Multidrug resistance (MDR) reversers: High activity and efficacy in a series of asymmetrical N, N -bis(alkanol)amine aryl esters

Article abstract of DOI:10.1016/j.ejmech.2014.09.084

As a continuation of our research on potent and efficacious P-gp-dependent multidrug resistance (MDR) reversers, several new N,N-bis(alkanol)amine aryl esters were designed and synthesized, varying the aromatic moieties or the length of the methylenic chain. The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay, where most of the new compounds were shown to be active. In particular the asymmetrical compounds, characterized by two linkers of different length, generally showed fairly high activities as MDR reversers. Some selected compounds (isomers 15-17) were further studied by evaluating their doxorubicin cytotoxicity enhancement (reversal fold, RF) on the K562/DOX cell line. The results of both pharmacological assays indicate that compounds 16 (GDE6) and 17 (GDE19) could be interesting leads for the development of new P-gp dependent MDR modulators.

Full text of DOI:10.1016/j.ejmech.2014.09.084

European Journal of Medicinal Chemistry 87 (2014) 398e412
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Multidrug resistance (MDR) reversers: High activity and efficacy in a
series of asymmetrical N,N-bis(alkanol)amine aryl esters
,
Silvia Dei a *, Marcella Coronnello b, Elisa Floriddia a, Gianluca Bartolucci a,
Cristina Bellucci a, Luca Guandalini a, Dina Manetti a, Maria Novella Romanelli a,
Milena Salerno c, Ivan Bello c, Enrico Mini b, Elisabetta Teodori a
a
ꢀ
Dipartimento NEUROFARBA-Sezione di Farmaceutica e Nutraceutica, Universita di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy
b
ꢀ
Dipartimento di Scienze della Salute, Universita di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy
Universite Paris 13, Sorbonne Paris Cite, Laboratoire CSPBAT, CNRS (UMR 7244), UFR-SMBH, 74 Rue Marcel Cachin, 93017 Bobigny, France
c
ꢁ
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2014
Received in revised form
12 September 2014
Accepted 26 September 2014
Available online 30 September 2014
As a continuation of our research on potent and efficacious P-gp-dependent multidrug resistance (MDR)
reversers, several new N,N-bis(alkanol)amine aryl esters were designed and synthesized, varying the
aromatic moieties or the length of the methylenic chain. The new compounds were tested on
doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay, where
most of the new compounds were shown to be active. In particular the asymmetrical compounds,
characterized by two linkers of different length, generally showed fairly high activities as MDR reversers.
Some selected compounds (isomers 15e17) were further studied by evaluating their doxorubicin cyto-
toxicity enhancement (reversal fold, RF) on the K562/DOX cell line. The results of both pharmacological
assays indicate that compounds 16 (GDE6) and 17 (GDE19) could be interesting leads for the develop-
ment of new P-gp dependent MDR modulators.
Dedicated to the memory of our colleague
and friend Professor Serena Scapecchi pre-
maturely deceased.
Keywords:
MDR reversers
© 2014 Elsevier Masson SAS. All rights reserved.
P-gp inhibitors
N,N-bis(alkanol)amine aryl esters
Chemosensitizers
Doxorubicin-resistant erythroleukemia
K562 cells (K562/DOX)
Pirarubicin uptake
1. Introduction
first formulated 40 years ago [2] and confirmed when P-glycopro-
tein (P-gp) was identified and studied [3]. Further studies have
Multidrug resistance (MDR) occurs when cancer cells become
resistant to chemotherapeutic drugs of different structure and
mechanism of action, resulting in the failure of cancer treatment
[1]. This kind of resistance is associated with the over-expression of
a family of ATP-dependent transporter proteins that extrude the
chemotherapeutic drug from the cells, lowering its concentration
below that necessary for anticancer action. This hypothesis was
shown that P-gp (ABCB1) is the most important member of a super
family of ATP-dependent transporter proteins that includes BCRP
(ABCG2) and MRP1 (ABCC1) as other representative players. P-gp,
the best studied member of the family, is a highly permissive
protein which transports a wide diversity of substrates. Intensive
work on this family of transporters has shown that multidrug
transporters are present in almost every cell where their physio-
logical role seems to be that of controlling the traffic of lipophilic
molecules and protecting the cell from toxic xenobiotics by
extruding them from the cell [4,5]. ABC proteins and especially P-gp
are expressed in many tissues, mostly in membrane barriers such as
BBB (bloodebrain barrier) [6]. Although a high resolution crystal
structure of human P-gp is still lacking, fundamental information
on its structure and mechanism of action has been collected from
the resolution of the 3D-structures of the bacterial homolog
Sav1866 [7] and murine P-gp (having 87% of homology with human
List of abbreviations: P-gp, P-glycoprotein; BCRP, breast cancer resistance pro-
tein; MRP1, multidrug resistance-associated protein 1; ABCB1, ATP binding cassette
protein B1; ABCG2, ATP binding cassette protein G2; ABCC1, ATP binding cassette
protein
ethylcarbodiimmide
C1;
DOX,
doxorubicin;
hydrochloride;
EDCl,
MTT,
1-(3-dimethylaminopropyl)-3-
3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide.
* Corresponding author.
E-mail address: silvia.dei@unifi.it (S. Dei).
http://dx.doi.org/10.1016/j.ejmech.2014.09.084
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
399
P-gp) [8,9] that has allowed the development of several useful
homology models [[10] and therein cited literatures].
Since their discovery and the elucidation of the mechanism of
action, P-gp and related proteins have been considered suitable
targets for circumventing transporter-dependent MDR [11].
Verapamil and cyclosporin
A were the early compounds
showing P-gp modulating activity and, together with many other
molecules, belong to the first generation of P-gp modulators.
However, their toxicity prevented their clinical use and, at present,
verapamil is only used as a standard in the search for new MDR
reversing molecules.
A large number of compounds showing MDR modulating ac-
tivity has been synthesized and studied since then (classified as
second- or third-generation) [12e14] and several have reached
clinical trials [15]. However, none has been approved for clinical
use. Their low potency, unsatisfactory toxicity and adverse changes
in the pharmacokinetic properties of the co-administered chemo-
therapeutic drug [16] have so far blocked their development and
some experts have expressed skepticism about the future of MDR
modulators [17,18]. Nevertheless, the search for new, safer, more
potent and efficacious multidrug transporter modulators is still of
interest, considering that such modulators seem to be useful for
different purposes. In fact, P-gp has been recently considered an
interesting target for CNS pathologies on the basis that it is an in-
tegral part of BBB [19,20]. It has been proposed that in Alzheimer's
Chart 1. General structures of designed compounds.
geometry; this residue is also present in previously synthesized
potent compounds [28,29].
Finally, the scaffold of quinine and quinidine (g and h, Chart 1)
were chosen as being well known P-gp transport modulators
[30,31]; since these residues were proved not to be productive, the
6-methoxyquinolinic moiety (f, Chart 1) was inserted to verify
whether a simplified structure was better.
disease, the extrusion through the BBB membrane of toxic A
b
amyloid, often because of age-related reduction of P-gp, is impaired
and that positive modulation of the transporter, for instance
through P-gp enhancers, would be beneficial in preventing the
As a second approach we decided also to explore the conse-
quences of varying the length of the spacers and of the relative
position of the nitrogen in the chain, abandoning the symmetry of
the linkers that characterized the previous series. To explore this
point, we synthesized new derivatives of the N,N-bis(alkanol)
amine series conserving the two aromatic ester portions (aee,
Chart 1), chosen by the same criteria as above, but connected by a
chain of nine components, eight methylenes and one nitrogen
atom, where the position of the nitrogen atom changes according to
the length of the two spacers (3- and 5-methylenes long). The new
compounds were compared to those having two spacers of iden-
tical length containing 4 methylenes that we synthesized in the
present work for the first time (12e26, Chart 1).
The reversal activity of the new compounds was evaluated by
the pirarubicin uptake assay in doxorubicin-resistant eryth-
roleukemia K562 cells; this assay was used as preliminary
screening. The compounds that showed the best activity in this test,
the set of isomers 15e17, were further studied by evaluating their
doxorubicin cytotoxicity enhancement (reversal fold, RF) on the
K562/DOX cell line.
disease [21,22]. However, the transport of Ab amyloid by P-gp re-
mains controversial [23,24]. On the contrary, the temporary nega-
tive modulation of the transporter could allow easier penetration of
drugs into the brain to exert their action [25]. On the whole, there
are several reasons for medicinal chemists to continue the search
for molecules that could be used to modulate and study P-gp and
related proteins.
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 portions by two identical poly-
methylenic chains of variable length as spacers. These compounds
were designed based on the information that the presence of
aromatic moieties and of one or more protonable nitrogen atoms
is an important property for the Pegp interaction. Molecular
flexibility was added as a further characteristic to allow the
molecules to choose the most productive binding modes, within
the large P-gp recognition site, which would result in a high af-
finity interaction. Actually, this approach provided good results
since most of those compounds proved to be very potent MDR
reversers [26,27].
Aiming to expand the structureeactivity relationships and to
optimize the potency and efficacy of compounds in this family, we
have synthesized new derivatives (1e11, Chart 1) carrying other
aromatic ester moieties. The spacers chosen, 3- or 5-methylenes
long, were those showing the best results in previous studies
[26,27].
The (E)-3-(3,4,5-trimethoxyphenyl)vinyl, 3,4,5-trimethoxy
phenyl and anthracene moieties (a, b and c, Chart 1) that were
present in the most potent of the previously synthesized com-
pounds [26,27] were combined with new aryl moieties (deh, Chart
1) chosen on the basis of the following rationale.
2. Chemistry
The reaction pathways used to synthesize the designed de-
rivatives (1e26) are reported in Scheme 1. The haloesters 27e35
were obtained by esterification of the suitable haloalkyl alcohol (3-
bromopropan-1-ol, 4-bromobutan-1-ol or 5-chloropentan-1-ol)
with the commercially available (E)-3-(3,4,5-trimethoxyphenyl)
acrylic acid, 3,4,5-trimethoxybenzoic acid or anthracene-9-
carboxylic acid. The desired esters were obtained by trans-
formation of the carboxylic acid in the corresponding acyl chloride
by reaction with SOCl2 in ethanol-free CHCl3, or by treatment of the
mixture of haloalkyl alcohol and acid with the activating agent EDCI
in the presence of DMAP in anhydrous CH2Cl2 (for details, see the
Experimental section). Derivatives 27e29 and 33 had already been
described by our group [26,27]; compound 32 had been obtained
The lipophilic fluorenyl moiety (d) is present in potent com-
pounds of the old sets studied [26,28]. The 3-(3,4,5-
trimethoxyphenyl)ethinyl moiety (e) can be seen as analogous to
the (E)-3-(3,4,5-trimethoxyphenyl)acrylic one (a) with altered

400
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
Final compounds 3 and 9, carrying the 6-methoxyquinolinic
moiety (residue f) were eventually obtained by reaction of 54
and 57 with the suitable acyl chloride obtained from the corre-
sponding 6-methoxyquinoline-4-carboxylic acid, which was
synthesized according to the literature [33] by oxidative demo-
lition of quinine sulfate. The other designed compounds 1e2, 4e8
and 10e26 were eventually obtained by reaction of 54e68 with
the proper carboxylic acid using EDCI and DMAP in anhydrous
CH2Cl2. The suitable acids are either commercially available ((E)-
3-(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic
acid, anthracene-9-carboxylic acid, 9H-fluorene-carboxylic acid),
synthesized
according
to
the
literature
(3-(3,4,5-
trimethoxyphenyl)propiolic acid [34,35]) or synthesized as re-
ported in Scheme 2. Commercially available quinine and quini-
dine were transformed in the corresponding ethyl acetate
derivatives 69 and 70, which yielded the acids 71 and 72 after
alkaline hydrolysis (Scheme 2).
2.1. Chemical stability
In order to evaluate the chemical stability of our compounds
during the biological tests, the derivatives 15e17, at a concentration
of 1 m
M, were maintained at 37 ꢀC in PBS for different durations.
The obtained samples were analyzed by LCeMS/MS as described in
the Experimental section. The results showed that, in the experi-
mental conditions, the degradation of all compounds tested was
negligible. Experimental data are reported in the Supplementary
material.
3. Biological studies
3.1. Modulation of pirarubicin uptake
The P-gp modulating properties of compounds 1e26 were
evaluated on the K562/DOX cell line. K562 is a human leukemia cell
line established from a patient with chronic myelogenous leukemia
in blast transformation [36]. K562/DOX cells resistant to doxoru-
bicin overexpress a unique membrane glycoprotein, P-gp [37e39].
The uptake of THP-adriamycin (pirarubicin) was measured by a
continuous spectrofluorometric signal of anthracycline at 590 nm
Scheme 1. (i) SOCl2, ethanol-free CHCl3 or EDCI, DMAP, an. CH2Cl2; for the details, see
the Experimental Section; (ii) NaI, acetone; (iii) H2N(CH2)mOH (m ¼ 3,4,5), CH3CN; (iv)
HCOOH/HCHO; (v) Ar1COCl, ethanol-free CHCl3 in the case of residue for Ar1COOH,
EDCI, DMAP, an. CH2Cl2 in the other cases; for the meaning of Ar and Ar1, see Tables 1
and 2.
(
lex ¼ 480 nm) after cell incubation, following the protocols re-
with a different procedure by He [32]. The chloropentane esters
33e35 were transformed in the corresponding iodo derivatives
36e38 with NaI in acetone, in order to achieve higher yields in the
following reaction. The bromo esters 27e32 and the iodo esters
36e38 were then transformed into the secondary amines 39e53 by
reaction with the corresponding aminoalcohol using standard
procedures. These compounds were alkylated by reductive
methylation with HCOOH/HCHO to give the corresponding tertiary
amines 54e68. The secondary amines 39, 41 and 42 and the cor-
responding methylated derivatives 54, 56 and 57 have been
described in our previous papers [26,27].
ported in previous papers [40,41]. The activity of the studied
compounds on pirarubicin uptake is expressed by: i) amax, which
represents the efficacy of the modulator and is the maximum in-
crease that can be obtained in the nuclear concentration of pirar-
ubicin in resistant cells 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); and ii) [I]0.5, which measures the potency of the modulator
and represents the concentration that causes a half-maximal in-
crease (
Tables 1 and 2).
a
¼ 0.5) in the nuclear concentration of pirarubicin (see
Scheme 2. (i) KOH, DMSO; BrCH2COOC2H5; (ii) KOH 10%.

