Peptidomimetic growth hormone secretagogue derivatives for positron emission tomography imaging of the ghrelin receptor

Article abstract of DOI:10.1016/j.ejmech.2018.08.062

The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis of positron emission tomography (PET) probes designed to target and image this receptor for disease diagnosis and staging. However, these probes have been restricted to small-molecule quinalizonones and peptide derivatives of the endogenous ligand ghrelin. We describe the design, synthesis and biological evaluation of a series of 4-fluorobenzoylated growth hormone secretagogues (GHSs) derived from peptidic (GHRP-1, GHPR-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal⁴]G-7039 and ipamorelin) families in order to test locations for the insertion of fluorine-18 for PET imaging. The peptidomimetic G-7039 was found to be the most suitable for ¹⁸F-radiolabelling as its non-radioactive 4-fluorobenzoylated analogue ([1-Nal⁴,Lys⁵(4-FB)]G-7039), had both a high binding affinity (IC₅₀ = 69 nM) and promising in vitro efficacy (EC₅₀ = 1.1 nM). Prosthetic group radiolabelling of the precursor compound [1-Nal⁴]G-7039 using N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) delivered the PET probe [1-Nal⁴,Lys⁵(4-[¹⁸F]-FB)]G-7039 in an average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of >34 GBq/μmol. This compound could be investigated as a PET probe for the detection of diseases that are characterised by overexpression of the ghrelin receptor.

