Hybridization Approach Applied to Umbelliferon and Vanilloids toward New Inhibitors of Carbonic Anhydrases IX and XII with In Vitro Antiproliferative and Anti-inflammatory Activities.

1
Introduction
Lung cancer is the most
commonly diagnosed cancer and the leading
cause of cancer-related death worldwide, accounting for approximately
12.4% of all new tumor cases. Nonsmall
cell lung cancer (NSCLC) represents about 85% of all lung cancer cases, with lung adenocarcinoma being the most prevalent
and lethal subtype, due to its molecular heterogeneity and typically
late-stage diagnosis. Standard treatments,
often including pemetrexed alone or in combination with chemotherapy
or radiotherapy, are often limited by drug resistance and side effects.
Although recent precision medicine introduced targeted treatments
for specific genetic mutations such as EGFR, ALK, and ROS1, significantly
improved outcomes for certain genetic subsets, a large proportion
of patients still lack effective therapeutic options.

In this scenario, a deeper understanding of the molecular
mechanisms
behind NSCLC development and progression and the identification of
new therapeutic targets remain crucial and challenging. Among these,
human (h) carbonic anhydrases (CAs, EC: 4.2.1.1) IX and XII have emerged.
CAs are a superfamily of metalloenzymes, responsible for the reversible
hydration of carbon dioxide (CO2) to bicarbonate (HCO3
–) and protons (H+) and involved
in many physiopathological pathways, including homeostasis, pH balance,
and CO2 fixation.
,
Isoforms IX and XII
are transmembrane isoenzymes, overexpressed in various solid tumors
under hypoxic conditions, including lung cancer,
−

that are involved
in regulating pH and sustaining the acidic tumor microenvironment,
thereby promoting lung cancer progression, metastasis, and therapy
resistance.
−

High expression of hCAsIX and XII has been linked to worse survival
in lung cancer patients, with the isoform IX being associated with
poor prognosis and aggressive NSCLC phenotypes. Additionally, hCA XII supports the function of P-glycoprotein
(Pgp), which is a key efflux pump responsible for drug resistance.
By maintaining Pgp activity, hCA XII enhances the clearance of chemotherapeutic
agents, promoting cancer cell survival and resistance to treatment.

Besides CAs, specific surface proteins
were found to contribute
to NSCLC progression. In particular, the epithelial cell adhesion
molecule (EpCAM) is frequently overexpressed in epithelial tumors
and promotes proliferation, migration, and the epithelial-to-mesenchymal
transition (EMT). CD9, a member of the tetraspanin family, is involved
in cell adhesion, signaling, and the regulation of exosome release,
and it shows context-dependent roles in cancer, with its downregulation
being associated with increased cell motility, high aggressivity,
and poor prognosis in NSCLC patients.
,

The
molecular alterations are sustained by a pro-inflammatory tumor
microenvironment, aligned with the complex transition from inflammation
to tumorigenesis. While it is well established that environmental
stimuli can trigger inflammation, the oncogenic changes driven by
chronic inflammation within the tissue microenvironment are less well
understood. In particular, the mechanisms by which these changes initiate
and promote pro-tumorigenic processes remain to be fully elucidated. Chronic inflammation of the bronchial epithelial
cells, which represents the first line of defense in the respiratory
tract, can initiate and sustain inflammatory responses upon exposure
to environmental insults such as pathogens, pollutants, or cigarette
smoke, and fostered by the transcription factor NF-kB signaling, lead
to the release of pro-inflammatory cytokines and chemokines, including
interleukins IL-6, IL-8, and Tumor-Necrosis Factor-α (TNF-α),
which recruit and activate immune cells, further amplifying the inflammatory
milieu.

In this complex landscape,
Nature has long served as a rich source
of pharmacologically active compounds, with several plant-derived
metabolites (e.g., coumarins, phenolic compounds) showing relevant
anti-inflammatory and anticancer properties, revealing a high potential
for application in the treatment of human diseases, as also largely
exploited by traditional herbal medicine.
−

Also, several
natural compounds have gained attention as multitatget anticancer
agents due to their structural diversity and ability to modulate key
tumor-related patways, Among these, 7-hydroxycoumarin, commonly known
as umbelliferon (UMB, 1, Figure
), is a coumarin derivative
distributed in numerous plant families such as Umbelliferae, Fabaceae,
and Rutaceae.
,

UMB and its isomer 6-hydroxycoumarin
(2, Figure
) have a
multifaceted biological profile, including antioxidant, anti-inflammatory,
and anticancer properties, which are exerted through multiple mechanisms,
ranging from oxidative stress modulation to apoptosis regulation.
,,
Of particular interest, certain
coumarins like 1 and 2 were found to act
as suicide inhibitors of CAs due to their esterase activity (as reported
for most representative α-, η-, and ι-CA classes
to date).
−

Specifically, the compact nucleus in 2H-chromene-2-one binds near the entrance of the CA catalytic site
and undergoes lactone hydrolysis, which frees the hydroxycinnamic
acid product, the “opened” form, that remains bound
to the enzyme zinc-bound water molecule, leading to the catalytic
activity inhibition due to steric hindrance.
−

Building
on this mechanism, several UMB derivatives
have been synthesized with the aim of improving CA inhibition and
anticancer activity.
−

For instance, monoterpene–coumarin hybrids designed using
the tail approach demonstrated selective inhibition of tumor-associated
CA isoforms and cytotoxicity against cancer cells. This strategy involves decorating the coumarin scaffold
with functional tails to enhance the target engagement and bioactivity.
Inspired by these findings, we propose to investigate the interconnection
between chronic inflammation and tumorigenesis, especially in lung
cancer, by designing and studying the effect of a series of hybrid
derivatives of UMB, selected as hCAs IX and XII-inhibiting
pharmacophore, and another natural compounds scaffold, that of vanilloids
(3–6, Figure
).
Like other natural phenolic compounds,
,,
vanilloids are also endowed with
antioxidant
and anti-inflammatory activity. The parent natural compound, Vanillin
(Van, Figure
), is reported to possess strong antitumor properties, likely
due to its activity on membrane-bound receptors such as the transient
receptor potential vanilloid type 1 (TRPV1) receptor as well as intracellular
targets including MARK4, CAMK4, and CK2, which are implicated in cancer
cell proliferation, apoptosis, and migration. Also, it was shown to
modulate cellular redox balance and DNA repair pathways. In this context, we designed and synthesized
a series of UMB–vanilloid conjugates through linear
aliphatic spacers or copper-catalyzed azide–alkyne cycloaddition
(CuAAC)-derived triazoles, aiming to combine the CA inhibitory properties
of the coumarin core with the bioactivity of vanilloids. Our strategy
was expected to enhance the pharmacological profile of the parent
natural compounds and allow for the modular introduction of structural
diversity to optimize target engagement in NSCLC. Aliphatic linkers
were
selected to explore conformational space and modulate the length between
the two main scaffolds, whereas triazoles can be useful to improve
aqueous solubility and metabolic stability.

2
Results
and Discussion
2.1
Design and Synthesis of
the Derivatives Library
A wide library of hybrids was designed
as hCAs IX and XII inhibitors
and synthesized (Figure
). The derivatives are composed of the UMB scaffold
and vanilloid moiety, linked together through methylene spacers, in
series A (7–24), or click chemistry-generated
1,2,3-triazoles, in series B (25–33).
In detail, series A derivatives
(7–24) were obtained by performing
two subsequent
microwave-assisted nucleophilic substitutions in the presence of potassium
carbonate in acetonitrile, as illustrated in Scheme
. Moreover, the proper vanilloid (3–6) was first reacted with the suitable alkyl dihalide a–e to afford intermediates 34–37, which undergo
reaction with UMB (1). As for series B derivatives, intermediates 34–37 were
converted into the corresponding azide (38–41)
and were reacted with propargyl umbelliferon (42), obtained
by reacting UMB (1) with propargyl bromide
(Scheme
). The latter
reacted with the proper azide (38–41) via Huisgen
CuAAC reaction, affording the 1,4-disubstituted triazole derivatives
(25–33).

2.2
Carbonic Anhydrase Inhibition
Assay
Hybrid compounds 7–33 were profiled
in vitro
through the stopped-flow CO2 hydration assay for their ability to modulate the hypoxic-tumor-related
hCAs IX and XII, along with the abundantly expressed isoforms I and
II. The obtained inhibition constant (K
I) values are reported in Table
and compared to the reference compounds UMB (1), vanilloids (3–6), the UMB intermediate 42, and the pan-CA inhibitor
acetazolamide (AAZ).
As can be observed in Table
, all the hybrids (7–33)
showed promising K
I values against the
two cancer-related isoforms, ranging from 69.6 to 941 nM for hCA IX
and 66.1 to 694 nM for isozyme XII (Table
). Moreover, they demonstrated high selectivity
versus hCAs IX and XI over hCAs I and II, being inactive at the highest
concentration tested (10,000 nM).Interestingly, UMB (1) displayed
a potent inhibitory activity and selectivity toward hCAs IX and XII,
with KI values of 24.9 and 45.1 nM, respectively, whereas
its regioisomer 2 was less active.

Moreover, parent vanilloids
3–6 did not exhibit inhibitory activity against
all four isoenzymes,
with Van (3) showing weak activity on hCAs
I and II, with KI values of 11.4 and 7.15 μM, respectively.
These data were also in accordance with phenolic compounds, usually
recognized as weak inhibitors of CAs.

Lastly, the propargylated UMB intermediate
(42) was found to be totally inactive on isoenzymes I
and II but showed interesting activity toward the cancer-related isoforms,
with K
I values slightly higher than hybrids 7–24, especially against hCA IX (K
I = 1350 nM), thereby presenting a good selectivity
over the physiologically relevant isoforms I and II.

By analyzing data for series A compounds,
robust
structure–activity relationship (SAR) considerations could
be performed:Regarding hCA
IX, the most potent compounds were 7, 8, 9, and 10, exhibiting K
I values ranging from 69.6 to 93.8 nM. These
results underscore the importance of the spacer between UMB and the vanilloid nucleus in the activity, as a shorter linker correlates
with a stronger inhibitory activity. In contrast, compounds 11 and 12, featuring a 3-methylene linker and
bearing
o

Van or etVan nucleus, respectively, showed reduced potency against hCA IX, with
KI values of 174.5 and 353 nM, respectively. Interestingly,
these findings highlight the critical role of the vanilloid portion
selection in designing effective hCA inhibitors. Compounds with longer
spacers (4-, 5-, or 6-methylene units), as in hybrids 13–15 and 18–24, showed K
I values of 434–829
nM, except 16 and 17, which exhibited higher
potency, with K
I values of 295 and 233
nM, respectively.

