Synthesis and biological evaluation of analogues of the potent ADAM8 inhibitor cyclo(RLsKDK) for the treatment of inflammatory diseases and cancer metastasis

Article abstract of DOI:10.1016/j.bmc.2016.06.042

The metalloproteinase ADAM8 serves as a pivotal catalyst in the development of inflammatory diseases and cancer metastasis. The cyclic peptide cyclo(RLsKDK) has been shown to inhibit the enzymatic activity of ADAM8 with high specificity and potency. Herein we report a structure–activity relationship (SAR) study of cyclo(RLsKDK) that involves the synthesis and biological evaluation of the lead compound and structural analogues thereof. This study provides insight into the ligand–receptor interactions that govern the binding of cyclo(RLsKDK) to the ADAM8 disintegrin domain and represents a stepping stone for the development of new treatments for inflammatory diseases and cancer metastasis.

Full text of DOI:10.1016/j.bmc.2016.06.042

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of analogues of the potent
ADAM8 inhibitor cyclo(RLsKDK) for the treatment of inflammatory
diseases and cancer metastasis
Victor Yim a,y, Anaïs F. M. Noisier a,y,à, Kuo-yuan Hung a, Jörg W. Bartsch b, Uwe Schlomann b,
Margaret A. Brimble a,c,
⇑
a School of Biological Sciences and The Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3a Symonds St, Auckland Central 1010, New Zealand
b Department of Neurosurgery, Marburg University, University Hospital, Baldingerstr., 35053 Marburg, Germany
c School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland 1010, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The metalloproteinase ADAM8 serves as a pivotal catalyst in the development of inflammatory diseases
and cancer metastasis. The cyclic peptide cyclo(RLsKDK) has been shown to inhibit the enzymatic activity
of ADAM8 with high specificity and potency. Herein we report a structure–activity relationship (SAR)
study of cyclo(RLsKDK) that involves the synthesis and biological evaluation of the lead compound and
structural analogues thereof. This study provides insight into the ligand–receptor interactions that
govern the binding of cyclo(RLsKDK) to the ADAM8 disintegrin domain and represents a stepping stone
for the development of new treatments for inflammatory diseases and cancer metastasis.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 23 May 2016
Revised 20 June 2016
Accepted 21 June 2016
Available online xxxx
Keywords:
ADAM8 inhibitors
Cancer metastasis
Cyclic peptides
Peptidomimetics
SAR study
1. Introduction
of the enzyme.8 ADAM8 activation is an autocatalytic process
which is triggered by either homodimeric binding of another
A disintegrin and metalloproteinase domain 8 (ADAM8) is a
catalytically active member of the ADAMs family of proteins, pri-
marily expressed in several tissues including the brain, bone and
spinal cord during embryonic development.1,2 Under inflammatory
conditions ADAM8 is up-regulated in most immune cells of the
central nervous system3 and in the lungs.4 ADAM8 is also associ-
ated with allergic responses5 and is overexpressed in various can-
cers including lung, prostate and pancreatic adenocarcinomas,
brain tumours, and renal cell carcinoma.6 Furthermore, the levels
of ADAM8 expression are strongly correlated with tumour malig-
nancy and invasive activity.7
ADAM8 or heterodimeric binding of other cell-adhesion molecules
to the disintegrin domain.9 Such dimerisation induces cleavage of
the pro-peptide, which in turn triggers a conformational change
of the metalloproteinase subdomain, thus exposing the catalyti-
cally active site (Fig. 1). The metalloproteinase subdomain then
cleaves the extracellular segments of membrane-bound proteins
such as cell adhesion molecules or extracellular matrix proteins
through a shedding process.1 Shedding of transmembrane proteins
releases extracellular molecules such as growth factors, hormones
and chemokines, which then trigger metabolic signalling pathways
that give rise to various physiological effects.10 Furthermore, pro-
tein shedding of its extracellular domain is a prerequisite to a reg-
ulated intramembrane proteolysis (RIP) event resulting in the
release of intracellular molecules that in turn induce intracellular
signalling pathways (Fig. 1).1
ADAM8 is a transmembrane protein, which possesses a mul-
tidomain architecture (Fig. 1). Its extracellular domain consists of
several subdomains, among which the pro-peptide, metallopro-
teinase and disintegrin subdomains are key to the overall activity
Owing to the significant role of ADAM8 in the mediation of
intra- and extracellular signalling pathways involved in inflamma-
tory conditions and cancer, inhibition of ADAM8 activity poses an
attractive strategy for the treatment of these diseases.
Unfortunately, previous efforts directed at inhibition of ADAM
activity failed to deliver useful therapeutic outcomes due to the
lack of specificity for a particular enzyme. Both Marimastat and
⇑
Corresponding author. Tel.: +64 937 375 99; fax: +64 937 374 22.
E-mail address: m.brimble@auckland.ac.nz (M.A. Brimble).
y
These authors contributed equally to this work.
Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028
à
Barcelona, Spain.
http://dx.doi.org/10.1016/j.bmc.2016.06.042
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yim, V.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.042

2
V. Yim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
metalloproteinase (MP)
pro-peptide
extracellular ligand
shedding
disintegrin
(DIS)
cysteine
extracellular
domain
activation
EGF repeats
-rich
transmembrane
domain
Intracellular
domain
ADAM8
transmembrane
protein
proteolysis
intracellular ligand
Figure 1. Structure of ADAM8 and its catalytic activity.
Batimastat are examples of broad-spectrum metalloproteinase
inhibitors for the treatment of cancer, for which clinical develop-
ment was terminated due to their deleterious side-effects.11
Previous work by Bartsch and Koller12 focused on a novel
approach based on the disruption of multimeric binding to the
ADAM8 disintegrin domain, potentially inhibiting ADAM8 prote-
olytic activity and downstream signalling with unique selectivity.
They postulated that peptides mimicking the structure of the inte-
grin binding loop contained in the disintegrin domain would pro-
vide potent ADAM8 inhibitors. The binding loop of an ADAM
protease is commonly comprised of six amino acids flanked by
conserved cysteine residues. Bartsch and Koller designed a short
cyclic peptide, cyclo(RLsKDK), based on the sequence of amino
acids located within the DIS loop of murine ADAM8 and demon-
strated that cyclo(RLsKDK) blocked ADAM8 activation with high
potency (IC50 = 182 nM) and high specificity, with in vivo efficacy
in a pancreatic cancer model (Fig. 2).13 The serine residue was
NH2
6Lys
5Asp
HO
O
O
O
N
H
1Arg
NH
cyclo(RLsKDK)
IC50 = 182 nM
O
NH
NH
HN
HN
4Lys
H2N
N
H
NH2
O
H
N
O
2Leu
3ser
O
HO
Figure 2. Structure of lead peptide cyclo(RLsKDK).
peptides were assembled by Fmoc solid-phase peptide synthesis
(SPPS) using aminomethyl polystyrene (PS) resin derivatised with
highly acid-labile 4-(4-hydroxymethyl-3-methoxyphenoxy)-buty-
ric acid (HMPB) linker.15 HMPB was chosen as only mild conditions
are required to effect peptide release from the resin, therefore
affording the side-chain protected peptides, necessary for in-solu-
tion cyclisation. When applicable, the b-amino acids were chosen
as the optimal point for cyclisation, thus avoiding troublesome
epimerisation during the head-to-tail macro-cyclisation step.
The desired C-terminal amino acid residues were attached to
the resin via an ester bond. The b-alanine, which served as a readily
available surrogate for the more expensive hSb, was used to estab-
lish a robust esterification protocol. Thus, Fmoc-b-alanine was
esterified with the linker-modified resin using DIC and 1 equiv of
DMAP under microwave irradiation (25 W, 50 °C), giving an
acceptable loading of 0.46 mmol/g (60% yield) as determined by
UV spectrophotometry.16 Fmoc-hSb(tBu)-OH was also successfully
coupled to the resin using these conditions (0.38 mmol/g, 52%
yield). The peptide containing the 3-carboxy-b-alanine residue
was prepared using the same esterification protocol where Fmoc-
Asp-OAll (All = allyl) was attached to the resin through its unpro-
incorporated as a
ine) to favour the formation of a b-turn,14 thus allowing the pre-
sentation of the peptide sequence in secondary structure
closely resembling the integrin-binding loop. The use of -amino
D-amino acid (where ‘s’ is used to denote D-ser-
a
D
acids also imparts the peptide with greater resistance to prote-
olytic degradation. Although the discovery of cyclo(RLsKDK) repre-
sents a key milestone in the treatment of inflammatory diseases
and cancer metastasis, the development of more potent and more
stable peptidomimetics is required to identify a valid drug candi-
date for clinical trials.
In this report, we describe the synthesis and SAR study of a
library of peptidomimetics based on the cyclo(RLs4KD6K) lead
motif. A systematic substitution approach was used to generate
two series of analogues. In the first series, the original
D-serine
was replaced by -serine, b-homoserine (hSb), b-alanine (Ab) and
L
3-carboxy-b-alanine in order to evaluate the contribution of the
hydroxyl group on the peptide activity. Through these substitu-
tions, we expected to probe the effect of modifying the
a-chain
length, and of altering the spatial conformation of the side-chain.
The second series of analogues focused on substitution of arginine
(R), leucine (L) and 6lysine (6K) with non-proteinogenic derivatives
to determine the impact of these structural substituents on the
binding. The aspartic acid (D) residue of cyclo(RLsKDK) was left
unmodified as it was previously shown to be crucial for binding.12
tected b-carboxylic acid. For esterification of Fmoc-
or Fmoc-
-Ser(tBu)-OH only 0.1 equiv of DMAP was employed to
prevent racemisation at the -carbon. The peptide was then elon-
L
-Ser(tBu)-OH
D
a
gated in an automated peptide synthesiser using HCTU as the cou-
pling reagent. When non-proteinogenic amino acids were
incorporated, the more potent HATU was used in conjunction with
microwave irradiation (25 W, 50 °C) to ensure complete acylation.