Full text of DOI:10.1016/j.ejmech.2018.08.062

Accepted Manuscript
Peptidomimetic growth hormone secretagogue derivatives for positron emission
tomography imaging of the ghrelin receptor
Milan M. Fowkes, Tyler Lalonde, Lihai Yu, Savita Dhanvantari, Michael S. Kovacs,
Leonard G. Luyt
PII:
S0223-5234(18)30734-7
10.1016/j.ejmech.2018.08.062
EJMECH 10673
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 July 2018
Revised Date: 16 August 2018
Accepted Date: 22 August 2018
Please cite this article as: M.M. Fowkes, T. Lalonde, L. Yu, S. Dhanvantari, M.S. Kovacs, L.G.
Luyt, Peptidomimetic growth hormone secretagogue derivatives for positron emission tomography
imaging of the ghrelin receptor, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.062.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Peptidomimetic growth hormone secretagogue derivatives for positron emission
tomography imaging of the ghrelin receptor
Milan M. Fowkes,a,b† Tyler Lalonde,a,b Lihai Yu,a,b Savita Dhanvantari,c Michael S. Kovacsc and
*
Leonard G. Luyta,b,c
aDepartment of Chemistry, Western University, 1151 Richmond Street, London, Ontario N6A 5B7,
Canada,
bLondon Regional Cancer Program, Lawson Health Research Institute, 790 Commissioners Road
East, London, Ontario N6A 4L6, Canada,
cImaging Program, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario N6A
4V2, Canada.
†Present address: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and
Musculoskeletal Sciences, University of Oxford, Roosevelt drive, Oxford, OX3 7FY, United Kingdom.
*Corresponding author. London Regional Cancer Program, Lawson Health Research Institute, 790 Commissioners
Road East, London, Ontario N6A 4L6, Canada.
E-mail address: lluyt@uwo.ca (L.G. Luyt).
Abbreviations: [18F]FB, [18F]fluorobenzoyl; [18F]SFB, N-succinimidyl-4-[18F]fluorobenzoate; [19F]FBA,
[19F]fluorobenzoic acid;
7-TM, 7-transmembrane; AEEA, 2-(2-(2-aminoethoxy)ethoxy)acetic acid; Aib, 2-
aminoisobutyric acid; cpm, counts per minute; D-1-Nal, D-1-naphthylalanine, D-2-Nal, D-2-naphthylalanine; D-2-Thi,
D-2-thienylalanine; D-Trp(2-Me); D-2-methyltryptophan, Inp, isonipecotic acid; d.c., decay-corrected; DIPEA, N,N-
diisopropylethylamine; Dpr, 2,3-diaminopropionic acid; EDC, N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide; Emax
,
maximal response; Fmoc-SPPS Fmoc-solid phase peptide synthesis; GH, growth hormone; GHRH, growth hormone
releasing hormone; GHSs, growth hormone secretagogues; HBSS, Hank’s balanced salt solution; HCTU, 1-
[Bis(dimethylamino)methylen]-5-chlorobenzotriazolium 3-oxide hexafluorophosphate; HEK293, human embryonic
kidney 293; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; Kryptofix K 2.2.2, 4,7,13,16,21,24-Hexaoxa-
1,10-diazabicyclo[8.8.8]hexacosane; MBHA, 4-Methylbenzhydrylamine hydrochloride salt resin; MeOTf, methyl
trifluoromethanesulfonate; NHS, N-hydroxysuccinimide; RP reverse phase; TBMe, tert-butyl methyl ether; TFA,
trifluoroacetic acid.
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Abstract
The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased
level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis
of positron emission tomography (PET) probes designed to target and image this receptor for
disease diagnosis and staging. However, these probes have been restricted to small-molecule
quinalizonones and peptide derivatives of the endogenous ligand ghrelin. We describe the design,
synthesis and biological evaluation of a series of 4-fluorobenzoylated growth hormone
secretagogues (GHSs) derived from peptidic (GHRP-1, GHPR-2 and GHRP-6) and peptidomimetic
(G-7039, [1-Nal4]G-7039 and ipamorelin) families in order to test locations for the insertion of
fluorine-18 for PET imaging. The peptidomimetic G-7039 was found to be the most suitable for
18F-radiolabelling as its non-radioactive 4-fluorobenzoylated analogue ([1-Nal4,Lys5(4-FB)]G-
7039), had both a high binding affinity (IC50 = 69 nM) and promising in vitro efficacy (EC50 = 1.1
nM). Prosthetic group radiolabelling of the precursor compound [1-Nal4]G-7039 using N-
succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) delivered the PET probe [1-Nal4,Lys5(4-[18F]-
FB)]G-7039 in an average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and
an average molar activity of > 34 GBq/µmol. This compound could be investigated as a PET probe
for the detection of diseases that are characterised by overexpression of the ghrelin receptor.
Keywords
Growth hormone secretagogues, peptidomimetics, positron emission tomography, ghrelin receptor,
18F-radiochemistry.
1. Introduction
Growth hormone secretagogues (GHSs) are a class of compounds that stimulate the
secretion of growth hormone (GH) by acting on the ghrelin receptor, also known as the growth
hormone secretagogue receptor type-1a (GHS-R1a)[1]. This receptor is predominantly found in the
hypothalamus and pituitary gland[1] and its secretion occurs through a route disparate from that of
growth hormone-releasing hormone (GHRH)[2]. The first GHSs to show GH release in vitro were a
series of Met-enkephalin analogues described by Bowers et al in 1980[3]. Further development of
these analogues led to a hexamer that released GH in vivo in a number of animals[4, 5], including
humans[6]. This peptide was later termed growth hormone-releasing peptide-6 (GHRP-6), and a
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number of GHSs were subsequently synthesised in order to find alternatives to recombinant human
GH therapy. These encompassed peptides (e.g. GHRP-1[7], hexarelin[8], KP-102 (later designated
GHRP-2)[9]), peptidomimetics (e.g. G-7039[10], [1-Nal]4G-7039[11] and ipamorelin[12]) and
small-molecules (e.g. L-692,429[13] and MK-0677[14]). The amino acid sequences of the peptidic
GHSs and the structures of the peptidomimetic and small-molecule GHSs are shown in Figure 1.
The endogenous ligand for GHS-R1a is ghrelin, a 28-mer peptide with an n-octanoyl group on the
Ser3 side-chain, which was discovered in 1999[15] and exhibits a multitude of biological activities,
such as the regulation of food intake and glucose metabolism[16, 17].
H-His-D-Trp-Ala-Trp-D-Phe-Lys-NH2
H-Ala-His-D-2-Nal-Ala-Trp-D-Phe-Lys-NH2
GHRP-6
GHRP-1
H-His-D-Trp(2-Me)-Ala-Trp-D-Phe-Lys-NH2
H-D-Ala-D-2-Nal-Ala-Trp-D-Phe-Lys-NH2
Hexarelin
GHRP-2
O
O
O
O
O
O
H
H
H
H
N
N
N
N
N
N
NH2
NH2
N
N
NH2
NH2
H
H
H
H
HN
O
O
HN
O
O
[1-Nal]4G-7039
G-7039
O
NH2
NH2
O
N
O
N
N
H2N
H
O
O
O
N
O
HN
H
H
N
N
H2N
N
N
H2N
H
H
HN
N
O
O
O
N
N
NH
N
S
O
O
Ipamorelin
L 692,429
MK-0677
Figure 1. Peptidic, peptidomimetic and small-molecule growth hormone secretagogues. All amino
acids are designated by the standard three-letter code. D-2-Nal, D-2-naphthylalanine; Inp,
isonipecotic acid; D-Trp(2-Me); D-2-methyltryptophan.
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The ghrelin receptor is expressed in a number of human malignancies including prostatic
carcinoma cell lines[18, 19] and tissues[20], breast carcinoma tumours and cell lines[21], testicular
tumours[22], and malignant ovarian cysts and tumours[23]. In addition, it has an elevated level of
expression in tissues from patients with atherosclerosis[24] and in biopsies from those with chronic
heart failure[25]. The ghrelin receptor thus represents a potential target for molecular imaging of
carcinoma and cardiovascular disease. Among the imaging modalities most commonly utilised in
the clinic, positron emission tomography (PET) combines a high spatial resolution with high
sensitivity[26]. A number of recent publications have thus described the development of PET
radiotracers for visualising the GHS-R1a receptor in disease[27-30]. For example, Hou and co-
workers synthesized the first picomolar quinazolinone binder of the ghrelin receptor and
successfully radiolabelled two nanomolar compounds in high radiochemical purity[30]. In another
study on small-molecule derivatives, in vivo imaging in mice with a [11C]radiotracer revealed a
higher specific uptake in the pancreas compared to other organs[28]. However, these studies[27-30]
and others[31-33] have primarily focused on either small-molecule quinazolinones[27, 28, 30, 31]
or ghrelin-derived compounds[29, 32, 33]. To the best of our knowledge, a growth hormone
secretagogue-based PET probe has not been reported to date. We reasoned that modification of
peptidic and peptidomimetic GHSs should result in a clinically translatable PET agent with high
target specificity, in vivo stability and favourable pharmacokinetic properties. Fluorine-18 was our
preferred choice of radioisotope as it is a small innocuous unit that can be easily installed in the
absence of a chelator using the [18F]fluorobenzoyl ([18F]FB) prosthetic group. This modification can
be initially trialled with 4-[19F]fluorobenzoic acid ([19F]FBA), in order to determine the optimal
location for the 18F-radioisotope without significantly affecting peptide/peptidomimetic binding to
the ghrelin receptor.
We describe herein the design, synthesis and biological evaluation of 4-fluorobenzoylated
derivatives of GHSs with peptidic (GHRP-1, GHRP-2 and GHRP-6) and peptidomimetic (G-7039,
[1-Nal4]G-7039 and ipamorelin) structures; the determination of their IC50 values for the ghrelin
receptor; the identification of the lead peptide [1-Nal4,Lys5(4-FB)]G-7039 with nanomolar IC50 and
EC50 values; and the 18F-radiolabelling of its precursor [1-Nal4]G-7039 to furnish the
peptidomimetic growth hormone secretagogue [1-Nal4,Lys5(4-[18F]-FB)]G-7039, which could be
applied to PET imaging of diseases via targeting of the ghrelin receptor.
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2. Results and Discussion
2.1 Design strategy for peptidic and peptidomimetic growth hormone secretagogues
Initially, we sought to determine the location for 18F insertion into the peptidic and
peptidomimetic GHSs without significantly affecting receptor binding affinity. A study of the
literature revealed that Huang and co-workers had designed a 3D pharmacophore for GHS activity
using peptidic and non-peptidic compounds without inclusion of the lysine side-chain[34]. This led
to the synthesis of a benzothiazepin compound with low nanomolar in vitro efficacy for the ghrelin
receptor[34]. In addition, we recently reported a ghrelin receptor homology model based on
neurotensin and opioid receptors with docking of the peptidomimetic G-7039[35]. The lysine
residue was found to be unimportant for G-7039-GHS-R1a binding owing to an unfavourable
interaction energy between its side-chain amino group and polar Glu197/Arg199 residues[35]. This
led us to select the lysine side-chain for 18F-radioisotope insertion in the peptidic (GHRP-1, GHRP-
2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) GHSs using the
[18F]FB prosthetic group via the pre-activated N-succinimidyl-4-[18F]fluorobenzoate
([18F]SFB)[36]. This modification was first tested by coupling a non-radioactive [18F]FB mimic
([19F]FBA) to the lysine side-chain of each individual GHS class using the orthogonal
allyloxycarbonyl (Alloc) protecting group by standard Fmoc-solid phase peptide synthesis (SPPS).
Any change in binding affinity for the ghrelin receptor (expressed in terms of the IC50) was
determined experimentally through a competitive receptor-ligand binding assay. Each parent
growth hormone secretagogue (Figure 1) would be modified in turn until an IC50 < 100 nM was
achieved.
2.2 Synthesis and characterisation of peptidic and peptidomimetic growth hormone
secretagogues
The peptidic and peptidomimetic GHSs were synthesised through manual or automated
Fmoc-SPPS and purified by reverse-phase (RP)-HPLC. Amino acids sequences for these GHSs are
shown in the supporting information (Table S1), with characterisation data listed in Table 1.
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HRMS
(calculated)
[M + H]+
712.3935
834.4103
979.4842
840.3667
860.4259
910.4416
866.3824
955.4943
840.4204
[M + Na]+
873.4524
1017.4511
[M + Na]+
831.4055
953.4223
798.4343*
920.4511
848.4499
970.4667
HRMS
%
%
Peptide/Peptidomimetic
(found)
[M + H]+
712.3959
834.4133
979.4868
840.3693
860.4284
910.4400
866.3850
955.4964
840.4173
[M + Na]+
873.4531
1017.4522
[M + Na]+
831.4070
953.4237
798.4339*
920.4529
848.4501
970.4693
Purity Yield
Ipamorelin
[Lys5(4-FB)]ipamorelin
[Lys5(AEEA-4-FB)]ipamorelin
[D-2-Thi4,Lys5(4-FB)]ipamorelin
[Inp1,Lys5(4-FB)]ipamorelin
[Inp1,D-2-Nal4,Lys5(4-FB)]ipamorelin
[Inp1,D-2-Thi4,Lys5(4-FB)]ipamorelin
GHRP-1
> 97
> 99
> 98
> 98
> 96
> 98
> 99
> 99
> 99
19
15
8
4
6
10
13
19
17
GHRP-2
GHRP-6
[Lys6(4-FB)]GHRP-6
> 97
> 96
14
7
[Dpr6]GHRP-6
[Dpr6(4-FB)]GHRP-6
G-7039*
[Lys5(4-FB)]G-7039
[1-Nal4]G-7039
> 99
> 98
> 99*
> 97
> 99
> 96
3
14
7*
6
23
9
[1-Nal4,Lys5(4-FB)]G-7039
Table 1. HRMS data, purities and yields for synthesised peptides and peptidomimetics. All amino
acids are designated by the standard three-letter code. 4-FB, 4-fluorobenzoyl; AEEA, 2-(2-(2-
aminoethoxy)ethoxy)acetic acid; D-2-Nal, D-2-naphthylalanine; D-2-Thi, D-2-thienylalanine; Dpr,
2,3-diaminopropionic acid; Inp, isonipecotic acid. *Characterisation data reported previously[35].
2.3 Structure-activity relationships of peptidic and peptidomimetic growth hormone
secretagogues
The IC50 values of the peptidic and peptidomimetic GHSs were determined through
receptor-ligand binding assays utilising human embryonic kidney 293 (HEK293)/ghrelin receptor
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cells and [125I]ghrelin as the competitive radioligand. In order to ascertain whether overexpression
of the receptor in HEK293 cells had been achieved, the endogenous ligand ghrelin was assayed
first. An IC50 value of 7.63 nM was obtained (Figure S1), indicating a satisfactory level of receptor
expression and maintaining consistency with previously reported values[37].
The initial peptidomimetic that was chosen for 19F-fluorobenzoylation was the pentapeptide
ipamorelin (H-Aib-His-D-2-Nal-D-Phe-Lys-NH2, compound 1, Table 2). This is because it
displayed a nanomolar binding affinity in previous work using HEK293/ghrelin receptor cells (Ki =
240 nM)[38].
Compound
Name
IC50, nM
483
Ipamorelin
[Lys5(4-FB)]ipamorelin
1
2
3
4
5
6
7
170
[Lys5(AEEA-4-FB)]ipamorelin
[D-2-Thi4,Lys5(4-FB)]ipamorelin
[Inp1,Lys5(4-FB)]ipamorelin
[Inp1,D-2-Nal4,Lys5(4-FB)]ipamorelin
[Inp1,D-2-Thi4,Lys5(4-FB)]ipamorelin
474
161
688
1920
1170
Table 2. IC50 values of ipamorelin and a series of derivatives thereof. The listed IC50 values were
determined in triplicate using HEK293/ghrelin receptor cells. For corresponding displacement
curves, the reader is referred to the supporting information.
Introduction of a fluorobenzoyl moiety into the lysine side-chain caused the IC50 value of
ipamorelin to decrease from 483 nM to 170 nM (compound 2, [Lys5(4-FB)]ipamorelin). This could
be a consequence of the fluorobenzoyl group strengthening hydrophobic interactions with aromatic
residues (e.g. Phe222 and Phe226) in the non-polar sub-pocket described in the open GHS-R1a
homology model of Pedretti and co-workers[39]. Ipamorelin also possesses the same three C-
terminal residues as G-7039 (the only difference being D-Phe4 as opposed to Phe4, respectively) and
thus the additional fluorobenzene ring could also be interacting with residues in one of the
hydrophobic sub-pockets (e.g. Phe286) detailed by Hou et al[35]. Extension of the lysine side-chain
through a short mini-PEG linker (2-(2-(2-aminoethoxy)ethoxy)acetic acid, AEEA) prior to 4-
fluorobenzoyl group coupling furnished [Lys5(AEEA-4-FB)]ipamorelin, compound 3. This had a
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higher IC50 value compared to compound 2 (cf. 474 nM for [Lys5(AEEA-4-FB)]ipamorelin to 170
nM for [Lys5(4-FB)]ipamorelin). This may result from the additional flexibility afforded by the
mini-PEG linker, which could cause a reduction in important hydrophobic interactions of the 4-
fluorobenzoyl moiety when compared to the less flexible compound 2. Replacement of the D-Phe
residue with D-2-thienylalanine (D-2-Thi) was expected to improve binding affinity. This strategy
was based on a study by Hansen and co-workers on dipeptide ipamorelin derivatives, where this
alteration caused the EC50 value to improve by an order of magnitude[40]. Unfortunately, this
modification furnished a compound with comparable affinity to the [Lys5(4-FB)]ipamorelin
analogue 2 with an IC50 = 161 nM ([D-2-Thi4,Lys5(4-FB)]ipamorelin, compound 4).
The primary amine at the N-terminus of ipamorelin was then exchanged for the secondary
amine isonipecotic acid (Inp) so as to make the subsequent compound ([Inp1,Lys5(4-
FB)]ipamorelin, compound 5) similar to the peptidomimetic G-7039, known to have an EC50 = 0.18
nM[10]. This led to a rise in the IC50 value (cf. [Lys5(4-FB)]ipamorelin, IC50 = 170 nM, compound
2 to [Inp1,Lys5(4-FB)]ipamorelin, IC50 = 688 nM, compound 5).
In order to probe the significance of the D-Phe residue in sub-pocket binding, this residue
was replaced with D-2-Nal in [Inp1,Lys5(4-FB)]ipamorelin (5) to give [Inp1,D-2-Nal4,Lys5(4-
FB)]ipamorelin (IC50 = 1920 nM, compound 6). This caused an approximately three-fold reduction
in binding affinity for the ghrelin receptor compared to the parent compound 5. This suggests that
D-Phe may be taking part in π-π or hydrophobic interactions with Phe119 in TM3[39, 41], playing
the same role as it does in the active tetra-peptide core of ghrelin[42]. The synthesis of compound 7
([Inp1,D-2-Thi4,Lys5(4-FB)]ipamorelin) and its corresponding IC50 value (1170 nM) could be seen
as an approximately two-fold reduction in binding affinity for the ghrelin receptor from switching
D-2-Thi for D-Phe in compound 5 ([Inp1,Lys5(4-FB)]ipamorelin); or an approximately seven-fold
decrease in binding resulting from insertion of an unfavourable secondary amine (Inp) into
compound 4 ([D-2-Thi4,Lys5(4-FB)]ipamorelin). In summary, the various synthesised derivatives of
ipamorelin did not yield the desired IC50 of < 100 nM, despite some improvements to its initial IC50
value.
Next, our attention turned to the peptidic GHSs (GHRP-1, GHRP-2 and GHRP-6) due to the
low nanomolar Ki values that were obtained for GHRP-2 and GHRP-6 (1.4 nM and 3.3 nM,
respectively) using HEK293/ghrelin receptor cells[38]. The IC50 values of GHRP-1, GHRP-2 and
GHRP-6 are shown in Table 3 (compounds 8-10, respectively).
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Compound
Name
GHRP-1
IC50, nM
181
8
GHRP-2
449
9
GHRP-6
73
10
11
12
13
[Lys6(4-FB)]GHRP-6
[Dpr6]GHRP-6
[Dpr6(4-FB)]GHRP-6
384
397
1060
Table 3. IC50 values of peptidic GHSs and their derivatives. The listed IC50 values were determined
in triplicate using HEK293/ghrelin receptor cells. For corresponding displacement curves, the
reader is referred to the supporting information.
It was found that GHRP-6, (H-His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) had the strongest
binding to the ghrelin receptor (IC50 = 73 nM, compound 10) compared to GHRP-1 (IC50 = 181 nM,
compound 8) and GHRP-2 (IC50 = 449 nM, compound 9). This peptide was therefore transformed
into the fluorobenzoylated analogue [Lys6(4-FB)]GHRP-6 (IC50 = 384 nM, compound 11).
Unfortunately, this alteration was not tolerated by the ghrelin receptor in spite of prior success with
ipamorelin (cf. compound 11, IC50 = 384 nM to compound 2, IC50 = 170 nM). One possible
explanation for this is that the lysine residue of GHRP-6 is involved in binding to polar sub-pocket
residues (e.g. Arg199)[39].
Endeavouring to increase affinity for the ghrelin receptor, the lysine of GHRP-6 was
replaced with 2,3-diaminopropionic acid (Dpr) to furnish [Dpr6]GHRP-6 (IC50 = 397 nM,
compound 12). This increase in IC50 could be a product of the shorter side-chain of Dpr which
could be decreasing lysine’s charge-transfer interactions with Arg199 in the polar sub-pocket. This
would also explain the greater loss in binding affinity after the attachment of the 4-fluorobenzoyl
group ([Dpr6(4-FB)]GHRP-6, IC50 = 1060 nM, compound 13). Not only does the lysine side-chain
appear to play an important role in receptor binding for GHRP-6 (a result contrary to that obtained
for ipamorelin), but the length of the alkyl chain also appears to be essential. Further derivatisation
of this class of peptides was not pursued owing to the high initial IC50 values of GHRP-1 (181 nM,
compound 8) and GHRP-2 (449 nM, compound 9) and the assumption that the lysine residue in
these peptides was also important for receptor interaction.
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Finally, two Genentech 5-mers G-7039 (H-Inp-D-2-Nal-D-2-Nal-Phe-Lys-NH2) and [1-
Nal4]G-7039 (H-Inp-D-2-Nal-D-2-Nal-1-Nal-Lys-NH2) were investigated as a consequence of their
reported low nanomolar in vitro efficacies (EC50 = 0.18 nM for G-7039[10] and 0.10 nM for [1-
Nal4]G-7039[11], respectively) and our recent computational study on the docking of G-7039 to
GHS-R1a, which suggested that the lysine residue was not critical for receptor binding[35]. The
parent compounds G-7039 (compound 14) and [1-Nal4]G-7039 (compound 16) and their
fluorobenzoyl congeners (compounds 15 and 17, respectively) can be viewed in Table 4.
Compound
Name
G-7039*
[Lys5(4-FB)]G-7039
[1-Nal4]G-7039
IC50, nM
5.2*
14
15
16
17
242
28
[1-Nal4,Lys5(4-FB)]G-7039
69
Table 4. IC50 values of Genentech peptidomimetics and their derivatives. The listed IC50 values
were determined in triplicate using HEK293/ghrelin receptor cells. For corresponding displacement
curves, the reader is referred to the supporting information. *Literature data[35].
Attaching G-7039 to para-fluorobenzoic acid had the effect of increasing the half-maximal
inhibitory concentration (5.2 nM to 242 nM for [Lys5(4-FB)]G-7039, compound 15), whilst a
smaller increase in the IC50 value was observed for [1-Nal4]G-7039 (28 nM to 69 nM for [1-
Nal4,Lys5(4-FB)]G-7039, compound 17). The smaller increase observed for [1-Nal4]G-7039
compared to G-7039 indicates that the introduction of an additional aromatic functionality
(fluorobenzene) along with the already present 1-Nal4 residue is simply strengthening hydrophobic
sub-pocket binding, whereas for G-7039 this modification is less favourable; perhaps due to the
reduction of salt-bridge interactions with Glu197 in the polar sub-pocket[35]. In spite of the
increase in IC50 value for [1-Nal4,Lys5(4-FB)]G-7039, this peptidomimetic represents the first lead
compound to be synthesised with the required nanomolar IC50 value (< 100 nM) making it the most
suitable candidate for further study of physical and biochemical characteristics prior to [18F]-
radiolabelling with [18F]SFB.
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2.4 Determination of lipophilicity of peptidic and peptidomimetic growth hormone
secretagogues
The logP value of [1-Nal4,Lys5(4-FB)]G-7039 as well as the other peptidic and
peptidomimetic GHSs were computed using ACD/LogP software (Table S2). Inserting the 4-
fluorobenzoyl moiety into any peptidomimetic parent compound resulted in an increase in
hydrophobicity for all derivatives (+ 2.25). Most of the parent compounds exhibited a favourable
logP value in the 1-3 unit range, except for G-7039 (5.28 ± 0.82) and [1-Nal4]G-7039 (6.51 ± 0.82).
This is virtue of the smaller size of these peptidomimetics compared to GHRP-1, GHRP-2 and
GHRP-6, and the prevalence of more hydrophobic aromatic residues compared to ipamorelin (D-2-
Nal and 1-Nal). Regrettably, the lead compound [1-Nal4,Lys5(4-FB)]G-7039 had a logP value of
8.76 ± 0.88, suggesting a lack of suitability for in vivo PET imaging due to potential solubility
issues. Nevertheless, [1-Nal4,Lys5(4-FB)]G-7039 possesses a considerably lower IC50 value
compared to the next best compound [D-2-Thi4, Lys5(4-FB)]ipamorelin (cf. 69 nM to 161 nM) and
was thus investigated in further in vitro assays.
2.5 Determination of efficacy of lead peptidomimetic [1-Nal4,Lys5(4-FB)]G-7039
The EC50 value of [1-Nal4,Lys5(4-FB)]G-7039 was calculated in terms of the release of
intracellular calcium ions (Figure 2). As expected, the control ligand ghrelin had a low in vitro
potency of 1.6 nM, whilst the EC50 value of [1-Nal4,Lys5(4-FB)]G-7039 was determined as 1.1 nM.
This low nanomolar efficacy signifies that the final compound remains a potent ghrelin receptor
agonist despite blocking of the lysine side-chain with the 4-fluorobenzoyl group.
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Figure 2. Dose-response curves for the lead peptidomimetic [1-Nal4,Lys5(4-FB)]G-7039 (red) and
the control ligand ghrelin (blue). The EC50 value for these compounds was determined in terms of
intracellular Ca2+ release. The percentage activation was normalised to the maximal response (Emax
)
of the control agonist ghrelin. Each assay concentration was performed in duplicate.
2.6 Stability of lead peptidomimetic [1-Nal4,Lys5(4-FB)]G-7039 in human serum
The lead peptidomimetic agonist [1-Nal4,Lys5(4-FB)]G-7039 was tested for stability in
human serum in order to assess its biological half-life. This serum half-life was found to be 718
minutes, indicating a good potential time-frame for in vivo PET imaging in small animal models of
carcinoma or cardiovascular disease. The [1-Nal4]G-7039 lead precursor was thus carried forward
for radiolabelling with [18F]SFB.
2.7 Synthesis of [18F]SFB and 18F-radiolabelling of the lead precursor [1-Nal4]G-7039
The synthesis of the [18F]SFB prosthetic group is shown in scheme 1. Acid catalysed
esterification of the 4-dimethylaminobenzoic acid starting material 18 furnished the intermediate t-
butyl ester 19. Methylation with trifluoromethanesulfonate (MeOTf) gave the triflate salt 20 in a
yield of 38% and ≥ 95% purity by UHPLC. Having synthesized the precursor salt 20, nucleophilic
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aromatic substitution with the [18F-] anion (acquired from the PET cyclotron by irradiation of
[18O]H2O) led to the formation of the radioactive compound 21.
Scheme 1. Synthesis of the [18F]SFB prosthetic group and subsequent 18F-radiolabelling of [1-
t
Nal4]G-7039. Reagents and Conditions: a) i) trifluoroacetic anhydride, THF, 0°C ii) BuOH, room
temperature, 2 h; b) MeOTf, N2, 0 °C, 1 h; c) 18F-, K2CO3, Kryptofix 2.2.2, DMSO, 120 °C, 10
minutes; d) 6M HCl, 120 °C, 10 minutes; e) NHS, EDC, MeCN, room temperature, 15-20 minutes;
f) [1-Nal4]G-7039, N,N-diisopropylethylamine (DIPEA), MeCN/H2O (1:1 ratio), 65 °C, 15-20
minutes.
Acid-catalysed de-esterification furnished [18F]FBA (22) with an average d.c. (decay-
corrected) radiochemical yield of 82%. The product was confirmed by co-injection with [19F]FBA
(see Figure S3 for representative radiochromatogram). Compound 22 was then coupled to N-
hydroxysuccinimide (NHS) using the coupling reagent N-(3-dimethylaminopropyl)-N'-
ethylcarbodiimide (EDC) which led to the formation of the [18F]SFB prosthetic group 23. The crude
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compound was purified by reverse-phase HPLC to give an average decay-corrected radiochemical
yield of 71% and radiochemical purity ≥ 99% (see Figure S4 for representative
radiochromatogram). The pure [18F]SFB was added to the peptide precursor [1-Nal4]G-7039 in a
mixture of MeCN/H2O using a small quantity of the hindered base DIPEA. After 15-20 minutes, the
crude [1-Nal4,Lys5(4-[18F]-FB)]G-7039 lead compound was obtained and purified by semi-
preparative HPLC. The final radiolabelled peptide [1-Nal4,Lys5(4-[18F]-FB)]G-7039 ([18F]17) was
obtained in an average decay-corrected radiochemical yield of 81%. For the entire synthesis, an
overall average d.c. radiochemical yield of 48% was achieved (n = 3), an average molar activity of
> 34 GBq/µmol and a radiochemical purity of ≥ 99%. Figure 3 displays a series of stacked
chromatograms including the peptidomimetic precursor [1-Nal4]G-7039 (16), the cold standard [1-
Nal4,Lys5(4-FB)]G-7039 (17) as well as the radiolabelled [1-Nal4,Lys5(4-[18F]-FB)]G-7039
peptidomimetic (18F]17).
Figure 3. Stacked HPLC Chromatograms for [1-Nal4]G-7039, [1-Nal4,Lys5(4-FB)]G-7039 (both λ
= 254 nm) and [1-Nal4,Lys5(4-[18F]-FB)]G-7039.
The retention times of the cold standard and “hot” peptide are almost identical ([1-
t
t
Nal4,Lys5(4-FB)]G-7039, R = 6.70 min compared to R = 6.65 min for the “hot” peptide),
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ACCEPTED MANUSCRIPT
indicating that the precursor [1-Nal4]G-7039 has been selectively radiolabelled at the amino group
of the lysine side-chain. Further evidence for successful coupling of [18F]SFB to [1-Nal4]G-7039
via the lysine side-chain was provided by a co-injection of a pre-mixed solution of the cold standard
and “hot” peptidomimetic (Figure 4).
Figure 4. Stacked HPLC chromatograms resulting from the co-injection of cold standard [1-
Nal4,Lys5(4-FB)]G-7039 (λ = 254 nm) and [1-Nal4,Lys5(4-[18F]-FB)]G-7039.
Once again, this showed very similar retention times between the two peptidomimetic
species ([1-Nal4, Lys5(4-FB)]G-7039 tR = 6.26 min and [1-Nal4,Lys5(4-[18F]-FB)]G-7039 tR = 6.17
min) lending further credence to regioselective 18F-fluorobenzoylation at the lysine side-chain.
3. Conclusions
In this study, several families of peptidic (GHRP-1, GHRP-2 and GHRP-6) and
peptidomimetic (ipamorelin, G-7039, [1-Nal4]G-7039) ghrelin receptor agonists were derivatised
through 4-fluorobenzoylation of the lysine side-chain. This led to a range of half-maximal
inhibitory concentrations ranging from 69 nM ([1-Nal4,Lys5(4-FB)]G-7039) to 1920 nM ([Inp1,D-2-
Nal4, Lys5(4-FB)]ipamorelin). The peptidomimetic [1-Nal4]G-7039 was identified as the most
15