Focusing our attention
on hCA XII, compound 7 showed strong inhibition with
a K
I of
66.1 nM, being the best-in-class of the series. Conversely,
compounds 8–24 were less potent (K
I values of 358–694 nM). In this case,
the distance between the two main moieties does not seem to significantly
impact the inhibitory activity of the series A derivatives. Interestingly,
compounds 16 and 17, which showed potent
inhibition of hCA IX, exhibited weak activity against hCA XII, with K
I values of 694 and 590 nM, respectively, thereby
making them the least effective compounds overall against this isoform.
These findings underscore the distinct structural requirements for
these hybrids to inhibit the two tumor-associated hCA isoforms.

The introduction of the triazole ring in
the spacer
in the series B compounds (25–33) increased
the distance between the UMB and vanilloid moieties,
resulting in slightly different results for hCAs IX and hCA XII.In particular, series B compounds displayed
a reduced inhibitory potency, with K
I values
ranging from 627 to 941 nM against hCA IX.

Better results were observed against hCA XII, with the
compounds showing K
I values ranging from
225 to 450 nM. Notably, isomers 31 and 32 exhibited distinct inhibitory profiles: the
iso

Van derivative (31) was significantly
more potent than the
o

Van 32, with half of the K
I value. This suggests
that the position of the aldehyde and methoxy groups plays a key role
in modulating hCA XII inhibition.

2.3
Molecular Modeling Studies
To evaluate
the ligand–protein interactions that could rationalize the
selectivity of the coumarin derivatives, molecular modeling analyses
based on docking and molecular dynamics (MD) simulations were carried
out. Compound 7 was selected as a representative ligand
of the series due to its high inhibitory activity against both hCA
IX and XII isoforms. Previous studies demonstrated that upon binding
to the catalytic site of hCAs, coumarin-based ligands are subjected
to hydrolysis at the level of their bicyclic core, which converts
them into the corresponding 2-hydroxy-cinnamic acids. Nevertheless,
the hydrolyzed compounds are still able to interact with the catalytic
pocket of the enzymes, indeed representing the actual hCA inhibitors.
,
Based on these considerations, molecular modeling studies were used
to investigate the potential binding mode into the studied hCA isoforms
of the 2-hydroxy-cinnamic acid obtained upon hydrolysis of compound 7. The ligand was docked into the binding site of hCA I, II,
IX, and XII using GOLD software with the PLP scoring function. Subsequently,
MD simulations were performed to account for protein flexibility and
consider the role of the solvent in the enzyme–ligand interactions
(see Materials and Methods for details). Figure S1 shows the energy-minimized structures of the ligand–protein
complexes obtained after the equilibration of the solvated systems,
performed as a preliminary step in the MD simulations. As observed
in Figure S1, the hydrolyzed form of compound 7 appeared to be able to bind to the catalytic site of all
four hCA isoforms, forming water-mediated interactions between its
carboxylic group and the prosthetic zinc ion of the enzymes. However,
during the production stage of the MD simulation, performed for evaluating
the stability of the equilibrated ligand–protein complexes,
the interactions of 7 with the catalytic pocket of hCA
I and II were progressively lost, and the ligand completely left the
binding site of the two proteins within the first 50 ns of MD, as
demonstrated by the analysis of the root-mean-square deviation (RMSD)
of the position of the ligand during the simulation with respect to
its initial coordinates (Figure S2). These
results, suggesting that the ligand cannot properly interact with
the catalytic site of hCAs I and II, are consistent with its lack
of activity against hCAs I and II (Table
). On the contrary, the binding disposition
of 7 into the catalytic pocket of hCAs IX and XII was
found to be stably maintained throughout 100 ns of simulations (Figure S2), in agreement with its activity and
selectivity for the two isoforms (Table
). With the aim of better evaluating and
refining the binding mode of the inhibitor into the catalytic site
of hCAs IX and XII, an additional 200 ns of MD simulation was performed
for the two corresponding ligand–protein complexes (7-hCA IX and 7-hCA XII). The RMSD analysis of the disposition
of the ligand during the whole simulation time (300 ns) confirmed
the stability of the ligand–protein interactions observed in
the two complexes, although the compound showed considerable adjustments
of its binding conformation during the MD, compared to the initial
conformation, with an average RMSD of about 4 Å (Figure S3). Interestingly, after about 120 ns
of MD, the binding conformation of the ligand into hCA IX partially
rearranged (as shown by the increase in the average RMSD to 5.7 Å)
to significantly converge into the binding mode observed in complex
with hCA XII, and the optimized conformation was then maintained for
the remaining simulation time (Figure S3). Figure
shows
the minimized average structure of hCAs XI and XII in complex with
the hydrolyzed form of compound 7 obtained from the second
half of the extended MD simulation.
As anticipated, the binding modes of the ligand
into the two hCA
isoforms appear similar, at least at the level of the 2-hydroxy-cinnamic
acid moiety. In fact, the ligand is anchored to both enzymes’
catalytic pockets thanks to its carboxylic group, which forms three
different H-bonds with the two threonine residues placed in proximity
of the prosthetic zinc ion. In particular, two H-bonds are established
with the backbone nitrogen and the hydroxyl group of T333 in hCA IX
(Figure
A) and the
homologue T227 in hCA XII (Figure
B), while a third H-bond is formed with the backbone
nitrogen of T332 in hCA IX (Figure
A) and the homologue T226 in hCA XII (Figure
B). Moreover, the ligand shows
water-mediated interactions with the hydroxyl group of T332/T226 and
the catalytic ion of both hCA isoforms. Finally, even the main hydrophobic
interactions formed by the 2-hydroxy-cinnamic portion of the inhibitor
in hCAs IX and XII are comparable, being represented by lipophilic
contacts with V235 and L331 in hCA IX (Figure
A), and with homologue residues V147 and
L225 in hCA XII (Figure
B). In addition to these ligand–protein interactions, shared
by the two complexes, in hCA IX, the hydrolyzed form of compound 7 shows a further water-mediated H-bond with the side chain
of Q224, established by its phenolic group, as well as hydrophobic
interactions with V262 and L266 formed by its vanilloid moiety (Figure
A). The lack of such
interactions in hCA XII is well compensated by the presence of a water-bridged
H-bond with T116 and van der Waals interactions with A157, S158, and
S161, which are both formed by the ligand’s vanilloid core
(Figure
B). Overall,
the similar binding modes predicted for the compound in hCA IX and
XII are consistent with the similar inhibitory activities determined
for 7 against the two enzymes. Moreover, the presence
of a water-mediated H-bond between the terminal aldehyde group of
the ligand and T116 might justify the drop of hCA XII inhibitory activity
observed when moving such a group from the meta to para position with respect to the alkoxy linker, as in compound 8, and/or when adding a further methylene group to the linker
connecting the vanilloid moiety with the coumarin core, as in compounds 9 and 10 (Table
). In addition, the evidence that compound 8 maintains the same hCA IX inhibitory activity observed for 7, and that 9 and 10 only show a
minor potency reduction against this isoform (Table
), is in agreement with the interactions
predicted for the vanilloid moiety of compound 7 within
hCA IX, consisting only in van der Waals interactions that are less
easily perturbed by the structural changes present in compounds 8–10 compared to 7.
Finally, the
analysis of the above-described ligand–protein
complexes, i.e.,
7-hCA IX and 7-hCA XII, in comparison with the structures of hCA I and
hCA II binding sites, suggested a rationale for justifying why the
ligand would not be able to properly interact with the catalytic pockets
of these latter isoforms. As shown in Figure S4A,B, in hCA I, the carboxylic group of the ligand cannot form the two
anchoring H-bonds established with T333 in hCA IX (Figure
A) and the homologue T227 in
hCA XII (Figure
B),
due to the presence of a histidine (H201 in hCA I) in place of the
threonine residues. Moreover, the presence in hCA I of four nonconserved
residues (i.e., F92, L132, A133, and A136) in the
region of the binding pocket interacting with the vanilloid moiety
of 7 would not let the ligand to assume a binding mode
similar to that predicted into either hCAs XI (Figure
A) or XII (Figure
B) and thus to maintain the same water-mediated
interactions. Although both the threonine residues anchoring the carboxylic
group of 7 to hCA IX and XII binding sites are conserved
in hCA II, four nonconserved residues (i.e., I91,
F130, G131, and V134) substantially reshape the entrance of the catalytic
pocket of this isoform (Figure S4C,D).
In particular, the side chain of F130 creates a sort of hill in the
middle of the cavity entrance, which would significantly clash with
both the vanilloid moiety and the cinnamic ring of 7,
in its binding mode predicted into hCA IX (Figure S4C). For this reason, the ligand could maintain neither a
similar binding conformation nor the water-bridged H-bond with Q224
observed in hCA IX (Figure
A). Similarly, the steric clashes with F130 would hamper the
ligand from assuming the binding conformation predicted in hCA XII
(Figure S4D); in addition, the presence
of I91 in hCA II, replacing T116 of hCA XII, would certainly prevent
the formation of the water-mediated H-bond formed by the vanilloid
group of 7 in hCA II (Figure
B). Overall, these considerations may justify
the lack of stable interaction between compound 7 and
hCAs I/II, and thus, the lack of inhibitory activity of the ligand
against the two isoforms.