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
401
Table 1
MDR-reversing activity of compounds 1e11.
[I]0.5 mMa
amax
b
N
n
Ar
Ar1
1
2
3
4
5
6
7
8
3
3
3
3
3
3
5
5
5
5
5
5
a
a
a
a
b
c
a
a
a
b
c
a
g
h
f
d
e
e
g
h
f
5.06 2.0
4.79 2.0
0.84 0.15
0.79 0.20
0.92 0.04
0.97 0.03
0.92 0.03
0.96 0.04
0.72 0.06
0.87 0.05
0.64 0.03
0.91 0.06
0.75 0.06
0.83 0.05
0.70 0.07
0.88 0.21
0.50 0.20
1.00 0.20
0.70 0.17
3.43 0.70
2.65 0.60
0.75 0.14
0.29 0.04
0.34 0.17
0.11 0.03c
1.6 0.30
9
10
11
e
e
d
ME32
Verapamil
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.
See Ref. [26].
a
¼ 0.5).
b
c
3.2. Antiproliferative effects and MDR reversal
(E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety with the scaffold of
two compounds that do modulate P-gp action such as quinine and
quinidine, failed to improve activity and, independently from the
length of the tether, compounds 1, 2, 7 and 8 were less potent than
verapamil, even if, when their structure is simplified as in the 6-
methoxyquinolinic moiety, the corresponding compounds (3, 9)
resulted more potent than the reference, in line with the activity of
the previously studied member of the family [26,27].
More interesting results were obtained with compounds 4e6
and 10e11, that contain (E)-3-(3,4,5-trimethoxyphenyl)vinyl,
3,4,5-trimethoxyphenyl and anthracene moieties combined with
fluorenyl and 3-(3,4,5-trimethoxyphenyl)ethinyl residues. As a
general trend, all the compounds bearing two 5-methylenic
chains are more potent than those with two 3-methylenic ones,
although the corresponding efficacies (amax) do not follow the
same trend. The most interesting compounds of the series are 10
and 11 as well as the previously synthesized compound ME32
[26].
The ability of selected compounds (15e17) to sensitize resistant
cells to doxorubicin was assessed on the K562/DOX cell line by
evaluating their reversal effects on the MDR phenotype. The com-
pounds were evaluated at 0.1, 1.0 and 3.0 mM concentrations in the
absence of doxorubicin to study their intrinsic cytotoxicity and in
the presence of doxorubicin to study their MDR-reversal activity on
both K562 and K562/DOX cell lines using the MTT (3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay
[42]. The potency of MDR modifiers was expressed by the reversal-
fold (RF) values, obtained as the ratio between the doxorubicin IC50
values on K562/DOX cells in the absence and in the presence of
modulators. The results are reported in Table 3 together with those
of the reference compound verapamil tested at 3.0
concentration.
mM
4. Results and discussion
These data confirmed our previous results [26,27] that the MDR
modulation activity of this family of molecules depends both on the
nature of the aromatic moieties and the length of the tethers. Since
one of the consequences of changing the length of the spacers is the
different distance of the nitrogen atom from the aromatic residues,
to collect more information on this aspect, we explored the effect of
making the molecular scaffold asymmetric, that is with two linkers
of different length. In this way the nitrogen atom is at different
distances from the two aromatic residues, according to the length
of the two different spacers.
As proof of concept, we prepared the compounds shown in
Table 2 (12, 13; 15, 16; 18, 19; 21, 22; 24, 25) where the two spacers
are 3- and 5-methylenes long, respectively. Since the two aromatic
moieties are different, two positional isomers are possible and they
were both synthesized. For comparison we also synthesized the
4.1. Modulation of pirarubicin uptake
The results of pirarubicin uptake modulation in the doxorubicin-
resistant erythroleukemia K562 cell line of the new compounds
were obtained using a previously described method [28] and are
reported in Table 1 (compounds 1e11) and 2 (compounds 12e26).
Verapamil, the gold standard of P-gp inhibition, was used as
reference compound. The previously synthesized compound ME32
[26] has been added in Table 1 for comparison with compound 4,
since it is its homolog with two 5-methylenic chains.
All the new molecules studied were able to modulate the ac-
tivity of P-gp, and, with few exceptions, were more potent than
verapamil. However, the data reported in Table
1 did not
completely fulfill our expectations. In particular, the combination of

402
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
Table 2
MDR-reversing activity of compounds 12e26.
Ma
amax
b
N
n
m
Ar
Ar1
[I]0.5
m
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
5
5
4
5
5
4
5
5
4
5
5
4
5
5
4
3
3
4
3
3
4
3
3
4
3
3
4
3
3
4
b
a
a
c
a
a
b
e
e
c
e
e
a
d
a
a
b
b
a
c
0.27 0.05
0.12 0.02
1.53 0.13
0.04 0.02
0.04 0.01
0.07 0.01
0.26 0.06
0.31 0.07
1.03 0.33
0.11 0.03
0.10 0.02
0.10 0.03
0.19 0.06
0.35 0.01
0.11 0.03
1.6 0.30
0.97 0.02
0.99 0.01
0.62 0.02
0.94 0.03
0.98 0.02
0.87 0.03
0.96 0.03
0.97 0.06
0.99 0.01
0.98 0.01
0.99 0.01
0.99 0.01
0.99 0.01
0.84 0.02
0.99 0.01
0.70 0.07
c
e
b
b
e
c
c
d
a
d
Verapamil
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.
a
¼ 0.5).
b
corresponding compounds having two 4-methylene spacers (14, 17,
20, 23, 26).
of nine components (eight methylenes and one nitrogen) con-
necting the two aromatic ester residues is very fruitful. In particular
the positional isomers 15e17 show potency in the low nanomolar
The data reported in Table 2 show that this second approach is
very promising, since with the exception of 14 and 20, all com-
pounds are active in the submicromolar range. Moreover, all de-
rivatives, except 14, show a high efficacy (amax), which is in most
cases close to 1, meaning that they can nearly completely reverse
pirarubicin extrusion in the test on the doxorubicin-resistant
erythroleukemia K562 cell line. These results indicate that a chain
range ([I]0.5 ¼ 0.04
mM for 15 and 16, and 0.07 mM for 17) and high
efficacies (amax ¼ 0.94, 0.99 and 0.87, respectively). These results
are unprecedented in the N,N-bis(alkanol)amine aryl esters series
and parallel the potency and efficacy of the conformationally
restrained analogous N,N-bis(cyclohexanol)amine aryl esters series
[28,29,43].
A thorough evaluation of the results reported in Table 2 in-
dicates that the asymmetric isomers of each set of compounds have
very similar activity while the corresponding symmetric isomer
behaves differently according to the set. Indeed, in two cases out of
five (sets 12e14, and 18e20) the symmetric compound is less
potent than both the asymmetric ones. In two cases (sets 15e17 and
21e23) the three compounds are equipotent and in another case
(set 24e26) the symmetric compound is more potent than the
asymmetric isomers.
These mixed results are not easily explainable. However, given
the extreme flexibility of this class of compounds and the variety of
possible interactions in the large P-gp recognition site, this fact
seems to confirm our already proposed hypothesis [27] that, very
likely, each N,N-bis(alkanol)amine aryl ester, both symmetrical and
asymmetrical, interacts with different binding modes.
Table 3
Effects of MDR-reversing agents 15e17 on doxorubicin cytotoxicity in K562 and
K562/DOX cells.
Compounds
K562 cells
IC50
M)a
K562/DOX cells
IC50
M)a
(
m
(
m
RFb
DOX
0.023 0.004
0.011 0.004
0.012 0.004
0.051 0.024
0.030 0.017
0.040 0.030
0.018 0.005
0.022 0.001
0.041 0.013
0.019 0.002
0.029d
2.00 0.24
0.62 0.19
0.32 0.10c
0.17 0.05c
0.30 0.06c
0.06 0.02c
0.035 0.008c
0.83 0.21
0.23 0.10c
0.063 0.01c
0.74 0.03
DOX þ 15 (0.1
DOX þ 15 (1.0
DOX þ 15 (3.0
DOX þ 16 (0.1
DOX þ 16 (1.0
DOX þ 16 (3.0
DOX þ 17 (0.1
DOX þ 17 (1.0
DOX þ 17 (3.0
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
mM)
3.2
6.3
11.8
6.7
33.3
57.1
2.4
8.7
31.8
2.7
This assumption seems supported by a docking experiment
made with the low energy conformations of derivatives 15e17 and
some of our previously synthesized compounds [26] to the active
DOX þ verapamil (3
a
mM)
Mean SE of at least 3 determinations performed with quadruplicate cultures at
each drug concentration tested and measured as described in Experimental section.
~
site of the human P-gp homology model [44] (G.M. Keseru, A.
b
Reversal fold of MDR was obtained as the ratio between the doxorubicin IC50
Tarksay, personal communication). The study failed to produce
significant results because of the uncertainty connected to the high
flexibility of our compounds and of the large binding pocket of the
transporter, making the docking calculations especially compli-
cated and unproductive.
values on K562/DOX cells in the absence and in the presence of modulators.
c
p < 0.05 vs control doxorubicin-treated; where not specified the IC50 value is not
significant compared to that of control cells (K562 and K562/DOX cells treated with
doxorubicin only).
d
Mean of 2 determinations performed with quadruplicate cultures.

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
403
In any case, some combinations of aromatic ester moieties and
spacers seem to be more fruitful than others. For instance, (E)-3-
(3,4,5-trimethoxyphenyl)acrylic and 9-anthracenecarboxylic ester
as well as a nine-atom spacer seem to benefit both potency and
efficacy of the compounds of this series.
To further clarify the role of the length and flexibility of the
spacers, we made other modifications such as that of varying the
number of methylenes of the two spacers or introducing a stereo-
genic center in one or both the chains of the most active com-
pounds. We will report the results in due time.
mortality of the resistant cells, thus restoring a significant cytotoxic
effect of doxorubicin.
In this assay the results are unprecedented in the N,N-bis(al-
kanol)amine aryl ester series and they parallel the potency and
efficacy of the conformationally restrained analogous series (N,N-
bis(cyclohexanol)amine aryl esters [28,29,43]). The RFs of 16 are
outstanding among our and other's MDR modulating molecules.
Another interesting result is that of 17 that shows a RF of 31.8 at the
3.0
m
M concentration.
As regards these three isomers, the pirarubicin uptake modu-
lation data (Table 2) and RF values (Table 3) show some differences.
The three derivatives showed similar [I]0.5 (0.04 M for 15 and 16,
and 0.07 M for 17) behavior in the pirarubicin uptake assay,
m
4.2. Antiproliferative effects and MDR reversal
m
whereas different behavior in the cytotoxicity assay.
We also studied some selected compounds (isomers 15e17) on
the MDR phenotype in the enhancement of the cytotoxic effects of
doxorubicin on K562/DOX cells overexpressing only P-gp.
The same trend was also found with the N,N-bis(cyclohexanol)
amine aryl ester series [28]. There, we propose a possible explana-
tion for this discrepancy, based on the quite different experimental
conditions of the two assays [28]. Actually, the two anthracyclines
used in the tests have different rates of cellular uptake, that of
pirarubicin being faster than doxorubicin. Thus their intracellular
concentrations depend on the kinetics of their P-glycoprotein
mediated efflux but also on the kinetics of their uptake by the cells
[45]. Moreover, cellular processes during the lengthy cytotoxicity
experiment (72 h) can alter the cytotoxic response of the cells.
At the three concentrations tested (0.1, 1.0 and 3.0 mM), the three
compounds have no intrinsic toxicity to the parental line, while
they have an intrinsic toxicity not exceeding 20% (at the higher
concentration), to the resistant cell line (data not shown). As re-
ported in Table 3, the compounds do not change the IC50 values of
doxorubicin on parental cells (Fig. 1A) significantly, showing good
in vitro selectivity for the resistant line (Fig.1B). In resistant cells the
RF values are concentration-dependent and indicate the ability of
these new molecules to restore the cytotoxicity of doxorubicin. At
the tested concentrations compounds 15e17 showed interesting
pharmacological profiles. As a matter of fact, compounds 15 and 17
5. Conclusion
In summary, we designed and synthesized several new N,N-
bis(alkanol)amine aryl esters by modulating the aromatic moieties or
the length of the methylenic chain. Most of the new compounds are
active on doxorubicin-resistant erythroleukemia K562 cells (K562/
DOX) in the pirarubicin uptake assay, showing in some cases
(asymmetric compounds) a low nanomolar potency and an efficacy
close to 100%, nearly completely reversing pirarubicin extrusion. The
pharmacological profile of the selected compounds 15e17 was
further characterized by evaluating their doxorubicin cytotoxicity
enhancementon the K562/DOX cell line. Altogether this new series of
P-gpmodulatingmoleculesseemsverypromisingandcompounds16
(GDE6) and 17 (GDE19) appear to be interesting leads for the devel-
opment of new P-gp-dependent MDR modulators. Further pharma-
cological characterization of compounds 16 and 17 is in progress.
have remarkable RF values at 1
M (11.8 and 31.8, respectively). Compound 16 has outstanding
activity since it is able to decrease the doxorubicin IC50 value 6.7,
33.3 and 57.1 times at 0.1, 1 and 3 M concentrations, respectively.
mM (6.3 and 8.7, respectively) and at
3
m
m
As shown in Fig. 1B, compounds 16 and 17 were able to cause 100%
6. Experimental section
6.1. Chemistry
All melting points were taken on a Büchi apparatus and are un-
corrected. Infrared spectra were recorded with a PerkineElmer
Spectrum RX I FT-IR spectrophotometer in Nujol mull for solids and
neat for liquids. NMR spectra were recorded on a Bruker Avance 400
spectrometer (400 MHz for 1H NMR, 100 MHz for 13C). Chromato-
graphic separations were performed on a silica gel column by gravity
chromatography (Kieselgel 40, 0.063e0.200 mm; Merck) or flash
chromatography (Kieselgel 40, 0.040e0.063 mm; Merck). Yields are
given after purification, unless otherwise stated. The combustion
analyses of compounds 1e26 are indicated by symbols, and the
analytical results are within 0.4% of the theoretical values. Com-
pounds were named following IUPAC rules as applied by ChemBio-
Draw Ultra 12.0 software. When reactions were performed in
anhydrousconditions, themixturesweremaintainedundernitrogen.
6.1.1. General procedure for the synthesis of haloesters 27e29,
31e33; 35
A 1 mmol portion of the appropriate carboxylic acid ((E)-3-
(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic acid
Fig. 1. Cytotoxicity curves related to the effects of doxorubicin alone and in combi-
nation with compounds 15e17 on K562 parental cells (A) and K562/DOX resistant
subline (B). The curves are representative of at least three experiments performed in
quadruplicate.