ACCEPTED MANUSCRIPT
suitable candidate for 18F-radiolabelling, as its 19F-congener ([1-Nal4,Lys5(4-FB)]G-7039,
compound 17) had a nanomolar binding affinity (IC50 = 69 nM), high in vitro potency (EC50 = 1.1
nM) and good serum stability (t1/2 = 718 min). This is most likely due to the presence of a core
framework of aromatic amino acids that are involved in strong hydrophobic interactions with
hydrophobic pockets of the ghrelin receptor. Prosthetic group radiolabelling of [1-Nal4]G-7039 with
[18F]SFB delivered the radiolabelled peptidomimetic in an overall average decay-corrected
radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of > 34 GBq/µmol.
Further study of this compound would be required to assess its potential for in vivo PET imaging of
diseases exhibiting heightened expression of the ghrelin receptor.
4. Materials and Methods
4.1 General information
All reagents were obtained from commercial suppliers and used without further purification.
Peptides were either synthesised manually or through the use of a Biotage SyroWave automated
peptide synthesizer. Peptide vessels were shaken using an IKA Vibrax VXR basic shaker with
centrifugation performed on a Beckman Coulter Allegra X-30R or Fisher GS-6R centrifuge. In
order to aid peptide dissolution, sonication of solutions was accomplished via a Bransonic 2510R-
MTH or Fisher F5-14 ultrasonic cleaner. A Fisher 2052 Isotemp machine was used to heat test
tubes in the Kaiser Test. Peptides were lyophilised using a Labconco FreeZone Freeze Dry System.
Accurate weighing was carried out on a Mettler-Toledo XP6 microbalance. UV traces were
obtained with a Waters 2487 UV/Vis Dual λ Absorbance Detector (170-900 nm) and low-resolution
mass spectra with a Micromass Quattro micro API mass spectrometer (ESI-LC-MS). Peptide
purification was achieved through HPLC (MeCN + 0.1% TFA, H2O + 0.1% TFA solvent system).
All peptides and small molecules obtained had a purity ≥ 95% as determined by HPLC or UHPLC
analysis. A RP preparative C-18 column (SunFire OBD, 19 x 150 mm or Agilent Zorbax 21.2 x 150
mm) was used for preparative HPLC, whilst a C-18 RP column (SunFire, 4.6 x 150 mm or Agilent
Zorbax, 4.6 x 150 mm) was used for analytical HPLC. Accurate mass spectrometry (HRMS) was
carried out on a Finnigan MAT 8400 mass spectrometer (EI) for small molecules and on a
1
13
Micromass LCT mass spectrometer (ESI-TOF) for peptides. H NMR and C NMR spectroscopy
were performed on a Mercury VX 400 machine at 400 and 100 MHz respectively. Chemical shifts
16

