Dual P-glycoprotein and CA XII inhibitors: A new strategy to reverse the P-gp Mediated Multidrug Resistance (MDR) in cancer cells y

Article abstract of DOI:10.3390/molecules25071748

A new series of N,N-bis(alkanol)amine aryl diesters was synthesized and studied as dual P-glycoprotein (P-gp) and carbonic anhydrase XII inhibitors (CA XII). These hybrids should be able to synergistically overcome P-gp mediated multidrug resistance (MDR) in cancer cells. It was reported that the effux activity of P-gp could be modulated by CA XII, as the pH reduction caused by CA XII inhibition produces a significant decrease in P-gp ATPase activity. The new compounds reported here feature both P-gp and CA XII binding moieties. These hybrids contain a N,N-bis(alkanol)amine diester scaffold found in P-glycoprotein ligands and a coumarin or benzene sulfonamide moiety to target CA XII. Many compounds displayed a dual activity against P-gp and CA XII being active in the Rhd 123 uptake test on K562/DOX cells and in the hCA XII inhibition test. On LoVo/DOX cells, that overexpress both P-gp and CA XII, some coumarin derivatives showed a high MDR reversal effect in Rhd 123 uptake and doxorubicin cytotoxicity enhancement tests. In particular, compounds 7 and 8 showed higher activity than verapamil and were more potent on LoVo/DOX than on K562/DOX cells overexpressing only P-gp. They can be considered as valuable candidates for selective P-gp/CA XII inhibition in MDR cancer cells.

Full text of DOI:10.3390/molecules25071748

molecules
Article
Dual P-Glycoprotein and CA XII Inhibitors: A New
Strategy to Reverse the P-gp Mediated Multidrug
†
Resistance (MDR) in Cancer Cells
Elisabetta Teodori 1 , Laura Braconi 1 , Silvia Bua , Andrea Lapucci , Gianluca Bartolucci
1
2
1
,
1
1
1,
1
Dina Manetti , Maria Novella Romanelli
, Silvia Dei * , Claudiu T. Supuran
and
Marcella Coronnello 2
1
Department of Neuroscience, Psychology, Drug Research and Child’s Health–Section of Pharmaceutical and
Nutraceutical Sciences, University of Florence, via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy;
elisabetta.teodori@unifi.it (E.T.); laura.braconi@unifi.it (L.B.); silvia.bua@unifi.it (S.B.);
gianluca.bartolucci@unifi.it (G.B.); dina.manetti@unifi.it (D.M.); novella.romanelli@unifi.it (M.N.R.);
claudiu.supuran@unifi.it (C.T.S.)
Department of Health Sciences—Section of Clinical Pharmacology and Oncology, University of Florence,
Viale Pieraccini 6, 50139 Firenze, Italy; andrea.lapucci@unifi.it (A.L.); marcella.coronnello@unifi.it (M.C.)
Correspondence: silvia.dei@unifi.it
2
*
†
Dedicated to the memory of Professor Fulvio Gualtieri, our great mentor who recently passed away.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Maria José Umbelino Ferreira
Received: 15 March 2020; Accepted: 8 April 2020; Published: 10 April 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A new series of N,N-bis(alkanol)amine aryl diesters was synthesized and studied as dual
P-glycoprotein (P-gp) and carbonic anhydrase XII inhibitors (CA XII). These hybrids should be able to
synergistically overcome P-gp mediated multidrug resistance (MDR) in cancer cells. It was reported
that the efflux activity of P-gp could be modulated by CA XII, as the pH reduction caused by CA XII
inhibition produces a significant decrease in P-gp ATPase activity. The new compounds reported
here feature both P-gp and CA XII binding moieties. These hybrids contain a N,N-bis(alkanol)amine
diester scaffold found in P-glycoprotein ligands and a coumarin or benzene sulfonamide moiety to
target CA XII. Many compounds displayed a dual activity against P-gp and CA XII being active in
the Rhd 123 uptake test on K562/DOX cells and in the hCA XII inhibition test. On LoVo/DOX cells,
that overexpress both P-gp and CA XII, some coumarin derivatives showed a high MDR reversal
effect in Rhd 123 uptake and doxorubicin cytotoxicity enhancement tests. In particular, compounds
7
and showed higher activity than verapamil and were more potent on LoVo/DOX than on K562/DOX
8
cells overexpressing only P-gp. They can be considered as valuable candidates for selective P-gp/CA
XII inhibition in MDR cancer cells.
Keywords: MDR reversers; P-gp modulators; CA XII inhibitors; K562/DOX; LoVo/DOX; hybrid
compounds; multitarget ligands; dual P-gp/CA XII inhibitory activity; selective chemosensitizers
1
. Introduction
The resistance of many tumor cells to structurally and mechanistically unrelated antineoplastic
drugs is the main failure reason of anticancer chemotherapy [1,2]. This phenomenon is called multidrug
resistance (MDR) and generally consists of acquired resistance as a result of chemotherapeutic treatment.
MDR is mainly due to overexpression of some energy dependent transmembrane proteins belonging to
the ABC (ATP Binding Cassette) transporter family [3,4]. These proteins act as extrusion pumps and are
responsible of an accelerated efflux of chemotherapeutic agents out of the cancer cells. As a consequence,
their cellular concentration decreases and the drug efficacy is reduced. Among the ABC proteins, three
transporters, P-glycoprotein (P-gp or ABCB1), the multidrug-resistance-associated protein-1 (MRP1 or
Molecules 2020, 25, 1748; doi:10.3390/molecules25071748
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1748
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ABCC1) and the breast cancer resistance protein (BCRP or ABCG2) are mainly associated with cancer
MDR being overexpressed in many cell lines [5].
P-glycoprotein is the most studied and characterized ABC transporter. It is overexpressed in many
tumors and its elevated expression has been considered the cause of the reduced chemotherapeutic
responses in various cancer types including both blood and solid cancers [
6,
7]. For this reason, P-gp has
been considered a possible target to overcome cancer multidrug resistance [
8] and, in the last years,
many P-gp modulators have been designed and synthesized. These compounds can be coadministered
with classical antitumor drugs that are substrates of P-gp, restoring their therapeutic efficacy in resistant
tumors [9]. Many compounds have been identified for this purpose, nevertheless few of them have
reached clinical trials [10]. The main obstacle is the widespread tissue distribution of P-gp and its
physiological role in the regulation of permeability of biological membranes, in controlling the secretion
of physiologically important lipophilic molecules and in the extrusion of xenobiotics that entered
the organism [11,12]. Therefore, P-gp modulators could interfere with the pharmacokinetics of other
substances, including coadministered chemotherapeutic agents [13]. For this reason, an ideal MDR
reverser should be a P-gp modulator able to inhibit selectively the efflux of anticancer drugs in tumor
cells without affecting the normal tissue membrane permeability.
Carbonic anhydrases (CAs, EC4.2.1.1) are metalloenzymes that catalyze the conversion of carbon
dioxide to bicarbonate and a proton. They play a crucial role for the maintenance of pH homeostasis
in the body and also have a metabolic function in several biosynthetic processes [14
CAs are involved in a multitude of physiological and pathological processes. Human CAs (hCAs)
comprise of 15 different -CA isoforms varying for their tissue distribution and cellular localization
membrane, cytosol and mitochondria) [14]. Among these isoforms, CA IX and XII are extracellular,
,15]. Therefore,
α
(
membrane bound CAs associated with tumor progression and metastases formation [15].
The overexpression of these two isoenzymes is induced in most hypoxic solid tumors by the
hypoxia inducible factor 1
nucleus leads to the formation of an active transcription factor, which induces the overexpression of
proteins involved in the proliferation and invasion of hypoxic tumor [19 22]. The overexpression
α (HIF-1α) [16–18]. The translocation of HIF-1α from the cytosol to the
–
of these proteins shifts the metabolism of tumor cells toward an anaerobic fermentation with the
prevalence of glycolysis with respect to oxidative phosphorylation. The glycolytic pathway leads
to the production of organic acids, which are extruded from the cells leading to an extracellular
acidification. This condition is also supported by the extrusion of protons, produced by carbon dioxide
hydration catalyzed by the transmembrane CA isoenzymes IX and XII, and the intracellular retention
of bicarbonate ions. Thus, in cancer cells CA IX and XII are responsible for maintaining an alkaline
intracellular pH and an extracellular acidosis that is beneficial for the growth of tumor cells but impairs
the growth of normal cells [23,24]. It has recently been shown [25] that even in normoxia the HIF-1α
factor accumulates in the cell cytoplasm and allows the transcription of some of its target genes
including CA IX and CA XII, acting as a pH sensor.
Kopecka and colleagues reported in recent work that the activity of the efflux transmembrane
transporter P-gp could be modulated by CA XII [26]. The P-gp efflux activity is known to be influenced
by intracellular pH, as the optimal pH at which P-gp operates is slightly alkaline. This condition is
maintained by the CA XII catalytic activity, which assures the suitable intra- and extracellular pH in
tumor cells overexpressing it. CA XII is highly expressed in some chemoresistant P-gp positive cancer
cells [26] and its activity is critical for the P-gp efflux function and contribution to MDR. In fact, there
are recent evidences that the reduction of pH, caused by the inhibition of CA XII, produces a significant
decrease in P-gp ATPase activity [26,27].
Compounds endowed with dual P-gp/CA XII inhibitory activity could thus be useful as
chemosensitizers able to overcome P-gp mediated MDR in cancer cells. This synergistic mechanism
should allow such compounds to be selective P-gp inhibitors for resistant tumor cells. Notably,
they may have low interference with the physiological activity of P-gp in the regulation of the
membrane permeability of normal tissues.

Molecules 2020, 25, 1748
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Acetazolamide and other classical CA inhibitors contain an aromatic sulfonamide moiety, which
coordinates the zinc ion buried at the bottom of the conical CA active site [28]. The aromatic and
heteroaromatic sulfonamides are non-selective inhibitors and, therefore, able to block both the
transmembrane isoforms CA IX and CA XII, and the physiological relevant cytosolic isoenzymes
CA I and CA II, thus leading to side effects [28]. Coumarin derivatives are a relatively new class of
CA inhibitors [29–31] that show high selectivity for inhibiting the tumor associated CA IX and CA
XII isoforms. This selectivity is explained by their particular binding mode; in fact, the hydrolyzed
coumarin moiety is able to obstruct the active site cavity, binding at its entrance, which is the most
variable part of the site among the various hCAs [32].
The aim of this study was to obtain compounds able to overcome the P-gp mediated MDR
in cancer cells by a synergistic mechanism of interference with CA XII and P-gp activity. For this
aim, we designed and synthesized a new series of hybrid inhibitors characterized by the presence
of both P-gp and CA XII binding moieties in order to possess a dual P-gp/CA XII inhibitory effect.
We incorporated coumarin or benzene sulfonamide moieties in a typical scaffold of P-glycoprotein
ligands that were previously studied by some of us. For many years one of our research group has been
involved in the study of P-gp ligands with the aim to discover potent MDR modulators [33–39]. For this
purpose, we designed and synthesized several compounds carrying the structural elements considered
important for P-gp interaction, such as high lipophilicity, the presence of hydrogen bond acceptor
groups, aromatic moieties and one or more protonable nitrogen atoms [40]. The structure of these
molecules is characterized by the presence of a basic nitrogen atom linked to aromatic ester residues by
spacers of different flexibility and length [33–39]. Most of these compounds are N,N-bis(alkanol)amine
aryl diesters that were potent and efficient P-gp-dependent multidrug resistance reversers on the
doxorubicin-resistant erythroleukemia K562 cell line (Structure A, Chart 1) [35–39].
Chart 1. General structures of the leads and newly synthesized compounds.
In this study we report a new series of N,N-bis(alkanol)amine aryl diesters (
two polymethylenic chains of variable length, carrying the (E)-3-(3,4,5-trimethoxyphenyl)vinyl,
,4,5-trimethoxyphenyl or the anthracene residues ( and ), combined with coumarin or benzene
sulfonamide moieties (Structures B and C, Chart 1).
1–28), incorporating
3
a,
b
c
The synthesized compounds were evaluated for their P-gp modulating activity by measuring
the uptake increase of the specific P-gp substrate rhodamine-123 (Rhd 123) on doxorubicin-resistant
erythroleukemia K562 cells (K562/DOX) that overexpress the membrane P-gp. The CA inhibition
efficacy of compounds
1–28 was evaluated on the tumor associated hCA IX and hCA XII and on the
physiological relevant cytosolic hCA I and II isoforms, to verify their CA isoforms selectivity profiles.