404
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
or anthracene-9-carboxylic acid) was transformed into the acyl
chloride by reaction with SOCl2 (2 mmol) in 5 mL of CHCl3 (free of
ethanol) at 60 ꢀC for 4e5 h.
Na2SO4, and the solvent was removed under reduced pressure
yielding a yellow oil which was used as such for the next reaction.
The reaction mixture was cooled to rt, and the solvent was
removed under reduced pressure; the mixture was then treated
twice with cyclohexane and the solvent removed under reduce
pressure. The acyl chloride obtained was dissolved in CHCl3 (free of
ethanol), and the suitable alcohol (3-bromopropan-1-ol, 4-
bromobutan-1-ol or 5-chloropentan-1-ol) (1 eq) was added. The
mixture was heated to 60 ꢀC. After 4 h, the reaction mixture was
cooled to rt, and treated with CH2Cl2. The resulting organic layer
was washed with 10% NaOH solution, dried with Na2SO4, and the
solvent was removed under reduced pressure. The substances ob-
tained were purified by flash chromatography in the case of 31 and
32, otherwise were used as such for the next reaction. Compounds
27e29, and 33 were already synthesized by our group with the
same procedure [26,27].
6.1.3.1. 1 (E)-5-iodopentyl 3-(3,4,5-trimethoxyphenyl)acrylate 36.
Yield: 87.7%. 1H NMR (CDCl3)
d: 7.60 (d, 1H, J ¼ 15.6 Hz, CH]CH);
6.74 (s, 2H, CH arom.); 6.34 (d, 1H, J ¼ 15.6 Hz, CH]CH); 4.21 (t, 2H,
J ¼ 6.6 Hz, CH2O); 3.88 (s, 9H, OCH3); 3.21 (t, 2H, J ¼ 6.6 Hz, CH2I);
1.92e1.85 (m, 2H, CH2); 1.75e1.71 (m, 2H, CH2); 1.56e1.54 (m, 2H,
CH2) ppm.
6.1.3.2. 5-Iodopentyl 3,4,5-trimethoxybenzoate 37. Yield: 90.0%. 1H
NMR (CDCl3)
d
: 7.30 (s, 2H, CH arom.); 4.32 (t, 2H, CH2O, J ¼ 6.4 Hz);
3.91 (s, 6H, OCH3); 3.90 (s, 3H, OCH3); 3.22 (t, 2H, CH2I, J ¼ 6.8 Hz);
1.94e1.76 (m, 4H, CH2); 1.61e1.58 (m, 2H, CH2) ppm.
6.1.3.3. 5-Iodopentyl anthracene-9-carboxylate 38. Yield: 93.0%. 1H
NMR (CDCl3) d: 8.54 (s, 1H, CH arom.); 8.03 (d, 4H, CH arom.,
J ¼ 8.4 Hz); 7.57e7.48 (m, 4H, CH arom.); 4.63 (t, 2H, CH2O,
J ¼ 6.6 Hz); 3.21 (t, 2H, CH2I, J ¼ 6.8 Hz); 1.94e1.88 (m, 4H, CH2);
1.64e1.58 (m, 2H, CH2) ppm.
6.1.1.1. 4-Bromobutyl-3,4,5-trimethoxybenzoate 31. Oil. Chromato-
graphic eluent: CH2Cl2 100. Yield: 53.3%. 1H NMR (CDCl3)
d 7.29 (s,
2H, CH arom.); 4.35 (t, 2H, CH2O, J ¼ 6.2 Hz); 3.90 (s, 9H, OCH3);
3.49 (t, 2H, CH2Br, J ¼ 6.4 Hz); 2.03e1.94 (m, 4H, CH2) ppm.
6.1.4. General procedure for the synthesis of hydroxyaminoesters
39e53
6.1.1.2. 4-Bromobutyl anthracene-9-carboxylate 32. Oil. Chromato-
The appropriate haloester (27e32, 36e38) (1 mmol) and the
suitable aminoalkylalcohol (3-aminopropan-1-ol, 4-aminobutan-
1-ol or 5-aminopentan-1-ol) (2 mmol) were dissolved in 1 mL of
anhydrous CH3CN. The mixture was heated at 80 ꢀC for 5e10 h. The
reaction mixture was cooled to room temperature, treated with
CH2Cl2 and the organic layer was washed with 10% NaOH solution.
After drying with Na2SO4, the solvent was removed under reduced
pressure and the residue purified by flash chromatography,
yielding a pale-yellow oil. Compounds 39, 41, and 42 were already
described by us [27].
graphic eluent: CH2Cl2/CH3OH/NH3 99:1:0.1. Yield: 81.5%. 1H NMR
(CDCl3)
d
: 8.54 (s, 1H, CH arom.); 8.03 (d, 4H, CH arom, J ¼ 8.4 Hz);
7.57e7.48 (m, 4H, CH arom.); 4.66 (t, 2H, CH2O, J ¼ 6.0 Hz); 3.49 (t,
2H, CH2Br, J ¼ 6.2 Hz); 2.08e1.97 (m, 4H, CH2) ppm.
6.1.1.3. 5-Chloropentyl anthracene-9-carboxylate 35. Oil. Yield:
93.2%. 1H NMR (CDCl3)
d: 8.54 (s, 1H, CH arom.); 8.03 (d, 4H, CH
arom. J ¼ 8.2 Hz); 7.57e7.48 (m, 4H, CH arom.); 4.64 (t, 2H, CH2O,
J ¼ 6.6 Hz); 3.56 (t, 2H, CH2Cl, J ¼ 6.6 Hz); 1.96e1.84 (m, 4H, CH2);
1.70e1.63 (m, 2H, CH2) ppm.
6 .1. 4 .1. 3 - ( ( 3 - H y d r o x y p r o p y l ) a m i n o ) p r o p y l 3 , 4 , 5 -
6.1.2. Preparation of 30 and 34
trimethoxybenzoate 40. Chromatographic eluent: CH2Cl2/MeOH/
6.1.2.1. (E)-4-bromobutyl 3-(3,4,5-trimethoxyphenyl)acrylate 30.
0.6 g (3.92 mmol) of 4-bromo-1-butanol was dissolved in an.
CH2Cl2 and the solution was maintained at 0 ꢀC. 1.40 g (5.88 mmol)
of (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, 0.383 g (3.14 mmol)
of DMAP and 1.35 g (7.06 mmol) of EDCI were added in this order.
The mixture was maintained at 0 ꢀC for 90 min and at rt for 48 h.
The organic layer was washed twice with water and twice with
NaHCO3 sat. solution and dried with Na2SO4. The solvent was
removed under reduced pressure, and the obtained compound was
purified by flash chromatography (CH2Cl2/CH3OH 99:1). Oil. Yield
NH4OH 95:5:0.5. Yield: 43.0%. 1H NMR (CDCl3)
d: 7.22 (s, 2H, CH
arom.); 4.31 (t, 2H, J ¼ 6.4 Hz, CH2O); 3.84 (s, 6H, OCH3); 3.83 (s, 3H,
OCH3); 3.71 (t, 2H, J ¼ 5.6 Hz, CH2OH); 3.24 (bs, 2H, OH þ NH); 2.81
(t, 2H, J ¼ 5.6 Hz, CH2N); 2.71 (t, 2H, J ¼ 7.2 Hz, CH2N); 1.94e1.83 (m,
2H, CH2); 1.68e1.61 (m, 2H, CH2) ppm.
6.1.4.2. 5-((5-Hydroxypentyl)amino)pentyl 3,4,5-trimethoxybenzoate
43. Chromatographic eluent: CH2Cl2/abs. ethanol/petroleum ether
NH4OH 340:65:60:8. Yield: 53.0%. 1H NMR (CDCl3)
d: 7.24 (s, 2H, CH
59.4%. 1H NMR (CDCl3)
2H, CH arom); 6.33 (d, 2H, J ¼ 16.0 Hz, CH]CH); 4.23 (t, 2H,
J ¼ 6.4 Hz, CH2O); 3.88 (s, 9H, OCH3); 3.46 (t, 2H, J ¼ 6.4 Hz, CH2Br);
2.03e1.96 (m, 2H, CH2); 1.93e1.84 (m, 2H, CH2) ppm.
d
: 7.59 (d, 1H, J ¼ 16.0 Hz, CH]CH), 6.74 (s,
arom.); 4.26 (t, 2H, CH2O, J ¼ 6.8 Hz); 3.86 (s, 6H, OCH3); 3.85 (s, 3H,
OCH3); 3.56 (t, 2H, CH2OH, J ¼ 6.4 Hz); 2.59e2.55 (m, 4H, CH2N);
2.25 (bs, 2H, OH þ NH); 1.78e1.71 (m, 2H, CH2); 1.54e1.30 (m, 10H,
CH2) ppm.
6.1.4.3. 5-((5-Hydroxypentyl)amino)pentyl anthracene-9-
6.1.2.2. 5-Chloropentyl 3,4,5-trimethoxybenzoate 34. With the same
procedure, compound 34 was obtained, and was purified by flash
chromatography (CH2Cl2/CH3OH 99:1). Oil. Yield: 80%. 1H NMR
carboxylate 44. Chromatographic eluent: CH2Cl2/abs. ethanol/pe-
troleum ether NH4OH 340:65:60:8. Yield: 63.1%. 1H NMR (CDCl3)
d:
8.51 (s, 1H, CH arom.); 8.04e8.00 (m, 4H, CH arom.); 7.56e7.46 (m,
4H, CH arom.); 4.61 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.60 (t, 2H, CH2OH,
J ¼ 6.4 Hz); 2.62e2.57 (m, 4H, CH2N); 2.02 (bs, 2H, OH þ NH);
1.93e1.87 (m, 2H, CH2); 1.60e1.32 (m, 10H, CH2) ppm.
(CDCl3)
d
: 7.29 (s, 2H, CH arom.); 4.33 (t, 2H, J ¼ 6.4 Hz, CH2O); 3.91
(s, 9H, OCH3); 3.57 (t, 2H, J ¼ 6.6 Hz, CH2Cl); 1.90e1.77 (m, 4H, CH2);
1.65e1.59 (m, 2H, CH2) ppm.
6.1.3. General procedure for the synthesis of iodo esters 36e38
A 1 mmol portion of the suitable chloroester (33e35) was dis-
solved in acetone. To this solution NaI (4 eq) was added, and the
resulting mixture was maintained 24 h at reflux in the dark. The
reaction mixture was cooled to room temperature, and the solvent
was removed under reduced pressure; the residue was dissolved in
CH2Cl2 and washed with water. The organic layer was dried with
6.1.4.4. (E)-3-((5-hydroxypentyl)amino)propyl 3-(3,4,5-
trimethoxyphenyl)acrylate 45. Chromatographic eluent: CH2Cl2/
MeOH/NH4OH 95:5:0.5. Yield: 63.7%. 1H NMR (CDCl3)
d: 7.61 (d, 1H,
CH]CH, J ¼ 16.0 Hz); 6.77 (s, 2H, CH arom.); 6.36 (d, 1H, CH]CH,
J ¼ 16.0 Hz); 4.30 (t, 2H, CH2O, J ¼ 6.2 Hz); 3.88 (s, 9H, OCH3); 3.66
(t, 2H, CH2OH, J ¼ 6.2 Hz); 2.75 (t, 2H, CH2N, J ¼ 7.0 Hz); 2.66 (t, 2H,

