Volume 22

Number 18
14 May 2024

Organic & Pages 3535-3762

Biomolecular
Chemistry
rsc.li/obc

ISSN 1477-0520

PAPER
Brenno A. D. Neto et al.
Revisiting Biginelli-like reactions: solvent effects,
mechanisms, biological applications and correction of
several literature reports
Organic &
Biomolecular Chemistry

PAPER

Revisiting Biginelli-like reactions: solvent effects,

Cite this: Org. Biomol. Chem., 2024,
mechanisms, biological applications and
22, 3630 correction of several literature reports†
Pedro S. Beck, a Arthur G. Leitão, a Yasmin B. Santana,a José R. Correa,a
Carime V. S. Rodrigues,a Daniel F. S. Machado,a Guilherme D. R. Matos, a
Luciana M. Ramos,b Claudia C. Gatto, a Sarah C. C. Oliveira,c
Carlos K. Z. Andrade a and Brenno A. D. Neto *a

This study critically reevaluates reported Biginelli-like reactions using a Kamlet–Abboud–Taft-based

solvent effect model. Surprisingly, structural misassignments were discovered in certain multicomponent
reactions, leading to the identification of pseudo three-component derivatives instead of the expected
MCR adducts. Attempts to replicate literature conditions failed, prompting reconsideration of the
described MCRs and proposed mechanisms. Electrospray ionization (tandem) mass spectrometry, NMR,
melting points, elemental analyses and single-crystal X-ray analysis exposed inaccuracies in reported
MCRs and allowed for the proposition of a complete catalytic cycle. Biological investigations using both
pure and “contaminated” derivatives revealed distinctive features in assessed bioassays. A new cellular
action mechanism was unveiled for a one obtained pseudo three-component adduct, suggesting simi-
larity with the known dihydropyrimidinone Monastrol as Eg5 inhibitors, disrupting mitosis by forming
monoastral mitotic spindles. Docking studies and RMSD analyses supported this hypothesis. The findings
Received 19th February 2024, described herein underscore the necessity for a critical reexamination and potential corrections of struc-
Accepted 17th April 2024
tural assignments in several reports. This work emphasizes the significance of rigorous characterization
DOI: 10.1039/d4ob00272e and critical evaluation in synthetic chemistry, urging a careful reassessment of reported synthesis and bio-
rsc.li/obc logical activities associated with these compounds.

Introduction context of MCRs. The presence of multiple (at least three)

active reagents, various mechanistic steps, and the possibility
Today, there is no doubt about the significance of multicom- of multiple mechanistic pathways render MCRs particularly
ponent reactions (MCRs) as essential tools within the synthetic susceptible to the influence of solvent effects.2 The signifi-
organic toolbox. MCRs offer the potential to generate diversity, cance of solvent effects in all areas of chemistry is undeniable,
complexity, and quickly assemble libraries of bioactive com- as discussed in several reviews.3–5 While no one disputes this
pounds. They may also align with green principles and the pro- importance, only a handful of studies have made dedicated
spect of sustainable processes. As recently highlighted,1 efforts to unravel these fundamental and vital effects on
however, solvent effects have often been overlooked in the MCRs.6–11 If we delve into quantitative effects, the available
studies are even more limited.12–15 No one questions the
importance of solvent screening for determining the optimal
a
University of Brasilia, Institute of Chemistry, Laboratory of Medicinal and reaction conditions that promote chemical transformations
Technological Chemistry. Campus Universitário Darcy Ribeiro, Brasília, DF, and enhance productivities. But to advance the realm of
70910-900, Brazil. E-mail: [email protected]
b MCRs, detailed investigations into solvent effects become para-
Universidade Estadual de Goiás (UEG), Anápolis, Goiás, 75001-970, Brazil
c
University of Brasilia, Institute of Biology, Laboratory of Allelopathy, Campus mount. As an illustration, we have recently demonstrated that
Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil a simple change in solvent, such as using methanol, can
† Electronic supplementary information (ESI) available: Additional optimization modulate the mechanism of the Ugi 4-component MCR,
experiments, copies of 1H, and 13C{1H} NMR, HRMS and IR spectra for all com- directing it towards an alternative catalytic cycle.16
pounds. All computed structures, relative and absolute energies of all structures,
Catalysis is regarded as a fundamental tool for aligning
reaction energy profiles, and Cartesian coordinates. CCDC 2323129. For ESI and
crystallographic data in CIF or other electronic format see DOI: https://doi.org/ MCRs with the principles of green chemistry.17 Considerable
10.1039/d4ob00272e effort should be dedicated to achieving more efficient reac-

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Organic & Biomolecular Chemistry Paper

tions, bypassing the use of hazardous and toxic solvents, mini- strated for the Biginelli MCR.13 Since then, no “catalyst-free”
mizing waste, and exploring both efficient and convenient condition has been advocated. This “catalyst-free” issue is par-
purification methods. While MCRs possess inherent environ- ticularly curious, considering that the advantages of catalysis
mentally friendly qualities, there is still a substantial need for for conducting this MCR had already been described since the
scientific endeavors to advance sustainable processes.18–20 The original Biginelli reports at the end of the 19th century.39–42
path toward greener catalyzed MCRs is illuminated by the elu- The mechanism of the reaction is still a topic of intense
cidation of solvent effects, which also play a crucial role in con- debate, with three different routes currently proposed to
siderations of selectivities (enantio- and diastereoselectivity), explain this MCR.43 This indicates that controversies continue
mechanistic route selection, and the recovery of catalytic to be associated with the Biginelli multicomponent
systems.1 Given that several solvents that were previously used transformation.
are now subject to stricter regulations worldwide,21–25 it is A groundbreaking study44 has revealed the remarkable bio-
imperative to replace such solvents with environmentally logical activity of a Biginelli adduct, specifically a DHPM (3,4-
friendly, bio-based, and non-toxic alternatives.26–32 This re- dihydropyrimidin-2(1H)-one or -thione derivative) known as
placement commences by utilizing solvents that possess these Monastrol (see Scheme 1). Monastrol demonstrated the ability
desirable features in basic research.33–36 to inhibit the mitotic kinesin Eg5, a motor protein crucial for
The Biginelli reaction stands as one of the most extensively spindle bipolarity, ultimately leading to the formation of
studied MCRs.37 Discovered in 1891 by Pietro Biginelli,38 this monoastral spindles in mitotic cells and subsequent cell
reaction has been a subject of ongoing debate and discussion. death. This discovery has fueled the development of numerous
In several reports, there are discrepancies, such as the year of DHPM derivatives, most of which are in racemic form and are
discovery being noted as 1893, when, in fact, it was in 1891, synthesized directly through the Biginelli MCR. These deriva-
and two years later, Biginelli published the comprehensive tives exhibit potent antitumoral activities, as documented
accounts of his eponymous reaction.38 Another intriguing elsewhere.45–55 The wide range of biological properties associ-
aspect is that the original structure proposed by Biginelli ated with DHPMs has been comprehensively reviewed.56–58
himself was not a heterocyclic compound but an open struc- Because of the appealing biological activities observed in
ture that required revisitation (see Scheme 1). various DHPM derivatives,59 their straightforward synthesis,
More recently, the reaction has been carried out under the and the rapid generation of libraries of bioactive compounds,
so-called “catalyst-free” conditions, but the beneficial effect variations of the Biginelli reaction, often referred to as
and importance of catalysis have been indisputably demon- Biginelli-like (or Biginelli-type) reactions, have been documen-
ted and extensively reviewed.60–66 These adaptations of the
Biginelli reaction encompass a range of strategies that involve
modifying a specific reagent among the three components or
replacing one (or two) of them,67–77 as illustrated by a few
examples in Scheme 2. Much like the Biginelli reaction, these
variations also exhibit their unique characteristics, including
distinct mechanistic pathways and others. However, these vari-
ations also exhibit enduring polemic issues.
In this study, we present our findings concerning solvent
effects on Biginelli-like MCRs, the mechanistic pathways of
these transformations, and associated side reactions. We also
advocate for a comprehensive correction of the vast existing lit-
erature on this topic. Within this context, we demonstrate that
certain structures attributed to MCRs have been inaccurately
assigned. Given that these structures are currently employed in
biological evaluations, yielding distinct yet positive effects,
there is an urgent need for their reassessment.

Results and discussion

Our investigation employed various analytical techniques and
models, including Kamlet–Taft solvent descriptors, NMR ana-
lysis, IR spectroscopy, single-crystal X-ray analysis, high-resolu-
Scheme 1 From top to bottom: the original synthesis and structure
tion mass spectrometry, and DFT calculations. These analyses
initially proposed for the first reported Biginelli reaction, the presently
accepted Biginelli MCR, and examples of bioactive derivatives (dihydro-
have exposed a significant issue within the scientific literature,
pyrimidin-2(1H)-thiones – DHPMs) directly obtained through the necessitating an immediate clarification and rectification of
Biginelli MCR. these studies.

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Paper Organic & Biomolecular Chemistry

Scheme 2 Examples of Biginelli-like multicomponent transformations: (I) 1,4-dihydropyrimidine (DHP) synthesis,78–81 (II) 2-amino-3-cyano-4H-
pyrans (ACP) derivatives,82,83 (III) 3,4-dihydro-2H-chromeno[4,3-d]pyrimidine-2,5(1H)-dione or thione (CPD) derivatives,82,84–102 and (IV) pyrano[3,2-
c]chromen-5(4H)-ones (PCC) compounds.103–106

We initiated the assessment of solvent effects using a

model reaction for the synthesis of CPD derivatives (Scheme 2-
III). Equimolar amounts of urea, 4-hydroxycoumarin, and
benzaldehyde were employed as starting reagents to produce
CPD-01 (with X = O and R1 = Ph, as shown in Scheme 2-III). In
pursuit of more sustainable catalytic approaches, we selected a
superacid imidazolium-based ionic liquid known for its high
activity in MCRs107 (see Scheme S1 and Table S1 in the ESI†
for the structure of the catalyst MSI3PW). Ionic liquids are cur-
rently regarded as sustainable alternatives in various industrial
processes, as extensively reviewed elsewhere.108 These organic
salts have been utilized in the chemical industry as solvents
and catalysts for nearly two decades,109 and their environmen-
tally friendly and advantageous physicochemical properties,
combined with their customizable characteristics, justify the
increasing industrial interest in using these compounds as
alternatives to toxic and hazardous solvents or catalysts.110–115
The reaction conditions were subsequently optimized in Fig. 1 Melting points descriptions for the claimed structure of CPD-01.
water as the solvent, considering various parameters such as See Table S2† for the references.
temperature, catalyst amount, and reaction time (see Fig. S1†)
to synthesize CPD-01. The optimal conditions were established
as follows: 1.00 mmol of each reagent, 0.5 mL of the solvent, parameters,117–120 were initially tested under the optimized
5 mol% of the catalyst, 80 °C, and a reaction time of 60 min. conditions (Table S1†) to depict mechanistic information, but
Table S1† provides a summary of the results obtained. During with no intention to use them as solvents for this MCR after
the reaction, a solid precipitated, which could be purified by reaching a greener condition. Reactions requiring excess
washing it with a mixture of ethanol and water (1 : 1 v/v). The reagents were also tested only to gain knowledge on the
solid was subsequently dried and initially characterized by its solvent effects over the reaction mechanism (Fig. S2†), but our
melting point, yielding a value in the range of 160–162 °C. goal was to establish an appropriate reaction condition to
The literature, however, contains various different values for avoid any excess and aiming at reaching the lowest possible E
the melting point of CPD-01 (as shown in Table S2† and factor (Table S1†).121–123
Fig. 1), prompting us to investigate the underlying reasons for In the Biginelli MCR38 (Scheme 1), a 1,3-dicarbonyl reagent
this discrepancy. As recently discussed in the context of is typically employed (e.g., ethyl or methyl acetoacetate).124–135
MCRs,116 melting point characterizations can potentially lead This reagent is reactive in its enol tautomeric form, but not in
to erroneous conclusions when not supported by additional the diketo form, as demonstrated in the catalyzed (HCl 10%)
analyses. version disclosed in the groundbreaking publication by
Eleven different solvents, including some toxic solvents Sherwood and co-workers.12 Catalyst-free versions, conducted
for gauging a wide range of Kamlet–Abboud–Taft in alternative green solvents, follow the same trend, where