After assembly of the peptide sequence and removal of the N-ter-
minal Fmoc protecting group, the linear peptides were cleaved
from the resin with a solution of TFA in CH2Cl2 (5/95 v/v) without
loss of side-chain protecting groups. The peptides were then
2. Results and discussion
2.1. Peptide synthesis
All the peptides reported herein were synthesised using the
general synthetic pathway described in Scheme 1. First, the linear
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V. Yim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
NHBoc
HO
O
R2
O
R4
O
H
N
H
N
H
N
i to v
O
O
O
N
H
H2N
N
H
O
N
H
n
HO
O
O
O
O
R1
R3
HMPB-PS resin
tBu
O
vi
HO
O
NHBoc
O
R4
H2N
O
N
H
R2
vii, viii
O
H
R4
O
O
NH
NH
H
N
H
N
N
OH
HN
HN
R2
H2N
N
H
N
H
R3
n
O
O
O
R1
O
R3
O
O
O
O
tBuO
n
n = 0 or 1
N
H
R1
cyclo(X 3X2X1KDX4)
Scheme 1. General synthetic pathway. Reagents and conditions: (i) Fmoc-AA-OH or Fmoc-Asp-OAll, DIC, DMAP, DMF/CH2Cl2 (20/80 v/v), microwave (25 W, 50 °C), 1 h; (ii)
piperidine/DMF (20/80 v/v), 2 ꢁ 5 min, rt; (iii) Fmoc-AA-OH, HCTU, NMM, DMF, 45 min, rt or Fmoc-AA-OH, HATU, iPr2NEt, DMF, microwave (25 W, 50 °C), 1 h; (iv) repeat (ii)
and (iii); (v) piperidine/DMF (20/80 v/v), 2 ꢁ 5 min, rt; (vi) TFA/CH2Cl2 (99.5/0.5 v/v), 8 ꢁ 5 min; (vii) HBTU, iPr2NEt, CH2Cl2/DMF, 5 days or HBTU, HOBt, iPr2NEt, CH2Cl2/DMF,
5 days; (viii) TFA/iPr3SiH/H2O, 2 h, rt (AA = amino acid).
Table 1
subjected to macrocyclisation at high dilution to avoid formation
of linear or cyclic oligomers.17 Two different conditions were
The IC50 values of synthetic peptides 1–19
employed for this key cyclisation step: for peptides containing
hSb, b-alanine, or Asp-OAll at the C-terminus, the reaction was
carried out with HBTU/iPr2NEt in a solution of DMF in CH2Cl2
(10/90 v/v) at a final peptide concentration of 0.9 mg/mL; for pep-
No.
Peptides
IC50 (nM)
Series 1: Peptidomimetics with different S substituents
1
2
3
4
5
Cyclo(RLsKDK) s =
Cyclo(RLSKDK) S =
D
-serine
-serine
182 23
240 45
157 32
1645 142
420 53
L
Cyclo(RLhSbKDK) hSb = b-homoserine
Cyclo(RLAbKDK) Ab = b-alanine
tides containing either L-serine or D-serine at the C-terminus, HOBt
was included in the reaction mixture to minimise racemisation.
Finally, the side-chain protecting groups were removed with a
cocktail solution of TFA/iPr3SiH/H2O (95/2.5/2.5 v/v), to afford the
crude cyclic products that were purified by RP-HPLC. For peptide
5 (Table 1), the allyl protecting group of the aspartic acid residue
was removed with Pd(PPh3)4 prior to orthogonal side-chain depro-
tection. A focused library of nineteen peptides (Table 1) was pre-
pared following this synthetic procedure.
Cyclo(RLS⁄KDK) S⁄ = (S)-3-carboxy-b-alanine
Series 2: Peptidomimetics containing Ab
6
7
8
Cyclo(R⁄LAbKDK) R⁄ = nitroarginine
2345 123
890 56
256 16
220 28
378 50
452 63
>5000
>5000
>5000
>10000
Cyclo(R⁄LAbKDK) R⁄ = citrulline
Cyclo(RL⁄AbKDK) L⁄ = tert-butylalanine
Cyclo(RL⁄AbKDK) L⁄ = homoleucine
Cyclo(RL⁄AbKDK) L⁄ = norleucine
9
10
11
12
13
14
15
Cyclo(RL⁄AbKDK) L⁄ = norvaline
Cyclo(RLAbKDK⁄) K⁄ = diaminopropionic acid
Cyclo(RLAbKDK⁄) K⁄ = diaminobutyric acid
Cyclo(RLAbKDK⁄) K⁄ = ornithine
2.2. Biological evaluation
Cyclo(RLAbKDK⁄) K⁄ = trimethyllysine
Series 3: Peptidomimetics containing hSb
The inhibitory activity of all synthetic peptides against the
release of CD23 antibody mediated by ADAM8 was measured using
an enzyme-linked immunosorbent assay (ELISA). The IC50 values
obtained for each peptide are reported in Table 1.