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
405
CH2N, J ¼ 7.0 Hz); 1.94e1.90 (m, 2H, CH2); 1.58e1.53 (m, 6H, CH2)
2.58 (t, 2H, CH2N, J ¼ 6.6 Hz); 1.89e1.85 (m, 2H, CH2); 1.67e1.43 (m,
ppm.
6H, CH2) ppm.
6.1.5. General procedure for the synthesis of
6 .1. 4 . 5 . 3 - ( ( 5 - H y d r o x y p e n t y l ) a m i n o ) p r o p y l 3 , 4 , 5 -
methylhydroxyaminoesters 54e68
trimethoxybenzoate 46. Chromatographic eluent: CH2Cl2/CH3OH/
NH4OH 90:5:0.5. Yield: 80.3%. 1H NMR (CDCl3)
d: 7.28 (s, 2H, CH
A 1 mmol portion of the appropriate hydroxyaminoester
(39e53) was dissolved in 5 mL of anhydrous ethanol and HCOOH
(17 mmol) and 37% HCHO solution (5 mmol) were added. The
mixture was heated to 80 ꢀC for 2e5 h and concentrated in vacuo.
The residue was then dissolved in CH2Cl2 and the organic layer was
washed with 10% NaOH solution. After drying with Na2SO4, the
solvent was removed under reduced pressure, giving a pure com-
pound as yellow oil which was used as such for the next reaction.
The compounds 54, 56 and 57 were already described by our group
[27].
arom.); 4.38 (t, 2H, CH2O, J ¼ 6.2 Hz); 3.90 (s, 9H, OCH3); 3.63 (t, 2H,
CH2OH, J ¼ 5.6 Hz); 2.75 (t, 2H, CH2N, J ¼ 6.6 Hz); 2.65 (t, 2H, CH2N,
J ¼ 6.4 Hz); 1.99e1.90 (m, 2H, CH2); 1.63e1.32 (m, 6H, CH2) ppm.
6.1.4.6. 3-((5-Hydroxypentyl)amino)propyl anthracene-9-
carboxylate 47. Chromatographic eluent: CH2Cl2/MeOH/NH4OH
95:5:0.5. Yield: 52.4%. 1H NMR (CDCl3)
d: 8.52 (s, 1H, CH arom.),
8.02 (d, 4H, CH arom., J ¼ 8.8 Hz); 7.56e7.46 (m, 4H, 4CH arom.);
4.70 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.56 (t, 2H, CH2OH, J ¼ 4.0 Hz); 2.87 (t,
2H, CH2N, J ¼ 7.2 Hz); 2.68 (t, 2H, CH2N, J ¼ 7.2 Hz); 2.30e2.12 (m,
2H, CH2); 1.62e1.39 (m, 6H, CH2) ppm.
6.1.5.1. 3-((3-Hydroxypropyl) (methyl)amino)propyl 3,4,5-
trimethoxybenzoate 55. Yield: 96.0%. 1H NMR (CDCl3)
d: 7.25 (s,
2H, CH arom.); 4.31 (t, 2H, CH2O, J ¼ 6.2 Hz); 4.02 (bs, 1H, OH); 3.84
(s, 6H, OCH3); 3.81 (s, 3H, OCH3); 3.72 (t, 2H, CH2OH, J ¼ 5.8 Hz);
2.56 (t, 2H, CH2N, J ¼ 5.8 Hz); 2.48 (t, 2H, CH2N, J ¼ 6.6 Hz); 2.24 (s,
3H, NCH3); 1.97e1.84 (m, 2H, CH2); 1.69e1.61 (m, 2H, CH2) ppm.
6.1.4.7. (E)-4-((4-hydroxybutyl)amino)butyl 3-(3,4,5-
trimethoxyphenyl)acrylate 48. Chromatographic eluent: CH2Cl2/
MeOH/NH4OH 95:5:0.5. Yield: 57.0%. 1H NMR (CDCl3)
d: 7.58 (d, 1H,
CH]CH, J ¼ 16.0 Hz); 6.74 (s, 2H, CH arom.), 6.32 (d, 1H, CH]CH,
J ¼ 16.0 Hz); 4.20 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.88 (s, 9H, OCH3); 3.57
(t, 2H, CH2OH, J ¼ 5.0 Hz); 2.68e2.64 (m, 4H, CH2N); 1.78e1.63 (m,
8H, CH2) ppm.
6.1.5.2. 5-((5-Hydroxypentyl) (methyl)amino)pentyl 3,4,5-
trimethoxybenzoate 58. Yield: 87.0%. 1H NMR (CDCl3)
d: 7.27 (s,
2H, CH arom.); 4.28 (t, 2H, J ¼ 6.4 Hz, CH2O); 3.88 (s, 9H, OCH3);
3.59 (t, 2H, J ¼ 6.6 Hz, CH2OH); 2.34e2.30 (m, 4H, CH2N); 2.18 (s,
3H, NCH3); 1.78e1.75 (m, 2H, CH2); 1.56e1.45 (m, 10H, CH2) ppm.
6.1.4.8. 4-((4-Hydroxybutyl)amino)butyl 3,4,5-trimethoxybenzoate
49. Chromatographic eluent: CH2Cl2/MeOH/NH4OH 95:5:0.5.
Yield: 59.1%. 1H NMR (CDCl3)
d: 7.27 (s, 2H, CH arom.); 4.31 (t, 2H,
6.1.5.3. 5-((5-Hydroxypentyl) (methyl)amino)pentyl anthracene-9-
CH2O, J ¼ 6.6 Hz); 3.89 (s, 9H, OCH3); 3.56 (t, 2H, CH2OH, J ¼ 5.0 Hz);
2.70e2.64 (m, 4H, CH2N); 1.82e1.79 (m, 2H, CH2); 1.67e1.60 (m,
6H, CH2) ppm.
carboxylate 59. Yield: 98.6%. 1H NMR (CDCl3)
d: 8.51 (s, 1H, CH
arom.); 8.01e7.98 (m, 4H, CH arom.); 7.56e7.46 (m, 4H, CH arom.);
4.59 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.57 (t, 2H, CH2OH, J ¼ 6.4 Hz);
2.83e2.75 (m, 4H, CH2N); 2.52 (s, 3H, NCH3); 1.92e1.85 (m, 2H,
CH2); 1.74e1.66 (m, 4H, CH2); 1.57e1.43 (m, 4H, CH2); 1.40e1.30 (m,
2H, CH2) ppm.
6.1.4.9. 4-((4-Hydroxybutyl)amino)butyl anthracene-9-carboxylate
50. Chromatographic eluent: CH2Cl2/CH3OH/NH4OH 98:2:0.2.
Yield: 36.6%. 1H NMR (CDCl3)
d: 8.53 (s, 1H, CH arom.); 8.03 (m, 4H,
CH arom.); 7.55e7.47 (m, 4H, CH arom.); 4.61 (t, 2H, CH2O,
J ¼ 6.6 Hz); 3.56 (t, 2H, CH2OH, J ¼ 5.0 Hz); 2.71e2.61 (m, 4H,
CH2N); 1.94e1.88 (m, 2H, CH2); 1.76e1.63 (m, 6H, CH2) ppm.
6.1.5.4. (E)-3-((5-hydroxypentyl) (methyl)amino)propyl 3-(3,4,5-
trimethoxyphenyl)acrylate 60. Yield: 99.0%. 1H NMR (CDCl3)
d:
7.62 (d, 1H, CH]CH, J ¼ 16.0 Hz); 6.78 (s, 2H, CH arom.); 6.36 (d, 1H,
CH]CH, J ¼ 16.0 Hz); 4.27 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.88 (s, 9H,
OCH3); 3.66 (t, 2H, CH2O, J ¼ 6.4 Hz); 2.49 (t, 2H, CH2N, J ¼ 7.0 Hz);
2.39 (t, 2H, CH2N, J ¼ 7.2 Hz); 2.25 (s, 3H, NCH3); 1.94e1.88 (m, 2H,
CH2) 1.63e1.38 (m, 6H, CH2) ppm.
6.1.4.10. (E)-5-((3-hydroxypropyl)amino)pentyl 3-(3,4,5-
trimethoxyphenyl)acrylate 51. Chromatographic eluent: CH2Cl2/
MeOH/NH4OH 95:5:0.5. Yield: 64.5%. 1H NMR (CDCl3)
d: 7.59 (d, 1H,
CH]CH, J ¼ 16.0 Hz); 6.75 (s, 2H, CH arom.); 6.34 (d, 1H, CH]CH,
J ¼ 16.0 Hz); 4.19 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.85 (s, 9H, OCH3); 3.80 (t,
2H, CH2OH, J ¼ 5.2 Hz); 2.87 (t, 2H, CH2N, J ¼ 5.6 Hz); 2.62 (t, 2H,
CH2N, J ¼ 6.8 Hz); 1.73e1.65 (m, 4H, CH2); 1.54e1.49 (m, 4H, CH2)
ppm.
6.1.5.5. 3-((5-Hydroxypentyl) (methyl)amino)propyl 3,4,5-
trimethoxybenzoate 61. Yield: 84.8%. 1H NMR (CDCl3)
d: 7.26 (s,
2H, CH arom.); 4.33 (t, 2H, CH2O, J ¼ 5.8 Hz); 3.88 (s, 9H, OCH3);
3.60 (t, 2H, CH2OH, J ¼ 6.4 Hz); 2.46 (t, 2H, CH2N, J ¼ 6.8 Hz); 2.34 (t,
2H, CH2N, J ¼ 6.2 Hz); 2.21 (s, 3H, NCH3); 2.00e1.86 (m, 2H, CH2);
1.60e1.30 (m, 6H, CH2) ppm.
6 .1. 4 .11. 5 - ( ( 3 - H y d r o x y p r o p y l ) a m i n o ) p e n t y l 3 , 4 , 5 -
trimethoxybenzoate 52. Chromatographic eluent: CH2Cl2/abs.
ethanol/petroleum ether NH4OH 340:65:60:8. Yield: 56.2%. 1H
6.1.5.6. 3-((5-Hydroxypentyl) (methyl)amino)propyl anthracene-9-
carboxylate 62. Yield: 98.9%. 1H NMR (CDCl3)
d: 8.52 (s, 1H, CH
NMR (CDCl3)
d
: 7.27 (s, 2H, CH arom.); 4.29 (t, 2H, CH2O, J ¼ 6.8 Hz);
3.89 (s, 6H, OCH3); 3.88 (s, 3H, OCH3); 3.78 (t, 2H, CH2OH,
J ¼ 5.2 Hz); 3.02 (bs, 2H, NH þ OH); 2.86 (t, 2H, CH2N, J ¼ 5.6 Hz);
2.63 (t, 2H, CH2N, J ¼ 7.0 Hz); 1.81e1.65 (m, 4H, CH2); 1.58e1.53 (m,
4H, CH2) ppm.
arom.); 8.03 (m, 4H, CH arom.); 7.55e7.46 (m, 4H, CH arom.); 4.67
(t, 2H, CH2O, J ¼ 6.4 Hz); 3.60 (t, 2H, CH2OH, J ¼ 6.4 Hz); 2.52 (t, 2H,
CH2N, J ¼ 7.4 Hz); 2.36 (t, 2H, CH2N, J ¼ 7.2 Hz); 2.08e2.03 (m, 2H,
CH2); 1.58e1.51 (m, 4H, CH2); 1.42e1.31 (m, 2H, CH2) ppm.
6.1.4.12. 5-((3-Hydroxypropyl)amino)pentyl anthracene-9-
6.1.5.7. (E)-4-((4-hydroxybutyl) (methyl)amino)butyl 3-(3,4,5-
carboxylate 53. Chromatographic eluent: CH2Cl2/abs. ethanol/pe-
trimethoxyphenyl)acrylate 63. Yield: 89.4%. 1H NMR (CDCl3)
d:
troleum ether NH4OH 340:65:60:8. Yield: 62.0%. 1H NMR (CDCl3)
d:
7.59 (d, 1H, CH]CH, J ¼ 16.0 Hz); 6.75 (s, 2H, 2CH arom.), 6.34 (d,
1H, CH]CH, J ¼ 16.0 Hz), 4.20 (t, 2H, CH2O, J ¼ 6.0 Hz), 3.89 (s, 9H,
OCH3); 3.55 (t, 2H, CH2OH, J ¼ 6.4 Hz); 2.45e2.38 (m, 4H, CH2N);
2.24 (s, 3H, NCH3); 1.75e1.61 (m, 8H, CH2) ppm.
8.50 (s, 1H, CH arom.); 8.05e7.99 (m, 4H, CH arom.); 7.56e7.46 (m,
4H, CH arom.); 4.60 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.77 (t, 2H, CH2OH,
J ¼ 5.4 Hz); 3.12 (bs, 2H, NH þ OH); 2.80 (t, 2H, CH2N, J ¼ 5.6 Hz);