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Organic & Biomolecular Chemistry Paper

only the enol tautomeric form is reactive, as demonstrated by The three possible and proposed mechanisms of
us13 and others.15 In these cases, the dipolarity/polarizability this Biginelli-like transformation (Scheme 3) already avail-
(π*) was the Kamlet–Taft (KT) descriptor with the most signifi- able88–91,93,141 (and some close variants82,142,143) could in
cant influence on the reaction outcome, particularly on achiev- principle help to understand the observed solvent effects,
ing higher productivities. This is because the concentration of although no experimental evidence has been so far provided
the enol tautomeric form dictates the final yields. Therefore, for this specific MCR transformation to the best of our
the application of a solvent with low values of π* (such as knowledge.
p-cymene as a green alternative)12 was crucial for restoring the Following the principles of organic chemistry transform-
reactive enol tautomeric form and advancing the MCR. ations, it is reasonable to exclude the enamine pathway (see
In the current evaluated Biginelli-like reaction, the “key Scheme 3) without the need for further investigation. This is
reagent” (i.e., 4-hydroxycoumarin) is predominantly found in particularly evident because the enol tautomer is predomi-
its enol tautomeric form to sustain the conjugated (aromatic) nantly present. Notably, the Kamlet–Taft β parameter emerges
structure of the molecule, which is considerably more as the primary solvent descriptor that influences the rate of
stable.136 The enol tautomeric form is found almost exclu- the Knoevenagel condensation, as documented in the existing
sively, regardless of both the solvent used and its Kamlet–Taft literature.144 Upon examining Table S1 (and Fig. S2†), it
parameters, and this is why this reagent is known for display- becomes apparent that higher values of α promote product for-
ing good-to-excellent nucleophilic character.137–139 This aspect mation, thus implying the iminium pathway as the preferred
circumvents the reactivity issue and expands the opportunity route for CPD formation, rather than the Knoevenagel route
to explore several other solvents. The three tautomeric forms of typically associated with solvents characterized by high β
4-hydroxycoumarin have been thoroughly reviewed elsewhere.140 values. In another study,145 it was indicated that depending on

Scheme 3 Three previously proposed reaction pathways for the Biginelli-type reaction studied herein which provide plausible explanations for
CPD-01 formation under acidic catalytic conditions.

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Paper Organic & Biomolecular Chemistry

the catalytic conditions, π* was identified as the key Kamlet– ratios in ESI analyses and enabling a more comprehensive
Taft parameter, and low-polarity solvents performed better. monitoring of catalyzed reactions.150 Comparable outcomes
However, α showed no discernible correlation. were noted when employing benzaldehyde instead of the
On the surface, the results from our experiments may charge-tagged aldehyde (Fig. 2).
appear logical and promising in elucidating the transform- During the reaction monitoring (Fig. 2), notable signals
ation (Table S1 and Fig. S2†). However, a study12 has pre- include the protonated aldehyde of m/z 107, the protonated
viously demonstrated that the values of the α descriptor are coumarin of m/z 163, the Knoevenagel intermediate of m/z
generally statistically insignificant in terms of Biginelli reac- 251, the bisureide derivative of m/z 209 (from the iminium
tion productivity. As a result, the findings presented here ion of m/z 149), and the presence of a dicoumarol deriva-
appear to contradict the expected behavior derived from a tive (m/z 413 and 435 for the sodiated derivative). Of inter-
robust solvent effect model, therefore making our results highly est is the ion of m/z 333 (sodiated) related to an advanced
questionable. The literature12,144,145 has indeed highlighted intermediate, from which cyclization should occur to yield
both β (favoring Knoevenagel condensations) and π* (favoring the final MCR adduct, i.e., the CPD-01. No other discern-
the iminium route) as critical descriptors for this transform- ible ions related to potential intermediates (Scheme 3)
ation or for the formation of any key intermediate (i.e., the were observed. All attempts to observe the CPD derivative,
iminium or the Knoevenagel intermediates). However, our both with and without a charged-tagged aldehyde, were
findings contrast with these published and well-conducted unsuccessful.
works, as we identify α from the Kamlet–Taft parameters as At this point, it became evident that the reaction did not
the primary factor influencing productivity in the synthesis of progress towards the formation of CPD scaffolds, not even
CPDs. through a concurrent mechanism (iminium- or Knoevenagel-
At this juncture, a comprehensive analysis of the results like, as illustrated in Scheme 3). Under the tested conditions
and a comparison with the existing literature became impera- (Table S1†), it seemed that only dicoumarol derivatives (DCs)
tive. NMR (1H and 13C, as shown in Fig. S3†), revealed certain were actually being produced (Scheme 4). The synthesis failure
issues. Notably, the chemical shifts in the NMR, especially for of CPD derivatives, previously mentioned in the literature151
the benzylic hydrogen, were excessively deshielded (exceeding but unexplored, resulted in the formation of only a DC deriva-
6.00 ppm when it should be approximately 4.00 ppm). tive. It also elucidates our results from elemental analysis
Additionally, while one would anticipate 15 carbon signals, (Table S3†), indicating that the low nitrogen content arises
only 14 were observable, potentially due to the overlapping of from urea contamination. The difference between the calcu-
one of the aromatic signals. High-resolution electrospray lated and obtained values for nitrogen content is indeed sig-
(tandem) mass spectrometry analyses – ESI-MS(/MS) – were nificant, as shown in Table S3.† It does not account, however,
conducted and yielded no signals corresponding to the for the nearly perfect match of the CHN content described
expected ionic species with a m/z of 293 (for the protonated in the literature for some reports (see Table S7† for the
CPD-01). We also attempted both high resolution MALDI and references).
EI ionization methods to detect signals of m/z 293 (for MALDI) These results also contribute to explaining the diversity of
or m/z 292 (for EI), but these procedures were unsuccessful. As melting points described (see Fig. 1 and Table S2†), indicat-
a final effort, elemental analyses were undertaken to deter- ing varying urea content as a contaminant of the claimed
mine the composition of the sample and compare it with the CPD-01, which was indeed DC-01. In due course, we will
literature. The results indicated a low content of nitrogen, sig- revisit the Kamlet–Taft analysis for this reaction. But we can
nificantly below what was expected for the claimed structure of anticipate that the formation of the DC derivative accounts
CPD-01 (Table S3†). To ensure the reproducibility of these for the significance of the α descriptor. In attempting to repli-
results, several CPD derivatives (see Scheme S2†) were syn- cate numerous previously reported conditions for the CPD
thesized and analyzed. All of these data are presented in synthesis (see details in the Experimental section), we con-
Table S3 and are compared with the available literature ( please sistently obtained the DC derivative. Intriguingly, one of
refer to the cited references and the ESI†). In our assessment, these reports describes an enantioselective CPD synthesis
however, the data did not align with the claimed structures of with high enantiomeric excess (ee) values, employing
the CPD derivatives. L-proline as the chiral catalyst. The lack of accessible HPLC
To explore the events unfolding during the reaction, we (or GC) for ee confirmation, coupled with the presented NMR
monitored it using ESI-MS(/MS), a technique renowned for its spectra, however, consistently supports the identification of
effectiveness in tracking MCRs.43 This method stands out for nonchiral DC derivatives.
its capability to provide continuous snapshots of the solution Simultaneously, our attempts to obtain PCC, ACP (see
phase, to facilitate a gentle transfer to the gas phase, and Scheme 2) and other Biginelli-like derivatives were ongoing.
detect transient intermediates and adducts.146–149 In this Despite some differences that we will elucidate in due course,
work, we also employed an aldehyde derivative with a charge we predominantly obtained only the DC derivative. These
tag in its structure to enhance detection and prevent the results point to a notable concern in the scientific literature
escape of any intermediate during the reaction (Fig. S4–S6†). regarding Biginelli-like reactions, predominantly when
This approach is recognized for enhancing signal-to-noise 4-hydroxycoumarin is involved as one of the components.

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Organic & Biomolecular Chemistry Paper

Fig. 2 ESI(+)-MS(/MS) monitoring of the MCR employing benzaldehyde, urea and 4-hydroxycoumarin. (A) 5 min of reaction. (B) 45 min of reaction.
(C) Detected structures and their calculated exact mass. Signals of m/z 209 and 231 are of very low intensities. The ion of m/z 305 corresponds to
the reversible addition of a methanol molecule in the gas phase. The asterisk denotes fragments from other ions.

Scheme 4 Attempts to form CPD-01 only resulted in the dicoumarol derivative DC-01, which was isolated but contaminated with urea. Several
catalytic conditions were tested and their details are provided in the Experimental section and Table S1.†

The situation regarding PCC derivatives was particularly form PCC-01 (see references in Scheme 2) are identical to
concerning, notably because the derivative PCC-01 (R1 and R2 DC-01, indicating a structural misassignment.
= Ph, Scheme 2) has been previously reported from a few auth- The case of ACP derivatives160–162 yielded promising results.
entic bimolecular reactions,152–159 and the comprehensive Despite the extensive volume of approximately 250 references
characterization of this derivative was readily available. Despite detailing the MCR formation of ACP derivatives, and the labor-
our efforts, all attempts to obtain PCC derivatives in an MCR ious task of scrutinizing each of them, we found alignment
fashion (see Table S8†) were unsuccessful, yielding only DC-01 with the DC structure description in only a few instances (as
in all attempts (Scheme 5). Interestingly, in one of the trials, a cited in Scheme 2). In a few other cases, the available charac-
crystal suitable for X-ray analysis formed in the reaction terizations did not allow for a more precise conclusion. This
medium, and DC-01 was obtained in high yield and undoubt- singular convergence indicates a degree of reliability in the
edly characterized. All available spectra (including spectro- characterization of this particular MCR class of compounds
scopic descriptions) from the three-component reaction to involving a coumarin as one of its components. For this multi-

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Paper Organic & Biomolecular Chemistry

Scheme 5 MCR attempts to form PCC-01 only resulted in the dicoumarol derivative DC-01. PCC-01 can be obtained through known bimolecular
reactions. DC-01 was obtained by mixing 1.00 mmol of each reagent, 5 mol% MSI3PW (catalyst), in 1 mL of the selected solvent at 80 °C for 60 min.
The bimolecular version of the reaction was carried out under stirring by mixing 1.00 mmol of each reagent and heating the mixture in a sealed flask
for 3 h at 120 °C.

component synthesis, however, we observed a pH dependence. Scheme 6 also depicts a reaction in which 5,5-dimethyl-1,3-
In our experiments, we observed a preference for ACP-01 for- cyclohexanedione was employed as the third component,
mation under basic conditions, whereas under acidic con- resulting in the preferential formation of the desired multi-
ditions, the DC-01 derivative was preferentially obtained component adduct TCC-01. Although a small amount of DC-01
(Scheme 6). was also generated, it could be readily removed through recrys-

Scheme 6 (Top) Concurrent formation of ACP-01 and DC-01 depending on the catalytic conditions. For example, using 1.0 mmol of each reagent,
EtOH as the solvent, and HCl (10 mol%) or MSI3PW (5 mol%) as the catalytic system, DC-01 is formed in 81% yield (considering 0.5 mmol of the cou-
marin) in 30 minutes at 80 °C. By switching the catalyst to t-BuOK, ACP-01 is obtained in 70% yield. (Center) Preferential formation of TCC-01
(10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-dione derivative) under acidic conditions at 80 °C in water for 2 h. (Bottom) Preferential for-
mation of DC-01 (1.0 mmol of each reagent), EtOH as the solvent, and SLS (10 mol%) or MSI3PW (5 mol%) as the catalyst, with no TCP-01 obtained.

