Design and synthesis of aminoester heterodimers containing flavone or chromone moieties as modulators of P-glycoprotein-based multidrug resistance (MDR)

Article abstract of DOI:10.1016/j.bmc.2017.11.016

In this study, a new series of heterodimers was synthesized. These derivatives are N,N-bis(alkanol)amine aryl esters or N,N-bis(ethoxyethanol)amine aryl esters carrying a methoxylated aryl residue combined with a flavone or chromone moiety. The new compounds were studied to evaluate their P-gp modulating activity on a multidrug-resistant leukemia cell line. Some of the new compounds show a good MDR reversing activity; interestingly this new series of compounds does not comply with the structure-activity relationships (SAR) outlined by previously synthesized analogs carrying different aromatic moieties. In the case of the compounds described in this paper, activity is linked to different features, in particular the characteristics of the spacer, which seem to be critical for the interaction with the pump. This fact indicates that the presence of a flavone or chromone residue influences the SAR of these series of products, and that flexible molecules can find different productive binding modes with the P-gp recognition site. These results support the synthesis of new compounds that might be useful leads for the development of drugs to control P-gp-dependent MDR.

Full text of DOI:10.1016/j.bmc.2017.11.016

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of aminoester heterodimers containing flavone
or chromone moieties as modulators of P-glycoprotein-based
multidrug resistance (MDR)
a
a
a
c
Silvia Dei a, , Maria Novella Romanelli , Dina Manetti , Niccolò Chiaramonte , Marcella Coronnello ,
⇑
Milena Salerno b, Elisabetta Teodori a
a 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
b Université Paris 13, Sorbonne Paris Cité, Laboratoire CSPBAT, CNRS (UMR 7244), UFR-SMBH, 74 rue Marcel Cachin, 93017 Bobigny, France
c Department of Health Sciences – Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a new series of heterodimers was synthesized. These derivatives are N,N-bis(alkanol)amine
aryl esters or N,N-bis(ethoxyethanol)amine aryl esters carrying a methoxylated aryl residue combined
with a flavone or chromone moiety. The new compounds were studied to evaluate their P-gp modulating
activity on a multidrug-resistant leukemia cell line. Some of the new compounds show a good MDR
reversing activity; interestingly this new series of compounds does not comply with the structure-activ-
ity relationships (SAR) outlined by previously synthesized analogs carrying different aromatic moieties.
In the case of the compounds described in this paper, activity is linked to different features, in particular
the characteristics of the spacer, which seem to be critical for the interaction with the pump. This fact
indicates that the presence of a flavone or chromone residue influences the SAR of these series of prod-
ucts, and that flexible molecules can find different productive binding modes with the P-gp recognition
site. These results support the synthesis of new compounds that might be useful leads for the develop-
ment of drugs to control P-gp-dependent MDR.
Received 25 July 2017
Revised 6 November 2017
Accepted 9 November 2017
Available online xxxx
Keywords:
P-gp modulators
MDR reversers
Heterodimers
Flavonoids
Doxorubicin-resistant human
erythroleukemia K562 cells (K562/DOX)
Pirarubicin uptake
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
the secretion of physiological lipophilic molecules and the extru-
sion of xenobiotics,5 but it is also the first efflux transporter discov-
Multidrug resistance (MDR) is the major cause of the failure in
antineoplastic treatment, and it is mainly due to the overexpres-
sion of some transmembrane proteins belonging to the ABC (ATP
Binding Cassette) transporters family.1 This family includes struc-
turally related membrane proteins that are made up of two
domains: the nucleotide binding domain (NBD) and the transmem-
brane spanning domain (TMD).2 ATP is hydrolysed in the NBD and
the resulting energy is used by the TMD to translocate substances
through the membrane by conformational changes.3
ered to be involved in drug resistance.6 In fact, P-gp is
overexpressed in many cancer cells as a result of chemotherapy
treatment causing an acquired resistance to a variety of anticancer
drugs.7,8 Moreover, in addition to the decrease in the intracellular
concentration of chemotherapeutic agents, P-gp might affect resis-
tance also through other mechanism: P-gp overexpressing cells
resulted less sensitive to caspase-dependent apoptosis induced
6
by a range of different stimuli.
Inhibition of the functions of P-gp is considered a potential
strategy for circumventing MDR. The use of a P-gp modulator in
co-administration with the classic antineoplastic drugs could
restore drug sensitivity of tumor cells in cancer therapy9–11; many
compounds have been identified for this purpose, nevertheless few
of them have reached clinical trials.12 The main problem associated
with the development of effective P-gp-mediated MDR inhibitors
seems due to their influence on the physiological role of P-gp, poor
specificity, low affinity for the binding site, and interference with
the pharmacokinetics of the associated chemotherapeutic agent.13
P-gp is the most studied ABC transporter.4 It is physiologically
present in cells of several human tissues where it plays an impor-
tant role by regulating the permeability of biological membranes,
Abbreviations: P-gp, P-glycoprotein; SAR, Structure-activity relationships; DOX,
Doxorubicin; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimmide hydrochlo-
ride; HOBt, 1-Hydroxybenzotriazole hydrate; DMAP, 4-dimethylaminopyridine.
⇑
Corresponding author.
E-mail address: silvia.dei@unifi.it (S. Dei).
https://doi.org/10.1016/j.bmc.2017.11.016
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Despite some doubts related to the future of P-gp inhibitors as a
different molecules can be accommodated in a plurality of binding
therapeutic approach to overcome MDR, the search for new
P-gp-interacting compounds is still of interest, not only in field of
cancer resistance.14 In fact, other potential uses of these agents
are emerging, such as that of enhancing drug penetration through
biologically protective barriers, such as the blood-brain and blood-
cerebrospinal fluid.15,16 As an example, the overexpression of P-gp
has been related to the occurrence of MDR in CNS diseases, such as
pharmacoresistant schizophrenia, thus reducing the effectiveness
of current therapeutic drugs.17 Moreover, the important role of
ABC transporter proteins like P-gp in stem cells has been evi-
denced.18 As a consequence, the increasing interest in the functions
and mechanism of action of P-gp indicates the need for new potent
and selective molecules to be used as pharmacological tools.
Recently, flavonoids were indicated as a promising family of
P-gp modulators. Flavonoids are constituents of fruits and vegeta-
bles and constitute the main group of polyphenolic compounds
present in human diet. These natural compounds have long been
associated with a variety of biochemical and pharmacological
properties, including antioxidative, anticarcinogenic, anti-inflam-
matory and antiviral activities.19 Their daily consumption is esti-
mated to be as high as 1 g, and it is therefore generally accepted
that flavonoids are not toxic. In the last 20 years, flavonoids have
attracted attention as putative MDR modulating agents, and their
interaction with P-gp was described by different researchers.19–22
Also some analogs, as functionalized chromones were described
as highly active compounds as MDR modulators.23
In the last decade, some synthetic flavonoid homodimers and
heterodimers as a new class of potent, safe and specific P-gp mod-
ulators were reported. Synthetic flavonoid dimers with polyethy-
lene glycol (PEG) as linker displayed, in drug-resistant human
breast cancer and leukemia cells, a higher P-gp modulating activ-
ity, compared to monomers.24,25 Introduction of an amine group
into the PEG linker improved both the aqueous solubility as well
as the modulating activity (Chart 1, structure I).26 SAR evaluation
indicated that flavonoid dimers with non-polar and hydrophobic
substituents generally showed a higher activity than that of dimers
decorated with polar and hydrophilic residues. Best results were
obtained when a benzyl group was introduced on the nitrogen.
In recent years we have studied several families of multidrug
resistance modulators including N,N-bis(alkanol)amine aryl esters
characterized by the presence of a basic nitrogen atom linked to
two different aromatic ester residues by two identical poly-
methylenic chains of variable length as spacers (Chart 1, structure
II).27,28 These compounds were designed on the bases of the infor-
mation that the presence of aromatic moieties and of one or more
protonable nitrogen atoms is an important property for the P-gp
interaction. In fact, although the atomic structure of human P-gp
has not yet been elucidated, current knowledge of the interaction
site suggest that it is a large, flexible drug binding domain where
modes, establishing
p-p, ion-p, hydrogen bonds and hydrophobic
interactions.29
In our design strategy, a high structural flexibility would allow
the molecules to choose the most productive binding mode, within
the large P-gp recognition site. Actually, this approach provided
good results since most of the synthesized compounds showed to
be very potent MDR reversers in a human leukemia cell line.27,28
Interestingly, a similar approach, labeled as ‘‘polyvalency”, had
been used successfully by Sauna and co-workers, who synthesized
several homodimers of the natural MDR inhibitor stipiamide.30
In this study we designed and synthesized a new series of
derivatives based on the polyvalency approach. Therefore we syn-
thesized new N,N-bis(alkanol)amine aryl esters and N,N-bis(ethox-
yethanol)amine aryl esters (1À17) carrying a methoxylated aryl
residue combined with
designed compounds are reported in Chart 2.
a flavone or chromone moiety. The
Diesters 1–10 (Chart 2, structure III) are characterized by the
presence of polymethylenic linkers and a N-methylated basic
portion; the spacers chosen, 3- or 5-methylenes long, were
those showing the best results in our previous studies.27,28 The
(E)-3-(3,4,5-trimethoxycynnamoyl) aryl moiety was chosen among
those that had previously given the best results27,28 and was
always maintained as first ester portion. This moiety was com-
bined with a second ester function carrying different flavonoids:
two hydrophilic hydroxychromones (Chart 2, b and d), two
hydrophobic methoxy-substituted chromones (a and c), and the
dihydroxyflavone residue (e). Introduction of methoxy groups
was designed since these residues often have a positive effect on
P-gp modulation. Derivative 11 was synthesized as an example
of a poly(ethyleneglycol) analog, which presents two linkers with
a length of 5 units. Since N-benzyl substitution on non-substituted
flavone derivatives gave the best results (Chart 1, structure I) in the
series of Chow and Chan,26 we also synthesized diesters 12–17
(Chart 2, structure IV), which are characterized by the presence
of a flavone acetic ester and different residues on the nitrogen
(methyl, benzyl or hydrogen); in this series both polymethylenic
and poly(ethyleneglycol) analogs were obtained, which always
show spacers of the same length.
The ability of the synthesized compounds to modulate P-gp was
evaluated by the pirarubicin uptake assay on doxorubicin-resistant
erythroleukemia K562 cells (K562/DOX cells), and their conforma-
tional behavior was investigated by means molecular dynamic
simulations.
2. Results and discussion
2.1. Chemistry
The reaction pathways used to synthesize the designed deriva-
tives (1À17) are reported in Schemes 1–7. All the derivatives were
obtained by the esterification of the suitable alcohol monoester
with the carboxylic acid carrying the appropriate chromone or
flavone residue.
R
I
structure
N
O
O
O
O
O
O
O
O
5-Methoxy-4-oxo-4H-chromene-2-carboxylic
5-hydroxy-4-oxo-4H-chromene-2-carboxylic
acid
19,
18,
and
acid
R = alkyl, arylalkyl, heteroarylalkyl moieties
CH3
5,7-dihydroxy-4-oxo-4H-chromene-2-carboxylic acid 20 were
obtained as previously described.23,31 The dimethoxy analog 23
was prepared as reported in Scheme 1: methylation on position
2 and 4 of 2,4,6-trihydroxyacetophenone gave 21 which was
reacted with diethyl oxalate and cyclized under acidic cyclization.
Ester 22 was then hydrolyzed obtaining acid 23 after acidification
of the obtained solution.
O
N
O
Ar1
II
Ar
structure
n = 2 - 7
(CH2)n
(CH2)n
O
O
Ar, Ar1 = aromatic or methoxy-substituted aromatic moieties
For the synthesis of the dihydroxy flavone acid derivative 26
(Scheme 2), the commercially available flavanone ( ) naringenin
Chart 1. General structures of the lead compounds.
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
OCH3
III
structure
CH3
H3CO
1 - 10
O
N
O
Ar
H3CO
(CH2)n
(CH2)n
O
n = 3,5
O
Ar =
O
O
O
H3CO
O
HO
O
O
O
O
HO
OH
O
H3CO
H3CO
O
OH
b
O
OH
c
e
a
d
O
OH
OCH3
CH3
H3CO
H3CO
11
N
O
O
O
O
OH
O
O
O
IV
structure
O
OCH3
R
N
H3CO
H3CO
O
12 - 17
O
O
O
X
X
O
O
X = CH2, O
R = H, CH3, benzyl
Chart 2. Structures of designed compounds.
