Development of a diketopiperazine-forming dipeptidyl Gly-Pro spacer for preparation of an antibody-drug conjugate

Article abstract of DOI:10.1039/c3md00075c

We developed a novel diketopiperazine-forming dipeptidyl spacer aimed at application in antibody-drug conjugates. Enzymatic cleavage of a peptide linked to the Gly-Pro spacer resulted in formation of diketopiperazine, which was stable and non-toxic, and release of the parent drug. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3md00075c

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Development of a diketopiperazine-forming dipeptidyl
Gly-Pro spacer for preparation of an antibody–drug
conjugate†
Cite this: Med. Chem. Commun., 2013,
4, 792
Shino Manabe, Hikaru Machida,b Yoshiyuki Aihara,a Masahiro Yasunaga,b
a
*
a
b
*
Yukishige Ito and Yasuhiro Matsumura
Received 5th March 2013
Accepted 5th March 2013
We developed a novel diketopiperazine-forming dipeptidyl spacer aimed at application in antibody–drug
conjugates. Enzymatic cleavage of a peptide linked to the Gly-Pro spacer resulted in formation of
diketopiperazine, which was stable and non-toxic, and release of the parent drug.
DOI: 10.1039/c3md00075c
www.rsc.org/medchemcomm
may interact with cellular nucleophiles, such as glutathione, a thiol
(Scheme 1).12 Some endopeptidases, such as prostate-specic
Introduction
Antibody–drug conjugates (ADCs) play an increasingly impor-
tant role in selective delivery of potent drugs to their intended
site of action in the body.1–3 There is a strong demand for effi-
cient ADC molecule design, especially for cancer chemotherapy,
because most chemotherapeutic medicines have a narrow safety
margin, resulting in severe adverse effects. Recently, several
ADCs have been developed, some of which have appeared
promising in clinical trials.4,5 An ADC consists of 3 components:
a monoclonal antibody that targets a tumor antigen, a cytotoxic
agent, and a linker (Fig. 1). The antibody is a pilot molecule for
delivery of an anti-cancer agent. The linker is further divided
into a specier and a spacer. The linker moiety is located
between the parent drug and the antibody and increases the rate
of enzymatic cleavage. The specier serves as a substrate for an
enzyme with site-specic activity. In the absence of a spacer,
enzymatic cleavage of the bond between the specier and the
parent drug may sometimes be inadequate, because of steric
hindrance caused by the parent drug.6,7 Once the bond between
the carrier and the linker is cleaved, the spacer should sponta-
neously release the drug in its active form. In our development
of ADCs,8–10 we focused on a novel linker development.
antigen, only cleave the amide bonds between amino acids;
therefore, the carbamate site in the p-aminobenzyloxycarbonyl
spacer is sometimes not cleaved by endopeptidases.13–15
In this study, we designed a novel spacer for use in a versatile
ADC strategy. The spacer consists of a proline-glycine (Gly-Pro)
dipeptide, and easily formed diketopiperazine by cyclization.
The rate of diketopiperazine formation depends on the
proportion of cis/trans amide isomers of the dipeptide.
Although most peptide sequences adopt only the energetically
favorable trans conformation, proline is unique in that it has a
cis-amide conformation.16,17 Thus, proline greatly enhances the
rate of diketopiperazine formation because of its great
propensity to adopt the cis-amide conformation.
The most widely used spacer is a p-aminobenzyloxycarbonyl
spacer.11 Once the amidebondbetween the specierandthe spacer
has been cleaved, the spacer degrades in a self-immolative manner,
and the parent drug is released. During degradation, a highly
reactive iminoquinone methide intermediate is formed, which
aRIKEN, Advanced Science Institute, Synthetic Cellular Chemistry Laboratory,
Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: smanabe@riken.jp; Fax: +81 48
462 4680; Tel: +81 48 467 9432
bInvestigative Treatment Division, Research Center for Innovative Oncology, National
Cancer Center Hospital East, Kashiwa, Chiba 277-0882, Japan. E-mail: yhmatsum@
east.ncc.go.jp; Fax: +81 4713 6857; Tel: +81 4713 6857
† Electronic supplementary information (ESI) available: Experimental details and
1H-NMR and 13C-NMR spectra. See DOI: 10.1039/c3md00075c
Fig. 1 Components of antibody–drug conjugates.
792 | Med. Chem. Commun., 2013, 4, 792–796
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sequence” for diketopiperazine formation during peptide
synthesis.18–22 Once an enzyme cleaves the peptide bond, the
Gly-Pro sequence forms diketopiperazine to release the parent