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Organic & Biomolecular Chemistry Paper

tallization during the purification step of TCC-01. This result Table 1 Solvent screening aimed at reducing DC-01 formation.
confirms the accuracy of the literature’s description163 regard- Reactions were carried out using 4-hydroxycoumarin (2.0 mmol) and
benzaldehyde (1.0 mmol), MSI3PW (5 mol%), 1.0 mL of the solvent, and
ing the formation of TCC derivatives through a multicompo-
60 min
nent approach, with DC-01 observed by us as a byproduct
formed in small amounts.
A comparable multicomponent Biginelli-like reaction,
wherein 2-aminobenzothiazole substitutes the 4-hydroxycou-
marin, has also been documented in the literature.84,164–169
Despite our attempts to reproduce various conditions for
synthesizing the MCR adduct (Scheme 6), all efforts yielded
DC derivatives, even when attempting to replicate the docu-
mented literature conditions, no TCP was obtained. Analysis of
the available NMR spectra (or spectral descriptions) strongly
suggests the formation of DC rather than any other MCR
adduct.
Considering all these findings, we decided to explore con-
ditions for synthesizing CPD-01. In this context, one of our Yields below 20% were arbitrarily highlighted. a Experiment repeated
objectives became to exploit the solvent effect to prevent the several times.
formation of the DC-01 derivative by the second coumarin
addition. This involved facilitating the addition of urea to the
Knoevenagel intermediate (Scheme 3) or promoting the dipoles. Considering the protonation of reagents and the pres-
addition of coumarin to the iminium intermediate (Scheme 3). ence of polar intermediates in the reaction conducted under
These efforts aimed at favoring the formation of the advanced acid catalysis, this descriptor should be taken into account in
intermediate through either of these two reaction pathways synthesizing DC-01 through a double coumarin addition to
(Scheme 3). Subsequently, the advanced intermediate could the aldehyde, via the formation of a Knoevenagel intermediate.
undergo cyclization, ultimately resulting in the synthesis of Table 1 also shows that the best results are obtained in sol-
CPD-01. vents with π* ranging from middle to high values.
This situation is indeed unusual, as typically, conditions The α descriptor returned a relatively significant correlation
are sought to improve yields and selectivities rather than lower (Fig. 3 and Table 1). For all good yields, however, solvents had
them. In this case, however, the lower the yield of the DC relatively high values of α. Water, with the highest α value
derivative, the better. Although we had previously ruled out among all tested solvents, produced the desired product in
the enamine pathway (Scheme 3), ironically, it is now the con- one of the highest yields and under the most appropriate con-
dition we aim to favor. The final cyclization step or an initial ditions, i.e., using no reagent excesses. The presence of polar
urea addition to the coumarin could, in principle, align with reagents/intermediates capable of solvent-solute interactions
our goal of obtaining CPD-01, even though it is now dependent through hydrogen bonds and the stabilization of charged
on the keto form of the coumarin reagent, which is found in a intermediates in an acidic medium also shed some light on
neglected concentration, as we discussed earlier in this work. the observed effect. The literature suggests that Michael
The results from this solvent exploration are shown in Table 1. additions are favored by high values of α,170 and the second
Results from Table 1 revealed intriguing patterns. Solely by step for DC formation involves a Michael addition to the
accounting for the solvent effect (Fig. 3), a significant drop in Knoevenagel intermediate. Another study171 suggests that α
yield, from 95% (Table 1, entry 9) to 1% (Table 1, entry 24), and β play a role in this type of addition in a polymerization
was observed, aligning with our expectations. The most reaction.
effective solvents in reducing DC-01 formation were those with In this context, the formation of DC relies on a combi-
low values of β descriptors (see Table S9†) because high values nation of several factors, where α, β, and π* contribute to
of β tend to favor the coumarin addition followed by water improved yields. The β descriptor facilitates Knoevenagel for-
elimination (Knoevenagel condensation). Our results now mation (initial stage), while α also aids in the Michael
align with the literature,144,145 as low values of β led to low addition (last stage). The π* descriptor is recognized to influ-
yields of DC-01, as intended, reaffirming the significance of ence the nucleophile reactivity in these reactions. However, in
the solvent in favoring a specific reaction pathway. Fig. 4 the present case, coumarin does not appear to be signifi-
initially shows that the β descriptor alone, however, has little cantly affected, but rather, the stabilization of intermediates
significance in the reaction outcome for obtaining DC-01, and and transition states (TSs) seems to play a role. A multivariate
this correlation is not statistically significant for any reaction analysis was then conducted, clearly demonstrating the
condition evaluated in this work. importance of considering all three parameters, as depicted
The analysis of π* does indicate a relatively significant cor- in Fig. 3D.
relation in the synthesis of DC-01. This parameter also reflects Now, back to our endeavors to synthesize CPD-01, in an
the solvent’s ability to aid in the stabilization of charges or effort to leverage the temperature effect, we opted for

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Paper Organic & Biomolecular Chemistry

Fig. 3 Correlations of reaction productivity (expressed as ln(P)) as a function of the Kamlet–Taft parameters: (A) α, (B) β, (C) π* and (D) multivariate
correlations considering α, β and π*. All data pertain to the isolated yields of the model reaction for DC-01 formation (benzaldehyde and coumarin
mixtures, 5 mol% of the catalyst MSI3PW at 80 °C for 60 min).

p-cymene and limonene as solvents (low values of β), given after 30 minutes during reaction monitoring. The
their high boiling points. Despite conducting reactions in the Knoevenagel intermediate (m/z 308) was observed at very low
temperature range of 80–140 °C, CPD-01 formation was not abundance, signifying two key aspects: (i) successful avoid-
observed at all. Utilizing p-cymene as the solvent, we employed ance of its formation exclusively by solvent selection, and
ESI-MS(/MS) reaction monitoring with a charge-tagged alde- (ii) when formed, immediate consumption, leading to the
hyde to observe the heterocyclic formation (Fig. 4). While DC derivative. This is highlighted by the low intensity appear-
DC-01 formation was avoided for a period of time, no CPD was ance of ions of m/z 470 and 492 (sodiated) related to the DC
detected. Similar outcomes were noted with limonene as the adduct. Similar outcomes were observed by switching the
solvent (Fig. S4 and S5†). We also explored reactions by solvent from p-cymene to limonene (Fig. S4 and S5†) or other
employing 4-hydroxy-6-methyl-2-pyrone as the reagent instead solvents with lower values of π*. Changing the coumarin to
of 4-hydroxycoumarin (Fig. S6†), but only the advanced inter- the pyrone reagent (Fig. S6†) or to 4-aminocoumarin (Fig. S7†)
mediate could be detected, not the final MCR adduct. yielded comparable results.
The reaction was initiated by mixing the charge-tagged In our ongoing efforts to address this issue, we explored the
aldehyde and urea to promote the iminium ion formation substitution of the 4-hydroxy group in the coumarin reagent
while avoiding the Knoevenagel adduct. As shown in Fig. 4, with urea (Scheme 7). This modification resulted in the for-
this strategy proved effective, favoring the bisureide deriva- mation of the 4-aminocoumarin derivative with yields exceed-
tive of m/z 266 formed through the second urea addition to ing 80%, along with a few minor byproducts. Among these
the aldehyde. This aligns with literature12 findings that indi- byproducts, we isolated less than 5 mg of a guanidine deriva-
cate a preference for the iminium pathway when p-cymene tive during a gram-scale synthesis. The guanidine-coumarin
is the solvent. Following 15 minutes, the third component derivative was characterized using ESI-MS/MS and employed to
(4-hydroxycoumarin) was introduced, promoting the for- monitor the reaction in the gas phase, with the goal of generat-
mation of the advanced intermediate of m/z 368, evident ing a CPD analogue structure similar to the original

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Organic & Biomolecular Chemistry Paper

Fig. 4 (Top) ESI(+)-MS monitoring using a charge-tagged aldehyde derivative in p-cymene aiming at the multicomponent synthesis of a CPD het-
erocycle. (A), (B), (C), (D), (E), (F), (G), (H), and (I) represent different reaction times, corresponding to 5, 10, 15, 20, 40, 60, 90, 120, and 240 min,
respectively. (Bottom) Structures detected in the experiment and their calculated exact masses. Please note that the ion of m/z 196 is formed by the
reversible methanol (the solvent in the ESI process) addition in the gas phase.

(Scheme 3) following an enamine-like reaction pathway. opted to conduct the reaction in water. This choice was motiva-
Application of the developed conditions led to the formation ted by the fact that the final DC products precipitate in this
of an unprecedented guanidine-containing CPD analogue, solvent, allowing for easy separation from the catalyst in the
which was monitored in the gas phase and characterized by aqueous medium and facilitating the reuse of the water-
MS/MS (Fig. 5). To the best of our knowledge, this is the first soluble catalytic system. The catalytic system notably demon-
report of a CPD-like structure coherently characterized by its strated sustained activity through at least three cycles with
fragmentation pattern. yields reaching up to 90%. This class of DC compounds is
Building upon our prior efforts (Table 1), we have now well-known for various biological activities,172–174 including
chosen to synthesize a few DC derivatives (Scheme 8) to verify antibacterial properties,175 inhibition of lipoxygenase enzymes,176
some biological properties. Although one of the tested ionic anticoagulant effects,177 antidiabetic effects,178 and many
liquids yielded slightly better results (Table 1, entry 9), we others, as recently reviewed.179

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Paper Organic & Biomolecular Chemistry

Scheme 7 Synthesis of a guanidine derivative (top) and a CPD analogue (bottom).

All synthesized DC derivatives (Scheme 8) were obtained in nant. This technique is often suggested as the initial step in
good yields. Based on all the data obtained in this work, a discovering potential new herbicides due to its high sensi-
plausible reaction mechanism could be proposed to explain tivity,181 with wheat coleoptile bioassays commonly used to
the formation of DC compounds in the presence of the three identify herbicidal compounds. The use of pure DC deriva-
components (urea or thiourea as contaminants) or simply by a tives (Fig. 6), synthesized without urea/thiourea, yielded
pseudo-multicomponent transformation using two equivalents expressive results. Comparable outcomes were observed
of coumarin, thereby also elucidating the unsuccessful when testing these DC derivatives contaminated with urea
formation of CPDs in these solvent-dependent equilibria or thiourea (Fig. S8†).
(Scheme 9). In the conducted assays, it was observed that the majority
A brief discussion on the biological activities of the pur- of DCs significantly inhibited coleoptile growth, with DC-03
ported CPD derivatives should also be included. While we do standing out and exhibiting an IC50 lower than that of
not question the reported biological activities, particularly con- Logran® (commercially available – see Fig. S8†), which was
sidering the well-established bioactivity of DC derivatives, a used as a positive control. These results suggest a remarkable
reassessment of the structure of these biologically active small potential for the biological activity of these adducts, as most
molecules may be warranted. This reevaluation could shed compounds demonstrated the inhibition of coleoptile growth.
light on biological responses such as antitubercular92 and anti- It is important to realize that this assay represents only a pre-
viral97 activities claimed for the supposed CPD derivatives. liminary stage, making it essential to conduct additional
CPDs activities against tumor cells have also been recently studies to comprehensively evaluate the herbicidal activity of
reviewed.180 these products.
Finally, we decided to assess the biological activities of We also evaluated DC-03 (both in its pure form and when
DC derivatives synthesized using the pseudo-three-com- contaminated) as an antitumoral agent. The aldehyde
ponent approach, as well as those synthesized through the employed in the synthesis of this DC derivative, namely pipero-
procedure aimed at evaluating the possible synthesis of nal, is identical and bears substituents at specific positions
CPDs (in the presence of urea/thiourea), to investigate any that have been proven to be essential for the biological activity
possible influence on the biological outcomes of an identi- of a recognized Biginelli DHPM adduct known as Piperastrol
fied contaminant. The selected initial assay for this study (structure shown in Scheme 1).182,183 The results obtained with
was the etiolated wheat coleoptile bioassay, renowned for its contaminated DC-03 (Fig. S9†) and pure DC-03 (Fig. 7) were
rapidity in providing preliminary results within 24 h and its similar and comparable to those observed with Monastrol
high sensitivity. Sensitivity was a crucial factor for our objec- (Scheme 1), the DHPM used as the positive control in the
tive of assessing the influence of urea/thiourea as a contami- experiment.

