Discovery of highly potent and neurotensin receptor 2 selective neurotensin mimetics

Article abstract of DOI:10.1021/jm200006c

The neurotensin receptor subtype 2 (NTS2) is involved in the modulation of tonic pain sensitivity and psychiatric diseases and is, therefore, regarded as a highly attractive pharmacological target protein. Aiming to discover NTS2 selective ligands, we her

Full text of DOI:10.1021/jm200006c

ARTICLE
pubs.acs.org/jmc
Discovery of Highly Potent and Neurotensin Receptor 2 Selective
Neurotensin Mimetics
J€urgen Einsiedel, Cornelia Held, Maud Hervet, Manuel Plomer, Nuska Tschammer, Harald H€ubner, and
Peter Gmeiner*
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen,
Germany
S Supporting Information
b
ABSTRACT:
The neurotensin receptor subtype 2 (NTS2) is involved in the modulation of tonic pain sensitivity and psychiatric diseases and is,
therefore, regarded as a highly attractive pharmacological target protein. Aiming to discover NTS2 selective ligands, we herein
describe the identification of screening hits and the chemical synthesis of structural variants leading to the highly potent and NTS2
selective peptideꢀpeptoid hybrids of type 3. The neurotensin mimetics 3a and 3eꢀg incorporating an N-(4-hydroxyphenethyl)-
glycine substructure exhibit single digit nanomolar affinity (Ki = 4.3ꢀ8.8 nM) and 1900ꢀ12000 fold selectivity over the neurotensin
receptor subtype 1 (NTS1). According to functional experiments, the test compounds 3a and 3eꢀg displayed an inhibition of
constitutive mitogen-activated protein kinase (MAPK) activity exceeding 2.6ꢀ4.6 times the inverse agonist activity of the
endogenous ligand neurotensin.
’ INTRODUCTION
derivatives with NTS2 selectivity showed analgesic effects in
mice.17 NTS2 is involved in the modulation of tonic pain
sensitivity18 and is, therefore, regarded as a highly attractive
pharmacological target protein. To further elucidate the physio-
logical role of NTS2 and to investigate intermolecular crosstalk
between NTS1 and NTS2,19 highly selective NTS2 ligands will
be necessary. As lead compounds, such molecular probes could
initiate the development of drug candidates avoiding the risk of
tumor promotion associated with the activation of NTS1. Very
recently, antipsychotic-like activity was reported for the meta-
bolically stabilized ligand 1 (NT79) showing significant NTS2
preference.20 According to recent structureꢀactivity relation-
ships (SAR) data, exchange of the parent peptide’s L-tyrosine unit
in position 11 by unnatural D-amino acids such as D-R-naphthy-
lalanine was accepted by NTS2 but led to a substantial attenua-
tion of NTS1 binding.17,21,22 These results lead to the suggestion
that modifications of both backbone and side chain structure in
position 11 might be key to subtype specific receptor recognition.
Aiming to discover NTS2 selective ligands, we extended our
previous investigations on the discovery and receptorꢀligand
interactions of neurotensin surrogates.23ꢀ25 On the basis of the
The tridecapeptide neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-
Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) is widely distributed in the
brain and in the gastrointestinal tract.1,2 Neurotensin exerts a
variety of physiological effects including hypothermia,3
analgesia,4 and antipsychotic properties.5 The C-terminal hex-
apeptide NT(8ꢀ13) with the sequence H-Arg-Arg-Pro-Tyr-Ile-
Leu-OH (2, Figure 1) proved to be the pharmacologically active
motif6 and has become the lead structure for the development of
neurotensin derived drugs and imaging agents (Figure 1).7,8 The
biological effects are mediated by two G-protein coupled recep-
tor (GPCR) subtypes including the high affinity, levocabastine-
insensitive receptor NTS1,9,10 and the low affinity, levocabastine-
sensitive subtype NTS2.11,12 The neurotensin receptor subtype 3
(NTS3) belongs to the Vps10p family of sorting receptors, which
contain a single transmembrane domain.13
The focus of neurotensin receptor research was mainly
directed to NTS1, which is expressed in the central nervous
system (CNS) and in peripheral organs.10 NTS1 appears to be
associated with the analgesic and antipsychotic effects of neuro-
tensin and was, therefore, evaluated as a drug target. As a negative
prognostic marker,14 NTS1 was also found in tumor tissue and
was reported to be involved in cancer growth.15,16 The sub-
type NTS2 has attracted growing interest because NT(8ꢀ13)
Received: January 4, 2011
Published: March 29, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
building blocks to give the respective peptideꢀpeptoid hybrids.
Starting from O-t-butyl protected tyramine (4a)26 and its
structural analogues 4bꢀ4d, N-alkylation was performed with
ethyl bromoacetate in presence of triethylamine to furnish the
N-alkylglycine derivatives 5aꢀd.27ꢀ30 Ester hydrolysis and sub-
sequent protection using 9-fluorenylmethyl N-hydroxysuccini-
midyl carbonate (Fmoc-OSu) gave access to the peptoid building
blocks 6aꢀd in 48ꢀ63% overall yield (Scheme 1).
