Multiple Multicomponent Reactions: Unexplored Substrates, Selective Processes, and Versatile Chemotypes in Biomedicine

Article abstract of DOI:10.1002/chem.201802877

Multiple multicomponent reactions rapidly assemble complex structures. Despite being very productive, the lack of selectivity and the reduced number of viable transformations restrict their general application in synthesis. Hereby, we describe a rationale for a selective version of these processes based in the preferential generation of intermediates which are less reactive than the initial substrates. In this way, applying the Groebke–Blackburn–Bienaymé reaction on a range of α-polyamino-polyazines, we prepared a family compact heterocyclic scaffolds with relevant applications in medicinal and biological chemistry (live cell imaging probes, selective binders for DNA quadruplexes, and antiviral agents against human adenoviruses). The approach has general character and yields complex molecular targets in a selective, tunable and direct manner.

Full text of DOI:10.1002/chem.201802877

A Journal of
Accepted Article
Title: Multiple Multicomponent Reactions: Unexplored Substrates,
Selective Processes and Versatile Chemotypes in Biomedicine
Authors: Rodolfo Lavilla, Ouldouz Ghashghaei, Samantha Caputo,
Miquel Sintes, Marc Revés, Nicola Kielland, Carolina
Estarellas, F. Javier Luque, Anna Aviñó, Ramón Eritja, José
Antonio Marrugal-Lorenzo, Ana Serna-Gallego, Jerónimo
Pachón, Javier Sánchez-Céspedes, Ryan Treadwell, Fabio
de Moliner, and Marc Vendrell
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201802877
Link to VoR: http://dx.doi.org/10.1002/chem.201802877
Supported by

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
Multiple Multicomponent Reactions: Unexplored Substrates,
Selective Processes and Versatile Chemotypes in Biomedicine
[
a]
[a]
[a]
[a]
[a]
Ouldouz Ghashghaei, Samantha Caputo, Miquel Sintes, Marc Revés, Nicola Kielland, Carolina
[
b]
[b]
[c]
[c]
[d]
Estarellas, F. Javier Luque, Anna Aviñó, Ramón Eritja, Ana Serna-Gallego, José Antonio
[
d]
[e]
[e]
[f]
Marrugal-Lorenzo, Jerónimo Pachón, Javier Sánchez-Céspedes, Ryan Treadwell, Fabio de
[f]
[f]
[a]
Moliner, Marc Vendrell and Rodolfo Lavilla*
4
,5
Abstract: Multiple multicomponent reactions rapidly assemble
complex structures. Despite being very productive, the lack of
selectivity and the reduced number of viable transformations restrict
their general application in synthesis. Hereby, we describe a
rationale for a selective version of these processes based in the
preferential generation of intermediates which are less reactive than
the initial substrates. In this way, applying the Groebke-Blackburn-
Bienaymé reaction on a range of -polyamino-polyazines, we
prepared a family compact heterocyclic scaffolds with relevant
applications in medicinal and biological chemistry (live cell imaging
probes, selective binders for DNA quadruplexes and antiviral agents
against human adenoviruses). The approach has general character
and yields complex molecular targets in a selective, tunable and
direct manner.
being paradigmatic examples.
Especially attractive in this context is Wessjohann’s
development of multiple MCRs, which further increases the
performance of the original formulation. In such impressive
transformations, bi(poly)functional components undergo
a
productive set of MCRs to assemble large adducts in a single
step. Through this approach, a variety of extremely complex
structures, including glycoconjugates, macrocyclic cages,
6
-9
peptoid-peptides, etc., was prepared.
This remarkable
achievement could be upgraded if different reactions were
selectively performed at the repeated functionalities. However,
at the present state, all reactive functional groups (mainly linked
through long alkyl chains) undergo the same transformation in
an indiscriminate manner. It is worth to mention the remarkable
exception of Orru’s chemically distinct di-isocyanides, which was
1
0
elaborated up to an 8CR. Sequential versions of multiple
MCRs have been conducted by either blocking an existing
functional group or by gradually generating new functionalities
Introduction
1
1,12
along the process.
Multicomponent reactions (MCRs), processes in which three or
more reactants interact to yield an adduct, are fundamental in
the development of synthetic methods, because of their
remarkable atom and step economies, molecular variability and
structural diversity. Isocyanides are pivotal in MCRs, leading
to the most fruitful processes, the Ugi and Passerini reactions
The design of selective multiple MCRs (Scheme 1A)
constitutes a step forward in the programmed synthesis of
complex compounds. In principle, such processes should be
viable, provided that the adduct from the first step would be less
reactive than the initial substrate. Thus, when the first MCR
takes place, the intermediate adduct would bear untouched
functional groups with altered electronic properties (ie, less
electrophilic) in comparison with those in the starting material, as
a consequence of the initial structural modification. In this way,
the reaction could proceed selectively, enabling a second MCR
with a different set of inputs. Also, in cases involving non-
symmetrical starting materials, the reactivity of the repeated
functional groups should be kinetically distinct (Scheme 1A).
