Synthesis and Biological Evaluation of RGD and isoDGR–Monomethyl Auristatin Conjugates Targeting Integrin αVβ3

Article abstract of DOI:10.1002/cmdc.201900049

This work reports the synthesis of a series of small-molecule–drug conjugates containing the α_Vβ₃-integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an “uncleavable” version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide–alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated α_vβ₃ receptor and were shown to retain nanomolar IC₅₀ values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with α_vβ₃ integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC₅₀ values in cell viability assays with both cancer cell lines tested (U87: 11.50±0.13 nm; M21: 6.94±0.09 nm) and is therefore a promising candidate for in vivo experiments.

Full text of DOI:10.1002/cmdc.201900049

Accepted Article
Title: " Synthesis and Biological Evaluation of RGD and isoDGR -
Monomethyl Auristatin Conjugates Targeting Integrin αVβ3
Authors: Umberto Piarulli, André Raposo Moreira Dias, lizeth Bodero,
Ana Martins, Daniela Arosio, Silvia Gazzola, Laura Belvisi,
Luca Pignataro, Christian Steinkühler, Alberto Dal Corso, and
Cesare Gennari
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To be cited as: ChemMedChem 10.1002/cmdc.201900049
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A Journal of
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10.1002/cmdc.201900049
ChemMedChem
A. Raposo Moreira Dias‡, L. Bodero‡, A.
Martins, D. Arosio, S. Gazzola, L.
Belvisi, L. Pignataro, C. Steinkühler, A.
Dal Corso, C. Gennari* and U. Piarulli*
Page No. – Page No.
Synthesis and Biological Evaluation
of RGD and isoDGR - Monomethyl
Auristatin Conjugates Targeting
Integrin αVβ3
One of eight: Out of the eight RGD and isoDGR - monomethyl Auristatin
conjugates synthesized, cyclo[DKP-isoDGR]-VA-MMAE has low nanomolar IC50
values in cell viability assays with both cancer cell lines tested (U87: 11.50 ± 0.13
nM; M21: 6.94 ± 0.09 nM).
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900049
ChemMedChem
Synthesis and Biological Evaluation of RGD and isoDGR -
Monomethyl Auristatin Conjugates Targeting Integrin αVβ3
André Raposo Moreira Dias,‡[a] Lizeth Bodero,‡[b] Ana Martins,[c] Daniela Arosio,[d] Silvia Gazzola,[b]
Laura Belvisi,[a,d] Luca Pignataro,[a] Christian Steinkühler,[c] Alberto Dal Corso,[a] Cesare Gennari*[a,d]
and Umberto Piarulli*[b]
Abstract: This work reports the synthesis of a series of small
molecule-drug conjugates containing the αVβ3-integrin ligand
cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable
Val-Ala (VA) linker or an “uncleavable” version devoid of this
sequence, and monomethyl Auristatin E (MMAE) or F (MMAF)
as cytotoxic agent. The conjugates were obtained via
a
straightforward synthetic scheme taking advantage of a copper-
catalyzed azide-alkyne cycloaddition as key-step. The
conjugates were tested for their binding affinity to the isolated
αvβ3 receptor, and shown to retain nanomolar IC50 values, in the
same range of the free ligands. The cytotoxic activity of the
conjugates was evaluated in cell viability assays with αvβ3
integrin over-expressing human glioblastoma (U87) and human
melanoma (M21) cells. The conjugates possess a markedly
lower cytotoxic activity compared to the free drugs, which is
consistent with an inefficient integrin-mediated internalization. In
almost all cases the conjugates featuring isoDGR as integrin
ligand exhibited higher potency than their RGD counterparts. In
particular, cyclo[DKP-isoDGR]-VA-MMAE conjugate has low
nanomolar IC50 values in cell viability assays with both cancer cell
lines tested (U87: 11.50 ± 0.13 nM; M21: 6.94 ± 0.09 nM) and is
therefore a promising candidate for in vivo experiments.
Figure 1. Molecular structures of monomethyl auristatin-E (1) and monomethyl
auristatin-F (2).