Solid phase supported peptide synthesis (SPPS) was done
starting from Fmoc-leucinyl loaded Wang resin. For the peptides
3a,d, we followed a conventional protocol, employing piperi-
dine/1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in dimethylfor-
mamide (DMF) to deprotect the Fmoc function. For acylation
reactions, we added the respective Fmoc protected amino acids
(AA) (5 equiv) and peptoid building blocks (3 equiv) in presence
of 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU). Peptides 3b,c were synthesized
with the help of microwave irradiation accelerating the Fmoc
deprotection and the (benzotriazol-1-yloxy)tripyrrolidinophos-
phonium hexafluorophosphate (PyBOP) promoted acylation
reaction. For the synthesis of the lysine derivatives 3eꢀg, we
chose PyBOP and 2-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tet-
ramethyl uronium hexafluorophosphate (HCTU) promoted,
microwave assisted coupling protocols and a HATU induced
proline incorporation (Scheme 2).
Biological Investigations. To identify subtype selective
NTS2 binders, a peptide library was investigated comprising
derivatives of NT(8ꢀ13) based alanine,31 D-amino acid,32,33 and
homo-β-amino acid scans.25 Additionally, a peptoid scan was
performed in comparison to the neurotensin mimetic [8Lys, 9Lys]
NT(8ꢀ13). Radioligand binding studies were done to evaluate
the peptide libraries for NTS1 and NTS2 affinity (Table 1).
Binding data were determined utilizing the radioligand
[3H]neurotensin and stably transfected Chinese hamster ovary
(CHO) cells expressing human NTS1 or [3H]NT(8ꢀ13) and
the human NTS2, which was transiently transfected in human
embryonic kidney (HEK 239) cells.
Figure 1. Structural modifications leading to the target compounds of
type 3.
Scheme 1a
Comparison of the selectivity ratios of the reference agents
9
neurotensin, NT(8ꢀ13), and [8Lys, Lys]NT(8ꢀ13), display-
ing a 2ꢀ10 fold preference for NTS1, with the test peptides of
the alanine and D-amino acid scans, confirmed our initial
suggestion that position 11 is crucial to subtype selectivity. Thus,
[11Ala]NT(8ꢀ13) and [11D-Tyr]NT(8ꢀ13) exhibited a 16- and
6.4-fold NTS2 preference. This pattern could be also observed
for the peptoid scan when the peptideꢀpeptoid hybrid incorpor-
ating an 11N-Tyr moiety displayed a ratio of Ki values of 30. Thus,
formal migration of the 4-hydroxybenzyl side chain from CR to
the backbone nitrogen of the reference peptide [8Lys,9Lys]-
NT(8ꢀ13) led to substantial attenuation of NTS1 binding
(NTS1: Ki = 30000 nM). It is noteworthy that structural
modifications in position 12 emerged as an alternative strategy.
Thus, substitution of 12Ile by D-Ile induced a 19-fold NTS2-
selectivity. Moreover, incorporation of homo-β-Ile into position
12 led to a selectivity factor of 46 and a Ki value of 5.4 nM for
NTS2, which is similar to the data that we received for the
reference peptide 1 (Ki = 34 nM, selectivity ratio = 250). We
intended to take advantage of efficient synthetic availability of
peptideꢀpeptoid hybrids and the thus facilitated practical access
to unnatural residues. Starting from the library member H-Lys-
Lys-Pro-NTyr-Ile-Leu-OH, our SAR studies involved structural
modifications in position 8 and 9 back to arginyl units of the
parent peptide and structural variation of the hydroxyphenyl
a Reagents and conditions: (a) ethyl bromoacetate, NEt3, THF, 0 °C to
RT, then RT, 2 h (65ꢀ97%); (b) (1) NaOH, MeOH, RT, 30 min, (2)
Fmoc-OSu, H2O/dioxane, pH 8.5ꢀ9.0, 0 °C to RT, then RT, 5 h
(63ꢀ79%).
above-mentioned studies, an alanine scan and systematic peptide
backbone modifications, we herein describe the identification of
screening hits and structural modifications leading to the highly
potent and NTS2 selective peptide-peptoid hybrids of type 3.
’ RESULTS AND DISCUSSION
Chemistry. Our plan of synthesis of the peptideꢀpeptoid
hybrids of type 3 involved the preparation of 9-fluorenylmethy-
loxycarbonyl (Fmoc)-protected N-alkylglycine derivatives
and subsequent solid phase supported incorporation of these
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Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
a Reagents and conditions: (a) For 3a,d: (1) piperidine/DBU/DMF, 2 min, (2) Fmoc-AA, HATU, DIPEA, NMP, RT, 2 h. For 3b,c: (1) piperidine/
DMF, μ-wave, (2) Fmoc-AA-OH, PyBOP, DIPEA, HOBt, DMF, μ-wave. For 3e: (1) piperidine/DMF, μ-wave, (2) Fmoc-AA-OH, PyBOP, DIPEA,
HOBt, DMF, μ-wave, except for proline; Fmoc-Pro-OH, HATU, DIPEA, μ-wave; For 3f,g: (1) piperidine/DMF, μ-wave, (2) peptide coupling; Fmoc-
AA-OH, HCTU, DIPEA, μ-wave, except for proline; Fmoc-Pro-OH, HATU, DIPEA, μ-wave. (b) TFA/phenol/H2O/TIS, RT, 2 h.