1
-3
[
a]
Dr. O. Ghashghaei, Dr. S. Caputo, M. Sintes, Dr. M. Revés, Dr. N.
Kielland, Prof. R. Lavilla. Laboratory of Medicinal Chemistry, Faculty
of Pharmacy and Institute of Biomedicine (IBUB), University of
Barcelona, Barcelona Science Park, Baldiri Reixac 10-12, Barcelona
08028, Spain and CIBER-BBN, Networking Centre for
Bioengineering, Biomaterials & Nanomedicine, Baldiri Reixac 10-12,
Barcelona 08028, Spain. Email: rlavilla@ub.edu
[
[
[
b]
c]
d]
Dr. C. Estarellas, Porf. F. J. Luque. Departament de Fisicoquímica,
Facultat de Farmàcia, and IBUB, Universitat de Barcelona, Prat de
la Riba 171, E-08921, Santa Coloma de Gramenet, Spain
Dr. A. Aviñó, Prof. R. Eritja. Department of Chemical & Biomolecular
Nanotechnology, Institute for Advanced Chemistry of Catalonia
Moreover, as the choice of multiple MCRs is rather limited to Ugi
13-15
reactions (apart form few reported processes),
of their scope would be highly beneficial.
the expansion
Furthermore, the use of heterocyclic inputs, privileged motifs
1
6
(
IQAC), CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain.
in drugs, yields meaningful MCR adducts.
Polyamino-
Dr. A. Serna-Gallego, J. A. Marrugal-Lorenzo. Clinical Unit of
Infectious Diseases, Microbiology and Preventive Medicine,
University Hospital Virgen del Rocío/ Institute of Biomedicine of
Seville (IBiS) /CSIC/University of Seville.
Prof. J. Pachón, Prof. J. Sánchez-Céspedes. Clinical Unit of
Infectious Diseases, Microbiology and Preventive Medicine,
University Hospital Virgen del Rocío/ Institute of Biomedicine of
Seville (IBiS) /CSIC/University of Seville & Department of Medicine,
University of Seville, Seville, Spain.
polyazines are attractive, yet unexplored MCR substrates. For
instance, diaminopyrimidines are relevant in medicinal
1
7
chemistry, while melamine plays a key role in materials
1
8
[
e]
f]
science.
The Groebke-Blackburn-Bienaymé reaction (GBBR), which
involves the acid-catalyzed interaction of α-aminoazines,
aldehydes and isocyanides to yield imidazoazines (Scheme
1
9-21
22
[
Prof. M. Vendrell, R. Treadwell, Dr. F. de Moliner. MRC/UoE Centre
for Inflammation Research, The University of Edinburgh, 47 Little
France Crescent, Edinburgh EH16 4TJ, UK.
1B),
important adducts in drug discovery, represents an
ideal candidate to embed in a novel highly ordered MCR
process. We hereby disclose our results on selective multiple
GBBRs (Scheme 1C).
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
Scheme 1. Selective Multiple Multicomponent Reactions (A) Concept of selective multiple MCRs. (B) Groebke-Blackburn-Bienaymé reaction (GBBR). (C) Present
work: selective multiple GBBRs.
2
5
9
3%) and 5g-m (13%-57%) respectively, were formed using
Results and Discussion
this protocol (Scheme 2A). Incidentally, on the course of a
GBBR upon adduct 4d, a lactamization took place giving the
pentacyclic adduct 5n (7%, Scheme 2E).
Multiple GBBRs: Reactivity and Scope
We first examined the GBBR of 4-chlorobenzaldehyde and
cyclohexyl isocyanide (two equivalents each) with 2,4-
diaminopyrimidine 1a. Initial screening of reaction conditions (SI)
showed that the expected multiple MCR adduct 5a (70%) was
formed under p-toluenesulfonic acid (PTSA) catalysis in DMF
To show the power of our approach, we prepared the 5CR
adducts 5h (50%) and 5j (40%), displaying the same
substituents in complementary positions, merely changing the
order in which the MCRs were performed (Scheme 2A). The
aldehyde/isocyanide scope of these multiple MCRs is basically
the same found in standard GBBRs.