ACCEPTED MANUSCRIPT
are referenced to residual solvent, reported in ppm on a δ scale and all coupling constants quoted in
hertz (Hz).
4.2 Manual Fmoc-SPPS
Rink amide MBHA resin (192 mg, 0.1 mmol, 1.0 equiv., 0.52 mmol g-1 loading) was
vortexed in DCM (2.0 ml, 1 min.), allowed to swell (15 min) and solvent removed. This was
followed by addition of DMF (2.0 ml), vortexing (1 min.) and removal of solvent. Deprotection of
the Fmoc group was then performed. A solution of 20% piperidine/DMF (1.5 ml, v/v) was added to
the resin and the subsequent mixture vortexed (2 min) and solvent removed. This was then repeated
a second time with vortexing for 15 minutes. After solvent had been removed, the resin was washed
of any unreacted by-products with DMF six times (2.0 ml, vortex 30 s). The desired amino acid or
small molecule (0.3 mmol, 3.0 equiv.) and coupling reagent (HCTU, 0.12 g, 0.3 mmol, 3.0 equiv.)
were then dissolved in DMF (1.5 ml) and added to the deprotected resin. After vortexing (30 s),
DIPEA (111 µl, 0.6 mmol, 6.0 equiv.) was added and the final mixture vortexed (1-2 h). The resin
was then washed with DMF (2.0 ml, 30 s vortex) a final four times. The deprotection/coupling
cycle was then repeated unless the final amino acid in the sequence had been added, in which case
the peptide was washed with DCM five times (2.0 ml, 30 s vortex) after washing with DMF and
stored in a refrigerator. Removal of the N-terminal Fmoc-group was carried out in the same fashion
as the deprotection cycle described previously, with resin washing occurring six times with DMF
(2.0 ml, 30 s vortex) and four times with DCM (2.0 ml, 30 s vortex). Successful synthesis of the
desired peptide was then ascertained via a microcleave prior to full cleavage of the peptide from the
solid-support. This was carried out as follows: a solution of 95% TFA: 2.5% (iPr)3SiH: 2.5% H2O
(300 µl) was added to a small number of resin beads (< 5 mg) and the subsequent mixture vortexed
(3 h). The clear liquid was then evaporated under a stream of N2. Analytical HPLC was then
performed to determine whether the correct peptide had been synthesised. If the correct peptide had
been obtained, a full cleavage was performed using a mixture of 95% TFA: 2.5% (iPr)3SiH: 2.5%
H2O (2.0 ml) for 5-7 h. The subsequent solution was cooled in an ice-bath alongside tert-butyl
methyl ether (TBMe, 40 ml). After 10 minutes, TBMe (20 ml) was added to the peptide solution,
leading to the formation of a white precipitate. The precipitate was cooled further (10 min) and then
centrifuged (7 min). Decanting of the supernatant was followed by addition of a second aliquot of
TBMe (20 ml), vortexing (30 s) and final centrifugation (7 min). After decanting, a white solid was
17

ACCEPTED MANUSCRIPT
obtained. This was then freeze-dried (20 min) to furnish crude peptide. Preparative HPLC was then
used to purify the product peptide.
4.3 Deprotection of the Alloc protecting group
The resin-bound peptide was vortexed in DCM (4.5 ml, 30 s) and allowed to swell (10 min).
Deprotection was carried out under a blanket of N2. The swollen resin-bound peptide was stirred (5
min) before addition of PhSiH3 (296 µl, 2.4 mmol, 24.0 equiv.). Further stirring (5 min) ensued
prior to treating with Pd(PPh3)4 (12 mg, 0.01 mmol, 0.1 equiv.). After 5 minutes, the solution was
vortexed (5 min), solvent removed and the brown-coloured resin washed four times with DCM (2.0
ml, 30 s vortex). The procedure was then repeated ab initio, with final resin washing occurring in
the following order: DCM, DMF, MeOH, DMF and DCM (all 2.0 ml, 30 s vortex).
4.4 Kaiser Test
A small number of resin beads (< 5 mg) were taken and treated with phenol: EtOH (200 µl,
8:2 v/v), 0.001 M KCN(aq.): pyridine (200 µl, 2:98 v/v, 0.001 M aqueous KCN) and ninhydrin in
EtOH (200 µl, 5% w/v), respectively. Tentagel resin (< 5 mg) was used as a control. Both test tubes
were heated to 70 °C. The presence of free amine was indicated by blue resin beads whilst yellow
or clear resin beads indicated the presence of protected amino groups.
4.5 Synthesis of peptidic- and peptidomimetic growth hormone secretagogues
All peptidomimetics were synthesised by the same general procedure described previously
unless otherwise noted.
4.5.1
H-Aib-His-D-2-Nal-D-Phe-Lys-NH2: Ipamorelin (1)
The product was purified by preparative HPLC (5-80% MeCN + 0.1% TFA). This furnished
1
a white powder (20.5 mg, 19%): H-NMR (400 MHz, CD3OD); D-2-Nal, D-Phe, His: δ 7.99 (s,
1H, ArH), 7.80-7.76 (m, 1H, ArH), 7.71 (d, J = 8.6 Hz, 2H, ArH), 7.56 (s, 1H, ArH), 7.44-7.37
(m, 2H, ArH), 7.32-7.17 (m, 6H, ArH), 6.92 (s, 1H, His Hδ), 4.58 (m, 3H, Hα), 3.30-3.21 (m,
1H, D-2-Nal Hβ), 3.15-3.00 (m, 2H, Hβ), 2.96-2.72 (m, 3H, Hβ), Lys: 4.11 (dd, J = 9.7, 4.2 Hz,
1H, Hα), 2.96-2.72 (m, 2H, Hε), 1.75-1.64 (m, 1H, Hβ), 1.54-1.40 (m, 3H, Hβ, 2Hδ), 1.05-0.93
18

