Introducing a squaramide-based self-immolative spacer for controlled drug release

Article abstract of DOI:10.1039/d0cc07683j

Herein we report the design, synthesis and assessment of the first example of a squaramide-based self-immolative system triggered by an enzymatic reduction. We have proved that the release of the alkylating agentN′,N′-(bis(2-chloroethyl)benzene)-1,4-diamine (ANM) provokes a dramatic reduction of the survival factor in glioblastoma cells, evidencing the suitability of the squaramide-based spacer for drug delivery applications.

Full text of DOI:10.1039/d0cc07683j

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Number 22
18 March 2021
Pages 2699–2828
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COMMUNICATION
Carmen Rotger et al.
Introducing a squaramide-based self-immolative spacer for
controlled drug release

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Introducing a squaramide-based self-immolative
spacer for controlled drug release†
Cite this: Chem. Commun., 2021,
57, 2736
Marta Ximenis,a Angel Sampedro,a Luis Martınez-Crespo,
Guillem Ramis,b
*
a
´
a
a
a
Received 23rd November 2020,
Accepted 9th February 2021
Francisca Orvay,
Antonio Costa
and Carmen Rotger
DOI: 10.1039/d0cc07683j
rsc.li/chemcomm
Herein we report the design, synthesis and assessment of the first straightforward conjugation to drugs.1 In this regard squaramides
example of a squaramide-based self-immolative system triggered are remarkable compounds since they are easy to synthesize,
by an enzymatic reduction. We have proved that the release of capable of interacting with other molecules and have been success-
the alkylating agent N0,N0-(bis(2-chloroethyl)benzene)-1,4-diamine fully used as linkers in bioconjugate active compounds.15
(ANM) provokes a dramatic reduction of the survival factor in
Besides, squaramides are kinetically stable for periods
glioblastoma cells, evidencing the suitability of the squaramide- longer than 100 days in the pH range of 3–10 at 37 1C.16,17
based spacer for drug delivery applications.
Alkyl squaramates are also known for their remarkable stability
in water, resisting hydrolysis at pH 7–9 for periods comprising
Efficient drug delivery is a challenging issue owing to the poor several hours to days depending on the structure.18
pharmacokinetic properties of many bioactive compounds.1
Herein, we introduce the first example of a minimalist self-
Prodrug design has become a successful strategy to upgrade immolative spacer based on the squaramide moiety (SQ-SIS)
their pharmacokinetic profile while voiding their pharmacological that disassembles through a cyclization reaction with the
activity and unwanted side effects.2,3 Ideally, prodrugs are enzy- simultaneous release of a leaving group (Fig. 1). Our design
matically activated at their target within the organism, yielding was inspired in our previous studies regarding the conforma-
the bioactive compound after the cleavage of labile bonds.4,5 tional properties of squaramides,19,20 where we demonstrated
However, the steric requirements for a successful enzymatic that N-methyl squaramides favour the E,Z conformation either
cleavage step are sometimes difficult to fulfill.6 The incorporation in the solid-state or in solution.21 Thus, we hypothesised that
of self-immolative spacers within the prodrug structure increases preorganization offered by this conformation would promote
the distance between the labile bond (trigger) and the drug the intramolecular attack of a nucleophile (trigger) located at
moiety, favoring enzymatic cleavage.7 In general, self-immolative
spacers spontaneously undergo a cascade of disassembly reac-
tions in response to an external stimulus, enabling a temporally
sequential release of drug molecules.8 These stimuli-responsive
molecules disassemble through two main mechanisms: elimina-
tion and cyclization processes.7 Many self-immolative spacers
have been employed to construct drug delivery systems, but also
sensors,9,10 molecular amplifiers,11–13 and stimuli-responsive
materials.14 Nevertheless, some self-immolative spacers have
complex structures, which can be an issue for their large scale
synthesis and commercial use. Therefore, there is a demand
for, new self-immolative spacers of affordable synthesis and
a Universitat de les Illes Balears, Cra. Valldemossa Km 7.5, Palma de Mallorca
07122, Spain. E-mail: carmen.rotger@uib.es
b
´
`
Institut Universitari d’Investigacio en Ciencies de la Salut (IUNICS),
´
`
Fig. 1 Schematic showing the rational design of the squaramide-based
self-immolative spacer SQ-SIS that disassembles through a cyclization
reaction. Chemical structures of the ethyl squaramates 1a and 1b, squaramides
2 and 3a-3c, and the cyclic squaramides 4a-4c.