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Organic & Biomolecular Chemistry Paper

Scheme 8 Synthesis of certain DC derivatives under optimized

conditions.

results using DOCK6184,185 and the Monastrol-bound Eg5 (PDB

1Q0B)186 indicated that both Monastrol and DC-03 bind to Eg5
with similar poses (Fig. 8) and binding scores of −45.55 and
−39.27 kcal mol−1, respectively.
Fig. 5 High resolution ESI(+)-MS/MS characterization of the guanidine- Due to its larger size, DC-03 can penetrate deeper regions
coumarin derivative (top) and of the guanidine-CPD analogue (bottom). in the binding site, as confirmed by DOCK6’s energy footprint
(Fig. 9). While Monastrol forms stronger electrostatic inter-
actions with the backbone of GLU118 and GLU116 (hydrogen
In the experiment, cell cycle arrest is observed in the bonds at 1.897 Å and 1.930 Å), DC-03 establishes weaker
monoastral mitotic spindle in the presence of both com- electrostatic interactions with ARG119 and GLY117 (at 3.266 Å
pounds (i.e., DC-03 and Monastrol). This result strongly and 2.549 Å). DC-03, however, maintains stability in the
suggests the inhibition of KSP/Eg5 kinesin activity in vitro, binding site through hydrophobic interactions with additional
leading to the appearance of monoastral spindles in accord- residues such as PHE239 and ALA218. Fig. 9C illustrates that
ance with the literature.44 This effect traps cells in the G2/M both inhibitors occupy identical binding sites, however, DC-03
phase and ultimately leads to cell death. The negative control exhibits additional interactions compared to the positive
shows the normal spindle apparatus during the metaphase of control Monastrol.
mitosis in the tumoral MCF-7 cells. It is observed as a bipolar Docking provides only a static snapshot of the system, and
position of centrosomes, with microtubules emanating from DOCK6’s Continuous Energy scoring function does not
opposite cell poles coupling opposing tension forces, align- account for conformational entropy and solvation/desolvation
ing chromosomes at the cell equator, and preparing them for effects. Ligand stability in the binding site can be more accu-
segregation to daughter cells. These features are inhibited by rately assessed through molecular dynamics (MD) simulations.
Monastrol and now, for the first time, described for a DC Two unrestrained 10 ns MD simulations were then conducted
derivative. using GROMACS 2020 to evaluate the stability of both ligands
One significant observation was the heightened density of within the binding site by measuring the RMSD with respect
microtubules in the monoastral mitotic spindle of cells treated to the initial production state. Each ligand was assigned
exclusively with the DC-03 small molecule. To better support GAFF2187,188 parameters and AM1-BCC189,190 partial charges
these findings, docking studies were then conducted. Docking using AmberTools22191 Antechamber.192 AmberTools’ tleap

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Paper Organic & Biomolecular Chemistry

Scheme 9 Plausible catalytic cycles and equilibria observed under catalytic acidic conditions that explain the preferential formation of DC deriva-
tives and the unsuccessful formation of CPD heterocycles. Note that these equilibria are solvent-dependent.

Fig. 6 Results of growth inhibition of coleoptile fragments for the treatments with selected DC derivatives.

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Organic & Biomolecular Chemistry Paper

Fig. 7 Cellular division (mitosis) inhibition of MCF-7 cells type by DC-03 (top) and by Monastrol, the positive control (center) and the negative
control (bottom). (A), (D) and (G) Nuclei stained with the commercially available DAPI (blue emitter). (B), (E) and (H) Anti-α-Tubulin monoclonal anti-
body (commercially available red emitter). (C), (F) and (I) Merged images from (A), (B), (D), (E) and (G), (H), respectively. Note that DC-03 induced
monoastral spindles in mitotic cells, similar to the positive control.

Fig. 8 Docked poses of Monastrol (A) and DC-03 (B) in Eg5 (PDB 1Q0B). CE represents DOCK6’s Continuous Energy score. Only relevant portions
of the residues and their side chains are displayed and hydrogen atoms bonded to carbon atoms are hidden. Magenta dashed lines indicate the most
significant electrostatic interactions between each ligand and the binding site. Panel (B) illustrates that DC-03 penetrates deeper into Eg5’s binding
pocket. (C) Overlay of Monastrol and DC-03 binding sites on Eg5.

was utilized to assign ff14SB parameters,193 protonate the stat to ensure proper sampling in the NPT ensemble. RMSD
protein, generate the receptor–ligand complex, and solvate the analysis of the MD trajectory revealed that both Monastrol and
system with TIP3P water.194 The resulting files were converted DC-03 maintain their poses in the binding site throughout the
to the GROMACS format using ParmEd.195 Production runs fol- 10 ns production run (Fig. 10). Backbone RMSDs also suggest
lowed one minimization and four equilibration stages (see that neither ligand induces significant structural changes in
details in the ESI†) and were conducted at 298 K and 1 bar Eg5’s backbone, supporting the hypothesis that DC-03 and
using a Langevin integrator and the Parrinello–Rahman baro- Monastrol may act similarly.

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Paper Organic & Biomolecular Chemistry

tions. The initial, seemingly contradictory results, however, led

us in a different direction, highlighting a crucial issue. A com-
prehensive examination of literature reports, along with their
spectroscopic and spectrometric data, and the appropriate
application of Kamlet–Taft parameters allowed us to identify
structural misassignments in a few Biginelli-like reactions. In
these cases, dicoumarol derivatives were likely forming instead
of the expected MCR adducts, and the presence of urea (or
thiourea) contaminants resulted in lower melting points. Our
attempts to reproduce the literature conditions also failed in
the cases indicated herein.
The use of ESI-MS(/MS) to monitor the reaction, with and
without charge-tagged reagents, prompted further inquiry into
the asserted MCRs and the proposed mechanisms for their
formation. The data we obtained supported our claims and
indicated the misassignment of several MCRs adducts. All
efforts to obtain CPD adducts only yielded DC derivatives con-
taminated with a third component from the reaction (i.e.,
urea). An analogue of a CPD structure was obtained in very low
Fig. 9 Energy footprint of the interaction between Monastrol and
DC-03 with the binding site (blue and red, respectively). Each vertical yields, allowing only for its MS/MS structural characterization,
line corresponds to a residue in the binding pocket, and the point where but it also highlighted the inaccuracy of the literature. A major
it intersects the colored curve represents its energy. The upper plot dis- conclusion is that CPD synthesis remains a synthetic chal-
plays van der Waals (VDW) energy components, while the lower plot lenge, and this structure needs to be obtained through
shows electrostatic energies. Monastrol and DC-03 exhibit similar VDW
different methodologies and synthetic sequences, as no MCR
energy footprints and share crucial electrostatic interactions in adjacent
residues (GLU116, GLY117, GLU118, ARG119). condition described so far has allowed its production. All ana-
lyses calculations shed light on the reaction mechanism and
the reasons for the synthetic failure of the methodologies
described so far.
Biological investigations with pure and “contaminated” DC
derivatives indicated that the presence of urea or thiourea did
not significantly affect the biological response in the investi-
gated bioassays. A new mechanism of cellular action was
revealed for a dicoumarol derivative, and the initial data
strongly suggested a similarity between the known DHPM
called Monastrol and DC-03 as Eg5 inhibitors, both of which
induce a monoastral mitotic spindle, thus preventing the
normal mitotic process. Docking studies and RMSD supported
this hypothesis. Further experiments are necessary to delve
deeper into this new class of Eg5 inhibitors, and they will be
disclosed in due course.
Finally, we recommend revisions, possibly the publication
of a corrigendum, for those articles with incorrect structural
assignments. These syntheses are currently being “repro-
duced” without critical evaluation and proper characterization.
These derivatives are being described as bioactive compounds
with different biological responses, necessitating a structural
correction and recalculation of dose-responses based on the
Fig. 10 RMSD analysis of Monastrol and dicoumarol derivative DC-03 actual structure of the bioactive compounds.
in Eg5. The average backbone RMSD was 1.709 Å for Eg5 + Monastrol
and 1.811 Å for Eg5 + DC-03. The average RMSDs for the ligands in the
binding pocket were, respectively, 0.351 Å and 0.383 Å.
Experimental section
General methods
Conclusions
All purchased chemicals were used as received without further
In summary, we applied a solvent effect model based on purification unless indicated. All solvents employed were of
Kamlet–Taft descriptors to assess various Biginelli-like reac- commercial grade, and when necessary, they underwent distil-

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Organic & Biomolecular Chemistry Paper