Due to their extremely high cytotoxicity, dolastatin analogs
cannot be used as drugs themselves and have been exploited
as optimal payloads for antibody-drug conjugates (ADCs) and
other tumor-targeting strategies.[ 2 ] ADCs have emerged as
powerful tools for limiting the main drawbacks of anticancer
drugs, such as severe side-effects and the insurgence of
multidrug resistance.[3] Indeed, among the four ADCs approved
and commercially available, brentuximab vedotin (AdcetrisTM
)
bears MMAE linked to an anti-CD30 monoclonal antibody via the
protease cleavable Val-Cit dipeptide linker.[4] MMAF differs from
MMAE for the presence of a charged C-terminal phenylalanine
residue, which limits its membrane permeability, attenuating off-
target toxicity.[2a] Although MMAF displays reduced cytotoxicity
compared to the more lipophilic MMAE, both drugs have shown
similar activity (IC50 values in the subnanomolar range) when
released intracellularly by ADCs.[2a, 5 ] Moreover, the different
physical-chemical properties of the two payloads were found to
have important implications in the relative mechanism of action
in vivo: cell-permeable MMAE is able to diffuse through
neighbouring cells, extending its toxic activity to other cells in the
tumor microenvironment (bystander-killing effect), while MMAF
is mostly retained inside the target cancer cell due to its poor
membrane permeability, resulting in increased intracellular
accumulation.[6]
Introduction
Monomethyl Auristatin E and F (MMAE and MMAF, 1 and 2 in
Figure 1) are synthetic analogs of dolastatin 10, a highly
cytotoxic pseudopeptide extracted from the sea hare Dolabella
auricularia. Similarly to the original natural product, these
analogs exert potent cytotoxic activity by strongly inhibiting
microtubule assembly and tubulin-dependent GTP hydrolysis,
resulting in cell cycle arrest and apoptosis.[1]
[a]
[b]
Dr. A. Raposo Moreira Dias, Prof. Dr. L. Belvisi, Dr. L. Pignataro,
Dr. A. Dal Corso, Prof. Dr. C. Gennari
Università degli Studi di Milano, Dipartimento di Chimica
Via C. Golgi, 19, 20133 Milan, Italy
E-mail: cesare.gennari@unimi.it
Dr. L. Bodero, Dr. S. Gazzola, Prof. Dr. U. Piarulli
Università degli Studi dell’Insubria, Dipartimento di Scienza e Alta
Tecnologia
In the last decades, low-molecular weight ligands (e.g. peptides,
peptidomimetics, aptamers, vitamins, and substrate analogs)
that can be easily accessed by chemical synthesis, have been
investigated as carriers for cytotoxic agents, aiming at
overcoming important limitations of ADCs, such as high
manufacturing costs, unfavorable pharmacokinetics (low tissue
diffusion and low accumulation rate) and possible
Via Valleggio, 11, 22100 Como, Italy
E-mail: umberto.piarulli@uninsubria.it
A. Martins, Dr. C. Steinkühler
[c]
[d]
Exiris Srl,
Via di Castel Romano, 100, 00128 Rome, Italy
Dr. D. Arosio
7
]
immunogenicity.[
Similarly to ADCs, the so-called small
CNR, Istituto di Scienze e Tecnologie Molecolari (ISTM)
Via C. Golgi, 19, 20133 Milan, Italy
molecule-drug conjugates (SMDCs) feature three fundamental
parts (i.e. ligand, cytotoxic drug and linker) and are generally
designed to deliver the payload in intracellular compartments of
* Corresponding author
‡Equal contributors
Supporting information for this article is given via a link at the end of
the document
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10.1002/cmdc.201900049
ChemMedChem
cancer cells (e.g. lysosomes), even though non-internalizing
SMDCs have also been developed.[8]
amanitin conjugates.[17] Cell viability assays indicated a slightly
increased cytotoxicity of the cyclo[DKP-isoDGR]-VA-α-amanitin
conjugate compared to the free drug.[17]
In order to further investigate the efficacy of our SMDCs, herein
we report the synthesis of novel conjugates bearing the αvβ3
integrin ligand 5 or 6 and MMAE or MMAF as payload (7-14,
Figure 3). We evaluated the binding affinity of conjugates 7-14
towards the isolated αvβ3 integrin receptor and their cytotoxic
activities on human glioblastoma (U87) and human melanoma
(M21) cell lines, which are known to over-express the αVβ3
integrin receptor.
Among the suitable targets for drug delivery, the transmembrane
αvβ3 integrin receptor is over-expressed on the cell surface of
various tumor types (such as melanoma, glioblastoma, renal cell
carcinoma, and tumors of lung, ovary, breast, prostate, and
colon),[9] where it is involved in different steps of tumor growth.