Molecules 2020, 25, 1748
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The expression levels of the two transmembrane proteins, P-gp and CA XII, were evaluated by
real time polymerase chain reaction (RT-PCR) analysis, on resistant K562/DOX and parental K562 cell
lines. The results showed a substantial increase in the mRNA expression level for the MDR1 gene
but only a low increase in the mRNA expression level for the CA XII gene on the resistant K562/DOX
line. Therefore, to prove the synergistic effect on the MDR reversal activity due to dual P-gp/CA XII
inhibition, the selected compounds were evaluated by the Rhd 123 uptake test, on human colorectal
carcinoma LoVo/DOX cells, which overexpress both P-gp and CA XII, as confirmed by the RT-PCR
analysis performed on both resistant and parental cell lines (LoVo/DOX, LoVo). The P-gp expression
level in both resistant K562/DOX and LoVo/DOX cells was also investigated using flow cytometric
analysis with a P-gp-specific antibody.
Finally, the selected compounds were evaluated by the doxorubicin cytotoxicity enhancement test
on K562/DOX and LoVo/DOX cells. Moreover, the susceptibility of derivatives
hydrolysis was assessed in phosphate buffer solution.
1–28 to spontaneous
2
. Results and Discussion
2
.1. Chemistry
Compounds 1–28 were prepared using a reaction pathway (Scheme 1) that has been previously
applied to the synthesis of analogues with the general formula A (Chart 1) [35–38]. In this work, some new
intermediates have been synthesized for preparing all the newly designed hybrids. Thus, chloroester 29 was
obtained by esterification of 6-chlorohexan-1-ol with the commercially available 3,4,5-trimethoxybenzoic
0
acid using EDC hydrochloride (N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride) and
DMAP (4-dimethylaminopyridine) in anhydrous CH Cl . The chloroalkyl ester was transformed in the
2
2
corresponding iodo derivative 30 using NaI in acetone, in order to achieve higher yields in the following
reactions. Treatment of 30 with 3-aminopropan-1-ol in acetonitrile gave the desired secondary amine
31. The same reaction performed on the already described 6-iodohexyl anthracene-9-carboxylate [38],
gave 32. These compounds were alkylated by reductive methylation with HCOOH/HCHO to give the
corresponding tertiary amines 33 and 34. Final compounds 1–14 were obtained by esterification of 33,
3
(
4
and the previously reported analogues 35
48) (described in Scheme 2) using EDC hydrochloride and HOBt (1-hydroxybenzotriazole hydrate)
in anhydrous CH CN. In turn, compounds 15 28 were prepared by esterification of 33–46 with
-sulfamoylbenzoyl chloride obtained from 4-sulfamoylbenzoic acid by reaction with SOCl2.
-((2-Oxo-2H-chromen-7-yl)oxy)acetic acid (48) was synthesized as reported in Scheme 2.
Alkylation of commercially available 7-hydroxy-2H-chromen-2-one with ethyl bromoacetate gave ester
7, which was hydrolyzed under alkaline conditions to obtain 48.
–46 [35–38] with 2-((2-oxo-2H-chromen-7-yl)oxy)acetic acid
–
3
4
2
4

Molecules 2020, 25, 1748
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Scheme 1.
Reagents and conditions: (i) 6-chlorohexan-1-ol, EDC hydrochloride, DMAP and
CH Cl ; (ii) NaI, acetone; (iii) H N(CH ) OH and CH CN; (iv) HCOOH/HCHO, EtOH;
2
2
2
2 3
3
(
(
v) 2-((2-Oxo-2H-chromen-7-yl)oxy)acetic acid (48), EDC hydrochloride, HOBt and CH CN;
vi) 4-sulfamoylbenzoyl chloride, ethanol-free CHCl3.
3
Scheme 2. Reagents and conditions: (i) BrCH COOC H , K CO , acetone and (ii) NaOH 10%.
2
2
5
2
3
2
.2. Chemical Stability
In order to evaluate the chemical stability of our compounds during biological tests, a study on
the susceptibility to spontaneous hydrolysis of derivatives 28 was planned. For this purpose, a series
1–
of experiments were carried out adding a known amount of analyte in phosphate buffer solution (PBS).
The samples were analyzed by LC–MS/MS method operating in the multiple reaction monitoring
(
MRM) mode [39]. The solution stability of each studied compound was verified by monitoring the
variation of the analyte concentration at different incubation times. The processed raw data and the
used LC–MS/MS methods are reported in the Supplementary Material.
The obtained results demonstrated that compounds 1–28 were stable in PBS showing a negligible
decay rate (k values close to 0). Therefore, extremely high half-life values can be calculated for these
derivatives. Since 240 min is the maximum time of observation in our experimental protocol, it is
possible to assume that the half-lives of these hybrids should be greater than 240 min.

Molecules 2020, 25, 1748
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2
.3. Rhodamine-123 Uptake Test on K562/DOX Cells
The ability of compounds 28 to inhibit the P-gp transport activity was evaluated by measuring
1–
the uptake of the specific P-gp substrate rhodamine-123 (Rhd 123) in K562/DOX cells, as described in
Material and Methods.
K562 is a highly undifferentiated erythroleukemia cell line originally derived from a patient with
chronic myelogenous leukemia [41]. The doxorubicin resistant K562/DOX cell line overexpresses
almost exclusively the transporter membrane protein P-gp [42].
The P-gp inhibitory activity of compounds 1–28, tested at 3 and 30
µM concentrations, are reported
in Table 1 together with those of verapamil, used as standard inhibitor.
Table 1. Rhd 123 uptake enhancement on K562/DOX cells and inhibitory activity on hCA I, II, IX and XII
isoforms of compounds
1
–28 and of the two reference compounds verapamil and acetazolamide (AAZ).
.
FR a
30 µM
b
Compounds
K (nM)
I
N
n
m
Ar
Structure 3 µM
hCA I
hCA II
hCA IX
hCA XII
1
2
3
4
5
6
7
8
9
3
3
5
5
6
6
7
7
3
6
7
3
6
7
3
3
5
5
6
6
7
7
3
6
7
3
6
7
3
5
3
5
3
4
2
7
5
3
2
5
3
2
3
5
3
5
3
4
2
7
5
3
2
5
3
2
a
a
a
a
a
a
a
a
b
b
b
c
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
C
C
C
C
C
C
C
C
C
C
1.44
1.57
1.00
1.99
3.67
1.39
2.08
2.33
1.00
1.16
1.11
1.00
1.43
1.25
1.00
1.08
1.13
1.00
1.06
1.01
1.30
2.85
1.00
1.42
1.44
2.10
1.08
1.45
3.30
1.00
4.76
5.81
2.58
5.45
5.85
1.98
4.48
4.15
3.95
2.18
1.58
2.53
2.45
4.22
2.30
1.83
3.90
4.99
3.23
2.58
3.70
3.20
1.66
2.88
3.50
4.45
2.03
4.25
4.71
1.00
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
142.5
47.4
54.6
26.6
26.7
40.2
29.7
24.2
133.3
103.1
97.1
150.8
82.7
125.8
178.4
210.6
215.3
226.5
39.7
33.9
68.4
171.9
26.6
25.3
9.3
8.9
48.8
18.9
6.4
36.4
9.1
7.1
9.2
8.9
8.3
45.7
9.1
42.9
91.0
94.3
33.1
62.6
58.6
41.7
35.2
96.7
63.7
57.6
8.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
c
c
a
a
a
a
a
a
a
a
b
b
b
c
55.1
447.2
56.6
62.6
509.6
84.5
151.7
554.8
203.7
424.8
8.4
14.0
533.2
73.7
284.4
48.9
445.0
127.5
8.9
83.5
42.7
90.2
117.0
241.2
28.2
338.7
616.7
237.9
81.2
422.4
475.5
23.4
31.3
23.5
c
c
20.6
Ver
AAZ
250.0
12.0
25.0
5.7
a
Inhibition of the P-gp transport activity on K562/DOX cells expressed as FR that is the ratio between the average
fluorescence intensity of rhodamine in the presence and in absence of modulators (FR = Rhd uptake + modulator/Rhd
b
uptake
±
−
modulator). Mean from 3 different assays, by a stopped flow technique (errors were in the range of
5–10% of the reported values).

Molecules 2020, 25, 1748
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The new compounds were preliminary studied to evaluate their intrinsic cytotoxicity at 3 and
30 µM concentrations by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)
assay [43] on the K562/DOX cells line. At the concentrations tested, all compounds showed a negligible
intrinsic toxicity (about 20%, data not shown).
The results were expressed as the FR (fluorescence ratio), which is the ratio between the average
fluorescence intensity of rhodamine in the presence and in the absence of modulators. Value 1 was
attributed to the average fluorescence intensity of the samples exposed only to rhodamine.
The data reported in Table 1 indicate that at 3
in the inhibition of the P-gp transport activity (FR values equal to or close to the unity), but
and 26 were able to increase the intracellular rhodamine uptake by at least twice o even more
FR = 1.99, 2.08, 2.33, 2.85 and 2.10, respectively), and compound was active as verapamil (FR = 3.67
and 3.30, respectively).
At 30 M concentration a significant increase in the intracellular rhodamine fluorescence intensity
was observed for all compounds and many of them showed FR values in the 4.15–5.85 range. Notably,
compounds and 18 showed FR values higher than that of verapamil, tested at the same
µM concentration most compounds were inactive
4, 7, 8,
2
2
(
5
µ
2, 4, 5
concentration (FR = 5.81, 5.45, 5.85 and 4.99, respectively, vs. FR = 4.71 of verapamil). On the contrary,
the standard CA inhibitor acetazolamide did not show any effect on K562/DOX cells at both the
concentrations used.
The results showed in Table 1 indicate that the introduction of a coumarin or a benzene sulfonamide
group maintains effective P-glycoprotein inhibitory ability, even if the potency is lower compared to
the previously synthesized analogues carrying on both sides the aromatic moieties
Chart 1 (General structure A) [38].
a–c reported in
Notably, compounds from the coumarin series (Structure B, Table 1) were in general more
potent than those from the benzene sulfonamide series (Structure C, Table 1; compare vs. 16
vs. 18 and vs. 19). On the other hand, considering the nature of the Ar group, compounds
2
,
4
5
bearing a (E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety were in general more active. Disappointingly,
and differently from what found in previous series [38], a clear influence of the length of the linkers on
the activity was not observed with the new derivatives reported here.
2
.4. CA Inhibitory Activity
The CA inhibitory efficacy of compounds 1–28 was evaluated on four human CA isoforms, the two
cytosolic hCA I and II and the transmembrane tumor-associate hCA IX and XII isoforms. The hCA
inhibition data of the new compounds were determined by a Stopped-Flow CO hydrase assay [44
]
2
and are reported in Table 1 together with those of the sulfonamide acetazolamide (AAZ), used as
a standard inhibitor.
Derivatives 1–14, characterized by the presence of a coumarin scaffold, inhibited both hCA IX and
XII isoforms at nanomolar concentrations, while they were inactive against the off-target isoforms hCA
I and II, in accord with the selectivity usually displayed by CA inhibitors decorated with this aromatic
moiety. In addition, this series of compounds showed lower Ki values on hCA XII (Ki = 6.4–48.8 nM)
than on hCA IX (Ki = 24.2–150.8 nM). Some compounds (
fold preference for hCA XII with respect to hCA IX: compounds
derivatives while carries a (E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety. The latter group can also
confer high potency, since (Ki 6.4 nM) was the most potent compound on hCA XII of this series.
1,
9,
10 and 11) displayed an interesting 12–15
9,
10 and 11 are 3,4,5-trimethoxyphenyl
1
5
On the contrary, derivatives with an anthracene moiety were, in general, less potent and selective,
with the exception of 13, which was nine times more active on hCA XII than on hCA IX. The linkers’
length seemed not to be crucial for the interaction with hCA XII: for instance, compounds
shortest linkers (n, m = 3) were equipotent with 8, which showed the longest linkers on both arms
n, m = 7). On hCA IX, if we take into account the compounds having the same Ar group ( , Ar =
was 3–6 times less potent
1 having the
(
1–
8
a)
only the short 3 + 3 linker seemed detrimental for activity, since compound
than 2–7.
1

Molecules 2020, 25, 1748
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As expected, compounds 15
–
28, incorporating a benzene sulfonamide moiety, were able to
inhibit all four isoforms, even if with different selectivity profiles, showing Ki values in the range of
0.6–241.2 nM and 8.0–96.7 nM ranges for hCA IX and XII isoforms, respectively. Almost all compounds
were more effective CA XII than CA IX inhibitors, with the exception of 23 and 24 (twice more potent
2
on hCA IX than on hCA XII), and of 20, 27 and 28, which showed similar K values on both isoforms.
i
The selectivity profiles towards hCA XII with respect to the two hCA I and II isoforms were
quite different. Compounds 16
4–20 times). In particular, compounds bearing an anthracene moiety (26
selective ones.
Compounds 19
respectively with an hCA II vs. hCA XII preference of 7, 3 and 6.5 times, respectively. Conversely,
some hCA XII vs. hCA II selectivity (2–20 times) was displayed by compounds 16 17 18 22 23 25
27 and 28. Also in this case compounds bearing an anthracene moiety (26 27 and 28) were all
quite selective.
In conclusion, the data reported in Table 1 show that many compounds acted as effective CA
inhibitors, although the Ar moiety ( ) or the linkers’ length did not clearly influence their activity
on the four hCA isoforms. Notably, several coumarin derivatives ( 11 and 13) and one
sulfonamide derivative (25) had Ki values on hCA XII in the low nanomolar range. On the contrary,
sulfonamides 19 20 and 24 displayed potent, although undesired, inhibitory activity on hCA II (Ki
values between 8.4 and 14 nM).
,
19
,
21
,
23,
26,
27 and 28 showed a preference for hCA XII vs. hCA I
(
,
27 and 28) were the most
,
20 and 24 were the most potent on hCA II, with Ki = 8.4, 14.0 and 8.9 nM,
,
,
,
,
,
,
2
6
,
,
a-c
1, 2, 5, 7–
,
2
.5. P-Glycoprotein and CA XII Expression in K562/DOX and LoVo/DOX Cells
The gene expression levels of P-gp and CA XII were checked by real-time PCR analysis in K562
leukemic and LoVo colon carcinoma cells, both in sensitive and resistant lines. This test was based on
the quantitative assessment of the presence of transcripts, i.e., messenger RNA molecules encoding
the two proteins [45]. The real-time PCR analysis indicated a substantial increase in the mRNA
expression level for the MDR1 gene on the resistant leukemic line, K562/DOX, with respect to the
parental counterpart (about 173 times). On the contrary, a low increase in the mRNA expression level
for the CA XII gene was observed on the resistant K562/DOX line compared with the parental one
(
about two times). The doxorubicin resistant colon carcinoma cell line LoVo/DOX, showed an increase
in the mRNA expression level 53 times higher than the parental counterpart for the MDR1 gene. In this
cell line an increase in the mRNA expression level for the CA XII gene of 11 times was also observed.
Details are reported in Material and Methods and Supplementary Material.
The P-glycoprotein expression level in resistant K562/DOX and LoVo/DOX cells was also checked
using flow cytometric analysis with the P-gp-specific monoclonal antibody Mab FITC conjugate CD243.
The obtained results, reported as a fluorescence ratio between the resistant line and the parental
counterpart, showed an increase in the protein expression of about 17.3 times in the K562/DOX line,
and of about 8.6 times in the LoVo/DOX line. Details are reported in Material and Methods and
Supplementary Material.
2
.6. Rhodamine-123 Uptake Test on LoVo/DOX Cells
Based on the results on hCA inhibitory activity and on K562/DOX Rhd 123 uptake tests, compounds
with the best profile in term of potency on P-gp and selectivity towards the hCA XII isoform were
selected (coumarin and , and benzene sulfonamide 21 25 and 26 derivatives) for further
evaluation on human colorectal carcinoma LoVo/DOX cells.
2,
5,
7
8
,
LoVo is a human colon adenocarcinoma cell line isolated from a metastatic nodule and its MDR
variant, LoVo/DOX, was obtained from LoVo (parental line) by exposure to increasing concentrations
of doxorubicin.
The selected compounds were preliminary studied at 3, 10 and 30
µM concentrations by the MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay [43] to evaluate their intrinsic

Molecules 2020, 25, 1748
9 of 26
cytotoxicity on LoVo/DOX cell line. The compounds displayed at 3 and 10
µM concentrations
a negligible intrinsic toxicity (about 20%), that increased at the higher dose (22%–42%). Therefore,
the 30 M concentration was not used in the further studies and the rhodamine-123 uptake test on the
µ
colon carcinoma model (LoVo/DOX cells) was performed at 3 and 10 µM.
The results, expressed as FR values, were reported as the histograms shown in Figure 1. At 3
the intracellular fluorescence intensity of rhodamine varied in the 1.32–6.70 range for compounds
µM,
2, 5,
7
and
8, but was negligible for 21
,
25 and 26 (FR values equal to or close to the unity; Figure 1, panel A).
At 10
µM, instead, all molecules showed a significant increase in the intracellular rhodamine uptake.
Especially, compounds 2, 5, 7 and 8 showed FR values of 10.50, 6.30, 12.60 and 9.90, respectively, that
resulted equal to or higher than that of verapamil (6.40) tested at the same concentration (Figure 1,
panel B).
Figure 1. Fluorescence ratio (FR) values in LoVo/DOX cells incubated with rhodamine in the presence
and in absence of modulators
2, 5, 7, 8, 21, 25 and 26 and verapamil at 3 µM (panel A) and 10 µM
(
panel ) concentrations. Value 1 was attributed to the average fluorescence intensity of the samples
B
exposed only to rhodamine.
These results confirmed the already observed behavior on K562/DOX cell line; in fact, also on
LoVo/DOX cells, the coumarin derivatives were in general more potent in P-gp transport inhibition
than compounds carrying the benzene sulfonamide moiety.