lation. All reactions were carried out in a temperature-con- benzenaminium (charge-tagged aldehyde), and 5 mL of
trolled oil bath. 1H (600 MHz) and 13C{1H} (150 MHz) NMR solvent ( p-cymene or limonene) were stirred for 20 min at
spectra were recorded in CDCl3 or DMSO-d6 solution with 80 °C. Then, 0.05 mmol of 4-hydroxycoumarin was added, and
tetramethylsilane (TMS) used as the internal standard. the mixture was kept at the same temperature for up to 24 h.
Multiplicities were denoted as follows: s (singlet), d (doublet), t This procedure was repeated, but with temperature raised
(triplet), td (triple doublet), q (quartet), and m (multiplet). from 80 to 140 °C. The reactions were monitored by HRMS
Chemical shifts are expressed in δ ppm and coupling con- and only DC-01 was observed.
stants ( J) are provided in Hz. Electrospray ionization (tandem) Attempt 6 (this work): in a sealed Schlenk tube, 1 mL of
mass spectrometry analyses were conducted on an instrument, water or p-cymene, 1 mmol of 4-hydroxycoumarin (0.16 g),
equipped with an ESI source, and with the accelerator TOF benzaldehyde (0.11 g), urea (0.06 g), and 10 mol% t-BuOK
analyzer. Fourier-transform infrared (FTIR) spectra were (0.12 g) were mixed. The reaction was maintained at 140 °C for
obtained using a Fourier Transform Infrared Spectrometer. 2 h. The reaction mixture was analyzed by HRMS and only
The samples were prepared in KBr pellets. Melting points were DC-01 was observed.
determined using an apparatus equipped with an oil bath, and
the heating rate for the analyses was set at 1 °C min−1. General procedure for the synthesis attempts of 7-phenyl-
6H,7H-benzo[4,5]thiazolo[3,2-a]chromeno[4,3-d]pyrimidin-6-
General procedure for the synthesis attempts of 4-phenyl-3,4- one (TPC-01)
dihydro-2H-chromeno[4,3-d]pyrimidine-2,5(1H)-dione Attempt 1: in a round-bottom flask, 1 mmol each of 4-hydroxy-
(CPD-01) based on previous reports (see Scheme 2 for coumarin (0.16 g), benzaldehyde (0.11 g), and benzothiazol-2-
references) amine (0.15 g) were combined with 10 mol% sodium lauryl
Attempt 1 (enantioselective attempt): equimolar ratios of sulfate as the catalyst (0.03 g) in 10 mL of water. The mixture
4-hydroxycoumarin (1 mmol, 0.16 g), benzaldehyde (1 mmol, was stirred for 5 h at 25 °C. After the reaction, the precipitate
0.11 g), and urea (1 mmol, 0.06 g) were mixed in a round- was filtered, dried, and recrystallized in ethanol. Only DC-01
bottom flask with 10 mol% L-proline as the chiral catalyst (43%, 182 mg) was observed.
(11 mg) and 1 mL of water. The mixture was microwave-irra- Attempt 2: in a round-bottom flask, 1 mmol of each reagent
diated at 70 °C for 10 min. The resulting solid was filtered, i.e., 4-hydroxycoumarin (0.16 g), benzaldehyde (0.11 g), and
washed with EtOH/H2O (1 : 1, v/v), dried, and recrystallized in benzothiazol-2-amine (0.15 g) were combined with 5 mol%
ethanol, yielding 59% (250 mg) of DC-01. (MSI)3PW as the catalyst (0.17 g) in 5 mL of ethanol. The
Attempt 2: in a round-bottom flask, 4-hydroxycoumarin mixture was stirred for 2 h at 50 °C. The precipitate was then
(1 mmol, 0.16 g), benzaldehyde (1 mmol, 0.11 g), and urea filtered, washed with hot water and ethanol, and recrystallized
(1 mmol, 0.06 g) were combined with 10 mol% sodium lauryl in ethanol. Only DC-01 (39%, 165 mg) was observed.
sulfate (30 mg) and 5 mL of water. The mixture was stirred for
5 h at 25 °C. The precipitate was filtered, washed with EtOH/ General procedure for the synthesis of 2-amino-5-oxo-4-phenyl-
H2O (1 : 1, v/v), dried, and recrystallized in ethanol, yielding 4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (APC-01)
34% (144 mg) of DC-01. Method A: in a round-bottom flask, 5 mmol of each reagent
Attempt 3: 4-hydroxycoumarin (1 mmol, 0.16 g), benz- i.e., benzaldehyde (0.53 g), malononitrile (0.33 g), and urea
aldehyde (1 mmol, 0.11 g), urea (1 mmol, 0.06 g), 20 mol% of (38 mg) as the catalyst were combined with 5 mL of a 1 : 1 (v/v)
SLS (60 mg), 2 drops of acetic acid, and 10 mL of water were EtOH/H2O solution. The mixture was stirred at 30 °C for
reacted in a Schlenk flask at 100 °C for 6 h. The solid was fil- 20 min, and then 5 mmol of 4-hydroxycoumarin (0.81 g) was
tered, washed with hot water (30 mL, 80 °C) and 5 mL of added. The reaction proceeded at 30 °C for 6 h. DC-01 (65%,
ethanol, dried, and recrystallized in ethanol, yielding 51% 1.38 g) and ACP-01 (35%, 0.55 g) were isolated.
(216 mg) of DC-01. Method B: in a sealed Schlenk tube, 1 mmol of benz-
Attempt 4 (this work): in a Schlenk tube, 1 mmol of aldehyde (0.11 g), malononitrile (0.07 g), and 4-hydroxycou-
4-hydroxycoumarin (0.16 g), benzaldehyde (0.11 g), urea marin (0.16 g) were combined with 5 mol% t-BuOK as the cata-
(0.06 g), and 5 mol% of the superacid catalyst (MSI)3PW lyst (16 mg) and 1 mL of ethanol. The mixture was heated to
(0.17 g) were reacted at 80 °C for 1 h. 1 mL of the following sol- 80 °C for 30 min. The resulting solid precipitate was filtered,
vents were also tested: water, ethanol, methanol, ethyl acetate, washed with EtOH/H2O solution, and recrystallized in ethanol.
t-butanol, acetonitrile, hexane, THF, dioxane, triethylamine, ACP-01 (80%, 253 mg) was isolated.
cyclohexane, octanol, 2,2,2-trifluoroethan-1-ol, acetone, chloro- Yield 1.24 g (78%, Method A) and 0.24 g (49%, Method B);
form, BMI·PF6, BMI·BF4, BMI·NTf2, dichloromethane, toluene, white solid; m.p. 257–259 °C; 1H NMR (600 MHz, DMSO-d6) δ
p-cymene, and limonene. After completion, the precipitate was ppm 7.91 (dd, J = 8.0, 1.5 Hz, 1H), 7.70 (ddd, J = 8.4, 7.4 Hz,
filtered, washed with hot water and ethanol, dried, and recrys- 1.6, 1H), 7.48 (ddd, J = 7.9, 7.5, 1.0 Hz, 1H), 7.45 (dd, J = 8.3,
tallized in ethanol. For all cases, DC-01 was obtained (see 0.6 Hz, 1H), 7.40 (s, 2H), 7.31 (tt, J = 3.5, 1.7 Hz, 2H), 7.24 (m,
Table 1). 4H), 4.44 (s, 1H); 13C{1H} NMR (150 MHz, DMSO-d6) δ ppm
Attempt 5 (this work): in a round-bottom flask, 0.05 mmol 159.6, 158.3, 153.5, 152.2, 143.4, 133.0, 128.6, 127.7, 127.2,
of urea (0.03 g), 0.05 mmol of 4-formyl-N,N,N-trimethyl- 124.7, 122.5, 119.3, 116.6, 113.0, 104.4, 58.0, 37.0; FTIR (cm−1)

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Paper Organic & Biomolecular Chemistry

3378, 3284, 3180, 2198, 1708, 1673, 1608, 1380, 1058; HRMS 3,3′-((4-Methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-
(ESI) m/z [M + H]+ calcd for [C27H21NO + H]+, 317.0921, found, 2-one) (DC-02)
317.0923. Yield 267 mg (89%); white solid; m.p. 247–248 °C. 1H NMR
General procedure for the synthesis of 10,10-dimethyl-7- (600 MHz, DMSO-d6) δ ppm 7.92 (d, J = 7.9 Hz, 2H), 7.60 (t, J =
phenyl-7,9,10,11-tetrahydro-6H,8H-chromeno[4,3-b]chromene- 7.9 Hz, 2H), 7.39 (dd, J = 8.2, 2.1 Hz, 2H), 7.34 (t, J = 7.9 Hz,
6,8-dione (TCC) 2H), 7.07 (d, J = 6.6 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.31 (s,
1H), 3.70 (s, 3H). 13C{1H} NMR (150 MHz, DMSO-d6) δ ppm
In a round-bottom flask, the following components were com- 164.9, 157.4, 152.2, 132.0, 131.2, 127.8, 123.9, 117.7, 116.0,
bined: (MSI)3PW (0.81 g, 5 mol%), 4-hydroxycoumarin (0.82 g, 113.6, 104.5, 55.0, 35.2. FTIR (cm−1) 3448, 3068, 3002, 2937,
5 mmol), 5,5-dimethyl-1,3-cyclohexanedione (0.70 g, 5 mmol), 2836, 2732, 2616, 1668, 1617, 1604, 1560, 1510, 1454, 1353,
benzaldehyde (0.50 g, 5 mmol), and water (10 mL). The result- 1309, 1257, 1178, 1093, 767 HRMS (ESI) m/z [M + H]+ calcd for
ing mixture underwent heating at 80 °C and continuous stir- [C26H18O7 + H]+ 443.1125 found, 443.1122.
ring at this temperature for 2 h. Subsequently, the reaction
mixture was allowed to cool to room temperature, followed by 3,3′-(Benzo[d][1,3]dioxol-5-ylmethylene)bis(4-hydroxy-2H-
filtration and thorough washing with hot water to isolate the chromen-2-one) (DC-03)
catalyst. The resulting crude product was subjected to purifi-
cation through recrystallization using an EtOH/CH2Cl2 solvent Yield 232 mg (90%); pale yellow solid; m.p. 258–260 °C; 1H
mixture (1 : 4, v/v). NMR (600 MHz, DMSO-d6) δ ppm 7.92 (d, J = 7.9, 1.5 Hz, 2H),
Yield 1.23 g (63%), white solid; mp 222–224 °C; 1H NMR 7.59 (td, J = 8.7, 3.2 Hz, 2H), 7.36 (d, J = 8.3, 2H), 7.32 (t, 8.3,
(600 MHz, CDCl3) δ ppm 7.81 (dd, J = 7.9, 1.5 Hz, 1H), 7.50 2H), 6.76 (d, J = 8.2 Hz, 1H), 6.71 (s, 1H), 6.62 (dt, J = 8.0, 1.5
(ddd, J = 8.7, 7.5, 1.5 Hz, 1H), 7.33–7.24 (m, 4H), 7.29–7.25 (m, Hz, 1H), 6.26 (s, 1H), 5.94 (s, 3H); 13C{1H} NMR (150 MHz,
2H), 7.20–7.17 (m, 1H), 4.90 (s, 1H), 2.63 (q, J = 17.6 Hz, 2H), DMSO-d6) δ ppm 165.2, 164.8, 152.3, 147.4, 145.3, 133.7, 132.0,
2.23 (q, J = 16.2 Hz, 2H), 1.11 (s, 3H), 1.03 (s, 3H); 13C{1H} 123.9, 123.8, 119.5, 117.9, 116.0, 107.8, 107.6, 104.4, 100.7,
NMR (150 MHz) δ ppm 196.1, 162.1, 160.7, 154.0, 152.7, 142.6, 35.8; FTIR (cm−1) 3448, 3081, 2898, 2736, 2615, 1662, 1616,
132.3, 128.7, 128.4, 127.2, 124.4, 122.6, 117.0, 115.3, 113.8, 1602, 1567, 1488, 1436, 1344, 1309, 1236, 1097, 1039, 763;
106.9, 50.8, 40.9, 33.5, 32.5, 29.3, 27.7; FTIR (cm−1) 3425, 3064, HRMS (ESI) m/z [M + H]+ calcd for [C26H16O8 + H]+, 457.0918,
2956, 2869, 1714, 1658, 1365, 1192, 1170, 1055, 761. HRMS found, 457.0916.
(ESI) m/z [M + H]+ calcd for [C24H20O4 + H]+, 373.1434; found,
373.1438. 3,3′-((4-Bromophenyl)methylene)bis(4-hydroxy-2H-chromen-2-
one) (DC-04)
General procedure for the synthesis of dicoumarol derivatives Yield 346 mg (89%); white solid; m.p. 267–268 °C; 1H NMR
(DCs) (600 MHz, DMSO-d6) δ ppm 7.89 (dd, J = 7.9, 1.4 Hz, 2H), 7.58
In a sealed Schlenk tube, 2 mmol of 4-hydroxycoumarin (t, J = 8.0 Hz, 2H), 7.38 (d, J = 9 Hz, 2H), 7.35 (d, J = 8.0 Hz,
(0.33 g), 1 mmol of respective aldehyde, 5 mol% of (MSI)3PW 2H), 7.31 (t, J = 7.9, 2H), 7.11 (d, J = 8.6 Hz, 2H), 6.29 (s, 1H);
catalyst (170 mg), and 1 mL of water as a solvent were added, 13
C{1H} NMR (150 MHz, DMSO-d6) δ ppm 165.8, 164.7, 152.3,
and the mixture was heated to 80 °C for 1 h. Upon completion 140.1, 131.9, 130.8, 129.2, 124.0, 123.7, 118.5, 118.4, 116.0,
of the reaction, the precipitate was filtered and washed with a 103.8, 35.8; FTIR (cm−1) 3428, 3070, 2728, 2609, 1668, 1617,
hot solution of EtOH/H2O (1 : 1, v/v). Subsequently, when 1604, 1560, 1488, 1351, 1307, 1093, 765. HRMS (ESI) m/z [M +
necessary, the desired product was recrystallized in ethanol to H]+ calcd for [C25H15BrO6 + H]+, 491.0125, found, 491.0120.
obtain the pure product. For a multigram synthesis, the same 3,3′-((4-(Dimethylamino)phenyl)methylene)bis(4-hydroxy-
procedure is applied but using 10 mmol of 4-hydroxycoumarin 2H-chromen-2-one) (DC-05). Yield 235 mg (85%); pink solid;
(1.60 g), 10 mmol of benzaldehyde (1.06 g), 5 mol% of m.p. 200–201 °C; 1H NMR (600 MHz, DMSO-d6) δ ppm 7.83
(MSI)3PW catalyst (1.74 g), and 5 mL of water. (dd, J = 7.9, 1.3 Hz, 2H), 7.52 (t, J = 7.9 Hz, 2H), 7.44 (d, J = 7.9
Hz, 2H), 7.31–7.23 (m, 7H), 6.30 (s, 1H), 3.15 (s, 6H); 13C{1H}
3,3′-(Phenylmethylene)bis(4-hydroxy-2H-chromen-2-one) NMR (150 MHz, DMSO-d6) δ ppm 167.5, 164.4, 152.5, 131.2,
(DC-01) 128.2, 124.1, 123.0, 119.6, 115.6, 103.0, 45.6, 36.0; FTIR (cm−1)
Yield 403 mg (88%) and 2.5 g (93%, in the multigram syn- 3434, 3045, 2917, 2792, 1673, 1606, 1540, 1400, 1351, 1043,
thesis); White solid; m.p. 230–231 °C; 1H NMR (600 MHz, 755; HRMS (ESI) m/z [M + H]+ calcd for [C27H21NO6 + H]+,
DMSO-d6) δ ppm 7.92 (d, J = 7.9 Hz, 2H), 7.60 (t, J = 8.3 Hz, 456.1442, found, 456.1439.
2H), 7.38 (d, J = 8.3 Hz, 2H), 7.33 (t, J = 7.9 Hz, 2H), 7.24 (t, J = Bioimaging. The breast cancer cell line (MCF-7) was main-
7.9 Hz, 2H), 7.17–7.14 (m, 3H); 13C{1H} NMR (150 MHz, tained following the recommendations of the American Type
DMSO-d6) δ ppm 165.1, 164.9, 152.2, 139.7, 132.0, 128.1, 126.7, Culture Collection (ATCC). DC-03 and Monastrol were diluted
125.7, 123.9, 123.8, 118.0, 116.0, 104.2, 36.0; FTIR (cm−1) 3446, to 100 μM in Dulbecco’s Modified Eagle’s medium (DMEM)
3068, 2736, 2615, 1660, 1616, 1604, 1567, 1495, 1450, 1346, supplemented with 0.1% dimethyl sulfoxide and 10% fetal calf
1299, 1182, 1093, 759; HRMS (ESI) m/z [M + H]+ calcd for serum. 7 × 104 cells were seeded on 13 mm cover slips inside a
[C25H16O6 + H]+, 413.1020, found, 413.1019. 24-well plate for 48 h to reach 75% confluence. The samples