drug in its active form.23 The diketopiperazine thus formed is
normally stable and non-toxic (Scheme 2).
Using this novel spacer, we designed a plasmin-cleavable pro-
drug for chemotherapy. Plasmin, a serine protease, plays an
important role in tumor invasion and metastasis.24,25 The proteo-
lytically active form of plasmin may be localized around the tumor
tissue because it is formed from the inactive proenzyme plas-
minogen by the urokinase-type plasminogen activator produced
by cancer and/or stroma cells.7,26–30 Plasmin activity is very rapidly
inhibited by inhibitors, such as a2-anti-plasmin, present within
the blood circulation. Thus, plasmin is a very promising enzyme
for use in a tumor-specic prodrug approach. Since the recogni-
tion sequence for cleavage by plasmin is a Val-Leu-Lys tripeptide,
we synthesized paclitaxel–peptide conjugates 1 and 2 (Fig. 2).
ThesecompoundshaveaGly-Prospacer-containingpentapeptide;
unnatural (D)-proline was used for the linker in compound 2 in
order to investigate the inuence of stereochemistry on drug-
release activity. Inordertocomparethe efficacyofthe novel linker,
we synthesized compound 3, containing the commonly used
p-aminobenzyloxycarbonyl spacer, and compound 4, without a
spacer. We chose paclitaxel as the parent drug because it is one of
the most useful anti-cancer agents clinically. The linker was
attached to the 2-hydroxy group, because it is expected to decrease
cytotoxic activity by modication of the 2-hydroxy function-
ality.31–37 For an initial evaluation of linkers, an acetyl group was
chosen for the N-terminus of paclitaxel-conjugated peptides,
instead of using maleimide or bromoacetamide for immobiliza-
tion to the antibody, in order to avoid the instability of maleimide
or bromoacetamide during evaluation of drug-release.
Scheme 1 The p-aminobenzyloxycarbonyl spacer degradation mechanism and
formation of a highly reactive intermediate; X ¼ NH or O.
In order to minimize the steric hindrance at the enzymatic
cleavage site, Gly was chosen for the amino acid site near the
cleavage site. The Gly-Pro sequence is well known as a “difficult
Since paclitaxel is sensitive to both acidic and basic condi-
tions, the methoxytrityl (Mmt) group, which is an acid-labile
amino-protecting group,38 was chosen for protecting the side-
chain amino group of lysine. The synthesis was achieved as
shown in Scheme 3. The sequential synthesis of a pentapeptide
Scheme 2 Drug-release mechanism of the Gly-Pro spacer.
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Fig. 2 Synthesized peptide–paclitaxel conjugates.
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and the proline. The Ac-Val-Leu-Lys(Mmt)-Gly-Pro-OBn
sequence 10 was prepared in accordance with the Fmoc peptide
synthesis strategy. Aer removal of the benzyl ester39 of tetra-
peptide 10, (L)-proline was added to the C-terminus. Again, aer
the hydrogenolysis of the benzyl ester of pentapeptide 11, the
pentapeptide was attached to paclitaxel by water-soluble car-
bodiimide$hydrochloride (WSCDI$HCl) in the presence of 4-
(dimethylamino)pyridine (DMAP). Finally, the Mmt group was
removed without intact paclitaxel. The paclitaxel–peptide
conjugate 1 was obtained in 71%. Unnatural (D)-proline-con-
taining paclitaxel–peptide was prepared in a similar manner
with a 27% overall yield from tetrapeptide 10.
The less nucleophilic aminobenzyl alcohol 16 was intro-
duced using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinone
(EEDQ); then, the peptide was prepared from the C-termini in a
sequential manner (Scheme 4). Aer activation of the hydroxyl
Scheme 3 Synthesis of compounds 1 and 2.
from C-termini was difficult because of diketopiperazine
formation at the dipeptide Fmoc-Gly-Pro-OBn-deprotection
step. The pentapeptide is divided into 2 parts, the tetrapeptide
Scheme 4 Synthesis of p-aminobenzyloxy spacer-containing paclitaxel–peptide
conjugate.
Scheme 5 Synthesis of paclitaxel–tripeptide conjugate 4.
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Dr S. Daikoku in Glycotrilogy ERATO Project for assistance with
the MS measurement and to Professor Hironobu Hojo at Tokai
University for discussions.
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Fig. 3 Paclitaxel releasing ability of compounds 1–4 in the presence of plasmin.
group of the peptide by p-nitrophenylcarbonate, the peptide was
introduced at the 2-position of paclitaxel.
The tripeptide Ac-Val-Leu-Lys(Mmt)-OH 24 was conjugated
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appropriate peptide sequence. This strategy would be applicable
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