2.4
In Vitro Investigations
on Human Cell Lines
2.4.1
Cell Viability Assessment
The effects
of UMB (1), parent vanilloids (3–6), and the most potent hybrids (7, 8, 9, 10, 11, 26, and 33) were assessed on human bronchial
epithelial (BEAS-2B) and human lung adenocarcinoma (A549) cell lines
by measuring their viability in response to increasing concentrations
of compounds up to 72 h via the MTT assay (Figure
).
Interestingly, etVan (6) significantly
reduces BEAS-2B cell viability within the first 24 h in an inverse
dose-dependent manner, while it promotes cell growth at later time
points in both BEAS-2B and A549 cells (Figure
A). These data were compared to those obtained
by testing two etVan-bearing hybrids from the series A (8) and B (33), showing distinct activity profiles. Compound 8 induces
a minor decrease in BEAS-2B cell vitality at the earliest exposure
time. Next, the activity becomes dose-dependent, BEAS-2B cell vitality
being significantly reduced to 50% at the highest concentration (100
μM) tested compared to that of the untreated control. In parallel, 8 impairs A549 cell growth exclusively within the first 24
h of exposure. Otherwise, compound 33 has no relevant
effects on the viability of BEAS-2B cells. However, it decreases A549
cell survival in an inverse dose-dependent manner, specifically in
the first 24 h after exposure.
The focused etVan subseries tested reveals intriguing
and divergent biological effects, particularly in relation to time-
and cell-type-specific responses. While the parent etVan showed time-dependent dual activity, its UMB-hybridization
products 8 and 33 exhibited distinct and
more selective cytotoxic profiles against the cancer cell line, highlighting
their promising role as anticancer agents.
As regards
iso

Van subseries,
the parent compound was tested along with the alkyl-linker-based hybrids 7 and 10 and the triazole derivative 26. BEAS-2B cell viability rises dose-dependently up to 24 h following
iso

Van (4) treatment.
After that, it decreases dose-dependently, although it is still higher
than the control sample (Figure
B). In contrast, A549 cells grow significantly after
48 h of exposure. Compound 7 decreases both BEAS-2B and
A549 cell viability exclusively within 24 h of exposure. Compound 10 significantly impairs BEAS-2B cell survival after 48 h
in a dose-dependent manner. Conversely, in A549 cells, it demonstrates
an inverse dose-dependent activity exclusively within the first 24
h, as well as compound 26, showing selectivity for A549
cells. As regards the
iso

Van subseries, the parent
iso

Van (4) mainly promotes cell viability, whereas hybrid 7 has early effects on cell viability in both cell lines,
and compounds 10 and 26 are selectively
cytotoxic toward BEAS-2B and A549 cells, respectively.
As regards Van compounds, we found that Van stimulates the
metabolic activity of BEAS-2B and A549 cells at all
time points, except for nonpathological cells at 72 h (Figure
A). Similarly, Van-hybrid 9 significantly increases BEAS-2B cell viability
after 48 h of exposure but has no relevant activity on A549 cells,
except at 24 h, where it slightly reduces cell survival. Also, we
compared the o
Van parent compound to
its hybrid 11. BEAS-2B cell vitality is significantly
enhanced within the first 24 h of exposure to
o

Van, followed by a time- and dose-dependent
reduction in cell viability. In contrast,
o

Van consistently enhances the A549 cell viability across
all time points analyzed (Figure
B). Similarly, compound 11 impairs BEAS-2B
cell viability in a time- and dose-dependent manner at 48 and 72 h,
while in A549 cells, it reduces viability only at 24 h. Interestingly, UMB (1) appears to be active at 24 h, decreasing
the viability of both BEAS-2B and A549 cells, except at 50 μM
on BEAS-2B cells (Figure
C). However, after 48 and 72 h, cell viability is slightly
higher than or lower than control levels.
In summary, the hybridization of UMB with vanilloids
seemed to significantly modulate the individual biological activities
of parent compounds, often enhancing selectivity on A549 cells over
that of BEAS-2B cells. We found that the parent compound UMB generally promotes cell viability in both cell lines, often exhibiting
dual time-dependent effects, whereas the tested hybrids determined
more specific and selective effective profiles, particularly against
the A549 cancer cell line. Thus, the structural modification performed
was able to boost the effect of the hydroxylated compounds UMB and vanilloids to more potent hCA inhibitors, highlighting their
potential as promising candidates for therapeutic development in hypoxic
tumors.

2.4.2
Cytotoxicity Occurrence in A549 Cells Exposed
to Selected Compounds (LDH Assay)
Then, compounds 9, 26, and 33 were selected because of their
biocompatibility on BEAS-2B and effectiveness on A549, especially
within the first 24 h. To assess whether the decrease in viability
might be a consequence of cytotoxicity occurrence in the presence
of selected compounds, the amount of lactate dehydrogenase (LDH) released
by A549 cells was measured in response to increasing concentrations
of compounds after a 24 h exposure (Figure
). Hybrids 26 and 33 at lower concentrations (10 μM) are the most effective in
causing cytotoxicity in an inverse dose-dependent manner, in alignment
with data on cell viability. The release of LDH into the culture medium
is nearly doubled compared to that of the control sample, with 1.71-
and 1.80-fold increases for 26 and 33, respectively.
In contrast, compound 9 exhibits greater cytotoxicity
at higher concentrations. However, none of the LDH increases registered
are statistically significant. Hence, cell cycle analyses were further
performed in the same experimental conditions to investigate if the
decrease of cell viability in the presence of 9, 26, and 33 might be caused by an arrest of cell
proliferation due to a cell block in one of the cell cycle checkpoints.

2.4.3
Cell Cycle Analyses
To establish
whether the selected compounds 9, 26, and 33 could induce cell cycle arrest at specific checkpoints
and decrease A549 cell proliferation, cell percentages found in G1,
S, and G2 phases (Figure
) were analyzed after 6 and 24 h from treatment. In Figure
, the bars related
to the control sample display a typical cell cycle profile, with proliferative
and active cells at 6 h (G1 phase = 51.95%; S phase = 30.56%; G2/M
phase = 17.49%) followed by an increase in cells in the S phase (37.00%)
and a decrease in cells in the G2 phase (11.16%) at 24 h. The G1 phase
does not vary. Treatment with compound 9 leads to a significant
increase in G1 cells (54.25% at 50 μM) and a minor decrease
in G2 cells (14.04% at 50 μM) after 6 h, followed by a moderate
reduction in G1 cells after 24 h. Exposure to compound 33 at higher concentrations (50 and 100 μM) results in a significantly
higher percentage of G1 cells compared with the control (54.80%),
with a decrease in G2 cells (13.44% at 100 μM). The G1 cell
percentage falls after 24 h, showing a dose-dependent behavior along
with a rise in S-phase cells. Compound 26 causes a remarkable
delay in the cell cycle in the G1 phase (56.17% at 100 μM) along
with a decrease in G2 cells after 6 h. Subsequently, at 24 h, the
G1 cell percentage decreases, while the S phase cell percentage increases.
The observed changes in cell cycle progression
upon treatment with
hybrids 9, 26, and 33 clearly
highlight the compounds’ ability to affect cell proliferation
by inducing cell cycle arrest, particularly in the G1 phase. Indeed,
such an increase in G1 cells can indicate a delay in the cycle progression,
while a decrease in G2 cells can correspond to an inhibition of mitosis.

2.4.4
Influence on CD9 and
EPCAM Expression
The effect of compounds 9, 33, and 26 at increasing concentrations (10,
50, and 100 μM)
on A549 cells was evaluated after 24 h of exposure by measuring the
expression of cell surface markers CD9 and epithelial cell adhesion
molecule (EPCAM) using specific fluorescently labeled antibodies and
DAPI staining. Briefly, CD9 is a tetraspanin mainly involved in cell
migration and metastasis. In parallel,
the transmembrane glycoprotein EPCAM is involved in cell adhesion
and is a well-known cell biomarker of lung cancer.

The fluorescence microscopy images shown in Figure
A,C indicate that
all three compounds produced a qualitatively similar pattern in the
expression of the analyzed markers. No appreciable differences were
observed between the compounds themselves; however, the concentration
had a significant impact on the variability of the CD9 fluorescence
intensity, suggesting a dose-dependent response for this marker. Interestingly,
and keeping in consideration the role of CD9 in reducing migration
of A549 cells, compound 33 revealed the strongest effect,
significantly decreasing the CD9 fluorescence intensity at 100 μM
compared to control and 10 μM. Compound 26 significantly
decreases CD9 expression at 100 μM compared to 10 μM (Figure
B). Similarly, the
compounds reveal the same effect on the EPCAM fluorescence intensity,
although the post hoc analysis failed to find significance
for multiple comparisons.

2.4.5
Anti-inflammatory Response in BEAS-2B Cells
Exposure of BEAS-2B cells to the conditioned medium from lipopolysaccharide
(LPS)-stimulated macrophages led to a time-dependent decrease in viability
(Figure
). Notably,
a significant reduction, approximately 50% compared to the control,
was observed after 48 h of treatment. In contrast, no significant
changes in the metabolic activity were detected within the first 24
h of exposure. While conditioned, treating cells with selected compounds 9, 33, and 26 effectively counteract
the conditioned medium-induced reduction in viability. Notably, compounds
can restore viability to levels comparable to those of untreated controls.
Among them, compounds 9 and 33 demonstrate
higher potency than 26, exhibiting significant efficacy
even at lower concentrations (10 and 50 μM).
Next, the LDH assay was performed
on the supernatants
collected
from BEAS-2B cells exposed to the conditioned medium and to the compounds
(Figure
A). As expected,
a significant increase in LDH release is observed after 48 h of treatment.
Consistent with the MTT assay results, all the compounds reduce the
LDH release in a dose-dependent manner, effectively counteracting
the cytotoxic effects of the conditioned medium. Microscopic analysis
of BEAS-2B cells exposed to the conditioned medium revealed marked
morphological alterations in accordance with an inflammatory and cytotoxic
response (as indicated by the red arrows; Figure
B). Indeed, cells appear more round-shaped
and with enhanced cytoplasmic elongations, whereas they display the
typical polygonal morphology of epithelial cells in the untreated
control. After 48 h of treatment, cells exhibited a loss of their
typical epithelial morphology, characterized by a uniform monolayer
of polygonal cells with well-defined borders and intact cell–cell
junctions. It was observed that there was a reduction in cell density
and loss of intercellular contacts. Treatment with selected compounds 9, 33, and 26 was effective in restoring
the disrupted epithelial integrity induced by the conditioned medium,
with a full recovery of normal cell shape and adhesion. Interestingly,
these findings seem to suggest that the tested compounds 9, 33, and 26 could exert a cytoprotective
effect against inflammation-induced cellular damage in BEAS-2B cells,
and, in particular, 9 and 33 demonstrate
anti-inflammatory properties at lower concentrations.

3
Conclusions
In summary, we developed
a series of UMB-vanilloid
hybrids as selective inhibitors of the tumor-associated hCAs IX and
XII. Most derivatives displayed potent and isoform-specific inhibitory
profiles and demonstrated significant anticancer and anti-inflammatory
activities in vitro. Interestingly, they were able to induce G1 arrest,
downregulate metastasis-related markers, and protect nonmalignant
bronchial epithelial cells from inflammatory stress. Their dual role
as anticancer and cytoprotective agents underscores the potential
of this hybridization approach, enabling the generation of multifunctional
scaffolds for NSCLC therapy and laying the foundation for further.