16
17
18
19
Cyclo(R⁄LhSbKDK) R⁄ = nitroarginine
1245 85
192 12
368 32
142 21
Cyclo(R⁄LhSbKDK) R⁄ = citrulline
Cyclo(R⁄LhSbKDK) R⁄ = homoarginine
Cyclo(RL⁄hSbKDK) L⁄ = homoleucine
We began our SAR study by investigating the effect of modify-
ing the non-proteinogenic
(RLsKDK) peptide sequence. Peptide 2 where the
was replaced by its -enantiomer showed a slight decrease in
potency. Little improvement was achieved by switching from
-serine to b-homoserine. Interestingly, both the complete removal
of the hydroxyl group as in peptide 4, or its substitution by a car-
boxylic acid moiety as in peptide 5 showed a detrimental effect on
activity. A respective 2.5 and 10-fold increase in IC50 was observed
for peptides 5 and 4 with respect to 3. The significant effect of the
hydroxyl group on peptide inhibitory activity could be attributed
to the existence of polar interactions between the ligand hydroxyl
side-chain and the receptor binding-surface.
Despite the decreased potency of peptide 4, b-alanine was
employed as a readily available surrogate of b-homoserine within
our second series of peptidomimetics. The focus of the study
shifted to investigating the activity of derivatives of 4 where argi-
nine, leucine and 6lysine were consecutively substituted with non-
proteinogenic analogues (see Series 2 in Table 1). Replacement of
arginine with nitroarginine (peptide 6) had a detrimental effect
D
-serine residue within the cyclo
D-amino acid
on the activity, while use of the neutral analogue citrulline (pep-
tide 7) led to an almost 2-fold increase in potency. These results
indicate that arginine substitution by residues acting as hydro-
gen-bond acceptors favored inhibition of ADAM8 activity as
opposed to charged and more sterically hindered ones. This trend
was further exemplified in our third series (vide infra). Remark-
ably, substitution of leucine by homoleucine afforded peptide 9,
which showed much improved activity (ꢀ7-fold lower IC50) than
its parent peptide 4. It was found from the biological data of pep-
tides 9–11 that residues with more extended and flexible
hydrophobic side-chains correlate with an increase in activity, thus
suggesting the presence of a hydrophobic pocket at the receptor-
binding site. Discrepancies in the results were observed when
L
D
steric hindrance was introduced in the
c-position of the chain.
While peptide 4 showed a ꢀ4-fold drop in activity, peptide 8 con-
taining a bulkier tert-butylalanine residue led to a ꢀ1.5-fold
improved IC50, compare to 11. All the analogues featuring modifi-
cations at 6lysine (peptides 12–15), showed a dramatic loss of
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V. Yim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
activity with IC50 values above 5000 nM. This highlights the impor-
tance of the KDK motif for binding to the integrin-binding loop of
the ADAM8 disintegrin domain.
RP-HPLC was performed on a Dionex P680 system using a Waters
XTerraÒ C18 column (5
min. A linear gradient of 0.1% TFA/H2O (solvent A) and 0.1% TFA/
CH3CN (solvent B) was used with detection at 210 nm. Preparative
RP-HPLC was performed on a Waters 600 System with a Waters
2487 dual wavelength absorbance detector using a Waters XTerraÒ
l
m, 4.6 ꢁ 150 mm) at a flow rate of 1 mL/
In our third series, we endeavoured to transpose both the argi-
nine and leucine analogues which gave the best results within the
b-alanine series to our new b-homoserine lead peptide 3. We
indeed hoped to observe a synergistic effect by introducing two
favourable substitutions simultaneously within the parent peptide
1. Interestingly, while peptides 17 and 19 showed increased activ-
ity compared to their b-alanine analogues 7 and 9, with a ꢀ4- and
ꢀ1.5-fold lower IC50, respectively, it did not produce the similar
10-fold increase in potency observed in our first series when
b-homoserine was employed in place of b-alanine. Despite peptide
19 being the most active analogue of our library, it only exhibited
slightly higher inhibition than peptide 3. In this series we also
further investigated the effect of modifying the arginine residue.
In b-homoserine-containing peptides 16 and 18, the nitroarginine
and the homoarginine were used in place of arginine, respectively.
As previously observed introducing charged and bulky residues at
this position had detrimental effects on the activity with a ꢀ8- and
ꢀ2-fold increase in IC50 with respect to 3. Although 17 showed
improved inhibition activity compared to its b-alanine analogue
7, it remained less active than 3, thus suggesting that the prevailing
interaction in the binding of 17 to its receptor occurs through the
side-chain of the b-homoserine rather than that of the citrulline.