function of the peptoid subunit by methoxyphenyl, phenyl, and
2-pyridyl groups. As a compensation of the migration of the
attaching point to the backbone nitrogen atom, a homologization
was part of our program because we suggested that a higher
flexibility of the side chain might allow the ligand to better adapt
to the binding pocket of the target receptor NTS2. In fact, the
receptor binding data clearly display that the homologization of
the side chain substantially increased NTS2 affinity. Thus, the
N-4-hydroxyphenethyl substituted peptideꢀpeptoid hybrid 3g
displayed excellent NTS2 binding (Ki = 4.3 nM). Interestingly,
this gain of affinity was restricted to NTS2 when NTS1 affinity
was nearly unchanged by the homologization. This resulted in a
7400-fold selectivity for NTS2 over NTS1. Similar NTS2 affinity
(NTS1, Ki = 28000 nM; NTS2, Ki = 8.8 nM) and selectivity was
observed for the NT(8ꢀ13) analogue 3a, incorporating two
original arginine residues in both positions 8 and 9. Thus, the
highly selective NTS2 recognition of the test compound of type 3
goes exclusively back to the peptoid unit in position 11. To
examine the relative contribution of both modifications, the
migration and the homologization of the tyrosine residue, the
peptide H-Arg-Arg-Pro-h-Tyr-Ile-Leu-OH was synthesized and
investigated for NTS1 and NTS2 affinity. The data resulted in a
Ki value of 17 nM for NTS2 and a selectivity ratio of 12. Thus,
homologization significantly contributes to a favorable NTS2
binding. However, the combination of both manipulations is
necessary to generate highly selective NTS2 receptor ligands.
Excellent binding and selectivity data were also determined for
the [8Arg,9Lys] and [8Lys,9Arg] analogues 3e and 3f, when the
8-lysinyl derivative 3f showed an unprecedented selectivity ratio
of 12000. Thus, both affinity and selectivity ratios of 3a and 3eꢀg
significantly exceed those for the NTS2 reference peptide 1.
Compared to the NT(8ꢀ13) derived peptoid hybrid 3a, struc-
tural variations of the phenoxy moiety leading to the test
compounds 3bꢀd resulted in an approximately 2ꢀ11 fold
decrease of affinity and a significant reduction of selectivity for
the para-unsubstituted derivatives 3c,d, indicating that a hydroxy
or methoxy group in para-position of residue 11 is beneficial for
the differentiation of a peptidic ligand between NTS2 and NTS1.
To investigate the intrinsic activity of the peptideꢀpeptoid
hybrids 3a and 3eꢀg in comparison to the endogenous ligand
neurotensin,34 a MAPK-driven luciferase reporter gene assay35,36
was performed by employing transiently transfected HEK 293
cells expressing human NTS1 and NTS2. In this test system, the
endogenous ligand neurotensin displayed an inhibition of con-
stitutive MAPK activity, which proved to be strongly increased in
NTS2-expressing cells (Supporting Information). Thus, neuro-
tensin behaves as an inverse agonist with an EC50 value of 110
nM (Figure 2A). In contrast, neurotensin induced an activation
of MAPK promoted luciferase expression (EC50 =1.6nM) inNTS1
expressing cell lines (Figure 2C), indicating agonist properties.
Compared to the parent peptide, the 11Tyr peptoid-peptide
hybrids 3a and 3eꢀg showed a superior impact on NTS2 activity.
Thus, 3a and 3g attenuated constitutive activity of NTS2-
expressing HEK 293 cells 2.6ꢀ4.6-fold stronger than the en-
dogenous peptide neurotensin (Figure 2A, Table 2), displaying
EC50 values between 480 and 840 nM. To validate that this effect
was diagnostic for an NTS2ꢀligand interaction, constitutive
activity of MAPK promoted luciferase expression was attenuated
by 3a and dose dependently regained by addition of the
neurotensin receptor antagonist 2-[[[5-(2,6-dimethoxyphenyl)-
1-[4-[[[3-(dimethylamino)propyl]methylamino]carbonyl]-2-
(1-methylethyl)phenyl]-1H-pyrazol-3-yl]carbonyl]amino]-
adamantane-2-carboxylic acid (SR142948A)37 (Figure 2B). As ex-
pected from the binding experiments, the NTS2 promoted effects of
the test compounds 3a and 3eꢀg proved to be subtype selective
over NTS1 (Emax < 10% at 10ꢀ5 M) (Table 2, Figure 2C). Thus,
the functional assay clearly indicated that the neurotensin mimetics
3a and 3eꢀg act as highly potent and subtype selective inverse
agonists that substantially inhibit constitutive activity of NTS2.38,39
’ CONCLUSION
Aiming to discover NTS2 selective ligands, we herein describe
