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

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.


Introduction
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. [1][2][3] Isocyanides are pivotal in MCRs, leading to the most fruitful processes, the Ugi and Passerini reactions being paradigmatic examples. 4,5 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, peptoid-peptides, etc., was prepared. [6][7][8][9] 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 elaborated up to an 8CR. 10 Sequential versions of multiple MCRs have been conducted by either blocking an existing functional group or by gradually generating new functionalities along the process. 11,12 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 nonsymmetrical starting materials, the reactivity of the repeated functional groups should be kinetically distinct (Scheme 1A). Moreover, as the choice of multiple MCRs is rather limited to Ugi reactions (apart form few reported processes), 13-15 the expansion of their scope would be highly beneficial.
Furthermore, the use of heterocyclic inputs, privileged motifs in drugs, yields meaningful MCR adducts. 16 Polyaminopolyazines are attractive, yet unexplored MCR substrates. For instance, diaminopyrimidines are relevant in medicinal chemistry, 17 while melamine plays a key role in materials science. 18 The Groebke-Blackburn-Bienaymé reaction (GBBR), which involves the acid-catalyzed interaction of α-aminoazines, aldehydes and isocyanides to yield imidazoazines (Scheme 1B), [19][20][21] important adducts in drug discovery, 22 represents an ideal candidate to embed in a novel highly ordered MCR process. We hereby disclose our results on selective multiple GBBRs (Scheme 1C).

Multiple GBBRs: Reactivity and Scope
We first examined the GBBR of 4-chlorobenzaldehyde and cyclohexyl isocyanide (two equivalents each) with 2,4diaminopyrimidine 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 (Scheme 2A). Using a variety of common aldehydes and 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 aldehydes and two isocyanides of distinct reactivity. 23,24 Although unsymmetrical adducts were detected in overstatistical 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 nonsymmetrical 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 (36%, Scheme 2A, X-Ray in SI), enabling selective multiple 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%-93%) 25 and 5g-m (13%-57%) respectively, were formed using 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).
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.
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 7CR, featuring the formation of nine bonds in a single step. The selective formation of melamine non-symmetrical adducts is not yet feasible and studies towards this goal are ongoing.

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) calculations 29,30 (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 position 4. The 2-amino tetrahedral adduct is favored by 4.6 kcal/mol relative to the corresponding amino-4 species. Finally, 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.
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.

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 accessible through classical synthesis, 31 and MCRs constitute a valuable platform to afford them. 32,33 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 electrondonating groups at specific sites to generate push-pull fluorophores with bright fluorescence emission in the orange-tored 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).
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.

ii) Antiviral Activity of Multiple GBBR Adducts
We determined the therapeutic potential of multiple GBBR scaffolds 22 against human adenoviruses (HAdV). While responsible for mild diseases in healthy individuals, HAdV infections are associated with high mortality in immunosuppressed patients. 37 The lack of approved specific antiviral drugs with efficacy and safety against HAdV further complicates the treatment of these patients. 38 In this context, representative GBBR adducts (Table 1 and Figure S11 in SI) were tested against HAdV using different susceptibility assays.
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 (doseindependent, 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.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65 iii) Affinity of Multiple GBBR Adducts to DNA Since DNA displays a variety of different structural types, it is crucial to have selective binders to study their roles as a preliminary step to develop medicines. 39 Especially relevant are G-quadruplexes, because of their wide presence in the genome, mainly involved in the maintenance of chromosomes and in the transcriptional regulation of genes. 40 Topology-dependent ligands, selectively binding quadruplexes, will therefore have a significant impact on biological and medicinal chemistry. 41 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.
In this context, we explored a subset of our GBBR adducts (5b, 11b, 12 and 18b) in the interaction with model DNA oligonucleotides. Competitive dialysis experiments 42 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 expression. 43 Melamine adduct 11b interacts with all 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).
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. 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.

Conclusions
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