406
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
6.1.5.8. 4-((4-Hydroxybutyl) (methyl)amino)butyl 3,4,5-
trimethoxybenzoate 64. Yield: 98.8%. 1H NMR (CDCl3)
: 7.28 (s,
2H, CH arom.); 4.31 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.89 (s, 9H, OCH3);
3.56 (t, 2H, CH2OH, J ¼ 5.0 Hz); 2.70e2.64 (m, 4H, CH2N); 2.23 (s,
3H, NCH3); 1.82e1.79 (m, 2H, CH2); 1.67e1.60 (m, 6H, CH2) ppm.
3.41e3.32 (m 1H, CH aliph.); 3.13e3.05 (m 1H, CH aliph.);
2.93e2.78 (m, 3H, CH aliph.); 2.28e2.13 (m, 2H, CH aliph.);
1.78e1.70 (m, 1H, CH aliph.); 1.54e1.42 (m, 2H, CH aliph.);
1.38e1.28 (m, 4H, CH aliph., CH2CH3) ppm.
d
6.1.7. Preparation of 71 and 72
6.1.5.9. 4-((4-Hydroxybutyl) (methyl)amino)butyl anthracene-9-
6.1.7.1. 2-((1R)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
carboxylate 65. Yield: 99.2%. 1H NMR (CDCl3)
d
: 8.53 (s, 1H, CH
vinylquinuclidin-2-yl)methoxy)acetic
acid
71. 300
mg
arom.); 8.04e8.02 (m, 4H, 4CH arom.); 7.56e7.27 (m, 4H, CH
arom.); 4.64 (t, 2H, CH2O, J ¼ 5.0 Hz); 3.54 (t, 2H, CH2OH,
J ¼ 6.2 Hz); 2.49e3.37 (m, 4H, NCH2); 2.20 (s, 3H, NCH3); 1.92e1.87
(m, 2H, CH2); 1.74e1.66 (m, 6H, CH2) ppm.
(0.731 mmol) of 69 were dissolved in 6.1 mL of a solution of KOH
10% in abs. ethanol. The mixture was maintained at rt for 40', and
concentrated in vacuo. The residue was treated with H2O, acidified
with HCl 2 N to pH 5, and evaporated. The residue was then treated
three times with CH2Cl2. Evaporation of the solvent yielded 320 mg
(100% yield) of 71 hydrochloride as white powder. ½aꢁD20 (CH3OH,
c ¼ 1) ¼ ꢂ146.1ꢀ. Mp: 208 ꢀC. IR (cmꢂ1) v: (Nujol) 3399 (OH); 2510
0
6.1.5.10. (E)-5-((3-hydroxypropyl) (methyl)amino)pentyl 3-(3,4,5-
trimethoxyphenyl)acrylate 66. Yield: 99.3%. 1H NMR (CDCl3)
d:
7.59 (d, 1H, CH]CH, J ¼ 16.0 Hz); 6.75 (s, 2H, CH arom.); 6.34 (d,
CH]CH, J ¼ 16.0 Hz); 1.19 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.87 (s, 9H,
OCH3); 3.79 (t, 2H, CH2OH, J ¼ 6.6 Hz); 2.59 (t, 2H CH2N, J ¼ 5.6 Hz);
2.37 (t, 2H, CH2N, J ¼ 7.4 Hz); 2.24 (s, 3H, NCH3); 1.75e1.66 (m; 4H;
CH2); 1.66e1.56 (m, 2H, CH2); 1.56e1.54 (m, 2H, CH2) ppm.
(Nþ); 1731 (CO). 1H NMR (CDCl3)
d: 8.87 (d, 1H, J ¼ 4.4 Hz, C2 H);
0
0
8.14 (d, 1H, J ¼ 9.4 Hz, C8 H); 7.70 (s, 1H, C5 H); 7.65 (d, 1H, J ¼ 4.4 Hz,
0
0
C3 H); 7.43 (d, 1H, J ¼ 9.4 Hz, C7 H); 6.45 (bs, 1H, CHO); 5.62e5.54
(m, 1H, CH]CH2); 5.07e5.02 (m, 2H, CH]CH2); 4.70e4.62 (m, 1H,
CH aliph.); 4.24 (d, 1H, J ¼ 16.4 Hz, COCHHO); 4.09 (s, 3H, OCH3);
4.02 (d,1H, J ¼ 16.4 Hz, COCHHO); 3.55 (t,1H, J ¼ 12.4 Hz, CH aliph.);
3.40 (t, 1H, J ¼ 9.2 Hz, CH aliph.); 3.24e3.16 (m, 2H, CH aliph.);
2.79e2.71 (m, 1H, CH aliph.); 2.30e2.21 (m, 2H, CH aliph.);
2.13e2.08 (bs, 1H, CH aliph.); 1.95e1.85 (m, 1H, CH aliph.); 1.23 (t,
1H, J ¼ 10.8 Hz, CH aliph.); ppm.
6.1.5.11. 5-((3-Hydroxypropyl) (methyl)amino)pentyl 3,4,5-
trimethoxybenzoate 67. Yield: 92.5%. 1H NMR (CDCl3)
d: 7.24 (s,
2H, CH arom.); 4.26 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.86 (s, 6H, OCH3);
3.84 (s, 3H, OCH3); 3.72 (t, 2H, CH2OH, J ¼ 5.0 Hz); 2.54 (t, 2H, CH2N,
J ¼ 5.4 Hz); 2.34 (t, 2H, CH2N, J ¼ 7.2 Hz); 2.19 (s, 3H, NCH3);
1.76e1.32 (m, 8H, CH2) ppm.
6.1.7.2. 2-((1S)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetic acid 72. With the same pro-
cedure, starting from 512 mg (1.23 mmol) of 70, 496 mg (96% yield)
of 72 were obtained, as hydrochloride.
6.1.5.12. 5-((3-Hydroxypropyl) (methyl)amino)pentyl anthracene-9-
carboxylate 68. Yield: 95.0%. 1H NMR (CDCl3)
d: 8.51 (s, 1H, CH
arom.); 8.05e8.00 (m, 4H, CH arom.); 7.56e7.46 (m, 4H, CH arom.);
4.62 (t, 2H, CH2O, J ¼ 6.8 Hz); 3.77 (t, 2H, CH2OH, J ¼ 5.2 Hz); 2.57 (t,
2H, CH2N, J ¼ 5.8 Hz); 2.38 (t, 2H, CH2N, J ¼ 7.4 Hz); 2.23 (s, 3H,
NCH3); 1.94e1.88 (m, 2H, CH2); 1.70e1.43 (m, 6H, CH2) ppm.
½aꢁ2D0 (CH3OH, c ¼ 1) ¼ þ237.5ꢀ. Mp: 181 ꢀC. IR (cmꢂ1) v: (nujol)
3399 (OH); 2510 (Nþ); 1731 (CO). 1H NMR (CDCl3)
d: 8.71 (d, 1H,
0
0
J ¼ 4.2 Hz, C2 H) 7.90 (d, 1H, J ¼ 9.2 Hz, C8 H); 7.49 (d, 1H, J ¼ 4.2 Hz,
0
0
0
C3 H); 7.24 (s, 1H, C5 H); 7.06 (d, 1H, J ¼ 9.2 Hz, C7 H); 6.44 (bs, 1H,
CHO); 6.09e5.99 (m, 1H, CH]CH2); 5.20e5.11 (m, 2H, CH]CH2);
4.72e4.65 (m, 1H, CH aliph.); 4.19 (d, 1H, J ¼ 15.6 Hz, COCHHO);
4.09 (s, 3H, OCH3); 3.74 (d, 1H, J ¼ 15.6 Hz, COCHHO); 3.70e3.62 (m,
1H, CH aliph.); 3.42e3.23 (m, 2H, (m, 2H, CH aliph.); 3.11e2.95 (m,
1H, CH aliph.); 2.62e2.46 (m, 2H, CH aliph.); 1.94 (bs,1H, CH aliph.);
1.88e1.79 (m,1H, CH aliph.); 1.68e1.60 (m, 1H, CH aliph.); 1.21e1.11
(m, 1H, CH aliph.); ppm.
6.1.6. Preparation of 69 and 70
6.1.6.1. Ethyl 2-((1R)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetate 69. 350 mg of KOH
(6.16 mmol) were dissolved in 15 mL of DMSO. After 10 min, 1 g
(3.08 mmol) of quinine was added, and the mixture was main-
tained at rt under N2 for 6 h. After the change of color of the so-
lution, 0.52 mL (4.62 mmol) of ethyl bromoacetate were added, and
the mixture was maintained at rt for 19 h, then it was treated with
water and extracted twice with CH2Cl2; the organic layer was then
washed three times with H2O, and dried on Na2SO4. The solvent
was removed under reduced pressure and the residue purified by
flash chromatography, using CH2Cl2/MeOH/NH4OH 95:5:0.5 as
eluent, yielding 300 mg (24.0%) of title compound (yellow-orange
0
6.1.8. General procedure for the synthesis of diester compounds
(1e26)
A solution of the suitable methylhydroxyaminoester 54e68
(0.250 mmol) in 5 mL of an. CH2Cl2 was cooled at 0 ꢀC and
0.360 mmol of the appropriate carboxylic acid, 0.083 mmol of 4-
dimethylaminopiridine (DMAP) and 0.500 mmol of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl)
were added. The reaction mixture was stirred for 1 h at 0 ꢀC and
48 h at room temperature. Then CH2Cl2 was added and the organic
layer was washed twice with a saturated solution of NaHCO3. After
drying with Na2SO4, the solvent was removed under reduced
pressure. The crude product was then purified by flash chroma-
tography using the appropriate eluting system, yielding the desired
compound as an oil. All the compounds were transformed into the
corresponding hydrochloride or oxalate as white solid. The salts
were crystallized from abs. ethanol/petroleum ether.
oil). 1H NMR (CDCl3)
d
: 8.73 (d, 1H, J ¼ 4.4 Hz, C2 H); 8.02 (d, 1H,
0
J ¼ 9.6 Hz, C8 H); 7.49e7.26 (m, 3H, CH arom.); 5.76e5.69 (m, 1H,
CH]CH2); 5.31e5.20 (m, 1H, CHO); 4.96e4.88 (m, 2H, CH]CH2);
4.19 (q, 2H, J ¼ 7.2 Hz, OCH2CH3); 4.05 (d, 1H, J ¼ 16.4 Hz, COCHHO);
3.91 (s, 3H, OCH3); 3.84 (d, 1H, J ¼ 16.4 Hz, COCHHO); 3.51e3.42 (m
1H, CH aliph.); 3.19e3.00 (m, 2H, CH aliph.); 2.74e2.53 (m, 2H, CH
aliph.); 2.27e2.21 (m 1H, CH aliph.); 1.94e1.47 (m, 5H, CH aliph);
1.24 (t, 3H, J ¼ 7.2 Hz, CH2CH3) ppm.
6.1.6.2. Ethyl 2-((1S)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetate 70. With the same proce-
dure, starting from 2 g (6.16 mmol) of quinidine, 512 mg (20% yield)
0
6.1.8.1. (E)-3-((3-(2-((1R)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetoxy)propyl) (methyl)amino)pro-
pyl 3-(3,4,5-trimethoxyphenyl)acrylate 1. Free base: chromato-
graphic eluent: CH2Cl2/MeOH/NH4OH 95/6/0.8. Yield: 26.0%. ½aꢁ2D0
0
of 70 were obtained. 1H NMR (CDCl3)
d
: 8.73 (d,1H, J ¼ 4.0 Hz, C2 H);
0
8.01 (d,1H, J ¼ 9.2 Hz, C8 H); 7.45e7.25 (m, 3H, CH arom.); 6.18e6.10
(m, 1H, CH]CH2); 5.34e5.26 (m, 1H, CHO); 5.10e5.02 (m, 2H, CH]
CH2); 4.17 (q, 2H, J ¼ 7.2 Hz, OCH2CH3); 4.05 (d, 1H, J ¼ 16.0 Hz,
COCHHO); 3.90 (s, 3H, OCH3); (d, 1H, J ¼ 16.0 Hz, COCHHO)
(CH3OH, c ¼ 1) ¼ ꢂ59.3ꢀ. 1H NMR (CDCl3)
d: 8.72 (d, 1H, C2 H,
J ¼ 4.4 Hz); 8.01 (d, 1H, C8 H, J ¼ 9.4 Hz); 7.56 (d, 1H, CH ¼ ,
0