ACCEPTED MANUSCRIPT
(m, 2H, Hγ), Aib: 1.48 (s, 3H, CH3) 1.44 (s, 3H, CH3) ppm. ESI-LC-MS m/z 356.9 [M + 2H]2+;
HRMS (ESI-MS) calcd. for C38H50N9O5 [M + H]+ 712.3935, found 712.3959.
4.5.2
H-Aib-His-D-2-Nal-D-Phe-Lys(4-FB)-NH2: [Lys5(4-FB)]ipamorelin (2)
Purification by preparative HPLC (20-70% MeCN + 0.1% TFA) yielded a white powder
(15.4 mg, 15%): 1H-NMR (400 MHz, CD3OD); D-2-Nal, D-Phe, His, 4-FB: δ 7.91 (s, 1H, ArH),
7.85-7.77 (m, 3H, ArH, 2F-ArH), 7.70 (d, J = 7.9 Hz, 2H, ArH), 7.54 (s, 1H, ArH), 7.45-7.40
(m, 2H, ArH), 7.29-7.19 (m, 6H, ArH), 7.14-7.07 (m, 2H, 2F-ArH), 6.93 (s, 1H, His Hδ), 4.65-
4.52 (m, 3H, Hα), 3.30-3.27 (m, 1H, Hβ), 3.23 (d, J = 4.0 Hz, 1H, Hβ), 3.10 (dd, J = 13.5, 7.8
Hz, 1H, Hβ), 3.04-2.74 (m, 3H, Hβ), Lys: 4.12 (dd, J = 9.8, 4.2 Hz, 1H, Hα), 3.04-2.74 (m, 2H,
Hε), 1.77-1.67 (m, 1H, Hβ), 1.58-1.42 (m, 3H, Hβ, 2Hδ), 1.14-1.03 (m, 2H, Hγ), Aib: 1.50 (s,
3H, CH3), 1.46 (s, 3H, CH3) ppm. ESI-LC-MS m/z 418.0 [M + 2H]2+; HRMS (ESI-MS) calcd. for
C45H53FN9O6 [M + H]+ 834.4103, found 834.4133.
4.5.3
H-Aib-His-D-2-Nal-D-Phe-Lys(AEEA-4-FB)-NH2:[Lys5(AEEA-4-
FB)]ipamorelin (3)
Peptide purification by preparative HPLC (20-60% MeCN + 0.1% TFA) delivered an off-
white powder (9.2 mg, 8%): 1H-NMR (400 MHz, CD3OD); His, D-2-Nal, D-Phe, 4-FB: δ 7.93 (s,
1H, ArH), 7.86-7.77 (m, 3H, ArH, 2F-ArH), 7.71 (d, J = 8.0 Hz, 2H, ArH), 7.55 (s, 1H, ArH),
7.46-7.41 (m, 2H, ArH), 7.31-7.18 (m, 6H, ArH), 7.17-7.10 (m, 2H, F-ArH), 6.94 (s, 1H, His
Hδ), 4.65-4.58 (m, 2H, Hα), 4.55 (t, J = 7.5 Hz, 1H, Hα), 3.16-2.76 (m, 6H, Hβ), Lys: 4.10 (dd, J
= 9.7, 4.2 Hz, 1H, Hα), 3.16-2.76 (m, 2H, Hε), 1.72-1.62 (m, 1H, Hβ), 1.52-1.43 (m, 1H, Hβ),
1.41-1.30 (m, 2H, Hδ), 1.07-0.97 (m, 2H, Hγ), AEEA linker: 3.93 (s, 2H, NHCOCH2O), 3.65-
3.61 (m, 6H, CH2), 3.53 (t, J = 5.6 Hz, 2H, CH2), Aib: 1.50 (s, 3H, CH3), 1.47 (s, 3H, CH3)
ppm. ESI-LC-MS m/z 490.4 [M + 2H]2+; HRMS (ESI-MS) calcd. for C51H64FN10O9 [M + H]+
979.4842, found 979.4868.
4.5.4
H-Aib-His-D-2-Nal-D-2-Thi-Lys(4-FB)-NH2: [D-2-Thi4,Lys5(4-
FB)]ipamorelin (4)
The title peptide was synthesized by automated peptide synthesis and purified by
1
preparative HPLC (20-80% MeCN + 0.1% TFA). This furnished a white solid (4.20 mg, 4%): H-
NMR (400 MHz, (CD3)2SO); δ 8.83 (s, 1H, ArH), 8.59 (d, J = 8.1 Hz, 1H, NH), 8.42 (t, J = 5.6
19

ACCEPTED MANUSCRIPT
Hz, 1H, NH), 8.29 (d, J = 9.3 Hz, 1H, NH), 8.20 (d, J = 8.2 Hz, 1H, NH), 8.09 (d, J = 8.9 Hz,
1H, NH), 7.99 (s, 2H, NH), 7.86-7.81 (m, 2H, F-ArH), 7.79-7.75 (m, 1H, ArH), 7.72 (dd, J =
8.7, 3.7 Hz, 2H, ArH), 7.65 (s, 1H, ArH), 7.41-7.37 (m, 2H, ArH), 7.35-7.31 (m, 2H, ArH, NH),
7.26 (dd, J = 4.8, 1.5 Hz, 1H, Thi-H), 7.22-7.16 (m, 2H, F-ArH), 7.05 (s, 2H, NH, ArH), 6.88-
6.84 (m, 2H, Thi-H), 4.73-4.66 (m, 1H, Hα), 4.62-4.51 (m, 2H, Hα), 4.19-4.12 (m, 1H, Lys-Hα),
3.21-3.12 (m, 4H, 2Hε, 2Hβ), 3.06-2.96 (m, 1H, Hβ), 2.87-2.77 (m, 2H, Hβ), 2.62-2.49 (m, 1H,
Hβ), 1.66-1.56 (m, 1H, Lys-Hβ), 1.50-1.36 (m, 3H, Lys-Hβ, 2Hδ), 1.26 (s, 3H, CH3), 1.20-1.10
(m, 2H, Hγ), 1.16 (s, 3H, CH3), ppm. ESI-LC-MS m/z 420.8 [M + 2H]2+; HRMS (ESI-MS) calcd.
for C43H51FN9O6S [M + H]+ 840.3667 found 840.3693.
4.5.5
H-Inp-His-D-2-Nal-D-Phe-Lys(4-FB)-NH2: [Inp1,Lys5(4-FB)]ipamorelin (5)
The title peptide was synthesized via automated peptide synthesis and purified by
preparative HPLC (20-70% MeCN + 0.1% TFA). The title compound was acquired as a white solid
1
(6.30 mg, 6%): H-NMR (400 MHz, (CD3)2SO); δ 8.79 (s, 1H, His Hε), 8.72 (s, 1H, NH), 8.44
(dd, J = 10.8, 6.4 Hz, 2H, NH), 8.19 (d, J = 7.8 Hz, 1H, NH), 8.17-8.10 (m, 2H, NH), 7.87-7.81
(m, 2H, F-ArH), 7.80-7.76 (m, 1H, ArH), 7.71 (d, J = 7.9 Hz, 2H, ArH), 7.63 (s, 1H, ArH),
7.42-7.35 (m, 2H, ArH), 7.33-7.29 (m, 2H, NH), 7.22-7.18 (m, 6H, ArH), 7.17-7.11 (m, 2H, F-
ArH), 7.06 (s, 1H, NH), 6.96 (s, 1H, His Hδ), 4.64-4.56 (m, 1H, Hα), 4.55-4.44 (m, 2H, Hα),
4.15-4.07 (m, 1H, Lys-Hα), 3.19-3.07 (m, 5H, CH2), 2.94 (dd, J = 13.6, 5.9 Hz, 1H, CH2), 2.87-
2.67 (m, 5H, CH2), 2.58-2.48 (m, 1H, CH2), 2.38-2.29 (m, 1H, Hα), 1.67-1.34 (m, 8H, CH2),
1.15-1.00 (m, 2H, CH2), ppm. ESI-LC-MS m/z 430.9 [M + 2H]2+; HRMS (ESI-MS) calcd. for
C47H55FN9O6 [M + H]+ 860.4259 found 860.4284.
4.5.6
H-Inp-His-D-2-Nal-D-2-Nal-Lys(4-FB)-NH2: [Inp1,D-2-Nal4,Lys5(4-
FB)]ipamorelin (6)
The title peptide was made by automated peptide synthesis and purified by preparative
1
HPLC (20-80% MeCN + 0.1% TFA). This delivered a white solid (11.3 mg, 10%): H-NMR (400
MHz, (CD3)2SO); δ 8.77 (s, 1H, His Hε), 8.51 (d, J = 7.5 Hz, 2H, NH), 8.36 (t, J = 5.5 Hz, 1H,
NH), 8.19-8.10 (m, 2H, NH), 8.05 (d, J = 8.7 Hz, 1H, NH), 7.85-7.79 (m, 3H, 2F-ArH, ArH),
7.79-7.74 (m, 3H, ArH), 7.73 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.68 (d, J = 2.4 Hz, 2H, ArH),
7.59 (s, 1H, ArH), 7.43-7.35 (m, 5H, ArH), 7.31-7.27 (m, 2H, NH), 7.20-7.13 (m, 2H, F-ArH),
7.05 (s, 1H, NH), 6.93 (s, 1H, His Hδ), 4.67-4.60 (m, 2H, Hα), 4.52-4.44 (m, 1H, Hα), 4.16-4.08
20