3646 | Org. Biomol. Chem., 2024, 22, 3630–3651 This journal is © The Royal Society of Chemistry 2024
Organic & Biomolecular Chemistry Paper

were divided into three groups and incubated under the fol- neutralize each receptor–ligand complex. The 10 ns production
lowing conditions for 48 h: (A) negative control incubated with step was preceded by one minimization and four equilibration
only culture medium plus 0.1% DMSO; (B) positive control steps. Energy minimization was run with a steepest descent
incubated with Monastrol at 100 μM; and (C) incubated with algorithm with an energy tolerance of 10 kJ mol−1 and step
DC-03 at 100 μM. After 48 h, the samples were washed three sizes of 0.01 kJ mol−1. The first equilibration step (NVT) was a
times with warm PBS (37 °C), pH 7.4, and subjected to fixation 100 ps simulation with a Langevin integrator set to generate
procedures in 3.7% formaldehyde for 30 minutes at room configurations at 298 K with a step size of 2 fs. All non-solvent
temperature. heavy atoms were kept in place by a 1000 kJ mol−1 position
Immunofluorescence assay. Fixed samples were permeabi- restraint. The second equilibration step (NPT_1) was set in the
lized in 0.1% Triton X-100 for 20 min and then incubated in a same way as the NVT equilibration step with the addition of a
blocking solution (PBS, pH 7.4, 1% skimmed milk, 2.5% Berendsen barostat to bring the density and the pressure of
bovine serum albumin, 8% fetal calf serum) for additional the simulation box to near-equilibrium conditions at 1 bar.
20 min. Subsequently, the samples were incubated with a The third equilibration step (NPT_2) used the Parrinello-
primary antibody (anti-α-tubulin) produced in mouse for 16 h Rahman barostat instead to ensure sampling is adequate in
at 4 °C. After three washes with PBS, pH 7.4, at room tempera- the isothermal–isobaric ensemble. The fourth equilibration
ture, the samples were incubated for 1 h at 37 °C with a sec- step (NPT_3) differed from NPT_2 by the restraints in the
ondary antibody, Alexa Fluor 594 anti-mouse (5 μg mL−1), in receptor. In NPT_3, only the backbone was restrained, and
the dark. Following three additional washes in PBS, pH 7.4, side chains were allowed to move freely. The production stage
the samples were incubated for 5 min with DAPI (300 nM) to (PROD) was run with a completely unrestrained system at
stain the cell nucleus. After three more washes in PBS, pH 7.4, 298 K and 1 bar using the same integrator and barostat as the
the samples were mounted on glass slides using ProLong Gold previous step. Excepting the minimization step, bonds to
Antifade agent and analyzed using a confocal laser scanning hydrogen were constrained using GROMACS’ implementation
microscope. of LINCS. van der Waals interactions were neglected beyond a
cutoff of 12 Å with a switch at 10 Å, and electrostatic inter-
Computational methods actions were calculated using the Particle Mesh Ewald (PME)
Docking simulations details. Preparation step: 1Q0B struc- method of order 4 with a real-space cutoff of 12 Å and grid
ture was downloaded from RCSB Protein Data Bank using spacing of 1.6 Å. Periodic boundary conditions were applied.
UCSF Chimera. The asymmetric unit consisted of a dimer
bound to two Monastrol molecules and two ADP molecules.
One monomer and excess crystalized water molecules were Author contributions
deleted, remaining a monomer, ADP complexed to Mg2+ and
adjacent water molecules, and Monastrol. The crystal structure All authors had important contributions to this work.
was initially relaxed using energy minimization and a short
heavy-atom restricted NVT MD run at 298 K in GROMACS 2020
to ensure that steric hindrance between the ligands and the Conflicts of interest
protein was minimized. The protein was parameterized with
ff14SB and the ligands with GAFF2 with AM1-BCC charges. The authors declare no competing financial interest.
Docking step. After ensuring that any steric hindrance was
minimized, Monastrol was removed from the binding pocket
and the surface of the protein was generated using DMS196 Acknowledgements
with a 1.4 Å radius probe atom. Docking spheres were gener-
ated inside the binding pocket with DOCK’s sphgen.197 1Q0B The authors are grateful for the financial support from CAPES,
energy grids were generated using the program grid.198 Each CNPq, FAPDF, FINATEC and UnB.
grid point had a coulombic energy term using a distance-
dependent dielectric of 4r and a 6–9 Lennard Jones term.
Dicoumarol was docked using DOCK6’s Grid scoring function References
and the best binding pose was minimized and rescored using
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This journal is © The Royal Society of Chemistry 2024 Org. Biomol. Chem., 2024, 22, 3630–3651 | 3651
 Supporting Information for

Revisiting Biginelli-like Reactions: Solvent Effects, Mechanisms, Biological

Applications and Correction of Several Literature Reports

Pedro S. Beck,a Arthur G. Leitao,a Yasmin B. Santana,a Jose R. Correa,a Carime V. S. Rodrigues,a Daniel

F. S. Machado,a Guilherme D. R. Matos,a Luciana M. Ramos,b Claudia C. Gatto,a Sarah C. C. Oliveira,a

Carlos K. Z. Andrade,a and Brenno A. D. Neto,*a

a University of Brasilia, Chemistry Institute, Laboratory of Medicinal and Technological Chemistry.

Campus Universitario Darcy Ribeiro, Brasília, DF, 70910-900, Brazil. E-mail: [email protected].

b Universidade Estadual de Goias (UEG), Anapolis, Goias, 75001-970, Brazil.

c University of Brasilia, Institute of Biology, Laboratory of Allelopathy, Campus Universitario Darcy

Ribeiro, Brasília, DF, 70910-900, Brazil.

S1
Table of Content

List of Schemes ................................................................................................................ 3

List of Tables .................................................................................................................... 4

List of Figures................................................................................................................... 5

Schemes ............................................................................................................................ 8

Tables ................................................................................................................................ 9

Figures ............................................................................................................................ 18

Determination of numerical values................................................................................. 48

References ...................................................................................................................... 49

S2
List of Schemes

Scheme S1. Synthesis reaction for the claimed structure of CPD-01 utilizing the

superacid imidazolium-based ionic liquid MSI3PW. ............................................................... 8

Scheme S2. Synthesis of the claimed CPD adducts and their respective yields based

on their theoretical masses. ............................................................................................................. 8

S3
List of Tables

Table S1. Kamlet-Taft descriptors, yields for the claimed CPD as a function of solvent

variation and reagents’ proportions, and E-factors. ............................................................... 9

Table S2. Melting point (mp) evaluation for the claimed CPD-01 structure reported

in the literature. ..................................................................................................................................10

Table S3. A comparative assessment of experimental versus theoretical elemental

analysis (CHN) data for alleged CPDs structures. Specifically, for claimed CPD-01,

the data reveals a composition wherein approximately two molecules of DC-01

correlate with nearly one molecule of urea/thiourea. ........................................................11

Table S4. Indicative of whether these articles provide descriptions and/or FTIR

spectra for the claimed CPD-01. ..................................................................................................12

Table S5. Indicative of whether these articles provide descriptions and/or mass

spectra for claimed CPD-01...........................................................................................................13

Table S6. Indicative of whether these articles provide descriptions and/or 1H and

13C NMR spectra for claimed CPD-01. .......................................................................................14

Table S7. Indicative of whether these articles show descriptions for elemental

analysis and accuracy with respect to the expected values for the claimed CPD-01

structure. ...............................................................................................................................................15

Table S8. Exploring reaction condition variations in the attempt to synthesize PCC-

01.a...........................................................................................................................................................16

Table S9. Kamlet-Taft parameters used for solvent effect analysis................................17

S4
List of Figures

Figure S1. Conditions optimization for the attempts to synthesize CPD-01. (Left)

Temperature optimization. (Center) Catalyst amount optimization. (Right) Time

optimization. ........................................................................................................................................18

Figure S2. Correlations of reaction productivity (expressed as ln(P)) as a function of

Kamlet-Taft parameters (,  and *). All data refer to isolated yields of the model

reaction (benzaldehyde, 4-hydroxycoumarin, and urea mixtures, 5 mol% of the

catalyst at 80 °C for 60 min). Reactions proportion (mmol) from top to bottom: (i)

Equimolar conditions i.e. 1.00 (benzaldehyde) : 1.00 (coumarin) : 1.00 (urea). (ii)

Aldehyde excess i.e. 2.00 (benzaldehyde) : 1.00 (coumarin) : 1.00 (urea). (iii)

Coumarin excess i.e. 1.00 (benzaldehyde) : 2.00 (coumarin) : 1.00 (urea). (iv) Urea

excess i.e. 1.00 (benzaldehyde) : 1.00 (coumarin) : 2.00 (urea). .....................................18

Figure S3. (A) 1H NRM (DMSO-d6, 300 MHz) for claimed CPD-01 adduct. (B) 13C

NRM (DMSO-d6, 75 MHz) for claimed CPD-01 adduct. These two spectra are

contaminated with urea. .................................................................................................................19

Figure S4. Reaction monitoring by HRMS using p-cymene as the solvent and at 80

°C. .............................................................................................................................................................20

Figure S5. Reaction monitoring by HRMS using limonene as the solvent and at 80

°C. .............................................................................................................................................................21

Figure S6. Reaction monitoring by HRMS using water as the solvent and at 80 °C.

...................................................................................................................................................................22

Figure S7. Reaction monitoring by HRMS using water as the solvent and at 100 °C.

