
83.7
± 0.3
66.4
± 0.1
0.29 ± 0.03
(lit. 0.51e)
> 99
22.9
± 0.2
> 1,000
< 1
< 1
n.d.
n.d.
< 1
< 1
< 1
< 1
< 1
n.d.
n.d.
< 1
(lit. 5100e)
2.6
± 0.5
60 ± 5
(lit. 160e)
98.3
± 0.8
86.8
± 0.3
0.88 ± 0.13
1.6 ± 0.1
n.d.
n.d.
> 99
> 99
< 1
> 99
n.d.
> 99
n.d.
n.d.
39.9
± 0.9
880 ± 260
(lit. 190e)
84.0
± 0.1
71.8
± 0.2
32.4
± 0.1
11 Propionyl-Arg-Arg-Pro-Tyr-Ile-Leu
1.0 ± 0.2
> 99
n.d.
92.5
± 0.9
12 Propionyl-Arg-Arg-Pro-Tyr-Tle-Leu
18 ± 2
n.d.
n.d.
> 99
n.d.
> 99
aDetermined by radioligand competition binding with [3H]13 at HT-29 cells; mean values from two (1, 2), three (3, 4) or four (6-9, 11, 12) independent
experiments, each performed in triplicate (for n > 2 Ki values are given ± SEM, in case of n = 2 individual Ki values are given in square brackets). bThe initial
concentration of the peptides in plasma/PBS (1:2 v/v) was 100 µM; presented are mean values ± SEM from three independent experiments (SEM not given if
no decomposition was observed). cKeller et al.42 dOrwig et al.34 eHärterich et al.33 n.d. = not determined.
C-terminal degradation. This is, on one hand, in agreement with
19-fold
increase
reports in the literature,24 on the other hand it is in disagreement
with other reports, which suggest an Ile12/Tle12 exchange as the
most crucial structural modification with respect to metabolic
stabilization.27-28
2.0
1.6
1.2
0.8
0.4
0.0
20
16
12
8
5.5-fold
increase
3.6-fold
increase
In conclusion, the synthesis and investigation of N-methylated
derivatives of NT(8-13) (1), N-terminally acylated derivatives
of 1 and analogs containing Tle12 instead of Ile12, revealed that
only the combination of appropriate N-terminal (e.g. N-
methylation of Arg8) and C-terminal (replacement of Ile12 by
Tle12) structural modifications in 1 affords highly stable (plasma
half-live > 48 h) congeners of 1 (compounds 7, 8 and 12).
Fortunately, two of the most stable compounds (7, 8) exhibited
the highest NTS1R affinities of the investigated analogs of 1.
This work answers open questions concerning the
controversially discussed impact of various structural
modifications of 1 on the enzymatic stability, thus supporting
the development of stable radiolabeled derivatives of 1, which
harbor the potential of being used as radiopharmaceuticals.
4.0-fold
increase
4
0
1
2
3
7
4
8
11 12
compound
Figure 2. Decrease in NTS1R affinity (increase in Ki) resulting
from the exchange of Ile12 by Tle12 in 1, 3, 4 and 11 (black bars)
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 13
giving 2, 7, 8 and 12 (grey bars), respectively. Note: the scales of
1
the Y-axes are different.
2
3
4
5
6
7
8
9
100
80
60
40
20
0
10 min
30 min
1 h
2 h
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6 h
24 h
48 h
1
2
3
4
5
6
9
11
7
8
12
Propionyl-[Tle12]NT(8-13)
Propionyl-
NT(8-13)
[Me-Arg9,Tle12]NT(8-13)
"N-methyl scan"
no other modification
[Me-Arg8,Tle12]NT(8-13)
Figure 3. Stabilities of 1-9, 11 and 12 in human plasma/PBS (1:2 v/v) at 37 °C investigated for up to 48 h. Data represent mean values ±
SEM from three independent experiments.
Funding Sources
ASSOCIATED CONTENT
This work was funded by the Deutsche Forschungsgemeinschaft
(research grant KE 1857/1-1 and 1-2, Graduate Training Program
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
General experimental conditions; experimental synthetic protocols
and analytical data of compounds 2-9, 11 and 12; radioligand
competition binding assay; experimental protocol for the
investigation of the stability of 1-9, 11 and 12 in human plasma;
Figures S1 and S2; RP-HPLC chromatograms of compounds 2-9,
11 and 12; 1H-NMR spectra of compounds 2-9, 11 and 12 in
DMSO-d6 and DMSO-d6/D2O (PDF)
(Graduiertenkolleg) GRK1910)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank Susanne Bollwein, Brigitte Wenzl, and Maria
Beer-Krön for excellent technical assistance, Christoph Müller for
the synthesis of compound 10 and Armin Buschauer for providing
laboratory equipment.
