Modular Assembly of Reversible Multivalent Cancer-Cell-Targeting Drug Conjugates

Article abstract of DOI:10.1002/anie.201703492

Herein is described a new modular platform for the construction of cancer-cell-targeting drug conjugates. Tripodal boronate complexes featuring reversible covalent bonds were designed to accommodate a cytotoxic drug (bortezomib), poly(ethylene glycol) (Peg) chains, and folate targeting units. The B-complex core was assembled in one step, proved stable under biocompatible conditions, namely, in human plasma (half-life up to 60 h), and underwent disassembly in the presence of glutathione (GSH). Stimulus-responsive intracellular cargo delivery was confirmed by confocal fluorescence microscopy, and a mechanism for GSH-induced B-complex hydrolysis was proposed on the basis of mass spectrometry and DFT calculations. This platform enabled the modular construction of multivalent conjugates with high selectivity for folate-positive MDA-MB-231 cancer cells and IC₅₀ values in the nanomolar range.

Full text of DOI:10.1002/anie.201703492

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Modular Assembly of Reversible Multivalent Targeting Drug
Conjugates
Authors: Fábio M. F. Santos, Ana I. Matos, Ana E. Ventura, João
Gonçalves, Luís F. Veiros, Helena Florindo, and Pedro Miguel
pimenta Gois
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703492
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10.1002/anie.201703492
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COMMUNICATION
Modular Assembly of Reversible Multivalent Targeting Drug
Conjugates
Fábio M. F. Santos,[a] Ana I. Matos,[a] Ana E. Ventura,[a] João Gonçalves,[a] Luís F. Veiros,[b] Helena F.
Florindo,[a] Pedro M. P. Gois [a]*
Abstract: Here is described a new modular platform to construct
cancer cell targeting drug conjugates. Tripodal boronate complexes,
featuring reversible covalent bonds, were design to accommodate, a
cytotoxic drug (bortezomib), polyethylene glycol (Peg) chains and
folate targeting units. The B-complex core was assembled in one
step, and proved stable in different biocompatible conditions, namely
human plasma (half-life up to 60 h) and reversible in the presence of
glutathione (GSH). The stimulus responsive intracellular cargo
delivery was confirmed by confocal fluorescence microscopy and a
mechanism for GSH induced B-complex hydrolysis was proposed
based on mass spectrometry and DFT calculations. This platform
enabled the modular construction of multivalent conjugates
exhibiting high selectivity for folate positive MDA-MB-231 cancer
cells and IC50’s in the nanomolar range.
and the biomolecule, groups that may control the conjugate
physicochemical properties (e.g. Peg ) and cleavable units that
promote the stimulus responsive (e.g. enzymes, pH, GSH)
release of the cargo at the target.[4] Hence, linkers often exhibit
structures with a high functional density, and their construction
involves a series of complex and costly synthetic steps that are
typically unsuited for a straightforward structural diversification
(Scheme 1A).[4b,c] Therefore, the engineering of composite
multifunctional molecules that enable the tuning of the construct
properties by simple variation of the components individual
properties, are highly desirable for the expedient discovery of
new TDCs with therapeutic usefulness.[4b,c]
Boronic acids (BAs) readily establish reversible covalent bonds
with Schiff base ligands to yield boronate complexes (B-
complexes), featuring
a modular and reversible tripodal
framework.[5] Based on this, we conceive that if B-complexes
would display suitable properties of stability and controlled
reversibility in biological settings, they could be used as a
platform to design multifunctional constructs to selectively target
and deliver cargo to cancer cells (Scheme 1B).