Universitat de les Illes Balears, and Institut d’Investigacio Sanitaria Illes Balears
(IdISBa), Cra. Valldemossa Km 7.5, Palma, Illes Balears, Spain
† Electronic supplementary information (ESI) available: Experimental details and
analytical data for all new compounds. See DOI: 10.1039/d0cc07683j
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the g-position of one alkyl squaramide substituent enhancing constants.23 The cyclization reaction is pH sensitive in all the
the disassembly process. We designed SQ-SIS to form a seven- studied intervals and gets faster when increasing the pH.
membered ring cyclic squaramide after the fragmentation
The conversion of the ethyl squaramate 1a into the cyclic
reaction. The cyclization step yield drops for the corresponding squaramide 4a is rapid even under moderate acidic conditions
six-membered analogues due to their inability to compensate (pH 5, kobs = 0.65 ꢁ 0.01 ꢂ 10ꢀ5 sꢀ1). At pH 7, the reaction gets
for the cyclobutenedione ring strain, opening up the CQC–N 5.6 times faster (kobs of 3.68 ꢁ 0.05 ꢂ 10ꢀ5 sꢀ1), and at pH 4 8,
bond angles and adopting a non-planar conformation.22 The the conversion was too fast to be measured, observing only the
low cyclization efficiency is more significant when in water band from product 4a. The cyclization reaction gets faster for
(pH Z7) the ester group hydrolysis competes with the cycliza- the N-Methylated analogue 1b and even at pH 7 could not be
tion (see the ESI,† and S18).18 Therefore, the kinetic control on measured, showing the effect of promoting the E,Z conforma-
the self-immolative reaction will be gained by the trigger tion on the reaction rate (see the ESI,† Fig. S18).
activation through an external stimulus and by tuning the
fugacity of the leaving group (Fig. 1).
The formation of the cyclic squaramide 4a was confirmed by
1H-NMR (buffered D2O-PBS at pH 7, 24 1C), following the
In our design, we decorated the SQ-SIS with a primary or appearance of a triplet at 3.7 ppm and a multiplet at 2.3 ppm
secondary alkyl amine group as a trigger since these groups are corresponding to the a-and b-CH2 of the cyclic compound,
conveniently capped with a protecting group until receiving the respectively, as well as the disappearance of the ethyl signals
corresponding stimulus. First, we evaluated the kinetic proper- at 4.8 and 1.6 ppm (see the ESI,† Fig. S19–S21).24 Similarly,
1
ties of the SQ-SIS by releasing different molecules of distinct squaramides 2 and 3b were also evaluated by UV and H-NMR
nucleofugacity as drug models. For this purpose, we synthe- spectroscopy, respectively (see the ESI,†Fig. 2b and c). Incuba-
sized by sequential condensation of diethyl squarate with the tion of 2 in buffered solutions from pH 3–9 at 37 1C led again to
corresponding amines, the ethyl squaramates 1a and 1b, and a pH-dependent kinetic curve. The kobs = 5.74 ꢁ 0.1 ꢂ 10ꢀ5 sꢀ1
the squaramides 2 and 3b (see the ESI,† Schemes S1 and S2) obtained for the cyclization reaction of the squaramide 2 at
bearing ethanol, n-butylamine, and p-anisidine as leaving pH 7.4 resulted in the same order of magnitude as the kobs
groups (Fig. 1) (see the ESI,† for synthetic details and com- obtained for the diethyl squaramate 1a. However, the fitting of
1
pound characterization).
the H-NMR data obtained from compound 3b at pH 8 gave a
The self-immolative behaviour of the ethyl squaramate 1a kobs = 1.44 ꢂ 10ꢀ5
s
ꢀ1, showing that the self-immolative
was evaluated at the pH range of 4.7–9 and 37 1C. The broad reaction is, in this case, remarkably slower than for compounds
band in the UV spectrum of 1a with a maximum at 275 nm was 1a and 2 (see the ESI,† Fig. S22 and S23). Thus, the temporary
used to follow the reaction progress, observing its disappear- evolution of the three compounds at pH 7 and 37 1C depends
ance and the correlated appearance of a new band at 293 nm on the nature of the leaving group and correlates well with the
corresponding to the cyclization product 4a (Fig. 2a).
pKa value of each group. After 8 h, the extent of the disassembly
The kinetic data were fitted to a pseudo-first-order rate law is insignificant (o1%) for compound 3b, but much higher for
using ReactLabTM Kinetics to obtain the corresponding kinetic compounds 2 (76%) and 1a (97%), showing these two have a
good profile for biological applications (Fig. 2d).