Molecular formula strings (CSV)
ABBREVIATIONS
AUTHOR INFORMATION
2-ClTrt, 2-Chlorotrityl; 2-ClTrt-Cl, 2-Chlorotrityl-chloride;
DIPEA, diisopropylethylamine; FCS, fetal calf serum; Fmoc, 9-
fluorenylmethoxycarbonyl; HBTU, O-(1H-benzotriazol-1-yl)-
Corresponding Author
* E-mail: max.keller@ur.de. Phone: (+49)941-9433329. Fax:
(+49)941-9434820
N,N,N′,N′-tetramethyluronium
1,1,1,3,3,3-hexafluoro-2-propanol;
hexafluorophosphate;
HOBt,
HFIP,
1-
Author Contributions
hydroxybenzotriazole; HT-29, human colorectal adenocarcinoma
cell line; IC50, inhibitor/antagonist concentration which suppresses
50% of an agonist induced effect, or displaces 50% of a labeled
ligand from the binding site; k, retention (or capacity) factor
(HPLC); Kd, dissociation constant obtained from a saturation
binding experiment; Ki, dissociation constant obtained from a
competition binding experiment; NT, neurotensin; NT(8-13),
neurotensin(8-13); NTS1R, neurotensin receptor 1; NTS2R,
L.S. performed any syntheses, radioligand competition
binding experiments and plasma stability studies. M.K.
initiated and planned the project. M.K. and G.B.
supervised the research. L.S., M.K. and G.B. wrote the
manuscript. All authors have given approval to the final
version of the manuscript.
ACS Paragon Plus Environment

Page 5 of 13
ACS Medicinal Chemistry Letters
neurotensin receptor 2; RP, reversed phase; SEM, standard error of
the mean; SPPS, solid-phase peptide synthesis; Tle, tert-
butylglycine.
1
2
3
4
5
6
Identified fragments after incubation in human plasma/PBS (1:2 v/v):
H2N
HN
NH
compd.
R1
R2
R3
R4
R5
A
F1 F2 F3 F4
OH
7
8
9
1
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
(S)-1-methylpropyl
tert-butyl
x
x
x
x
O
H
H
N
O
O
R4
O
3
CH3
H
H
H
(S)-1-methylpropyl
(S)-1-methylpropyl
(S)-1-methylpropyl
(S)-1-methylpropyl
tert-butyl
x
x
x
x
R1
N
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
H
CH3
H
H
H
N
C1
N
N
R
N
COOH
5
H
CH3
H
H
x
x
3
O
A
R5
C2
C3
HN
HN
6
H
CH3
H
CH3
H
x
7
H
H
NH2
8
H
CH3
H
H
H
tert-butyl
F1
9
H
H
H
CH3 (S)-1-methylpropyl
x
x
x
x
x
x
F2
11
12
CO-C2H5
CO-C2H5
H
H
H
H
H
(S)-1-methylpropyl
tert-butyl
x
F3
F4
H
H
H
Figure 4. Major enzymatic cleavage sites (C1-C3) of compounds 1-9, 11 and 12 as well as corresponding fragments F1-F4, identified by
LC-HRMS analysis after incubation in human plasma at 37 °C for up to 48 h.
Biodistribution and catabolism of 18F-labeled neurotensin(8-13)
analogs. Nucl. Med. Biol. 2002, 29, 61-72.
REFERENCES
(11) Alshoukr, F.; Rosant, C.; Maes, V.; Abdelhak, J.; Raguin, O.;
Burg, S.; Sarda, L.; Barbet, J.; Tourwé, D.; Pelaprat, D.; Gruaz-
Guyon, A. Novel neurotensin analogues for radioisotope
targeting to neurotensin receptor-positive tumors. Bioconjugate
Chem. 2009, 20, 1602-1610.
(12) García-Garayoa, E.; Bläuenstein, P.; Blanc, A.; Maes, V.;
Tourwé, D.; Schubiger, P. A. A stable neurotensin-based
radiopharmaceutical for targeted imaging and therapy of
neurotensin receptor-positive tumours. Eur. J. Nucl. Med. Mol.
Imaging 2009, 36, 37-47.