The lack of a significant synergic effect observed in this series sug-
gests that structural modifications which maximise the interaction
of one residue, concurrently hinder the interaction of the other
residue with the receptor.
Prep MS C18 column (10
l
m, 19 ꢁ 300 mm) at a flow rate of
10 mL/min. Gradient systems were adjusted according to the
elution profiles and peak profiles obtained from the analytical
RP-HPLC chromatograms.
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hex-
afluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium
hexafluorophosphate
(HBTU)
and
O-(6-
Chlorobenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluo-
rophosphate (HCTU) were purchased from Advanced ChemTech.
N,N-dimethylformamide (DMF) and acetonitrile (CH3CN) (synthe-
sis grade) were purchased from Scharlau, and CH2Cl2 (synthesis
grade) from ECP. Diisopropylethylamine (iPr2NEt), piperidine,
triisopropylsilane (iPr3SiH), 4-(dimethylamino)pyridine (DMAP),
N-methylmorpholine (NMM) were purchased from Sigma–Aldrich.
N,N-Diisopropylcarbodiimide (DIC) was purchased from GL Bio-
chem. 4-(4-hydroxymethyl-3-methoxyphenoxy) butyric acid
(HMPB) was purchased from Merck. Trifluoroacetic acid (TFA)
was purchased from Oakwood Chemicals. L-Amino acids were used
unless otherwise stated. Fmoc-amino acids were purchased from
GL Biochem, ChemImpex Inc., or PolyPeptide Laboratory. The cata-
lyst Pd(PPh3)4 and aminomethyl PS resin19 were synthesised
18
according to published procedures.
In conclusion, we have identified two new strategies for the
design of potent ligands of ADAM8 disintegrin domain. One con-
sists of taking advantage of the receptor binding site involving
the polar interaction observed with peptide 3 while the second
focused on exploiting the hydrophobic pocket which is believed
to be responsible for the improved binding of peptides 8–10.
3.2. General peptide synthesis procedures
Solid phase peptide synthesis (0.2 mmol scale) was performed
on aminomethyl PS resin (1.0 mmol/g) based on the Fmoc based
strategy. HMPB linker was attached to the resin using general
method A, and coupling of the first amino acid residue to the
HMPB-PS resin was performed according to general method B or
C. The degree of attachment of the first amino acid residue to the
resin was determined using UV spectrophotometry.16 The desired
peptide sequences were synthesised using general method D on
TributeTM peptide synthesiser, and peptide coupling of unnatural
amino acids was performed manually according to general method
E. The linear peptides were cleaved from the resin using general
method F, and the crude products were cyclised using general
method G. The side-chain protecting groups were removed from
the cyclised peptides according to general method H, and the allyl
protecting group was removed according to general method I. The
crude peptides were purified according to general method J.
2.3. Conclusion
During this SAR study of the potent cyclo(RLsKDK) ADAM8 inhi-
bitor, two prevalent modes of interactions between the ligand and
the receptor were identified. Two peptidomimetics; cyclo(RLhSb-
KDK) 3 and cyclo(RhLAbKDK) 9; were found to present slightly
increased activity compared to the lead peptide. Although the
activity could not be significantly improved, these compounds
could serve as potential new lead structures replacing cyclo
(RLsKDK) for future SAR studies. Furthermore, it was established
that Based on these promising results, additional work focusing
on the design of a ligand capable of taking advantage of both the
interaction sites described herein is on-going in our laboratory.
3.2.1. General method A: attachment of HMPB linker to
aminomethyl PS resin
Aminomethyl PS resin (0.2 mmol) was swollen in CH2Cl2/DMF
(1/1, v/v) for 20 min and the solvent was drained. HMPB (4 equiv)
was dissolved in DMF/CH2Cl2 (3 mL, 5/95 v/v), and DIC (4 equiv)
was added. The mixture was added to the resin, and the reaction
was agitated for 2 h. The solution was drained, and the resin was
washed with DMF (3ꢁ), CH2Cl2 (3ꢁ) and then air dried.
3. Experimental
3.1. General information
All reagents were purchased as reagent grade and used without
further purification. HPLC solvents were purchased as HPLC grade
and used without further purification. UV measurements were
obtained using a Jenway 7315 spectrophotometer. Electrospray
ionisation mass spectra (ESI-MS) were recorded on an Agilent
Technologies 1120 Compact LC connected to a HP Series 1100
MSD spectrometer or a Bruker micrOTOF-Q II spectrometer. Sam-
ples were introduced using direct flow injection at 0.2 mL/min into
an ESI source in positive mode, using 0.1% formic acid/H2O and
0.1% formic acid/CH3CN (1/1, v/v). Major and significant fragments
were quoted in the form x m/z (mass to charge ratio). Analytical
3.2.2. General method B: attachment of Fmoc-hSb(OtBu)-OH,
Fmoc-b-Ala-OH, or Fmoc-Asp-OAll to HMPB-PS resin
To a solution of Fmoc protected amino acid (2 equiv) in DMF/
CH2Cl2 (3 mL, 20/80 v/v) was added DIC (2 equiv), and the mixture
was added to the HMPB-PS resin. DMAP (1 equiv) was added to the
resin, and the reaction was stirred under MW irradiation (25 W,
50 °C) for 1 h. The solution was drained, and the resin was washed
with DMF (3ꢁ) and CH2Cl2 (3ꢁ) and then air dried.