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
407
0
0
0
J ¼ 16.0 Hz); 7.39 (m, 2H, C3 H and C5 H); 7.35 (d, 1H, C7 H,
J ¼ 9.4 Hz); 6.72 (s, 2H, CH arom); 6.31 (d, 1H, CH ¼ , J ¼ 16.0 Hz);
5.78e5.65 (m, 1H, CH]CH2); 5.32e5.27 (m, 1H, CHO); 4.96e4.87
(m, 2H, CH]CH2); 4.20e4.14 (m, 4H, OCH2); 4.05 (d, 1H, COCHHO,
J ¼ 16.0 Hz); 3.91 (s, 3H, OCH3); 3.86 (d, 1H, COCHHO, J ¼ 16.0 Hz);
3.85 (s, 6H, OCH3); 3.84 (s, 3H, OCH3); 3.54e3.45 (m, 1H, CH aliph.);
3.18e3.03 (m, 2H, CH aliph.); 2.76e2.57 (m, 2H, CH aliph.); 2.39 (t,
2H, NCH2, J ¼ 7.2 Hz); 2.35 (t, 2H, NCH2, J ¼ 7.0 Hz); 2.28e2.21 (m,
1H, CH aliph.); 2.17 (s, 3H, NCH3); 1.94e1.59 (m, 8H, CH aliph.);
6.1.8.4. (E)-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
propyl)amino)propyl 9H-fluorene-9-carboxylate 4. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98/2/0.2. Yield: 33.0%.
1H NMR (CDCl3)
d
: 7.74 (d, 2H, CH arom., J ¼ 7.2 Hz); 7.65 (d, 2H, CH
arom., J ¼ 7.6 Hz); 7.58 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 7.40 (t, 2H, CH
arom., J ¼ 7.2 Hz); 7.32 (t, 2H, CH arom., J ¼ 7.6 Hz); 6.47 (s, 2H, 2CH
arom.); 6.34 (d, 1H, CH ¼ , 16.0 Hz); 4.24e4.20 (m, 4H, OCH2); 3.87
(s, 9H, OCH3); 2.44e2.34 (m, 4H, NCH2); 2.19 (s, 3H, NCH3);
1.81e1.75 (m, 4H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.94 (C);
1.56e1.47 (m, 1H, CH aliph.) ppm. 13C-APT-NMR (CDCl3)
d
: 169.71
153.49 (C); 144.7 (CH ¼ ); 129.89 (CH arom.); 128.11 (CH arom);
127.32 (CH arom.); 125.85 (CH arom.); 120.03 (CH arom.); 117.30
(CH ¼ ); 105.24 (CH arom.); 63.55 (CH2O); 62.83 (CH2O); 60.96
(OCH3); 56.15 (OCH3), 54.16 (NCH2); 53.81 (NCH2); 53.55 (CH
fluor.); 41.90 (NCH3); 26.54 (CH2); 26.39 (CH2) ppm.
(C); 166.91 (C); 158.08 (C); 153.43 (C); 147.35 (CH arom); 144.62
(CH ¼ ); 143.15 (C); 141.46 (CH ¼ ); 140.14 (C); 131.91 (CH arom);
129.89 (C); 127.53 (C); 122.06 (CH arom.); 121.89 (CH arom.); 117.32
(CH ¼ ); 114.58 (CH2 ¼ ); 105.21 (CH arom.); 101.15 (CH arom.);
66.17 (CH2O); 63.34 (CH2O); 62.90 (CH2O); 60.15 (CH); 56.82 (CH2);
56.20 (OCH3); 56.12 (OCH3); 55.85 (OCH3); 54.24 (NCH2); 54.02
(NCH2); 43.14 (CH2); 42.00 (NCH3); 39.67 (CH); 27.73 (CH); 27.38
(CH2); 26.74 (CH2); 26.55 (CH2) ppm.
Hydrochloride: mp 60e62 ꢀC. Anal: C33H38ClNO7 (C, H, N).
6.1.8.5. 3-(Methyl(3-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
propyl)amino)propyl 3,4,5-trimethoxybenzoate 5. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98/2/0.2. Yield: 48.0%.
Oxalate: mp 121e124 ꢀC. Anal: C43H55N3O13 (C, H, N).
1H NMR (CDCl3)
d: 7.26 (s, 2H, CH arom.); 6.79 (s, 2H, CH arom.);
6.1.8.2. (E)-3-((3-(2-((1S)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetoxy)propyl) (methyl)amino)pro-
pyl 3-(3,4,5-trimethoxyphenyl)acrylate 2. Free base: chromato-
graphic eluent: CH2Cl2/MeOH/NH4OH 95/6/0.8. Yield: 15.1%. ½aꢁ2D0
0
4.34 (t, 2H, CH2O, J ¼ 6.8 Hz); 4.26 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.87 (s,
6H, OCH3); 3.86 (s, 3H, OCH3); 3.84 (s, 3H, OCH3); 3.81 (s, 6H,
OCH3); 2.52e2.43 (m, 4H, NCH2); 2.23 (s, 3H, NCH3); 1.98e1.81 (m,
4H, 2CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.16 (C); 154.07 (C);
(CH3OH, c ¼ 1) ¼ þ53.4ꢀ. 1H NMR (CDCl3)
d
: 8.73 (d, 1H, C2 H,
153.15 (C); 152.91 (C); 142.19 (C); 140.95 (C); 125.35 (C); 114.14 (C);
110.36 (CH arom.); 106.79 (CH arom.); 86.70 (C); 79.84 (C); 64.37
(CH2O); 63.40 (CH2O); 60.96 (OCH3); 60.86 (OCH3); 56.22 (OCH3);
54.11 (NCH2); 54.01 (NCH2); 42.00 (NCH3); 26.70 (CH2); 26.42 (CH2)
ppm.
0
J ¼ 4.4 Hz); 8.01 (d, 1H, C8 H, J ¼ 9.2 Hz); 7.56 (d, 1H, CH ¼ ,
J ¼ 16.0 Hz); 7.46e7.25 (m, 3H, CH arom.); 6.73 (s, 2H, CH arom);
6.31 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 6.19e6.08 (m, 1H, CH]CH2);
5.62e5.51 (m, 1H, CHO); 5.17e5.07 (m, 2H, CH]CH2); 4.23e4.16
(m, 4H, CH2O); 4.09 (d, 1H, COCHHO, J ¼ 16.0 Hz); 3.95 (s, 3H,
OCH3); 3.87 (d, 1H, COCHHO, J ¼ 16.0 Hz); 3.86 (s, 6H, OCH3); 3.85
(s, 3H, OCH3); 3.60e3.50 (m, 1H, CH aliph); 3.19e2.73 (m, 4H, CH
aliph); 2.44e2.21 (m, 6H, NCH2 and CH aliph); 2.18 (s, 3H, NCH3);
1.88e1.71 (m, 5H, CH aliph); 1.64e1.46 (m, 2H, CH aliph); 1.29e1.22
Hydrochloride: low melting solid. Anal: C29H38ClNO10 (C, H, N).
6.1.8.6. 3-(Methyl(3-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
propyl)amino)propyl anthracene-9-carboxylate 6. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98/2/0.2. Yield: 44.2%.
(bs, 1H, CH aliph) ppm. 13C-APT-NMR (CDCl3)
d: 169.53 (C); 166.92
1H NMR (CDCl3)
d: 8.51 (s, 1H, CH arom.); 8.06e7.89 (m, 4H, CH
(C); 158.17 (C); 153.43 (C); 147.28 (CH arom.); 144.72 (CH ¼ );
144.60 (C); 140.14 (C); 139.92 (CH ¼ ); 131.89 (CH arom); 129.90 (C);
127.27 (C); 122.24 (CH arom); 122.02 (CH arom); 117.33 (CH ¼ );
115.23 (CH2 ¼ ); 105.21 (CH arom.); 101.20 (CH arom); 66.42
(CH2O); 63.38 (CH2O); 62.92 (CH2O); 59.91 (CH); 56.50 (OCH3);
56.25 (OCH3); 56.09 (OCH3); 54.25 (NCH2); 54.04 (NCH2); 49.99
(CH2); 49.27 (CH2); 42.01 (NCH3); 39.77 (CH); 28.27 (CH); 26.74
(CH2); 26.55 (CH2); 25.88 (CH2) ppm. ½aꢁD20 (CH3OH, c ¼ 1) ¼ þ53.4ꢀ.
Oxalate: mp 110e113 ꢀC. Anal: C43H55N3O13 (C, H, N).
arom.); 8.57e7.43 (m, 4H, CH arom.); 6.79 (s, 2H, CH arom.); 4.68 (t,
2H, CH2O, J ¼ 6.4 Hz); 4.29 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.87 (s, 3H,
OCH3); 3.80 (s, 6H, OCH3); 2.59 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.53 (t, 2H,
CH2N, J ¼ 7.2 Hz); 2.29 (s, 3H, NCH3); 2.12e2.04 (m, 2H, CH2);
1.98e1.87 (m, 2H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 130.98 (C);
129.31 (CH arom.); 128.63 (CH arom.); 128.40 (C); 127.01 (CH
arom.); 125.50 (CH arom.); 124.95 (CH arom.); 114.13 (C); 110.42
(CH arom.); 86.91 (C); 79.34 (C); 64.13 (CH2O); 63.87 (CH2O); 60.99
(OCH3); 56.22 (OCH3); 54.17 (NCH2); 53.97 (NCH2); 41.69 (NCH3);
26.35 (CH2); 26.02 (CH2) ppm.
6.1.8.3. (E)-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
propyl)amino)propyl 6-methoxyquinoline-4-carboxylate 3. Free
base: chromatographic eluent: CH2Cl2/MeOH/NH4OH 95/5/0.5.
Hydrochloride: mp 86e87 ꢀC. Anal: C34H36ClNO7 (C, H, N).
6.1.8.7. (E)-5-((5-(2-((1R)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetoxy)pentyl) (methyl)amino)pen-
tyl 3-(3,4,5-trimethoxyphenyl)acrylate 7. Free base: chromato-
graphic eluent: CH2Cl2/MeOH/NH4OH 95/6/0.8. Yield: 49.1%. ½aꢁ2D0
0
Yield: 15.2%. 1H NMR (CDCl3)
d: 8.82 (d, 1H, C2H, J ¼ 4.6 Hz); 8.21 (d,
1H, C5H, J ¼ 2.8 Hz); 8.01 (d, 1H, C8eH, J ¼ 9.2 Hz); 7.89 (d, 1H, C3H,
J ¼ 4.6 Hz); 7.54 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 7.38 (dd, 1H, C7H,
J ¼ 9.6 Hz, J ¼ 2.8 Hz); 6.70 (s, 2H, CH arom); 6.29 (d, 1H, CH,
J ¼ 16.0 Hz); 4.48 (t, 2H, COCH2, J ¼ 6.4 Hz); 4.25 (t, 2H, COCH2,
J ¼ 6.6 Hz); 3.95 (s, 3H, OCH3); 3.86 (s, 3H, OCH3); 3.85 (s, 6H,
OCH3); 2.54 (t, 2H, NCH2, J ¼ 7.0 Hz); 2.48 (t, 2H, CH2N, J ¼ 7.2 Hz);
2.26 (s, 3H, NCH3); 2.01e1.96 (m, 2H, CH2); 1.88e1.85 (m, 2H, CH2)
(CH3OH, c ¼ 1) ¼ þ85.3ꢀ. 1H NMR (CDCl3)
d: 8.71 (d, 1H, C2 -H,
0
J ¼ 4.0 Hz); 8.00 (d, 1H, C8 H, J ¼ 9.2 Hz); 7.55 (d, 1H, CH ¼ ,
J ¼ 16.0 Hz); 7.46e7.27 (m, 3H, CH arom.); 6.72 (s, 2H, CH arom.);
6.31 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 6.16e6.09 (m, 1H, CH]CH2);
5.48e5.39 (m, 1H, CHO); 5.12e5.06 (m, 2H, CH]CH2); 4.23e3.99
(m, 5H, CH2O, and COCH2O); 3.95e3.78 (m, 13H, OCH3, COCH2O);
3.47e3.37 (m, 1H, CH aliph.); 3.14e3.05 (m, 1H, CH aliph.);
2.98e2.67 (m, 3H, CH aliph.); 2.34e2.08 (m, 9H, NCH2, CH aliph.,
ppm. 13C-APT-NMR (CDCl3)
d: 166.96 (C); 166.34 (C); 159.16 (C);
153.38 (C); 146.92 (CH arom.); 145.62 (C); 144.75 (CH ¼ ); 140.03
(C); 132.69 (C); 131.37 (CH arom.); 129.84 (C); 129.82 (C); 126.73
(C); 122.77 (CH arom.); 122.65 (CH arom.); 117.20 (CH ¼ ); 105.14
(CH arom.); 103.18 (CH arom.); 64.01 (CH2O); 62.83 (CH2O); 60.97
(OCH3); 56.12 (OCH3); 55.59 (OCH3); 54.21 (NCH2); 54.16 (NCH2);
42.07 (NCH3); 26.70 (CH2); 26.67 (CH2) ppm.
NCH3); 1.78e1.21 (m, 16H, CH aliph.) ppm. 13C-APT-NMR (CDCl3)
d:
169.61 (C); 166.96 (C); 158.02 (C); 153.41 (C); 147.27 (CH arom.);
144.56 (CH ¼ ); 143.60 (C); 140.37 (CH2 ¼ ); 140.12 (C); 131.80 (CH
arom.); 129.92 (C); 127.34 (C); 121.94 (CH arom.); 121.91 (CH
arom.); 117.43 (CH ¼ ); 114.83 (CH2 ¼ ); 105.21 (CH arom.); 101.19
(CH arom.); 66.39 (CH2O); 65.11 (CH2O); 64.88 (CH2O); 59.94 (CH);
Hydrochloride: mp 71e73 (dec) ꢀC. Anal: C30H37ClN2O8 (C, H,
N).

408
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
57.99 (NCH2); 57.59 (NCH2); 56.45 (OCH3); 56.17 (OCH3); 55.84
(OCH3); 50.05 (CH2); 49.37 (CH2); 42.11 (NCH3); 40.05 (CH); 28.48
(CH2); 28.70 (CH2); 28.25(CH); 26.90 (CH2); 26.83 (CH2); 26.21
(CH2); 23.97 (CH2); 23.81 (CH2) ppm.
Hydrochloride: mp 90e91 ꢀC. Anal: C33H46ClNO10 (C, H, N).
6.1.8.11. 5-(Methyl(5-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
pentyl)amino)pentyl anthracene-9-carboxylate 11. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 95/5/0.5. Yield: 10.3%.
Oxalate: mp 80e82 ꢀC. Anal: C47H63N3O13 (C, H, N).
1H NMR (CDCl3)
d: 8.54 (s, 1H, CH arom.); 8.02 (t, 4H, CH arom.,
6.1.8.8. (E)-5-((5-(2-((1S)-(6-methoxyquinolin-4-yl) ((1S,4S,5S)-5-
vinylquinuclidin-2-yl)methoxy)acetoxy)pentyl) (methyl)amino)pen-
tyl 3-(3,4,5-trimethoxyphenyl)acrylate 8. Free base: chromato-
graphic eluent: CH2Cl2/MeOH/NH4OH 95/6/0.8. Yield: 48.3%. ½aꢁ2D0
0
J ¼ 7.6 Hz); 7.57e7.48 (m, 4H, CH arom.); 6.84 (s, 2H, CH arom.);
4.63 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.21 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.88 (s,
3H, OCH3); 3.84 (s, 6H, OCH3); 2.50e2.35 (m, 4H, NCH2); 2.27 (s, 3H,
NCH3); 1.93e1.88 (m, 2H, CH2); 1.70e1.30 (m, 10H, CH2) ppm.
Hydrochloride: mp 97e98 ꢀC. Anal: C38H44ClNO7 (C, H, N).
(CH3OH, c ¼ 1) ¼ ꢂ54.2ꢀ. 1H NMR (CDCl3)
d: 8.73 (d, 1H, C2 H,
0
J ¼ 4.4 Hz); 8.01 (d, 1H, C8 H, J ¼ 9.6 Hz); 7.57 (d, 1H, CH ¼ ,
J ¼ 16.0 Hz); 7.46e7.25 (m, 3H, CH arom.); 6.73 (s, 2H, CH arom.);
6.32 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 5.76e5.67 (m, 1H, CH]CH2);
5.47e5.38 (m, 1H, CHO); 4.95e4.89 (m, 2H, CH]CH2); 4.21e4.04
(m, 5H, CH2O, and COCHHO); 3.94 (s, 3H, OCH3); 3.86 (s, 6H, OCH3);
3.85 (s, 3H, OCH3); 3.80 (d,1H, COCHHO, J ¼ 15.8 Hz); 3.66e3.57 (m,
1H, CH aliph.); 3.28e3.03 (m, 2H, CH aliph.); 2.81e2.57 (m, 2H, CH
aliph.); 2.47e2.23 (m, 5H, NCH2, CH aliph.); 2.17 (s, 3H, NCH3);
1.98e1.78 (m, 3H, CH aliph.); 1.75e1.23 (m, 14H, CH aliph.) ppm.
6.1.8.12. (E)-5-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
propyl)amino)pentyl 3,4,5-trimethoxybenzoate 12. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98:3:0.3. Yield: 53.0%.
1H NMR (CDCl3)
d
: 7.57 (d, 1H, CH]CH, J ¼ 16.0 Hz); 7.27 (s, 2H, CH
arom.); 6.73 (s, 2H, CH arom.); 6.32 (d, 1H, CH]CH, J ¼ 16.0 Hz);
4.29 (t, 2H, CH2O, J ¼ 6.8 Hz); 4.22 (t, 2H, CH2O, J ¼ 6.4 Hz) 3.88 (s,
6H, 2OCH3); 3.87 (s, 3H, OCH3); 3.86 (s, 6H, OCH3); 3.85 (s, 3H,
OCH3); 2.45 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.36 (t, 2H, NCH2, J ¼ 7.2 Hz);
2.22 (s, 3H, NCH3) 1.90e1.75 (m, 4H, 2CH2); 1.56e1.50 (m, 4H, 2CH2)
13C-APT-NMR (CDCl3)
d: 169.75 (C); 166.99 (C); 158.10 (C); 153.42
(C); 147.29 (CH arom); 144.76 (C); 144.51 (CH ¼ ); 143.08 (C); 141.33
(CH2 ¼ ); 140.09 (C); 131.89 (CH arom; 129.93 (C); 127.34 (C); 122.12
(CH arom); 121.91 (CH arom); 117.44 (CH ¼ ); 114.64 (CH2 ¼ );
105.27 (CH arom.); 105.17 (CH arom.); 66.50 (CH2O); 64.92 (CH2O);
64.54 (CH2O); 60.13 (CH); 57.68 (NCH2); 57.60 (NCH2); 56.77 (CH2);
56.16 (OCH3); 56.08 (OCH3); 55.87 (OCH3); 43.03 (CH2); 42.18
(NCH3); 39.62 (CH); 28.71 (CH2); 28.47 (CH2); 27.72 (CH); 27.30
(CH2); 26.93 (CH2); 26.87 (CH2); 23.97 (CH2); 23.92 (CH2) ppm.
Oxalate: mp 129e132 ꢀC. Anal: C47H63N3O13 (C, H, N).
ppm. 13C-APT-NMR (CDCl3)
d: 166.94 (C); 166.24 (C); 153.41 (C);
152.90 (C); 144.72 (CH); 129.88 (C); 125.45 (C); 117.29 (CH); 106.77
(CH); 105.19 (CH); 65.11 (CH2O); 62.97 (CH2O); 60.95 (OCH3); 60.90
(OCH3); 57.58 (NCH2); 56.23 (OCH3); 56.14 (OCH3); 54.26 (NCH2);
42.09 (NCH3); 28.72 (CH2); 26.95 (CH2); 26.65 (CH2); 23.91 (CH2)
ppm.
Hydrochloride: mp 165-67 ꢀC. Anal: C31H44ClNO10 (C, H, N).
6.1.8.13. (E)-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
pentyl)amino)propyl 3,4,5-trimethoxybenzoate 13. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 95:5:0.5. Yield: 70.4%.
6.1.8.9. (E)-5-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
pentyl)amino)pentyl 6-methoxyquinoline-4-carboxylate 9. Free
base: chromatographic eluent: CH2Cl2/diethyl ether/petroleum
ether/abs. EtOH/NH4OH 360/360/900/180/9.9. Yield: 47.7%. 1H NMR
1H NMR (CDCl3)
d
: 7.57 (d, 1H, CH]CH, J ¼ 15.6 Hz); 7.26 (s, 2H, CH
arom.); 6.73 (s, 2H, CH arom.); 6.32 (d, 1H, CH]CH, J ¼ 15.6 Hz);
4.34 (t, 2H, CH2O, J ¼ 6.6 Hz); 4.17 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.88 (s,
18H, OCH3); 2.47 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.35 (t, 2H, NCH2,
J ¼ 7.2 Hz); 2.20 (s, 3H, NCH3); 1.95e1.89 (m, 2H, CH2); 1.72e1.65
(m, 2H, CH2); 1.65e1.63 (m, 2H, CH2); 1.52e1.50 (m, 2H, CH2) ppm.
(CDCl3)
d
: 8.83 (d, 1H, C2H, J ¼ 4.4 Hz); 8.21 (d, 1H, C5H, J ¼ 2.8 Hz);
8.02 (d, 1H, C8H, J ¼ 9.2 Hz); 7.90 (d, 1H, C3H, J ¼ 4.4 Hz); 7.57 (d, 1H,
CH ¼ , J ¼ 16.0 Hz); 7.39 (dd, 1H, C7H, J ¼ 9.2 Hz, J ¼ 2.8 Hz); 6.73 (s,
2H, CH arom.); 6.32 (d, 1H, CH ¼ , J ¼ 16.0 Hz); 4.42 (t, 2H, CH2O,
J ¼ 6.6 Hz); 4.18 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.95 (s, 3H, OCH3); 3.87 (s,
6H, OCH3); 3.86 (s, 3H, OCH3); 2.35e2.31 (m, 4H, NCH2); 2.21 (s, 3H,
NCH3); 1.89e1.80 (m, 2H, CH2); 1.75e1.67 (m, 2H, CH2); 1.60e1.47
13C-APT-NMR (CDCl3)
d: 167.00 (C); 166.19 (C); 153.42 (C); 152.91
(C); 144.61 (CH); 142.17 (C); 140.07 (C); 129.93 (C); 125.41 (C);
117.42 (CH); 106.78 (CH); 105.20 (CH); 64.54 (CH2O); 63.59 (CH2O);
60.95 (OCH3); 60.90 (OCH3); 57.64 (NCH2); 56.22 (OCH3); 56.14
(OCH3); 54.22 (NCH2); 42.22 (NCH3); 28.71 (CH); 27.08 (CH); 26.74
(CH); 23.91 (CH) ppm.
(m, 6H, CH2); 1.45e1.37 (m, 2H, CH2) ppm. 13C-APT-NMR (CDCl3)
d:
167.02 (C); 166.43 (C); 159.15 (C); 153.40 (C); 146.96 (CH arom);
145.66 (C); 144.62 (CH ¼ ); 140.01 (C); 132.79 (C); 131.42 (CH
arom); 129.92 (C); 126.73 (C); 122.79 (CH arom); 122.70 (CH arom);
117.41 (CH ¼ ); 105.14 (CH arom.); 103.16 (CH arom); 65.67 (CH2O);
64.55 (CH2O); 60.96 (OCH3); 57.71 (NCH2); 57.66 (NCH2); 56.13
(OCH3); 55.57 (OCH3); 42.26 (NCH3); 28.71 (CH2); 28.66 (CH2);
27.01 (CH2); 24.09 (CH2); 23.99 (CH2) ppm.
Hydrochloride: mp 63e64 ꢀC. Anal: C31H44ClNO10 (C, H, N).
6.1.8.14. (E)-4-(methyl(4-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
butyl)amino)butyl 3,4,5-trimethoxybenzoate 14. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 95:5:0.5. Yield: 28.1%.
1H NMR (CDCl3)
d
: 7.58 (d, 1H, CH]CH, J ¼ 15.6 Hz); 7.28 (s, 2H, CH
Hydrochloride: mp 77e80 (dec) ꢀC. Anal: C34H45ClN2O8 (C, H, N).
arom.); 6.74 (s, 2H, CH arom.); 6.33 (d, 1H, CH]CH, J ¼ 15.6 Hz);
4.32 (t, 2H, CH2O, J ¼ 6.6 Hz), 4.20 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.92 (s,
18H, OCH3), 2.40e2.35 (m, 4H, NCH2); 2.21 (s, 3H, NCH3); 1.80e1.69
6.1.8.10. 5-(Methyl(5-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
pentyl)amino)pentyl 3,4,5-trimethoxybenzoate 10. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 95/5/0.5. Yield: 54.9%.
(m, 4H, CH2); 1.63e1.60 (m, 4H, CH2) ppm. 13C-APT-NMR (CDCl3)
d:
166.90 (C); 166.16 (C); 153.39 (C); 152.88 (C); 144.58 (CH); 142.17
(C); 140.08 (C); 129.88 (C); 125.40 (C); 117.38 (CH); 106.80 (CH);
105.22 (CH); 65.01 (CH2O); 64.37 (CH2O); 60.88 (OCH3); 60.83
(OCH3); 57.35 (NCH2); 57.30 (NCH2); 56.19 (OCH3); 56.11 (OCH3);
42.02 (NCH3); 26.73 (CH2); 23.83 (CH2) ppm.
1H NMR (CDCl3)
d: 7.28 (s, 2H, CH arom.); 6.82 (s, 2H, CH arom.);
4.30 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.22 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.89 (s,
9H, OCH3); 3.88 (s, 3H, OCH3); 3.87 (s, 6H, OCH3); 2.42e2.32 (m, 4H,
NCH2); 2.23 (s, 3H, NCH3); 1.82e1.68 (m, 4H, CH2); 1.60e1.35 (m,
8H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.24 (C); 154.18 (C);
Hydrochloride: mp 60e61 ꢀC. Anal: C31H44ClNO10 (C, H, N).
153.14 (C); 152.90 (C); 125.44 (C); 114.19 (C); 110.32 (CH); 106.75
(CH); 86.63 (C); 79.89 (C); 65.98 (CH2O); 65.06 (CH2O); 61.01
(OCH3); 60.92 (OCH3); 57.54 (NCH2); 57.45 (NCH2); 56.24 (OCH3);
42.00 (NCH3); 28.70 (CH2); 28.39 (CH2); 26.62 (CH2); 23.94 (CH2);
23.78 (CH2) ppm.
6.1.8.15. (E)-5-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
propyl)amino)pentyl anthracene-9-carboxylate 15. Free base:
chromatographic eluent: CH2Cl2/MeOH/NH4OH 98:3:0.3. Yield:
42.0%. 1H NMR (CDCl3)
d: 8.51 (s, 1H, CH arom.); 8.02 (t, 4H, CH