ACCEPTED MANUSCRIPT
(m, 1H, Lys-Hα), 3.17-2.93 (m, 7H, CH2), 2.88-2.66 (m, 4H, CH2), 2.53-2.47 (m, 1H, CH2),
2.34-2.25 (m, 1H, Hα), 1.63-1.34 (m, 6H, CH2), 1.32-1.23 (m, 2H, CH2), 1.07-0.95 (m, 2H,
CH2), ppm. ESI-LC-MS m/z 455.9 [M + 2H]2+; HRMS (ESI-MS) calcd. for C51H57FN9O6 [M + H]+
910.4416 found 910.4400.
4.5.7
H-Inp-His-D-2-Nal-D-2-Thi-Lys(4-FB)-NH2: [Inp1,D-2-Thi4,Lys5(4-
FB)]ipamorelin (7)
The title peptide was prepared by automated peptide synthesis and purified by preparative
1
HPLC (20-80% MeCN + 0.1% TFA). This furnished a white powder (14.0 mg, 13%): H-NMR
(400 MHz, CD3OD); His, D-2-Nal, D-2-Thi, 4-FB: δ 8.23 (s, 1H, His Hε), 7.86-7.81 (m, 2H, F-
ArH), 7.80-7.77 (m, 1H, ArH), 7.73 (dd, J = 8.8, 3.3 Hz, 2H, ArH), 7.59 (s, 1H, ArH), 7.45-
7.39 (m, 2H, ArH), 7.27 (dd, J = 8.5, 1.6 Hz, 1H, ArH), 7.22 (dd, J = 5.0, 1.2 Hz, 1H, Thi-
ArH), 7.15-7.08 (m, 2H, F-ArH), 6.92 (s, 1H, His Hδ), 6.91-6.85 (m, 2H, Thi-ArH), 4.61 (dd, J
= 10.3, 4.4 Hz, 1H, Hα), 4.53-4.47 (m, 2H, Hα), 4.22 (dd, J = 9.8, 4.2 Hz, 1H, Lys-Hα), 3.39-
3.29 (m, 6H, CH2), 3.26-3.22 (m, 1H, CH2), 3.01-2.80 (m, 5H, CH2), 2.53-2.44 (m, 1H, Inp-Hα),
1.87-1.50 (m, 8H, CH2), 1.35-1.22 (m, 2H, CH2), ppm. ESI-LC-MS m/z 433.8 [M + 2H]2+;
HRMS (ESI-MS) calcd. for C45H53FN9O6S [M + H]+ 866.3824 found 866.3850.
4.5.8
H-Ala-His-D-2-Nal-Ala-Trp-D-Phe-Lys-NH2: GHRP-1 (8)
The product was purified by preparative HPLC (15-80% MeCN + 0.1% TFA). This yielded
1
a white powder (26.4 mg, 19%): H-NMR (400 MHz, CD3OD); His, D-2-Nal, Trp, D-Phe: δ 8.41
(s, 1H, His Hε), 7.81-7.71 (m, 3H, ArH), 7.65 (s, 1H, ArH), 7.49 (d, J = 7.8 Hz, 1H, ArH), 7.46-
7.40 (m, 2H, ArH), 7.35 (dd, J = 8.4, 1.7 Hz, 1H, ArH), 7.31 (d, J = 8.1 Hz, 1H, ArH), 7.24-7.13
(m, 3H, ArH), 7.10-7.04 (m, 4H, ArH), 7.00 (t, J = 7.5 Hz, 1H, ArH), 6.89 (d, J = 0.8 Hz, 1H, His
Hδ), 4.67-4.60 (m, 2H, Hα), 4.38 (t, J = 7.4 Hz, 1H, Hα), 4.32 (t, J = 7.7 Hz, 1H, Hα), 3.25-2.68 (m,
8H, Hβ), Ala, Ala: 4.17-4.10 (m, 1H, Hα), 3.94 (q, J = 7.0 Hz, 1H, Hα), 1.35 (d, J = 7.1 Hz, 3H,
CH3), 1.10-1.01 (m, 3H, CH3), Lys: 4.17-4.10 (m, 1H, Hα), 3.25-2.68 (m, 2H, Hε), 1.81-1.70 (m,
1H, Hβ), 1.58-1.45 (3H, Hβ, 2Hδ), 1.10-1.01 (m, 2H, Hγ), ppm. ESI-LC-MS m/z 478.5 [M + 2H]2+;
HRMS (ESI-MS) calcd. for C51H63N12O7 [M + H]+ 955.4943 found 955.4964.
21

ACCEPTED MANUSCRIPT
4.5.9
H-D-Ala-D-2-Nal-Ala-Trp-D-Phe-Lys-NH2: GHRP-2 (9)
Purification by preparative HPLC (25-70% MeCN + 0.1% TFA) delivered the title peptide
as a white powder (19.7 mg, 17%): 1H-NMR (400 MHz, CD3OD); D-2-Nal, Trp, D-Phe: δ 7.76 (d,
J = 9.2 Hz, 2H, ArH), 7.73 (s, 1H, ArH), 7.65 (s, 1H, ArH), 7.51-7.47 (m, 1H, ArH), 7.44-7.37 (m,
2H, ArH), 7.35 (dd, J = 8.4, 1.7 Hz, 1H, ArH), 7.25-7.14 (m, 4H, ArH), 7.09-7.01 (m, 4H, ArH),
7.00 (s, 1H, ArH), 4.77 (dd, J = 10.9, 4.6 Hz, 1H, Hα), 4.49 (t, J = 6.8 Hz, 1H, Hα), 4.36 (t, J = 7.6
Hz, 1H, Hα), 3.11 (t, J = 6.7 Hz, 2H, Hβ), 3.07 (d, J = 4.5 Hz, 1H, Hβ), 2.88-2.70 (m, 3H, Hβ), D-
Ala, Ala: 4.23 (q, J = 7.1 Hz, 1H, Hα), 3.79 (q, J = 7.0 Hz, 1H, Hα), 1.27 (d, J = 7.2 Hz, 3H, CH3),
1.00 (d, J = 7.1 Hz, 3H, CH3), Lys: 4.16 (dd, J = 10.3, 4.1 Hz, 1H, Hα), 2.88-2.70 (m, 2H, Hε),
1.82-1.72 (m, 1H, Hβ), 1.56-1.43 (m, 3H, Hβ, 2Hδ), 1.07-0.96 (m, 2H, Hγ), ppm. ESI-LC-MS m/z
409.9 [M + 2H]2+; HRMS (ESI-MS) calcd. for C45H55N9O6Na [M + Na]+ 840.4204 found 840.4173.
4.5.10
H-His-D-Trp-Ala-Trp-D-Phe-Lys-NH2: GHRP-6 (10)
Purification by preparative HPLC (15-80% MeCN + 0.1% TFA) gave a white powder (19.7
mg, 14%): 1H-NMR (400 MHz, CD3OD); His, D-Trp, Trp, D-Phe: δ 8.46 (s, 1H, His Hε), 7.54 (d,
J = 7.8 Hz, 1H, ArH), 7.46 (d, J = 7.8 Hz, 1H, ArH), 7.28 (t, J = 7.7 Hz, 2H, ArH), 7.23-7.13 (m,
4H, ArH), 7.12-6.92 (m, 8H, 7ArH, His Hδ), 4.47 (t, J = 7.4 Hz, 2H, Trp Hα), 4.35 (t, J = 6.7 Hz,
1H, Hα), 4.28 (t, J = 7.9 Hz, 1H, Hα), 3.22-3.14 (m, 3H, Hβ), 3.14-3.02 (m, 3H, Hβ), 2.83 (d, J = 8.6
Hz, 2H, Hβ), Lys: 4.03 (dd, J = 10.3, 3.9 Hz, 1H, Hα), 2.75 (t, J = 6.8 Hz, 2H, Hε), 1.76-1.63 (m,
1H, Hβ), 1.51-1.39 (m, 3H, Hβ, 2Hδ), 0.97-0.88 (m, 2H, Hγ), Ala: 3.91 (q, J = 7.2 Hz, 1H, Hα), 0.86
(d, J = 7.3 Hz, 3H, CH3) ppm. ESI-LC-MS m/z 437.4 [M + 2H]2+; HRMS (ESI-MS) calcd. for
C46H57N12O6 [M + H]+ 873.4524 found 873.4531.
4.5.11
H-His-D-Trp-Ala-Trp-D-Phe-Lys(4-FB)-NH2: [Lys6(4-FB)]GHRP-6 (11)
The product was purified by preparative HPLC (15-80% MeCN + 0.1% TFA). This yielded
1
a white powder (9.60 mg, 7%): H-NMR (400 MHz, CD3OD); His, D-Trp, Trp, D-Phe, 4-FB: δ
8.50 (s, 1H, His Hε), 7.88-7.80 (m, 2H, F-ArH), 7.57 (d, J = 7.9 Hz, 1H, ArH), 7.49 (d, J = 7.8
Hz, 1H, ArH), 7.30 (t, J = 8.2 Hz, 2H, ArH), 7.26-6.95 (m, 14H, 13ArH, His Hδ), 4.53-4.46 (m,
2H, Hα), 4.37-4.30 (m, 2H, Hα), 3.26-3.04 (m, 6H, Hβ), 2.87 (d, J = 8.0 Hz, 2H, Hβ), Lys: 4.04
(dd, J = 10.2, 4.0 Hz, 1H, Hα), 3.26-3.04 (m, 2H, Hε), 1.81-1.70 (m, 1H, Hβ), 1.58-1.43 (m, 3H,
2Hδ, Hβ), 1.06-0.97 (m, 2H, Hγ), Ala: 3.94 (q, J = 7.3 Hz, 1H, Hα), 0.87 (d, J = 7.3 Hz, 3H,
22