O
O
H3CO
OH
HO
OH
CH3
H3CO
O
H3CO
O
OEt
OH
a
b, c
d, e
CH3
H3CO
O
OH
O
H3CO
O
H3CO
O
22
21
23
Reagents: a) CH3I, K2CO3 acetone; b) (COOEt)2, EtONa, EtOH; c) EtOH, HCl conc.; d) NaHCO3 10%; e) HCl.
Scheme 1.
O
OH
O
OEt
HO
O
O
HO
O
O
O
a
b
24
OH
OH
( ) naringenin
O
O
O
OEt
c, d
OH
HO
O
O
HO
O
O
26
25
OH
OH
Reagents: a) BrCH2COOEt, K2CO3, acetone; b) I2, pyridine; c) NaHCO3 10%; d) HCl.
Scheme 2.
was alkylated with ethyl bromoacetate to give 24 and oxidized
with I2 in pyridine to give 25. Alkaline hydrolysis of the ethyl ester
and acidification led to the desired acid 26.
Alcohol 36, already described by us,27 was obtained with a more
productive procedure (Scheme 4). The chloro derivative 3327 was
transformed in the corresponding iodo derivative 34 with NaI in
acetone, in order to achieve higher yields in the following reaction;
34 was then alkylated with the aminoalcohol 3527,33 to give com-
pound 36.
The synthesis of the benzyl-substituted intermediates is
reported in Scheme 5. Reaction of secondary amine 38 with iodo-
derivative 37 and Et3N in acetonitrile gave alcohol 40. In the same
way 41 was obtained from 39 and 34. Intermediates 3834 and 3935
had been already described, but were obtained by us in a different
In order to obtain the non-substituted flavone acid derivative
30, the suitable allyl-protected flavone 27 was synthesized as
reported in the literature.25 As shown in Scheme 3, cleavage of
the allyl protecting group by a catalytic amount of tetrakis(triph-
enylphosphine)palladium (0) led to 28,25 which was characterized
in this study. The deprotected phenolic function was alkylated
with ethyl bromoacetate; the obtained ester 29 was transformed
into 30 in the usual way.
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
O
O
O
a
b
O
CH2
OH
O
28
27
O
O
O
O
c, d
O
CH3
OH
O
30
O
29
O
O
Reagents: a) Pd(PPh3)4, K2CO3, MeOH; b) BrCH2COOEt, K2CO3, DMF; c) NaHCO3 10%; d) HCl. Compound 27
was obtained as reported in ref. 25.
Scheme 3.
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
a
O
Cl
O
I
O
O
O
O
33
34
OCH3
b
H3CO
H3CO
CH3
N
O
OH
CH3
HN
O
O
OH
O
O
36
35
33
Reagents: a) NaI, acetone; b) Et3N, CH3CN. Compound
was obtained as reported
in ref. 27; compound 35 was obtained as reported in ref. 27 and 33.
Scheme 4.
a
+
Cl
OH
X
NH2
38 X = CH2
39
X = O
OH
OCH3
HN
H3CO
H3CO
X
I
O
X
b
O
OCH3
37 X = CH2
H3CO
H3CO
34
X = O
N
OH
O
X
X
O
40 X = CH2
41
X = O
was obtained as reported in ref. 32.
37
Reagents: a) K2CO3, CH3CN; b) TEA, CH3CN. Compound
Scheme 5.
way, by reacting the suitable chloroalcohol and benzylamine in the
presence of K2CO3 in anhydrous CH3CN.
substituted alcohols 40 and 41, compounds 13 and 16 were
obtained respectively.
Final compounds 1–13, 15 and 16 were eventually obtained by
reaction of alcohols 31,27 32,27 36, 40 or 41 with the proper car-
boxylic acid using the activating agent EDCI in the presence of
HOBt in anhydrous CH2Cl2 (Scheme 6; for details, see the Experi-
mental Section 4). In particular, starting from the alcohol 31, final
compounds 1–5 were obtained; using the alkyl alcohol 32,
compounds 6–10 and 12 were synthesized. The ethoxy alcohol
36 led to derivatives 11 and 15; at last, starting from the N-benzyl
In the case of the final compounds 14 and 17, which carry a
non-substituted nitrogen, as first the aminic function was pro-
tected, as shown in Scheme 7. Alkyl alcohol 4227 and ethoxy alco-
hol 4327 were treated with (BOC)2O in THF yielding protected
derivatives 44 and 45.27 These intermediates were reacted with
acid 30 and the activating agent EDCI in the presence of DMAP to
give 46 and 47 respectively; cleavage of the BOC protecting group
with trifluoroacetic acid led to the compounds 14 and 17.
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
R
N
a
R
N
O
O
Ar
OH
O
Y
Y
Y
Y
O
O
O
Y
Ar
R
N
1
31 Y = (CH2)3
R = CH3
R = CH3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
a
b
c
d
e
a
b
c
d
e
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
32
2
Y = (CH2)5
36
3
Y = (CH2)2O(CH2)2 R = CH3
4
40 Y = (CH2)5
R = benzyl
41
5
Y = (CH2)2O(CH2)2 R = benzyl
6
7
8
9
10
11
(CH2)2O(CH2)2
d
12 (CH2)5
f
f
f
f
13
15
(CH2)5
(CH2)2O(CH2)2
benzyl
CH3
16 (CH2)2O(CH2)2
benzyl
18-20 23 26 30
), EDCI, HOBT. For the meaning of Ar, see Table 1. Compounds
Reagents: a) ArCOOH (
,
,
,
31 and 32 were obtained as reported in ref. 32.
Scheme 6.
OCH3
H3CO
H
N
O
OH
H3CO
Y
Y
a
O
OCH3
42 Y = CH2
H3CO
H3CO
43
Y = O
t-BOC
N
O
OH
Y
Y
O
44 Y = CH2
45
Y = O
O
O
b
OCH3
H3CO
H3CO
46
Y = CH2
t-BOC
N
47 Y = O
O
O
Y
Y
O
O
O
O
c
OCH3
H3CO
H3CO
14 Y = CH2
17
O
H
Y = O
O
N
O
Y
Y
O
O
O
30
Reagents: a) (BOC)2O, THF; b) , EDCI, DMAP, CH2Cl2, THF; c) CF3COOH, CH2Cl2. Compounds
42,
43 45
and
were obtained as reported in ref. 27.
Scheme 7.
2.2. Biological studies: modulation of pirarubicin uptake
The uptake of THP-adriamycin (pirarubicin) was measured by
means of a continuous spectrofluorometric signal of anthracycline
at 590 nm (kex = 480 nm) after cell incubation, following the proto-
cols reported in previous papers.40,41 The P-gp modulating activity
of the studied compounds on the pirarubicin uptake test is
expressed by: i) [I]0.5, which measures the potency of the modula-
tor and represents the concentration that causes a half-maximal
The ability of the synthesized compounds to modulate P-gp was
evaluated on K562/DOX cells. K562 is a human leukemia cell line
established from a patient with chronic myelogenous leukemia in
blast transformation.36 K562/DOX doxorubicin resistant cells over-
express only the membrane glycoprotein P-gp.37–39 Before evaluat-
ing the biological activity of the derivatives, their intrinsic toxicity
was assessed on both K562 and K562/DOX cell lines using the MTT
increase (a = 0.5) in the nuclear concentration of pirarubicin, and
ii) max, which represents the efficacy of the modulator and is
a
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide)
the maximum increase in the nuclear concentration of pirarubicin
assay. The compounds tested at 1.0 and 3.0 mM concentrations
have no intrinsic toxicity both to the parental and resistant cell
line. Data and experimental details are reported in the Supplemen-
tary material.
in resistant cells that can be obtained with a given compound. The
value of
a varies between 0 (in the absence of the inhibitor) and 1
(when the amount of pirarubicin in resistant cells is the same as in
sensitive cells).
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
The results obtained are reported in Table 1 together with those
of verapamil, the gold standard of P-gp inhibition, used as refer-
ence compound.
active than their ethoxyethylic analogs (compare 12 vs 15, 13 vs 16,
14 vs 17). As regard the influence of the N-residue, in the poly-
methylenic set of compounds, the methyl group confers the best
As regards the polymethylenic derivatives 1–10, it clearly
emerges that, regardless of the flavone or chromone residue which
is combined with the (E)-3-(3,4,5-trimethoxycynnamoyl)moiety,
the length of the linker is essential for the activity. In fact all the
compounds carrying two 3-carbon alkyl chains are almost inactive,
showing an efficacy between 4% and 30%. Therefore for compounds
1–5, [I]0.5 values could not be evaluated.
More interesting results were instead obtained with the analogs
carrying two 5-carbon alkyl chains. Derivatives 6–10 are able to
completely reverse P-gp-dependent pirarubicin extrusion (amax
close to 1) with potency values ([I]0.5) in the micromolar range.
In particular, the hydroxychromone compound 7 and the dihy-
droxyflavone derivative 10 show the highest reversing activity in
the series, with potencies higher than that of verapamil ([I]0.5 value
activity (12, [I]0.5 = 0.34
[I]0.5 = 0.43 M), while the activity is less pronounced in the case
of the secondary amine (14, [I]0.5 = 1.32 M). In the ethoxylated
series, only the N-methyl substituted derivative 15 shows a
remarkable efficacy ( max = 0.86), with a potency of 1.23 M.
lM), followed by the N-benzyl group (13,
l
l
a
l
These data are somehow unexpected. In fact, in the series of N-
methyl derivatives previously synthesized in our laboratory,27,28,32
compounds with two 3-carbon alkyl chains showed in most cases a
good MDR modulating activity, even if compounds bearing two 5-
carbon alkyl chains were often more potent. As regard the effica-
cies, the different series of compounds showed mixed results,
depending from the inserted aryl residue. Surprisingly, in the series
synthesized in this study the presence of the shorter linker (com-
pounds 1–5) completely or nearly completely abolishes the activ-
ity, regardless of the different size of the aryl group Ar1 (Table 1).
On the contrary, activity is maintained in the presence of the
longer chain (compounds 6–10, 12–14), and as expected the
MDR modulation activity of this set of derivatives depends on
the nature of the aromatic ester. Evaluation of the effect of the fla-
vonoid residue is not straightforward: insertion of the monohy-
droxy chromone b leads to the most interesting compound in
this series, but also the dihydroxy flavone e has a positive influence
of 0.35 lM and 0.96 lM with respect to 1.6 lM). In this series,
insertion of an ethoxylated chain of the same number of atoms
(see compound 11 vs compound 9) completely abolishes the activ-
ity (amax value: not measurable for 11 and 0.99 for 9).
A similar behavior is maintained in the series of compounds
12–17, carrying the flavone residue f and different residues on
the nitrogen. In this case, regardless from the group present on
the basic nitrogen, the polymethylenic derivatives are always more
Table 1
P-gp modulating activity of compounds 1–17. Verapamil is reported as comparison.
OCH3
H3CO
R
O
N
O
Ar1
H3CO
OCH3
(CH2)n
(CH2)n
O
O
A
R
H3CO
H3CO
N
O
O
Ar1
O
O
O
O
B
O
O
O
O
O
O
H3CO
O
HO
O
HO
O
O
O
OH O
H3CO
OH
b
H3CO
OH O
O
f
c
e
d
a
Ma
amax
b
N
Structure
n
R
Ar1
[i]0.5
l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
A
A
A
A
A
A
A
A
A
A
B
A
A
A
B
B
B
3
3
3
3
3
5
5
5
5
5
CH3
a
b
c
d
e
a
b
c
d
e
d
f
–
–
–
–
–
0.12 0.03
0.06 0.02
0.04 0.008
0.20 0.03
0.30 0.02
0.98 0.02
0.94 0.03
0.99 0.01
0.99 0.01
0.99 0.01
n.ac
0.99 0.01
0.99 0.01
0.69 0.17
0.86 0.13
0.27 0.01
0.26 0.01
0.70 0.07
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
benzyl
H
CH3
benzyl
H
3.50 0.5
0.35 0.05
1.32 0.57
2.43 0.48
0.96 0.20
–
0.34 0.07
0.43 0.12
1.32 0.22
1.23 0.17
–
5
5
5
f
f
f
f
17
verapamil
f
–
1.6 0.3
a
Concentration of the inhibitor that causes a 50% increase in nuclear concentration of pirarubicin (
Efficacy of MDR-modulator and maximum increase that can be obtained in the nuclear concentration of pirarubicin in resistant cells. Results are expressed as the mean
a
= 0.5).
b
SE of three independent experiments done at least three times.
c
Not active.