Molecules 2020, 25, 1748
10 of 26
2
.7. Enhancement of Doxorubicin Cytotoxicity
Compounds 21 25 and 26 were further evaluated, at 3 and 10
2,
5,
7,
8,
,
µM concentrations, to
assess their effects on the doxorubicin cytotoxicity enhancement on K562/DOX and LoVo/DOX cell lines.
Doxorubicin is a P-gp substrate and is usually inactive in tumors overexpressing the pump.
The extent of resistance to doxorubicin of K562/DOX and LoVo/DOX cells was established and
expressed by the resistance index, as reported in Material and Methods.
In this test, doxorubicin was used at the low toxic concentrations that caused a 20% cell growth
inhibition (IC ) on the two resistant lines (IC = 0.5 µM for K562/DOX cells and 0.3 µM for LoVo/DOX
20
20
cells).
On K562/DOX cells, all compounds at 3
IC20 cytotoxicity, except compound 5, which reduced the cell growth from 80% to 51.7%. At 10
µ
M had low or no effect in the enhancement of doxorubicin
µM, all
tested compounds increased doxorubicin cytotoxicity with different extent. Coumarin derivatives
5, 7
and 8 reduced the cell growth below 50%, in particular compound 5 reduced it to 25.1% (Table 2).
Table 2. Cells’ growth experiments performed on K562/DOX and LoVo/DOX cells in the presence of
0 µM (K562/DOX) or 0.3 µM (LoVo/DOX) concentrations of doxorubicin alone or in association with
.5
compounds 2, 5, 7, 8, 21, 25 and 26 at 3 and 10 µM concentrations.
Cell Growth (%) a
Compounds
K562/DOX cells
LoVo/DOX cells
doxo
doxo (IC ) + 2 (3 µM)
80.0 ± 3.6
85.6 ± 4.7
65.0 ± 5.2
51.7 ± 3.0
25.1 ± 12.0
78.1 ± 4.9
48.0 ± 5.0
82.0 ± 5.9
49.0 ± 2.3
78.8 ± 10.2
68.7 ± 3.6
80.3 ± 5.9
76.0 ± 3.7
77.9 ± 6.1
74.0 ± 2.6
69.0 ± 0.03
62.0 ± 0.1
80.0 ± 2.5
82.7 ± 10.2
41.5 ± 7.3
58.0 ± 2.4
51.0 ± 1.9
61.0 ± 3.3
34.9 ± 8.1
68.0 ± 2.9
32.2 ± 10.7
82.0 ± 2.7
59.0 ± 3.3
81.7 ± 2.0
73.7 ± 0.015
82.0 ± 2.0
58.2 ± 5.9
54.0 ± 0.06
51.0 ± 0.09
20
doxo (IC ) + 2 (10 µM)
20
doxo (IC ) + 5 (3 µM)
20
doxo (IC ) + 5 (10 µM)
20
doxo (IC ) + 7 (3 µM)
20
doxo (IC ) + 7 (10 µM)
20
doxo (IC ) + 8 (3 µM)
20
doxo (IC ) + 8 (10 µM)
20
doxo (IC ) + 21 (3 µM)
20
doxo (IC ) + 21 (10 µM)
20
doxo (IC ) + 25 (3 µM)
20
doxo (IC ) + 25 (10 µM)
20
doxo (IC ) + 26 (3 µM)
20
doxo (IC ) + 26 (10 µM)
20
doxo (IC ) + Ver (3 µM)
20
doxo (IC ) + Ver (10 µM)
20
a
Data are the mean
Material and Methods.
±
SE of at least three determinations performed with quadruplicate cultures as described in
On LoVo/DOX cells, compounds
respectively, and the other derivatives had no effect.
At the higher concentration (10 M) almost all compounds were able to increase the doxorubicin
cytotoxicity. Notably, compounds and caused a reduction of the cells’ growth from 80% to
1.5%, 51.0%, 34.9% and 32.2%, respectively. The date reported in Table 2 indicate that, in general,
5, 7 and 8 at 3 µM reduced the cell growth to 58.0, 61.0 and 68.0%,
µ
2,
5
,
7
8
4
the ability of these compounds on the doxorubicin cytotoxicity enhancement was higher on LoVo/DOX
cells than on K562/DOX cells.
3
. Material and Methods
3
.1. Chemistry
All melting points were taken on a Büchi apparatus and were uncorrected. NMR spectra were
1
13
recorded on a Bruker Avance 400 spectrometer (400 MHz for H NMR, 100 MHz for C NMR) using
1
13
residual solvent such as chloroform (δ = 7.26) as the internal standard. H and C chemical shifts are

Molecules 2020, 25, 1748
11 of 26
expressed in ppm (
δ) referring to TMS. Spectral data are reported using the following abbreviations:
s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublets, t = triplet and m = multiplet
and coupling constants are reported in Hz, followed by integration. Chromatographic separations
were performed on a silica gel column by flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck).
Yields were given after purification, unless otherwise stated.
The high-resolution mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan
LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). The accurate
-
1
mass measure was carried out by introducing, via syringe pump at 10
(
µ
L min , the sample solution
-
1
1.0 µg mL in mQ water: acetonitrile 50:50), and the signal of the positive ions was acquired.
The proposed experimental conditions allowed to monitoring the protonated molecules of studied
+
compounds ([M + H] species), that they were measured with a proper dwell time to achieve 60,000
units of resolution at full width at half maximum (FWHM). The elemental composition of compounds
was calculated on the basis of their measured accurate masses, accepting only results with an attribution
error less than 2.5 ppm and a not integer RDB (double bond/ring equivalents) value, in order to consider
only the protonated species [46].
Compounds were named following IUPAC rules as applied by ChemBioDraw Ultra 14.0 software
(Cambridgesoft, Perkin Elmer, Milan, Italy). When reactions were performed in anhydrous conditions,
the mixtures were maintained under nitrogen. Free bases 15–28 were transformed into the hydrochloride
by treatment with a solution of acetyl chloride (1.1 equivalents) in anhydrous CH OH. The salts were
3
crystallized from abs. ethanol/petroleum ether.
6
-Chlorohexyl 3,4,5-trimethoxybenzoate 29. A solution of 909 mg (4.28 mmol) of 3,4,5-trimethoxybenzoic
◦
acid in 25 mL of anhydrous CH Cl was cooled at 0 C and 0.33 mL (2.86 mmol) of 6-chlorohexan-1-ol,
2
2
0
.28 g (2.28 mmol) of DMAP and 0.98 g (5.14 mmol) of EDC hydrochloride were added in this order.
◦
The reaction mixture was stirred at 0 C for 1 h and at room temperature for 48 h, then treated with
CH Cl . The organic layer was washed three times with water and twice with a saturated solution of
2
2
NaHCO . After drying with Na SO , the solvent was removed under reduced pressure. The crude
3
2
4
product was then purified by flash chromatography using CH Cl /CH OH 98:2 as eluent, yielding
2
2
3
1
9
43 mg (100%) of the desired compound as an oil. H-NMR (400 MHz, CDCl )
δ
: 7.22 (s, 2H, CH);
3
4
.24 (t, J = 6.8 Hz, 2H, OCH ); 3.84 (s, 6H, OCH ); 3.83 (s, 3H, OCH ); 3.47 (t, J = 6.4 Hz, 2H, CH Cl);
2
3
3
2
1
.75–1.69 (m, 4H, CH ); 1.49–1.39 (m, 4H, CH ) ppm.
2 2
6
-Iodohexyl 3,4,5-trimethoxybenzoate 30. To a solution of 1.069 g (3.24 mmol) of compound 29 in 15 mL of
acetone, NaI (2.90 g, 19.4 mmol) was added, and the resulting mixture was maintained at reflux in the
dark for 19 h. The reaction mixture was cooled to room temperature, and the solvent was removed
under reduced pressure; the residue was dissolved in CH Cl and washed with water. The organic
2
2
layer was dried with Na SO , and the solvent was removed under reduced pressure yielding 1.27 g
2
4
1
(
(
93%) of the desired compound 30 as an oil, which was used as such for the next reaction. H-NMR
400 MHz, CDCl3) : 7.21 (s, 2H, CH); 4.24 (t, J = 6.4 Hz, 2H, OCH ); 3.83 (s, 6H, OCH ); 3.82 (s, 3H,
δ
2
3
OCH ); 3.11 (t, J = 6.8 Hz, 2H, CH I); 1.77–1.69 (m, 4H, CH ); 1.40–1.35 (m, 4H, CH ) ppm.
3
2
2
2
6-((3-Hydroxypropyl)amino)hexyl 3,4,5-trimethoxybenzoate 31. To a solution of 1.27 g (3.00 mmol) of
compound 30 in 20 mL of anhydrous CH CN, 0.42 g (3.00 mmol) of K CO and 0.70 mL (9.00 mmol)
3
2
3
◦
of 3-aminopropan-1-ol were added. The mixture was heated at 80 C for 18 h. Then the solvent was
removed under reduced pressure; the residue was treated with CH Cl and the organic layer was
2
2
washed with 10% NaOH solution. After drying with Na SO , the solvent was removed under reduced
2
4
pressure and the residue purified by flash chromatography using CH Cl /CH OH/NH OH 90:10:1 as
2
2
3
4
1
eluent. The desired compound 31 (0.950 g, 86%) was obtained as an oil. H-NMR (400 MHz, CDCl ) δ:
3
7
.22 (s, 2H, CH); 4.24 (t, J = 6.8 Hz, 2H, OCH ); 3.85 (s, 6H, OCH ); 3.83 (s, 3H, OCH ); 3.73 (t, J = 5.2 Hz,
2 3 3
2
H, CH OH); 3.45 (bs, 2H, NH and OH); 2.82 (t, J = 5.6 Hz, 2H, NCH ); 2.56 (t, J = 7.2 Hz, 2H, NCH );
2
2
2
1
.72–1.63 (m, 4H, CH ); 1.49–1.33 (m, 6H, CH ) ppm.
2 2

Molecules 2020, 25, 1748
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6-((3-Hydroxypropyl)amino)hexyl anthracene-9-carboxylate 32. Following the same procedure described
for compound 31, starting from 1.44 g (3.34 mmol) of 6-iodohexyl anthracene-9-carboxylate [38],
1
compound 32 (0.766 g, 60%) was obtained as an oil. H-NMR (400 MHz, CDCl )
δ: 8.44 (s, 1H, CH);
3
7
.98–7.93 (m, 4H, CH); 7.50–7.40 (m, 4H, CH); 4.55 (t, J = 6.8 Hz, 2H, OCH ); 3.67 (t, J = 5.2 Hz, 2H,
2
CH OH); 2.70 (t, J = 5.6 Hz, 2H, NCH ); 2.49 (t, J = 6.8 Hz, 2H, NCH ); 1.83–1.77 (m, 2H, CH ); 1.63–1.57
2
2
2
2
(
m, 2H, CH ); 1.42–1.31 (m, 6H, CH ) ppm.
2 2
6-((3-Hydroxypropyl)(methyl)amino)hexyl 3,4,5-trimethoxybenzoate 33. Compound 31 (0.238 g, 0.65 mmol)
was dissolved in 5 mL of anhydrous ethanol, then HCOOH (0.40 mL) and 37% HCHO solution
◦
(
0.09 mL, 3.23 mmol) were added. The mixture was heated at 80 C for 4–5 h and concentrated in vacuo.
The residue was then dissolved in CH Cl and the organic layer was washed with 10% NaOH solution.
2
2
After drying with Na SO , the solvent was removed under reduced pressure, and the residue purified
2
4
by flash chromatography using CH Cl /CH OH/NH OH 90:10:1 as eluent. The desired compound 33
2
2
3
4
1
(
0.169 g, 68%) was obtained as an oil. H-NMR (400 MHz, CDCl )
δ: 7.24 (s, 2H, CH); 4.25 (t, J = 6.8 Hz,
3
2
2
H, OCH ); 3.86 (s, 6H, OCH ); 3.85 (s, 3H, OCH ); 3.74 (t, J = 5.2 Hz, 2H, CH OH); 2.55 (t, J = 5.6 Hz,
2
3
3
2
H, NCH ); 2.33 (t, J = 7.2 Hz, 2H, NCH ); 2.20 (s, 3H, NCH ); 1.74–1.63 (m, 4H, CH ); 1.51–1.30 (m, 6H,
2
2
3
2
CH ) ppm.
2
6-((3-Hydroxypropyl)(methyl)amino)hexyl anthracene-9-carboxylate 34. Following the same procedure
described for compound 33, starting from 0.224 (0.59 mmol) of 32, compound 34 (0.226 g, 98%) was
1
obtained as an oil. H-NMR (400 MHz, CDCl )
δ: 8.39 (s, 1H, CH); 8.01 (d, J =8.8 Hz, 2H, CH); 7.90
3
(
d, J = 8.8 Hz, 2H, CH); 7.50–7.38 (m, 4H, CH); 4.56 (t, J = 6.8 Hz, 2H, OCH ); 3.71 (t, J = 5.2 Hz, 2H,
2
CH OH); 2.43 (t, J = 5.6 Hz, 2H, NCH ); 2.23 (t, J = 7.2 Hz, 2H, NCH ); 2.12 (s, 3H, NCH ); 1.85–1.78
2
2
2
3
(
m, 2H, CH ); 1.61–1.50 (m, 2H, CH ); 1.49–1.28 (m, 6H, CH ) ppm.
2 2 2
Ethyl 2-((2-oxo-2H-chromen-7-yl)oxy)acetate 47. To a solution of 7-hydroxy-2H-chromen-2-one (240 mg,
.97 mmol) in 8 mL of acetone, K CO (228 mg, 1.63 mmol) was added. The mixture was stirred at
0
2
3
room temperature for 15 min, and then ethyl bromoacetate (0.18 mL, 1.63 mmol) dissolved in 6 mL
◦
acetone was added dropwise. The reaction mixture was heated at 58 C for 6 h and, after cooling,
water (10 mL) was added. The suspension was filtered under vacuum and the solid obtained was
◦
dried under vacuum. The title compound was obtained as a white solid. Yield: 89%, mp 107–110 C.
H-NMR (400 MHz, CDCl3)
1
δ: 7.64 (d, J = 9.6 Hz, 1H, CH); 7.40 (d, J = 8.4 Hz, 1H, CH); 6.89 (dd, J = 8.4,
2
.2 Hz, 1H, CH); 6.78 (d, J = 2.2 Hz, 1H, CH); 6.28 (d, J = 9.6 Hz, 1H, CH); 4.68 (s, 2H, OCH ); 4.30
2
(
q, J = 7.2 Hz, 2H, OCH ); 1.32 (t, J = 7.2 Hz, 3H, CH ) ppm.
2
3
2
2
-((2-Oxo-2H-chromen-7-yl)oxy)acetic acid 48. Ten milliliters of 10% NaOH solution were added to
25 mg (0.95 mmol) of 47. The mixture was heated at 100 C for 3 h. After cooling, the solution was
◦
acidified with HCl 2N and the solid was filtered under vacuum and then dried. The title compound
◦
1
was obtained as a white solid. Yield: 100%, mp 220–223 C. H-NMR (400 MHz, DMSO-d )
δ
: 7.98
d, J = 9.6, Hz 1H, CH); 7.62 (d, J = 8.4 Hz, 1H, CH); 6.94 (s, 1H, CH); 6.93 (d, J = 8.4 Hz, 1H, CH); 6.28
d, J = 9.6 Hz, 1H, CH); 4.81 (s, 2H, OCH ) ppm.
6
(
(
2
3
.1.1. General Procedure for the Synthesis of Diester Compounds 1–14
To a solution of 48 (0.26 mmol) in 25 mL of anhydrous CH CN, 0.33 mmol of EDC hydrochloride
3
and 0.33 mmol of HOBt were added. The mixture was stirred at room temperature for 1 h, and then
the suitable (hydroxyalkyl) methylaminoester 33–46 (0.22 mmol) dissolved in 5 mL of anhydrous
CH CN was added. The reaction mixture was stirred for 4 h at room temperature and the solvent was
3
removed under reduce pressure. Then CH Cl was added and the organic layer was washed twice
2
2
with a saturated solution of NaHCO . After drying with Na SO , the solvent was removed under
3
2
4
reduced pressure. The crude product was then purified by flash chromatography, using the proper
eluting system, yielding the desired compound as an oil.