Recent developments of human biology facilitate a more clear
understanding of the intricate pathogenesis of complex diseases
such as cancer.[1] The evolution of normal cells to a neoplastic
state is a multifaceted biological process in which normal cells
acquire capabilities of sustaining proliferative signalling, evading
growth suppressors, resisting cell death, enabling replicative
immortality, inducing angiogenesis, activating invasion and
metastasis.[1a] Therefore the most recent strategies to tackle
cancer, aim at interrupting one or more of these stages using
multifunctional constructs, in which, the biological activity of the
individual components is preserved and tuned depending on the
construct structure. Despite conceptually simple, the assembly
of multifunctional constructs is often hampered by the
overwhelming complexity of the synthetic process.[2]
Targeting drug conjugates (TDCs), like antibody (ADCs) and
small-molecules drug conjugates (SMDC), are multifunctional
constructs that combine the lethality of potent cytotoxic drugs
with the targeting ability of specific biomolecules that elicit a high
affinity for antigens overexpressed in cancer cells.[3] In these
conjugates, the linker technology used to connect both functions
contributes decisively for the therapeutic usefulness of these
constructs, by enabling the biomolecule functionalization without
altering its pharmacokinetic properties, by maintaining the
conjugate integrity in circulation and triggering the release of the
active drug only upon reaching the target.[4] Given these
requirements, the linker engineering often contemplates the
incorporation of reactive handles to selectively attach the drug
Scheme 1. (A) Linear construction of TDCs; (B) Modular and reversible
assembly of multifunctional TDCs promoted by boron.
To test this idea, we prepared several B-complexes based on a
one-pot
three-component
reaction
in
which
4-
methoxysalicylaldehyde and phenyl BA were reacted with L-
phenylalanine, anthranilic acid or 2-aminophenol in equimolar
amounts. This simple protocol, that requires no chromatographic
isolation step, afforded the corresponding complexes 1, 3 and 4
in yields up to 90 % (Scheme 2, see also sections 1.2-1.4 SI).
Once prepared, the stability of these cores was evaluated at
physiological pH 7.4 and compounds 1, 3 and 4 displayed half-
lifes lower than two hours in this media (Scheme 2, see also
sections 2.2, 2.4 and 2.5 SI). Inspection of the boronated cores
suggested that the poor stability of these molecules could be
due to a rapid hydrolysis promoted by the addition of water to
the electrophilic imine C-atom.[6] Consequently, to overcome this
excess of reactivity, analogous complexes were synthesized
starting from 2-hydroxy-4-methoxyacetophenone. This proved to
[a]
F. M. F. Santos, A. I. Matos, A. E. Ventura, Prof. J. B. Gonçalves,
Prof. H. F. Florindo, Prof. P. M. P. Gois
Research Institute for Medicines (iMed.ULisboa), Faculty of
Pharmacy, Universidade de Lisboa, Lisbon, Portugal.
E-mail: pedrogois@ff.ulisboa.pt
[b]
Prof. L. F. Veiros
Centro de Química Estrutural, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001, Lisboa
Portugal.
Supporting information for this article is given via a link at the end of
the document
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10.1002/anie.201703492
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be a slightly more challenging transformation and, despite our
many attempts, the preparation of the complex from anthranilic
acid was not achieved. Despite this, the introduction of a methyl
substituent on the imine C-atom of 2 and 5 (Scheme 2, see also
sections 1.2 and 1.4 SI) drastically improved their stability at pH
7.4, in particular B-complex 5, prepared from 2-aminophenol,
displayed a half-life of 39.8 h and this performance was
maintained at a lysosomal pH 4.8 (t1/2 = 38 h) and in human
plasma (t1/2 = 39.6 h) (Scheme 2, see also section 2.6-2.8 SI).