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
on the activity. The presence of the lipophilic non-substituted fla-
vone f confers a good activity to the molecule, confirming the
results previously reported.26
of both lipophilic/hydrophilic balance and ability to establish
H-bonds. This evidence seems to suggest, for the compounds
described in this work, a particular way of interaction with the
large P-gp recognition site. Furthermore, the flexibility of the
designed molecules allow each compound to bind in its own
way, finding the best productive binding mode. This fact is a fur-
ther proof of the validity of the polyvalent binding approach.
If we extend the analysis to compounds 11 and 15–17, the
results indicate that P-gp modulation is mainly related on the nat-
ure of the chain, rather than on its length. The ethoxyethylic
derivatives indeed are always less potent with respect to the poly-
methylenic ones, even if the two spacers contain the same number
of atoms. These data contrast with the results of Chow and Chan,26
,
2.3. Molecular modeling studies
since the very active flavonoid dimers described always carry two
ethoxyethylic chains. It must however be kept in mind that the
pharmacological assays and the cell lines employed in the case of
these flavonoid compounds are different from those used in our
studies. If we consider the effect of the substituent on the basic
nitrogen, the presence of the methyl group seems to confer the
best characteristics to the molecules, while the benzyl residue,
which is reported to have a positive effect in the flavonoid
heterodimers,26 in our series has not such a good influence, in par-
ticular when combined with an ethoxylated spacer. In the case of
the secondary amines 14 and 17 the results are further less
interesting.
The fact that this new series of compounds does not comply
with the SAR outlined by our previous derivatives opens a new sce-
nario. For our ‘‘hybrid” heterodimers the activity is linked to differ-
ent features, in particular the characteristics of the spacer, which
seem to be critical for the interaction with the pump. In fact, com-
pounds bearing two 5-carbon alkyl chains always show P-gp mod-
ulating activity. Likely enough, the presence of a flavone or
chromone residue modifies the molecular characteristics in terms
In order to investigate the different conformational behavior of
ethoxyethylic and polymethylenic derivatives, the conformational
effect of the O/CH2 substitution in compounds 12–17 was evalu-
ated by means of molecular dynamic simulations. This methodol-
ogy was chosen owing to the high conformational flexibility of
the compounds, which makes a conformational search difficult
either by means of systematic dihedral rotation or simulated
annealing.
The compounds, in the neutral and protonated forms, under-
went a 10 ns dynamic simulation in the vacuum. All molecules
adopt bended conformations showing the (E)-3-(3,4,5)-
trimethoxycynnamyl and flavone moieties close in space. A set of
seven distances has been selected to monitor changes in the con-
formational behavior possibly due to the O/CH2 substitution; for
compounds 12–17 in the neutral form the mean values of d1-d7
(Table 2) recorded in the last 3 ns of the dynamic simulation, do
not show a clear trend, suggesting a similar dynamic behavior for
the two sets of compounds 12–14 and 15–17. On the contrary, a
difference was found for the protonated forms. In fact, an
Table 2
Distances (Å) between selected atoms throughout a 3 ns dynamic simulation.a
d4
R
N
CH3
N
d6
d7
X
X
X
X
O
O
O
O
O
O
O
O
O
d5
12ac: X = CH2
15ac: X = O
d1
d2
MeO
OMe
OMe
O
d3
O
N
X
d1
d2
d3
d4
d5
d6
d7
12
13
14
15
16
17
CH2
CH2
CH2
O
O
O
CH2
CH2
CH2
O
O
O
9.8 0.7
9.2 0.6
8.9 2.1
8.0 0.5
8.8 2.9
8.6 1.5
9.5 2.2
9.3 2.8
2.8 0.1
2.7 0.1
2.7 0.1
10.0 1.8
8.6 1.7
9.1 1.9
–
4.9 1.2
6.0 0.7
5.6 2.3
5.1 0.7
5.3 1.2
7.8 2.9
5.9 0.4
6.0 0.7
7.8 2.9
5.4 2.1
5.7 1.3
8.9 3.3
–
7.8 1.0
10.0 0.5
9.1 1.7
9.1 1.7
8.7 1.9
7.1 1.2
12.0 1.0
12.3 0.9
8.2 1.3
5.6 1.8
4.8 0.8
4.0 0.5
6.4 1.8
7.7 2.1
5.1 1.1
5.3 0.9
7.1 1.5
6.6 0.4
7.3 1.4
7.7 1.2
7.1 1.5
4.9 1.0
10.9 0.4
10.8 0.4
9.4 1.3
6.5 0.9
6.6 0.9
6.7 0.7
5.6 1.2
6.2 1.9
6.5 1.6
6.1 1.1
6.5 0.5
5.6 0.5
6.8 0.6
6.0 0.6
6.2 0.6
6.2 0.7
6.0 0.8
5.3 1.2
2.7 0.1
5.8 0.4
5.1 0.7
2.9 0.2
7.0 0.7
6.3 0.8
3.4 0.7
3.8 0.5
7.6 0.8
7.4 0.2
6.7 0.9
6.6 0.8
5.7 0.8
5.9 0.9
7.6 0.7
8.2 0.7
7.9 0.5
3.0 0.3
3.0 0.2
2.8 0.1
7.4 0.8
6.1 0.1
4.5 1.8
4.2 1.4
10.0 1.1
8.4 08
9.0 0.8
10.3 1.6
8.1 0.6
8.0 0.7
8.5 0.5
9.1 1.1
9.2 0.8
8.5 0.6
–
12H+
13H+
14H+
15H+
16H+
17H+
12ac
15ac
12acH+
15acH+
CH2
O
CH2
O
–
–
–
–
–
–
–
–
–
a
Values are the mean SD recorded during the last 3 ns of a 10 ns simulation performed in vacuum. H+ means that the molecule has been calculated in the protonated
form.
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8
S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Conformations of 12 (right) and 15 (left), sampled from the dynamic simulation, showing the different H-bond (green).
intramolecular hydrogen bond has been observed between the NH+
group and the flavone C@O for compounds 12–14, while for 15–17
the H-bond is formed between the NH+ group and the C@O of the
acetate moiety; accordingly, mean values below 3 Å have been
found for d2 (compounds 12–14) and d7 (compounds 15–17),
and the H-bond interactions were found stable within the time
window of observation (see Fig. S2 in the Supplementary material).
In Fig. 1 two conformations of 12 (right) and 15 (left), highlighting
the different H-bond. In addition, a second H-bond is formed for
both the N-unsubstituted derivatives 14 and 17 involving the
C@O of the trimethoxycinnamyl moiety (d6 < 3 Å, Table 2).
conformation adopted by the molecules in the protonated forms,
where a difference was found. Some of these compounds show
an interesting P-gp modulating activity, which can hopefully be
increased by inserting different residues; these results support
the synthesis of new compounds that might be useful leads for
the development of drugs to control P-gp-dependent MDR.
4. Experimental
4.1. Chemistry
To avoid a possible interference of the intramolecular p-stack-
All melting points were taken on a Büchi apparatus and are
uncorrected. Infrared spectra were recorded with a Perkin-Elmer
Spectrum RX I FT-IR spectrophotometer in Nujol mull for solids
and neat for liquids. 1D and 2D (COSY and HSQC) NMR spectra
were recorded on a Bruker Avance 400 spectrometer (400 MHz
for 1H NMR, 100 MHz for 13C NMR). Chromatographic separations
were performed on a silica gel column by gravity chromatography
(Kieselgel 40, 0.063–0.200 mm; Merck) or flash chromatography
(Kieselgel 40, 0.040–0.063 mm; Merck). Yields are given after
purification, unless otherwise stated.
ing, calculations were repeated for 12 and 15 in which both aro-
matic appendages have been deleted, transforming the ester
moieties into simpler acetates. Visual inspection of the dynamic
simulation showed that these molecules adopt both bended and
extended conformations, with the former being more frequent.
Again, in the protonated molecules some distances were shorter
than in the neutral forms due to intramolecular H-bonds, which
however were less stable than in the parent molecules (data not
shown). No clear difference between O- and CH2-containing com-
pounds has been noticed.
Therefore, the performed dynamic simulations suggest a differ-
ent preference for the carbonyl groups involved in H-bond with the
protonated nitrogen atom between the polymethylenic com-
pounds 12–14 and the corresponding ethoxyethylic derivatives
15–17. Further studies are needed to understand if this difference
is the reason for the different activity of the two sets of
compounds.
ESI-MS spectra were obtained using a Varian 1200L triple quad-
rupole system (Palo Alto, CA, USA) equipped by Electrospray
Source (ESI) operating in both positive and negative ions.
The data were acquired in scan mode between the range 150–
800 m/z. In the instrumental conditions used, the most abundant
ion species expected for the analytes will be [M+H]+ or [MÀH]À.
Compounds 1–17 were obtained in a purity ꢀ95%. Their com-
bustion analyses are indicated by symbols, and the analytical
results are within 0.4% of the theoretical values. Compounds were
named following IUPAC rules as applied by Reaxys database. When
reactions were performed in anhydrous conditions, the mixtures
were maintained under nitrogen. Free bases 1–17 were trans-
formed into the hydrochloride by treatment with a solution of
acetyl chloride (1.1 eq) in anhydrous CH3OH, or into the oxalate
by treatment with 1.05 eq of oxalic acid in ethyl acetate. The salts
were crystallized from abs. ethanol/petroleum ether.
3. Conclusions
In this study, a new series of heterodimers was synthesized.
These derivatives are N,N-bis(alkanol)amine aryl esters or N,N-bis
(ethoxyethanol)amine aryl esters, containing a flavone or chro-
mone moiety. Some of the new compounds show a good P-gp mod-
ulating activity on the pirarubicin uptake test; interestingly this
new series of compounds does not comply with the SAR outlined
by our previous derivatives. In the case of the compounds
described in this paper, different structure-activity relationships
can be drawn, confirming that the presence of a flavone or chro-
mone residue influences the SAR of these series of products, and
that flexible molecules can find different productive binding modes
with the P-gp recognition site. Molecular dynamic simulations
suggest that the O/CH2 substitution elicits some effects on the
4.1.1. 1-(2-Hydroxy-4,6-dimethoxyphenyl)ethan-1-one 21
2,4,6-Trihydroxyacetophenone (2 g, 10.7 mmol), methyl iodide
(2.70 mL, 42.9 mmol) and K2CO3 (2.96 g, 21.4 mmol) were
dissolved in 50 mL of acetone. The mixture was heated to reflux
for 24 h in the dark. The reaction mixture was filtered under vac-
uum, and the filtrate was evaporated under reduced pressure.
The crude product was purified by flash chromatography using
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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
cyclohexane/ethyl acetate 95:5 as eluting system; 1.13 g of title
compound 21 were obtained. Yield: 36%. 1H NMR (CDCl3) d: 6.05
(d, J = 2.4 Hz, 1H, CH arom.); 5.92 (d, J = 2.4 Hz, 1H, CH arom.);
3.85 (s, 3H, OCH3); 3.81 (s, 3H, OCH3); 2.60 (s, 3H, CH3) ppm.
Hz, 2H, CH arom.); 6.93 (d, J = 9.0 Hz, 2H, CH arom.); 6.67
(d, J = 2.4 Hz, 1H, CH arom); 6.65 (s, 1H, CHCO); 6.37 (d, J = 2.4
Hz, CH arom); 4.82 (s, 2H, OCH2CO); 4.28 (q, J = 7.2 Hz, 2H,
CH2CH3); 1.29 (t, J = 7.2 Hz, 3H, CH2CH3) ppm.