(13) Maschauer, S.; Einsiedel, J.; Hocke, C.; Hübner, H.; Kuwert, T.;
Gmeiner, P.; Prante, O. Synthesis of a 68Ga-labeled peptoid-
peptide hybrid for imaging of neurotensin receptor expression in
vivo. ACS Med. Chem. Lett. 2010, 1, 224-228.
(14) Maschauer, S.; Einsiedel, J.; Haubner, R.; Hocke, C.; Ocker, M.;
Hübner, H.; Kuwert, T.; Gmeiner, P.; Prante, O. Labeling and
Glycosylation of Peptides Using Click Chemistry: A general
approach to 18F-glycopeptides as effective imaging probes for
positron emission tomography. Angew. Chem., Int. Ed. 2010, 49,
976-979.
(15) Alshoukr, F.; Prignon, A.; Brans, L.; Jallane, A.; Mendes, S.;
Talbot, J.-N.; Tourwé, D.; Barbet, J.; Gruaz-Guyon, A. Novel
DOTA-neurotensin analogues for 111In scintigraphy and 68Ga
PET imaging of neurotensin receptor-positive tumors.
Bioconjugate Chem. 2011, 22, 1374-1385.
(16) Maschauer, S.; Ruckdeschel, T.; Tripal, P.; Haubner, R.;
Einsiedel, J.; Hübner, H.; Gmeiner, P.; Kuwert, T.; Prante, O. In
vivo monitoring of the antiangiogenic effect of neurotensin
receptor-mediated radiotherapy by small-animal positron
emission tomography: a pilot study. Pharmaceuticals 2014, 7,
464-481.
(17) Jia, Y.; Shi, W.; Zhou, Z.; Wagh, N. K.; Fan, W.; Brusnahan, S.
K.; Garrison, J. C. Evaluation of DOTA-chelated neurotensin
analogs with spacer-enhanced biological performance for
neurotensin-receptor-1-positive tumor targeting. Nucl. Med. Biol.
2015, 42, 816-823.
(1) Myers, R. M.; Shearman, J. W.; Kitching, M. O.; Ramos-
Montoya, A.; Neal, D. E.; Ley, S. V. Cancer, chemistry, and the
cell: molecules that interact with the neurotensin receptors. ACS
Chem. Biol. 2009, 4, 503-525.
(2) Vincent, J.; Mazella, J.; Kitabgi, P. Neurotensin and neurotensin
receptors. Trends Pharmacol. Sci. 1999, 20, 302-309.
(3) Carraway, R.; Leeman, S. E. The amino acid sequence of a
hypothalamic peptide, neurotensin. J. Biol. Chem. 1975, 250,
1907-1911.
(4) Tanaka, K.; Masu, M.; Nakanishi, S. Structure and functional
expression of the cloned rat neurotensin receptor. Neuron 1990,
4, 847-854.
(5) Mazella, J.; Zsürger, N.; Navarro, V.; Chabry, J.; Kaghad, M.;
Caput, D.; Ferrara, P.; Vita, N.; Gully, D.; Maffrand, J. P.;
Vincent, J. P. The 100-kDa neurotensin receptor is gp95/sortilin,
a non-G-protein-coupled receptor. J. Biol. Chem. 1998, 273,
26273-26276.
(6) Maoret, J.-J.; Pospai, D.; Rouyer-Fessard, C.; Couvineau, A.;
Laboisse, C.; Voisin, T.; Laburthe, M. Neurotensin receptor and
its mRNA are expressed in many human colon cancer cell lines
but not in normal colonic epithelium: binding studies and RT-
PCR experiments. Biochem. Biophys. Res. Commun. 1994, 203,
465-471.
(7) Reubi, J. C.; Waser, B.; Friess, H.; Büchler, M.; Laissue, J.
Neurotensin receptors: a new marker for human ductal pancreatic
adenocarcinoma. Gut 1998, 42, 546-550.
(8) Reubi, J. C.; Waser, B.; Schaer, J.; Laissue, J. A. Neurotensin
receptors in human neoplasms: high incidence in Ewing's
sarcoma. Int. J. Cancer 1999, 82, 213-218.
(9) Souazé, F.; Dupouy, S.; Viardot-Foucault, V.; Bruyneel, E.;
Attoub, S.; Gespach, C.; Gompel, A.; Forgez, P. Expression of
neurotensin and NT1 receptor in human breast cancer: a potential
role in tumor progression. Cancer Res. 2006, 66, 6243-6249.