Since the discovery that endogenous ligands bind integrin αvβ3
through the tripeptide sequences Arg-Gly-Asp (RGD)[ 10 ] and
11
]
isoAsp-Gly-Arg
(isoDGR),[
many
peptides
and
peptidomimetics containing these motifs have been designed,
leading to high-affinity αvβ3 integrin ligands.[12 ]
Our research group entered this field by developing cyclic RGD
Results and Discussion
and isoDGR integrin ligands containing
a
bifunctional
diketopiperazine (DKP) scaffold.[13] Notably, the integrin ligands
cyclo[DKP-RGD] and cyclo[DKP-isoDGR] (3 and 4, Figure 2)
displayed low-nanomolar IC50 values in competitive binding
assays against the purified αVβ3 receptor as well as a marked
selectivity towards the αVβ3 receptor compared to αVβ5.[13d]
Synthesis of conjugates 7 – 14
Conjugates 7-14 (Figure 3), including two cyclo[DKP-RGD]-
MMAE (7 and 9), two cyclo[DKP-isoDGR]-MMAE (8 and 10), two
cyclo[DKP-RGD]-MMAF (11 and 13) and two cyclo[DKP-
isoDGR]-MMAF (12 and 14), were synthesized as discussed
below. In these conjugates, the integrin ligands are bound to
MMAE and MMAF payloads via: (i) a p-aminobenzylcarbamate
(PABC) self-immolative spacer and a lysosomally cleavable VA
linker or an “uncleavable” version devoid of this sequence, (ii) a
triazole linkage, and (iii) a PEG-4 spacer. The synthesis of
cyclo[DKP-RGD]-MMAE/MMAF (7, 9, 11 and 13) and
cyclo[DKP-isoDGR]-MMAE/MMAF (8, 10, 12 and 14) conjugates
was performed through the linear synthetic strategy shown in
Scheme 1A/B. The Fmoc-protected VA-p-aminobenzyl alcohol
(15) was synthesized according to
a previously reported
procedure.[15] Upon Fmoc removal from 15, the resulting free
amine was treated with 4-pentynoic acid, leading to compound
16 in high yield. The latter was then converted into the
corresponding 4-nitrophenyl carbonate (17). The secondary
amine of MMAE and MMAF was derivatized with the linker by
treatment with 4-nitrophenyl carbonate 17, leading to
carbamates 18 and 19. The final conjugates 7-8 and 11-12 were
obtained through a copper-catalyzed azide-alkyne cycloaddition
(CuAAC)[18 ] between alkynes 18-19 and the azide-functionalized
ligands cyclo[DKP-RGD]-PEG-4-N3 20[15] and cyclo[DKP-
isoDGR]-PEG-4-N3 21.[17]
Figure 2. A: ligand cyclo[DKP-RGD] (3) and its functionalized version (5); B:
ligand cyclo[DKP-isoDGR] (4) and its functionalized version (6).
Later on, compounds 3 and 4 were functionalized with a
benzylamine moiety (5 and 6, Figure 2) allowing their
conjugation to different bioactive molecules.[14,15,16 ] Conjugates
of the functionalized integrin ligands 5 and 6 with the antimitotic
drug paclitaxel (PTX) were synthesized featuring
a self-
immolative spacer and the lysosomally-cleavable Val-Ala (VA)
linker.[15,16] More recently, ligands 5 and 6 were bound to the
RNA polymerase II inhibitor α-amanitin via a 6’-ether, giving
cyclo[DKP-RGD]-VA-α-amanitin and cyclo[DKP-isoDGR]-VA-α-
Figure 3. MMAE/MMAF conjugates: cyclo[DKP-RGD]-VA-MMAE (7), cyclo[DKP-isoDGR]-VA-MMAE (8), cyclo[DKP-RGD]-Unc-MMAE (9), cyclo[DKP-isoDGR]-
Unc-MMAE (10), cyclo[DKP-RGD]-VA-MMAF (11), cyclo[DKP-isoDGR]-VA-MMAF (12), cyclo[DKP-RGD]-Unc-MMAF (13), cyclo[DKP-isoDGR]-Unc-MMAF (14).
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10.1002/cmdc.201900049
ChemMedChem
For the preparation of conjugates bearing an uncleavable linker
(9-10 and 13-14, Scheme 1B), 4-pentynoic acid was reacted
with the secondary amine of MMAE and MMAF affording tertiary
amides 22 and 23 in good yields. Then, the CuAAC with
functionalized ligands 20 and 21 afforded the uncleavable
conjugates 9-10 and 13-14. All final compounds were purified by
semipreparative HPLC and lyophilized before biological assays.
Table 1. Inhibition of biotinylated vitronectin binding to the isolated αVβ3
receptor.