Molecules 2020, 25, 1748
13 of 26
(E)-3-(methyl(3-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)propyl)amino)propyl 3-(3,4,5-trimethoxyphenyl)acrylate 1.
From (hydroxyalkyl)methylaminoester 35 [35]. Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield:
2
2
3
1
3
(
6
7%. H-NMR (400 MHz, CDCl )
δ
: 7.63 (d, J = 9.4 Hz, 1H, CH); 7.58 (d, J = 16.0 Hz, 1H, CH=CH); 7.38
d, J = 8.4 Hz, 1H, CH); 6.87 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.77 (d, J = 2.2 Hz, 1H, CH); 6.74 (s, 2H, CH);
.33 (d, J = 16.0 Hz, 1H, CH=CH); 6.26 (d, J = 9.4 Hz, 1H, CH); 4.68 (s, 2H, OCH ); 4.30–4.22 (m, 4H,
3
2
OCH ); 3.88 (s, 6H, OCH ); 3.87 (s, 3H, OCH ); 2.47–2.35 (m, 4H, NCH ); 2.22 (s, 3H, NCH ); 1.87–1.81
2
3
3
2
3
13
(
(
(
m, 4H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.97 (C); 166.95 (C); 160.84 (C); 155.62 (C); 153.44
C); 144.73 (CH); 143.16 (CH); 129.88 (C); 128.97 (CH); 117.31 (CH); 113.78 (CH); 133.33 (C); 112.80
CH); 105.27 (CH); 101.70 (CH); 65.34 (CH ); 64.02 (CH ); 62.85 (CH ); 60.95 (OCH ); 56.17 (OCH );
2
2
2
2
3
3
5
2 2 3 2 2
;
4.20 (CH ); 53.85 (CH ) 41.95 (NCH ); 26.67 (CH ); 26.47 (CH ) ppm. ESI-HRMS (m/z) calculated for
+
[M + H] ion species C H NO = 570.2334, found 570.2340.
30 36 10
(
E)-3-(methyl(5-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)pentyl)amino)propyl 3-(3,4,5-trimethoxyphenyl)acrylate 2.
1
From 36
CDCl3)
.85 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.74 (d, J = 2.2 Hz, 1H, CH); 6.72 (s, 2H, CH); 6.31 (d, J = 16.0 Hz,
H, CH=CH); 6.23 (d, J = 9.6 Hz, 1H, CH); 4.66 (s, 2H, OCH ); 4.23–4.16 (m, 4H, OCH ); 3.85 (s, 6H,
[37]. Oil. Chromatographic eluent: CH Cl /CH OH 93:7. Yield: 29%. H-NMR (400 MHz,
2 2 3
δ: 7.60 (d, J = 9.6 Hz, 1H, CH); 7.56 (d, J = 16.0 Hz, 1H, CH=CH); 7.36 (d, J = 8.4 Hz, 1H, CH);
6
1
2
2
OCH ); 3.84 (s, 3H, OCH ); 2.42 (t, J = 6.8 Hz, 2H, NCH ); 2.30 (t, J = 6.8 Hz, 2H, NCH ); 2.19 (s, 3H,
3
3
2
2
NCH ); 1.88–1.83 (m, 2H, CH ); 1.71–1.60 (m, 2H, CH ); 1.50–1.40 (m, 2H, CH ); 1.38–1.28 (m, 2H,
3
2
2
2
13
CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.98 (C); 166.92 (C); 160.81 (C); 155.63 (C); 153.41 (C);
44.65 (CH); 143.18 (CH); 129.87 (C); 128.95 (CH); 117.33 (CH); 113.70 (CH); 113.29 (C); 112.79 (CH);
05.26 (CH); 101.71 (CH); 65.63 (CH ); 65.32 (CH ); 62.98 (CH ); 60.92 (OCH ); 57.45 (CH ); 56.15
2
1
1
2
2
2
3
2
(
(
OCH ); 54.19 (CH ); 42.08 (NCH ); 28.42 (CH ); 26.87 (CH ); 26.67 (CH ); 23.67 (CH ) ppm. ESI-HRMS
3 2 3 2 2 2 2
+
m/z) calculated for [M + H] ion species C H NO = 598.2647, found 598.2651.
32
40
10
(
E)-5-(methyl(3-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)propyl)amino)pentyl 3-(3,4,5-trimethoxyphenyl)acrylate 3.
1
From 37 [37]. Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield: 33%. H-NMR (400 MHz,
2
2
3
CDCl3)
.82 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.72 (d, J = 2.2 Hz, 1H, CH); 6.70 (s, 2H, CH); 6.30 (d, J = 15.6 Hz,
H, CH=CH); 6.21 (d, J = 9.6 Hz, 1H, CH); 4.64 (s, 2H, OCH ); 4.22 (t, J = 6.4 Hz, 2H, OCH ); 4.15
δ: 7.59 (d, J = 9.6 Hz, 1H, CH); 7.53 (d, J = 15.6 Hz, 1H, CH=CH); 7.34 (d, J = 8.4 Hz, 1H, CH);
6
1
2
2
(
(
(
(
t, J = 6.4 Hz, 2H, OCH ); 3.83 (s, 6H, OCH ); 3.82 (s, 3H, OCH ); 2.34 (t, J = 6.8 Hz, 2H, NCH ); 2.29
2 3 3 2
t, J = 7.2 Hz, 2H, NCH ); 2.15 (s, 3H, NCH ); 1.82–1.75 (m, 2H, CH ); 1.69–1.64 (m, 2H, CH ); 1.52–1.32
2
3
2
2
13
m, 4H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.95 (C); 166.97 (C); 160.77 (C); 155.61 (C); 153.38
2
C); 144.58 (CH); 143.20 (CH); 140.04 (C); 129.90 (C); 128.98 (CH); 117.41 (CH); 113.68 (CH); 113.29 (C);
1
12.73 (CH); 105.21 (CH); 101.68 (CH); 65.29 (CH ); 64.47 (CH ); 64.06 (CH ); 60.90 (OCH ); 57.51 (CH );
2
2
2
3
2
5
6.12 (OCH ); 53.88 (CH ); 41.97 (NCH ); 28.64 (CH ); 26.81 (CH ); 26.37 (CH ); 23.81 (CH ) ppm.
3
2
3
+
2
2
2
2
ESI-HRMS (m/z) calculated for [M + H] ion species C H NO = 598.2647, found 598.2642.
32
40
10
(
E)-5-(methyl(5-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)pentyl)amino)pentyl 3-(3,4,5-trimethoxyphenyl)acrylate 4.
1
From 38 [35]. Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield: 46%. H-NMR (400 MHz,
2
2
3
CDCl3)
.87 (dd, J = 8.8, 2.2 Hz, 1H, CH); 6.76 (d, J = 2.2 Hz, 1H, CH); 6.74 (s, 2H, CH); 6.33 (d, J = 16.0 Hz,
H, CH=CH); 6.25 (d, J = 9.6 Hz, 1H, CH); 4.67 (s, 2H, OCH ); 4.22–4.15 (m, 4H, OCH ); 3.87 (s, 6H,
δ: 7.62 (d, J = 9.6 Hz, 1H, CH); 7.57 (d, J = 16.0 Hz, 1H, CH=CH); 7.38 (d, J = 8.8 Hz, 1H, CH);
6
1
2
2
OCH ); 3.86 (s, 3H, OCH ); 2.35–2.29 (m, 4H, NCH ); 2.20 (s, 3H, NCH ); 1.75–1.63 (m, 4H, CH );
3
3
2
3
2
13
1
.55–1.30 (m, 8H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.99 (C); 167.01 (C); 160.86 (C); 160.82
2
(
C); 155.66 (C); 153.43 (C); 144.61 (CH); 143.20 (CH); 140.21 (C); 129.93 (C); 128.97 (CH); 117.44 (CH);
113.76 (CH); 113.32 (C); 112.85 (CH); 105.24 (CH); 101.70 (CH); 65.66 (CH ); 65.34 (CH ); 64.54 (CH );
2 2 2
6
0.95 (OCH ); 57.64 (CH ); 57.54 (CH ); 56.16 (OCH ); 42.15 (NCH ); 28.70 (CH ); 28.45 (CH ); 26.88
3
2
2
3
3
2
2
+
(
CH ); 26.80 (CH ); 23.97 (CH ); 23.77 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
C H NO = 626.2960, found 626.2951.
34
44
10