the identification of screening hits and the chemical synthesis
of structural variants leading to the highly potent and NTS2
selective peptideꢀpeptoid hybrids of type 3. The neurotensin
mimetics 3a and 3eꢀg incorporating an N-(4-hydroxy-
phenethyl)glycine substructure exhibit single digit nanomolar
affinity (Ki = 4.3ꢀ8.8 nM) and 1900ꢀ12000-fold selectivity over
the subtype NTS1. According to functional experiments, the test
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ARTICLE
Table 1. Receptor Binding Data of Peptide Library Members and the Lead Modification Products 3aꢀg in Comparison to the
Reference Agents Neurotensin, NT(8-13) (2), 1, and [8Lys, 9Lys]NT(8-13) Employing Human NTS1 and NTS2a
Ki value ( SEM/SD b [nM]
selectivity
hNTS1 c
[3H]neurotensin
hNTS2 d
[3H]NT(8ꢀ13)
compd
ratio Ki (NTS1)/Ki (NTS2)
Alanine Scan
H-Ala-Arg-Pro-Tyr-Ile-Leu-OH
H-Arg-Ala-Pro-Tyr-Ile-Leu-OH
H-Arg-Arg-Ala-Tyr-Ile-Leu-OH
H-Arg-Arg-Pro-Ala-Ile-Leu-OH
H-Arg-Arg-Pro-Tyr-Ala-Leu-OH
H-Arg-Arg-Pro-Tyr-Ile-Ala-OH
4.6 ( 0.57 b
11 ( 4.3 b
56 ( 10
24 ( 3.7
63 ( 8.3
83 ( 10
0.082
0.46
0.41
16
26 ( 8.2
1300 ( 470
100 ( 11 b
1100 ( 550 b
63 ( 12
1.6
1300 ( 290
0.85
D-Amino Acid Scan
H-D-Arg-Arg-Pro-Tyr-Ile-Leu-OH
H-Arg-D-Arg-Pro-Tyr-Ile-Leu-OH
H-Arg-ArgD-Pro-Tyr-Ile-Leu-OH
H-Arg-Arg-Pro-D-Tyr-Ile-Leu-OH
H-Arg-Arg-Pro-Tyr-D-Ile-Leu-OH
H-Arg-Arg-Pro-Tyr-Ile-D-Leu-OH
0.61 ( 0.16 b
5.4 ( 1.5 b
51 ( 2.8 b
0.11
1.4
71 ( 34 b
220 ( 64 b
590 ( 150 b
140 ( 45
0.37
6.4
890 ( 320
13000 ( 710 b
1300 ( 560 b
690 ( 100
3700 ( 1200
19
0.35
Homo-β-amino Acid Scan
H-h-β-Arg-Arg-Pro-Tyr-Ile-Leu-OH
H-Arg-h-β-Arg-Pro-Tyr-Ile-Leu-OH
H-Arg-Arg-h-β-Pro-Tyr-Ile-Leu-OH
H-Arg-Arg-Pro-h-β-Tyr-Ile-Leu-OH
H-Arg-Arg-Pro-Tyr-h-β-Ile-Leu-OH
H-Arg-Arg-Pro-Tyr-Ile-h-β-Leu-OH
0.23 ( 0.047
3.4 ( 0.99 b
47 ( 11
0.61 ( 0.13
16 ( 2.1b
210 ( 46
44 ( 30b
0.38
0.21
0.22
0.19
46
8.4 ( 0.28b
250 ( 49
5.4 ( 0.99
25 ( 0.0b
8.4 ( 1.9
0.34
Peptoid Scan
NLys-Lys-Pro-Tyr-Ile-Leu-OH
1.6 ( 0.23
17 ( 3.4
0.74 ( 0.026
21 ( 7.9
2.2
0.81
30
H-Lys-NLys-Pro-Tyr-Ile-Leu-OH
H-Lys-Lys-Pro-NTyr-Ile-Leu-OH
H-Lys-Lys-Pro-Tyr-NIle(1)-Leu-OHe
H-Lys-Lys-Pro-Tyr-NIle(2)-Leu-OHe
H-Lys-Lys-Pro-Tyr-Ile-NLeu-OH
30000 ( 5900
3300 ( 1000
3000 ( 960
3700 ( 1300
1000 ( 280
330 ( 130
1200 ( 460
1600 ( 520
10
2.5
2.3
Lead Modification
28000 ( 7400
3a
8.8 ( 1.4
38 ( 9.0
20 ( 5.0
100 ( 24
6.7 ( 1.9
4.4 ( 1.6
4.3 ( 0.63
17 ( 2.1b
3200
1200
130
3b
47000 ( 14000
2600 ( 230
3c
3d
10000 ( 2300
80000 ( 20000
8300 ( 1900
32000 ( 7000
210 ( 57b
100
3e
12000
1900
7400
12
3f
3g
H-Arg-Arg-Pro-h-Tyr-Ile-Leu-OH
Reference Agents
neurotensin
0.59 ( 0.16 f
0.24 ( 0.024
8500 ( 2300
2.7 ( 0.42b
4.9 ( 0.37
1.2 ( 0.17 f
34 ( 9.5
0.12
0.20
250
NT(8ꢀ13) (2)
1
H-Lys-Lys-Pro-Tyr-Ile-Leu-OH
6.1 ( 0.71b
0.44
a Ki values ( SEM are the means of 3ꢀ16 individual experiments each done in triplicate. b Ki values ( SD derived from two experiments. c Membranes from
CHO cells stably expressing human NTS1. d Homogenates from HEK cells transienly expressing hNTS2. e Due to the application of (RS)-2-butylamine for
the peptoid synthesis, we obtained two separable isomers of the respective Ile-peptoid, which were HPLC separated.25 f KD values ( SEM.
compounds 3a and 3eꢀg displayed an inhibition of constitutive
MAPK activity exceeding 2.6ꢀ4.6 times the inverse agonist
activity of the endogenous ligand neurotensin. As lead
compounds, molecular probes of type 3 will initiate the devel-
opment of drug candidates avoiding the risk of tumor promotion
associated with the activation of NTS1. The objective of this
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Table 2. Functional Activity of 3a, 3eꢀg, the Reference
Neurotensin, and the Antagonist SR142948A Derived from
the Modulation of MAPK-Driven Luciferase Activity in
hNTS1 and hNTS2 Expressing Cells
hNTS1
EC50 [nM]a
hNTS2
EC50 [nM]a
b
b
compd
Emax
Emax
3a
3e
3f
0.12
0.07
0
480
840
690
590
3.2
2.6
4.6
3.1
0
3g
0
SR142948A
0
neurotensin
1.6
1.0
110
1.0
a EC50 values in [nM] derived from the mean curves of 3ꢀ11 indepen-
dent experiments each done in triplicate. b Ligand efficacy expressed as
E
max in [fold reference].
under dry N2. Evaporations of product solutions were done in vacuo
with a rotatory evaporator. Column chromatography was performed
using 60 μm silica gel. For thin layer chromatography (TLC), silica gel
60 μm plates were used (ultraviolet (UV), I2 or ninhydrin detection).