4.1.2. Ethyl 5,7-dimethoxy-4-oxo-4H-chromene-2-carboxylate 22
A solution of 21 (884 mg, 4.51 mmol) in ethyl oxalate (2.4 mL,
18.04 mmol) was added to a freshly prepared solution of EtONa
in abs. EtOH (obtained by addition of 623 mg (27.1 mmol) of
sodium to 10 mL of abs. EtOH). The mixture was heated to reflux
for 10 h, then it was cooled to room temperature and water was
added. The solvent was evaporated, and the obtained brown resi-
due was treated with HCl 2N and extracted with ethyl acetate.
The organic layer was washed with brine, dried on Na2SO4 and
evaporated under vacuum. The crude residue was dissolved in
15 mL of EtOH 96% and heated to reflux for 15 min; conc. HCl
(0.6 mL) was added, and the obtained solution was maintained to
reflux for 1 h. The reaction mixture was cooled to room tempera-
ture, and the solvent was evaporated. The crude brown product
was purified by flash chromatography, using cyclohexane/ethyl
acetate 50:50 as eluting system, yielding 800 mg (71%) of a light
yellow solid (mp 151–153 °C). 1H NMR (CDCl3) d: 6.98 (s, 1H,
CH-CO); 6.60 (d, 1H, J = 2.4 Hz, CH arom.); 6.38 (d, J = 2.4 Hz, 1H,
CH arom.); 4.43 (q, J = 7.2 Hz, 2H, CH2CH3); 3.94 (s, 3H, OCH3);
3.89 (s, 3H, OCH3); 1.41 (t, J = 7.2 Hz, 3H, CH2CH3) ppm.
4.1.6. 2-[4-(5,7-Dihydroxy-4-oxo-4H-chromen-2-yl)phenoxy]acetic
acid 26
With the same procedure described for 23, and starting from
149 mg (0.42 mmol) of 25 and 15 ml of a solution of 10% NaHCO3,
130 mg of 26 as yellow solid were obtained, that did not need fur-
ther purification (mp 159–161 °C). Yield: 94%. 1H NMR (CD3OD) d:
7.90 (d, J = 8.8 Hz, 2H, CH arom.); 6.95 (d, J = 8.8 Hz, 2H, CH arom.);
6.68 (d, J = 2.4 Hz, 1H, CH arom.); 6.67 (s, 1H, CHCO); 6.39
(d, J = 2.4 Hz, 1H, CH arom.); 4.81 (s, 2H, OCH2CO) ppm.
4.1.7. 2-(4-Hydroxyphenyl)-4H-chromen-4-one 28
A catalytic amount of Pd(PPh3)4 (280 mg, 0.24 mmol) was
added to a solution of 2725 (2.26 g, 8.1 mmol) and K2CO3 (4.49 g,
32.5 mmol) in 40 mL of anhydrous MeOH at reflux. The reaction
mixture was stirred at reflux for 4 h; then it was filtered to remove
K2CO3. The obtained brown solution was diluted with water, acid-
ified to pH 3 using 2 N HCl in ice and extracted twice with diethyl
ether. The organic layer was dried on Na2SO4, evaporated and puri-
fied by a filtration on silica gel with CH2Cl2/MeOH 9:1. Yield: 1.90 g
(98.2%). 1H NMR (CD3OD) d: 8.12 (d, J = 8.0 Hz, 1H, CH arom.); 7.91
(d, J = 8.4 Hz, 2H, CH arom,); 7.78 (t, J = 8.0 Hz, 1H, CH arom.); 7.69
(d, J = 8.0 Hz, 1H, CH arom.); 7.47 (t, J = 8.0 Hz, 1H, CH arom.); 6.94
(d, J = 8.4 Hz, 2H, CH arom.); 6.79 (s, 1H, CHCO) ppm.
4.1.3. 5,7-Dimethoxy-4-oxo-4H-chromene-2-carboxylic acid 23
To 200 mg (0.57 mmol) of 22, 20 mL of a solution of 10%
NaHCO3 were added. The mixture was heated at 80 °C for 3 h. After
cooling, the solution was acidified with conc. HCl and extracted
with ethyl acetate. The organic layer was washed with water, dried
over Na2SO4 and evaporated under vacuum, yielding 170 mg (95%)
of a pale yellow solid (mp 241–243 °C). 1H NMR (CD3OD) d: 6.86 (s,
1H, CH-CO); 6.74 (d, J = 2.4 Hz, 1H, CH arom.); 6.56 (d, J = 2.4 Hz,
1H, CH arom.); 3.94 (s, 3H, OCH3); 3.91 (s, 3H, OCH3) ppm.
4.1.8. Ethyl 2-[4-(4-oxo-4H-chromen-2-yl)phenoxy]acetate 29
K2CO3 (1.10 g, 8.0 mmol) and ethyl bromoacetate (1.06 mL, 9.6
mmol) were added to a solution of 1.90 g (8.0 mmol) of 28 in 50 mL
of DMF. The mixture was stirred at room temperature for 24 h,
then it was filtered under vacuum by adding a little amount of
MeOH. The gold-yellow solution was evaporated, yielding 2.52 g
(97.4%) of title compound as light-yellow solid (mp 131–132 °C).
1H NMR (CDCl3) d: 8.14 (d, J = 7.6 Hz, 1H, CH arom.); 7.81 (d, J =
8.4 Hz, 2H, CH arom.); 7.59 (t, J = 7.6 Hz, 1H, CH arom.); 7.47 (d,
J = 8.0 Hz, 1H, CH arom.); 7.34 (t, J = 8.0 Hz, 1H, CH arom.);6.96
(d, J = 8.4 Hz, 2H, CH arom.); 6.67 (s, 1H, CHCO); 4.63 (s, 2H, OCH2-
CO); 4.22 (q, J = 7.2 Hz, 2H, CH2CH3); 1.25 (t, J = 7.2 Hz, 3H, CH2CH3)
ppm.
4.1.4. Ethyl 2-[4-(5,7-dihydroxy-4-oxo-3,4-dihydro-2H-1-
benzopyran-2-yl)phenoxy]acetate 24
To a solution of 200 mg of ( )naringenin (0.73 mmol) in 7 ml of
acetone, K2CO3 (101.5 mg, 0.73 mmol) was added. The mixture
was stirred at room temperature for 15 min, then ethyl bromoac-
etate (0.08 ml, 0.73 mmol) dissolved in 3 mL acetone was added
dropwise. The reaction mixture was stirred 24 h at room tempera-
ture, filtered under vacuum, and the filtrate was evaporated under
reduced pressure. The crude product was purified by flash chro-
matography using CH2Cl2/MeOH 99:1 as eluting system. 158 mg
of 24 were obtained as a white solid (mp 185–186 °C), yield 60%.
MS m/z (%): 359.1 (100) [M+H]+; 1H NMR (CD3OD) d: 7.32 (d,
J = 8.4 Hz, 2H, CH arom.); 6.81 (d, J = 8.4 Hz, 2H, CH arom.);
6.06–6.03 (m, 2H, CH arom.); 5.41–5.37 (m, 1H, CHO); 4.74 (s, 2H,
OCH2CO); 4.25 (q, J = 7.2 Hz, 2H, CH2CH3); 3.20–3.13 (m, 1H,
CHHCO); 2.77–2.72 (m, 1H, CHHCO); 1.28 (t, J = 7.2 Hz, 3H, CH2CH3)
ppm.
4.1.9. 2-[4-(4-Oxo-4H-chromen-2-yl)phenoxy]acetic acid 30
To 2.52 g (7.8 mmol) of 29 100 mL of a solution of 10% NaHCO3
were added. The mixture was stirred at 100 °C for 5 h and then 18
h at room temperature. After cooling, the solution was acidified
with HCl 2 N. A yellow solid was obtained which was collected
by under vacuum/suction filtration. Yield 2.13 g (92.6%) (mp
219–221 °C). 1H NMR (CD3OD) d: 8.13 (d, J = 8.4 Hz, 1H, CH arom.);
8.03 (d, J = 8.8 Hz, 2H, CH arom.); 7.80 (t, J = 8.4 Hz, 1H, CH arom.);
7.73 (d, J = 7.6 Hz, 1H, CH arom.); 7.50 (t, J = 7.6 Hz, 1H, CH arom.);
7.12 (d, J = 8.8 Hz, 2H, CH arom.); 6.85 (s, 1H, CHCO); 4.78 (s, 2H,
OCH2CO) ppm.
4.1.5. Ethyl 2-[4-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)phenoxy]
acetate 25
4.1.10. 2-(2-Iodoethoxy)ethyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-
enoate 34
To a solution of 150 mg (0.42 mmol) of 24 in 8 mL of anhydrous
pyridine, 106.34 mg (0.42 mmol) of I2 were added. The mixture
was kept at 90 °C for 6 h then it was cooled to room temperature
and poured on ice. The solution was extracted twice with ethyl
acetate, then the organic layer was washed three times with a sat-
urated solution of Na2S2O3, once with a saturated solution of NaCl,
and dried on Na2SO4. Evaporation of the solvent and distillation of
the pyridine yielded 149 mg of 25 as yellow solid, mp 185–187 °C.
The compound was not further purified. Yield: 100%. ESI-MS:
[MÀH]À species at m/z 355. 1H NMR (CD3OD) d: 7.88 (d, J = 9.0
NaI (367 mg, 2.45 mmol) was added to a solution of 211 mg
(0.61 mmol) of 3327 in 10 mL of acetone. The reaction was stirred
at reflux in the dark for 20 h. The mixture was then cooled to room
temperature, the solvent was evaporated, and the residue was
treated with water and extracted three times with CH2Cl2. The
organic layer was dried on Na2SO4 and evaporated to give a
light-yellow oil (231 mg, 87%) that was no further purified. 1H
NMR (CDCl3) d: 7.59 (d, J = 16.0 Hz, 1H, CH@CH); 6.71 (s, 2H, CH
arom.); 6.35 (d, J = 16.0 Hz, 1H, CH@CH); 4.33 (t, J = 4.8 Hz, 2H,
Please cite this article in press as: Dei S., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.016

10
S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
CH2O); 3.83 (s, 9H, OCH3); 3.76–3.73 (m, 4H, CH2OCH2); 3.23 (t, J =
6.8 Hz, 2H, CH2I) ppm.
4.1.15. 2-(2-{Benzyl[2-(2-hydroxyethoxy)ethyl]amino}ethoxy)ethyl
(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate 41
Iododerivative 34 (492 mg, 1.13 mmol), aminoalcohol 39 (220
mg, 1.13 mmol) and anhydrous triethylamine (0.45 mL, 3.40
mmol) were dissolved in 10 mL of anhydrous acetonitrile. The mix-
ture was stirred at 82 °C for 24 h, then it was evaporated. The
obtained residue was dissolved in CH2Cl2, and the organic layer
was washed three times with a solution of 10% NaOH, dried on
Na2SO4 and evaporated to give the crude product. After purifica-
tion by flash chromatography, using CH2Cl2/MeOH/NH4OH
95:5:0.5 as eluting system, 225 mg (39.6%) of 41 as yellow oil were
obtained. 1H NMR (CDCl3) d: 7.60 (d, J = 16.0 Hz, 1H, CH@CH);
7.33–7.21 (m, 5H, CH arom.); 6.73 (s, 2H, CH arom.); 6.36 (d, J =
16.0 Hz, 1H, CH@CH); 4.33 (t, J = 4.4 Hz, 2H, CH2OCO); 3.89 (s,
6H, OCH3); 3.86 (s, 3H, OCH3); 3.71–3.51 (m, 12H, OCH2 and NCH2-
Ph and CH2OH); 2.79–2.75 (m, 4H, CH2N) ppm.
4.1.11. 2-(2-{[2-(2-Hydroxyethoxy)ethyl](methyl)amino}ethoxy)ethyl
(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate 3627
227 mg (0.52 mmol) of 34 were reacted with 70 mg (0.59
mmol) of 3527,33 and 0.2 mL of anhydrous Et3N in 4 mL of anhy-
drous acetonitrile. The reaction mixture was stirred at 60 °C for
18 h. Then CH2Cl2 was added, and the solution was washed twice
with basic water, dried on Na2SO4 and evaporated to give the crude
product that was purified by flash chromatography, using CH2Cl2/
MeOH/NH4OH 95:5:0.5 as eluting system. 100 mg (50.0%) of title
compound were obtained. 1H NMR (CDCl3) d: 7.61 (d, J = 16.0 Hz,
1H, CH@CH); 6.75 (s, 2H, CH arom.); 6.38 (d, J = 16.0 Hz, 1H,
CH@CH); 4.35 (t, J = 4.8 Hz, 2H, CH2OCO); 3.87 (s, 9H, OCH3);
3.72 (t, J = 4.8 Hz, 2H, CH2OH); 3.68–3.57 (m, 8H, CH2OCH2); 2.69
(t, J = 5.6 Hz, 2H, CH2N); 2.65 (t, J = 5.2 Hz, 2H, CH2N); 2.33 (s, 3H,
NCH3) ppm.