ACCEPTED MANUSCRIPT
CH3) ppm. ESI-LC-MS m/z 498.4 [M + 2H]2+; HRMS (ESI-MS) calcd. for C53H59FN12O7Na [M +
Na]+ 1017.4511 found 1017.4522.
4.5.12
H-His-D-Trp-Ala-Trp-D-Phe-Dpr-NH2: [Dpr6]GHRP-6 (12)
Purification by preparative HPLC (15-80% MeCN + 0.1% TFA) furnished the title peptide
1
as a white powder (4.7 mg, 3 %): H-NMR (400 MHz, CD3OD); 8.49 (s, 1H His Hε), 7.55 (d, J =
7.9 Hz, 1H, ArH), 7.46 (d, J = 7.9 Hz, 1H, ArH), 7.30 (t, J = 8.6 Hz, 2H, ArH), 7.21-7.12 (m, 3H,
ArH), 7.11-7.04 (m, 3H, ArH), 7.02-6.92 (m, 5H, ArH), 6.89 (d, J = 1.0 Hz, 1H, His Hδ), 4.62 (t, J
= 6.8 Hz, 1H, Hα), 4.52 (t, J = 7.9 Hz, 1H, Hα), 4.42-4.33 (m, 2H, Hα), 4.28 (t, J = 6.7 Hz, 1H, Dpr-
Hα), 4.00 (q, J = 7.3 Hz, 1H, Ala-Hα), 3.24-3.11 (m, 3H, CH2), 3.12-2.92 (m, 5H, CH2), 2.89-2.76
(m, 2H, CH2), 0.94 (d, J = 7.3 Hz, 3H, Ala-CH3), ppm. ESI-LC-MS m/z 831.4 [M + H]+; HRMS
(ESI-MS) calcd. for C43H51N12O6 [M + H]+ 831.4055 found 831.4070.
4.5.13
H-His-D-Trp-Ala-Trp-D-Phe-Dpr(4-FB)-NH2: [Dpr6(4-FB)]GHRP-6 (13)
Preparative HPLC (25-70% MeCN + 0.1% TFA) gave the title compound as a white solid
(19.8 mg, 14%): 1H-NMR (400 MHz, CD3OD); His, D-Trp, Trp, D-Phe, 4-FB: δ 8.58 (d, J = 1.3
Hz, 1H, His Hε), 7.82-7.75 (m, 2H, F-ArH), 7.52 (d, J = 7.9 Hz, 1H, ArH), 7.40 (d, J = 7.9 Hz,
1H, ArH), 7.27 (dd, J = 8.6, 1.5 Hz, 2H, ArH), 7.15-7.07 (m, 5H, ArH), 7.06-6.98 (m, 6H,
ArH), 6.97-6.89 (m, 3H, 2ArH, His Hδ), 4.54-4.45 (m, 1H, Hα), 4.44-4.30 (m, 3H, Hα), 3.22-
2.98 (m, 6H, Hβ), 2.93-2.74 (m, 2H, Hβ), Dpr: 4.54-4.45 (m, 1H, Hα), 3.67 (dd, J = 13.8, 5.6
Hz, 1H, Hβ), 3.54 (dd, J = 13.8, 7.8 Hz, 1H, Hβ), Ala: 3.93 (q, J = 7.3 Hz, 1H, Hα), 0.86 (d, J =
7.3 Hz, 3H, CH3) ppm. ESI-LC-MS m/z 477.4 [M + 2H]2+; HRMS (ESI-MS) calcd. for C50-
H54FN12O7 [M + H]+ 953.4223 found 953.4237.
4.5.14
H-Inp-D-2-Nal-D-2-Nal-Phe-Lys-NH2: G-7039* (14)
Purification of the peptide proceeded through preparative HPLC (25-80% MeCN + 0.1%
1
TFA). The title compound was obtained as a white powder (6.70 mg, 7%): H-NMR (400 MHz,
CD3OD); 7.80-7.76 (m, 1H, ArH), 7.74 (dd, J = 6.1, 2.2 Hz, 2H, ArH), 7.70 (dd, J = 8.5, 3.5 Hz,
3H, ArH), 7.56 (s, 1H, ArH), 7.50 (s, 1H, ArH), 7.46-7.35 (m, 4H, ArH), 7.26 (dd, J = 8.5, 1.5 Hz,
1H, ArH), 7.20-7.10 (m, 6H, ArH), 4.66-4.56 (m, 2H, Hα), 4.50 (dd, J = 9.3, 5.4 Hz, 1H, Hα), 4.30
(dd, J = 9.5, 4.7 Hz, 1H, Lys-Hα), 3.20-2.80 (m, 10H, CH2), 2.78-2.64 (m, 2H, CH2), 2.33-2.24 (m,
23

ACCEPTED MANUSCRIPT
1H, Inp-Hα), 1.92-1.82 (m, 1H, CH2), 1.74-1.65 (m, 1H, CH2), 1.64-1.44 (m, 5H, CH2), 1.44-1.24
(m, 3H, CH2), ppm. ESI-LC-MS m/z 399.8 [M + 2H]2+; HRMS (ESI-MS) calcd. for C47H56N7O5 [M
+ H]+ 798.4343 found 798.4339. HRMS data and yield has been reported previously[35], whereas
the 1H-NMR data has not.
4.5.15
H-Inp-D-2-Nal-D-2-Nal-Phe-Lys(4-FB)-NH2: [Lys5(4-FB)]G-7039 (15)
Preparative HPLC (35-80% MeCN + 0.1% TFA) furnished the title compound as a white
solid (5.90 mg, 6%): 1H-NMR (400 MHz, (CD3)2SO); δ 8.50 (d, J = 8.2 Hz, 1H, NH), 8.45 (t, J =
5.5 Hz, 1H, NH), 8.39 (s, 1H, NH), 8.15-8.09 (m, 2H, NH), 8.04 (d, J = 8.4 Hz, 1H, NH), 7.86-
7.82 (m, 2H, F-ArH), 7.82-7.76 (m, 2H, ArH), 7.75-7.66 (m, 4H, ArH), 7.58 (s, 1H, ArH), 7.48
(s, 1H, ArH), 7.45-7.38 (m, 4H, ArH), 7.31 (dd, J = 8.5, 1.5 Hz, 1H, ArH), 7.28 (s, 1H, ArH),
7.26 (s, 1H, NH), 7.24-7.17 (m, 5H, ArH), 7.15-7.08 (m, 2H, ArH), 7.04 (s, 1H, NH), 4.66-4.60
(m, 1H, Hα), 4.60-4.53 (m, 2H, Hα), 4.20-4.14 (m, 1H, Lys-Hα), 3.25-3.14 (m, 2H, CH2), 3.13-
3.02 (m, 3H, CH2), 2.98-2.91 (m, 1H, CH2), 2.90-2.83 (m, 1H, CH2), 2.81-2.56 (m, 5H, CH2),
2.30-2.22 (m, 1H, Inp-Hα), 1.75-1.65 (m, 1H, CH2), 1.62-1.55 (m, 1H, CH2), 1.54-1.43 (m, 6H,
CH2), 1.38-1.19 (m, 2H, CH2), ppm. ESI-LC-MS m/z 452.5 [M + 4H - F]2+; HRMS (ESI-MS)
calcd. for C54H59FN7O6 [M + H]+ 920.4511 found 920.4529.
4.5.16
H-Inp-D-2-Nal-D-2-Nal-1-Nal-Lys-NH2: [1-Nal4]G-7039 (16)
Peptide purification by preparative HPLC (25-80% MeCN + 0.1% TFA) delivered a white
powder (24.9 mg, 23%): 1H-NMR (400 MHz, CD3OD); δ 8.12 (d, J = 8.4 Hz, 1H, ArH), 7.84 (d,
J = 7.8 Hz, 1H, ArH), 7.78-7.72 (m, 2H, ArH), 7.68 (d, J = 8.5 Hz, 3H, ArH), 7.62-7.46 (m, 5H,
ArH), 7.43-7.35 (m, 5H, ArH), 7.30 (d, J = 6.4 Hz, 1H, ArH), 7.27-7.21 (m, 2H, ArH), 7.02
(dd, J = 8.4, 1.5 Hz, 1H, ArH), 4.69 (dd, J = 9.4, 5.4 Hz, 1H, Hα), 4.65-4.55 (m, 2H, Hα), 4.32
(dd, J = 9.4, 4.8 Hz, 1H, Lys-Hα), 3.65 (dd, J = 14.4, 5.4 Hz, 1H, CH2), 3.20-3.10 (m, 2H, CH2),
3.05-2.94 (m, 3H, CH2), 2.92-2.80 (m, 5H, CH2), 2.76-2.62 (m, 2H, CH2), 2.30-2.23 (m, 1H,
Inp-Hα), 1.92-1.81 (m, 1H, CH2), 1.75-1.29 (m, 9H, CH2), ppm. ESI-LC-MS m/z 424.8 [M +
2H]2+; HRMS (ESI-MS) calcd. for C51H58N7O5 [M + H]+ 848.4499, found 848.4501.
24