4
Experimental Section
4.1
Synthesis of the Derivatives Library
4.1.1
General
Chemistry
All commercially
available chemicals and solvents were used as purchased. Chromatographic
separations were performed on columns packed with silica gel (230–400
mesh, for the flash technique). Reaction monitoring was performed
through thin-layer chromatography (TLC) by using 0.2 mm-thick silica
gel–aluminum-backed plates (60 F254). TLC spot visualization
was performed under short and long wavelengths (254 and 365 nm, respectively)
of ultraviolet irradiation and stained with ninhydrin or basic permanganate. 1H and 13C NMR spectra were recorded on a Varian
Oxford 300 MHz NMR operating at 300 and 75 MHz, respectively. The
NMR spectra for the newly synthesized compounds are reported in the
Supporting Information, in Figures S5–S58. Spectra are reported in parts per million (δ scale) and internally
referenced to the CDCl3 or DMSO-d
6 signal, at δ 7.26 and 2.50 ppm, respectively. Data
are shown as follows: chemical shift, multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, qi = quintet, m = multiplet,
br = broad signal, and ap = apparent), integration, and coupling constants
(J) in Hertz (Hz). Chemical shifts for carbon are
reported in parts per million (δ scale) and referenced to the
carbon resonances of CDCl3 or DMSO-d
6 at δ 77.0 or 39.0 ppm, respectively. The 1H NMR spectra confirmed the anticipated number of hydrogens in each
compound. Melting points were measured on a Stuart melting point apparatus
SMP1 (Fisher Scientific Italia, Segrate (MI), Italy) and are uncorrected
(temperatures are reported in °C). Elemental analyses for C,
H, and N were recorded on a PerkinElmer 240 B microanalyzer (PerkinElmer
Italia, Milan, Italy), and the analytical results are within ±0.4%
of the theoretical values for all compounds (purity >95%). Microwave-assisted
reactions were performed with Biotage Initiator+ (Biotage Sweden AB,
Uppsala, Sweden) in a 10 mL vial suitable for an automatic single-mode
reactor (2.45 GHz high-frequency microwaves, power range 0–300
W). The internal vial temperature was controlled by an IR sensor.

4.1.2
Procedure and Characterization Data for
Derivatives 7–42
4.1.2.1
General
Procedure for the Synthesis of
Compounds 7–24 (Series A)
Bromo intermediates 34–37. The proper alkyl halide selected from 1-bromo-2-chloroethane,
1-bromo-3-chloropropane, 1,4-dibromobutane, 1,5-dibromopentane, and
1,6-dibromohexane (1.5 equiv) was added to the colorless solution
of the suitable vanilloid (3–6) (1.0 equiv) in
ethanol (0.4 M). Then, an aqueous solution of K2CO3 (4.0 equiv, 1.0 M) was added to the stirring reaction mixture,
and the final homogeneous solution was placed in a microwave reactor
and exposed to microwave irradiation at 120 °C for 2–3
h (irradiation power reaches its maximum at the beginning of the reaction;
then it decreases to lower and quite constant values). After the reaction
had reached completion, the reaction mixture was cooled with pressurized
air, poured into cold H2O, and extracted with EtOAc three
times. The combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure.
The reaction crude was then purified through flash column chromatography
on silica gel with a suitable mixture of n-hexane/EtOAc
to afford the intermediates 34–37. Experimental
data were in agreement with the literature.
Alkyl Hybrids 7–24. The suitable bromo derivative 34–37 (1.5 equiv) was dissolved in DMF (0.4 M). Then, UMB (1) (1.0 equiv) and K2CO3 (4.0
equiv) were added to the stirring reaction mixture and placed in a
microwave reactor and exposed to microwave irradiation at 100 °C
for 2 h. Once the reaction was completed, the mixture was poured into
H2O and extracted with EtOAc three times (3 × 15 mL).
The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure to afford the crude
products. These were then subjected to column chromatography on silica
gel with different n-hexane/EtOAc mixtures and recrystallization
from ethanol (when solid) to afford compounds 7–24.
4-Methoxy-3-(2-((2-oxo-2H-chromen-7-yl)­oxy)­ethoxy)­benzaldehyde (7). White solid, mp 148–150 °C, 43% yield.
1

H NMR (300 MHz, CDCl3) δ: 3.96 (s, 3H, OCH3), 4.45–4.47 (m, 4H,
OCH2CH2O), 6.26 (d, J = 9.3
Hz, 1H, Ar), 6.89–6.94 (m, 2H, Ar), 7.01 (d, J = 9.0 Hz, 1H, Ar), 7.38 (d, J = 8.4 Hz, 1H, Ar),
7.49–7.51 (m, 2H, Ar), 9.86 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 56.2,
67.0, 67.4, 101.8, 110.9, 111.0, 112.9, 113.1, 113.4, 127.5, 128.8,
130.0, 143.4, 148.5, 155.0, 155.8, 161.2, 161.8, 190.7. Anal. Calcd
for C19H16O6: C, 67.05; H, 4.74.
Found: C, 67.25; H, 4.82.
3-Ethoxy-4-(2-((2-oxo-2H-chromen-7-yl)­oxy)­ethoxy)­benzaldehyde (8). White solid,
mp 139–141 °C, 67% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.44 (t, J = 7.0 Hz, 3H, OCH2CH3), 4.14 (q, J = 7.0 Hz, 2H, OCH2CH3), 4.44–4.50
(m, 4H, OCH2CH2O), 6.26 (d, J = 9.3 Hz, 1H, Ar), 6.89–6.91
(m, 2H, Ar), 7.05 (d, J = 7.5 Hz, 1H, Ar), 7.37–7.46
(m, 3H, Ar), 7.63 (d, J = 9.3 Hz, 1H, Ar), 9.85 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 14.6, 64.6, 67.0, 67.6, 101.8, 111.2, 112.9, 113.0,
113.4, 126.1, 128.8, 130.8, 143.3, 149.5, 153.6, 155.8, 161.0, 161.7,
190.9. Anal. Calcd for C20H18O6:
C, 67.79; H, 5.12. Found: C, 67.96; H, 5.20.
3-Methoxy-4-(3-((2-oxo-2H-chromen-7-yl)­oxy)­propoxy)­benzaldehyde (9). White solid, mp 135–137 °C, 80% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.35–2.43
(m, 2H, OCH2
CH
2CH2O), 3.92 (s, 3H, OCH3), 4.25
(t, J = 5.8 Hz, 2H, OCH
2CH2CH2O), 4.31 (t, J = 6.1 Hz, 2H, OCH2CH2
CH
2O), 6.22 (d, J = 9.3 Hz, 1H, Ar), 6.82–6.86
(m, 2H, Ar), 7.00 (d, J = 8.4 Hz, 1H, Ar), 7.34–7.45
(m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 9.84 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 28.8, 56.0, 64.8, 65.3, 101.5, 109.4, 111.6, 112.7,
112.8, 113.2, 126.6, 128.8, 130.3, 143.3, 149.9, 153.7, 155.9, 161.1,
161.9, 190.8. Anal. Calcd for C20H18O6: C, 67.79; H, 5.12. Found: C, 67.53; H, 5.01.
4-Methoxy-3-(3-((2-oxo-2H-chromen-7-yl)­oxy)­propoxy)­benzaldehyde (10). White solid, mp 141–142 °C, 77% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.32–2.40
(m, 2H, OCH2
CH
2CH2O), 3.95 (s, 3H, OCH3), 4.25
(q, J = 7.2 Hz, 4H, OCH
2CH2
CH
2O), 6.23
(d, J = 9.3 Hz, 1H, Ar), 6.84–6.87 (m, 2H,
Ar), 6.98 (d, J = 8.1 Hz, 1H, Ar), 7.36 (d, J = 9.0 Hz, 1H, Ar), 7.45 (d, J = 9.3 Hz,
2H, Ar), 7.62 (d, J = 9.3 Hz, 1H, Ar), 9.83 (s, 1H,
CHO).
13