4.1.16. General procedure for the synthesis of diester compounds 1–13,
15 and 16
To a solution of the suitable acid (18,23 19,23 20,31 23, 26 or 30)
(1 eq.) in anhydrous THF, EDCI (1.2 eq.), HOBt (1.2 eq.) and a solu-
tion of the proper alcohol (31,32 32,32 36, 40, 41) (1 eq.) in anhy-
drous CH3CN or CH2Cl2 were added. The mixture was stirred at
room temperature for 48 h, then it was evaporated. The obtained
residue was dissolved in CH2Cl2, and the organic layer was washed
twice with water and once with a saturated solution of NaHCO3,
dried on Na2SO4 and evaporated to give the desired ester. As
reported hereinafter, when necessary the oily product was purified
by flash chromatography using the appropriate eluting system. All
the compounds were transformed into the corresponding
hydrochloride or oxalate. The salts were crystallized from abs.
ethanol/petroleum ether and were obtained as white solids.
4.1.12. 5-(Benzylamino)pentan-1-ol 3834
To a solution of benzylamine (0.66 mL, 6.03 mmol) and 5-
chloropentan-1-ol (0.42 mL, 4.02 mmol) in 5 mL of anhydrous ace-
tonitrile, 555 mg of K2CO3 (4.02 mmol) were added. The mixture
was stirred at 80 °C for 48 h and concentrated in vacuo. The residue
was dissolved with CH2Cl2, and the organic layer was washed twice
with a solution of 10% NaOH, dried on Na2SO4 and evaporated to
give the crude product that was purified by flash chromatography,
using CH2Cl2/MeOH/NH4OH 95:5:0.5 as eluting system. 160 mg
(20.6%) of title compound as a yellow oil were obtained. 1H NMR
(CDCl3) d: 7.33–7.26 (m, 5H, CH arom.); 3.78 (s, 2H, NCH2Ph);
3.61 (t, J = 6.4 Hz, 2H, CH2OH); 2.65 (t, J = 6.8 Hz, 2H, NCH2); 1.88
(s, 1H, NH); 1.61–1.50 (m, 4H, CH2); 1.47–1.42 (m, 2H, CH2) ppm.
4.1.16.1. (3-({3-[(E)-5-Methoxy-4-oxo-4H-chromene-2-carbonyloxy]
propyl}(methyl)amino)propyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-
2-enoate 1. Free base: chromatographic eluent: CH2Cl2/MeOH/NH4OH
95:5:0.5. Yield 93%. 1H NMR (CDCl3) d: 7.58 (t, J = 8.4 Hz, 1H,
CH arom.); 7.56 (d, J = 16.0 Hz, 1H, CH@CH); 7.12 (d, J = 8.4 Hz, 1H,
CH arom.); 6.98 (s, 1H, CHCO); 6.81 (d, J = 8.4 Hz, 1H, CH arom.);
6.72 (s, 2H, CH arom.); 6.32 (d, J = 16.0 Hz, 1H, CH@CH); 4.45 (t, J =
6.4 Hz, 2H, CH2O); 4.26 (t, J = 6.4 Hz, 2H, CH2O); 3.96 (s, 3H, OCH3);
3.88 (s, 6H, OCH3); 3.87 (s, 3H, OCH3); 2.55–2.50 (m, 4H, NCH2);
2.27 (s, 3H, NCH3); 1.99–1.89 (m, 4H, CH2) ppm. 13C NMR (CDCl3)
d: 178.01 (C); 166.94 (C); 160.00 (C); 159.01 (C); 157.98 (C);
153.38 (C); 149.55 (C); 144.71 (CH@CH); 134.72 (CH); 129.87 (C);
117.25 (CH); 116.42 (CH@CH); 110.53 (CH); 106.84 (CH); 103.21
(CH); 65.02 (CH2O); 62.74 (CH2O); 60.96 (OCH3); 56.48 (OCH3);
56.15 (OCH3); 54.22 (NCH2); 53.79 (NCH2); 41.89 (NCH3); 26.63
(CH2); 26.35 (CH2) ppm.
4.1.13. 5-[Benzyl-(5-hydroxypentyl)amino]pentyl (2E)-3-(3,4,5-
trimethoxyphenyl)prop-2-enoate 40
Compounds 38 (160 mg, 0.83 mmol), 3732 (360 mg, 0.83 mmol)
and anhydrous triethylamine (0.36 ml, 2.72 mmol) were dissolved
in 15 mL of anhydrous acetonitrile. The mixture was stirred at
reflux for 27 h, then it was evaporated. The obtained residue was
dissolved in CH2Cl2, and the organic layer was washed three times
with a solution of 10% NaOH, dried on Na2SO4 and evaporated to
give the crude product. After purification by flash chromatography,
using CH2Cl2/MeOH/NH4OH 95:5:0.5 as eluting system, 124 mg
(30.0%) of 40 as pale-yellow oil were obtained. 1H NMR (CDCl3)
d: 7.59 (d, J = 16.0 Hz, 1H, CH@CH); 7.31–7.22 (m, 5H, CH arom.);
6.75 (s, 2H, CH arom.); 6.34 (d, J = 16.0 Hz, 1H, CH@CH); 4.18 (t,
J = 6.4 Hz, 2H, CH2OCO); 3.89 (s, 6H, OCH3); 3.88 (s, 3H, OCH3);
3.61–3.56 (m, 4H, NCH2Ph and CH2OH); 2.45–2.41 (m, 4H,
CH2N); 1.70–1.64 (m, 4H, CH2); 1.55–1.47 (m, 4H, CH2); 1.43–
1.33 (m, 4H, CH2) ppm.
Oxalate: mp 87–89 °C. Anal: C32H37NO14 (C, H, N).
4.1.16.2. 3-({3-[(E)-5-Hydroxy-4-oxo-4H-chromene-2-carbonyloxy]
propyl}(methyl) amino)propyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-
2-enoate 2. Free base: yield 93%. 1H NMR (CDCl3) d: (t, J = 8.4 Hz,
1H, CH arom.); 7.55 (d, J = 16.0 Hz, 1H, CH@CH); 7.02 (s, 1H,
CHCO); 7.01 (d, J = 8.4 Hz, 1H, CH arom.); 6.82 (d, J = 8.4 Hz, 1H,
CH arom.); 6.72 (s, 2H, CH arom.); 6.31 (d, J = 16.0 Hz, 1H, CH@CH);
4.48 (t, J = 6.4 Hz, 2H, CH2O); 4.26 (t, J = 6.4 Hz, 2H, CH2O); 3.87 (s,
9H, OCH3); 2.53–2.47 (m, 4H, NCH2); 2.25 (s, 3H, NCH3); 2.00–1.84
(m, 4H, CH2) ppm. 13C NMR (CDCl3) d: 183.72 (C); 166.92 (C);
160.68 (C); 159.89 (C); 153.42 (C); 153.00 (C); 149.56 (C); 144.73
(CH@CH); 136.48 (CH); 129.83 (C); 117.22 (CH@CH); 113.46
(CH); 112.04 (CH); 107.71 (CH); 105.26 (CH); 65.39 (CH2O);
62.77 (CH2O); 60.95 (OCH3); 56.16 (OCH3); 54.22 (NCH2); 53.75
(NCH2); 41.97 (NCH3); 26.73 (CH2); 26.42 (CH2) ppm.
4.1.14. 2-[2-(Benzylamino)ethoxy]ethan-1-ol 3935
To a solution of benzylamine (0.88 mL, 8.04 mmol) and 2-(2-
chloroethoxy)ethanol (0.84 mL, 8.04 mmol) in 5 mL of anhydrous
acetonitrile, 1.11 g of K2CO3 (8.04 mmol) were added. The mixture
was stirred at reflux for 48 h and concentrated in vacuo. The resi-
due was dissolved with CH2Cl2, and the organic layer was washed
three times with a solution of 10% NaOH, dried on Na2SO4 and
evaporated to give the crude product that was purified by flash
chromatography, using CH2Cl2/MeOH/NH4OH 93:7:0.3 as eluting
system. 323 mg (20.6%) of 39 as a yellow oil were obtained. 1H
NMR (CDCl3) d: 7.34–7.25 (m, 5H, CH arom.); 3.83 (s, 2H, NCH2Ph);
3.69 (t, J = 4.2 Hz, 2H, OCH2CH2OH); 3.63 (t, J = 5.2 Hz, 2H,
OCH2CH2N); 3.55 (t, J = 4.2 Hz, 2H, CH2OH); 2.83 (t, J = 5.2 Hz, 2H,
NCH2CH2O) ppm.
Oxalate: mp 106–107 °C. Anal: C31H35NO14 (C, H, N).
Please cite this article in press as: Dei S., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.016

S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
4.1.16.3. 3-({3-[(E)-5,7-Dimethoxy-4-oxo-4H-chromene-2-carbony-
loxy]propyl}(methyl)amino)propyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 3. Free base: chromatographic eluent: CH2Cl2/MeOH
95:5. Yield 54%. 1H NMR (CDCl3) d: 7.50 (d, J = 16.0 Hz, 1H,
CH@CH); 6.87 (s, 1H, CHCO); 6.68 (s, 2H, CH arom.); 6.50 (d, J =
2.0 Hz, 1H, CH arom.); 6.28 (d, J = 2.0 Hz, 1H, CH arom.); 6.26 (d,
J = 16.0 Hz, 1H, CH@CH); 4.39 (t, J = 6.4 Hz, 2H, CH2O); 4.20 (t, J =
6.4 Hz, 2H, CH2O); 3.85 (s, 3H, OCH3); 3.83 (s, 6H, OCH3); 3.82 (s,
6H, OCH3); 2.49–2.42 (m, 4H, NCH2); 2.21 (s, 3H, NCH3); 1.91–
1.79 (m, 4H, CH2) ppm. 13C NMR (CDCl3) d: 176.94 (C); 166.88
(C); 164.75 (C); 160.82 (C); 160.53 (C); 159.65 (C); 153.33 (C);
149.73 (C); 144.63 (CH@CH); 140.01 (C); 129.83 (C); 117.21
(CH@CH); 116.60 (CH); 109.97 (C); 105.20 (CH); 96.69 (CH);
93.07 (CH); 64.91 (CH2O); 62.71 (CH2O); 60.88 (OCH3); 56.34
(OCH3); 56.11 (OCH3); 55.85 (OCH3); 54.18 (NCH2); 53.73
(NCH2); 41.86 (NCH3); 26.62 (CH2); 26.33 (CH2) ppm.
(C); 149.57 (C); 144.59 (CH); 134.72 (CH@CH); 129.95 (C);
117.47 (CH@CH); 116.45 (CH); 110.61 (CH); 106.89 (CH); 105.27
(CH); 66.77 (CH2O); 64.56 (CH2O); 60.96 (OCH3); 57.67 (NCH2);
57.52 (NCH2); 56.52 (OCH3); 56.18 (OCH3); 42.17 (NCH3); 28.72
(CH2); 28.42 (CH2); 26.83 (CH2); 23.99 (CH2); 23.84 (CH2) ppm.
Oxalate: mp 88–89 °C. Anal: C36H45NO14 (C, H, N).