Melting temperatures are uncorrected. Infrared (IR) spectra were
registered from a thin film on a NaCl crystal. Nuclear magnetic
resonance (NMR) data were acquired with the help of a 360 MHz (if
not otherwise stated) or a 600 MHz spectrometer, if not stated otherwise
in CDCl3; 13C NMR spectra were recorded at 90 MHz if not stated
otherwise in CDCl3. Chemical shifts are given in δ relative to tetra-
methylsilane (TMS) in parts per million (ppm). Mass spectra were
acquired using electronic ionization (EI), atmospheric pressure chem-
ical ionization (APCI) or electron spray ionization (ESI) techniques.
ESI-ToF high mass accuracy and resolution experiments were per-
formed on a Bruker maXis MS (Bruker Daltonics, Bremen, Germany) in
the laboratory of the Chair of Bioinorganic Chemistry (Prof. Dr. Ivana
Ivanoviꢀc-Burmazoviꢀc), FAU. CHN analyses were conducted in the
analytical laboratory of the Division of Organic Chemistry, FAU. High
performance liquid chromatography (HPLC) analysis revealed a purity
>95% for all SAR compounds.
N-[2-(tert-Butyloxyphenyl)ethyl]glycine Ethyl Ester27,28
(5a). Ethyl bromoacetate (448.0 mg, 2.68 mmol), 2-(4-tert-butyloxy-
phenyl)ethylamine (4a, 569.0 mg, 2.95 mmol),26a and triethylamine
(0.74 mL, 5.36 mmol) were dissolved in tetrahydrofuran (THF) (4 mL)
at 0 °C and then stirred at room temperature (RT) for 4 h. The mixture
was filtered and the residue washed with THF. The filtrate was
concentrated in vacuo, and the residue was purified by flash column
chromatography (hexane/ethyl acetate gradient 9:1 f 7:3 f 5:5) to
afford 5a (594.0 mg, 79%) as a colorless oil. IR (film, NaCl) 3451, 3334,
1739 cmꢀ1. 1H NMR δ 1.25 (t, J = 7.1 Hz, 3H), 1.32 (s, 9H), 1.64 (brs,
1H), 2.77 (m, 2H), 2.86 (m, 2H), 3.41 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H),
6.91 (m, 2H), 7.01 (m, 2H). 13C NMR (90 MHz) δ 14.2, 29.0, 35.8,
50.8, 51.0, 60.7, 78.2, 124.2, 129.0, 134.5, 153.7, 172.3. APCI-MS 280
[M þ H]þ.
Figure 2. Functional activity of the test compound 3a compared to the
reference NT employing a MAPK-driven reporter gene assay in hNTS1-
and hNTS2-expressing HEK 293 cells. (A) Inhibition of the constitutive
activity on luciferase expression in hNTS2 expressing cells as a measure
of intrinsic activity of 3a and NT. (B) Dose dependent attenuation of the
intrinsic effect of 3 μM 3a mediated by the antagonist SR142948A as a
diagnostic measure of NTS2 specific activation. (C) Activation of
luciferase expression in hNTS1 expressing cells mediated by 3a in
comparison to NT. Mean curves of 7ꢀ11 pooled, independent experi-
ments each done in triplicate.
work was to discover NTS2 selective ligands. We anticipate that
the peptoid moiety in position 11 of final products of type 3 will
be advantageous for in vivo stability.40ꢀ42 Because our molecular
probes still contain peptidic partial structures, further chemical
manipulations will be necessary to evaluate pharmacokinetic
profiles and to achieve oral bioavailability.
N-(9-Fluorenylmethyloxycarbonyl)-N-[2-(4-tert-butyloxy-
phenyl)ethyl]glycine (6a).27,28. To a solution of N-[2-(4-tert-buty-
loxyphenyl)ethyl]glycine ethyl ester (5a, 365.5 mg, 1.28 mmol) in
MeOH (4 mL), 4N NaOH (0.64 mL, 2.56 mmol) was added and
stirring was performed for 30 min. The mixture was concentrated in
vacuo, and then water (4 mL) was added and the pH was adjusted to
9.0ꢀ9.5 by employing 2N HCl. Subsequently, the mixture was cooled to
0 °C and a solution of Fmoc-OSu (451.7 mg, 1.34 mmol) in dioxan
(8 mL) was added. The pH was readjusted to 9.0ꢀ9.5, and stirring at RT
was performed for 5 h. After addition of water, the solution was extracted
with diethylether, and the organic layer was discarded. Thereafter, the
’ EXPERIMENTAL SECTION
Chemistry. Reagents and dry solvents were obtained from com-
mercial sources and were used as received. Reactions were conducted
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aqueous layer was acidified to pH 7.5ꢀ8.0 and extracted with ethyl
acetate. The combined organic layers were dried using Na2SO4 and
concentrated in vacuo. Recrystallization (diethylether/hexane) afforded
6a (400.0 mg, 66%) as a white solid, melting point (mp) 123ꢀ124 °C.
followed by washings with DMF (5ꢁ). Peptide coupling was done
employing 5 equiv of each Fmoc-AA/PyBOP/DIPEA and 7.5 equiv
1-hydroxybenzotriazole (HOBt), dissolved in a minimum amount of
DMF (irradiation: 15 ꢁ 10 s, 50 W). In between each irradiation step,
cooling of the reaction mixture to a temperature of ꢀ10 °C was achieved
by sufficient agitation in an ethanolꢀice bath.