C NMR (75 MHz, CDCl3) δ: 28.8, 56.2, 64.9, 65.2, 101.3, 110.0, 110.5, 110.7,
112.5, 113.1, 113.2, 127.1, 128.7, 130.0, 143.4, 148.8, 154.9, 155.9,
161.2, 162.1, 190.9. Anal. Calcd for C20H18O6: C, 67.79; H, 5.12. Found: C, 67.56; H, 5.19.
3-Methoxy-2-(3-((2-oxo-2H-chromen-7-yl)­oxy)­propoxy)­benzaldehyde (11). White solid, mp 94–96 °C, 85% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.28–2.26
(m, 2H, OCH2
CH
2CH2O), 3.82 (s, 3H, OCH3), 4.27–4.32
(m, 4H, OCH
2CH2
CH
2O), 6.25 (d, J = 9.6 Hz,
1H, Ar), 6.85–6.89 (m, 2H, Ar), 7.14 (d, J = 4.2 Hz, 2H, Ar), 7.37–7.42 (m, 2H, Ar), 7.64 (d, J = 9.3 Hz, 1H, Ar), 10.4 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 29.8,
56.0, 65.0, 71.1, 101.5, 112.6, 112.7, 113.2, 118.0, 119.4, 124.3,
128.8, 130.0, 143.4, 151.4, 152.9, 155.9, 161.2, 162.1, 190.0. Anal.
Calcd for C20H18O6: C, 67.79; H,
5.12. Found: C, 67.96; H, 5.19.
3-Ethoxy-4-(3-((2-oxo-2H-chromen-7-yl)­oxy)­propoxy)­benzaldehyde (12). White solid, mp 153–154 °C, 80% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.45
(t, J = 7.0 Hz, 3H, OCH2
CH
3), 2.33–2.41 (m, 2H,
OCH2
CH
2CH2O), 4.13 (q, J = 7.0 Hz, 2H, OCH
2CH3), 4.24–4.31 (m, 4H, OCH
2CH2
CH
2O), 6.23 (d, J = 9.3 Hz, 1H, Ar), 6.83–6.86
(m, 2H, Ar), 6.99 (d, J = 7.8 Hz, 1H, Ar), 7.34–7.43
(m, 4H, Ar), 7.62 (d, J = 9.6 Hz, 1H, Ar), 9.83 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 14.7, 28.8, 64.5, 64.8, 65.3, 101.5, 110.8, 112.1,
112.6, 112.8, 113.2, 126.4, 128.8, 130.3, 143.3, 149.3, 154.0, 155.9,
161.1, 162.0, 190.9. Anal. Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.21; H, 5.40.
3-Methoxy-4-(4-((2-oxo-2H-chromen-7-yl)­oxy)­butoxy)­benzaldehyde (13). White solid, mp 129–130 °C, 89% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.02–2.10 (m, 4H, OCH2
CH
2
CH
2CH2O),
3.91 (s, 3H, OCH3), 4.12 (t, J = 6.0 Hz,
2H, OCH
2CH2CH2CH2O), 4.19 (t, J = 5.8 Hz, 2H, OCH2CH2CH2
CH
2O), 6.25 (d, J = 9.3 Hz, 1H, Ar), 6.79–6.83
(m, 2H, Ar), 6.97 (d, J = 8.1 Hz, 1H, Ar), 7.34–7.45
(m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H), 9.84 (s, 1H,
CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.6, 25.9, 29.7, 56.0, 68.1, 68.6, 101.3, 109.3,
111.4, 112.5, 112.9, 113.1, 126.7, 128.7, 130.1, 143.4, 149.8, 153.9,
155.9, 161.2, 162.2, 190.9. Anal. Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.20; H, 5.39.
4-Methoxy-3-(4-((2-oxo-2H-chromen-7-yl)­oxy)­butoxy)­benzaldehyde (14). White solid, mp 77–78 °C, 52% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.04–2.07 (m, 4H, OCH2
CH
2
CH
2CH2O),
3.94 (s, 3H, OCH3), 4.11–4.18 (m, 4H, OCH
2CH2CH2
CH
2O), 6.24 (d, J = 9.3 Hz,
1H, Ar), 6.81–6.85 (m, 2H, Ar), 6.97 (d, J = 8.1 Hz, 1H, Ar), 7.34–7.47 (m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H), 9.84 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.6,
26.0, 56.2, 68.2, 68.5, 101.3, 110.3, 110.2, 110.6, 113.0, 126.9,
128.7, 143.4, 148.9, 190.8. Anal. Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.28; H, 5.39.
3-Methoxy-2-(4-((2-oxo-2H-chromen-7-yl)­oxy)­butoxy)­benzaldehyde (15). White solid, mp 83–85 °C, 45% yield.
1

H NMR (300 MHz, CDCl3) δ: 2.01–2.04 (m, 4H, OCH2
CH
2
CH
2CH2O),
3.89 (s, 3H, OCH3), 4.11 (t, J = 5.9 Hz,
2H, OCH
2CH2CH2CH2O), 4.20 (t, J = 6.2 Hz, 2H, OCH2CH2CH2
CH
2O), 6.25 (d, J = 9.3 Hz, 1H, Ar), 7.13–7.15
(m, 2H, Ar), 7.35–7.43 (m, 2H, Ar), 7.63 (d, J = 9.9 Hz, 1H), 10.45 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.7, 26.7, 56.0,
68.1, 74.3, 101.4, 112.5, 112.9, 113.0, 118.1, 119.3, 124.1, 128.8,
130.0, 143.4, 151.7, 153.0, 155.9, 161.2, 162.2, 190.2. Anal. Calcd
for C21H20O6: C, 68.47; H, 5.47.
Found: C, 68.60; H, 5.53.
3-Ethoxy-4-(4-((2-oxo-2H-chromen-7-yl)­oxy)­butoxy)­benzaldehyde (16). White solid,
mp 122–123 °C, 64% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.46 (t, J = 7.0 Hz, 3H, OCH2CH3), 2.06–2.13
(m, 4H, OCH2
CH
2
CH
2CH2O), 4.10–4.20 (m,
6H, OCH
2CH2CH2
CH
2O and OCH
2CH3), 6.25 (d, J = 9.3 Hz,
1H, Ar), 6.81–6.83
(m, 2H, Ar), 6.97 (d, J = 8.1 Hz, 1H, Ar), 7.34–7.44
(m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 9.84 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 14.7, 25.6, 25.7, 64.5, 68.1, 68.6, 101.4, 110.9,
111.8, 112.5, 112.9, 113.1, 126.4, 128.7, 130.2, 143.3, 149.2, 154.2,
155.9, 161.2, 162.2, 190.9. Anal. Calcd for C22H22O6: C, 69.10; H, 5.80. Found: C, 69.01; H, 5.87.
3-Methoxy-4-((5-((2-oxo-2H-chromen-7-yl)­oxy)­pentyl)­oxy)­benzaldehyde (17). White solid, mp 103–104 °C, 78% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.66–1.72 (m, 2H, OCH2CH2
CH
2CH2CH2O), 1.89–1.99 (m, 4H, OCH2
CH
2CH2
CH
2CH2O), 3.91 (s, 3H, OCH3), 4.05 (t, J = 6.1 Hz, 2H, OCH
2CH2CH2CH2CH2O), 4.14 (t, J = 6.4 Hz, 2H, OCH2CH2CH2CH2
CH
2O), 6.23 (d, J = 9.3 Hz, 1H, Ar), 6.78–6.83 (m, 2H, Ar), 6.97
(d, J = 8.4 Hz, 1H, Ar), 7.34 (d, J = 8.1 Hz, 1H, Ar), 7.40–7.45 (m, 2H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 9.84 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 22.6,
28.6, 28.7, 56.0, 68.3, 68.8, 101.3, 109.3, 111.4, 112.5, 112.9, 113.0,
126.7, 128.7, 130.0, 143.4, 149.8, 154.0, 155.9, 161.2, 162.3, 190.9.
Anal. Calcd for C22H22O6: C, 69.10;
H, 5.80. Found: C, 69.27; H, 5.89.
4-Methoxy-3-((5-((2-oxo-2H-chromen-7-yl)­oxy)­pentyl)­oxy)­benzaldehyde (18). White solid, mp 138–139 °C, 79% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.68–1.71
(m, 2H, OCH2CH2
CH
2CH2CH2O), 1.88–1.98 (m, 4H, OCH2
CH
2CH2
CH
2CH2O), 3.94 (s, 3H, OCH3), 4.05 (t, J = 6.1 Hz, 2H, OCH
2CH2CH2CH2CH2O), 4.11 (t, J = 6.4 Hz, 2H, OCH2CH2CH2CH2
CH
2O), 6.23 (d, J = 9.3 Hz, 1H, Ar), 6.79–6.84
(m, 2H, Ar), 6.98
(d, J = 8.1 Hz, 1H, Ar), 7.35 (d, J = 8.7 Hz, 1H, Ar), 7.40 (d, J = 1.5 Hz, 1H, Ar),
7.44 (dd, J = 8.1, 1.8 Hz, 1H, Ar), 7.62 (d, J = 9.6 Hz, 1H, Ar), 9.83 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 22.6,
28.7, 56.2, 68.3, 68.7, 101.4, 110.3, 110.6, 112.4, 112.9, 113.0,
126.8, 128.7, 130.1, 143.4, 149.0, 154.8, 155.9, 161.2, 162.3, 190.9.
Anal. Calcd for C22H22O6: C, 69.10;
H, 5.80. Found: C, 68.98; H, 5.73.
3-Methoxy-2-((5-((2-oxo-2H-chromen-7-yl)­oxy)­pentyl)­oxy)­benzaldehyde (19). White solid, mp 75–76 °C, 58% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.66–1.74
(m, 2H, OCH2CH2
CH
2CH2CH2O), 1.84–1.95 (m, 4H, OCH2
CH
2CH2
CH
2CH2O), 3.89 (s, 3H, OCH3), 4.05 (t, J = 6.4 Hz, 2H, OCH2), 4.16 (t, J = 6.4
Hz, 2H, OCH2), 6.23 (d, J = 9.3 Hz, 1H,
Ar), 6.80 (ddd, J = 8.0, 7.5, 2.4 Hz, 2H, Ar), 7.11–7.15
(m, 2H, Ar), 7.34–7.43 (m, 2H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 10.45 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 22.5, 28.8,
29.8, 56.0, 68.4, 74.6, 101.4, 112.5, 112.9, 113.0, 118.1, 119.2,
124.0, 128.7, 130.0, 143.4, 151.9, 153.0, 155.9, 161.2, 162.3, 190.3.
Anal. Calcd for C22H22O6: C, 69.10;
H, 5.80. Found: C, 69.24; H, 5.91.
3-Ethoxy-4-((5-((2-oxo-2H-chromen-7-yl)­oxy)­pentyl)­oxy)­benzaldehyde (20). White solid, mp 88–89 °C, 52% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.45
(t, J = 6.7 Hz, 3H, OCH2
CH
3), 1.67–1.73 (m, 2H,
OCH2CH2
CH
2CH2CH2O), 1.90–1.98 (m, 4H, OCH2
CH
2CH2
CH
2CH2O), 4.05 (t, J = 6.4 Hz, 2H, OCH
2CH3), 4.10–4.17 (m, 4H, OCH
2CH2CH2CH2
CH
2O), 6.24 (d, J = 9.3 Hz, 2H, Ar), 6.78–6.83
(m, 2H, Ar), 6.96 (d, J = 8.4 Hz, 1H, Ar), 7.34–7.43
(m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 9.83 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 14.7, 22.7, 28.6, 28.7, 64.5, 68.3, 68.9, 101.3,
110.9, 111.8, 112.5, 112.5, 112.9, 113.0, 126.5, 128.7, 130.0, 126.5,
128.7, 130.0, 143.4, 149.2, 154.3, 155.9, 161.2, 162.3, 190.9. Anal.
Calcd for C23H24O6: C, 69.68; H,
6.10. Found: C, 69.77; H, 6.18.
3-Methoxy-4-((6-((2-oxo-2H-chromen-7-yl)­oxy)­hexyl)­oxy)­benzaldehyde (21). White solid, mp 92–93 °C, 71% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.57–1.59
(m, 4H, OCH2CH2
CH
2
CH
2CH2CH2O), 1.83–1.93 (m, 4H, OCH2
CH
2CH2CH2
CH
2CH2O), 3.91
(s, 3H, OCH3), 4.02 (t, J = 6.1 Hz, 2H,
OCH
2CH2CH2CH2CH2CH2O), 4.12 (t, J = 6.7 Hz, 2H, OCH2CH2CH2CH2CH2
CH
2O), 6.24
(d, J = 9.3 Hz, 1H, Ar), 6.78–6.83 (m, 2H,
Ar), 6.96 (d, J = 8.4 Hz, 1H, Ar), 7.33–7.44
(m, 3H, Ar), 7.63 (d, J = 9.6 Hz, 1H, Ar), 9.84 (s,
1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.7, 25.7, 28.9, 56.0, 68.4, 68.9, 101.3, 109.3,
111.4, 112.4, 112.9, 126.8, 128.7, 129.9, 143.4, 149.8, 154.1, 155.9,
161.3, 162.3, 190.9. Anal. Calcd for C23H24O6: C, 69.68; H, 6.10. Found: C, 69.81; H, 6.01.
4-Methoxy-3-((6-((2-oxo-2H-chromen-7-yl)­oxy)­hexyl)­oxy)­benzaldehyde (22). White solid, mp 99–101 °C, 89% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.56–1.59
(m, 4H, OCH2CH2
CH
2CH2CH2O), 1.83–1.93 (m, 4H, OCH2
CH
2CH2CH2
CH
2CH2O), 3.94
(s, 3H, OCH3), 4.02
(t, J = 6.4 Hz, 2H, OCH
2CH2CH2CH2CH2CH2O), 4.09 (t, J = 6.7 Hz, 2H, OCH2CH2CH2CH2CH2
CH
2O), 6.24 (d, J = 9.3 Hz,
1H, Ar), 6.79–6.84 (m, 2H, Ar), 6.97 (d, J = 8.1 Hz, 1H, Ar), 7.36–7.45 (m, 3H, Ar), 7.63 (d, J = 9.3 Hz, 1H, Ar), 9.83 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.7,
25.7, 28.9, 56.2, 68.4, 68.8, 101.3, 110.0, 110.2, 110.6, 112.4, 113.0,
126.7, 128.7, 130.1, 143.4, 149.1, 154.8, 155.9, 161.2, 162.4, 190.9.
Anal. Calcd for C23H24O6: C, 69.68;
H, 6.10. Found: C, 69.50; H, 6.02.
3-Methoxy-2-((6-((2-oxo-2H-chromen-7-yl)­oxy)­hexyl)­oxy)­benzaldehyde (23). White solid, mp 102–104 °C, 70% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.54–1.59
(m, 4H, OCH2CH2
CH
2
CH
2CH2CH2O), 1.82–1.88 (m, 4H, OCH2
CH
2CH2CH2
CH
2CH2O), 3.89
(s, 3H, OCH3), 4.07 (t, J = 6.5 Hz, 2H,
OCH
2CH2CH2CH2CH2CH2O), 4.13 (t, J = 6.5 Hz, 2H, OCH2CH2CH2CH2CH2
CH
2O), 6.24
(d, J = 9.3 Hz, 1H, Ar), 6.79–6.85 (m, 2H,
Ar), 7.11–7.14 (m, 2H, Ar), 7.34–7.43 (m, 2H, Ar), 7.63
(d, J = 9.6 Hz, 1H, Ar), 10.45 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.7, 25.8, 28.9, 30.0, 56.0, 68.4, 74.8, 101.3, 112.4,
112.9, 118.1, 119.1, 124.0, 128.7, 130.0, 143.5, 152.0, 153.1, 155.9,
161.3, 162.3, 190.3. Anal. Calcd for C23H24O6: C, 69.68; H, 6.10. Found: C, 69.59; H, 6.03.
3-Ethoxy-4-((6-((2-oxo-2H-chromen-7-yl)­oxy)­hexyl)­oxy)­benzaldehyde (24). White solid, mp 107–109 °C, 89% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.46
(t, J = 7.1 Hz, 3H, OCH2CH3), 1.56–1.60 (m, 4H, OCH2CH2
CH
2
CH
2CH2CH2O), 1.84–1.92 (m, 4H, OCH2
CH
2CH2CH2
CH
2CH2O), 4.02
(t, J = 6.5 Hz, 2H, OCH2CH3), 4.09–4.14 (m, 4H, OCH
2CH2CH2CH2CH2
CH
2O), 6.24 (d, J = 9.3 Hz,
1H, Ar), 6.79–6.83 (m, 2H, Ar), 6.96 (d, J = 8.4 Hz, 1H, Ar), 7.34–7.43 (m, 3H, Ar), 7.64 (d, J = 9.6 Hz, 1H, Ar), 9.83 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 14.7,
25.7, 28.8, 28.9, 64.5, 68.4, 68.9, 101.3, 110.8, 111.7, 112.4, 113.0,
126.6, 128.7, 129.9, 143.3, 149.1, 154.4, 191.0. Anal. Calcd for C24H26O6: C, 70.23; H, 6.38. Found: C,
70.29; H, 6.41.