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
409
arom., J ¼ 7.6 Hz); 7.59 (d, 1H, CH]CH, J ¼ 16.0 Hz); 7.55e7.45 (m,
4H, CH arom.); 6.73 (s, 2H, CH arom.); 6.33 (d, 1H, CH]CH,
J ¼ 15.6 Hz); 4.62 (t, 2H, CH2O, J ¼ 6.6 Hz); 4.23 (t, 2H, CH2O,
J ¼ 6.4 Hz); 3.87e3.85 (m, 9H, OCH3); 2.48 (t, 2H, NCH2, J ¼ 7.2 Hz);
2.40 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.24 (s, 3H, NCH3); 1.94e1.86 (m, 4H,
1H NMR (CDCl3)
d: 7.28 (s, 2H, CH arom.); 6.82 (s, 2H, CH arom.);
4.30 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.22 (t, 2H, CH2O, J ¼ 6.4 Hz);
3.89e3.84 (m, 18H, OCH3); 2.48 (t, 2H, NCH2, J ¼ 7.4 Hz); 2.34 (t, 2H,
NCH2, J ¼ 7.2 Hz); 2.22 (s, 3H, NCH3); 1.82e1.61 (m, 8H, CH2) ppm.
13C-APT-NMR (CDCl3)
d: 154.17 (C); 153.16 (C); 152.93 (C); 125.38
CH2); 1.58e1.54 (m, 4H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.95
(C); 114.20 (C); 110.36 (CH arom.); 106.79 (CH arom.); 86.72 (C);
79.91 (C); 66.03 (CH2O); 63.55 (CH2O); 61.01 (OCH3); 60.91 (OCH3);
57.51 (NCH2); 56.24 (OCH3); 42.12 (NCH3); 28.42 (CH2); 26.89
(CH2); 26.66 (CH2); 23.74 (CH2) ppm.
(C); 153.42 (C); 144.72 (CH); 130.99 (C); 129.90 (C); 129.24 (CH);
128.63 (CH); 128.37 (C); 126.93 (CH); 125.46 (CH); 124.99 (CH);
117.33 (CH); 105.23 (CH); 65.79 (CH2O); 62.95 (CH2O); 60.97
(OCH3); 57.53 (NCH2); 56.14 (OCH3); 54.23 (NCH2); 42.08 (NCH3);
28.71 (CH2); 26.85 (CH2); 26.66 (CH2); 23.98 (CH2) ppm.
Hydrochloride: mp 67e68 ꢀC. Anal: C31H42ClNO10 (C, H, N).
Hydrochloride: mp 85e87 ꢀC. Anal: C36H42ClNO7 (C, H, N).
6.1.8.20. 4-(Methyl(4-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
butyl)amino)butyl 3,4,5-trimethoxybenzoate 20. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 95:5:0.5. Yield: 30.6%.
6.1.8.16. (E)-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
pentyl)amino)propyl anthracene-9-carboxylate 16. Free base:
chromatographic eluent: CH2Cl2/CH3OH/NH4OH 95:5:0.5. Yield:
1H NMR (CDCl3)
d: 7.26 (s, 2H, CH arom.); 6.80 (s, 2H, CH arom.);
4.31 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.22 (t, 2H, CH2O, J ¼ 6.4 Hz);
48.8%. 1H NMR (CDCl3)
d: 8.50 (s, 1H, CH arom.); 8.01 (t, 4H, CH
3.88e3.82 (m, 18H, OCH3); 2.45e2.39 (m, 4H, NCH2); 2.24 (s, 3H,
arom, J ¼ 9.4 Hz); 7.57 (d, 1H, CH]CH, J ¼ 15.6 Hz); 7.54e7.46 (m,
5H, CH arom.); 6.72 (s, 2H, CH arom.); 6.33 (d, 1H, CH]CH,
J ¼ 16.0 Hz); 4.67 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.17 (t, 2H, CH2O,
J ¼ 6.6 Hz); 3.86e3.84 (m, 9H, OCH3); 2.57 (t, 2H, NCH2, J ¼ 7.0 Hz);
2.40 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.27 (s, 3H, NCH3); 2.09e2.05 (m, 2H,
CH2); 1.70e1.67 (m, 2H, CH2); 1.54e1.48 (m, 4H, CH2) ppm. 13C-APT-
NCH3); 1.80e1.60 (m, 8H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.22
(C); 154.13 (C); 153.16 (C); 152.92 (C); 142.19 (C); 140.93 (C); 125.38
(C); 114.17 (C); 110.37 (CH arom.); 106.80 (CH arom.); 86.69 (C);
79.86 (C); 65.83 (CH2O); 64.94 (CH2O); 60.98 (OCH3); 60.89 (OCH3);
56.29 (NCH2); 56.28 (NCH2); 56.27 (OCH3); 56.25 (OCH3); 41.76
(NCH3); 26.70 (CH2); 26.43 (CH2); 23.58 (CH2); 23.43 (CH2) ppm.
Hydrochloride: mp 66e67 ꢀC. Anal: C31H42ClNO10 (C, H, N).
NMR (CDCl3) d: 167.03 (C); 153.41 (C); 144.63 (CH); 130.99 (C);
129.94 (C); 129.28 (CH); 128.63 (CH); 128.39 (C); 126.94 (CH);
125.47 (CH); 124.99 (CH); 117.44 (CH); 105.23 (CH); 64.50 (CH2O);
64.11 (CH2O); 60.96 (OCH3); 57.58 (NCH2); 56.14 (OCH3); 54.20
(NCH2); 42.02 (NCH3); 28.68 (CH2); 26.82 (CH2); 26.53 (CH2); 23.90
(CH2) ppm.
6.1.8.21. 5-(Methyl(3-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
propyl)amino)pentyl anthracene-9-carboxylate 21. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98:3:0.3. Yield: 35.0%.
1H NMR (CDCl3)
d: 8.51 (s, 1H, CH arom.); 8.02 (t, 4H, CH arom.,
Hydrochloride: mp 64e65 ꢀC. Anal: C36H42ClNO7 (C, H, N).
J ¼ 9.6 Hz); 7.55e7.46 (m, 4H, CH arom.); 6.81 (s, 2H, CH arom.);
4.62 (t, 2H, CH2O, J ¼ 6.8 Hz); 4.27 (t, 2H, CH2O, J ¼ 6.4 Hz); 3.87 (s,
3H, OCH3); 3.82 (s, 6H, OCH3); 2.43 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.36 (t,
2H, NCH2, J ¼ 6.8 Hz); 2.20 (s, 3H, NCH3); 1.94e1.84 (m, 4H, CH2);
6.1.8.17. (E)-4-(methyl(4-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
butyl)amino)butyl anthracene-9-carboxylate 17. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 95:5:0.5. Yield: 13.4%.
1.62e1.45 (m, 4H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 166.95 (C);
1H NMR (CDCl3)
d: 8.51 (s, 1H, CH arom.); 8.02 (t, 4H, CH arom.,
153.42 (C); 130.99 (C); 129.90 (C); 129.24 (CH); 128.63 (CH); 128.37
(C); 126.93 (CH); 125.46 (CH); 124.99 (CH); 110.26 (CH); 65.79
(CH2O); 62.95 (CH2O); 60.97 (OCH3); 57.53 (NCH2); 56.14 (OCH3);
54.23 (NCH2); 42.08 (NCH3); 28.71 (CH2); 26.85 (CH2); 26.66 (CH2);
23.98 (CH2) ppm.
J ¼ 9.0 Hz); 7.59 (d, 1H, CH]CH, J ¼ 16.0 Hz); 7.55e7.45 (m, 4H, CH
arom.); 6.73 (s, 2H, CH arom.); 6.33 (d, 1H, CH]CH, J ¼ 16.0 Hz);
4.64 (t, 2H, CH2O, J ¼ 6.6 Hz); 4.20 (t, 2H, CH2O, J ¼ 6.6 Hz);
3.87e3.85 (m, 9H, OCH3), 2.49e2.38 (m, 4H, NCH2); 2.23 (s, 3H,
NCH3); 1.98e1.89 (m, 2H, CH2); 1.78e1.63 (m, 4H, CH2); 1.63e1.57
Hydrochloride: mp 64e65 ꢀC. Anal: C36H40ClNO7 (C, H, N).
(m, 2H, CH2) ppm. 13C-APT-NMR (CDCl3)
d: 167.00 (C); 153.42 (C);
144.63 (CH); 131.00 (C); 129.94 (C); 129.24 (CH); 128.63 (CH);
128.39 (C); 126.93 (CH); 125.64 (CH); 125.00 (CH); 117.44 (CH);
105.21 (CH); 65.79 (CH2O); 64.45 (CH2O); 60.96 (OCH3); 57.36
(NCH2); 57.25 (NCH2); 56.14 (OCH3); 42.09 (NCH3); 29.71 (CH2);
26.78 (CH2); 23.92 (CH2) ppm.
6.1.8.22. 3-(Methyl(5-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
pentyl)amino)propyl anthracene-9-carboxylate 22. Free base:
chromatographic eluent: CH2Cl2/CH3OH/NH4OH 99:1:0.1. Yield:
48.4%. 1H NMR (CDCl3)
d: 8.51 (s, 1H, CH arom.); 8.02 (t, 4H, CH
arom., J ¼ 7.6 Hz); 7.55e7.25 (m, 4H, CH arom.); 4.67 (t, 2H, CH2O,
J ¼ 6.6 Hz); 4.20 (t, 2H, CH2O J ¼ 6.6 Hz); 3.82 (s, 9H, OCH3); 2.53 (t,
2H, NCH2, J ¼ 7.2 Hz); 2.36 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.24 (s, 3H,
NCH3); 2.08e2.03 (m, 2H, CH2); 1.52e1.43 (m, 6H, CH2) ppm. 13C-
Hydrochloride: mp 67e68 ꢀC. Anal: C36H42ClNO7 (C, H, N).
6.1.8.18. 5-(Methyl(3-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
propyl)amino)pentyl 3,4,5-trimethoxybenzoate 18. Free base: chro-
matographic eluent: CH2Cl2/MeOH/NH4OH 98:3:0.3. Yield: 57.2%.
APT-NMR (CDCl3) d: 169.68 (C); 154.21 (C); 153.13 (C); 130.97 (C);
129.26 (CH); 128.63 (CH a); 128.37 (C); 126.95 (C); 125.48 (CH);
125.01 (CH); 114.23 (C); 110.31 (C); 66.09 (CH2O); 64.22 (CH2O);
61.01 (OCH3); 57.64 (NCH2); 56.20 (OCH3); 54.23 (NCH2); 42.18
(NCH3); 28.43 (CH2); 27.00 (CH2); 26.72 (CH2); 23.77 (CH2) ppm.
Hydrochloride: mp 65e66 ꢀC. Anal: C36H40ClNO7 (C, H, N).
1H NMR (CDCl3)
d: 7.27 (s, 2H, CH arom.); 6.81 (s, 2H, CH arom.);
4.29 (t, 2H, CH2O, J ¼ 6.4 Hz); 4.26 (t, 2H, CH2O, J ¼ 6.8 Hz); 3.88 (s,
6H, OCH3); 3.87 (s, 3H, OCH3); 3.85 (s, 3H, OCH3); 3.83 (s, 6H,
OCH3); 2.43 (t, 2H, NCH2, J ¼ 7.2 Hz); 2.33 (t, 2H, NCH2, J ¼ 7.2 Hz);
2.20 (s, 3H, NCH3) 1.88e1.74 (m, 4H, CH2); 1.56e1.38 (m, 4H, CH2)
ppm. 13C-APT-NMR (CDCl3)
d: 153.16 (C); 152.91 (C); 114.18 (C);
6.1.8.23. 4-(Methyl(4-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
butyl)amino)butyl anthracene-9-carboxylate 23. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 98:2:0.2. Yield: 37.1%.
110.38 (CH); 106.82 (CH); 65.12 (CH2O); 64.54 (CH2O); 60.88
(OCH3); 57.60 (NCH2); 56.24 (OCH3); 54.03 (NCH2); 42.07 (NCH3);
28.72 (CH2); 27.01 (CH2); 26.45 (CH2); 23.90 (CH2) ppm.
1H NMR (CDCl3)
d: 8.53 (s, 1H, CH arom.); 8.04 (t, 4H, CH arom.,
Hydrochloride: mp 61e62 ꢀC. Anal: C31H42ClNO10 (C, H, N).
J ¼ 7.6 Hz); 7.55e7.49 (m, 4H, CH arom.); 6.83 (s, 2H, 2CH arom.);
4.65 (t, 2H, CH2O, J ¼ 6.6 Hz); 4.24 (t, 2H, CH2O, J ¼ 6.6 Hz); 3.86 (s,
9H, OCH3); 2.44e2.35 (m, 4H, NCH2); 2.22 (s, 3H, NCH3); 1.94e1.90
6.1.8.19. 3-(Methyl(5-((3-(3,4,5-trimethoxyphenyl)propioloyl)oxy)
pentyl)amino)propyl 3,4,5-trimethoxybenzoate 19. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 99:1:0.1. Yield: 11.3%.
(m, 2H, CH2); 1.74e1.66 (m, 6H, CH2) ppm. 13C-APT-NMR (CDCl3)
d:
153.15 (C); 130.99 (C); 129.51 (CH arom.); 129.24 (C); 128.75 (CH