(
Scheme 2A). Using a variety of common aldehydes and
Next, we explored the range of the aminoazine component.
Diaminoquinazoline 1b (Scheme 2B) underwent a regioselective
GBBR yielding mono-adduct 6a (65%). In a different reaction,
substrate 1b afforded the symmetrical bis-adduct 7a (78%).
Moreover, a second GBBR, performed upon 6a, gave the non-
symmetrical compound 7b (43%, Scheme 2B).
Diamino-pyridazine 1c afforded the mono GBBR adducts 8a-
d (24%-60%). The symmetrical and non-symmetrical double
GBBR adducts 9a (38%) and 9b (36%) were also prepared
(Scheme 2C). Interestingly, with substrate 1c, the di-GBBR
adducts were only formed using aromatic isocyanides, whereas
bulkier ones (tert-butyl) failed to react, likely due to the steric
clash of the proximal amines in the putative structure.
Remarkably, the mono GBBR adducts 8, upon interaction with
an isocyanide, an aldehyde and a carboxylic acid underwent a
standard Ugi reaction to yield the new scaffold 10. Adduct 10a
isocyanides, the double GBBR adducts 5a-5f (36%-95%,
Scheme 2A) were prepared. The innate selectivity of the
aminoazine 1a was then studied using mixtures of two
23,24
aldehydes and two isocyanides of distinct reactivity.
Although unsymmetrical adducts were detected in over-
statistical ratios, the complexity of the mixtures precluded any
practical use (SI).
Thus, we addressed a sequential approach by forming one
mono-GBBR adduct first, then reacting this intermediate with a
distinct aldehyde-isocyanide pair to yield the double non-
symmetrical compound. Equimolar amounts of aminoazine 1a,
aldehyde 2a and isocyanide 3a were reacted with Yb(OTf)
3
catalysis in Acetonitrile under microwave irradiation, leading to
the selective formation of mono-GBBR adduct 4a (80%, Scheme
2A, structure confirmed by X-Ray, SI). Then, this compound
underwent a second GBBR with aldehyde 2b and isocyanide 3a
under PTSA catalysis in DMF affording the expected product 5g
(
14%, unoptimized) was directly isolated in a one-pot process
(
36%, Scheme 2A, X-Ray in SI), enabling selective multiple
involving a spontaneous GBBR-Ugi sequence in the presence of
AcOH (first catalyzing the GBBR and then reacting in the Ugi
step).
MCRs with full control on all four diversity points of scaffold 5. In
this way, a variety of mono- and di-GBBR adducts 4a-g (23%-
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
Scheme 2. The scope of the multiple GBBRs A) GBBRs and double GBBRs of 2,4-diaminopyrimidine (1a) and the synthesized library. B) 2,4-Diaminoquinazoline
1b) in multiple GBBRs. C) GBBR, double GBBRs and GBBR-Ugi upon 3,6-diaminopyridazine (1c) and the synthesized library. D) Melamine (1d) triple GBBR
processes. E) Lactam 5n.
(
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
Adducts 10b (77%) and 10c (61%), featuring five diversity points, Post-Condensation Transformations
were thus prepared from the corresponding mono adducts
Post-transformations of MCR adducts can diversify the initial
scaffolds, leading to complex and valuable chemotypes. In this
way, our GBBR cores were modified through a number of
representative reactions. The pyridine rings in adduct 5b were
alkylated with iodomethane to give salt 12 (78%, Figure 1A).
This selective alkylation reflects the higher reactivity of the
DMAP-like moieties. Additionally, 2,5-diketopiperazine 13 (43%),
(
Scheme 2C).
Finally, we tackled melamine (1d), a key key reactant with
widespread use. However, its reactivity is troublesome due to its
poor solubility in most organic solvents, thus having remained
unexplored in MCRs. Using our PTSA method, we generated
the triple adducts 11a-e (31%-57%, Scheme 2D, 11a X-Ray
crystallography in SI). Scaffold 11 is a novel tripodal, compact,
N-fused tetracyclic nucleus, conveniently synthesized in a formal
2
6
a relevant scaffold in medicinal chemistry, was generated from
2
7
the GBBR-Ugi adduct 10c (Figure 1B).
N-fused polycyclic structures, resembling N-doped
7CR, featuring the formation of nine bonds in a single step. The
2
8
selective formation of melamine non-symmetrical adducts is not
yet feasible and studies towards this goal are ongoing.
nanographene ribbons, are valuable in materials science,
although their preparation entails complex multistep sequences.