4.1.2.2
General Procedure for
the Synthesis of
Compounds 25–33 (Series B)
Azido intermediates
(38–41). The proper bromo derivative 34–37 (1.0 equiv) was dissolved in DMF (1.0 M), and NaN3 (1.2
equiv) was added. The reaction mixture was allowed to stir for 2 h
at room temperature. After completion, the reaction mixture was quenched
with H2O and extracted with EtOAc three times. The combined
organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Compounds were
obtained with no further purification and were directly used in the
next step.
Propargylated umbelliferon (42). To UMB (1) (1.0 equiv) in ACN (0.4 M), propargyl
bromide (80 wt % in toluene) (1.5 equiv) and K2CO3 (4.0 equiv) were added, and the reaction mixture was allowed to
reflux for 12 h. After completion, the reaction mixture was poured
into cold H2O and extracted with EtOAc three times. The
combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The crude
reaction mixture was then purified through flash column chromatography
on silica gel by eluting with an 8:2 n-hexane/EtOAc
mixture. Characterization data are in accordance with previous literature.

Triazole hybrids 25–33. The suitable azido
derivative 38–41 (1.2 equiv), propargylated UMB 42 (1.0 equiv), CuSO4 5H2O (0.01
equiv), and sodium ascorbate (0.1 equiv) were dissolved in a mixture
of 1:1 H2O and tBuOH (0.25 M) and stirred
at room temperature overnight. After completion, the reaction mixture
was poured into cold H2O and extracted with EtOAc three
times. The combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure.
The crude reaction mixture was then purified through flash column
chromatography on silica gel with suitable n-hexane/EtOAc
mixtures and, in some cases, recrystallized from ethanol to afford
final compounds 25–33.
3-Methoxy-4-(4-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­butoxy)­benzaldehyde (25). White solid, mp 53–55 °C, 81% yield.
1

H NMR (300 MHz, DMSO) δ: 1.68–1.75
(m, 2H, OCH2
CH
2CH2CH2N), 1.93–2.02 (m, 2H, OCH2CH2
CH
2CH2N), 3.81 (s, 3H, OCH3), 4.09 (t, J =
6.2 Hz, 2H, OCH
2CH2CH2CH2N), 4.55 (t, J = 6.9 Hz, 2H,
OCH2CH2CH2
CH
2N), 5.25 (s, 2H, CH2–Triazole), 6.27
(d, J = 9.3 Hz, 1H, Ar), 7.00 (dd, J = 9.0, 2.4 Hz, 1H, Ar), 7.12–7.15 (m, 2H, Ar), 7.37 (s, 1H,
Ar), 7.51 (dd, J = 8.1, 2.4 Hz, 1H, Ar), 7.62 (d, J = 8.7 Hz, 1H, Ar), 7.97 (d, J = 9.3 Hz,
1H, Ar), 8.29 (s, 1H, Triazole), 9.81 (s, 1H, CHO).
13

C NMR (75 MHz, DMSO) δ: 25.9, 27.0, 49.6,
62.2, 68.4, 102.1, 112.6, 113.0, 113.4, 125.3, 125.3, 126.4, 130.0,
142.4, 144.7, 153.9, 161.6, 191.8. Anal. Calcd for C24H23N3O6: C, 64.10; H, 5.16; N, 9.35. Found:
C, 64.27; H, 5.22; N, 9.43.
4-Methoxy-3-(4-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­butoxy)­benzaldehyde (26). White solid, mp 133–135 °C, 71% yield.
1

H NMR (300 MHz, CDCl3) δ: 1.87
(quint, J = 6.2 Hz, 2H, OCH2
CH
2CH2CH2N), 2.17 (quint, J = 7.1 Hz, 2H, OCH2CH2
CH
2CH2N), 3.94 (s, 3H, OCH3), 4.11 (t, J = 6.1 Hz, 2H, OCH
2CH2CH2CH2N), 4.53 (t, J = 7.0 Hz, 2H, OCH2CH2CH2
CH
2N), 5.24 (s, 2H, CH2–Triazole), 6.24 (d, J = 9.3 Hz, 1H, Ar),
6.92–6.99 (m, 3H, Ar), 7.36–7.40 (m, 2H, Ar), 7.46 (dd, J = 8.2, 1.8 Hz, 1H, Ar), 7.63 (d, J =
10.2 Hz, 1H, Ar), 7.82 (s, 1H, Triazole), 9.82 (s, 1H, CHO).
13

C NMR (75 MHz, CDCl3) δ: 25.7, 27.6, 50.0, 56.1, 62.4, 68.4, 102.2, 110.3, 110.7,
112.7, 113.0, 113.5, 123.3, 127.0, 128.9, 130.1, 142.8, 143.3, 148.8,
154.7, 155.7, 161.0, 161.4, 190.8. Anal. Calcd for C24H23N3O6: C, 64.10; H, 5.16; N, 9.35. Found:
C, 64.00; H, 5.11; N, 9.24.
4-Methoxy-3-((5-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­pentyl)­oxy)­benzaldehyde (27). Yellow solid, mp 107–108 °C, 70% yield.
1