4.1.16.7. 5-({5-[(E)-5-Hydroxy-4-oxo-4H-chromene-2-carbonyloxy]
pentyl}(methyl)amino)pentyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-
2-enoate 7. Free base: chromatographic eluent: CH2Cl2/MeOH
95:5. Yield 73%. 1H NMR (CDCl3) d: 7.59–7.53 (m, 2H, CH arom.
and CH@CH); 7.04 (d, J = 8.0 Hz, 1H, CH arom.); 7.03 (s, 1H, CH
arom.); 6.83 (d, J = 8.0 Hz, 1H, CH arom.); 6.74 (s, 2H, CH arom.);
6.33 (d, J = 16.0 Hz, 1H, CH@CH); 4.39 (t, J = 6.4 Hz, 2H, CH2O);
4.19 (t, J = 6.4 Hz, 2H, CH2O); 3.88 (s, 6H, OCH3); 3.87 (s, 3H,
OCH3); 2.39–2.36 (m, 4H, NCH2); 2.23 (s, 3H, NCH3); 1.84–1.68
(m, 6H, CH2); 1.61–1.42 (m, 6H, CH2) ppm. 13C NMR (CDCl3) d:
167.01 (C); 160.70 (C); 153.44 (C); 153.00 (C); 149.56 (C); 144.63
(CH@CH); 136.52 (CH); 129.93 (C); 117.43 (CH@CH); 113.51
(CH); 112.08 (CH); 107.77 (CH); 105.27 (CH); 67.09 (CH2O);
64.53 (CH2O); 60.97 (OCH3); 57.64 (NCH2); 57.48 (NCH2); 56.18
(OCH3); 42.12 (NCH3); 29.70 (CH2); 28.71 (CH2); 28.38 (CH2);
26.84 (CH2); 23.97 (CH2); 23.80 (CH2) ppm.
Oxalate: mp 120–121 °C. Anal: C33H39NO15 (C, H, N).
4.1.16.4. 3-({3-[(E)-5,7-Dihydroxy-4-oxo-4H-chromene-2-carbony-
loxy]propyl}(methyl)amino)propyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 4. Free base: chromatographic eluent: CH2Cl2/MeOH
95:5. Yield 71%. 1H NMR (CDCl3) d: 7.52 (d, J = 16.0 Hz, 1H,
CH@CH); 6.75 (s, 1H, CHCO); 6.70 (s, 2H, CH arom.); 6.31 (d, J =
16.0 Hz, 1H, CH@CH); 6.30 (d, J = 2.0 Hz, 1H, CH arom.); 6.20 (d, J
= 2.0 Hz, 1H, CH arom.); 4.39 (t, J = 6.4 Hz, 2H, CH2O); 4.25 (t, J =
6.4 Hz, 2H, CH2O); 3.86 (s, 6H, OCH3); 3.85 (s, 3H, OCH3); 2.67–
2.61 (m, 4H, NCH2); 2.35 (s, 3H, NCH3); 2.02–1.93 (m, 4H, CH2)
ppm. 13C NMR (CDCl3) d: 181.70 (C); 167.21 (C); 165.41 (C);
162.26 (C); 159.87 (C); 157.58 (C); 153.38 (C); 151.67 (C); 145.17
(CH@CH); 140.09 (C); 129.74 (C); 116.85 (CH); 113.34 (CH@CH);
105.78 (C); 105.25 (CH); 100.35 (CH); 95.03 (CH); 64.57 (CH2O);
62.58 (CH2O); 60.97 (OCH3); 56.15 (OCH3); 54.11 (NCH2); 53.49
(NCH2); 41.24 (NCH3); 25.96 (CH2); 25.78 (CH2) ppm.
Oxalate: mp 106–107 °C. Anal: C35H43NO14 (C, H, N).
4.1.16.8. 5-({5-[(E)-5,7-Dimethoxy-4-oxo-4H-chromene-2-carbony-
loxy]pentyl}(methyl)amino)pentyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 8. Free base: chromatographic eluent: CH2Cl2/MeOH
95:5. Yield 68%. 1H NMR (CDCl3) d: 7.57 (d, J = 16.0 Hz, 1H,
CH@CH); 6.92 (s, 1H, CH arom.); 6.73 (s, 2H, CH arom.); 6.59 (d,
J = 2.0 Hz, 1H, CHCO); 6.37 (d, J = 2.0 Hz, 1H, CH arom.); 6.32 (d, J
= 16.0 Hz, 1H, CH@CH); 4.34 (t, J = 6.4 Hz, 2H, CH2O); 4.18 (t, J =
6.4 Hz, 2H, CH2O); 3.92 (s, 3H, OCH3); 3.87 (s, 6H, OCH3); 3.85 (s,
6H, OCH3); 2.49–2.45 (m, 4H, NCH2); 2.33 (s, 3H, NCH3); 1.82–
1.46 (m, 12H, CH2) ppm. 13C NMR (CDCl3) d: 181.60 (C); 167.46
(C); 167.03 (C); 162.20 (C); 160.07 (C); 157.91 (C); 153.40 (C);
151.78 (C); 144.75 (CH@CH); 140.10 (C); 129.83 (C); 117.21
(CH@CH); 113.32 (CH); 105.42 (C); 105.22 (CH); 100.90 (CH);
95.45 (CH); 66.41 (CH2O); 64.27 (CH2O); 60.90 (OCH3); 57.12
(NCH2); 56.85 (NCH2); 56.30 (OCH3); 56.10 (OCH3); 55.72
(OCH3); 41.12 (NCH3); 28.49 (CH2); 28.09 (CH2); 25.42 (CH2);
25.31 (CH2); 23.76 (CH2); 23.68 (CH2) ppm.
Oxalate: mp 175–177 °C. Anal: C31H35NO15 (C, H, N).
4.1.16.5. 3-{[3-({2-[4-(5,7-Dihydroxy-4-oxo-4H-chromen-2-yl)phe-
noxy] acetyl}oxy)propyl](methyl)amino}propyl (2E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-enoate 5. Free base: chromatographic
eluent: CH2Cl2/MeOH 95:5. Yield 12%. 1H NMR (CDCl3) d: 7.68 (d,
J = 8.8 Hz, 2H, CH arom.); 7.58 (d, J = 16.0 Hz, 1H, CH@CH); 6.91
(d, J = 8.8 Hz, 2H, CH arom.); 6.77 (s, 2H, CH arom.); 6.56–6.52
(m, 2H, CH arom. and CHCO); 6.36 (d, J = 16.0 Hz, 1H, CH@CH);
6.33–6.28 (m, 1H, CH arom.); 4.64 (s, 2H, OCH2CO); 4.29 (t, J =
6.4 Hz, 2H, CH2O); 4.23 (t, J = 6.4 Hz, 2H, CH2O); 3.86 (s, 9H,
OCH3); 2.54–2.47 (m, 4H, NCH2); 2.27 (s, 3H, NCH3); 1.89–1.87
(m, 4H, CH2) ppm. 13C NMR (CDCl3) d: 182.34 (C); 168.14 (C);
167.14 (C); 164.45 (C); 163.22 (C); 162.17 (C); 160.56 (C); 157.42
(C); 153.38 (C); 145.03 (CH@CH); 139.99 (C); 129.81 (C); 128.30
(CH); 122.24 (C); 117.05 (CH@CH); 116.30 (CH); 105.91 (C);
105.16 (CH); 103.67 (CH); 98.26 (CH); 93.31 (CH); 65.14 (OCH2-
CO); 63.85 (CH2O); 62.84 (CH2O); 61.01 (OCH3); 56.13 (OCH3);
54.11 (NCH2); 53.80 (NCH2); 41.71 (NCH3); 26.18 (CH2); 26.08
(CH2) ppm. Oxalate: mp 112–113 °C. Anal: C38H41NO16 (C, H, N).
Oxalate: mp 110–111 °C. Anal: C37H47NO15 (C, H, N).
4.1.16.9. 5-({5-[(E)-5,7-Dihydroxy-4-oxo-4H-chromene-2-carbony-
loxy]pentyl}(methyl)amino)pentyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 9. Free base: chromatographic eluent: CH2Cl2/
MeOH/NH4OH 90:10:0.5. Yield 71%. 1H NMR (CDCl3) d: 7.55 (d, J =
16.0 Hz, 1H, CH@CH); 6.81 (s, 1H, CHCO); 6.72 (s, 2H, CH arom.);
6.33 (d, J = 2.0 Hz, 1H, CH arom.); 6.30 (d, J = 16.0 Hz, 1H, CH@CH);
6.19 (d, J = 2.0 Hz, 1H, CH arom.); 4.29 (t, J = 6.4 Hz, 2H, CH2O); 4.16
(t, J = 6.4 Hz, 2H, CH2O); 3.86 (s, 6H, OCH3); 3.85 (s, 3H, OCH3);
2.62–2.58 (m, 4H, NCH2); 2.42 (s, 3H, NCH3); 1.77–1.63 (m, 8H,
CH2); 1.44–1.40 (m, 4H, 2CH2) ppm. 13C NMR (CDCl3) d: 181.59
(C); 167.48 (C); 167.05 (C); 162.18 (C); 160.06 (C); 157.90 (C);
153.41 (C); 151.78 (C); 144.78 (CH@CH); 140.11 (C); 129.87 (C);
117.25 (CH@CH); 113.37 (CH); 105.40 (C); 105.28 (CH); 100.94
(CH); 95.48 (CH); 66.46 (CH2O); 64.22 (CH2O); 60.94 (OCH3);
57.00 (NCH2); 56.86 (NCH2); 56.17 (OCH3); 41.12 (NCH3); 28.49
(CH2); 28.09 (CH2); 25.42 (CH2); 25.31 (CH2); 23.76 (CH2); 23.68
(CH2) ppm.
4.1.16.6. 5-({5-[(E)-5-Methoxy-4-oxo-4H-chromene-2-carbonyloxy]
pentyl}(methyl)amino)pentyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-
2-enoate 6. Free base: chromatographic eluent: CH2Cl2/diethyl
ether/petroleum ether/abs.EtOH/NH4OH 120:120:300:60:3.3.
Yield 81%. 1H NMR (CDCl3) d: 7.60 (t, J = 8.4 Hz, 1H, CH arom.);
7.58 (d, J = 16.0 Hz, 1H, CH@CH); 7.15 (d, J = 8.4 Hz, 1H, CH arom.);
6.98 (s, 1H, CHCO); 6.83 (d, J = 8.4 Hz, 1H, CH arom.); 6.74 (s, 2H,
CH arom.); 6.33 (d, J = 16.0 Hz, 1H, CH@CH); 4.36 (t, J = 6.8 Hz,
2H, CH2O); 4.19 (t, J = 6.8 Hz, 2H, CH2O); 3.98 (s, 3H, OCH3); 3.88
(s, 6H, OCH3); 3.87 (s, 3H, OCH3); 2.37–2.33 (m, 4H, NCH2); 2.22
(s, 3H, NCH3); 1.80–1.43 (m, 12H, CH2) ppm. 13C NMR (CDCl3) d:
178.05 (C); 167.01 (C); 160.02 (C); 159.04 (C); 157.92 (C); 153.44
Oxalate: mp 160–167 °C. Anal: C35H43NO15 (C, H, N).
4.1.16.10. 5-{[5-({2-[4-(5,7-Dihydroxy-4-oxo-4H-chromen-2-yl)phe-
noxy] acetyl}oxy)pentyl](methyl)amino}pentyl (2E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-enoate 10. Free base: chromatographic
Please cite this article in press as: Dei S., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.016

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S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
eluent: CH2Cl2/MeOH 95:5. Yield 18%. 1H NMR (CD3OD) d: 7.85 (d,
J = 8.4 Hz, 2H, CH arom.); 7.57 (d, J = 16.0 Hz, 1H, CH@CH); 6.92 (d,
J = 8.4 Hz, 2H, CH arom.); 6.88 (s, 2H, CH arom.); 6.64 (d, J = 2.0 Hz,
1H, CH arom.); 6.62 (s, 1H, CHCO); 6.43 (d, J = 16.0 Hz, 1H,
CH@CH); 6.34 (d, J = 2.0 Hz, 1H, CH arom.); 4.87 (s, 2H, OCH2CO);
4.26 (t, J = 6.4 Hz, 2H, CH2O); 4.21 (t, J = 6.4 Hz, 2H, CH2O); 3.84
(s, 6H, OCH3); 3.79 (s, 3H, OCH3); 2.99–2.91 (m, 4H, NCH2); 2.72
(s, 3H, NCH3); 1.78–1.68 (m, 12H, CH2) ppm.