Molecules 2020, 25, 1748
14 of 26
(
E)-6-(methyl(3-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)propyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate
5
.
1
From 39 [38]. Oil. Chromatographic eluent: CH Cl /CH OH 96:4. Yield: 80%. H-NMR (400 MHz,
2 2 3
CDCl3)
.85 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.74 (d, J = 2.2 Hz, 1H, CH); 6.72 (s, 2H, CH); 6.32 (d, J = 16.0 Hz,
H, CH=CH); 6.24 (d, J = 9.6 Hz, 1H, CH,); 4.66 (s, 2H, OCH ); 4.24 (t, J = 6.4 Hz, 2H, OCH ); 4.16
δ: 7.61 (d, J = 9.6 Hz, 1H, CH); 7.56 (d, J = 16.0 Hz, 1H, CH=CH); 7.36 (d, J = 8.4 Hz, 1H, CH);
6
1
2
2
(
t, J = 6.4 Hz, 2H, OCH ); 3.85 (s, 6H, OCH ); 3.84 (s, 3H, OCH ); 2.35 (t, J = 7.2 Hz, 2H, NCH );
2 3 3 2
2
.28 (t, J = 7.6 Hz, 2H, NCH ); 2.16 (s, 3H, NCH ); 1.84–1.77 (m, 2H, CH ); 1.70–1.64 (m, 2H, CH );
2 3 2 2
13
1
.47–1.30 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.96 (C); 167.01 (C); 160.84 (C); 160.79
2
(
C); 155.64 (C); 153.41 (C); 144.57 (CH); 143.20 (CH); 140.06 (C); 129.93 (C); 128.98 (CH); 117.45 (CH);
113.73 (CH); 113.31 (C); 112.77 (CH); 105.21 (CH); 101.70 (CH); 65.31 (CH ); 64.55 (CH ); 64.15 (CH );
2 2 2
6
0.93 (OCH ); 57.62 (CH ); 56.14 (OCH ); 53.91 (CH ); 42.01 (NCH ); 28.71 (CH ); 27.09 (CH ); 26.40
3
2
3
2
3
2
2
+
(CH ); 25.90 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H NO = 612.2803,
2 2 33 42 10
found 612.2794.
(
E)-6-(methyl(4-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)butyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 6.
1
From 40 [38]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 97:3:0.3. Yield: 100%. H-NMR
2 2 3 4
(400 MHz, CDCl3)
δ: 7.61 (d, J = 9.6 Hz, 1H, CH); 7.55 (d, J = 16.0 Hz, 1H, CH=CH); 7.36 (d, J = 8.4 Hz,
1H, CH); 6.84 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.73 (d, J = 2.2 Hz, 1H, CH); 6.71 (s, 2H, CH); 6.31
(
d, J = 16.0 Hz, 1H, CH=CH); 6.23 (d, J = 9.6 Hz, 1H, CH); 4.65 (s, 2H, OCH ); 4.21–4.14 (m, 4H, OCH );
2 2
3
.85 (s, 6H, OCH ); 3.84 (s, 3H, OCH ) 2.32–2.27 (m, 4H, NCH ); 2.16 (s, 3H, NCH ); 1.69–1.62 (m, 4H,
3
3
2
3
1
3
CH ); 1.51–1.29 (m, 8H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ: 167.98 (C); 166.98 (C); 160.85 (C);
2
2
160.77 (C); 155.59 (C); 153.38 (C); 144.54 (CH); 143.26 (CH); 139.99 (C); 129.91 (C); 129.00 (CH); 117.44
(
CH); 113.66 (CH); 113.29 (C); 112.73 (CH); 105.16 (CH); 101.69 (CH); 65.50 (CH ); 65.28 (CH ); 64.53
2
2
(
2
6
CH ); 60.92 (OCH ); 57.57 (CH ); 56.96 (CH ); 56.12 (OCH ); 41.88 (NCH ); 28.68 (CH ); 27.11 (CH );
2 3 2 2 3 3 2 2
+
6.44 (CH ); 25.89 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H NO
=
10
2
2
34 44
26.2960, found 626.2966.
(
E)-7-(methyl(2-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)ethyl)amino)heptyl 3-(3,4,5-trimethoxyphenyl)acrylate 7.
1
From 44
CDCl3)
.88 (dd, J = 8.8, 2.2 Hz, 1H, CH); 6.78 (d, J = 2.2 Hz, 1H, CH); 6.74 (s, 2H, CH); 6.33 (d, J = 16.0 Hz,
H, CH=CH); 6.27 (d, J = 9.6 Hz, 1H, CH); 4.70 (s, 2H, OCH ); 4.31 (t, J = 5.6 Hz, 2H, OCH ); 4.18
[38]. Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield: 10%. H-NMR (400 MHz,
2 2 3
δ: 7.63 (d, J = 9.6 Hz, 1H, CH); 7.58 (d, J = 16.0 Hz, 1H, CH=CH); 7.38 (d, J = 8.8 Hz, 1H, CH);
6
1
2
2
(
t, J = 5.6 Hz, 2H, OCH ); 3.88 (s, 6H, OCH ); 3.87 (s, 3H, OCH ); 2.64 (t, J = 5.6 Hz 2H, NCH ); 2.38
2 3 3 2
(
t, J = 7.2 Hz, 2H, NCH ); 2.26 (s, 3H, NCH ); 1.71–1.65 (m, 4H, CH ); 1.47–1.30 (m, 6H, CH ) ppm.
C-NMR (100 MHz, CDCl3) δ: 167.93 (C); 167.05 (C); 160.89 (C); 160.74 (C); 153.43 (C); 144.64 (CH);
2
3
2
2
1
3
1
1
43.21 (CH); 129.93 (C); 129.03 (CH); 117.43 (CH); 113.80 (CH); 113.38 (C); 112.79 (CH); 105.22 (CH);
01.84 (CH); 65.35 (CH ); 65.26 (CH ); 64.52 (CH ); 60.97 (OCH ); 57.53 (CH ); 56.17 (OCH ); 54.99
2
2
2
3
2
3
(
CH ); 52.50 (NCH ); 29.02 (CH ); 28.66 (CH ); 27.07 (CH ); 25.86 (CH ) ppm. ESI-HRMS (m/z)
2 3 2 2 2 2
+
calculated for [M + H] ion species C H NO = 612.2803, found 612.2794.
33
42
10
(
E)-7-(methyl(7-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)heptyl)amino)heptyl 3-(3,4,5-trimethoxyphenyl)acrylate 8.
1
From 41 [35]. Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield: 31%. H-NMR (400 MHz,
2
2
3
CDCl3)
.84 (dd, J = 8.8, 2.2 Hz, 1H, CH); 6.75 (d, J = 2.2 Hz, 1H, CH); 6.73 (s, 2H, CH); 6.33 (d, J = 16.0 Hz, 1H,
CH=CH); 6.24 (d, J = 9.6 Hz, 1H, CH); 4.66 (s, 2H, OCH ); 4.20–4.15 (m, 4H, OCH ); 3.86 (s, 6H, OCH );
δ: 7.61 (d, J = 9.6 Hz, 1H, CH); 7.56 (d, J = 16.0 Hz, 1H, CH=CH); 7.37 (d, J = 8.8 Hz, 1H, CH);
6
2
2
3
3
.85 (s, 3H, OCH ); 2.34–2.28 (m, 4H, NCH ); 2.20 (s, 3H, NCH ); 1.70–1.62 (m, 4H, CH ); 1.50–1.20
3 2 3 2
13
(
m, 16H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ: 168.00 (C); 167.03 (C); 160.87 (C); 160.82 (C); 155.65
2
(C); 153.41 (C); 144.54 (CH); 143.22 (CH); 140.06 (C); 129.94 (C); 128.97 (CH); 117.48 (CH); 113.72 (CH);
1
13.30 (C); 112.82 (CH); 105.21 (CH); 101.70 (CH); 65.73 (CH ); 65.33 (CH ); 64.63 (CH ); 60.94 (OCH );
2
2
2
3
5
7.77 (CH ); 57.74 (CH ); 56.14 (OCH ); 42.15 (NCH ); 29.20 (CH ); 29.08 (CH ); 28.69 (CH ); 28.43
2
2
3
3
2
2
2
(
(
CH ); 27.44 (CH ); 27.38 (CH ); 27.04 (CH ); 27.02 (CH ); 25.92 (CH ); 25.72 (CH ) ppm. ESI-HRMS
2 2 2 2 2 2 2
+
m/z) calculated for [M + H] ion species C H NO = 682.3586, found 682.3573.
38
52
10

Molecules 2020, 25, 1748
15 of 26
3
-(methyl(5-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)pentyl)amino)propyl 3,4,5-trimethoxy benzoate
9
. From 42 [37]
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 90:10:1. Yield: 86%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 7.61 (d, J = 9.2 Hz, 1H, CH); 7.36 (d, J =8.4 Hz, 1H, CH); 7.25 (s, 2H, CH); 6.85 (dd, J = 8.4,
2
.2 Hz, 1H, CH); 6.73 (d, J = 2.2 Hz, 1H, CH); 6.23 (d, J = 9.2 Hz, 1H, CH); 4.66 (s, 2H, OCH ); 4.33
2
(
t, J = 6.4 Hz, 2H, OCH ); 4.16 (t, J = 6.4 Hz, 2H, OCH ); 3.86 (s, 9H, OCH ); 2.56 (t, J = 6.8 Hz, 2H,
2 2 3
NCH ); 2.41 (t, J =7.2 Hz, 2H, NCH ); 2.29 (s, 3H, NCH ); 2.00–1.95 (m, 2H, CH ); 1.67–1.60 (m, 2H,
2
2
3
2
13
CH ); 1.52–1.46 (m, 2H, CH ); 1.35–1.27 (m, 2H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ: 168.01 (C);
2
2
2
1
1
6
66.15 (C); 160.88 (C); 160.80 (C); 155.61 (C); 152.91 (C); 143.27 (CH); 142.19 (C); 129.00 (CH); 125.24 (C);
13.69 (CH); 113.28 (C); 112.86 (CH); 106.78 (CH); 101.63 (CH); 65.50 (CH ); 65.30 (CH ); 63.30 (CH );
2
2
2
0.90 (OCH ); 57.19 (CH ); 56.24 (OCH ); 53.96 (CH ); 41.70 (NCH ); 28.36 (CH ); 26.37 (CH ); 26.18
3
2
3
2
3
2
2
+
(CH ); 23.62 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H NO = 572.2490,
2 2 30 38 10
found 572.2479.
6
-(methyl(3-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)propyl)amino)hexyl 3,4,5-trimethoxy benzoate 10. From 33
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 90:10:1. Yield: 83%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 7.61 (d, J = 9.6 Hz, 1H, CH); 7.37 (d, J = 8.4 Hz, 1H, CH); 7.27 (s, 2H, CH); 6.85 (d, J = 8.4 Hz,
1
3
H, CH); 6.75 (s, 1H, CH); 6.24 (d, J = 9.6 Hz, 1H, CH); 4.67 (s, 2H, OCH ); 4.29–4.23 (m, 4H, OCH );
2
2
.88 (s, 9H, OCH ); 2.40 (t, J = 7.2 Hz, 2H, NCH ); 2.33 (t, J = 7.2 Hz, 2H, NCH ); 2.20 (s, 3H, NCH );
3
2
2
3
13
1
.83–1.73 (m, 4H, CH ); 1.47–1.34 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.92 (C); 166.18
2
2
(C); 160.77 (C); 155.60 (C); 152.87 (C); 143.18 (CH); 142.14 (C); 128.97 (CH); 125.45 (C); 113.69 (CH);
1
13.29 (C); 112.72 (CH); 106.80 (CH); 101.68 (CH); 65.29 (CH ); 65.09 (CH ); 63.99 (CH ); 60.85 (OCH );
2
2
2
3
5
7.49 (CH ); 56.22 (OCH ); 53.85 (CH ); 41.81 (NCH ); 28.68 (CH ); 27.02 (CH ); 26.89 (CH ); 26.22
2
3
2
3
2
2
2
+
(
CH ); 25.89 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H NO = 586.2647,
2 2 31 40 10
found 586.2643.
7
-(methyl(2-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)ethyl)amino)heptyl 3,4,5-trimethoxy benzoate 11. From 45 [38]
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 89%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 7.58 (d, J = 9.6 Hz, 1H, CH); 7.33 (d, J = 8.4 Hz, 1H, CH); 7.24 (s, 2H, CH); 6.83 (d, J = 8.4 Hz,
1
4
H, CH); 6.73 (s, 1H, CH); 6.20 (d, J = 9.6 Hz, 1H, CH); 4.66 (s, 2H, OCH ); 4.29 (t, J = 5.6 Hz, 2H, OCH );
2
2
.24 (t, J = 6.4 Hz, 2H, OCH ); 3.85 (s, 9H, OCH ); 2.67 (t, J = 5.2 Hz, 2H, NCH ); 2.40 (t, J =7.6 Hz, 2H,
2
3
2
13
NCH ); 2.27 (s, 3H, NCH ); 1.73–1.69 (m, 2H, CH ); 1.45–1.26 (m, 8H, CH ) ppm. C-NMR (100 MHz,
2
3
2
2
CDCl3) δ: 167.86 (C); 166.08 (C); 160.70 (C); 155.52 (C); 152.82 (C); 143.21 (CH); 142.07 (C); 128.96 (CH);
125.43 (C); 113.54 (CH); 113.23 (C); 112.60 (CH); 106.74 (CH); 101.72 (CH); 65.21 (CH ); 65.06 (CH );
2 2
6
2.40 (CH ); 60.76 (OCH ); 57.53 (CH ); 56.15 (OCH ); 55.10 (CH ); 42.00 (NCH ); 29.01 (CH ); 28.58
2
3
2
3
2
3
2
+
(
CH ); 27.08 (CH ); 26.46 (CH ); 25.84 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
C H NO = 586.2647, found 586.2638.
31
40
10
3
-(methyl(5-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)pentyl)amino)propyl anthracene-9-carboxylate 12. From 43 [37].
1
Oil. Chromatographic eluent: CH Cl /CH OH 95:5. Yield: 81%. H-NMR (400 MHz, CDCl )
δ
: 8.51
s, 1H, CH); 8.05–8.00 (m, 4H, CH); 7.56–7.46 (m, 5H, CH); 7.30 (d, J = 8.8 Hz, 1H, CH); 6.82 (dd, J = 8.8,
2
2
3
3
(
2
4
2
.2 Hz, 1H, CH); 6.73 (d, J = 2.2 Hz, 1H, CH); 6.23 (d, J = 9.2 Hz, 1H, CH); 4.68 (t, J = 6.4 Hz, 2H, OCH2);
.64 (s, 2H, OCH ); 4.19 (t, J = 6.4 Hz, 2H, OCH ); 2.54 (t, J =7.2 Hz, 2H, NCH ); 2.35 (t, J = 7.2 Hz,
2
2
2
H, NCH ); 2.25 (s, 3H, NCH ); 2.11–2.02 (m, 2H, CH ); 1.68–1.61 (m, 2H, CH ); 1.53–1.45 (m, 2H,
2
3
2
2
13
CH ); 1.39–1.30 (m, 2H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 169.60 (C); 167.99 (C); 160.87 (C);
2
2
1
(
(
2
5
60.77 (C); 155.59 (C); 143.19 (CH); 130.97 (C); 129.29 (CH); 128.93 (CH); 128.64 (CH); 128.38 (C); 127.98
C); 126.96 (CH); 125.48 (CH); 124.97 (CH); 113.66 (CH); 113.24 (C); 112.74 (CH); 101.68 (CH); 65.55
CH ); 65.31 (CH ); 64.04 (CH ); 57.40 (CH ); 54.13 (CH ); 41.89 (NCH ); 28.38 (CH ); 26.59 (CH );
6.39 (CH ); 23.67 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H NO =
2 2 35 36 7
82.2486, found 582.2489.
2
2
2
2
2
3
+
2
2
6
-(methyl(3-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)propyl)amino)hexyl anthracene-9-carboxylate 13. From 34. Oil.
1
Chromatographic eluent: CH Cl /CH OH/NH OH 90:10:1. Yield: 98%. H-NMR (400 MHz, CDCl ) δ:
2
2
3
4
3