H NMR (300 MHz, DMSO) δ: 1.32–1.42
(m, 2H, OCH2CH2
CH
2CH2CH2N), 1.75 (quint, J = 6.2 Hz, 2H, OCH2
CH
2CH2CH2CH2N), 1.86 (quint, J = 7.0 Hz, 2H, OCH2CH2CH2CH2
CH
2N), 3.83 (s, 3H,
OCH3), 3.98 (t, J = 6.4 Hz, 2H, OCH
2CH2CH2CH2CH2N), 4.39 (t, J = 7.0 Hz, 2H, OCH2CH2CH2CH2
CH
2N), 5.24 (s, 2H, CH2–Triazole), 6.28
(d, J = 9.3 Hz, 2H, Ar), 7.00 (dd, J = 8.8, 2.4 Hz, 1H, Ar), 7.13–7.16 (m, 2H, Ar), 7.35 (d, J = 1.8 Hz, 1H, Ar), 7.53 (dd, J = 8.1,
1.8 Hz, 1H, Ar), 7.61 (d, J = 8.7 Hz, 1H, Ar), 7.97
(d, J = 9.3 Hz, 1H, Ar), 8.29 (s, 1H, Triazole),
9.80 (s, 1H, CHO).
13

C NMR (75
MHz, DMSO) δ: 28.7, 56.2, 64.8, 65.2, 101.3, 110.0, 110.4, 110.7,
112.5, 113.0, 113.2, 143.4, 148.8, 154.8, 155.8, 161.2, 162.0, 190.8.
Anal. Calcd for C25H25N3O6: C, 64.79; H, 5.44; N, 9.07. Found: C, 64.61; H, 5.50; N, 9.15.
3-Methoxy-2-((5-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­pentyl)­oxy)­benzaldehyde (28). Colorless oil, 82% yield.
1

H NMR (300 MHz, DMSO) δ: 1.33–1.43 (m, 2H, OCH2CH2
CH
2CH2CH2N), 1.73 (quint, J = 6.1 Hz, 2H, OCH2
CH
2CH2CH2CH2N), 1.88 (quint, J = 7.1 Hz,
2H, OCH2CH2CH2
CH
2CH2N), 3.35 (s, 3H, OCH3), 4.03
(t, J = 6.4 Hz, 2H, OCH
2CH2CH2CH2CH2N),
4.39 (t, J = 7.0 Hz, 2H, OCH2CH2CH2CH2
CH
2N), 5.24 (s, 2H, CH2–Triazole), 6.26 (d, J = 9.3 Hz, 1H, Ar), 6.98 (dd, J = 8.7,
2.4 Hz, 1H, Ar), 7.13–7.17 (m, 2H, Ar), 7.23 (dd, J = 7.8, 1.8 Hz, 1H, Ar), 7.34 (dd, J = 7.5, 1.8
Hz, 1H, Ar), 7.60 (d, J = 8.1 Hz, 1H, Ar), 7.95 (d, J = 9.6 Hz, 1H, Ar), 8.29 (s, 1H, Triazole), 10.27 (s, 1H,
CHO).
13

C NMR (75 MHz, DMSO)
δ: 22.8, 29.2, 29.8, 49.8, 56.5, 62.2, 74.4, 102.0, 113.0, 113.1,
113.4, 118.7, 119.3, 124.7, 125.2, 129.8, 129.9, 142.3, 144.7, 151.6,
153.3, 155.7, 160.7, 161.6, 190.4. Anal. Calcd for C25H25N3O6: C, 64.79; H, 5.44; N, 9.07. Found:
C, 64.96; H, 5.49; N, 9.00.
3-Ethoxy-4-((5-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­pentyl)­oxy)­benzaldehyde (29). Pale-yellow solid, mp 67–69 °C, 67% yield.
1

H NMR (300 MHz, DMSO) δ: 1.29
(t, J = 6.7 Hz, 3H, OCH2
CH
3), 1.33–1.40 (m, 2H, OCH2CH2
CH
2CH2CH2N), 1.76 (quint, J = 6.2 Hz, 2H, OCH2
CH
2CH2CH2CH2N), 1.87 (quint, J = 7.1 Hz, 2H, OCH2CH2CH2
CH
2CH2N), 4.01–4.08 (m, 4H, OCH
2CH2CH2CH2CH2N and OCH
2CH3), 4.39
(t, J = 7.0 Hz, 2H, OCH2CH2CH2CH2
CH
2N), 5.24 (s, 2H, CH2–Triazole), 6.29 (d, J = 9.3 Hz, 1H, Ar), 6.98 (dd, J = 8.8,
2.4 Hz, 1H, Ar), 7.12–7.15 (m, 2H, Ar), 7.34 (d, J = 1.8 Hz, 1H, Ar), 7.50 (dd, J = 8.4, 1.8 Hz, 1H,
Ar), 7.62 (d, J = 9.0 Hz, 1H, Ar), 7.98 (d, J = 9.3 Hz, 1H, Ar), 8.28 (s, 1H, Triazole), 9.80 (s, 1H,
CHO).
13

C NMR (75 MHz, DMSO)
δ: 14.7, 23.2, 28.2, 29.9, 50.3, 62.4, 64.5, 68.7, 102.1, 110.9,
111.8, 112.7, 113.0, 113.5, 122.8, 125.5, 128.9, 130.2, 143.0, 143.2,
149.2, 154.2, 161.0, 190.9. Anal. Calcd for C26H27N3O6: C, 65.40; H, 5.70; N, 8.80. Found: C,
65.20; H, 5.59; N, 8.93.
3-Methoxy-4-((6-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­hexyl)­oxy)­benzaldehyde (30). Colorless oil, 81% yield.
1

H NMR (300 MHz, DMSO) δ: 1.36–1.44 (m, 2H, OCH2CH2
CH
2CH2CH2CH2N), 1.47–1.57 (m, 2H, OCH2CH2CH2
CH
2CH2CH2N), 1.79–1.90 (m, 2H, OCH2
CH
2CH2CH2CH2CH2N), 1.92–2.01 (m, 2H, OCH2CH2CH2CH2
CH
2CH2N), 3.88 (s, 3H, OCH3), 4.05
(t, J = 6.5 Hz, 2H, NCH2), 4.37 (t, J = 7.1 Hz, 2H, OCH2), 5.21 (s, 2H, CH2–Triazole), 6.22 (d, J = 9.6 Hz, 1H, Ar),
6.88–6.93 (m, 3H, Ar), 7.34–7.41 (m, 3H, Ar), 7.60 (d, J = 9.9 Hz, 1H, Ar), 7.64 (s, 1H, Triazole), 9.80 (s, 1H,
CHO).
13

C NMR (75 MHz, DMSO)
δ: 25.3, 26.1, 28.6, 30.1, 50.3, 56.0, 62.3, 68.7, 102.1, 109.3,
111.4, 112.7, 112.9, 113.4, 122.8, 126.7, 128.9, 130.0, 142.9, 143.3,
149.8, 154.0, 155.7, 161.0, 190.8. Anal. Calcd for C26H27N3O6: C, 65.40; H, 5.70; N, 8.80. Found:
C, 65.24; H, 5.62; N, 8.96.
4-Methoxy-3-((6-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­hexyl)­oxy)­benzaldehyde (31). White solid, mp 63–65 °C, 67% yield.
1

H NMR (300 MHz, DMSO) δ: 1.21–1.32
(m, 2H, CH2), 1.36–1.46 (m, 2H, CH2),
1.64–1.73 (m, 2H, CH2), 1.77–1.87 (m, 2H,
CH2), 3.84 (s, 3H, OCH3), 3.97 (t, J = 6.4 Hz, 2H, NCH2), 4.36 (t, J = 7.0
Hz, 2H, OCH2), 5.23 (s, 2H, CH2–Triazole),
6.27 (d, J = 9.9 Hz, 1H, Ar), 6.98 (dd, J = 8.7, 2.4 Hz, 1H, Ar), 7.12–7.15 (m, 2H, Ar), 7.34 (d, J = 1.8 Hz, 1H, Ar), 7.53 (dd, J = 8.4,
1.8 Hz, 1H, Ar), 7.61 (d, J = 8.1 Hz, 1H, Ar), 7.96
(d, J = 9.3 Hz, 1H, Ar), 8.27 (s, 1H, Triazole),
9.80 (s, 1H, CHO).
13

C NMR (75
MHz, DMSO) δ: 25.3, 26.0, 28.8, 30.0, 49.8, 56.4, 62.2, 68.5,
102.0, 111.0, 111.9, 113.0, 113.1, 113.4, 125.2, 126.4, 130.0, 142.3,
144.7, 148.9, 155.7, 160.7, 161.6, 191.9. Anal. Calcd for C26H27N3O6: C, 65.40; H, 5.70; N, 8.80.
Found: C, 65.65; H, 5.78; N, 8.63.
3-Methoxy-2-((6-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­hexyl)­oxy)­benzaldehyde (32). White solid, mp 99–101 °C, 62% yield.
1

H NMR (300 MHz, DMSO) δ: 1.21–1.31
(m, 2H, OCH2CH2
CH
2CH2CH2CH2N), 1.37–1.47
(m, 2H, OCH2CH2CH2
CH
2CH2CH2N), 1.67 (quint, J = 6.2 Hz, 2H, OCH2
CH
2CH2CH2CH2CH2N), 1.83 (quint, J = 7.1 Hz, 2H, OCH2CH2CH2CH2
CH
2CH2N), 3.34 (s, 3H, OCH3), 4.03
(t, J = 6.4 Hz, 2H, OCH
2CH2CH2CH2CH2CH2N), 4.36 (t, J = 7.0 Hz, 2H, OCH2CH2CH2CH2CH2
CH
2N), 5.23 (s, 2H, CH2–Triazole),
6.27 (d, J = 9.3 Hz, 1H, Ar), 7.00 (dd, J = 8.8, 2.4 Hz, 1H, Ar), 7.13–7.19 (m, 2H, Ar), 7.25 (dd, J = 7.8, 1.8 Hz, 1H, Ar), 7.34 (dd, J =
7.5, 1.8 Hz, 1H, Ar), 7.62 (d, J = 8.7 Hz, 1H, Ar),
7.97 (d, J = 9.3 Hz, 1H, Ar), 8.28 (s, 1H, Triazole),
10.28 (s, 1H, CHO).
13

C NMR (75
MHz, DMSO) δ: 25.2, 26.0, 29.7, 30.1, 49.8, 56.5, 62.2, 74.5,
102.0, 113.0, 113.1, 113.4, 118.7, 119.3, 124.6, 125.2, 129.8, 130.0,
142.3, 144.8, 151.6, 153.3, 155.7, 161.6, 190.5. Anal. Calcd for C26H27N3O6: C, 65.40; H, 5.70;
N, 8.80. Found: C, 65.22; H, 5.59; N, 8.91.
3-Ethoxy-4-((6-(4-(((2-oxo-2H-chromen-7-yl)­oxy)­methyl)-1H-1,2,3-triazol-1-yl)­hexyl)­oxy)­benzaldehyde (33). Colorless oil, 74% yield.
1