Figure 1. Post-Transformations of Multiple GBBR Adducts. A) Pyridinium salt 12. B) Diketopiperazine 13. C and D) Ullmann-type adducts 14 and 15. E, F and G)
Suzuki-Miyaura cross-coupling adducts 16a-c and molecular packing arrangement of compound 16c. New bonds marked in magenta, added residues in pink.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
In this context, GBBR adducts were subjected to metal-
position 4. The 2-amino tetrahedral adduct is favored by 4.6
kcal/mol relative to the corresponding amino-4 species. Finally,
catalyzed
amination upon adduct 5f yielded the heptacyclic compound 14
67%, Figure 1C). The same transformation upon 11b
cross-couplings.
Intramolecular
Ullmann-type
loss of
a water molecule leads to two almost isostable
(
protonated imine conformers, which are more stable (5.7
kcal/mol) than the 4-amino counterparts. The second step
involves the cyclization of the adduct formed upon addition of
the isocyanide to the imine derivative.
successfully gave the decacyclic radial derivative 15 (47%,
Figure 1D). Tetracyclic adducts 11c-d, fitted with aryl iodide
residues, reacted with arylboronic acids via multiple Suzuki-
Miyaura cross-couplings to access star-shaped compounds 16a-
c (31-49%, Figure 1E-G). Remarkably, compounds 16b-c have
nanometric dimensions, the latter reaching almost 30 Å length.
Its X-Ray diffraction shows a regular molecule with a planar core
and some degree of conformational freedom around the
cyclohexyl residues (Figure 1G and SI). The crystal packing
features an interesting docking arrangement of the central π
system with intertwining of the triaryl groups (Figure S2 in SI).
These examples notably demonstrate the power of the
methodology to build functionalized nanosized entities through a
direct bottom-up approach.
This process preferentially takes place through intermediate
I12a, leading to an adduct where the nitrilium carbon atom may
face either the N1 or N3 in the pyrimidine ring, the former
conformer being favored by 2.6 kcal/mol. Cyclization occurs via
the attack of the azine nitrogens to the nitrilium C atom through
an almost barrierless process for N1, whereas for N3 the
transition is less favourable. Therefore, the conformational
preference of the intermediate (I22,1) dictates the formation of the
final product 4. Overall, the selectivity appears to arise from the
combined effect of the preferential formation of one imine (from
the amino group at position 2) and its subsequent isocyanide
addition/cyclization via the azine N1. These results support the
feasibility of selective multiple MCRs involving difunctionalized
substrates.
Computational Studies of Mechanism Pathways
The regioselective formation of mono-GBBR adducts 4
(
Scheme 2A) is key for the synthetic usefulness of the approach.
This outcome is the result of two crucial steps, the first one
involving the imine formation since the preferential attack of the
amino groups at positions 2 or 4 may be relevant to the
selectivity (Scheme 3). To this end, M062X/6-31+G(d)
Applications in Chemical Biology and Medicinal Chemistry
To determine the usefulness of our methodology to access
and tune valuable scaffolds, we explored the performance of the
synthesized compounds in a number of disciplines related to
biomedicine. As a proof of concept, we intended to show that the
reported approach would rend functional compounds, amenable
to fast optimization.
29,30
calculations
(SI) were performed to identify the transition
states (TSs) formed along the corresponding pathways. The TSs
formed via addition of the amino group in position 2 are ≈ 3
kcal/mol more stable than the ones generated upon attack at
Scheme 3. Mechanistic Rationale for the Selective Multiple GBBRs: Reaction pathways from the interaction between 2,4-diaminopyrimidine (1a), benzaldehyde
2) and cyclohexyl isocyanide. The relative free energies (kcal/mol) are shown in blue.
(
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
1
2
Figure 2. GBBR Adducts as Fluorophores: A) Chemical tunability of the fluorescence emission of different GBBR adducts. B) Selective formation of the 5-
membered BODIPY-like fluorophore 18a and its X-Ray diffraction. C) Probe 18b. D, E, F) Comparative photophysical analysis of the adduct 18a (in green) and its
precursor 17 (in black).
i) Bioimaging Studies of Selected Multiple GBBR Adducts
The need for novel functional fluorophores has prompted the
development of new synthetic strategies to prepare probes not
fluorophores with bright fluorescence emission in the orange-to-
red region (10a, Figure 2A). Notably, a number of GBBR
adducts behaved as activatable fluorophores, with emission
intensities depending on the microenvironment (Table S2,
Figures S6-S7 in SI).
31
accessible through classical synthesis, and MCRs constitute a
3
2,33
valuable platform to afford them.