After establishing the stability of these cores in different
biological conditions, we studied their stimulus responsive
hydrolysis. Cancer cells typically exhibit an increased
concentration of GSH (micromolar range) in the cytoplasm, and
for that reason, GSH has been extensively targeted to promote
conjugates dissociation upon internalization.[7] Based on this, the
stability of 5 at pH 7.4 was studied in the presence of 10 equiv.
of GSH. As expected, the presence of GSH compromised the
structural integrity of the B-complex and the half-life of 5 was
reduced by 7 h in these conditions (Scheme 2, see also section
2.10 SI). Taking in consideration the stability and controlled
reversibility exhibited by B-complex 5, hydroxyacetophenone
and aminophenol were selected as the components to test the
assemblage of a multifunctional TDC featuring a cytotoxic drug,
a small Peg chain and a targeting unit.
methodologies, the hydroxyacetophenone and the aminophenol
components were modified to incorporate a small Peg chain (6)
and an azide function (7) for post-functionalization with a folate-
cyclooctyne unit (9) (Scheme 3, see also sections 1.5-1.7 SI).[10]
With all components at hand, the assembly reaction was
attempted using Btz, 6 and 7, though the desired product was
never formed. We reasoned this lack of reactivity to be due to a
combination of stereo constraints imposed by the Btz structure
and the Peg chain in 6 (see section 6.1 SI). Consequently, the 4-
substituted hydroxyacetophenone
8 was synthesized and
reacted with 7 and Btz. Pleasingly, these components readily
afforded 10, which was isolated in 25 % yield. Next, 9 was used
to install the folate unit directly onto the B-complex 10 via a
strain promoted alkyne-azide cycloaddition – SPAAC. The
reaction proceeded smoothly in DMSO and the formation of 11
was confirmed by ESI-MS (Scheme 3, see sections 1.8-1.9 SI).
Scheme 2. One pot synthesis of B-complexes 1-5 and yields obtained.
Evaluation of their stability at pHs 7.4, 4.8 and in human plasma. * B-complex
5 was also evaluated at pH 7.4 in the presence of 10 equiv. of GSH.
Scheme 3. Assemblage of B-complex 10 (CH3CN, 75 ºC, 18h, 25 %) and
subsequent folate functionalization via SPAAC (DMSO, 25 ºC, 17 h, 85 %) to
yield compound 11.
Bortezomib (Btz) is a potent proteasome inhibitor approved by
FDA for the treatment of myeloma and lymphoma cancers.
However, this drug is far from reaching its true therapeutic
potential, because the BA function readily forms reversible
covalent complexes with vicinal Lewis base donors of proteins,
peptides and sugars, which contributes to the emergency of
unfavourable pharmacokinetic properties and off-target
toxicities.[8] Therefore, Btz was selected as the cytotoxic
component, not only because it exhibits a BA function that is
essential to generate the B-complex, but also because Btz
would benefit from an improved selectivity endowed by the
construct vectorization to cancer cells.
Regarding the targeting unit, folic acid was chosen to direct the
SMDCs to cancer cells. Folic acid is an essential vitamin for cell
functioning and is commonly used as a recognition moiety,
because many cancer cell lines over-express receptors for this
small vitamin due to their fast cell division and growth.[9]
Once prepared, the selectivity and cytotoxicity of B-complex 11
was evaluated against MDA-MB-231 human breast cancer cells
that overexpress folate receptors (FR), using
a 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo
phenyl)-2H-tetrazolium (MTS) cell proliferation assay. The study
started with the evaluation of Btz, which was shown to be quite
cytotoxic (IC50 of 14.2 nM) against this cancer cell line. Next, the
cytotoxicity of B-complexes 10 and 11 was determined in the
same conditions. In this assay, 10 was completely inactive up to
a concentration of 100 nM while B-complex 11, featuring a folate
targeting unit, exhibit an improved potency (IC50 of 67.5 nM)
against this cancer cell line (Scheme 4, see also section 3.2.1
SI). Considering that the free folic acid 9 was also non cytotoxic
against MDA-MB-231 cancer cells (see section 3.2.1 SI), the
observed activity of 11, clearly suggests that folic acid is
mediating the B-complex internalization into the cancer cell
where Btz is released. Encouraged by these results, the
platform modularity was explored to tune the construct structure
and activity.