4H, CH2); 1.41–1.29 (m, 4H, CH2) ppm. 13C NMR (CDCl3) d:
178.30 (C); 168.34 (C); 167.01 (C); 163.06 (C); 160.51 (C); 156.18
(C); 153.44 (C); 144.61 (CH@CH); 140.13 (C); 133.64 (CH);
129.92 (C); 128.79 (CH); 128.16 (CH); 128.06 (CH); 125.68 (CH);
125.15 (CH); 123.94 (C); 117.97 (CH); 117.44 (CH@CH); 115.09
(CH); 106.50 (CH); 105.26 (CH); 65.59 (CH2O); 65.24 (CH2O);
64.57 (CH2O); 60.96 (OCH3); 58.61 (NCH2Ph); 56.16 (OCH3);
53.62 (CH2N); 53.52 (CH2N); 28.63 (CH2); 28.38 (CH2); 26.66
(2CH2); 23.77 (CH2); 23.57 (CH2) ppm.
Oxalate: mp 165–166 °C. Anal: C42H49NO16 (C, H, N).
Hydrochloride: mp 58–60 °C. Anal: C46H52ClNO10 (C, H, N).
4.1.16.11. 2-{2-[(2-{2-[(E)-5,7-Dihydroxy-4-oxo-4H-chromene-2-car-
bonyloxy] ethoxy}ethyl)(methyl)amino]ethoxy}ethyl (2E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-enoate 11. Free base: chromatographic
eluent: CH2Cl2/petroleum ether/abs.EtOH/NH4OH 340:60:65:8.
Yield 79%. 1H NMR (CDCl3) d: 7.53 (d, J = 16.0 Hz, 1H, CH@CH);
6.71 (s, 1H, CHCO); 6.69 (s, 2H, CH arom.); 6.29 (d, J = 16.0 Hz,
1H, CH@CH); 6.28 (d, J = 2.0 Hz, 1H, CH arom.); 6.20 (d, J = 2.0 Hz,
1H, CH arom.); 4.38 (t, J = 6.4 Hz, 2H, CH2O); 4.28 (t, J = 6.4 Hz,
2H, CH2O); 3.85 (s, 9H, OCH3); 3.78–3.70 (m, 2H, OCH2); 3.69–
3.61 (m, 6H, OCH2); 2.83–2.75 (m, 4H, NCH2); 2.42 (s, 3H, NCH3)
ppm. 13C NMR (CDCl3) d: 181.54 (C); 166.89 (C); 166.06 (C);
162.06 (C); 159.78 (C); 157.52 (C); 153.37 (C); 151.27 (C); 145.17
(CH@CH); 140.07 (C); 129.77 (C); 116.86 (CH@CH); 113.48 (CH);
105.50 (C); 105.29 (CH); 100.63 (CH); 95.18 (CH); 69.07 (CH2O);
68.91 (CH2O); 68.45 (CH2O); 68.32 (CH2O); 68.22 (CH2O); 68.06
(CH2O); 65.76 (CH2O); 63.64 (CH2O); 56.94 (NCH2); 56.78
(NCH2); 56.20 (OCH3); 56.06 (OCH3); 42.50 (NCH3) ppm.
4.1.16.14. 2-{2-[Methyl-({2-[2-({2-[4-(4-oxo-4H-chromen-2-yl)phe-
noxy]acetyl}oxyethoxy]ethyl})amino]ethoxy}ethyl (2E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-enoate 15. Free base: chromatographic
eluent: CH2Cl2/MeOH/NH4OH 93:7:0.3 Yield 6.2%. 1H NMR (CDCl3)
d: 8.21 (d, J = 7.6 Hz, 1H, CH arom.); 7.88 (d, J = 8.8 Hz, 2H, CH
arom.); 7.68 (t, J = 7.6 Hz, 1H, CH arom.); 7.62–7.53 (m, 2H, CH
arom. and CH@CH); 7.41 (t, J = 7.6 Hz, 1H, CH arom.); 7.03 (d, J =
8.8 Hz, 2H, CH arom.); 6.75 (s, 2H, CH arom.); 6.74 (s, 1H, CHCO);
6.37 (d, J = 16.0 Hz, 1H, CH@CH); 4.76 (s, 2H, OCH2CO); 4.39–4.34
(m, 4H, 2CH2OCO); 3.88 (s, 9H, OCH3); 3.74–3.49 (m, 8H, CH2O);
2.72–2.64 (m, 4H, CH2N); 2.36 (s, 3H, NCH3) ppm. ESI-MS:
[MÀH]+ species at m/z 706.
Hydrochloride: low melting solid. Anal: C38H44ClNO12 (C, H, N).
4.1.16.15.
2-{2-[Benzyl({2-[2-({2-[4-(4-oxo-4H-chromen-2-yl)phe-
Oxalate: mp 53–56 °C. Anal: C33H39NO17 (C, H, N).
noxy]acetyl}oxy)ethoxy]ethyl})amino]ethoxy}ethyl (2E)-3-(3,4,5-tri-
methoxyphenyl)prop-2-enoate 16. Free base: chromatographic
eluent: CH2Cl2/MeOH/NH4OH 95:5:0.5. Yield 73.0%. 1H NMR
(CDCl3) d: 8.20 (d, J = 8.0 Hz, 1H, CH arom.); 7.87 (d, J = 8.8 Hz,
2H, CH arom.); 7.66 (t, J = 8.0 Hz, 1H, CH arom.); 7.59 (d, J = 16.0 Hz,
1H, CH@CH); 7.53 (d, J = 8.0 Hz, 1H, CH arom.); 7.39 (t, J = 8.0 Hz,
1H, CH arom.); 7.35–7.20 (m, 5H, CH arom.); 7.00 (d, J = 8.8 Hz,
2H, CH arom.); 6.73 (s, 1H, CHCO); 6.72 (s, 2H, CH arom.); 6.36 (d, J
= 16.0 Hz, 1H, CH@CH); 4.69 (s, 2H, OCH2CO); 4.35–4.31 (m, 4H,
CH2OCO); 3.87 (s, 6H, OCH3); 3.85 (s, 3H, OCH3); 3.71–3.64 (m, 4H,
OCH2); 3.62 (s, 2H, NCH2Ph); 3.60–3.55 (m, 4H, OCH2); 2.79–2.73
(m, 4H, CH2N) ppm. 13C NMR (CDCl3) d: 178.33 (C); 168.24 (C);
166.85 (C); 163.06 (C); 160.43 (C); 156.19 (C); 153.43 (C); 145.09
(CH@CH); 133.64 (CH); 129.81 (C); 128.24 (CH); 128.07 (CH);
125.69 (CH); 125.15 (CH); 123.94 (C); 117.97 (CH); 117.04 (CH@CH);
115.11 (CH); 106.53 (CH); 105.31 (CH); 69.00 (OCH2); 68.54 (OCH2);
65.11 (OCH2); 64.44 (OCH2); 63.59 (OCH2); 60.96 (OCH3); 59.74
(NCH2Ph); 56.16 (OCH3); 53.83 (CH2N) ppm.
4.1.16.12.
5-{Methyl-[5-({2-[4-(4-oxo-4H-chromen-2-yl)phenoxy]
acetyl}oxy) pentyl]amino}pentyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 12. Free base: chromatographic eluent: CH2Cl2/
diethyl
ether/petroleum
ether/abs.EtOH/NH4OH
120:120:300:60:3.3. Yield 78.5%. 1H NMR (CDCl3) d: 8.17 (d, J =
8.0 Hz, 1H, CH arom.), 7.84 (d, J = 9.2 Hz, 2H, CH arom.); 7.64 (t, J =
8.0 Hz, 1H, CH arom.); 7.55 (d, J = 16.0 Hz, 1H, CH@CH); 7.49 (d,
J = 8.0 Hz, 1H, CH arom.); 7.36 (t, J = 8.0 Hz, 1H, CH arom.); 6.98
(d, J = 9.2 Hz, 2H, CH arom.); 6.71 (s, 2H, CH arom.); 6.69 (s, 1H,
CHCO); 6.31 (d, J = 16.0 Hz, 1H, CH@CH); 4.67 (s, 2H, OCH2CO);
4.20–4.16 (m, 4H, CH2OCO); 3.84 (s, 9H, OCH3); 2.31–2.26 (m,
4H, CH2N); 2.17 (s, 3H, NCH3); 1.70–1.63 (m, 4H, CH2); 1.52–1.25
(m, 8H, CH2); ppm. 13C NMR (CDCl3) d: 178.23 (C); 168.33 (C);
166.97 (C); 163.01 (C); 160.48 (C); 156.13 (C); 153.40 (C); 144.58
(CH@CH); 133.62 (CH); 129.90 (C); 128.02 (CH); 125.62 (CH);
125.12 (CH); 125.04 (C); 123.89 (C); 117.95 (CH); 117.42 (CH@CH);
115.06 (CH); 106.43 (CH); 105.22 (CH); 65.52 (CH2O); 65.21
(CH2O); 64.50 (CH2O); 60.92 (OCH3); 57.62 (CH2N); 57.53
(CH2N); 56.13 (OCH3); 56.10 (OCH3); 42.13 (NCH3); 28.68 (CH2);
28.45 (CH2); 26.87 (CH2); 26.82 (CH2); 23.93 (CH2); 23.75 (CH2)
ppm.
Hydrochloride: low melting solid. Anal: C44H48ClNO12 (C, H, N).
4.1.17. 5-{[(tert-Butoxy)carbonyl](5-hydroxypentyl)amino}pentyl
(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate 44
To a solution of 4232 (278 mg, 0.68 mmol) in 10 mL of THF stir-
red at 0 °C, a solution of 185 mg of di-tert-butyl dicarbonate (0.85
mmol) in 5 mL of THF and triethylamine (0.19 mL, 1.36 mmol)
were added, and the mixture was stirred 30 min in ice-bath and
18 h at room temperature. The solution was concentrated in vacuo
and the oily residue was dissolved in CH2Cl2; the organic solution
was washed twice with water, dried on Na2SO4, and evaporated
yielding 275 mg (yield: 79.5%) of title compound as a yellow oil
that was not further purified. 1H NMR (CDCl3) d: 7.58 (d, J = 15.8
Hz, 1H, CH@CH); 6.75 (s, 2H, CH arom.); 6.33 (d, J = 15.8 Hz, 1H,
CH@CH); 4.19 (t, J = 6.4 Hz, 2H, CH2OCO); 3.89 (s, 6H, OCH3);
3.87 (s, 3H, OCH3); 3.64–3.61 (m, 2H, CH2OH); 3.20–3.14 (m, 4H,
CH2N); 1.74–1.69 (m, 4H, CH2); 1.45 (s, 9H, t-butyl); 1.74–1.32
(m, 8H, CH2) ppm.
Hydrochloride: mp 105–107 °C. Anal: C40H48ClNO10 (C, H, N).
4.1.16.13.
5-{Benzyl[5-({2-[4-(4-oxo-4H-chromen-2-yl)phenoxy]
acetyl}oxy)pentyl] amino}pentyl (2E)-3-(3,4,5-trimethoxyphenyl)
prop-2-enoate 13. Free base: chromatographic eluent: CH2Cl2/
MeOH/NH4OH 95:5:0.5. Yield 50.8%. 1H NMR (CDCl3) d: 8.20 (d, J
= 7.6 Hz, 1H, CH arom.); 7.87 (d, J = 8.8 Hz, 2H, CH arom.); 7.66
(t, J = 7.6 Hz, 1H, CH arom.); 7.58 (d, J = 16.0 Hz, 1H, CH@CH);
7.52 (d, J = 7.6 Hz, 1H, CH arom.); 7.39 (t, J = 7.6 Hz, 1H, CH arom.);
7.31–7.20 (m, 5H, CH arom.); 7.00 (d, J = 8.8 Hz, 2H, CH arom.);
6.74 (s, 1H, CHCO); 6.72 (s, 2H, CH arom.); 6.33 (d, J = 16.0 Hz,
1H, CH@CH); 4.68 (s, 2H, OCH2CO); 4.20–4.15 (m, 4H, CH2OCO);
3.87 (s, 6H, OCH3); 3.85 (s, 3H, OCH3); 3.53 (s, 2H, NCH2Ph);
2.42–2.38 (m, 4H, CH2N); 1.67–1.60 (m, 4H, CH2); 1.53–1.45 (m,
Please cite this article in press as: Dei S., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.016

S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
4.1.18. 5-{[(tert-Butoxy)carbonyl][5-({2-[4-(4-oxo-4H-chromen-2-yl)
phenoxy]acetyl}oxy)pentyl]amino}pentyl (2E)-3-(3,4,5-
trimethoxyphenyl)prop-2-enoate 46
Hz, 1H, CH arom.); 7.39 (t, J = 8.0 Hz, 1H, CH arom.); 7.02 (d, J =
8.8 Hz, 2H, CH arom.); 6.74 (s, 3H, CHCO and CH arom.); 6.33 (d,
J = 15.6 Hz, 1H, CH@CH); 4.70 (s, 2H, OCH2CO); 4.23–4.16 (m, 4H,
CH2OCO); 3.87 (s, 9H, OCH3); 2.64–2.59 (m, 4H, CH2N); 1.71–
1.33 (m, 12H, CH2) ppm. 13C NMR (CDCl3) d: 178.33 (C); 168.36
(C); 167.00 (C); 163.09 (C); 160.51 (C); 156.19 (C); 153.44 (C);
144.67 (CH@CH); 133.66 (CH); 129.91 (C); 128.08 (CH); 125.68
(CH); 125.17 (CH); 123.92 (C); 117.97 (CH); 117.39 (CH@CH);
115.10 (CH); 106.50 (CH); 105.28 (CH); 65.44 (OCH2); 65.25
(OCH2); 64.42 (OCH2); 60.95 (OCH3); 56.17 (OCH3); 49.64
(CH2N); 49.56 (CH2N); 29.31 (CH2); 29.21 (CH2); 28.65 (CH2);
28.41 (CH2); 23.79 (CH2); 23.61 (CH2) ppm.