Molecules 2020, 25, 1748
16 of 26
8
(
1
.38 (s, 1H, CH); 7.96 (d, J = 8.4 Hz, 2H, CH); 7.90 (d, J = 8.4 Hz, 2H, CH); 7.47–7.37 (m, 5H, CH); 7.16
d, J = 8.8 Hz, 1H, CH); 6.70 (dd, J = 8.8, 2.2 Hz, 1H, CH); 6.64 (d, J = 2.2 Hz, 1H, CH); 6.10 (d, J = 9.6 Hz,
H, CH); 4.57–4.52 (m, 4H, OCH ); 4.17 (t, J = 6.4 Hz, 2H, OCH ); 2.27 (t, J = 6.8 Hz, 2H, NCH ); 2.21
2
2
2
(
(
(
1
6
t, J = 6.8 Hz, 2H, NCH ); 2.09 (s, 3H, NCH ); 1.85–1.69 (m, 4H, CH ); 1.46–1.35 (m, 4H, CH ); 1.33–1.26
2 3 2 2
13
m, 2H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 169.71 (C); 167.94 (C); 160.85 (C); 160.75 (C); 155.60
C); 143.16 (CH); 142.69 (C); 141.90 (C); 130.98 (C); 129.22 (CH); 128.95 (CH); 128.63 (CH); 128.36 (C);
28.16 (C); 126.93 (CH); 125.47 (CH); 124.99 (CH); 113.70 (CH); 113.28 (C); 112.69 (CH); 101.70 (CH);
5.84 (CH ); 65.30 (CH ); 64.06 (CH ); 57.52 (CH ); 53.87 (CH ); 41.91 (NCH ); 28.75 (CH ); 27.04
2
2
2
2
2
2
3
2
+
(
CH ); 26.95 (CH ); 26.30 (CH ); 26.04 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
C H NO = 596.2643, found 596.2652.
36
38
7
7
-(methyl(2-(2-((2-oxo-2H-chromen-7-yl)oxy)acetoxy)ethyl)amino)heptyl anthracene-9-carboxylate 14. From 46 [38].
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 96:4:0.4. Yield: 68%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
.80 (dd, J = 8.4, 2.2 Hz, 1H, CH); 6.73 (d, J = 2.2 Hz, 1H, CH); 6.20 (d, J = 9.6 Hz, 1H, CH); 4.64 (s, 2H,
OCH ); 4.60 (t, J = 6.8 Hz, 2H, OCH ); 4.29 (t, J = 5.6 Hz, 2H, OCH ); 2.62 (t, J = 5.6 Hz, 2H, NCH );
δ: 8.48 (s, 1H, CH); 8.03–7.98 (m, 4H, CH); 7.54–7.44 (m, 5H, CH); 7.28 (d, J = 8.4 Hz, 1H, CH);
6
2
2
2
2
2
.36 (t, J = 7.2 Hz, 2H, NCH ); 2.24 (s, 3H, NCH ); 1.90–1.83 (m, 2H, CH ); 1.50–1.25 (m, 8H, CH ) ppm.
C-NMR (100 MHz, CDCl3) δ: 169.71 (C); 167.94 (C); 160.85 (C); 160.73 (C); 155.59 (C); 143.15 (CH);
2
3
2
2
1
3
1
1
5
30.98 (C); 129.19 (CH); 128.91 (CH); 128.61 (CH); 128.35 (C); 126.90 (CH); 125.45 (CH); 124.99 (CH);
13.68 (CH); 113.26 (C); 112.71 (CH); 101.75 (CH); 65.87 (CH ); 65.29 (CH ); 63.11 (CH ); 57.91 (CH );
2
2
2
2
5.48 (CH ); 42.49 (NCH ); 29.14 (CH ); 28.72 (CH ); 27.24 (CH ); 27.07 (CH ); 26.07 (CH ) ppm.
2
3
2
+
2
2
2
2
ESI-HRMS (m/z) calculated for [M + H] ion species C H NO = 596.2643, found 596.2631.
36
38
7
3
.1.2. General Procedure for the Synthesis of Diester Compounds 15–28
A 1 mmol portion of 4-sulfamoylbenzoic acid was transformed into the acyl chloride by reaction
◦
with SOCl (2 mmol) in 5 mL of CHCl (free of ethanol) at 60 C for 5 h. The reaction mixture was cooled
2
3
to room temperature, and the solvent was removed under reduced pressure; the mixture was then
treated twice with cyclohexane and the solvent removed under reduced the pressure. The acyl chloride
obtained was dissolved in CHCl (free of ethanol), and the suitable (hydroxyalkyl)methylaminoester
3
3
3
–
46 (1 eq) was added. The mixture was stirred for 17 h at room temperature and the solvent was
removed under reduce pressure. Then CH Cl was added and the organic layer was washed twice
2
2
with a saturated solution of NaHCO . After drying with Na SO , the solvent was removed under
3
2
4
reduced pressure. The crude product was then purified by flash chromatography using the proper
eluting system, yielding the desired compound as an oil.
All the compounds were transformed into the corresponding hydrochloride as a white solid.
The salts were crystallized from abs. ethanol/petroleum ether.
(
E)-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)propyl)amino)propyl 4-sulfamoyl benzoate 15
.
From (hydroxyalkyl) methylaminoester 35 [35]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH
2
2
3
4
1
9
3:7:0.3. Yield: 63%. H-NMR (400 MHz, CDCl )
δ
: 8.02 (d, J = 8.4 Hz, 2H, CH); 7.88 (d, J = 8.4 Hz,
3
2
H, CH); 7.54 (d, J = 16.0 Hz, 1H, CH=CH); 6.69 (s, 2H, CH); 6.30 (d, J = 16.0 Hz, 1H, CH=CH); 4.35
(
2
t, J = 6.4 Hz, 2H, OCH ); 4.22 (t, J = 6.0 Hz, 2H, OCH ); 3.83 (s, 6H, OCH ); 3.82 (s, 3H, OCH );
.51–2.46 (m, 4H, NCH ); 2.23 (s, 3H, NCH ); 1.95–1.81 (m, 4H, CH ) ppm. C-NMR (100 MHz, CDCl3)
2 3 2
2 2 3 3
13
δ
: 167.04 (C); 165.14 (C); 153.31 (C); 146.14 (C); 144.75 (CH); 139.89 (C); 133.72 (C); 130.10 (CH); 129.94
(
C); 126.29 (CH); 117.29 (CH); 105.28 (CH); 64.04 (CH ); 62.70 (CH ); 60.95 (OCH ); 56.16 (OCH );
2 2 3 3
5
4.00 (CH ); 53.69 (CH ); 41.99 (NCH ); 26.47 (CH ); 26.43 (CH ) ppm. ESI-HRMS (m/z) calculated for
2 2 3 2 2
+
[M + H] ion species C H N O S = 551.2058, found 551.2050. Hydrochloride: low melting solid.
26 35 2 9
(
E)-5-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)propyl)amino)pentyl 4-sulfamoyl benzoate 16
.
1
From 36 [37]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 21%. H-NMR
2
2
3
4
(400 MHz, CDCl3)
δ: 8.09 (d, J = 8.4 Hz, 2H, CH); 7.95 (d, J = 8.4 Hz, 2H, CH); 7.58 (d, J = 16.0 Hz,
1
H, CH=CH); 6.73 (s, 2H, CH); 6.33 (d, J = 16.0 Hz, 1H, CH=CH); 4.33 (t, J = 6.8 Hz, 2H, OCH ); 4.22
2

Molecules 2020, 25, 1748
17 of 26
(
t, J = 6.4 Hz, 2H, OCH ); 3.86 (s, 6H, OCH ); 3.85 (s, 3H, OCH ); 2.48 (t, J = 7.2 Hz, 2H, NCH ); 2.39
2 3 3 2
(
t, J = 7.2 Hz, 2H, NCH ); 2.24 (s, 3H, NCH ); 1.89–1.74 (m, 4H, CH ); 1.59–1.41 (m, 4H, CH ) ppm.
C-NMR (100 MHz, CDCl3) δ: 167.06 (C); 165.15 (C); 153.39 (C); 146.05 (C); 144.83 (CH); 140.09 (C);
2
3
2
2
1
3
133.97 (C); 130.17 (CH); 129.90 (C); 126.37 (CH); 117.25 (CH); 105.35 (CH); 65.67 (CH ); 62.88 (CH );
2 2
6
0.94 (OCH ); 57.43 (CH ); 56.18 (OCH ); 53.93 (CH ); 41.99 (NCH ); 28.48 (CH ); 26.76 (CH ); 26.48
3
2
3
2
3
2
2
+
(
CH ); 23.84 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H N O S = 579.2371,
2 2 28 39 2 9
◦
found 579.2380. Hydrochloride: mp 98–100 C.
(
E)-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)pentyl)amino)propyl 4-sulfamoyl benzoate 17
.
1
From 37 [37]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 100%. H-NMR
2
2
3
4
(400 MHz, CDCl3)
δ: 8.10 (d, J = 8.4 Hz, 2H, CH); 7.96 (d, J = 8.4 Hz, 2H, CH); 7.58 (d, J = 16.0 Hz,
1
H, CH=CH); 6.73 (s, 2H, CH); 6.34 (d, J = 16.0 Hz, 1H, CH=CH); 4.40 (t, J = 6.4 Hz, 2H, OCH ); 4.15
2
(
t, J = 6.8 Hz, 2H, OCH ); 3.87 (s, 6H, OCH ); 3.86 (s, 3H, OCH ); 2.48 (t, J = 6.8 Hz, 2H, NCH );
2 3 3 2
2
.35 (t, J = 7.2 Hz, 2H, NCH ); 2.22 (s, 3H, NCH ); 1.96–1.90 (m, 2H, CH ); 1.71–1.64 (m, 2H, CH );
2 3 2 2
13
1
(
6
.51–1.40 (m, 4H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.23 (C); 165.14 (C); 153.35 (C); 146.22
C); 144.81 (CH); 139.97 (C); 133.79 (C); 130.14 (CH); 129.93 (C); 126.35 (CH); 117.33 (CH); 105.27 (CH);
4.53 (CH ); 64.06 (CH ); 60.94 (OCH ); 57.50 (CH ); 56.15 (OCH ); 53.75 (CH ); 42.14 (NCH ); 28.66
2
2
2
3
2
3
2
3
+
(
CH ); 26.87 (CH ); 26.39 (CH ); 23.81 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
◦
C H N O S = 579.2371, found 579.2364. Hydrochloride: mp 83–86 C.
28
39
2
9
(
E)-5-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)pentyl)amino)pentyl 4-sulfamoyl benzoate 18
.
1
From 38 [35]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 97:3:0.3. Yield: 19%. H-NMR
2
2
3
4
(400 MHz, CDCl3)
δ: 8.11 (d, J = 8.4 Hz, 2H, CH); 7.96 (d, J = 8.4 Hz, 2H, CH); 7.58 (d, J = 16.0 Hz,
1
H, CH=CH); 6.74 (s, 2H, CH); 6.33 (d, J = 16.0 Hz, 1H, CH=CH); 4.33 (t, J = 6.4 Hz, 2H, OCH ); 4.17
2
(
t, J = 6.8 Hz, 2H, OCH ); 3.87 (s, 6H, OCH ); 3.85 (s, 3H, OCH ); 2.40–2.32 (m, 4H, NCH ); 2.22 (s, 3H,
2 3 3 2
13
NCH ); 1.83–1.66 (m, 4H, CH ); 1.58–1.37 (m, 8H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.15
C); 165.17 (C); 153.41 (C); 146.25 (C); 144.76 (CH); 140.14 (C); 133.94 (C); 130.17 (CH); 129.92 (C); 126.36
CH); 117.36 (CH); 105.36 (CH); 65.79 (CH ); 65.63 (CH ); 64.53 (CH ); 60.92 (OCH ); 57.41 (CH );
3
2
2
(
(
2
2
2
3
2
5
7.38 (CH ); 56.17 (OCH ); 42.05 (NCH ); 28.65 (CH ); 28.53 (CH ); 26.64 (CH ); 23.96 (CH ); 23.90
2 3 3 2 2 2 2
+
(
CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H N O S = 607.2684, found
2 30 43 2 9
◦
07.2672. Hydrochloride: mp 83–85 C.
6
(
E)-3-(methyl(6-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)hexyl)amino)propyl 4-sulfamoyl benzoate 19
.
1
From 39 [38]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 90:10:1. Yield: 40%. H-NMR
2
2
3
4
(
400 MHz, CDCl3) δ: 8.07 (d, J = 8.4 Hz, 2H, CH); 7.94 (d, J = 8.4 Hz, 2H, CH); 7.57 (d, J = 16.0 Hz, 1H,
CH=CH); 6.73 (s, 2H, CH); 6.33 (d, J = 16.0 Hz, 1H, CH=CH); 4.99 (bs, 2H, NH ); 4.35 (t, J = 6.4 Hz, 2H,
2
OCH ); 4.12 (t, J = 6.8 Hz, 2H, OCH ); 3.85 (s, 6H, OCH ); 3.83 (s, 3H, OCH ); 2.49 (t, J = 7.2 Hz, 2H,
2
2
3
3
NCH ); 2.35 (t, J = 7.2 Hz, 2H, NCH ); 2.22 (s, 3H, NCH ); 1.95–1.88 (m, 2H, CH ); 1.69–1.60 (m, 2H,
2
2
3
2
1
3
CH ); 1.49–1.32 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.28 (C); 165.16 (C); 153.35 (C);
2
2
1
46.38 (C); 144. 83 (CH); 139.98 (C); 133.70 (C); 130.15 (CH); 129.91 (C); 126.34 (CH); 117.31 (CH); 105.26
(
CH); 64.63 (CH ); 63.96 (CH ); 60.93 (OCH ); 57.51 (CH ); 56.15 (OCH ); 53.69 (CH ); 42.00 (NCH );
2
2
3
2
3
2
3
2
8.63 (CH ); 27.01 (CH ); 26.93 (CH ); 26.22 (CH ); 25.87 (CH ) ppm. ESI-HRMS (m/z) calculated for
2 2 2 2 2
+
◦
[M + H] ion species C H N O S = 593.2527, found 593.2522. Hydrochloride: mp 73–76 C.
29
41
2
9
(
E)-4-(methyl(6-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)hexyl)amino)butyl 4-sulfamoyl benzoate 20
.
1
From 40 [38]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 49%. H-NMR
2
2
3
4
(
400 MHz, CDCl3) δ: 8.06 (d, J = 8.8 Hz, 2H, CH); 7.92 (d, J = 8.8 Hz, 2H, CH); 7.57 (d, J = 16.0 Hz, 1H,
CH=CH); 6.73 (s, 2H, CH); 6.33 (d, J = 16.0 Hz, 1H, CH=CH); 5.50 (bs, 2H, NH ); 4.32 (t, J = 6.4 Hz, 2H,
2
OCH ); 4.15 (t, J = 6.8 Hz, 2H, OCH ); 3.85 (s, 6H, OCH ); 3.84 (s, 3H, OCH ) 2.40–2.31 (m, 4H, NCH );
2
2
3
3
2
2
.20 (s, 3H, NCH ); 1.79–1.72 (m, 2H, CH ); 1.70–1.55 (m, 4H, CH ); 1.50–1.30 (m, 6H, CH ) ppm.
C-NMR (100 MHz, CDCl3) δ: 167.22 (C); 165.21 (C); 153.35 (C); 146.39 (C); 144.75 (CH); 139.95 (C);
3
2
2
2
1
3
1
33.72 (C); 130.15 (CH); 129.92 (C); 126.31 (CH); 117.36 (CH); 105.21 (CH); 65.53 (CH ); 64.61 (CH );
2 2