ACCEPTED MANUSCRIPT
4.5.17
H-Inp-D-2-Nal-D-2-Nal-1-Nal-Lys(4-FB)-NH2: [1-Nal4,Lys5(4-FB)]G-7039
(17)
The product was purified by preparative HPLC (25-90% MeCN + 0.1% TFA) which yielded
1
a white powder (9.80 mg, 9%): H-NMR (400 MHz, (CD3)2SO); δ 8.64 (d, J = 8.3 Hz, 1H, NH),
8.46 (t, J = 5.5 Hz, 1H, NH), 8.33 (s, 1H, NH), 8.25 (d, J = 8.4 Hz, 1H, ArH), 8.13 (d, J = 8.0
Hz, 1H, NH), 8.09 (d, J = 7.5 Hz, 1H, NH), 8.03 (d, J = 8.5 Hz, 1H, NH), 8.01-7.97 (m, 1H,
NH), 7.89 (d, J = 8.2 Hz, 1H, ArH), 7.86-7.80 (m, 2H, F-ArH), 7.79-7.72 (m, 3H, ArH), 7.71-
7.66 (m, 2H, ArH), 7.58-7.50 (m, 4H, ArH), 7.44 (d, J = 6.9 Hz, 1H, ArH), 7.42-7.34 (m, 5H,
ArH), 7.31-7.25 (m, 2H, ArH), 7.21-7.15 (m, 3H, 2F-ArH, ArH), 7.07 (s, 1H, NH), 7.02 (d, J =
8.4 Hz, 1H, ArH), 4.72-4.65 (m, 1H, Hα), 4.62-4.50 (m, 2H, Hα), 4.19 (dd, J = 13.3, 8.4 Hz, 1H,
Lys-Hα), 3.61 (dd, J = 14.3, 3.9, 1H, CH2), 3.24-3.00 (m, 5H, CH2), 2.97-2.89 (m, 1H, CH2),
2.82-2.49 (m, 5H, CH2), 2.29-2.19 (m, 1H, Inp-Hα), 1.78-1.66 (m, 1H, CH2), 1.66-1.54 (m, 1H,
CH2), 1.53-1.40 (m, 5H, CH2), 1.40-1.16 (m, 2H, CH2), ppm. ESI-LC-MS m/z 477.4 [M + 4H -
F]2+; HRMS (ESI-MS) calcd. for C58H61FN7O6 [M + H]+ 970.4667 found 970.4693.
4.6 Synthesis of 4-(tert-butoxycarbonyl)-N,N,N-trimethylbenzenammonium triflate (20)
4-dimethylaminobenzoic acid (1.00 g, 6.05 mmol, 1.0 equiv.) was added to THF (50 ml)
and the resultant mixture stirred and cooled to 0°C. After 15 minutes, trifluoroacetic anhydride
(1.85 ml, 13.3 mmol, 2.2 equiv.) was added dropwise and the consequent blue solution stirred (35
min). Addition of tBuOH (11.4 ml, 119 mmol, 19.7 equiv.) was followed by further stirring at room
temperature (2 h). The solution was then poured into saturated NaHCO3 (250 ml) and extracted
with DCM (3x 100 ml, 3x 50 ml). The combined organic layers were dried (MgSO4), filtered by
gravity and residual solvent removed by rotary evaporation. This delivered a black oil which was
eluted through a silica pad (DCM, 60 ml) and solvent removed by concentration in vacuo. The final
yellow oil (1.12 g, crude) was then re-dissolved in DCM (dry, 30 ml) and cooled to 0°C under a
blanket of N2. This was followed by the addition of MeOTf (0.86 ml, 7.61 mmol, 1.5 equiv.) and
stirring at 0°C (1 h). The reaction mixture was then poured into an ice-cold solution of Et2O (200
ml) which caused instant precipitation of the product salt as a white crystalline solid (740 mg,
1
38%): H-NMR (400 MHz, CD3COCD3); δ 8.24 (d, J = 9.3 Hz, 2H), 8.18 (d, J = 9.3 Hz, 2H),
3.91 (s, 9H, NMe3), 1.60 (s, 9H, OC(CH3)3); 13C-NMR (100 MHz, CD3COCD3); δ 205.5 (CO)
163.6 (ArC), 133.7 (ArC), 131.0 (ArC), 120.9 (ArC), 81.7, 56.9, 27.3 (CH3CO) ppm. ESI-MS
25

ACCEPTED MANUSCRIPT
m/z 165.1 [C10H13O2]+; HRMS (ESI-MS) calcd for C14H22NO2 [M-CF3O3S]+ 236.1651, found
236.1658.
4.7 Receptor-ligand binding assay
Competitive binding assays were carried out using HEK293/ghrelin receptor cells with
[125I]ghrelin as the competitive radioligand. A solution of binding buffer (50 ml, pH 7.4) was made
up by adding 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, 0.3 g, 25 mM), MgCl2
(0.051 g, 5 mM), CaCl2 (7.4 x 10-3 g, 1 mM), EDTA (0.015 g, 2.5 mM) and BSA (0.2 g, 0.4%) to
distilled water. The resultant solution was filtered through a 0.22 µm syringe filter and kept on ice.
An aliquot of frozen cells was thawed to room temperature (1.5 x 106 cells/pellet),
centrifuged (3000 rpm, 10 min, room temperature) and the subsequent cell pellet re-suspended in
binding buffer (2 ml) and placed on ice. A stock solution of peptide/peptidomimetic was diluted in
binding buffer to acquire final concentrations between 10-5 to 10-11 M and added (30 µl) to these
cells (50 µl) in triplicate.
[125I]-ghrelin (10 µl) was added to binding buffer (3 ml) and vortexed. An aliquot of this
[125I]-ghrelin solution (20 µl) was counted on a Cobra II Auto-Gamma gamma counter and initial
volume adjusted to obtain 14000-17000 counts per minute (cpm) per 20 µl aliquot. [125I]-ghrelin
(20 µl) was added to each peptide/peptidomimetic-cell mixture, to give a final volume of 300 µl in
binding buffer. Each solution concentration was vortexed, followed by agitation (550 rpm, 20 min,
°
°
37 C). After 20 minutes, samples were spun (13 000 rpm, 5 min, 4 C) and placed on ice. The
supernatant was removed and the cell pellet rinsed with ice-cold Tris-HCl (200 µl, 50 mM, pH 7.4).
The samples were spun again (13,000 rpm, 5 min, 4 °C), cooled on ice, supernatant removed and
cell pellet counted using a Cobra II Auto-Gamma gamma counter.
All binding assays were performed in triplicate for each concentration of
peptide/peptidomimetic. Binding buffer (300 µl) and [125I]-ghrelin (20 µl) alone were used as
background controls. [125I]-ghrelin (20 µl) and cells (50 µl) in binding buffer (230 µl) were used to
calculate the percentage of bound [125I]-ghrelin displaced by each concentration of
peptide/peptidomimetic.
4.8 Computation of partition coefficients (LogP) for peptides and peptidomimetics
The n-octanol/water partition coefficients for all peptides and peptidomimetics were
calculated using ACD/LogP prediction software from ACD/ChemSketch (Freeware) 2017.2.1.
26

ACCEPTED MANUSCRIPT
4.9 Calcium flux dose-response assay
This assay was used to determine the EC50 value for [1-Nal4,Lys5(4-FB)]G-7039 in terms of
intracellular Ca2+ release, with ghrelin used as the control ligand. The peptidomimetic compound
was dissolved in DMSO (1 ml) at a concentration of 250 µM and solubility tested in Hank’s
balanced salt solution (HBSS): 1.26 mM CaCl2, 0.493 mM MgCl2·6H2O, 0.407 mM MgSO4·7H2O,
5.33 mM KCl, 0.441 mM KH2PO4, 4.17 mM NaHCO3, 137.9 mM NaCl, 0.338 mM Na2HPO4, 5.56
mM D-Glucose, 20 mM HEPES, pH 7.4 with a final concentration of 1.2 % DMSO. Following
successful solubility, human recombinant ghrelin receptor calcium-optimised stable cell lines
(chem-1 cells) were loaded with a fluorescent calcium dye (Fluo-8 NW from ABD Bioquest 21080)
and calcium flux detected in response to the agonist by a Molecular Devices FLIPRTETRA
instrument in a modified HBSS buffer (20 mM HEPES, 2.5 mM Probenecid, pH 7.4). Fluorescence
values underwent baseline correction and the percentage activation was normalised to the maximum
response (Emax) of the control ligand ghrelin. The agonist assay was performed for a total of 180
seconds and each assay concentration was performed in duplicate.
4.10
Serum stability study
A Waters Oasis HLB plate was used for both collection and extraction. The peptidomimetic
[1-Nal4,Lys5(4-FB)]G-7039 was incubated in a solution of 25% human serum in PBS for 24 h.
Aliquots were removed at 0, 20, 60, 240, 420, and 1440 minutes, with each time-point performed in
triplicate. These aliquots were then mixed with 4% (v/v) ammonium hydroxide (NH4OH) in water
to denature peptidomimetic-serum (albumin) interactions. [1-Nal4,Lys5(4-FB)]G-7039 was then
extracted from human serum using 20% MeOH/2% HCO2H in water and quantified by UHPLC-
MS. The amount was then expressed as the percentage of the area under the curve (AUC) in the UV
chromatogram relative to time 0, plotted against time (minutes) and data fitted to a curve of
exponential decay to determine the serum half-life (t1/2).
4.11
Radiochemistry
4.11.1 General information
All reagents and solvents used for radiosynthesis were purchased from Sigma-Aldrich
unless otherwise specified. 18F-fluoride was produced via the 18O(p,n) 18F reaction in a GE
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PETtrace 880 cyclotron (Lawson Health Research Institute, London, Ontario, Canada). A Biotage
V-10 evaporator was used to remove solvent. A Waters HPLC with a 1525 binary pump system
(solvent A = MeCN + 0.1% TFA, solvent B = H2O + 0.1% TFA) and two detectors (Waters 2487
Dual λ absorbance detector set at 254 nm and a radioactive flow count detector) were utilised for
product analysis and purification. An analytical column (Agilent Eclipse XDB-C18, 4.6 × 150 mm,
5 µm) was used for determining radiochemical purity and molar activity of [1-Nal4,Lys5(4-[18F]-
FB)]G-7039. A semi-preparative column (Agilent Zorbax SB-C18, 9.4 × 150 mm, 5 µm) was used
for the purification of [18F]SFB and [1-Nal4,Lys5(4-[18F]-FB)]G-7039.
4.11.2
Synthesis of [18F]FBA
Scheme 1 shows the synthetic pathway to [18F]FBA. Aqueous [18F]fluoride was trapped on a
Waters Sep-Pak Accell Plus Light (46 mg) QMA Carbonate cartridge pre-activated with MilliQ
water (10 ml). The initial radioactivity of the cartridge was measured using a dose calibrator (980-
1300 MBq) A solution of acetonitrile/H2O (1 ml, 25:75, v/v) containing potassium carbonate (2.1
mg, 0.015 mmol, 2.6 eq.) and 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (6.0
mg, 0.016 mmol, 2.8 eq.) was used to elute [18F]fluoride into the reaction vial. The solvent was
removed azeotropically under vacuum at 50 °C. [18F]fluoride was then dried a further two times
under the aforementioned conditions by adding anhydrous acetonitrile (1 ml). 4-Tert-
butoxycarbonyl)-N,N,N-trimethylbenzenammonium triflate (2.0 mg, 0.0057 mmol, 1.0 eq.) in
anhydrous DMSO (400 µl) was added to dried [18F]F- and the subsequent solution heated for 10
min at 120 °C. Aqueous HCl (1 ml, 6 M) was added and the resultant mixture heated further (10
min, 120 °C). Water (2 ml) was added to dilute the reaction mixture containing crude [18F]FBA.
This solution was then trapped on two inter-connected Waters Sep-Pak C-18 light cartridges
sequentially pre-treated with ethanol (15 ml) and water (15 ml). Elution of product then occurred
using anhydrous acetonitrile (800 µl) to deliver the title compound [18F]FBA in an average d.c.
radiochemical yield of 82% (n = 3). This compound was used in the next step without further
purification. To confirm that the correct product had been acquired, a co-injection of 4-[19F]-FBA
and [18F]-FBA in water was performed (20 µl, 30-70% MeCN + 0.1% TFA gradient system, see
Figure S3 supporting information).
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