Compound
Structure
IC50 (nM)[a]
3
4
cyclo[DKP-RGD]
4.5 ± 1.1
9.2 ± 1.1
cyclo[DKP-isoDGR]
7
cyclo[DKP-RGD]-VA-MMAE
cyclo[DKP-isoDGR]-VA-MMAE
cyclo[DKP-RGD]-Unc-MMAE
cyclo[DKP-isoDGR]-Unc-MMAE
cyclo[DKP-RGD]-VA-MMAF
cyclo[DKP-isoDGR]-VA-MMAF
cyclo[DKP-RGD]-Unc-MMAF
cyclo[DKP-isoDGR]-Unc-MMAF
58.5 ± 10.5
36.2 ± 0.2
40.0 ± 16.1
14.5 ± 0.6
57.8 ± 26.0
43.9 ± 2.1
30.2 ± 12.9
10.7 ± 2.8
8
αVβ3 Integrin receptor competitive binding assays
9
Conjugates 7-14 were evaluated for their ability to inhibit
biotinylated vitronectin binding to the isolated αVβ3 receptor. The
competitive binding assay was performed by incubating the
immobilized integrin receptor with solutions of the cyclo[DKP-
RGD]-MMAE/MMAF (7, 9, 11, and 13), cyclo[DKP-isoDGR]-
MMAE/MMAF (8, 10, 12 and 14) conjugates at different
concentrations (10-12 -10-5 M) in the presence of biotinylated
vitronectin (1 µg mL-1) and measuring bound vitronectin. The
calculated half-maximal inhibitory concentrations (IC50) listed in
Table 1 demonstrate that conjugates 7-14 retain good binding
affinity for integrin αvβ3, with IC50 values in the nanomolar range,
similar or only slightly worse compared to the free ligands 3 and
4.
10
11
12
13
14
[a] IC50 values were calculated as the concentration of compound required
for 50% inhibition of biotinylated vitronectin binding to the αvβ3 receptor, as
estimated by GraphPad Prism software. All values are the arithmetic mean
± the standard deviation (SD) of duplicate determinations.
Scheme 1. Synthesis of conjugates 7-14. Reagents and conditions: a) 1) piperidine, DMF, RT, 2 h; 2) 4-pentynoic acid, HATU, HOAt, iPr2NEt, DMF, RT,
overnight; b) 4-nitrophenyl chloroformate, pyridine, RT, THF, 2 h; c) MMAE, HOBt, iPr2NEt, RT, DMF:pyridine (4:1), 65 h; d) MMAF·HCl, HOBt, iPr2NEt, RT,
DMF:pyridine (4:1), 65 h; e) (αvβ3-integrin ligand)-PEG-4-N3, CuSO4∙5H2O, NaAsc, DMF/H2O (1:1), 35 ºC, overnight; f) 4-pentynoic acid, MMAE, HATU, HOAt,
iPr2NEt, DMF, RT, overnight; g) 4-pentynoic acid, MMAF·HCl, HATU, HOAt, iPr2NEt, DMF, RT, overnight. HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HOAt
=
1-hydroxy-7-azabenzotriazole; HOBt
=
1-hydroxybenzotriazole; iPr2NEt
=
N,N-
diisopropylethylamine; RT = room temperature; NaAsc = sodium ascorbate.
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10.1002/cmdc.201900049
ChemMedChem
Cell viability assays
Finally, our data show that in almost all cases the compounds
featuring cyclo[DKP-isoDGR] as integrin ligand exhibited higher
potency than their analogs equipped with cyclo[DKP-RGD]. This
is evident when comparing cyclo[DKP-isoDGR]-VA-MMAE (8)
and cyclo[DKP-RGD]-VA-MMAE (7) both in U87 and M21
cancer cell lines (3.4-fold and 7.9-fold more potent, respectively).
The antiproliferative activity of compounds 7-14 was evaluated
against αvβ3 integrin over-expressing human glioblastoma (U87)
and human melanoma (M21) cells.[19,20] The cells were treated
for 72 hours with increasing doses of free MMAE (1), MMAF (2)
and MMAE/MMAF conjugates 7-14 and the cell viability was
then evaluated by MTT assay. The calculated IC50 values are
shown in Table 2.
Conclusions
Conjugation of anticancer drugs to αvβ3 integrin ligands
Table 2. Evaluation of anti-proliferative activity of free MMAE (1) and MMAF
(2), MMAE/MMAF conjugates 7-14 in U87 and M21 cancer cell lines after 72h
incubation.
represents
a
promising approach in tumor-targeted
chemotherapy. In this work, we synthesized eight conjugates 7-
14 bearing the αVβ3-integrin ligands cyclo[DKP-RGD] 3 or
cyclo[DKP-isoDGR] 4 and the potent tubulin polymerization
inhibitor MMAE (1) or MMAF (2), widely investigated as
payloads in antibody-drug conjugates (ADCs). Conjugates 7, 8
and 11, 12 contain the lysosomally cleavable VA linker, whereas
conjugates 9, 10 and 13, 14 present an uncleavable linker.
Conjugates 7-14 were found to inhibit the biotinylated vitronectin
binding to the purified αvβ3 receptor in nanomolar concentrations,
retaining the excellent integrin binding affinity of the free ligands.