The positive effect of the squaramide N-methyl group on the
cyclization step promoting the squaramide E,Z became again
evident when we compared the kinetic constants obtained by
1H-NMR (pH 8, 37 1C) of squaramides 3a (kobs = 0.02 ꢂ 10 ꢀ5 sꢀ1
)
and 3c (kobs = 0.07 ꢂ 10 ꢀ5 sꢀ1) showing a threefold increment in
the cyclization rate for the N-methylated squaramide. Addition-
ally, the trigger nucleophilicity also influences the overall cycliza-
tion reaction. Thus, 3b (kobs = 1.44 ꢂ 10ꢀ5 sꢀ1) having a secondary
amine as a nucleophile disassembles roughly 20 fold faster than
squaramide 3c bearing a primary amine (see the ESI,† Fig. S23).
To gain control over the trigger activation, we used
p-nitrobenzylcarbamate (p-NBzC), an enzymatically removable
protecting group, to cap the trigger. The p-NBzC group is easily
reduced by nitroreductases (NTR) in the presence of NADH as a
reducing agent.25 This protecting group, for which no asso-
ciated toxicity has been reported, has been successfully applied
in combined prodrug therapies.10,26,27 Therefore, we synthe-
sized squaramide 5a and studied its enzyme triggered self-
immolative reaction exposing the squaramide at pH 7.4 and
37 1C to the NTR/NADH tandem (5 mg mLꢀ1 of NTR and 35 mM
NADH), and monitored the self-immolative reaction by HPLC
for 48 h. The enzymatic reduction and p-NBzC removal were
Fig. 2 Representative UV changes observed for the cyclization of (a) 1a
(PBS pH 5.7, 37 1C) and (b) 2 (PBS, pH 9 at 37 1C) (blue) to give 4a and 4b,
respectively (red). (c) Disappearance of 3b (blue) and appearance of 4b
(red) followed by 1H-NMR (PBS pH 8–10 DMSO-d6, 37 1C). (d) Percentage
of cyclized squaramides 4a-4b over time, comparing compounds 1a
(black), 2 (blue) and 3c (red) at pH 7 and 37 1C.
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Fig. 4 (a) Chemical structure of compounds 6, 15, and temozolomide
(TMZ). (b) DNA alkylation activity of 15 (A) and 6 (B and C) against plasmid
pmax-GFP by agarose gel electrophoresis. DNA was incubated for 48 h at
pH 7.4 and 37 1C. Lane P, DNA control; lanes 1–3, 25, 50, and 100 mM of 15;
lanes 4–6, 25, 50, and 100 mM of 6 with the reduction tandem NRT/NADH;
lanes 7–9, 25, 50, and 100 mM of 6 alone. (c) Representative clonogenic
plates of U87-MG glioblastoma cells after 10 days comparing untreated
cells (A) with cells treated with 10 mM of temozolomide (B), 15 (C), and 6
(D) for 1 h. (d) Representation of the Survival Factor index for LN229 (black)
and U87-MG (grey) cells obtained from the clonogenic assay.
Fig. 3 (a) 5a releases p-anisidine in response to the NTR/NADH tandem.
(b) Time course plot of the disassembly reaction. The p-anisidine release
rate was measured by the formation of cyclic squaramide 4b (blue) using
an HPLC-PDA detector set at 293 nm.
The crude reaction mixture was analysed by ESI-HRMS (ꢀ)
mode. The analysis of the mass spectra showed the mass peak
corresponding to the single strand monoalkylated DDD-15
adduct [3813.5982 (z = 1)], and peaks of multi charged species
of the same adduct [1271.1994 (z = 3) and 953.3995 (z = 4)].