The versatility of the novel
multiple GBBRs allows the fine tuning of the structural and
spectral properties of the adducts. Particularly, we controlled
their red-shifted fluorescence emission wavelengths by
extending their electronic conjugation with connected aryl
groups (12 vs 5b and 9a, Figure 2A). Our approach also
enabled the introduction of electron-withdrawing and electron-
donating groups at specific sites to generate push-pull
The excellent photophysical features of the BODIPY scaffold
3
4,35
in bioimaging,
encouraged us to explore the generation of
novel BODIPY fluorophores arising from α-pyridyl-GBBR
adducts. Remarkably, model GBBR compound 17 reacted with
BF
displays a BF
nitrogen, suggesting that the formation of the 5-membered ring
3
to selectively render complex 18a (Figure 2B), which
2
bridge linking the imino group and the pyridine
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
Figure 3. Brightfield and fluorescence confocal microscope images of human A549 epithelial cells upon incubation with compound 18a (2 µM, green signal) and
the commercially available trackers LysoTracker Red (red signal) and MitoTracker Red (red signal). Scale bar: 10 µm.
3
6
is faster than the conventional 6-membered BODIPY cycle.
Remarkably, five members of this chemset displayed significant
anti-HAdV activity in the plaque assay, when evaluated at a
concentration of 10 µM (Table 1, SI). Among these, compounds
5h and 5n presented a dose-dependent activity, with IC50 values
of 1.19 and 3.42 µM, respectively, and high selectivity indexes
(94 and 62, Table 1). The most cytotoxic molecule, 5b presented
the lowest selectivity index. Interestingly, compound 10b (dose-
independent, inactive below 10 µM) displayed a relevant virus
yield reduction (157-fold, Table 1). In relation with frequently
used antivirals, cidofovir, the drug of choice for HAdV infections,
showed a significantly lower potency (IC50 ≈24 µM). These
results are encouraging given the observed structure-activity
relationships. In particular, GBBR adducts 5g and 5h, sharing
the same diaminopyrimidine scaffold, display very different
bioactivity (Table 1, SI). Also adduct 10b is considerably more
active than the close analogue 9a. This trend strongly suggests
that the bioactivity profile could be further improved through
programmed exploration around this chemistry.
Analogously, the BODIPY-like compound 18b was obtained
from the double GBBR adduct 5d (Figure 2C).
Notably, compound 18b showed pH-dependent fluorescence
emission, with a pKa of 5.0 and brighter fluorescence in basic
media, unlike the majority of BODIPY dyes. Next, the
photophysical features of the probe 18a were analyzed and
compared with those of its precursor. Besides exhibiting a
remarkably longer emission wavelength (453 nm to 514 nm,
Figure 2D), compound 18a featured
a
40-fold higher
fluorescence quantum yield, presumably due to the increased
rigidification of the BODIPY core (Figure 2E). Unlike its
precursor 17 (or 18b), the fluorescence emission of compound
8a did not show meaningful pH dependence, asserting its value
as a bright green fluorophore for bioimaging assays covering a
broad range of pH values (Figure 2F, SI).
We also confirmed the compatibility of probe 18a for live-cell
imaging by incubating human lung A549 epithelial cells and
acquiring images under a confocal fluorescence microscope.
Fluorophore 18a showed excellent cell permeability and
preferential accumulation in the mitochondria, as demonstrated
by co-incubation with the commercially available LysoTracker
and MitoTracker dyes (Figure 3, SI). These results indicate the
suitability of multiple GBBRs to generate novel fluorescent
scaffolds with excellent features for bioimaging studies.
1
Table 1. Inhibitory HAdV activity of multiple GBBR adducts
[
a]
[b]
Adduct
IC50 (µM)
2,46±0,00
1,19±0,08
3,42±1,33
CC50 (µM)
22,90±2,24
112,50±2,94
215,22±8,53
62,80±12,53
132,2±1,41
S.I
9
V.Y.R
5b
5h
5n
115,42±59,76
11,54±5,98
24,88±12,87
11,54±5,98
157,72±0,00
94
63
---
---
ii) Antiviral Activity of Multiple GBBR Adducts
We determined the therapeutic potential of multiple GBBR
2
2
[
c]
scaffolds
responsible for mild diseases in healthy individuals, HAdV
infections are associated with high mortality in
against human adenoviruses (HAdV). While
10a
0b
≤ 10
≤ 10
[
c]
1
3
7
immunosuppressed patients. The lack of approved specific
antiviral drugs with efficacy and safety against HAdV further
complicates the treatment of these patients. In this context,
representative GBBR adducts (Table 1 and Figure S11 in SI)
were tested against HAdV using different susceptibility assays.