Next, we addressed the preparation of the remaining
components of the boronated core. Following reported
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10.1002/anie.201703492
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Antibody (Ab) drug conjugates are a powerful class of TDCs that
use Abs (e.g. Immunoglobulin G - IgG) as the tumor cell
recognition moiety.[4a] IgGs typically feature bivalent Fab regions
that are responsible for the high specificity and affinity of the Ab
against a determine epitope. Inspired by this design, we
envisioned that the boronated core could also be modified to
After establishing the potential of this molecular arrangement to
engage in an effective targeted delivery of Btz, we studied the
drug delivery mechanism. Hence, B-complex 16 featuring a non-
fluorescent coumarin substituted phenyl BA was prepared (see
section 1.14 SI) and used to label MDA-MB-231 and 4T1 cancer
cells (Scheme 5). It is well established that BAs readily undergo
oxidative boronate cleavage in the presence of intracellular
reactive oxygen species, namely at cancer cells’ cytoplasm.[12]
Hence, B-complex 16 was designed to signal the hydrolysis of
the BA component via the oxidation of the BA to phenol that self-
emotively generates the quinone methide and releases the
fluorescent coumarin probe as shown in Scheme 5. To test this
hypothesis, 16 (1 µM) was added to MDA-MB-231 and 4T1 cells
and incubated for 10, 30 and 180 minutes. Images obtained by
confocal microscopy clearly show in MDA-MB-231 cells, the
fluorescent coumarin probe distributed throughout the cell
cytoplasm, and confirmed the successful B-complex 16 entry
into the cells, as well as the hydrolysis of the BA component
already at the first time point recorded (Scheme 5, see also
section 4 SI). Gratifyingly, a similar internalization pattern was
not observed when using the 4T1 cells (section 4 SI).
install
a
bivalent
folate
recognition
moiety
using
hydroxyacetophenone 12 and aminophenol 13 featuring small
Peg chains and azide functions.[11] As shown in Scheme 4, 12
and 13 were readily synthesized and used in the successful
assembly of B-complex 14 that was isolated in 8% yield (see
also sections 1.10-1.12 SI). The post-functionalization of 14 with
the folate-cyclooctyne 9 afforded B-complex 15 (see section
1.13 SI), which was then tested against MDA-MB-231 cancer
cells. As shown in Scheme 4, the incorporation of a bivalent
folate recognition moiety maintained the conjugate 15 ability to
deliver Btz and a IC50 of 62 nM (Scheme 4, see also section
3.2.1 SI).
To determine the selectivity induced by the presence of the folic
acid targeting unit, both B-complexes 11 and 15 were tested
against a 4T1 mouse breast carcinoma cell line with a very low
expression of folate receptors. Differently from Btz that proved
to be highly cytotoxic against this cell line (IC50 of 22.8 nM), both
constructs did not perturb the cells viability in up to a
concentration of 100 nM, being only cytotoxic at higher
concentrations (1-100 µM) (see section 3.2.2 SI). These results
indicate that B-complexes 11 and 15 are somewhat less potent
than Btz though, comparing with the free drug, both exhibited an
improved selectivity towards overexpressed FR cell lines.
Scheme 5. Synthesis of complex 16 and confocal fluorescence microscopy
analysis of its incubation in MDA-MB-231 human breast cancer cells. Plasma
membrane was labelled with WGA-Alexa Fluor® 594.