Additionally, the monoisotopic mass of the cyclic squaramide
4b [181.0972 (z = 1)] was detected in ESI (+) mode (see the ESI,†
and Fig. S27). The formation of the same DDD-15 adduct and
its multicharged species were detected when the free drug
15 was incubated with DDD (see Fig. S25, ESI†). DNA alkylation
was also detected when compound 6 was incubated without the
reduction tandem (see Fig. S26, ESI†). However in this case,
along with the corresponding adduct DDD-6 [4180.8627 (z = 1)],
unreacted DDD and 6 were also detected. Therefore, we have
detected the effective enzymatic release of compound 15
through the self-immolative cyclization step.
The DNA alkylation was also observed running a gel retarda-
tion assay after the incubation of compound 6 with the plasmid
pmax-GFP (3.4 kb) as a DNA source (Fig. 4).§ The plasmid was
incubated for 48 h at pH 7.4 and 37 1C with increasing
concentrations of 6 (0.25–1 ꢂ 10ꢀ4 M) and the reduction
tandem NRT/NADH. The plasmid was incubated under the
same conditions alone and with increasing concentrations of
the free 15 (0.25 to 1 ꢂ 10ꢀ4 M) and 6 as control experiments.
The agarose gel electrophoresis run from the reaction mixtures
showed evident retardation on the DNA mobility in all the
samples because of the plasmid alkylation. Similar DNA alkyla-
tion ratios were obtained in samples containing compounds 15
(lanes 1–3) and 6 with the reduction tandem (lanes 4–6). When
incubated alone, compound 6 does not alkylate the plasmid to
the same extent, which suggests that the activity of the drug is
almost instantaneous to give squaramide 5b. The intra-
molecular cyclization of 5b went on within hours to yield the
cyclic squaramide 4b (Fig. 3b).‡
Once we had optimized the different elements of the self-
immolative prodrug system, we synthesized compound 6 to
evaluate the behaviour of the SQ-SIS by releasing a bioactive
compound. Based on the previous experiments, we selected
N0,N0-(bis(2-chloroethyl)benzene)-1,4-diamine 15 as a drug,
which is a representative member of the Aniline Nitrogen
Mustard (ANM) family (Scheme 1). The development of
mustard-based hybrids is a known strategy for the discovery
of anticancer drugs.28,29 Squaramide 6 was obtained in two
synthetic steps (see the ESI†). First, diethyl was subsequently
condensed with the p-NBzC protected N1,N3-dimethylpropane-
1,3-diamine, and then with 15 (see the ESI,† Schemes S3 and S4).
To determine the reactivity of compound 6 towards DNA, we
performed a crosslinking assay using the Drew–Dickerson
Dodecamer (DDD), a prototypical B-DNA.30 Squaramide 6
(40 mM, 37 1C, pH, 7.4) and NTR/NADH (5 mg mLꢀ1 and
35 mM respectively) were incubated with DDD (10 mM).
Scheme 1 Self-immolative disassembly of compound 6 triggered by
enzymatic reduction of the nitro group.
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reduced by the conjugation with the squaramide ring. This Notes and references
experiment also shows that the active free drug 15 has been
‡ No toxicity was found for concentrations of 4b up to 200 mM.
§ Plasmid pmax-GFP encodes the green fluorescent protein.
efficiently released in samples containing the NTR/NADH enzy-
matic system.
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Finally, we tested the self-immolative system 6 against two
glioblastoma cultured cell lines (LN229 and U87-MG) by running
a clonogenic assay, an in vitro cell survival assay based on the
ability of a single cell to grow into a colony, to reveal the long-term
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In conclusion, we have reported the first example of a
squaramide-based self-immolative module SQ-SIS with suitable
properties for drug release applications. SQ-SIS resists hydro-
lysis in physiological conditions and, after receiving an external
stimulus, evolves through a cyclization reaction releasing dif-
ferent types of leaving groups. Still, aromatic amines are the
most suitable compounds to conjugate with the module due to
its moderate release rate at a useful pH interval for biological
applications. Conjugation of SQ-SIS with p-nitrobenzylcarbamate,
and the aromatic nitrogen mustard 15, yields the enzymatic
triggered self-immolative prodrug 6. Under enzymatic reduction
conditions, 6 disassembles, releasing the alkylating agent 15.
In vitro experiments and the clonogenic assays with U87-MG
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cells treated with 6 suggests that the squaramide-based delivery
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We gratefully acknowledge the financial support from
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Conflicts of interest
There are no conflicts to declare.
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