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V. Yim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
3.2.3. General method C: attachment of Fmoc-
Fmoc-
-Ser(OtBu)-OH to HMPB-PS resin
L
-Ser(OtBu)-OH or
3.3. Characterisation data
3.3.1. Cyclo(RLsKDK) s = -serine (1)
Peptide 1 (13.0 mg, 9%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.4 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
728.4; Found 728.4.
D
To a solution of Fmoc protected amino acid (4 equiv) in DMF/
CH2Cl2 (3 mL, 20/80 v/v) was added DIC (4 equiv), and the mixture
was added to the HMPB-PS resin. DMAP (0.1 equiv) was added to
the resin, and the reaction was agitated for 2 h. The solution was
drained, and the resin was washed with DMF (3ꢁ), CH2Cl2 (3ꢁ)
and then air dried.
D
3.3.2. Cyclo(RLSKDK) S = L-serine (2)
3.2.4. General method D: automated Fmoc SPPS
Peptide 2 (8.0 mg, 6%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.3 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
728.4; Found 728.4.
Couplings of Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp
(OtBu)-OH and Fmoc-Leu-OH (5 equiv) were carried out at room
temperature in the presence of HCTU (4.6 equiv) and NMM
(10 equiv) in DMF for 45 min. The Fmoc protecting group was
removed using piperidine/DMF (3 mL, 20/80 v/v, 2 ꢁ 5 min).
3.3.3. Cyclo(RLhSbKDK) hSb = b-homoserine (3)
Peptide 3 (3 mg, 2%) was obtained as a white solid in >99% pur-
ity according to analytical RP-HPLC. Rt = 5.8 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
742.5; Found 742.3.
3.2.5. General method E: manual coupling of unnatural amino
acid
To a solution of Fmoc protected unnatural amino acid (2 equiv)
in DMF (2 mL) was added HATU (1.85 equiv) and iPr2NEt (4 equiv),
and the solution was added to the resin. After stirring under MW
irradiation (25 W, 50 °C) for 1 h, the resin was drained, washed
with DMF (3ꢁ), CH2Cl2 (3ꢁ) and then air dried. The Fmoc protect-
ing group was removed using piperidine/DMF (3 mL, 20/80 v/v,
2 ꢁ 5 min).
3.3.4. Cyclo(RLAbKDK) Ab = b-alanine (4)
Peptide 4 (9.5 mg, 7%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 5.7 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
712.4; Found 712.3.
3.2.6. General method F: cleavage of peptide from resin
The resin containing the desired linear peptide was agitated in
TFA/CH2Cl2 (3 mL, 0.5/99.5 v/v) for 5 min, drained and the same
procedure was repeated eight times. The combined filtrates
(ca. 25 mL) were evaporated under a flow of N2 gas, and the crude
peptide was immediately dissolved in CH3CN and lyophilised.
3.3.5. Cyclo(RLS⁄KDK) S⁄ = (S)-3-carboxy-b-alanine (5)
Peptide 5 (0.3 mg, 0.1%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 7.1 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
756.4; Found 756.1.
3.3.6. Cyclo(R⁄LAbKDK) R⁄ = nitroarginine (6)
3.2.7. General method G: macrocyclisation
Peptide 6 (5 mg, 3%) was obtained as a white solid in >95% pur-
ity according to analytical RP-HPLC. Rt = 6.6 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
757.4; Found 757.4.
The linear peptide and HBTU (3 equiv) were dissolved in CH2-
Cl2/DMF (9/1, v/v), and the mixture was slowly added to a solution
of iPr2NEt (5 equiv) in CH2Cl2/DMF (9/1, v/v) using a syringe pump
at a rate of 0.5 mL/h to reach a final peptide concentration of
0.9 mg/mL. The solution was concentrated on a rotary evaporator,
and the crude cyclic peptide was recovered by lyophilisation. For
the synthesis of peptides 2 and 3, HOBt (3 equiv) was added to
the CH2Cl2/DMF solution containing the peptide and HBTU to pre-
vent racemisation during cyclisation.
3.3.7. Cyclo(R⁄LAbKDK) R⁄ = citrulline (7)
Peptide 7 (19 mg, 13%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 5.4 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
713.4; Found 713.4.
3.2.8. General method H: cleavage of side-chain protecting
groups
3.3.8. Cyclo(RL⁄AbKDK) L⁄ = tert-butylalanine (8)
Peptide 8 (3.8 mg, 3%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.7 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
726.5; Found 726.5.