In addition, conjugates 7-14 were evaluated in vitro for their
activity against the αvβ3 over-expressing U87 and M21 cancer
cell lines. Overall, the cell viability data shown in Table 2 indicate
that the conjugates bearing both peptidomimetic ligands
IC50 (nM)[a]
IC50 (nM)[a]
Compound
U87
M21
Free Drugs
MMAE (1)
MMAF (2)
0.076 ± 0.08
94.40 ± 0.06
0.065 ± 0.088
> 1000
Conjugates with cyclo[DKP-RGD]
cyclo[DKP-RGD]-VA-MMAE (7)
38.99 ± 0.11
> 1000
55.19 ± 0.06
320.6 ± 0.10
> 1000
cyclo[DKP-RGD]-Unc-MMAE (9)
cyclo[DKP-RGD]-VA-MMAF (11)
cyclo[DKP-RGD]-Unc-MMAF (13)
Conjugates with cyclo[DKP-isoDGR]
cyclo[DKP-isoDGR]-VA-MMAE (8)
cyclo[DKP-isoDGR]-Unc-MMAE (10)
cyclo[DKP-isoDGR]-VA-MMAF (12)
cyclo[DKP-isoDGR]-Unc-MMAF (14)
385.90 ± 0.09
> 5000
cyclo[DKP-RGD] and cyclo[DKP-isoDGR] possess
a lower
> 1000
anticancer activity compared to the free drugs, which is
consistent with an inefficient integrin-mediated internalization of
the conjugates by the targeted cancer cell.
11.50 ± 0.13
685.50 ± 0.08
165.90 ± 0.05
763.70 ± 0.08
6.94 ± 0.09
399.8 ± 0.08
936.2 ± 0.07
> 1000
However, the isoDGR-bearing conjugates display a systematic
higher anticancer activity compared to the RGD counterparts,
suggesting the possibility, which will be explored in future
experiments, that the cyclo[DKP-isoDGR] is able to promote a
more significant internalization with respect to the cyclo[DKP-
RGD] ligand. In particular, the cyclo[DKP-isoDGR]-VA-MMAE
conjugate (8) shows a higher activity than cyclo[DKP-RGD]-VA-
MMAE (7) upon incubation with both U87 and M21 cancer cell
lines (3.4- and 7.9-fold, respectively). The low nanomolar IC50
values in both cancer cell lines (U87: 11.50 ± 0.13 nM; M21: 6.94
± 0.09 nM) suggest that the cyclo[DKP-isoDGR]-VA-MMAE
conjugate (8) is a promising candidate for in vivo experiments,
which will be important to obtain evidences of conjugate
accumulation at the tumor site. Further studies will include the
use of extracellularly cleavable linkers to exploit the passive
diffusion properties of MMAE. Indeed, the latter is an emerging
strategy in the drug delivery field, supported by recent evidences
of promising anticancer activity of non-internalizing ADCs[22] and
SMDCs.[8e,23]
[a] IC50 values were calculated as reported in the Supporting Information, from
viability curves using GraphPad Prism software. All values are the arithmetic
mean ± the standard deviation (SD) of triplicate determinations.
Under these experimental conditions, MMAE (1) proved to be
much more potent than MMAF (2), confirming literature data.[1,2a]
In general, the antiproliferative activity of the integrin-targeted
conjugates was found to be lower than the parent free drugs. In
the case of the MMAE conjugates with uncleavable linker (9 and
10), the loss of potency is greater than three orders of
magnitude (≥ 5 x 103). When the linker is cleavable (7 and 8),
the potency is still reduced, but to a lesser extent, with the
cyclo[DKP-isoDGR]-VA-MMAE conjugate
8
showing low
nanomolar IC50 values, only two orders of magnitude worse than
the free drug.
Antibody-MMAF conjugates display IC50 values in the
subnanomolar range because of assisted internalization.
Moreover, antibody-MMAF (and not MMAE) conjugates tolerate
Experimental Section
MMAE and MMAF drugs are commercially available from Levena
Biopharma (USA) and Oskar Tropitzsch GmbH (Germany). Ligands
cyclo[DKP-RGD] (3) and cyclo[DKP-isoDGR] (4), their functionalized
analogs 5 and 6, Fmoc-Val-Ala-PABA (15) and azide-PEG-4-ligands 20
and 21 were prepared according to the cited literature. The synthetic
procedures for the preparation of compounds 7–14 are reported in the
Supporting Information, along with the 1H NMR and 13C NMR spectra,
the HPLC traces and HRMS spectra. The inhibition assays of biotinylated
vitronectin binding to the αvβ3 receptor for compounds 7-14 are reported
21 ]
non-cleavable linkers coupled to the drug N-terminus.[2a,
These effects are clearly absent in Table 2, where the IC50
values of the integrin ligand-MMAF conjugates are at best in the
submicromolar range. These results are consistent with the
observation that the cyclo[DKP-RGD] ligand accumulates on the
cell membrane of αVβ3 integrin-expressing cancer cells, while it
is poorly internalized by receptor-mediated endocytosis.[8e]
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10.1002/cmdc.201900049
ChemMedChem
in the Supporting Information. Also, the cell antiproliferative studies on
U87 and M21 cell lines are described in the Supporting Information.