H NMR (300 MHz, DMSO) δ: 1.42–1.47 (m, 5H, OCH2CH3 and CH2), 1.50–1.60 (m, 2H,
CH2), 1.81–1.90 (m, 2H, CH2), 1.92–2.02
(m, 2H, CH2), 4.07 (t, J = 6.4 Hz, 2H,
NCH2), 4.14 (q, 2H, OCH2CH3), 4.39
(t, J = 7.0 Hz, 2H, OCH2), 5.24 (s, 2H,
CH2–Triazole), 6.25 (d, J = 9.6
Hz, 1H, Ar), 6.91–6.95 (m, 3H, Ar), 7.36–7.42 (m, 3H,
Ar), 7.61 (s, 1H, Triazole), 7.64 (d, J = 1.8, 1H,
Ar) 9.81 (s, 1H, CHO).
13

C NMR (75 MHz, DMSO) δ: 14.7, 25.3, 26.1, 28.7, 30.1, 50.3, 62.4,
64.5, 68.7, 102.1, 111.0, 111.8, 112.7, 113.5, 122.8, 126.5, 128.9,
130.0, 143.2, 149.2, 161.0, 161.3, 190.9. Anal. Calcd for C27H29N3O6: C, 65.97; H, 5.95; N, 8.55.
Found: C, 66.13; H, 6.03; N, 8.69.

4.2
CA Inhibition
Assay
An Applied Photophysics
stopped-flow instrument measured the CA-catalyzed CO2 hydration
activity. The indicator used was phenol
red (0.2 mM), with measurements taken at an absorbance maximum of
557 nm. HEPES (10 mM, pH 7.5) supplemented with 0.1 M Na2SO4 served as the reaction buffer, and the CA-catalyzed
CO2 hydration reaction was monitored for 10–100
s. The CO2 concentrations varied from 1.7 to 17 mM. The
uncatalyzed rates were also measured and subtracted. Stock solutions
of inhibitors (10 mM) were provided in distilled–deionized
water containing 10% DMSO, and dilutions of up to 0.001 μM were
prepared with the reaction buffer. Inhibitor and enzyme solutions
were preincubated together for 6 h at room temperature prior to the
assay in order to allow for the formation of the E–I complex.
The inhibition constants were obtained by nonlinear least-squares
methods using PRISM 3 and the Cheng–Prusoff equation, representing the mean from at least three different
determinations. The enzyme concentrations were in the range of 4–15
nM. All CA isoforms were recombinant ones obtained in-house, as reported earlier.
,

4.3
In Silico
Studies
4.3.1
X-ray Structures
The crystal structures
of hCA I (PDB code 1AZM), hCA II (PDB code 4E3H), hCA IX (PDB code 5FL4), and hCA XII (PDB code 1JCZ) were downloaded from the Protein Data Bank. Automated docking was carried out for the hydrolyzed
form of compound 7, whose structure was built using MolBook
Pro software. All docking calculations
were performed using the GOLD 5.1 program,
,
using the PLP scoring function. The “allow early termination”
command was deactivated, whereas the possibility for the ligand to
flip ring corners was activated. The remaining GOLD default parameters
were used for the calculations, in which the ligand was subjected
to 100 genetic algorithm runs, and the corresponding docking results
were clustered using a RMSD cutoff of 2.0 Å. For each docking
calculation, the top-scoring pose of the best cluster of solutions
was taken into account.

4.3.2
Molecular Dynamics Simulations
MD simulations were carried out with AMBER, version 22. Each complex
was subjected to an MD procedure based on already successfully applied
protocols, using the ff14SB force field at 300 K.
,
General Amber force field (GAFF) parameters were used for the ligand,
whose partial charges were calculated with the AM1-BCC method, as
implemented in the Antechamber suite of AMBER 22. Prior to MD simulations,
each complex was located in a rectangular parallelepiped water box
and solvated with a 15 Å water cap using the TIP3P explicit solvent
model for water. Sodium ions were then added as counterions for the
neutralization of the solvated systems. Each system was subjected
to two stages of energy minimization, each composed of 5000 steps
of steepest descent, followed by conjugate gradient until a convergence
of 0.05 kcal/(mol·Å2) was reached. In the first
stage, the whole protein was blocked with a position restraint of
500 kcal/(mol·Å2) to uniquely minimize the position
of the water molecules, while in the second stage, the entire system
was energy-minimized by applying a harmonic potential of 10 kcal/(mol·Å2) only to the protein α carbons. The minimized complexes
were then used as the starting point for a total of 100 ns of the
MD simulation. A 0.5 ns constant-volume simulation in which the temperature
of the system was raised from 0 to 300 K was initially performed.
In the second step, the system was equilibrated through a 3 ns constant-pressure
simulation, maintaining the temperature at a constant value of 300
K with the use of a Langevin thermostat. An additional 96.5 ns of
constant-pressure MD was then performed, for a total of 100 ns of
simulation. In all three MD steps, a harmonic potential of 10 kcal/(mol·Å2) was applied to the protein α carbons. All simulations
were performed using particle mesh Ewald (PME) electrostatics with
a cutoff of 10 Å for nonbonded interactions and periodic boundary
conditions. A simulation step of 2.0 fs was employed as all bonds
involving hydrogen atoms were kept rigid using the SHAKE algorithm.
Further 200 ns MD simulations were performed for 7-CA
XII and 7-CA IX complexes, using the same parameters
employed for the third constant-pressure MD step. This additional
MD stage was performed using the hydrogen mass repartition (HMR) scheme and a time step of 4.0 fs, since this technique
proved to be useful to reduce the simulation time while maintaining
an unbiased MD protocol.
,
The analysis of the
MD trajectories was performed with the Cpptraj software included in
the AMBER suite.

4.4
Cell-Based Assays
4.4.1
Cell Cultures
Human bronchial epithelial
(BEAS-2B; CRL-3588) and human lung carcinoma (A549; CCL-185) cell
lines were purchased from ATCC. BEAS-2B and A549 cells were cultured
in complete RPMI 1640 (Merck, Darmstadt, Germany) at 37 °C and
5% CO2. The medium was supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 1% penicillin–streptomycin, and 1%
sodium pyruvate.

4.4.2
Cell Treatment
Cells were seeded
according to the different experimental techniques and left to adhere
for 24 h. Next, the medium was removed, and cells were treated with
selected compounds 7–11, 26, and 33, which were dissolved in dimethyl sulfoxide
(DMSO) to achieve a range of concentrations from 0 μM (untreated
control = CTRL) to 100 μM. The percentage of DMSO was maintained
below 0.3%. The viability of the cells was assessed after 24, 48,
and 72 h.

4.4.3
Cell Viability (MTT Assay)
BEAS-2B
and A549 cells were seeded in 96-well culture-treated plates (Falcon,
Corning Incorporated, Brooklyn, NY, USA) at 0.5 × 104 cells/well. Untreated cells were set as the experimental control
(100% of cell metabolic activity). Exposure times varied from 24 to
72 h. At the established time points (24, 48, and 72 h), exposure
media were replaced with a fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) 0.5 mg/mL (Merck, Darmstadt, Germany) and processed
as elsewhere reported. The optical density
in each well was measured by using a spectrophotometer (Thermo Fisher
Scientific, Waltham, MA, USA) at a wavelength of 540 nm. Each
experiment was performed twice in triplicate per experimental condition
(n = 6).

4.4.4
Cytotoxicity Assay (LDH
Test)
To
assess the cytotoxicity in A549 cells after treatments, the amount
of lactate dehydrogenase released into the culture medium was measured
using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison,
WI, USA) according to the manufacturer’s instructions. Cell supernatants were harvested from the same
cultures processed for the MTT assay after 24 h of treatment, and
the measured optical density values were normalized to those obtained
from the MTT assay.

4.4.5
Cell Cycle Analysis
A549 cells
were seeded in 12-well plates at 0.5 × 105 cells/well
and treated for 6 and 24 h, as previously described. At each time
point, cells were detached using StemPro Accutase, collected by centrifugation,
and then fixed overnight at 4 °C in 70% v/v cold ethanol. After
fixation, cells were processed as described elsewhere. The PI fluorescence was detected by a flow cytometer
equipped with a 488 nm laser (CytoFlex flow cytometer, Beckman Coulter,
CA, USA) in the FL-3 channel. At least 10,000 events/sample were collected
and analyzed with the CytExpert Software, version 2.3 (Beckman Coulter,
CA, USA), and the percentages of cells in the G1, S, or G2 phase of
the cell cycle were calculated using the ModFit LT Software, version
5.0 (Verity Software House, Topsham, ME, USA).

4.4.6
Imaging and Analysis
Images were
acquired using the high-content imaging microscope Operetta CLS (Revvity,
Waltham, Massachusetts, U.S.), using a 20× water objective. Thirty-five
fields were imaged for each replicate, and mean values were considered.
Harmony Software was used for image processing and analysis. Briefly,
cells were segmented to obtain the nuclei population (setting the
channel on the DAPI and using method B) and the related cytoplasm
localization (setting the channel on the Alexa 488 and using Method
A). Then, the cell mean fluorescence intensity was calculated for
each condition. Mean values for each well were then used for statistical
analysis.

4.4.7
Establishment of Pro-inflammatory
Conditions
To establish an inflamed environment, differentiated
macrophages
were treated with LPS 0.5 μg/mL (lipopolysaccharide from E. coli purchased from Merck, Darmstadt, Germany,
stock solution 1 mg/mL in water) for 24 h. BEAS-2B cells were treated
with the medium collected from LPS-stimulated macrophage cultures
and afterward exposed to treatments. Cells were fixed with 4% glutaraldehyde
and stained with crystal violet. Images were acquired and analyzed
by the Leica Application Suite LAS EZ version 3.4 (Leica, Wetzlar,
Germany).

4.4.8
Statistical Analysis
Statistics
were performed using a two-way analysis of variance (ANOVA) followed
by Dunnett and Tukey’s multiple comparison tests by means of
the Prism 8.0 software (GraphPad, San Diego, CA, USA). Results are
presented as mean values ± standard deviations. Values of p ≤ 0.05 were considered statistically significant.

Supplementary Material