[
a] Selectivity index. [b] Virus yield reduction (fold-reduction). [c] The IC50
could not be determined. 10a and 10b do not show a dose-dependent
effect, although they are active at 10 µM.
3
8
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
iii) Affinity of Multiple GBBR Adducts to DNA
sequences, being significantly more intense with the single
strand T20. Compound 5b showed a non-specific, weak affinity
for several sequences (SI). However, its dimethylated salt 12
remarkably displayed a potent and selective affinity for the
hybrid quadruplex 24blc (Figure 4B). The cationic nature of 12
may explain its stronger interactions in comparison with those of
adduct 5b. The charged BODIPY-adduct 18b showed affinity
again for 24bcl (Figure 4A).
Since DNA displays a variety of different structural types, it is
crucial to have selective binders to study their roles as a
39
preliminary step to develop medicines. Especially relevant are
G-quadruplexes, because of their wide presence in the genome,
mainly involved in the maintenance of chromosomes and in the
4
0
transcriptional regulation of genes.
Topology-dependent
ligands, selectively binding quadruplexes, will therefore have a
41
significant impact on biological and medicinal chemistry. Flat
N-heteroaromatic motifs with polar or cationic groups are
frequently found in active binders, stabilizing the G-tetrads by -
stacking and electrostatic interactions, although they are often
non-selective for a defined substructure.
To study the interaction of compound 12 with quadruplexes,
we carried out fluorescence titrations of sequences GG1 and
24bcl (hybrid quadruplex) with double strand DS26 as the
negative control (Figure S15 in SI). The titration curves showed
that raising the relative oligonucleotide-drug concentration up to
18-fold, resulted in a significant increase (≈5-fold) in the
fluorescence of the combination 24bcl-12. A similar, yet less
potent, behaviour (≈3-fold), was detected with GG1. However, in
the case of interaction with DS26, no significant change was
observed (Figure 4C). Considering that oligonucleotides are
non-fluorescent, and that the drug concentration is constant, the
observed increase in fluorescence arises from the formation of
oligomer-drug complexes.
In this context, we explored a subset of our GBBR adducts
(
5b, 11b, 12 and 18b) in the interaction with model DNA
4
2
oligonucleotides. Competitive dialysis experiments
were
performed with ten oligonucleotides representing prototypical
DNA structures (Table S4 in SI): single-strands, double-strands
and, especially, G-quadruplexes, representing different
topologies (parallel, antiparallel and hybrid), some of which are
sensitive to binders causing downregulation of oncogene
4
3
expression. Melamine adduct 11b interacts with all DNA
Figure 4. DNA Binding Studies of Multiple GBBR adducts: A, B) Competitive dialysis assays of ten oligonucleotide sequences with compounds 18b and 12. C)
Fluorescence titration of a 0.2 μM solution of the GBBR adduct 12 after addition of increasing amounts of oligonucleotides DS26, GG1 and 24bcl (from 0 to 10
μM) in potassium phosphate buffer.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
These results indicate that compound 12 has selective affinity to
quadruplex DNA structures, especially 24bcl (Figure 4B, C) and
may serve as a lead to develop new selective binders, a
promising way to generate novel anticancer drugs.
Conflict of interest
The authors declare no conflict of interest.
Conclusions
Keywords: azines •biological activity • isocyanides•
multicomponent reactions • novel scaffolds
In summary, we have developed a rationale for selective
multiple MCRs, by performing GBBRs involving heterocyclic
di/triamines, substantially expanding the scope for these
processes. Their feasibility is based on sequential processes
exploiting the higher reactivity along preferred evolutionary
pathways, discriminating between nearly identical functional
groups. The mechanism of these selective transformations was
established by means of computational methods. This
sequential mode allows the programmed incorporation of
substituents at up to 5/6 diversity points. As a proof of concept,
the resulting adducts, which would be impossible or extremely
challenging to prepare by alternative synthetic pathways, display
remarkable properties as antivirals, fluorescent probes, selective
DNA binders and nanometric blocks. Moreover, their fast and
controlled synthesis would enable the straightforward tuning of
their properties. The general character of this approach and the
applications of the ensuing scaffolds will significantly expand the
reach of selective multiple MCRs in biology and medicine
[
1]
Eds: J. Zhu, Q. Wang, M. Wang. Multicomponent Reactions in Organic
Synthesis, Wiley-VCH, Weinheim 2014.
[
2] E. Ruijter, R. Scheffelaar, R. V. Orru, Angew. Chem. Int. Ed. 2011, 50,
234-6246; Angew. Chem. 2011, 123, 6358-6371.