Molecules 2020, 25, 1748
18 of 26
6
0.94 (OCH ); 57.45 (CH ); 57.03 (CH ); 56.14 (OCH ); 42.05 (NCH ); 28.66 (CH ); 27.14 (CH ); 26.84
3 2 2 3 3 2 2
+
(
CH ); 26.60 (CH ); 25.86 (CH ); 23.62 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
◦
C H N O S = 607.2684, found 607.2683. Hydrochloride: mp 89–91 C.
30
43
2
9
(
E)-2-(methyl(7-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)heptyl)amino)ethyl 4-sulfamoyl benzoate 21
.
1
From 44 [38]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 45%. H-NMR
2
2
3
4
(400 MHz, CDCl3)
δ: 8.11 (d, J = 8.8 Hz, 2H, CH); 7.95 (d, J = 8.8 Hz, 2H, CH); 7.59 (d, J = 15.6 Hz,
1
H, CH=CH); 6.75 (s, 2H, CH); 6.34 (d, J = 15.6 Hz, 1H, CH=CH); 4.43 (t, J = 5.6 Hz, 2H, OCH ); 4.14
2
(
(
(
(
t, J = 6.4 Hz, 2H, OCH ); 3.87 (s, 6H, OCH ); 3.86 (s, 3H, OCH ); 2.76 (t, J = 5.6 Hz, 2H, NCH ); 2.43
2 3 3 2
t, J = 7.2 Hz, 2H, NCH ); 2.31 (s, 3H, NCH ); 1.66–1.62 (m, 2H, CH ); 1.49–1.45 (m, 2H, CH ); 1.33–1.25
2
3
2
2
13
m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.37 (C); 165.14 (C); 153.40 (C); 146.19 (C); 144.92
CH); 140.07 (C); 133.83 (C); 130.29 (CH); 129.90 (C); 126.36 (CH); 117.29 (CH); 105.29 (CH); 64.73 (CH2);
3.46 (CH ); 60.96 (OCH ); 57.80 (CH ); 56.17 (OCH ); 55.47 (CH ); 42.73 (NCH ); 29.13 (CH ); 28.66
2
6
2
3
2
3
2
3
2
+
(
CH ); 27.20 (CH ); 27.17 (CH ); 25.92 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
◦
C H N O S = 593.2527, found 593.2524. Hydrochloride: mp 70–73 C.
29
41
2
9
(
E)-7-(methyl(7-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)heptyl)amino)heptyl 4-sulfamoyl benzoate 22
.
1
From 41 [35]. Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 22%. H-NMR
2
2
3
4
(400 MHz, CDCl3)
δ: 8.15 (d, J = 8.4 Hz, 2H, CH); 8.00 (d, J = 8.4 Hz, 2H, CH); 7.61 (d, J = 15.6 Hz,
1
H, CH=CH); 6.77 (s, 2H, CH); 6.37 (d, J = 15.6 Hz, 1H, CH=CH); 4.36 (t, J = 6.8 Hz, 2H, OCH ); 4.20
2
(
t, J = 6.4 Hz, 2H, OCH ); 3.90 (s, 6H, OCH ); 3.89 (s, 3H, OCH ); 2.40–2.35 (m, 4H, NCH ); 2.26 (s, 3H,
2 3 3 2
13
NCH ); 1.82–1.65 (m, 4H, CH ); 1.52–1.30 (m, 16H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 167.13
C); 165.18 (C); 153.42 (C); 146.07 (C); 144.64 (CH); 139.96 (C); 134.09 (C); 130.22 (CH); 129.96 (C); 126.42
CH); 117.46 (CH); 105.29 (CH); 65.78 (CH ); 64.66 (CH ); 60.95 (OCH ); 57.57 (CH ); 56.17 (OCH );
3
2
2
(
(
2
2
3
2
3
4
2
6
2.02 (NCH ); 29.14 (CH ); 28.67 (CH ); 28.53 (CH ); 27.37 (CH ); 27.36 (CH ); 26.77 (CH ); 25.95 (CH );
3 2 2 2 2 2 2 2
+
5.89 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H N O S = 663.3310, found
2
34 51
2
9
◦
63.3298. Hydrochloride: mp 68–70 C.
3
-(methyl(5-((4-sulfamoylbenzoyl)oxy)pentyl)amino)propyl 3,4,5-trimethoxybenzoate 23. From 42 [37]
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 22%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 8.11 (d, J = 8.8 Hz, 2H, CH); 7.97 (d, J = 8.8 Hz, 2H, CH); 7.28 (s, 2H, CH); 4.37–4.32
(
(
(
m, 4H, CH ); 3.90 (s, 9H, OCH ); 2.62 (t, J = 6.8 Hz, 2H, NCH ); 2.51 (t, J = 6.8 Hz, 2H, NCH ); 2.35
2
3
2
2
s, 3H, NCH ); 2.05–1.98 (m, 2H, CH ); 1.83–1.76 (m, 2H, CH ); 1.65–1.58 (m, 2H, CH ); 1.51–1.43
3
2
2
2
13
m, 2H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 166.25 (C); 165.15 (C); 152.91 (C); 146.07 (C);
2
1
5
33.93 (C); 130.21 (CH); 126.41 (CH); 125.20 (C); 106.87 (CH); 65.56 (CH ); 63.28 (CH ); 60.92 (OCH );
2
2
3
7.29 (CH ); 56.28 (OCH ); 54.00 (CH ); 41.74 (NCH ); 28.42 (CH ); 26.33 (CH ); 26.16 (CH ); 23.82
2
3
2
3
2
2
2
+
(
CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species C H N O S = 553.2214, found
2 26 37 2 9
◦
53.2218. Hydrochloride: mp 52–55 C.
5
6
-(methyl(3-((4-sulfamoylbenzoyl)oxy)propyl)amino)hexyl 3,4,5-trimethoxybenzoate 24
.
From 33.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 55%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 8.01 (d, J = 8.4 Hz, 2H, CH); 7.88 (d, J = 8.4 Hz, 2H, CH); 7.21 (s, 2H, CH); 5.56 (bs, 2H, NH2);
4
2
.31 (t, J = 6.0 Hz, 2H, OCH ); 4.19 (t, J = 6.8 Hz, 2H, OCH ); 3.82 (s, 6H, OCH ); 3.81 (s, 3H, OCH );
2
2
3
3
.45 (t, J = 7.2 Hz, 2H, NCH ); 2.31 (t, J = 7.2 Hz, 2H, NCH ); 2.18 (s, 3H, NCH ); 1.90–1.85 (m, 2H,
2
2
3
3
1
CH ); 1.69–1.65 (m, 2H, CH ); 1.42–1.28 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ: 166.39 (C);
2
2
2
165.13 (C); 152.86 (C); 146.37 (C); 142.11 (C); 133.70 (C); 130.13 (CH); 126.33 (CH); 125.41 (C); 106.85
(
CH); 65.20 (CH ); 63.98 (CH ); 60.87 (OCH ); 57.50 (CH ); 56.25 (OCH ); 53.77 (CH ); 41.94 (NCH );
2
2
3
2
3
2
3
2
8.64 (CH ); 27.03 (CH ); 26.89 (CH ); 26.23 (CH ); 25.90 (CH ) ppm. ESI-HRMS (m/z) calculated for
2 2 2 2 2
+
◦
[M + H] ion species C H N O S = 567.2371, found 567.2380. Hydrochloride: mp 53–56 C.
27
39
2
9
7
-(methyl(2-((4-sulfamoylbenzoyl)oxy)ethyl)amino)heptyl 3,4,5-trimethoxybenzoate 25
.
From 45 [38].
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 40%. H-NMR (400 MHz,
2
2
3
4
CDCl3) δ: 8.08 (d, J = 8.4 Hz, 2H, CH); 7.93 (d, J = 8.4 Hz, 2H, CH); 7.27 (s, 2H, CH); 4.43 (t, J = 5.6 Hz,

Molecules 2020, 25, 1748
19 of 26
2
H, OCH ); 4.26 (t, J = 6.8 Hz, 2H, OCH ); 3.89 (s, 6H, OCH ); 3.87 (s, 3H, OCH ); 2.78 (t, J = 5.6 Hz, 2H,
2 2 3 3
NCH ); 2.44 (t, J = 7.2 Hz, 2H, NCH ); 2.32 (s, 3H, NCH ); 1.74–1.71 (m, 2H, CH ); 1.50–1.46 (m, 2H,
2
2
3
2
13
CH ); 1.40–1.30 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 166.48 (C); 165.14 (C); 152.88
2
2
(
6
C); 146.32 (C); 142.14 (C); 133.65 (C); 130.24 (CH); 126.31 (CH); 125.40 (C); 106.86 (CH); 65.29 (CH2);
3.35 (CH ); 60.88 (OCH ); 57.81 (CH ); 56.26 (OCH ); 55.40 (CH ); 42.63 (NCH ); 29.12 (CH ); 28.63
2
3
2
3
2
3
2
+
(
CH ); 27.19 (CH ); 27.02 (CH ); 25.93 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion species
2 2 2 2
◦
C H N O S = 567.2371, found 567.2368. Hydrochloride: mp 60–63 C.
27
39
2
9
3
-(methyl(5-((4-sulfamoylbenzoyl)oxy)pentyl)amino)propyl anthracene-9-carboxylate 26. From 43 [37]
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 41%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 8.52 (s, 1H, CH); 8.10 (d, J = 8.4 Hz, 2H, CH); 8.02 (d, J = 8.4 Hz, 4H, CH); 7.93 (d, J = 8.4 Hz,
2
H, CH); 7.55–7.47 (m, 4H, CH); 4.66 (t, J = 6.4 Hz, 2H, OCH ); 4.32 (t, J = 6.4 Hz, 2H, OCH ); 2.60
2
2
(
1
t, J = 7.2 Hz, 2H, NCH ); 2.44 (t, J = 7.2 Hz, 2H, NCH ); 2.29 (s, 3H, NCH ); 2.12–2.05 (m, 2H, CH );
2
2
3
2
13
.79–1.72 (m, 2H, CH ); 1.60–1.40 (m, 4H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 169.68 (C); 165.17
2
2
(C); 146.00 (C); 133.88 (C); 130.94 (C); 130.20 (CH); 129.39 (CH); 128.67 (CH); 128.35 (C); 127.79 (C);
1
4
27.05 (CH); 126.37 (CH); 125.52 (CH); 124.89 (CH); 65.56 (CH ); 63.95 (CH ); 57.32 (CH ); 53.98 (CH );
2 2 2 2
+
1.73 (NCH ); 28.42 (CH ); 26.06 (CH ); 23.82 (CH ) ppm. ESI-HRMS (m/z) calculated for [M + H] ion
3
2
2
2
◦
species C H N O S = 563.2210, found 563.2211. Hydrochloride: mp 82–84 C.
31
35
2
6
6
-(methyl(3-((4-sulfamoylbenzoyl)oxy)propyl)amino)hexyl anthracene-9-carboxylate 27
.
From 34.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 29%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 8.51 (s, 1H, CH); 8.08 (d, J = 8.4 Hz, 2H, CH); 8.01 (d, J = 8.4 Hz, 4H, CH); 7.93 (d, J = 8.4 Hz,
2
2
H, CH); 7.55–7.46 (m, 4H, CH); 5.08 (bs, 2H, NH ); 4.59 (t, J = 6.8 Hz, 2H, OCH ); 4.37 (t, J = 6.4 Hz,
2
2
H, OCH ); 2.51 (t, J = 7.2 Hz, 2H, NCH ); 2.38 (t, J = 7.2 Hz, 2H, NCH ); 2.24 (s, 3H, NCH ); 1.97–1.82
2
2
2
3
13
(m, 4H, CH ); 1.51–1.35 (m, 6H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 169.85 (C); 165.09 (C);
2
2
146.22 (C); 142.61 (C); 141.80 (C); 133.63 (C); 130.96 (C); 130.18 (CH); 129.28 (CH); 128.64 (CH); 128.32
(
(
C); 128.01 (C); 127.00 (CH); 126.37 (CH); 125.50 (CH); 124.91 (CH); 65.85 (CH ); 63.71 (CH ); 57.23
2 2
CH ); 53.71 (CH ); 41.60 (NCH ); 28.62 (CH ); 26.86 (CH ); 26.39 (CH ); 25.92 (CH ); 25.84 (CH ) ppm.
2
2
3
2
2
2
2
2
+
ESI-HRMS (m/z) calculated for [M + H] ion species C H N O S = 577.2367, found 577.2361.
Hydrochloride: mp 81–84 C.
32
37
2
6
◦
7
-(methyl(2-((4-sulfamoylbenzoyl)oxy)ethyl)amino)heptyl anthracene-9-carboxylate 28. From 46 [38]
.
1
Oil. Chromatographic eluent: CH Cl /CH OH/NH OH 93:7:0.3. Yield: 33%. H-NMR (400 MHz,
2
2
3
4
CDCl3)
δ: 8.52 (s, 1H, CH); 8.11 (d, J = 8.0 Hz, 2H, CH); 8.01 (d, J = 8.0 Hz, 4H, CH); 7.93 (d, J = 8.0 Hz,
2
2
H, CH); 7.56–7.47 (m, 4H, CH); 5.31 (bs, 2H, NH ); 4.60 (t, J = 6.8 Hz, 2H, OCH ); 4.46 (t, J = 5.6 Hz,
2
2
H, OCH ); 2.80 (t, J = 5.6 Hz, 2H, NCH ); 2.47 (t, J = 7.2 Hz, 2H, NCH ); 2.35 (s, 3H, NCH ); 1.88–1.82
2
2
2
3
13
(
m, 2H, CH ); 1.54–1.30 (m, 8H, CH ) ppm. C-NMR (100 MHz, CDCl3)
δ
: 169.93 (C); 165.08 (C); 146.27
2
2
(
C); 133.47 (C); 130.96 (C); 130.28 (CH); 129.28 (CH); 128.64 (CH); 128.32 (C); 128.01 (C); 127.00 (CH);
1
26.31 (CH); 125.50 (CH); 124.92 (CH); 65.97 (CH ); 62.86 (CH ); 57.65 (CH ); 55.18 (CH ); 42.34 (NCH );
2
2
2
2
3
2
[
9.03 (CH ); 28.64 (CH ); 27.10 (CH ); 26.53 (CH ); 26.00 (CH ) ppm. ESI-HRMS (m/z) calculated for
2
2
2
2
2
+
◦
M + H] ion species C H N O S = 577.2367, found 577.2374. Hydrochloride: mp 71–73 C.
32 37 2 6
3
3
.2. Stability Tests
.2.1. Chemicals
Acetonitrile (Chromasolv), formic acid and ammonium formate (MS grade), NaCl, KCl, Na HPO
2
4
2
H O, KH PO (Reagent grade) and verapamil hydrochloride (analytical standard, used as an internal
2 2 4
−1
standard) were purchased by Sigma-Aldrich (Milan, Italy). MilliQ water 18 M
from Millipore’s Simplicity system (Milan-Italy).
Ω
cm was obtained
−
1
−1
Phosphate buffer solution (PBS) was prepared by adding 8.01 g L of NaCl, 0.2 g L of KCl, 1.78
g L 1 of Na HPO 2H O and 0.27 g L of KH PO .
−
−1
2
4
2
2
4