IR (film, NaCl) 3413, 1697 cmꢀ1 1H NMR (two rotamers were
.
observed) δ 1.31 (s, 9H), 2.51 and 2.78 (2ꢁ t, each J = 7.1 Hz, 2H),
3.30 and 3.49 (2ꢁ t, each J = 7.1 Hz, 2H), 3.69 and 3.81 (2ꢁ s, 2H), 4.21
(m, 1H), 4.45 and 4.57 (2ꢁ m, 2H), 6.81ꢀ6.85 (m, 2H), 6.89 and 7.03
(2ꢁ m, 2H), 7.26ꢀ7.39 (m, 4H), 7.51 and 7.58 (2ꢁ d, each J = 7.4 Hz,
2H), 7.72 (d, J = 7.4 Hz, 2H). 13C NMR (90 MHz) δ 29.3, 33.7, 34.0,
47.2, 47.3, 49.0, 49.5, 50.3, 50.8, 67.2, 67.5, 77.2, 78.1, 119.9, 120.0,
124.3, 124.4, 124.7, 124.8, 127.0, 127.1, 127.6, 127.7, 129.0, 129.1, 133.2,
133.6, 141.3, 141.4, 143.8, 153.8, 155.6, 156.6, 174.5, 174.7. APCI-MS
474 [M þ H]þ. Anal. Calcd for C29H31NO5 (351.40): C, 73.55; H, 6.60;
N, 2.96. Found: C, 73.14; H, 6.22; N, 2.65.
Elongation Method 3 (EM3). Microwave irradiation was done as
described for EM2. For the synthesis of 3e, peptide coupling was
performed employing 5 equiv of the corresponding Fmoc-AA/
PyBOP/DIPEA and 7.5 equiv HOBt, the peptoid (3 equiv) was coupled
with 3 equiv PyBOP/DIPEA and 4.5 equiv HOBt in DMF. For the
synthesis of 3f,g, 5 equiv of the each Fmoc-AA/HCTU and 10 equiv
DIPEA (for the peptoid building block 3/3/6 equiv) in DMF were
applied. Fmoc-Pro-OH (5 equiv) was routinely coupled 2ꢁ with HATU
(5 equiv) and DIPEA (10 equiv) in DMF.
[11N-(2-(4-Hydroxyphenyl)ethyl)-Gly]NT(8ꢀ13) (3a). The
peptideꢀpeptoid hybrid was synthesized according to EM1. Purification
[C18]: eluent: 0.1% TFA in acetonitrile (A) and 0.1% TFA in H2O
(B) applying a linear gradient starting from 5% A in 95% B to 35% A in
65% B in 14.0 min, FR 21.0 mL/min, tr 12.6 min. Purity: S1B, 98.0%
(tr: 13.7 min); S2A, 98.0% (tr: 14.7 min). [M þ H]þ: calcd, 831.5;
found, 831.6. HRESI-TOF: calcd for C39H67N12O8, 831.5205; found,
831.5192.
Synthesis of the PeptideꢀPeptoid Hybrids. The peptide
synthesis was done according to standard protocols as described below.
The synthesis was performed starting from commercially available
Fmoc-Leu-Wang resin. R-Amino acids were incorporated as their
commercially available derivatives: Fmoc-Ile-OH, Fmoc-Pro-OH, and
Fmoc-Arg(Pbf)-OH. Elongation of the peptide chain was done by
repetitive cycles of Fmoc deprotection either without (elongation
method 1) or with the help of microwave irradiation (elongation
method 2 and 3) and subsequent coupling of the AA derivative. After
the last acylation step, the N-terminal Fmoc-residue was deprotected,
the resin was 10ꢁ rinsed with CH2Cl2 and dried in vacuo. The cleavage
from the resin was performed using a mixture of trifluoroacetic acid
(TFA)/phenol/H2O/triisopropylsilane (TIS) 88:5:5:2 for 2 h, followed
by a filtration of the resin. After evaporation of the solvent in vacuo and
precipitation in t-butylmethylether, the crude peptides were purified
using preparative RP-HPLC: Agilent 1100 preparative series, column
Zorbax Eclipse XDB-C8, 21.2 mm ꢁ 150 mm, 5 μm particles [C8],
employing solvent systems, linear gradient and flow rate [FR] as
specified below or ZORBAX 300SB-C18, 21.2 mm ꢁ 250 mm, 7 μm
column [C18] employing solvent systems, linear gradient and flow rate
[FR] as specified below.
After the separation, the peptides were lyophilized and peptide purity
and identity were assessed by analytical HPLC (Agilent 1100 analytical
series, equipped with QuatPump and VWD detector; column Zorbax
Eclipse XDB-C8 analytical column, 4.6 mm ꢁ 150 mm, 5 μm, flow rate
0.5 mL/min) coupled to a Bruker Esquire 2000 mass detector equipped
with an ESI-trap. System 1 (S1): xꢀy % CH3OH in H2O þ 0.1%
HCO2H in 18 min (S1A: 10ꢀ40% in 18 min, 40ꢀ95% in 2 min,
95ꢀ95% in 2 min, S1B 10ꢀ55%), system 2 (S2): xꢀy % CH3CN in
H2O þ 0.1% HCO2H in 26 min (S2A: 3ꢀ40%, S2B 3ꢀ20%).