L. Belvisi, L. Pignataro, A. Dal Corso, C. Gennari, Chem. Eur. J.
2019, 25, 1696-16700.
Acknowledgements
[9] J. S. Desgrosellier, D. A. Cheresh, Nat. Rev. Cancer 2010, 10, 9-
22; b) F. Danhier, A. Le Breton, V. Préat, Mol. Pharm. 2012, 9, 2961-
2973; c) N. Franceschi, H. Hamidi, J. Alanko, P. Sahgal, J. Ivaska, J.
Cell Sci. 2015, 128, 839-852.
We thank the European Commission (Marie Skłodowska-Curie
ITN MAGICBULLET 642004) for PhD fellowships (to A.R.M.D.,
L. B. and A. M.) and financial support. We also gratefully
acknowledge Ministero dell’Università e della Ricerca (PRIN
2015 project 20157WW5EH) for financial support.
[10] M. D. Pierschbacher, E. Ruoslahti, Nature 1984, 309, 30-33.
[11] Biochemical studies have shown that a spontaneous post-
translational modification, occurring at the Asn-Gly-Arg (NGR) motif
of the extracellular matrix protein fibronectin, leads to the isoAsp-
Gly-Arg (isoDGR) sequence, see: F. Curnis, R. Longhi, L. Crippa, A.
Cattaneo, E. Dondossola, A. Bachi, A. Corti, J. Biol. Chem. 2006,
281, 36466-36476; b) F. Curnis, A. Sacchi, A. Gasparri, R. Longhi,
A. Bachi, C. Doglioni, C. Bordignon, C. Traversari, G.-P. Rizzardi, A.
Corti, Cancer Res. 2008, 68, 7073-7082; c) A. Corti, F. Curnis, J.
Cell Sci. 2011, 124, 515-522; d) M. Ghitti, A. Spitaleri, B. Valentinis,
S. Mari, C. Asperti, C. Traversari, G. P. Rizzardi, G. Musco, Angew.
Chem. Int. Ed. 2012, 51, 7702-7705; Angew. Chem. 2012, 124,
7822–7825.
Keywords: antitumor agents • peptidomimetics • drug delivery •
integrins • auristatins
References
[1] a) G. R. Pettit, Y. Kamano, C. L. Herald, A. A. Tuinman, F. E.
Boettner, H. Kizu, J. M. Schmidt, L. Baczynskyj, K. B. Tomer, R. J.
Bontems, J. Am. Chem. Soc. 1987, 109, 6883-6885; b) R. Bai, G. R.
Pettit, E. Hamel, Biochem. Pharmacol. 1990, 40, 1859-1864.
[12] a) K.-E. Gottschalk, H. Kessler, Angew. Chem. Int. Ed. 2002,
41, 3767-3774; Angew. Chem. 2002, 114, 3919–3927; b) L. Auzzas,
F. Zanardi, L. Battistini, P. Burreddu, P. Carta, G. Rassu, C. Curti, G
. Casiraghi, Curr. Med. Chem. 2010, 17, 1255-1299; c) T. G. Kapp,
F. Rechenmacher, S. Neubauer, O. V. Maltsev, E. A. Cavalcanti-
Adam, R. Zarka, U. Reuning, J. Notni, H.-J. Wester, C. Mas-Moruno,
J. Spatz, B. Geiger, H. Kessler, Sci. Rep. 2017, 7, 39805.
[2] a) S. O. Doronina, B. A. Mendelsohn, T. D. Bovee, C. G.
Cerveny, S. C. Alley, D. L. Meyer, E. Oflazoglu, B. E. Toki, R. J.
Sanderson, R. F. Zabinski, A. F. Wahl, P. D. Senter, Bioconjugate
Chem. 2006, 17, 114-124; b) J. A. Francisco, C. G. Cerveny, D. L.
Meyer, B. J. Mixan, K. Klussman, D. F. Chace, S. X. Rejniak, K. A.
Gordon, R. DeBlanc, B. E. Toki, C.-L. Law, S. O. Doronina, C. B.
Siegall, P. D. Senter, A. F. Wahl, Blood 2003, 102, 1458-1465; c) S.