6
[3]
[4]
C. Hulme in Multicomponent Reactions, (Eds:J. Zhu and H. Bienaymé)
Wiley-VCH, Weinheim, 2005, pp. 311-341.
Ed: V. Nenajdenko, Isocyanide Chemistry: Applications in Synthesis
and Material Science, Wiley-VCH, Weinheim, 2012.
[
[
5]
6]
A. Dömling, Chem. Rev. 2006, 106, 17-89.
L. A. Wessjohann, R. A. Neves Filho, D. G. Rivera in Isocyanide
Chemistry: Applications in Synthesis and Material Science, (Ed: V.
Nenajdenko). Wiley-VCH, Weinheim, 2012, pp. 233-262.
L. A. Wessjohann, D. G. Rivera, O. E. Vercillo, Chem. Rev. 2009, 109,
796-814.
[
7]
[8]
L. A. Wessjohann, R. A. Neves Filho, A. R. Puentes, M. C. Morejon, in
Multicomponent Reactions in Organic Synthesis, J. Zhu, Q. Wang, and
M. Wang, eds. Wiley-VHC, Weinheim, 2014 pp. 231-264.
[
[
9] For a recent result, see: L. A. Wessjohann, O. Kreye, D. G. Rivera,
Angew. Chem. Int. Ed. 2017, 56, 3501-3505; Angew. Chem. 2017, 129,
3555-3559.
Experimental Section
10] N. Elders, D. van der Born, L. J. Hendrickx, B. J. Timmer, A. Krause, E.
Janssen, F. J. de Kanter, E. Ruijter, R. V. Orru, Angew. Chem. Int. Ed.
2009, 48, 5856-5859.; Angew. Chem. 2009, 121, 5970-5973.
Full account of the experimental procedures is provided in the
Supplementary Information. It includes experimental protocols,
compound characterization data, copies of the spectra, X-ray description
of some adducts, details on the computational studies, bioimaging
experiments, antiviral studies, and DNA binding determinations.
[11] D. G. Rivera, L. A. Wessjohann, J. Am. Chem. Soc. 2009, 131, 3721-
3732.
[12] S. Brauch, S. S. Van Berkel, B. Westermann, Chem. Soc. Rev. 2013,
42, 4948-4962.
[
[
[
13] F. Leon, D. G. Rivera, L. A. Wessjohann, J. Org. Chem. 2008, 73,
762-1767.
1
14] P. Janvier, M. Bois‐Choussy, H. Bienaymé, J. Zhu, Angew. Chem. Int.
Ed. 2003, 42, 811-814; Angew. Chem. 2003, 115, 835-838.
15] N. Christinat, R. Scopelliti, K. Severin, J. Org. Chem. 2007, 72, 2192-
2200.
Acknowledgements
We thank the funding of DGICYT-Spain (CTQ2015-67870-P,
CTQ2014-52588-R, SAF2017-88107-R, ERA-NET PCIN-2015-
[16] N. Isambert, R. Lavilla, Chem. Eur. J. 2008, 14, 8444-8454.
[
[
17] B. Roth, C. C. Cheng, Progr. Med. Chem. 1982, 19, 269-331.
18] G. Slater, L. M. Perdigꢀo, P. H. Beton, N. R. Champness, Acc. Chem.
Res. 2014, 47, 3417-3427.
2
24), and Generalitat de Catalunya (SGR 2014-137, 187, 1189
and 2017-1439), Instituto de Salud Carlos III, MINECO, the
Spanish Network for Research in Infectious Diseases (REIPI
RD16/0016/0009)- cofinanced by "A way to achieve Europe"
ERDF. S.C., on leave from the University of Genoa, thanks a U.
Genoa scholarship to support her stage. F.J.L. thanks the
Consorci de Serveis Universitaris de Catalunya (Molecular
Recognition) and the ICREA Academia. M. V. acknowledges
funding from the Biotechnology and Biological Sciences
Research Council (BB/M025160/1). We thank Prof. Juan C. de
la Torre (Scripps Research Institute, San Diego), Dr. Antonio L.
Llamas-Saiz (U. Santiago de Compostela), Prof. Carmen
Escolano (University of Barcelona) and Dr. Shoaleh Ghashghaie
for useful contributions.
[
[
19] K. Groebke, L. Weber, F. Mehlin, Synlett 1998, 661-663.
20] C. Blackburn, B. Guan, P. Fleming, K. Shiosaki, S. Tsai, Tetrahedron
Lett. 1998, 39, 3635-3638.
[21] H. Bienayme, K. Bouzid, Angew. Chem. Int. Ed. 1998, 37, 2234-2237;
Angew. Chem. 1998, 110, 2349-2352.