Molecules 2020, 25, 1748
20 of 26
3
.2.2. Preparation of Samples
Each sample was prepared adding 10
µL of working solution 1 to 100
µL of the tested matrix (PBS)
in micro centrifuge tubes. The obtained solutions correspond to 1 µM of analyte.
Each set of samples was incubated in triplicate at four different times, 0, 30, 60 and 120 min at
7 C. Therefore, the degradation profile of each analyte was represented by a batch of 12 samples
◦
3
(
4 incubation times
×
3 replicates). After the incubation, the samples were added with 300 µL of
ISTD solution and centrifuged (room temperature for 5 min at 10,000 rpm). The supernatants were
transferred in auto sampler vials and dried under a gentle stream of nitrogen.
The dried samples were dissolved in 1.0 mL of 10 mM of formic acid in mQ water: acetonitrile
80:20 solution. The obtained sample solutions were analyzed by LC–MS/MS methods described in the
Supplementary Material.
3
.3. Biology and Enzymology
3
.3.1. Cell lines and Cultures
The K562 cell line is a highly undifferentiated erythroleukemia originally derived from a patient
with chronic myelogenous leukemia [41]. The K562 leukemia cells and the P-gp expressing K562/DOX
cells were obtained from Prof. J. P. Marie (Hopital Hotel-Dieu, Paris, France). These cells were cultured
in RPMI 1640 medium with GlutaMAX I (GIBCO) medium supplemented with 10% fetal calf serum
◦
(
FCS; GIBCO) at 37 C in a humidified incubator with 5% CO . To maintain the resistance, every month,
2
resistant cells were cultured for three days with 400 nM doxorubicin. K562/DOX cells overexpress
almost exclusively the membrane glycoprotein P-gp [42].
Human colon adenocarcinoma cell line (LoVo) was isolated from a metastatic nodule, and its MDR
variant, LoVo/DOX, were obtained from LoVo (parental line) by exposure to increasing concentrations
of doxorubicin and maintained in vitro in Ham’s F12 medium supplemented with 10% fetal bovine
◦
serum and vitamins (Life Technology, Monza, Italy) at 37 C in a 5% CO atmosphere [47]. These
2
two cell lines have been kindly offered by Professor D’Incalci of the Department of Oncology, IRCCS
(Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy).
3
.3.2. Rhodamine-123 (Rhd 123) Uptake
The inhibition of P-gp activity was evaluated by measuring the uptake of the P-gp substrate
rhodamine-123 (Rhd 123) in K562/DOX and LoVo/DOX cells, in the absence or in the presence of
compounds by a flow cytometric test. Briefly, K562/DOX cells were sedimented and diluted to obtain
5
a cell suspension at 5
×
10 cells/mL in complete RPMI 1640 medium; LoVo/DOX cells suspension
was obtained by incubating monolayer cell cultures with EDTA and trypsin, then cells were diluted
to obtain a cell suspension at 5
5
×
10 cells/mL in complete Ham’s F12. Cells were loaded with Rhd
◦
123, 5.0
µ
M, for 30 min at 37 C in a humidified atmosphere with 5% CO in the presence of the tested
2
compounds at 3.0, 10 and 30 µM concentrations, or of the reference compound verapamil at 3.0 µM
concentration. An aliquot of cells (control) was incubated with the fluorochrome in the absence of
inhibitors. All compounds were added 15 min before Rhd 123.
At the end of the uptake, cells were sedimented, washed twice in ice-cold PBS, placed in PBS on
ice, and kept in the dark until flow cytometric analysis. Samples were analyzed on a FACScantoflow
cytometer (Becton Dickinson, San Jose CA, USA) equipped with two lasers at 488/633 nm and
FACSDIVA software. The green fluorescence of Rhd 123 was collected by a 530-nm band pass filter
and at least 10,000 events were acquired.
The histograms were generated by program GraphPad Prism 5 (GraphPad Prism software, Inc.,
San Diego, CA, USA); the data were obtained from the ratio between the average fluorescence intensity
of the samples preincubated with the compounds under study and subsequently exposed to rhodamine,
and the fluorescence of the sample exposed only to the rhodamine.

Molecules 2020, 25, 1748
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3
.3.3. Intrinsic Cytotoxicity
The intrinsic toxicity of the compounds under study was determined by an MTT assay [43] after
exposing the parental cells (K562 and LoVo) and resistant cells (K562/DOX and LoVo/DOX) to three
concentrations (3, 10 and 30 M) for 72 h. The percentage of growth compared to the untreated control
µ
was transformed into histograms with the GraphPad Prism 5 program (Graph Pad software, Adalta,
Arezzo, Italy).
3
.3.4. CA Inhibition Assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used to assay
the catalytic/inhibition of various CA isozymes [44]. Phenol Red (at a concentration of 0.2 mM) was
used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as
a buffer and, 0.1 M Na SO or NaClO (for maintaining constant the ionic strength; these anions are not
2
4
4
inhibitory in the used concentration), following the CA-catalyzed CO hydration reaction for a period of
2
◦
5
–10 s. Saturated CO solutions in water at 25 C were used as a substrate. Stock solutions of inhibitors
2
were prepared at a concentration of 10 µM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done
with the assay buffer mentioned above. At least 7 different inhibitor concentrations have been used for
measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10
min at room temperature prior to the assay, in order to allow for the formation of the E–I complex.
Triplicate experiments were done for each inhibitor concentration, and the values reported throughout
the paper were the mean of such results. The inhibition constants were obtained by non-linear
least-squares methods using the Cheng–Prusoff equation, as reported earlier [29], and represent the
mean from at least three different determinations. All CA isozymes used here were recombinant
proteins obtained as reported earlier by our group [48,49].
3
.3.5. Reverse Transcription RT-PCR
The expression levels of MDR1 and CAXII were analyzed by quantitative PCR (qRT-PCR)
using a RotorGene 3000 (Qiagen, Hilden, Germany) instrument. Primers were purchased from IDT
Germany). Of the total RNA 500 ng was retro-transcribed using iScript (Bio-Rad, Hercules, CA, USA)
(
and amplified with specific primers: for MDR1, Fw CAGCTATTCGAAGAGTGGGCACAAAC and Rv
GCCTCTGCATCAGCTGGACTGTTG. For CAXII, Fw CAAAGGCCAGGAAGCATTCGTCCC and
Rv ATGTGTGTGCAGTACAGGGCTGTCTC. PCR amplification was carried out by SsoAdvancedTM
®
Universal SYBR Green Supermix (Bio-Rad, USA) according to the manual’s instruction. In the
present analysis 18s rRNA was confirmed to be stable and was used as the normalizer Fw
CGGCTACCACATCCAAGGAA and Rv GTTATTTTTCGTCACTACCTCCCCGGG. The RT-qPCR was
◦
◦
◦
performed using the following procedure: 98 C for 2 min, 40 cycles of 98 C for 5 s and 60 C for 10 s.
◦
The program was set to reveal the melting curve of each amplicon from 60 to 95 C with a read every
◦
0
.5 C.
3
.3.6. Flow cytometric P-Glycoprotein Expression
6
Flow cytometric analysis was carried out on leukemic cells and on cell suspensions (10 cells/mL)
of colon carcinoma cells obtained by incubating monolayer cell cultures with EDTA and trypsin.
For the P-gp determination on the cell surface, the cells were incubated for 30 min at RT with MAb
FITC conjugate CD243. After washing with ice-cold phosphate buffer saline (PBS, pH 7.2) with 1%
bovine serum albumin (BSA, Sigma), cells were immediately analyzed. Dead cells were excluded
from the analysis by gate on cells size before the acquisitions. Ten thousand events were acquired
and fluorescence was analyzed with a FACScanto flow cytometer (Becton Dickinson) equipped with
a 15 mW, 488 nm, air-cooled argon ion laser. The fluorescence emissions were collected through
a 530 nm band-pass filter for fluorescein.

Molecules 2020, 25, 1748
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For quantitative evaluation of P-gp expression, the mean fluorescence channel (MFC) was
calculated by FCS5 express software (De Novo software). P-gp content in arbitrary units was expressed
as the ratio of the MFC of labeled resistant cells on the MFC of parental ones.
3
.3.7. Doxorubicin IC50 and IC20 Evaluation on K562/DOX and LoVo/DOX Cell Lines
In regards to the leukemic line, K562/DOX, the values of doxorubicin IC50 and IC20 were
extrapolated from the cytotoxicity curves previously performed [38].
For the LoVo/DOX cell line, the doxorubicin growth inhibition was determined by the MTT
4
assay and the IC50 and IC20 values were determined. Approximately 10 cells/well were plated onto
9
6-well plates and allowed to attach for 24 h before treatment with a range of drug concentrations
−
11
−5
from 3 × 10
to 3 × 10
M
for 3 days; each concentration was repeated four times. At the end of the
incubation the cells were processed according to the MTT protocol [43] and the optical density of the
chromogenic product was measured at 570 nm. IC50 and IC20 values were determined graphically from
relative survival curves obtained by GraphPad Prism 5 software (GraphPad, San Diego, CA, USA).
The resistance index to doxorubicin of the two resistant cell lines was obtained from the ratio
between the doxorubicin IC50
(
µ
M) values on the resistant and parental lines. The resistance index was
0.25 M and 0.034 0.008 M on K562/DOX and K562 cells,
M and 0.063 0.0066 M on LoVo/DOX
73 for K562/DOX cells [38] (IC50 = 2.47
±
µ
±
µ
respectively) and 13.5 for LoVo/DOX cells (IC50 = 0.85
and LoVo cells, respectively).
±
0.084
µ
±
µ
3
.3.8. Enhancement of Doxorubicin Toxicity in the Presence of Compounds 2, 5, 7, 8, 21, 25 and 26.
Doxorubicin was used at the concentration corresponding to the IC20 of the resistant lines, which
were 0.5
µM and 0.3 µM for K562/DOX and LoVo/DOX cell lines, respectively. Compounds 2, 5, 7, 8,
2
1, 25 and 26 were studied at two concentrations, 3 and 10 µM.
To evaluate the enhancement of doxorubicin toxicity in the presence of the tested compounds,
4
the cells, in exponential growth phase, were seeded at 10 cells/well and solutions of either compounds
or doxorubicin, or a solution of doxorubicin in combination with the compounds, were added to the
◦
wells repeated in quadruplicate. Then the plates were incubated at 37 C for 72 h in a humidified
atmosphere with 5% CO /95% air. The MTT working solution was then added and plates were further
2
incubated for 3 h. Following incubation cells and formazan crystals were inspected microscopically.
The supernatant was then carefully removed by slow aspiration and the formazan crystals were
dissolved in 150 mL of acidified isopropanol solution. The absorbance of the solution was then read on
an automated plate reader at a wavelength of 570 nm. The increase in the toxicity of doxorubicin was
quantified by the ratio between the growth of the cell sample treated with IC20 of doxorubicin and that
of the cell sample treated with IC20 of doxorubicin in combination with the various concentrations of
the tested compounds.
4
. Conclusions
In this work a new series of P-glycoprotein and CA XII inhibitor hybrids was synthesized and
studied for their ability to reverse the P-gp mediated MDR in cancer cells, which overexpressed both
the transmembrane P-gp and CA XII proteins. A dual P-gp and CA XII inhibitory activity could be
useful for a synergistic antitumor mechanism with consequent selective inhibition of P-glycoprotein
overexpressed in resistant cancer cells.
The synthesized compounds are characterized by the presence of both P-gp and CA XII binding
moieties, such as a N,N-bis(alkanol)amine diester scaffold combined with a coumarin or benzene
sulfonamide moieties endowed with a dual P-gp and CA XII inhibitory activity.
Many compounds showed multitarget activity against the two transmembrane proteins P-gp
and CA XII. Derivatives bearing a coumarin residue, displayed high and selective hCA XII inhibitory
activity and a moderate P-gp inhibitory activity on K562/DOX cells, which overexpressed only P-gp.
Interestingly, some coumarin compounds showed a synergistic MDR reversal effect; indeed, they were

Molecules 2020, 25, 1748
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more active in the P-gp inhibition on LoVo/DOX cells, which overexpressed both P-gp and CA XII, than
on K562/DOX cells, which overexpressed only P-gp, in both rhodamine-123 uptake and doxorubicin
cytotoxicity enhancement tests.
In particular, the coumarin derivatives
moiety linked to a 7-methylenes spacer (n = 7, Ar =
more potent on LoVo/DOX than on K562/DOX cells both in the Rhd 123 uptake test and in the doxorubicin
cytotoxicity enhancement assay. Moreover, when tested at 10 M concentrations, these two compounds
7
and
8, which incorporate a (E)-3-(3,4,5-trimethoxyphenyl)vinyl
a, see Table 1) showed the best inhibitory profile, being
µ
showed higher activity than verapamil in both tests on LoVo/DOX line. In fact, they were able to increase the
Rhd 123 intracellular fluorescence intensity with FR = 12.60 and 9.90, respectively, vs. FR = 6.40 of verapamil,
and to reduce the cell growth rate from 80% to 34.9% and 32.2%, respectively, vs. 51.0% of verapamil. These
two compounds were very potent and selective for hCA XII with respect to the physiological relevant
cytosolic isoenzymes hCA I and hCA II (hCA XII, Ki = 9.1 and 7.1 nM for 7 and 8, respectively, and hCA I
and hCA II, Ki = >10,000 nM).
Taking all these results into account, we discovered a new class of hybrid derivatives with
an innovative synergistic inhibitory activity mechanism on P-glycoprotein and carbonic anhydrases.
More work is needed to optimize the interaction of these molecules with both targets and to study their
pharmacokinetic properties. Notably, compounds 7 and 8 can be considered valuable candidates as
chemosensitizers able to overcome P-gp mediated MDR in cancer cells with a synergistic mechanism
that make them potentially selective inhibitors of P-glycoprotein overexpressed in resistant cancer cells.
Supplementary Materials: The following are available online. Figure S1: mRNA expression level of MDR1 and
CA XII genes, Figure S2: Fluorescence curves obtained with a FACScanto flow cytometer, Table S1: Elution
gradient of mobile phase used for LC–MS/MS analyses, Table S2: MRM parameters, Table S3: Linear regression
data, linearity coefficients, precision and LOD values for each analyte.
Author Contributions: Conceptualization, E.T., M.N.R., S.D. and C.T.S., Funding acquisition, E.T., M.N.R. and
S.D., Investigation, L.B., S.B., A.L., G.B., D.M. and M.C., Methodology, L.B., Project administration, E.T., L.B. and
S.D.; Supervision, E.T., S.D. and C.T.S., Validation, L.B., S.B., A.L. and G.B., Writing—original draft, E.T., L.B., G.B,
S.D. and M.C., Writing—review and editing, E.T., D.M., M.N.R., S.D. and C.T.S. All authors have read and agreed
to the published version of the manuscript.
Funding: This project was supported grants from the University of Florence (Fondo Ricerca Ateneo RICATEN17
and RICATEN18).
Conflicts of Interest: The authors declare no conflict of interest.
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