O. Doronina, B. E. Toki, M. Y. Torgov, B. A. Mendelsohn, C. G.
Cerveny, D. F. Chace, R. L. DeBlanc, R. P. Gearing, T. D. Bovee, C.
B. Siegall, Nat. Biotechnol. 2003, 21, 778-784; d) S. C. Jeffrey, J. B.
Andreyka, S. X. Bernhardt, K. M. Kissler, T. Kline, J. S. Lenox, R. F.
Moser, M. T. Nguyen, N. M. Okeley, I. J. Stone, Bioconjugate Chem.
2006, 17, 831-840; e) S. O. Doronina, T. D. Bovee, D. W. Meyer, J.
B. Miyamoto, M. E. Anderson, C. A. Morris-Tilden, P. D. Senter,
Bioconjugate Chem. 2008, 19, 1960-1963; f) M. A. Fanale, Lancet
Oncol. 2017, 18, 1566-1568.
[13] a) A. S. M. da Ressurreição, A. Vidu, M. Civera, L. Belvisi, D.
Potenza, L. Manzoni, S. Ongeri, C. Gennari, U. Piarulli, Chem. Eur.
J. 2009, 15, 12184-12188; b) M. Marchini, M. Mingozzi, R. Colombo,
I. Guzzetti, L. Belvisi, F. Vasile, D. Potenza, U. Piarulli, D. Arosio, C.
Gennari, Chem. Eur. J. 2012, 18, 6195-6207; c) M. Mingozzi, A. Dal
Corso, M. Marchini, I. Guzzetti, M. Civera, U. Piarulli, D. Arosio, L.
Belvisi, D. Potenza, L. Pignataro,C. Gennari, Chem. Eur. J. 2013,
19, 3563-3567; d) S. Panzeri, S. Zanella, D. Arosio, L. Vahdati, A.
Dal Corso, L. Pignataro, M. Paolillo, S. Schinelli, L. Belvisi, C.
Gennari, U. Piarulli, Chem. Eur. J. 2015, 21, 6265-6271.
[14] a) R. Colombo, M. Mingozzi, L. Belvisi, D. Arosio, U. Piarulli, N.
Carenini, P. Perego, N. Zaffaroni, M. De Cesare, V. Castiglioni, E.
Scanziani, C. Gennari, J. Med. Chem. 2012, 55, 10460-10474; b) M.
Mingozzi, L. Manzoni, D. Arosio, A. Dal Corso, M. Manzotti, F.
Innamorati, L. Pignataro, D. Lecis, D. Delia, P. Seneci, C. Gennari,
Org. Biomol. Chem. 2014, 12, 3288-3302.
[3] S. Agnello, M. Brand, M. F. Chellat, S. Gazzola, R. Riedl, Angew.
Chem. Int. Ed., 2018, DOI: 10.1002/anie.201802416; Angew.
Chem. 2018, DOI: 10.1002/ange.201802416.
[4] J. Katz, J. E. Janik, A. Younes, Clin. Cancer Res. 2011, 17, 6428-
6436.
[15] a) A. Dal Corso, M. Caruso, L. Belvisi, D. Arosio, U. Piarulli, C.
Albanese, F. Gasparri, A. Marsiglio, F. Sola, S. Troiani, B. Valsasina,
L. Pignataro, D. Donati, C. Gennari, Chem. Eur. J. 2015, 21, 6921-
6929; b) S. Zanella, M. Mingozzi, A. Dal Corso, R. Fanelli, D. Arosio,
M. Cosentino, L. Schembri, F. Marino, M. De Zotti, F. Formaggio, L.
Pignataro, L. Belvisi, U. Piarulli, C. Gennari, ChemistryOpen 2015, 4,
633-641; c) A. Pina, A. Dal Corso, M. Caruso, L. Belvisi, D. Arosio,
S. Zanella, F. Gasparri, C. Albanese, U. Cucchi, I. Fraietta, A.
Marsiglio, L. Pignataro, D. Donati, C. Gennari, ChemistrySelect
2017, 2, 4759-4766; d) A. Raposo Moreira Dias, A. Pina, A. Dal
Corso, D. Arosio, L. Belvisi, L. Pignataro, M. Caruso, C. Gennari,
Chem. Eur. J. 2017, 23, 14410-14415; e) P. L. Rivas, I. Ranđelović,
A. Raposo Moreira Dias, A. Pina, D. Arosio, J. Tóvári, G. Mező, A.
Dal Corso, L. Pignataro, C. Gennari, Eur. J. Org. Chem. 2018, 2902-
2909.