The side-chain protecting groups on the crude cyclic peptide
were removed by agitating in a solution of TFA/iPr3SiH/H2O
(4 mL, 95/2.5/2.5 v/v) for 2 h. The crude peptide was precipitated
from cold Et2O, centrifuged, the supernatant decanted, and the
same procedure was repeated. The resulting solid was dissolved
in H2O and lyophilised.
3.3.9. Cyclo(RL⁄AbKDK) L⁄ = homoleucine (9)
Peptide 9 (4.5 mg, 3%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.9 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
726.5; Found 726.4.
3.2.9. General method I: removal of allyl protecting group
The cyclic peptide was dissolved in argon degassed CH2Cl2
(1 mL). Pd(PPh3)4 (0.04 equiv) and PhSiH3 (4 equiv) were added
to the solution, and the reaction mixture was stirred under argon
for 1 h, filtered, and the filtrate was concentrated under reduced
pressure.
3.3.10. Cyclo(RL⁄AbKDK) L⁄ = norleucine (10)
Peptide 10 (1.7 mg, 1%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 10.4 min (XTerraÒ
C18, 5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
711.4; Found 712.3.
3.2.10. General method J: purification
The crude peptide was dissolved in H2O and purified by prepar-
ative RP-HPLC on a XTerraÒ C18 column using a gradient of 0–60%
B at 1% B/min with a flow rate of 10 mL/min. The purity of the pep-
tide was determined by flow injection (ESI+, 100 V) and analytical
RP-HPLC (XTerraÒ C18 column, 5–95% B, 3% B/min, 1 mL/min).
3.3.11. Cyclo(RL⁄AbKDK) L⁄ = norvaline (11)
Peptide 11 (1.7 mg, 1%) was obtained as a white solid in >95%
purity according to analytical RP-HPLC. Rt = 6.3 min (XTerraÒ C18,
Please cite this article in press as: Yim, V.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.042

6
V. Yim et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
697.4; Found 698.2.
1:2 dilutions ranging from 400 to 12.5 units/mL. Blank and sample
duplicates (each 100 L) were also applied, after that 50 L of
l
l
diluted biotinylated anti-CD23 monoclonal antibody (1/100)
was added. After application of all required solutions the plate
was incubated for 2 h at room temperature. The multiwell plate
was then emptied and washed 4 times with washing buffer to
remove the unbound biotin conjugates and re-incubated with
3.3.12. Cyclo(RLAbKDK⁄) K⁄ = diaminopropionic acid (12)
Peptide 12 (2.5 mg, 2%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 9.3 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
669.4; Found 670.4.
100
temperature. After additional washing of the plate, TMB substrate
solution (100 L/well) was added for 10–20 min until colour devel-
lL per well of diluted streptavidin–HRP (1/100) for l h at room
3.3.13. Cyclo(RLAbKDK⁄) K⁄ = diaminobutyric acid (13)
Peptide 13 (6.4 mg, 5%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.0 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
683.4; Found 684.5.
l
opment. Absorbance in each individual well was determined using
an ELISA plate reader (BMG Labtech, Offenburg, Germany) at
405 nm. The amount of soluble CD23 released from COS7 cells
when using the cyclic peptides is reduced to an extent that reflects
the contribution of ADAM8 to the overall cleavage of CD23 in these
cells.5
3.3.14. Cyclo(RLAbKDK⁄) K⁄ = ornithine (14)
Peptide 14 (1.4 mg, 1%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 5.8 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
697.4; Found 698.4.
Acknowledgment
The authors thank Cancer Research UK for financial support.
3.3.15. Cyclo(RLAbKDK⁄) K⁄ = trimethyllysine (15)
Peptide 15 (2.0 mg, 1%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.0 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
754.5; Found 754.4.
Supplementary data
Supplementary data (structures and LC–MS profiles of synthetic
peptides) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2016.06.042.
3.3.16. Cyclo(R⁄LhSbKDK) R⁄ = nitroarginine (16)
Peptide 16 (6.4 mg, 4%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 6.8 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
786.4; Found 787.4.
References and notes
1. Edwards, D. R.; Handsley, M. M.; Pennington, C. J. Mol. Aspects Med. 2008, 29,
258.
2. Kelly, K.; Hutchinson, G.; Nebenius-Oosthizen, D.; Smith, A. J. H.; Bartsch, J. W.;
Horiuchi, K.; Rittger, A.; Manova, K.; Docherty, A. J. P.; Blopel, C. P. Dev. Dyn.
2005, 232, 221.
3. Schlomann, U.; Rathke-Hartlieb, S.; Yamamoto, S.; Jockusch, H.; Bartsch, J. W. J.
Neurosci. 2000, 20, 7964.
4. King, N. E.; Zimmermann, N.; Pope, S. M.; Fulkerson, P. C.; Nikolaidis, N. M.;
Mishra, A.; Witte, D. P.; Rothenberg, M. E. Am. J. Respir. Cell Mol. Biol. 2004, 31,
257.