(10) Bergmann, R.; Scheunemann, M.; Heichert, C.; Mäding, P.;
Wittrisch, H.; Kretzschmar, M.; Rodig, H.; Tourwé, D.; Iterbeke,
K.; Chavatte, K.; Zips, D.; Reubi, J. C.; Johannsen, B.
(18) Maschauer, S.; Einsiedel, J.; Hübner, H.; Gmeiner, P.; Prante, O.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 13
18F- and 68Ga-labeled neurotensin peptides for PET imaging of
neurotensins for NTS1-receptor-targeted tumor imaging with
neurotensin receptor 1. J. Med. Chem. 2016, 59, 6480-6492.
(19) Deng, H.; Wang, H.; Zhang, H.; Wang, M.; Giglio, B.; Ma, X.;
Jiang, G.; Yuan, H.; Wu, Z.; Li, Z. Imaging neurotensin receptor
in prostate cancer with 64Cu-labeled neurotensin analogs. Mol.
Imaging 2017, 16, 1536012117711369.
99mTc. J. Med. Chem. 2006, 49, 4767-4776.
1
2
3
4
5
6
7
8
(32) Maina, T.; Nikolopoulou, A.; Stathopoulou, E.; Galanis, A. S.;
Cordopatis, P.; Nock, B. A. [99mTc]Demotensin 5 and 6 in the
NTS1-R-targeted imaging of tumours: synthesis and preclinical
results. Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 1804-1814.
(33) Härterich, S.; Koschatzky, S.; Einsiedel, J.; Gmeiner, P. Novel
insights into GPCR—Peptide interactions: Mutations in
extracellular loop 1, ligand backbone methylations and molecular
modeling of neurotensin receptor 1. Bioorg. Med. Chem. 2008,
16, 9359-9368.
(34) Orwig, K. S.; Lassetter, M. R.; Hadden, M. K.; Dix, T. A.
Comparison of N-terminal modifications on neurotensin(8-13)
analogues correlates peptide stability but not binding affinity
with in vivo efficacy. J. Med. Chem. 2009, 52, 1803-1813.
(35) Boules, M.; Liang, Y.; Briody, S.; Miura, T.; Fauq, I.; Oliveros,
A.; Wilson, M.; Khaniyev, S.; Williams, K.; Li, Z.; Qi, Y.;
Katovich, M.; Richelson, E. NT79: A novel neurotensin analog
with selective behavioral effects. Brain Res. 2010, 1308, 35-46.
(36) Einsiedel, J.; Held, C.; Hervet, M.; Plomer, M.; Tschammer, N.;
Hübner, H.; Gmeiner, P. Discovery of highly potent and
neurotensin receptor 2 selective neurotensin mimetics. J. Med.
Chem. 2011, 54, 2915-2923.
(20) Lang, C.; Maschauer, S.; Hübner, H.; Gmeiner, P.; Prante, O.
Synthesis and evaluation of
a
18F-labeled diarylpyrazole
glycoconjugate for the imaging of NTS1-positive tumors. J. Med.
Chem. 2013, 56, 9361-9365.
(21) Schulz, J.; Rohracker, M.; Stiebler, M.; Goldschmidt, J.; Grosser,
O. S.; Osterkamp, F.; Pethe, A.; Reineke, U.; Smerling, C.;
Amthauer, H. Comparative evaluation of the biodistribution
profiles of a series of nonpeptidic neurotensin receptor-1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
antagonists reveals
a promising candidate for theranostic
applications. J. Nucl. Med. 2016, 57, 1120-1123.
(22) Baum, R. P.; Singh, A.; Schuchardt, C.; Kulkarni, H. R.; Klette,
I.; Wiessalla, S.; Osterkamp, F.; Reineke, U.; Smerling, C. 177Lu-
3BP-227 for neurotensin receptor 1-targeted therapy of
metastatic pancreatic adenocarcinoma: first clinical results. J.
Nucl. Med. 2018, 59, 809-814.
(23) Pedersen, J. H.; Beck, H.; Shokouh-Amiri, M.; Fischer, A. Effect
of neurotensin in the dumping syndrome. Scand. J.
Gastroenterol. 1986, 21, 478-482.
(37) Held, C.; Plomer, M.; Hübner, H.; Meltretter, J.; Pischetsrieder,
(24) García-Garayoa, E.; Allemann-Tannahill, L.; Bläuenstein, P.;
Willmann, M.; Carrel-Rémy, N.; Tourwé, D.; Iterbeke, K.;
Conrath, P.; Schubiger, P. A. In vitro and in vivo evaluation of
new radiolabeled neurotensin(8-13) analogues with high affinity
for NT1 receptors. Nucl. Med. Biol. 2001, 28, 75-84.