To 275 mg (0.54 mmol) of alcohol 44 dissolved in 25 mL of
anhydrous CH2Cl2, a solution of 240 mg (0.81 mmol) of acid 30 in
25 mL of anhydrous THF, 53 mg (0.43 mmol) of DMAP and 186
mg (0.97 mmol) of EDCI were added. The reaction mixture was
stirred at room temperature for 70 h, then it was evaporated. The
obtained residue was dissolved in CH2Cl2, and the organic layer
was washed twice with water and one time with a saturated solu-
tion of NaHCO3, dried on Na2SO4 and evaporated to give the crude
ester. After purification by flash chromatography, using CH2Cl2/
MeOH/NH4OH 95:5:0.5 as eluting system, 189 mg (44.5% yield)
of title compound as colorless oil were obtained. 1H NMR (CDCl3)
d: 8.21 (d, J = 8.0 Hz, 1H, CH arom.); 7.88 (d, J = 9.2 Hz, 2H, CH
arom.); 7.67 (t, J = 8.0 Hz, 1H, CH arom.); 7.56–7.49 (m, 2H, CH
arom. and CH@CH); 7.41 (t, J = 8.0 Hz, 1H, CH arom.); 7.02 (d, J =
9.2 Hz, 2H, CH arom.); 6.74 (s, 2H, CH arom.); 6.73 (s, 1H, CHCO);
6.32 (d, J = 16.0 Hz, 1H, CH@CH); 4.69 (s, 2H, OCH2CO); 4.23–4.18
(m, 4H, CH2OCO); 3.87 (s, 6H, OCH3); 3.86 (s, 3H, OCH3); 3.18–
3.13 (m, 4H, CH2N); 1.78–1.67 (m, 4H, CH2); 1.56–1.49 (m, 4H,
CH2); 1.44 (s, 9H, t-butyl); 1.40–1.29 (m, 4H, CH2) ppm.
Hydrochloride: 110–112 °C. Anal: C39H46ClNO10 (C, H, N).
4.1.20.2.
2-[2-({2-[2-({2-[4-(4-Oxo-4H-chromen-2-yl)phenoxy]
acetyl}oxy)ethoxy]ethyl}amino)ethoxy]ethyl (2E)-3-(3,4,5-trimethox-
yphenyl)prop-2-enoate 17. Free base: chromatographic eluent:
CH2Cl2/MeOH/NH4OH 95:5:0.5. Yield 87.6%. 1H NMR (CDCl3) d:
8.20 (d, J = 8.0 Hz, 1H, CH arom. 7.87 (d, J = 8.8 Hz, 2H, CH arom.);
7.66 (t, J = 8.0 Hz, 1H, CH arom.); 7.59 (d, J = 16.0 Hz, 1H, CH@CH);
7.53 (d, J = 8.0 Hz, 1H, CH arom.); 7.40 (t, J = 8.0 Hz, 1H, CH arom.);
7.02 (d, J = 8.8 Hz, 2H, CH arom.); 6.73 (s, 3H, CHCO and CH arom.);
6.37 (d, J = 16.0 Hz, 1H, CH@CH); 4.73 (s, 2H, OCH2CO); 4.37–4.33
(m, 4H, CH2OCO); 3.85 (s, 9H, OCH3); 3.74–3.57 (m, 8H, OCH2);
2.85–2.80 (m, 4H, CH2N) ppm. 13C NMR (CDCl3) d: 178.33 (C);
168.27 (C); 166.86 (C); 163.06 (C); 160.44 (C); 156.17 (C); 153.42
(C); 145.12 (CH@CH); 133.64 (CH); 129.80 (C); 128.08 (CH);
125.67 (CH); 125.15 (CH); 123.91 (C); 117.97 (CH); 117.01
(CH@CH); 115.12 (CH); 106.49 (CH); 105.30 (CH); 70.57 (OCH2);
69.11 (OCH2); 68.66 (OCH2); 65.12 (OCH2); 64.37 (OCH2); 63.55
(OCH2); 60.95 (OCH3); 56.15 (OCH3); 49.19 (CH2N); 49.15 (CH2N)
ppm.
4.1.19. 2-(2-{[(tert-Butoxy)carbonyl]({2-[2-({2-[4-(4-oxo-4H-
chromen-2-yl)phenoxy]acetyl}oxy)ethoxy]ethyl})amino}ethoxy)ethyl
(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate 47
To 99 mg (0.19 mmol) of alcohol 4527 dissolved in 15 mL of
anhydrous CH2Cl2, a solution of 86 mg (0.10 mmol) of acid 30 in
15 mL of anhydrous THF, 19 mg (0.29 mmol) of DMAP and 66 mg
(0.35 mmol) of EDCI were added. The reaction mixture was stirred
at room temperature for 70 h, then it was evaporated. The obtained
residue was dissolved in CH2Cl2, and the organic layer was washed
twice with water and one time with a saturated solution of
NaHCO3, dried on Na2SO4 and evaporated to give the crude ester.
After purification by flash chromatography, using CH2Cl2/MeOH/
NH4OH 95:5:0.5 as eluting system, 72 mg (47.2% yield) of title
compound as yellow oil were obtained. 1H NMR (CDCl3) d: 8.20
(d, J = 7.6 Hz; 1H, CH arom.); 7.87 (d, J = 8.8 Hz, 2H, CH arom.);
7.66 (t, J = 7.6 Hz, 1H, CH arom.); 7.59 (d, J = 15.8 Hz, 1H, CH@CH);
7.53 (d, J = 8.0 Hz, 1H, CH arom.); 7.40 (t, J = 8.0 Hz, 1H, CH arom.);
7.02 (d, J = 8.8 Hz, 2H, CH arom.); 6.75 (s, 2H, CH arom.); 6.73 (s,
1H, CHCO); 6.36 (d, J = 15.8 Hz, 1H, CH@CH); 4.73 (s, 2H, OCH2CO);
4.37–4.32 (m, 4H, CH2OCO); 3.87 (s, 3H, OCH3); 3.86 (s, 6H, OCH3);
3.72–3.53 (m, 8H, OCH2); 3.50–3.42 (m, 4H, CH2N); 1.44 (s, 9H, t-
butyl) ppm.
Hydrochloride: 122–123 °C. Anal: C37H42ClNO12 (C, H, N).
4.2. Biology
4.2.1. Cell lines and cultures
The K562 is an undifferentiated erythroleukemia cell line orig-
inally derived from
a patient with chronic myelogenous
leukemia.36 The K562 leukemia cells and the P-gp over-expressing
K562/DOX cells were obtained from Prof. J.P. Marie (Hopital Hotel-
Dieu, Paris, France). The cells were cultured following a previously
reported protocol.42
4.2.2. Drugs and chemicals
Purified verapamil and pirarubicin were purchased by Sigma-
Aldrich (Milan – Italy). Concentrations were determined by dilut-
4.1.20. General procedure for the synthesis of diester compounds 14
and 17
ing stock solutions to approximately 10À5 M and using e480
=
To a solution of the suitable BOC-protected ester (46 or 47) (0.1
mmol) in anhydrous CH2Cl2, 26 eq of trifluoroacetic acid were
added. The mixture was stirred 45 min at room temperature, then
it was evaporated. The obtained residue was dissolved in CH2Cl2,
and the organic layer was washed three times with a saturated
solution of NaHCO3, dried on Na2SO4 and evaporated to give the
crude ester, which was purified by flash chromatography using
the appropriate eluting system, as reported hereinafter. The com-
pounds were transformed into the corresponding hydrochloride
as white solid. The salts were crystallized from abs. ethanol/petro-
leum ether.
11,500 MÀ1 cmÀ1. Stock solutions were prepared just before use.
Buffer solutions were HEPES buffer containing 5 mM HEPES, 132
mM NaCl, 3.5 mM CaCl2, 5 mM glucose, at pH 7.3.
The uptake of pirarubicin in cells was followed by monitoring
the decrease in the fluorescence signal at 590 nm (kex = 480 nm)
according to the previously described method.43,44
4.3. Molecular dynamics simulations
A 10 ns MD simulation was performed for all molecules using
GROMACS v5.1 program45. The DS ViewerPro 6.0 program46 was
used to build the initial conformations of molecules. The partial
atomic charge of the structures, were calculated with CHIMERA47
using AM1-BCC method and the topology was created with
4.1.20.1. 5-{[5-({2-[4-(4-Oxo-4H-chromen-2-yl)phenoxy]acetyl}oxy)
pentyl]amino}pentyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate
14. Free base: chromatographic eluent: CH2Cl2/MeOH/NH4OH
97:3:0.3. Yield 88.7%. 1H NMR (CDCl3) d: 8.20 (d, J = 8.0 Hz, 1H,
CH arom.); 7.88 (d, J = 8.8 Hz, 2H, CH arom.); 7.67 (t, J = 8.0 Hz,
1H, CH arom.); 7.57 (d, J = 15.6 Hz, 1H, CH@CH); 7.53 (d, J = 8.0
ACPYPE48 based on the routine Antechamber49
.
The OPLS-AA/L all-atom force field50 parameters were applied
to all the structures.
Please cite this article in press as: Dei S., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.016

14
S. Dei et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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The simulation was performed in vacuum. To remove bad con-
Gland Biol Neoplasia. 2009;14:3–9.
tacts, energy minimization was performed using the steepest des-
cent algorithm until convergence is achieved or for 50,000
maximum steps. In the consecutive steps, equilibrations of the sys-
tem were conducted in two phases: (1) canonical NVT ensemble, a
100 ps position-restrained of molecules at 300 K was carried out
using a Temperature coupling thermostat (velocity rescaling with
a stochastic term) to ensure the proper stabilization of the temper-
ature51; (2) isothermalisobaric NPT ensemble, a 100 ps position-
restrained of molecules at 300 K and 1 bar was carried out without
using barostat pressure coupling to stabilize the system. These
were then followed by a 10 ns MD run without position restraints
at 300 K. the Lincs algorithm52 was used for bond constraints to
maintain rigid bond lengths.
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Modulation of P-glycoprotein-mediated multidrug resistance by flavonoid
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The initial velocity was randomly assigned taken from Max-
well–Boltzman distribution at 300 K and computed with a time
step of 2 fs, and the coordinates were recorded every 0.1 ns.
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Acknowledgments
The authors thank Dr. Cristina Bellucci for her excellent technical
assistance and prof. Fabrizio Melani for helpful discussion. This
work was supported by grants from the University of Florence
(Fondo Ricerca Ateneo RICATEN15).
a
series of N, N-bis(alkanol)amine aryl esters as P-glycoprotein (Pgp)
dependent multidrug resistance (MDR) inhibitors.
2010;53:1755–1762.
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simultaneous binding of two drugs to a multidrug binding protein. EMBO J.
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Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.11.016.
30. Sauna ZE, Andrus MB, Turner TM, Ambudkar SV. Biochemical basis of
polyvalency as
a strategy for enhancing the, efficacy of P-glycoprotein
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