Elongation Method 1 (EM1). Fmoc deprotection: the resin was
treated 5ꢁ with 2% DBU/2% piperidine in DMF for 2 min and rinsed
with CH2Cl2 (10ꢁ). HATU (3ꢀ5 equiv) and the carboxylic acids (3ꢀ
5 equiv) were dissolved in N-methylpyrrolidone (NMP) (least volume
possible). After addition of diisopropylethylamine (DIPEA) (6ꢀ
10 equiv), the mixture was added to the resin and agitated for 8ꢀ16
h, followed by several CH2Cl2 washes. For the introduction of the
peptoid moiety, 3 equiv of the building blocks was employed. Fmoc-Pro-
OH and the first Fmoc-Arg(Pbf)-OH were attached using a double
coupling, initially employing 5 equiv, then 3 equiv of the carboxylic acid
derivative. If possible, complete acylation was monitored with the help of
the Kaiser Test. When the test indicated incomplete coupling, the
procedure was repeated.
[9Lys, 11N-(2-(4-Hydroxyphenyl)ethyl)-Gly]NT(8ꢀ13) (3e).
The peptide was synthesized according to EM3. Purification [C8]:
MeOH (A) and 0.1% HCO2H in H2O (B) applying a linear gradient
starting from 10% A in 90% B to 50% A in 50% B in 18.0 min. FR:
10.0 mL/min. tr: 8.8 min. Purity: S1B >99% (tr: 13.3 min); S2A 99%
(tr 14.8 min). [M þ H]þ: calcd, 803.5; found, 803.6.
[8Lys, 11N-(2-(4-Hydroxyphenyl)ethyl)-Gly]NT(8ꢀ13) (3f).
The peptide was synthesized according to EM3. Purification [C8]:
MeOH (A) and 0.1% HCO2H in H2O (B) applying a linear gradient
starting from 10% A in 90% B to 50% A in 50% B in 18.0 min. FR:
10.0 mL/min. tr: 9.3 min. purity: S1B, 98.6% (tr: 14.0 min); S2A, 96.8%
(tr: 15.4 min). [M þ H]þ: calcd, 803.5; found, 803.6.
[8Lys, 9Lys, 11N-(2-(4-Hydroxyphenyl)ethyl)-Gly]NT(8ꢀ13)
(3g). The peptide was synthesized according to EM3. Purification [C8]:
MeOH (A) and 0.1% HCO2H in H2O (B) applying a linear gradient
starting from 10% A in 90% B to 50% A in 50% B in 18.0 min. FR:
10.0 mL/min. tr: 8.9 min. Purity: S1B 96.9% (tr: 12.8 min); S2A 95.8%
(tr: 14.2 min). [M þ H]þ: calcd, 775.5; found, 775.6.
Cloning and Transfection. The hNTS1 and hNTS2 cDNA was
purchased from the UMR cDNA Resource Center subcloned into a
pcDNA3.1(þ) eukaryotic expression vector. To create a cell line stably
expressing hNTS1 the pcDNA3.1 hNTS1 expression vector was used to
clone CHO FRT cells (Invitrogene, Darmstadt, GE). Cells were grown
in DMEM-F12 supplemented with 10% fetal bovine serum (FBS),
2 mM L-glutamine, 1% Pen-Strep (all from Invitrogene), and 400 μg/mL
hygromycin B (Calbiochem Merck Chemicals, Nottongham, UK). For
receptor binding experiments with NTS2, HEK 293 cells were transi-
ently transfected according the calcium phosphate precipitation
method.43 For the reporter-gene assay on functional activity of NTS1
and NTS2 HEK 293 cells were transiently transfected with the lipo-
fectine method using the TransIT-293 transfection reagent (Mirus Bio
Corporation, Madison, WI). In brief, transient transfection was done
with HEK 293 cells, which were maintained in DMEM-F12 supple-
mented with 10% FBS, 2 mM L-glutamine, and 1% Pen-Strep and kept in
a humid atmosphere at 37 °C and 5% CO2. For the calcium phosphate
precipitation method, 30 μg of pcDNA3.1 hNTS2 was added to each
15 cm dish, cells were grown for 48 h and harvested and worked up as
described previously.44 For the reporter-gene assay HEK 293 cells were
grown in 10 cm dishes to about 60% confluency and lipofectine
transfection was done by adding a mixture of 6 μg gene of interest
(pcDNA3.1 hNTS1 or pcDNA3.1 hNTS2), 5 μg pFR-Luc reporter
Elongation Method 2 (EM2). Microwave assisted (Discover
microwave oven, CEM Corp.) peptide synthesis was carried out in
silanized glass tubes loosely sealed with a silicon septum. Remark: the
development of overpressure was avoided by using DMF as the solvent
and intermittent cooling. Fmoc deprotection: the resin was treated 1ꢁ
with 20% piperidine in DMF (microwave irradiation: 5 ꢁ 5 s, 100 W),
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Journal of Medicinal Chemistry
ARTICLE
plasmid, and 0.5 μg pFA2-Elk1 trans-activator plasmid (PathDetect Elk1
Trans-Reporting System, Stratagene, Santa Clara, CA) to the cells.