4-Aminocoumarin was used instead of 4-hydroxycoumarin. ...........................................23

Figure S8. Wheat coleoptile bioassay testing the synthesized DC derivatives

contaminated with urea or thiourea (proportion of  2 DC : 1 urea/thiourea). Logran

S5
is the positive control. CDPs 01, 03, 05, 07 and 08 are indeed and respectively DCs

01, 02, 03, 04 and 05 contaminated with urea. CPDs 02, 04 and 06 are respectively

DCs 01, 02, 03, contaminated with thiourea. .........................................................................24

Figure S9. Cellular division (mitosis) inhibition by DC-03 contaminated with urea

(proportion of  2 DC : 1 urea/thiourea). (A) Nuclei stained with the commercially

available DAPI (blue emitter). (B) Anti--Tubulin monoclonal antibody

(commercially available red emitter). (C) Merged images from (A)-(B). Remarkably,

despite the presence of those contaminant, DC-03 induced monoastral spindles in

mitotic cells. .........................................................................................................................................25

Figure S10. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-01. (B)13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of DC-01. ................................................................................26

Figure S11. 2D COSY NMR (600 MHz, DMSO-d6) for DC-01. ...........................................27

Figure S12. 2D HSQC NMR (600 MHz and 150 MHz, DMSO-d6) for DC-01. ..............28

Figure S13. 2D HMBC (600 MHz and 150 MHz, DMSO-d6) for DC-01. ........................29

Figure S14. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-02. (B) 13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of DC-02. ................................................................................30

Figure S15. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-03. (B)13C NMR

spectrum (150 MHz, DMSO-d6) of DC-03. ................................................................................31

Figure S16. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-04. (B)13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of DC-04. ................................................................................32

Figure S17. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-05. (B)13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of DC-05. ................................................................................33

Figure S18. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of APC-01. (B)13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of APC-01. .............................................................................34

S6
Figure S19. (A) 1H NMR spectrum (600 MHz, CDCl3) of TCC-01. (B)13C{1H} NMR

spectrum (150 MHz, CDCl3) of TCC-01. ....................................................................................35

Figure S20. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of 4-aminocoumarin.

(B)13C{1H} NMR spectrum (150 MHz, DMSO-d6) of 4-aminocoumarin. ......................36

Figure S21. FTIR (KBr) spectrum for DC-01. ........................................................................37

Figure S22. FTIR (KBr) spectrum for DC-02. ........................................................................37

Figure S23. FTIR (KBr) spectrum for DC-03. ........................................................................38

Figure S24. FTIR (KBr) spectrum for DC-04. ........................................................................38

Figure S25. FTIR (KBr) spectrum for DC-05. ........................................................................39

Figure S26. FTIR (KBr) spectrum for 4-aminocoumarin. .................................................39

Figure S27. FTIR (KBr) spectrum for APC-01. ......................................................................40

Figure S28. FTIR (KBr) spectrum for TCC-01. ......................................................................40

Figure S29. (A) ESI(+)-MS for DC-01. (B) ESI(+)-MS/MS for DC-01. ..........................41

Figure S30. (A) ESI(+)-MS for DC-02. (B) ESI(+)-MS/MS for DC-02. ..........................42

Figure S31. (A) ESI(+)-MS for DC-03. (B) ESI(+)-MS/MS for DC-03. ..........................43

Figure S32. (A) ESI(+)-MS for DC-04. (B) ESI(+)-MS/MS for DC-04. ..........................44

Figure S33. (A) ESI(+)-MS for DC-05. (B) ESI(+)-MS/MS for DC-05. ..........................45

Figure S34. (A) ESI(+)-MS for APC-01. (B) ESI(+)-MS/MS for APC-01. .....................46

Figure S35. (A) ESI(+)-MS for TCC-01. (B) ESI(+)-MS/MS for TCC-01. ......................47

S7
Schemes

Scheme S1. Synthesis reaction for the claimed structure of CPD-01 utilizing the superacid

imidazolium-based ionic liquid MSI3PW.

Scheme S2. Synthesis of the claimed CPD adducts and their respective yields based on their

theoretical masses.

S8
Tables

Table S1. Kamlet-Taft descriptors, yields for the claimed CPD as a function of solvent variation and

reagents’ proportions, and E-factors.

Isolated yields (%)

Reagents’ proportion (mmol)
Coumarin : Aldehyde : Urea
Kamlet-Taft
Solvent 1:1:1 2:1:1 1:2:1 1:1:2 E factor†
descriptors
 0.00
AcOEt  0.45 74% 67% 72% 62% 47
* 0.55
 0.19
MeCN  0.31 63% 85% 65% 53% 41
* 0.75
 1.17
Water  0.47 94% 94% 90% 71% 36
* 1.09
 0.63
[C4C1Im][BF4]  0.38 69% 93% 83% 77% 38
* 1.04
 0.62
[C4C1Im][NTf2]  0.23 80% 90% 88% 59% 39
* 0.90
 0.63
[C4C1Im][PF6]  0.19 71% 94% 75% 64% 37
* 1.02
 0.04
CH2Cl2  -0.01 74% 79% 80% 75% 44
* 0.79
 0.83
EtOH  0.75 77% 90% 75% 58% 39
* 0.51
 -0.08
Hexane  0.00 53% 63% 59% 44% 55
* 0.00
 0.93
MeOH  0.66 65% 92% 82% 52% 38
* 0.58
 0.00
PhMe  0.11 55% 72% 75% 50% 47
* 0.54
†
Calculated for the best yield of each solvent.

S9
Table S2. Melting point (mp) evaluation for the claimed CPD-01 structure reported in the literature.

Year of publication mp (°C) Reference

2003 - 1
2006 162 2
2008 162-163 3
2014 210-212 4
2015 265-267 5
2016 162-163 6
2016 160-162 7
2017 160-162 8
2018 160-162 9
2018 160-162 10
2018 206 11
2018 160-162 12
2018 238-240 13
2019 165-167 14
2019 160-162 15
2020 163-165 16
2020 211-213 17
2020 160-162 18
2022 162-163 19
2022 160-162 20
2023 214-216 21
2023 210-212 22

S10
Table S3. A comparative assessment of experimental versus theoretical elemental analysis (CHN)

data for alleged CPDs structures. Specifically, for claimed CPD-01, the data reveals a composition

wherein approximately two molecules of DC-01 correlate with nearly one molecule of urea/thiourea.

Experimental Data Theorical Data

Carbon Hydrogen Nitrogen Carbon Hydrogen Nitrogen
Structure
(%) (%) (%) (%) (%) (%)
CPD-01 64.62 3.92 2.90 69.86 4.14 9.58
CPD-02 63.59 3.47 0.48 66.22 3.92 9.08
CPD-03 68.65 3.95 0.13 67.08 4.38 8.69
CPD-04 53.33 2.85 0.06 63.89 4.17 8.28
CPD-05 67.29 3.36 0.07 64.29 3.6 8.33
CPD-06 64.04 3.25 0.01 61.36 3.43 7.95
CPD-07 56.01 2.82 1.32 55.01 2.99 7.55
CPD-08 70.53 4.49 2.98 68.05 5.11 12.53

S11
Table S4. Indicative of whether these articles provide descriptions and/or FTIR spectra for the

claimed CPD-01.

Year of publication Experimental description Available Spectrum Reference

2003 - - 1
2006 Yes No 2
2008 Yes No 3
2014 Yes No 4
2015 Yes No 5
2016 Yes No 6
2016 Yes No 7
2017 Yes No 8
2018 Yes No 9
2018 No No 10
2018 Yes No 11
2018 No No 12
2018 Yes Yes 13
2019 No No 14
2019 Yes No 15
2020 Yes No 16
2020 No Yes 17
2020 Yes No 18
2022 Yes No 19
2022 Yes Yes 20
2023 Yes Yes 21
2023 No No 22

S12
Table S5. Indicative of whether these articles provide descriptions and/or mass spectra for claimed

CPD-01.

Year of Experimental Ionization Source Available

Reference
publication Description / (m/z detected) Spectrum
2003 - - 1
2006 No No 2
Not available /
2008 Yes No 3
(292.0)
2014 No No 4
2015 No No 5
2016 No No 6
ESI-(+) /
2016 Yes No 7
(292.3)
2017 No No 8
2018 No No 9
ESI-(+) /
2018 Yes Yes 10
(292.3)
2018 No No 11
ESI-(+) /
2018 Yes Yes 12
(292.3)
Not available /
2018 Yes No 13
(292)
2019 No No 14
ESI-(+) /
2019 Yes Yes 15
(292.3)
ESI-(+) /
2020 Yes No 16
(293.3)
2020 No No 17
2020 No No 18
Not available /
2022 Yes No 19
(292.0)
2022 No No 20
2023 No No 21
2023 No No No 22

S13
Table S6. Indicative of whether these articles provide descriptions and/or 1H and 13C NMR spectra

for claimed CPD-01.

1H
NMR 13C NMR

Year Experimental Available Experimental Available Reference

Description spectrum Description spectrum
2003 - - - - 1
2006 Yes No No No 2
2008 Yes No No No 3
2014 Yes No Yes No 4
2015 Yes No Yes No 5
2016 Yes No No No 6
2016 Yes Yes* Yes Yes 7
2017 Yes No No No 8
2018 Yes No Yes No 9
2018 Yes Yes* Yes Yes* 10
2018 Yes No Yes No 11
2018 Yes Yes* Yes Yes* 12
2018 Yes Yes Yes No 13
2019 Yes Yes No No 14
2019 Yes Yes* Yes Yes* 15
2020 Yes No Yes No 16
2020 No No No No 17
2020 Yes No Yes No 18
2022 Yes No No No 19
2022 No No No No 20
2023 Yes Yes No No 21
2023** No No No No 22
* Indicate the articles that likely have duplicate spectra among themselves. ** Available
spectra for similar structures, but not for CPD-01.

S14
Table S7. Indicative of whether these articles show descriptions for elemental analysis and accuracy

with respect to the expected values for the claimed CPD-01 structure.

Year of Experimental
Accuracy Calculate (%) Found (%) Reference
publication Description
2003 - - - - 1
C 69.88 C 69.80
2006 Yes Yes H 4.10 H 4.01 2
N 9.58 N 9.49
C 69.86 C 69.85
2008 Yes Yes H 4.10 H 4.14 3
N 9.58 N 9.56
2014 No - - - 4
C 69.86 C 69.67
2015 Yes Yes H 4.14 H 4.11 5
N 9.58 N 9.61
C 69.86 C 69.64
2016 Yes No*(a) H 4.14 H 3.96 6
N 9.58 N 9.69
2016 No - - - 7
2017 No - - - 8
2017 No - - - 9
C 69.86 C 70.02
2018 Yes Yes H 4.14 H 4.18 10
N 9.58 N 9.69
2018 No - - - 11
C 69.86 C 70.02
2018 Yes Yes H 4.14 H 4.18 12
N 9.58 N 9.69
C 69.86 C 70.00
2018 Yes Yes H 4.14 H 4.27 13
N 9.58 N 9.60
2019 No - - - 14
C 69.86 C 70.02
2019 Yes Yes H 4.14 H 4.18 15
N 9.58 N 9.69
C 69.86
2020 Yes Yes Not available H 4.14 16
N 9.58
2020 No - - - 17
C 69.86 C 69.92
2020 Yes Yes H 4.14 H 4.22 18
N 9.58 N 9.70
C 69.86 C 69.85
2022 Yes No*(b) H 4.10 H 4.14 19
N 9.58 N 5.30
2022 No - - - 20
2023 No - - - 21
2023 No - - - 22
* = Chemical formula calculated based on experimental CHN: (a) C17H11N2O3; (b) C31H22N2O7.
The results of elemental analyses in red highlight that the values have been triplicated in
different publications. The value highlighted in blue indicates that the obtained value is
exactly equal to the theoretical value.