(25) Kitabgi, P.; De Nadai, F.; Rovère, C.; Bidard, J. N. Biosynthesis,
maturation, release, and degradation of neurotensin and
neuromedin N. Ann. N. Y. Acad. Sci. 1992, 668, 30-42.
(26) Tyler-McMahon, B. M.; Boules, M.; Richelson, E. Neurotensin:
peptide for the next millennium. Regul. Pept. 2000, 93, 125-136.
(27) Bruehlmeier, M.; García-Garayoa, E.; Blanc, A.; Holzer, B.;
Gergely, S.; Tourwé, D.; Schubiger, P. A.; Bläuenstein, P.
Stabilization of neurotensin analogues: effect on peptide
catabolism, biodistribution and tumor binding. Nucl. Med. Biol.
2002, 29, 321-327.
(28) García-Garayoa, E.; Bläuenstein, P.; Bruehlmeier, M.; Blanc, A.;
Iterbeke, K.; Conrath, P.; Tourwé, D.; Schubiger, P. A.
Preclinical evaluation of a new, stabilized neurotensin(8-13)
pseudopeptide radiolabeled with 99mTc. J. Nucl. Med. 2002, 43,
374-383.
(29) Bläuenstein, P.; García-Garayoa, E.; Rüegg, D.; Blanc, A.;
Tourwé, D.; Beck-Sickinger, A.; Schubiger, P. A. Improving the
tumor uptake of 99mTc-labeled neuropeptides using stabilized
peptide analogues. Cancer Biother. Radiopharm. 2004, 19, 181-
188.
M.; Gmeiner, P. Development of a metabolically stable
neurotensin receptor 2 (NTS2) ligand. ChemMedChem 2013, 8,
75-81.
(38) Mascarin, A.; Valverde, I. E.; Mindt, T. L. Structure-activity
relationship studies of amino acid substitutions in radiolabeled
neurotensin conjugates. ChemMedChem 2016, 11, 102-107.
(39) Bittermann, H.; Einsiedel, J.; Hübner, H.; Gmeiner, P. Evaluation
of lactam-bridged neurotensin analogues adjusting (Pro10) close
to the experimentally derived bioactive conformation of NT(8-
13). J. Med. Chem. 2004, 47, 5587-5590.
(40) Kokko, K. P.; Hadden, M. K.; Orwig, K. S.; Mazella, J.; Dix, T.
A. In vitro analysis of stable, receptor-selective neurotensin[8-
13] analogues. J. Med. Chem. 2003, 46, 4141-4148.
(41) Barroso, S.; Richard, F.; Nicolas-Ethève, D.; Reversat, J. L.;
Bernassau, J. M.; Kitabgi, P.; Labbé-Jullié, C. Identification of
residues involved in neurotensin binding and modeling of the
agonist binding site in neurotensin receptor 1. J. Biol. Chem.
2000, 275, 328-336.
(42) Keller, M.; Kuhn, K. K.; Einsiedel, J.; Hübner, H.; Biselli, S.;
Mollereau, C.; Wifling, D.; Svobodova, J.; Bernhardt, G.;
Cabrele, C.; Vanderheyden, P. M.; Gmeiner, P.; Buschauer, A.
Mimicking of Arginine by functionalized N ω-carbamoylated
arginine as a new broadly applicable approach to labeled
bioactive peptides: high affinity angiotensin, neuropeptide Y,
neuropeptide FF, and neurotensin receptor ligands as examples.
J. Med. Chem. 2016, 59, 1925-1945.
(30) Maes, V.; García-Garayoa, E.; Bläuenstein, P.; Tourwé, D. Novel
99mTc-labeled
neurotensin
analogues
with
optimized
biodistribution properties. J. Med. Chem. 2006, 49, 1833-1836.
(31) Nock, B. A.; Nikolopoulou, A.; Reubi, J. C.; Maes, V.; Conrath,
P.; Tourwé, D.; Maina, T. Toward stable N4-modified
ACS Paragon Plus Environment

Page 7 of 13
ACS Medicinal Chemistry Letters
1
2
Table of Contents Graphic
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
83x33mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 9 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
83x67mm (300 x 300 DPI)
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 10 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
139x90mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 11 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
177x59mm (300 x 300 DPI)
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 12 of 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
83x127mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 13 of 13
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
82x44mm (600 x 600 DPI)
ACS Paragon Plus Environment