5. Fourie, A. M.; Coles, F.; Moreno, V.; Karlsson, L. J. Biol. Chem. 2003, 278, 30469.
6. (a) Ishikawa, N.; Daigo, Y.; Yasui, W.; Inai, K.; Nishimura, H.; Tsuchiya, E.;
Kohno, N.; Nakamura, Y. Clin. Cancer Res. 2004, 10, 8363; (b) Roemer, A.;
Schwettmann, L.; Jung, M.; Stephan, C.; Roigas, J. A. N.; Kristiansen, G.; Loening,
S. A.; Lichtinghagen, R.; Jung, K. J. Urol. 2004, 172, 2162.
7. Wildeboer, D.; Naus, S.; Sang, Q.-X. A.; Bartsch, J. W.; Pagenstecher, A. J.
Neuropathol. Exp. Neurol. 2006, 65, 516.
8. Moss, M. L.; Bartsch, J. W. Biochemistry 2004, 43, 7227.
9. Schlomann, U.; Wildeboer, D.; Webster, A.; Antropova, O.; Zeuschner, D.;
Knight, C. D.; Docherty, A. J.; Lambert, M.; Skelton, L.; Jockusch, H.; Bartsch, J.
W. J. Biol. Chem. 2002, 277, 48210.
10. Reiss, K.; Saftig, P. Semin. Cell Dev. Biol. 2009, 20, 126.
11. (a) Rothenberg, M. L.; Nelson, M. R.; Hande, K. R. Stem Cells 1999, 17, 237; (b)
Sparano, J. A.; Bernardo, P.; Stephenson, P.; Gradishar, W. J.; Ingle, J. N.; Zucker,
S.; Davidson, N. E. J. Clin. Oncol. 2004, 22, 4683.
12. Bartsch, J.; Koller, G. Protease Inhibition. WO Patent 2,009,047,523, 2009.
13. Schlomann, U.; Koller, G.; Conrad, C.; Ferdous, T.; Golfi, P.; Garcia, A. M.;
Höfling, S.; Parsons, M.; Costa, P.; Soper, R.; Bossard, M.; Hagemann, T.;
Roshani, R.; Sewald, N.; Ketchem, R. R.; Moss, M. L.; Rasmussen, F. H.; Miller, M.
A.; Lauffenburger, D. A.; Tuveson, D. A.; Nimsky, C.; Bartsch, J. W. Nat. Commun.
2015, 6, 6175. http://dx.doi.org/10.1038/ncomms7175 (in press).
14. Weisshoff, H.; Prasang, C.; Henklein, P.; Frommel, C.; Zschunke, A.; Mugge, C.
Eur. J. Biochem. 1999, 259, 776.
15. Egner, B. J.; Cardno, M.; Bradley, M. J. Chem. Soc., Chem. Commun. 1995, 21,
2163.
16. Chan, W. C.; White, P. D. In Fmoc Solid Phase Peptide Synthesis; Oxford
University Press, 2000; pp 62–63.
17. Kaur, H.; Heapy, A. M.; Brimble, M. A. Synlett 2012, 23, 2284.
18. Coulson, D. R.; Satek, L. C.; Grim, S. O. In Inorg. Synth; Cotton, F. A., Ed.; John
Wiley & Sons, 2007; pp 121–124.
3.3.17. Cyclo(R⁄LhSbKDK) R⁄ = citrulline (17)
Peptide 17 (0.9 mg, 0.6%) was obtained as a white solid in >99%
purity according to analytical RP-HPLC. Rt = 7.8 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
742.4; Found 743.5.
3.3.18. Cyclo(R⁄LhSbKDK) R⁄ = homoarginine (18)
Peptide 18 (0.8 mg, 0.5%) was obtained as a white solid in >95%
purity according to analytical RP-HPLC. Rt = 6.4 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
756.5; Found 756.1.
3.3.19. Cyclo(R⁄LhSbKDK) R⁄ = homoleucine (19)
Peptide 19 (2.3 mg, 2%) was obtained as a white solid in >95%
purity according to analytical RP-HPLC. Rt = 7.2 min (XTerraÒ C18,
5–95% B, 3% B/min, 1.0 mL/min); m/z (ESI-MS): [M+H]+ Calcd
755.5; Found 756.1.
3.4. Inhibition of ADAM8-mediated CD23 release from cells by
cyclic peptides
The ability of cyclic peptides to inhibit ADAM8-mediated shed-
ding of CD23 from COS7 cells was measured using an ELISA as
described.13 COS7 cells co-transfected with CD23 and ADAM8
cDNA were seeded out in 12-well plates at a density of 2 ꢁ 105 -
cells/well with or without peptides (range between 10 nM and
10 lM) for 6 h. After 6 h, the amounts of CD23 released in the
supernatants were determined using a commercial CD23 ELISA
19. Harris, P. W. R.; Brimble, M. A. Synthesis 2009, 20, 3460.
(R&D Systems, UK). The standards were prepared as eight serial
Please cite this article in press as: Yim, V.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.042