S15
Table S8. Exploring reaction condition variations in the attempt to synthesize PCC-

01.a

Temperature
Entry Solvent Time Catalyst Product Yield
(°C)
1 Ethanol 50 30 min HCl DC-01 51%
2 Ethanol 60 30 min HCl DC-01 53%
3 Ethanol 70 30 min HCl DC-01 52%
4 Ethanol 80 30 min HCl DC-01 44%
5 Ethanol 90 30 min HCl DC-01 48%
6 Ethanol 100 30 min HCl DC-01 44%
7 Ethanol 60 2h HCl DC-01 47%
8 Ethanol 60 3h HCl DC-01 44%
9 Ethanol 60 4h HCl DC-01 44%
10 Ethanol 60 5h HCl DC-01 45%
11 Ethanol 60 6h HCl DC-01 48%
12 Ethanol 70 6h HCl DC-01 43%
13 Ethanol 80 6h HCl DC-01 42%
14 Ethanol 90 6h HCl DC-01 43%
15 Ethanol 100 6h HCl DC-01 47%
16 Ethanol 60 24 h HCl DC-01 44%
17 Ethanol 100 24 h HCl DC-01 38%
18 Ethanol 60 48 h HCl DC-01 48%
19 Ethanol 100 48 h HCl DC-01 38%
20 Ethanol 60 30 min (MSI)3PW DC-01 73%
21 Ethanol 60 24 h (MSI)3PW DC-01 81%
22 Ethanol 100 30 min (MSI)3PW DC-01 55%
23 Ethanol 100 24 h (MSI)3PW DC-01 88%
24 Ethanol 60 30 min KOtBu DC-01 65%
25 Ethanol 60 24 h KOtBu DC-01 80%
26 t-Butanol 60 30 min KOtBu DC-01 66%
27 t-Butanol 60 24 h KOtBu DC-01 65%
28 Solventless 60 15 min MWIb DC-01 40%
a b
No condition allowed the formation of ACP-01. Microwave irradiation.

S16
Table S9. Kamlet-Taft parameters used for solvent effect analysis.

Solvent   
1,4-Dioxane 0.00 0.37 0.55
Acetone 0.08 0.43 0.71
AcOEt 0.00 0.45 0.55
BMI.BF4 0.63 0.38 1.04
BMI.NTf2 0.62 0.23 0.90
BMI.PF6 0.63 0.38 1.04
CF3CH2OH 1.51 0.00 0.73
CHCl2 0.04 -0.01 0.79
CHCl3 0.20 0.10 0.58
Cyclohexane 0.00 0.00 0.00
EtOH 0.83 0.75 0.51
H2O 1.17 0.47 1.09
Hexane -0.08 0.00 0.00
Limonene 0.00 0.00 0.16
MeCN 0.19 0.31 0.75
MeOH 0.93 0.66 0.58
n-OcOH 0.77 0.81 0.4
p-Cymene 0.00 0.13 0.39
t-BuOH 0.68 0.93 0.41
THF 0.00 0.55 0.58
Toluene 0.00 0.11 0.54
Triethylamine 0.00 0.71 0.14

S17
Figures

Figure S1. Conditions optimization for the attempts to synthesize CPD-01. (Left) Temperature

optimization. (Center) Catalyst amount optimization. (Right) Time optimization.

Figure S2. Correlations of reaction productivity (expressed as ln(P)) as a function of Kamlet-Taft

parameters (,  and *). All data refer to isolated yields of the model reaction (benzaldehyde, 4-

hydroxycoumarin, and urea mixtures, 5 mol% of the catalyst at 80 °C for 60 min). Reactions

proportion (mmol) from top to bottom: (i) Equimolar conditions i.e. 1.00 (benzaldehyde) : 1.00

(coumarin) : 1.00 (urea). (ii) Aldehyde excess i.e. 2.00 (benzaldehyde) : 1.00 (coumarin) : 1.00 (urea).

(iii) Coumarin excess i.e. 1.00 (benzaldehyde) : 2.00 (coumarin) : 1.00 (urea). (iv) Urea excess i.e. 1.00

(benzaldehyde) : 1.00 (coumarin) : 2.00 (urea).

S18
Figure S3. (A) 1H NRM (DMSO-d6, 300 MHz) for claimed CPD-01 adduct. (B) 13C{1H} NRM (DMSO-d6,

75 MHz) for claimed CPD-01 adduct. These two spectra are contaminated with urea. Note the

number of hydrogens and carbons do not fit the claimed CPD-01 structure.

S19
Figure S4. Reaction monitoring by HRMS using p-cymene as the solvent and at 80 °C.

S20
 2CR – 5 min 2CR – 10 min

3CR – 15 min 3CR – 30 min

3CR – 45 min 3CR – 60 min

3CR – 90 min 3CR – 180 min

Figure S5. Reaction monitoring by HRMS using limonene as the solvent and at 80 °C.

S21
Figure S6. Reaction monitoring by HRMS using water as the solvent and at 80 °C.

S22
Figure S7. Reaction monitoring by HRMS using water as the solvent and at 100 °C. 4-Aminocoumarin

was used instead of 4-hydroxycoumarin.

S23
 CPD-01 CPD-02
20 20

Growth Inibhition (%)

Growth Inibhition (%)

0 0

-20 -20

-40 -40

-60 -60

-80 -80

-100 -100
1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5 1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5
Concentration (mol L-1) Concentration (mol L-1)

CPD-03 CPD-04
20 20
Growth Inibhition (%)

Growth Inibhition (%)

0 0

-20 -20

-40 -40

-60 -60

-80 -80

-100 -100
1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5 1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5
Concentration (mol L-1) Concentration (mol L-1)

CPD-05 CPD-06
20 20
Growth Inibhition (%)

Growth Inibhition (%)

0 0

-20 -20

-40 -40

-60 -60

-80 -80

-100 -100
1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5 1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5
Concentration (mol L-1) Concentration (mol L-1)

CPD-07 CPD-08
20 20
Growth Inibhition (%)

Growth Inibhition (%)

0 0

-20 -20

-40 -40

-60 -60

-80 -80

-100 -100
1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5 1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5
Concentration (mol L-1) Concentration (mol L-1)

Logran
20
Growth Inibhition (%)

-20

-40

-60

-80

-100
1 x 10-3 3 x 10-4 1 x 10-4 3 x 10-5 1 x 10-5
Concentration (mol L-1)

Figure S8. Wheat coleoptile bioassay testing the synthesized DC derivatives contaminated with urea

or thiourea (proportion of  2 DC : 1 urea/thiourea). Logran is the positive control. CDPs 01, 03, 05,

07 and 08 are indeed and respectively DCs 01, 02, 03, 04 and 05 contaminated with urea. CPDs 02,

04 and 06 are respectively DCs 01, 02, 03, contaminated with thiourea.

S24
Figure S9. Cellular division (mitosis) inhibition by DC-03 contaminated with urea (proportion of  2

DC : 1 urea/thiourea). (A) Nuclei stained with the commercially available DAPI (blue emitter). (B)

Anti--Tubulin monoclonal antibody (commercially available red emitter). (C) Merged images from

(A)-(B). Remarkably, despite the presence of those contaminant, DC-03 induced monoastral spindles

in mitotic cells.

S25
 (A)

(B)

Figure S10. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-01. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of DC-01.

S26
Figure S11. 2D COSY NMR (600 MHz, DMSO-d6) for DC-01.

S27
Figure S12. 2D HSQC NMR (600 MHz and 150 MHz, DMSO-d6) for DC-01.

S28
Figure S13. 2D HMBC (600 MHz and 150 MHz, DMSO-d6) for DC-01.

S29
 (A)

(B)

Figure S14. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-02. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of DC-02.

S30
 (A)

(B)

Figure S15. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-03. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of DC-03.

S31
 (A)

(B)

Figure S16. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-04. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of DC-04.

S32
 (A)

(B)

Figure S17. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of DC-05. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of DC-05.

S33
 (A)

(B)

Figure S18. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of APC-01. (B)13C{1H} NMR spectrum (150

MHz, DMSO-d6) of APC-01.

S34
 (A)

(B)

Figure S19. (A) 1H NMR spectrum (600 MHz, CDCl3) of TCC-01. (B)13C{1H} NMR spectrum (150 MHz,

CDCl3) of TCC-01.

S35
 (A)

(B)

Figure S20. (A) 1H NMR spectrum (600 MHz, DMSO-d6) of 4-aminocoumarin. (B)13C{1H} NMR

spectrum (150 MHz, DMSO-d6) of 4-aminocoumarin.

S36
 Transmittance (%)
3446 2736, 2615
3068

1182

1495 1093

1346, 1299

1567
759
1616
1660

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S21. FTIR (KBr) spectrum for DC-01.

2732, 2616
Transmittance (%)

3448 3068, 3002, 2937, 2836

1454

1309
1093
1178
1510
1560
1257
1617, 1604
1353
767
1668

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S22. FTIR (KBr) spectrum for DC-02.

S37
 2736, 2615

Transmittance (%)
3081
2898 1436

1236
3448
1567
1488
1344, 1309

763
1616, 1602
1097, 1039

1662

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S23. FTIR (KBr) spectrum for DC-03.

Transmittance (%)

3070 2728, 2609

3428

1093
1488

1560

1617, 1604
1351, 1307
765
1668

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S24. FTIR (KBr) spectrum for DC-04.

S38
 2917, 2792

Transmittance (%)
3045

1043
3434
1400, 1351

1540
755

1606

1673

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S25. FTIR (KBr) spectrum for DC-05.

Transmittance (%)

3390 3208 1457

817, 750
1193
1550
1644 1118, 937

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S26. FTIR (KBr) spectrum for 4-aminocoumarin.

S39
 Transmittance (%) 3284

2198
3378 3180
1608 1058

1380

1708 1673

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S27. FTIR (KBr) spectrum for APC-01.

2869
3064
Transmittance (%)

2956
3425

1055

761

1192, 1170
1365

1714 1658

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Figure S28. FTIR (KBr) spectrum for TCC-01.

S40
 (A)

(B)

Figure S29. (A) ESI(+)-MS for DC-01. (B) ESI(+)-MS/MS for DC-01.

S41
 (A)

(B)

Figure S30. (A) ESI(+)-MS for DC-02. (B) ESI(+)-MS/MS for DC-02.

S42
 (A)

(B)

Figure S31. (A) ESI(+)-MS for DC-03. (B) ESI(+)-MS/MS for DC-03.

S43
 (A)

(B)

Figure S32. (A) ESI(+)-MS for DC-04. (B) ESI(+)-MS/MS for DC-04.

S44
 (A)

(B)

Figure S33. (A) ESI(+)-MS for DC-05. (B) ESI(+)-MS/MS for DC-05.

S45
 (A)

(B)

Figure S34. (A) ESI(+)-MS for APC-01. (B) ESI(+)-MS/MS for APC-01.

S46
 (A)

(B)

Figure S35. (A) ESI(+)-MS for TCC-01. (B) ESI(+)-MS/MS for TCC-01.

S47
Determination of numerical values

Productivity is formally expressed as the molar ratio of the produced output to the

unreacted limiting reagent. This can be determined through the following equation:

𝑛𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑌𝑖𝑒𝑙𝑑
𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = ≡
(1 − 𝑌𝑖𝑒𝑙𝑑) ∗ 𝑛𝐿𝑖𝑚𝑖𝑡𝑖𝑛𝑔𝑅𝑒𝑎𝑐𝑡𝑎𝑛𝑡 1 − 𝑌𝑖𝑒𝑙𝑑

Example: In the synthesis of DC-01, 0.297 g of the compound was obtained

from a reaction involving 2 mmol of 4-hydroxycoumarin and 1 mmol of

benzaldehyde. This yield corresponds to approximately 0.72 mmol, resulting in a

72% yield. Calculating the productivity using the productivity equation, the value for

this synthesis is 2.57, as demonstrated below.

0.72 𝑚𝑚𝑜𝑙 0.72

≡ = 2.57
(1 − 0.72) ∗ 1 𝑚𝑚𝑜𝑙 1 − 0.72

The impact of the solvent was investigated by correlating the Kamlet-Taft

solvatochromic parameters of each solvent with the natural logarithm of

productivity.

S48
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S49