[5] a) M. S. Sutherland, R. J. Sanderson, K. A. Gordon, J. Andreyka,
C. G. Cerveny, C. Yu, T. S. Lewis, D. L. Meyer, R. F. Zabinski, S. O.
Doronina, P. D. Senter, C. L. Law, A. F. Wahl, J. Biol. Chem. 2006,
281, 10540-10547. b) Y. Wang, S. Fan, W. Zhong, X. Zhou, S. Li,
Int. J. Mol. Sci. 2017, 18, 1860.
[6] a) F. Li, K. K. Emmerton, M. Jonas, X. Zhang, J. B. Miyamoto, J.
R. Setter, N. D. Nicholas, N. M. Okeley, R. P. Lyon, D. R. Benjamin,
C. L. Law, Cancer Res. 2016, 76, 2710-2719; b) Y. V. Kovtun, V. S.
Goldmacher, Cancer Lett. 2007, 255, 232-240; c) K. Temming, D. L.
Meyer, R. Zabinski, P. D. Senter, K. Poelstra, G. Molema,R. J. Kok,
Mol. Pharm. 2007, 4, 686-694.
[7] S. Cazzamalli, A. Dal Corso, F. Widmayer, D. Neri, J. Am. Chem.
Soc. 2018, 140, 5, 1617-1621.
[8] a) N. Krall, J. Scheuermann, D. Neri, Angew. Chem. Int. Ed.
2013, 52, 1384-1402; Angew. Chem. 2013, 125, 1424-1443; b) G.
Casi, D. Neri, J. Med. Chem. 2015, 58, 8751-8761; c) M.
Srinivasarao.; C. V. Galliford; P. S. Low, Nat. Rev. Drug Discov.
2015, 14, 203–219. d) M. Srinivasarao, P. S. Low, Chem. Rev. 2017,
117, 12133-12164; e) A. Raposo Moreira Dias, A. Pina, A. Dean, H.-
G. Lerchen, M. Caruso, F. Gasparri, I. Fraietta, S. Troiani; D. Arosio,
[16] S. Zanella, S. Angerani, A. Pina, P. López Rivas, C. Giannini, S.
Panzeri, D. Arosio, M. Caruso, F. Gasparri, I. Fraietta, C. Albanese,
A. Marsiglio, L. Pignataro, L. Belvisi, U. Piarulli, C. Gennari, Chem.
Eur. J. 2017, 23, 7910-7914.
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900049
ChemMedChem
[17] L. Bodero, P. López Rivas, B. Korsak, T. Hechler, A. Pahl, C.
Müller, D. Arosio, L. Pignataro, C. Gennari, U. Piarulli, Beilstein J.
Org. Chem. 2018, 14, 407-415.
[18] a) 1,3-Dipolar Cycloadditions Chemistry, R. Huisgen, Ed., Wiley
(New York), 1984, Vol. 1, pp. 1-176; b) R. Huisgen, Pure Appl.
Chem. 1989, 61, 613-628; c) H. C. Kolb, M. G. Finn, K. B.
Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004-2021; Angew.
Chem. 2001, 113, 2056-2075; d) H. C. Kolb, K. B. Sharpless, Drug
Discov. Today 2003, 8, 1128-1137.
[19] a) S. L. Goodman, H. J. Grote, C. Wilm, Biol. Open 2012, 1,
329-340; b) N. V. Currier, S. E. Ackerman, J. R. Kintzing, R. Chen,
M. F. Interrante, A. Steiner, A. K. Sato, J. R. Cochran, Mol. Cancer
Ther. 2016, 15, 1291-1300.
[20] E. B. Voura, R. A. Ramjeesingh, A. M.P. Montgomery, C.-H.
Siu, Mol Biol Cell. 2001, 9, 2699-2710.
[21] B. P. Gray, L. Kelly, D. P. Ahrens, A. P. Barry, C. Kratschmer,
M. Levy, B. A. Sullenger, Proc. Natl. Acad. Sci. 2018, 115, 4761-
4766.
[ 22 ] a) M. Yasunaga, S. Manabe, D. Tarin, Y. Matsumura,
Bioconjugate Chem. 2011, 22, 1776-1783; b) E. Perrino, M. Steiner,
N. Krall, G. J. Bernardes, F. Pretto, G. Casi, D. Neri, Cancer Res.
2014, 74, 2569–2578; c) A. Dal Corso, R. Gébleux, P. Murer, A.
Soltermann, D. Neri, J. Control. Release 2017, 264, 211-218.
[23] S. Cazzamalli, B. Ziffels, F. Widmayer, P. Murer, G. Pellegrini,
F. Pretto, S. Wulhfard, D. Neri, Clin. Cancer Res. 2018, 24, 3656–
3667.
This article is protected by copyright. All rights reserved.