Upon confirming the BA release inside the cancer cell, GSH was
studied as the promoter of the B-complex disassembling. With
this objective, 20 was prepared (see section 1.15 SI) and
evaluated in plasma (see section 2.9 SI). This compound proved
to be more stable than the analogue 5 constructed with phenyl
BA, and exhibit a half-life of 60 h in these conditions. Next, B-
complex 20 was incubated with GSH in ammonium acetate
buffer pH 7.4 at 37 ºC and the reaction was monitored by ESI-
MS over 72 h (see section 5 SI). As shown in Scheme 6 (see
section 5 SI), analysis of the reaction mixture immediately
indicated the presence of an adduct combining the masses of 20
and GSH (m/z 912, Scheme 6). Signals with masses of m/z 367,
489, and 258 were also detected and correspondingly assigned
to structures 23, 24 and 25 (Scheme 6). Taking this in
consideration, we proposed that the mechanism by which the
drug is released, probably initiates with GSH thiol addition to the
150
125
100
75
50
25
0
25
50
75
100
Concentration (nM)
B-complex 10
B-complex 11
B-complex 14
B-complex 15
Scheme 4. Construction of B-complex 14 (CH3CN, 75 ºC, 18 h, 8 %). Folate
functionalization via SPAAC (DMSO, 25 ºC, 17 h, 99 %) to yield difolic SMDC
15. Cell viability determined by MTS assay, after 48h incubation of MDA-MB-
231 (ATCC® HTB-26TM) with Btz (5-100 nM), and B-complexes 10-11, 14-15
(25-100 nM).
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electrophilic imine carbon centre of 20 to form intermediate 21
present in the reaction mixture (m/z 912). DFT calculations
performed on a simplified model,[13] suggest that thiol addition
destabilizes the complex by increasing the electronic density on
the B-atom bond, weakening the B-O bond of the aminophenol
(Scheme 6, see also section 6.2 SI). This process leads to the
opening of the 5-member ring and to the formation of
intermediate 22 in which Btz (m/z of 367) is more solvent
exposed and consequently more susceptible to hydrolysis. This
corresponds to the less stable intermediate along the path, 15
kcal/mol above the reagents. This mechanism is also supported
by the presence of compound 24 (m/z of 489), which probably
derives from an intramolecular addition of the amino group on to
the GSH amide and consequent release of a glycine residue.
Hydrolysis of 24 forms the imine 25 that is also clearly present in
the reaction mixture (m/z 258). The free energy balance
calculated for the entire process indicates a fairly exergonic
reaction with ΔG = –6 kcal/mol.
spectrometry and DFT calculations, was proposed a reaction
mechanism for the GSH induced hydrolysis of the B-complex
that involves the GSH thiol addition to the electrophilic imine
carbon. Considering the boronated core straightforward
modularity, stability in human plasma and stimulus
responsiveness, the availability of cytotoxic drugs that maybe
simply borylated (e.g. SN38), this platform evidences unique
properties to be used in the construction of multivalent
therapeutic conjugates and as a protective group for BAs that
blocks the undesired reactivity of this function.
Acknowledgements
The authors thank Fundação para a Ciência e a Tecnologia (FCT),
Portugal
(grants:
SFRH/BD/94779/2013,
PTDC/QEQ-
QOR/1434/2014,
UID/DTP/04138/2013,
SAICTPAC/0019/2015
(iMed.ULisboa), UID/QUI/00100/2013 (LFV). Patent rights granted to
Hovione SA PT109941.
Keywords: Boronic acids • Multivalent • Bortezomib • SMDC
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In summary, here is presented for the first time a modular
platform to construct cancer cell targeting drug conjugates, using
BAs to promote the assembly of functional components featuring
a cytotoxic drug (Btz), Peg chains and azide handles. The
multivalent boronated core was assembled in a single step (up
to 25 % isolated yield) and the azide component was effectively
post-functionalized with folate targeting units via a SPAAC
reaction. This molecular architecture afforded conjugates with
high selectivity against folate positive MDA-MB-231 cancer cells
and IC50 in the nanomolar the range. The boronated core was
shown to be quite stable in human plasma (up to 60 h half-life),
though reversible in the presence of GSH. The intracellular
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COMMUNICATION
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COMMUNICATION
Fábio M. F. Santos, Ana I. Matos, Ana
E. Ventura, João Gonçalves, Luís F.
Veiros, Helena F. Florindo, Pedro M. P.
Gois*
Page No. – Page No.
Modular Assembly of Reversible
Multivalent Targeting Drug
Conjugates
Text for Table of Contents
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