Receptor Binding Experiments. Receptor binding data were
determined according to protocols as described previously.45 In detail,
hNTR1 binding was measured using homogenats of membranes from
CHO cells stably expressing human NTR1 at a final concentration of
2 μg/well and the radioligand [3H]neurotensin (specific activity 116 Ci/
mmol; PerkinElmer, Rodgau, Germany) at a concentration of 0.5 nM.46
Specific binding of the radioligand was determined at a KD value of
0.59 ( 0.16 nM and a Bmax of 1150ꢀ7290 fmol/mg protein. Unspecific
binding was determined in the presence of 10 μM neurotensin. NTS2
binding was done using homogenats of membranes from HEK 293,
which were transiently transfected with the pcDNA3.1 vector containing
the human NTS2 gene (UMR) by the calcium phosphate method.43
Membranes were incubated at a final concentration of 20 μg/well
together with 0.5 nM of [3H]NT(8ꢀ13) (specific activity 136 Ci/
mmol; custom synthesis of [leucine-3H]NT(8ꢀ13) by GE Healthcare,
Freiburg, Germany) at a KD value of 1.2 ( 0.17 nM and a Bmax
of 325ꢀ2560 fmol/mg protein. Specific binding of the radioligand
was determined at a KD value of 0.47ꢀ0.54 nM and a Bmax of
5050ꢀ7030 fmol/mg protein. Unspecific binding was determined in
the presence of 10 μM NT(8ꢀ13) and the protein concentration was
generally established by the method of Lowry using bovine serum
albumin as standard.47
Luciferase Reporter-Gene Assay. The luciferase reporter gene
assay was performed according to the manufacturer’s instructions
(PathDetect Elk1 Trans-Reporting System, Stratagene, USA). Briefly,
HEK 293 cells were transfected with the TransIT-293 transfection
reagent (Mirus Bio Corporation, Madison, WI). Twenty-four hours
after transfection, cells were trypsinized and washed once to remove
FBS. Cells were resuspended in DMEM-F12 supplemented with 1%
FBS, 2 mM L-glutamine, and 1% Pen-Strep and seeded into white half
area 96-well plates (50000 cells/well). The test compounds were added
at the indicated concentrations at a final volume of 50 μL and incubated
for 24 h at 37 °C and 5% CO2. To quantify an increase in luciferase
expression, 50 μL of Bright-Glo reagent (Promega, Mannheim,
Germany) was added to each well. After 2 min of incubation at RT
and constant agitation to achieve complete cell lysis, luminescence was
measured with a multiplate reader (Viktor3V, Perkin-Elmer, Rodgau
Germany).
experiments are presented. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ49 9131 85-29383. Fax: þ49 9131 85-22585. E-mail:
peter.gmeiner@medchem.uni-erlangen.de.
’ ACKNOWLEDGMENT
This work was supported by the Deutsche Forschungsge-
meinschaft (DFG). Iris Torres is acknowledged for skilful
technical assistance. We thank Prof. Ivana Ivanoviꢀc-Burmazoviꢀc
and Leanne Nye for high resolution ESI-TOF mass spectra. M.P.
thanks the Universit€at Bayern e.V. for a Ph.D. grant.
’ ABBREVIATIONS USED
NTS1, neurotensin receptor 1; NTS2, neurotensin receptor 2;
NTS3, neurotensin receptor 3; GPCR, G-protein coupled re-
ceptor; CNS, central nervous system; SAR, structureꢀactivity
relationships; AA, amino acid; CHO, Chinese hamster ovary;
HEK, human embryonic kidney; SPPS, solid phase peptide
synthesis; Fmoc, 9-fluorenylmethyloxycarbonyl; THF, tetrahy-
dofuran; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate; PyBOP, (benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate; HCTU, 2-(1H-
6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexa-
fluorophosphate; HPLC, high performance liquid chromatogra-
phy; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; Boc, tert-butyloxycar-
bonyl; HOBt, 1-hydroxybenzotriazole; NMP, N-methylpyrroli-
done; DMF, dimethylformamide; DIPEA, diisopropylethyl-
amine; TFA, trifluoroacetic acid; TIS, triisopropylsilane; MAPK,
mitogen-activated protein kinase; TLC, thin layer chromatogra-
phy; IR, infrared; UV, ultraviolet; NMR, nuclear magnetic reso-
nance; TMS, tetramethylsilane; EI-MS, electron ionization mass
spectroscopy; APCI, atmospheric pressure chemical ionization;
ESI, electron spray ionization; RT, room temperature; MeOH,
methanol; mp, melting point; CHN, combustion/elemental
analysis; FBS, fetal bovine serum; RLU, relative luminescence
units
Data Analysis. Data analysis of the competition curves from the
radioligand binding experiments was accomplished by nonlinear regres-
sion analysis using the algorithms in PRISM (GraphPad Software, San
Diego, CA). EC50 values derived from the resulting dose response curves
were transformed into the corresponding Ki values utilizing the equation
of Cheng and Prusoff.48
Data analysis of the reporter-gene assay started when transforming
the displayed luminescence values which were given as relative lumines-
cence units (RLU) into normalized data. For NTS1 activation the
efficacy (Emax) of the test compound was expressed as fold effect of the
reference NT. For hNTS2 expressing cells the basal constitutive activity
(see Supporting Information) was defined as difference of RLU com-
pared to mock transfected cells expressing neither hNTS1 nor hNTS2.
The maximum attenuating effect of NT on RLU was defined as 100%
and the effects of the test compounds were set as fold of NT. The
resulting curves were analyzed by nonlinear regression analysis to
determine EC50 values representing the concentration corresponding
to 50% of maximal efficacy.
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