[
[
[
22] N. Devi, R. K. Rawal, V. Singh, Tetrahedron 2015, 71, 183-232.
23] R. Appel, H. Mayr, J. Am. Chem. Soc. 2011, 133, 8240-8251.
24] V. V. Tumanov, A. A. Tishkov, H. Mayr, Angew. Chem., Int. Ed. 2007, 46,
3563-3566; Angew. Chem. 2007, 119, 3633-3636.
[
[
25] All yields correspond to unoptimized reactions. Tuning these MCRs
processes is perfectly feasible but may require specific conditions for
each transformation. Hereby, we report the obtained results using the
general procedures, although particular modifications may lead to
improved yields.
26] D. Borthwick, Chem. Rev. 2012, 112, 3641-3716.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
[
27] L. Banfi, A. Basso, R. Riva, in Synthesis of Heterocycles via
Multicomponent Reactions I, (Eds: R. V. Orru, E. Ruijter). Topics in
Heterocyclic Chemistry. Springer, Heidelberg, 2010, pp. 1-39.
[35] A. Loudet. K. Burgess, Chem. Rev. 2007, 107, 4891-4932.
[36] W. Li, W. Lin, J. Wang, X. Guan, Org. Lett. 2013, 15, 1768-1771.
[37] P. Martínez-Aguado, A. Serna-Gallego, J. A. Marrugal-Lorenzo, I.
Gómez-Marín, J. Sánchez-Céspedes, Drug Discov. Today 2015, 20,
1235-1242.
[
[
[
[
28] H. Wang, T. Maiyalagan, X. Wang, X. ACS Catal. 2012, 2, 781-794.
29] Y. Zhao, D. G. Truhlar, Theor. Chem. Acc. 2008, 120, 215-241.
30] P. C. Hariharan, J. A. Pople, Theor. Chim. Acta 1973, 28, 213-218.
31] F. de Moliner, N. Kielland, R. Lavilla, M. Vendrell, Angew. Chem. Int. Ed.
[38] M. M. Y. Waye, C. W. Sing, Pharmaceuticals 2010, 3, 3343-3354.
[39]
A. Ali, S. Bhattacharya, Bioorg. Med. Chem. 2014, 22, 4506-4521.
2
017, 56, 3758-3769; Angew. Chem. 2017, 129, 3812-3823.
[40] D. Rhodes, H. J. Lipps, Nucleic Acids Res. 2015, 43, 8627-8637.
[41] G. W. Collie, G. N. Parkinson, Chem. Soc. Rev. 2011, 40, 5867-5892.
[42] R. Ferreira, A. Aviñó, R. Pérez-Tomás, R. Gargallo, R. Eritja, J. Nucleic
Acids 2010, Article ID 489060.
[
[
32] L. Levi, T. J. Müller, T. Chem. Soc. Rev. 2016, 45, 2825-2846.
33] Incidentally, GBBRs have allowed the preparation of a chemset of
fluorophores in droplet arrays: O. N. Burchak, L. Mugherli, M. Ostuni, J.
J. Lacapere, M. Y. Balakirev, J. Am. Chem. Soc. 2011, 133, 10058-
[43] K. I. E. McLuckie, Z. A. E. Waller, D. A. Sanders, D. Alves, R.
Rodriguez, J. Dash, G. J. McKenzie, A. R. Venkitaraman, S.
Balasubramanian, J. Am. Chem. Soc. 2011, 133, 2658-2663.
1
0061.
[
34] N. Boens, V. Leen, W. Dehaen, Chem. Soc. Rev. 2012, 41, 1130-1172.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201802877
FULL PAPER
FULL PAPER
Multiple & selective: A rationale for
selective multiple Multicomponent
O. Ghashghaei, S. Caputo, M. Sintes, M.
Revés, N. Kielland, C. Estarellas, F. J.
Luque, A. Aviñó, R. Eritja, A. Serna-
Gallego, J. A. Marrugal-Lorenzo, J.
Pachón, J. Sánchez-Céspedes, R.
Treadwell, F. de Moliner, M. Vendrell, R.
Lavilla*
Reactions
implemented in Groebke-Blackburn-
Bienaymé transformations with
was
developed
and
a
range of polyamino-polyazines to yield
novel chemotypes, fitted with up to 5
diversity points. The generated
adducts
performance as live cell fluorescent
probes, selective G-quadruplex
binders, and antiviral agents.
display
impressive
Page No. – Page No.
Multiple Multicomponent Reactions:
Unexplored Substrates, Selective
Processes and Versatile Chemotypes
in Biomedicine
This article is protected by copyright. All rights reserved.