410
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
arom.); 128.63 (CH arom.); 127.22 (CH arom.); 125.61 (CH arom.);
124.99 (CH arom.); 114.23 (C); 110.38 (CH arom.); 86.63 (C); 79.95
(C); 65.98 (CH2O); 65.80 (CH2O); 57.26 (NCH2); 56.23 (OCH3); 56.08
(OCH3); 42.02 (NCH3); 26.77 (CH2); 26.46 (CH2); 23.95 (CH2); 23.73
(CH2) ppm.
DOX cells were obtained from Prof. J.P. Marie (Hopital Hotel-Dieu,
Paris, France). These cells were cultured following the previously
reported protocol [43].
6.2.2. Drugs and chemicals
Hydrochloride: mp 63e64 ꢀC. Anal: C36H40ClNO7 (C, H, N).
Purified pirarubicin was provided by SigmaeAldrich (Milan e
Italy). Concentrations were determined by diluting stock solutions
to approximately 10ꢂ5 M and using ε480 ¼ 11,500 Mꢂ1 cmꢂ1. 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.
6.1.8.24. (E)-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
pentyl)amino)propyl 9H-fluorene-9-carboxylate 24. Free base:
chromatographic eluent: CH2Cl2/CH3OH/NH4OH 99:1:0.1. Yield:
28.3%. 1H NMR (CDCl3)
d
: 7.76 (d, 2H, CH arom., J ¼ 7.6 Hz); 7.66 (d,
2H, CH arom., J ¼ 7.6 Hz); 7.58 (d, 1H, CH]CH, J ¼ 16.0 Hz); 7.42 (t,
2H, CH arom., J ¼ 7.6 Hz); 7.34 (t, 2H, CH arom., J ¼ 7.6 Hz); 6.76 (s,
2H, CH arom.); 6.35 (d, 1H, CH]CH, J ¼ 16.0 Hz); 4.88 (s, 1H, CH
fluor.,); 4.22e4.18 (m, 4H, CH2O); 3.88 (s, 9H, OCH3); 2.36e2.17 (m,
4H, NCH2); 2.17 (s, 3H, NCH3); 1.83e1.67 (m, 4H, CH2); 1.51e1.35 (m,
Doxorubicin hydrochloride (DOX), verapamil, dimethylsulph-
oxide (DMSO) and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl
tetrazolium bromide (MTT) were purchased from SigmaeAldrich
(MilaneItaly). MTT stock solution was prepared following the
previously described method [43].
4H, CH2) ppm. 13C-APT-NMR (CDCl3)
d
: 170.85 (C); 167.03 (C);
Test compounds were prepared in DMSO at 10ꢂ2 M, DOX stock
solutions and verapamil were prepared in water at 10ꢂ2 M. Drugs
and test compounds were then diluted with complete medium to
obtain the 10ꢃ desired final maximum test concentrations.
Verapamil was used as a standard chemomodulator and was
153.43 (C); 144.62 (CH ¼ ); 141.43 (C); 140.73 (C); 140.08 (C); 129.95
(C); 128.11 (CH arom.); 127.32 (CH arom.); 125.61 (CH arom.);
120.03 (CH arom.); 117.47 (CH ¼ ); 105.23 (CH arom.); 64.59
(CH2O); 63.76 (CH2O); 60.96 (OCH3); 57.62 (NCH2); 56.15 (OCH3);
54.02 (NCH2); 53.54 (CH fluor.); 42.13 (NCH3); 28.71 (CH2); 26.99
(CH2); 26.53 (CH2); 23.92 (CH2) ppm.
evaluated at 3 mM.
Hydrochloride: mp 58e59 ꢀC. Anal: C35H42ClNO7 (C, H, N).
6.2.3. Cellular drug accumulation and MTT assay
The uptake of pirarubicin in cells was followed by monitoring
the decrease in the fluorescence signal at 590 nm (lex ¼ 480 nm)
according to the previously described method [46].
To evaluate the reverting activity of test compounds, the cells, in
exponential growth phase (3 ꢃ 105 cells/mL), were seeded at 3000
cells/well and either solutions of test compounds or solution of
doxorubicin or combination of a solution of doxorubicin and test
compounds were added to the wells and the plates were incubated
at 37 ꢀC for 72 h in 5% CO2 incubator. Test compounds were eval-
6.1.8.25. (E)-5-(methyl(3-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
propyl)amino)pentyl 9H-fluorene-9-carboxylate 25. Free base:
chromatographic eluent: CH2Cl2/CH3OH/NH4OH 98:2:0.2. Yield:
33.9%. 1H NMR (CDCl3)
d: 7.75 (d, 2H, CH arom., J ¼ 7.4 Hz); 7.68 (d,
2H, CH arom., J ¼ 7.4 Hz); 7.60 (s, 1H, CH]CH, J ¼ 16.0 Hz); 7.44 (t,
2H, CH arom., J ¼ 7.4 Hz); 7.35 (t, 2H, CH arom., J ¼ 7.4 Hz); 6.77 (s,
2H, CH arom.); 6.37 (d, 1H, CH]CH, J ¼ 16.0 Hz); 4.89 (s, 1H, CH
fluor.); 4.26e4.18 (m, 4H, CH2O); 3.90 (s, 9H, OCH3); 2.41e2.34 (m,
4H, NCH2); 2.22 (s, 3H, NCH3); 1.75e1.57 (m, 8H, CH2) ppm. 13C-
uated for MDR reversal activity at 0.1, 1.0 and 3
sponding doxorubicin concentrations tested were between 0.003
and 100 M. The MTT working solution was added and plates were
mM and the corre-
APT-NMR (CDCl3) d: 167.01 (C); 153.44 (C); 144.70 (CH]CH); 141.43
(C); 140.72 (C); 129.92 (C); 128.11 (CH arom.); 127.33 (CH arom.);
125.59 (CH arom.); 120.03 (CH arom.); 117.38 (CH ¼ ); 105.24 (CH
arom.); 65.23 (CH2O); 64.38 (CH2O); 60.67 (OCH3); 57.15 (NCH2);
57.08 (NCH2); 56.16 (OCH3); 53.54 (CH fluor.); 41.88 (NCH3); 26.73
(CH2); 26.53 (CH2); 23.64 (CH2); 23.42 (CH2) ppm.
m
further 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 DMSO. The absorbance of the solution
was then read on an automated plate reader at a wavelength of
540 nm.
Hydrochloride: mp 60e61 ꢀC. Anal: C35H42ClNO7 (C, H, N).
6.1.8.26. (E)-4-(methyl(4-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)
butyl)amino)butyl 9H-fluorene-9-carboxylate 26. Free base: chro-
matographic eluent: CH2Cl2/CH3OH/NH4OH 99:1:0.1. Yield: 39.9%.
6.3. Statistical analysis
1H NMR (CDCl3)
d
: 7.77 (d, 2H, CH arom. J ¼ 7.6 Hz); 7.67 (d, 2H, CH
All experiments were carried out in quadruplicate and were
independently done at least 3 times (if not otherwise reported). All
results are presented as means
performed using the one-way Anova test and Bonferroni's multiple
comparison test (GraphPad Prism software, Inc. CA, USA).
arom. J ¼ 7.2 Hz); 7.60 (d, 1H, CH]CH, J ¼ 16.0 Hz); 7.43 (t, 2H, CH
arom., J ¼ 7.2 Hz); 7.32 (t, 2H, arom., J ¼ 7.6 Hz); 6.76 (s, 2H, CH
arom.); 6.37 (d, 1H, CH]CH, J ¼ 16.0 Hz); 4.89 (s, 1H, CH fluor.);
4.23e4.15 (m, 4H, CH2O); 3.89 (s, 9H, OCH3); 2.39e2.34 (m, 4H,
NCH2); 2.22 (s, 3H, NCH3); 1.70e1.48 (m, 8H, CH2) ppm. 13C-APT-
SE and statistical analysis was
NMR (CDCl3)
d
: 167.08 (C); 153.44 (C); 144.76 (CH]CH); 141.42 (C);
6.4. Chemical stability
140.72 (C); 129.92 (C); 128.11 (CH arom.); 127.37 (CH arom.); 126.03
(CH arom.); 120.03 (CH arom.); 117.38 (CH ¼ ); 105.24 (CH arom.);
65.25 (CH2O); 64.38 (CH2O); 60.70 (OCH3); 57.15 (NCH2); 57.12
(NCH2); 56.16 (OCH3); 53.60 (CH fluor.); 41.90 (NCH3); 26.72 (CH2);
26.55(CH2); 23.67 (CH2); 23.41 (CH2) ppm.
6.4.1. Materials and methods
Acetonitrile (CHROMASOLV), formic acid (MS grade), NaCl, KCl,
Na2HPO4 2H2O, KH2PO4 (reagent grade) and metoprolol tartrate
(analytical standard, used as internal standard) were purchased
Hydrochloride: mp 62e63 ꢀC. Anal: C35H42ClNO7 (C, H, N).
from SigmaeAldrich (Milan e Italy). MilliQ water 18 MU was ob-
tained from Millipore's Simplicity system. Phosphate buffer solu-
tion (PBS) was prepared by adding 8.01 g/L of NaCl, 0.2 g/L of KCl,
1.78 g/L of Na2HPO4 2H2O and 0.27 g/L of KH2PO4. The internal
6.2. Biology
6.2.1. Cell lines and cultures
standard solution was prepared at 5 mM of metoprolol tartrate in
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/
acetonitrile (ISTD solution). G-Therm 015 thermostatic oven was
used to maintained the samples at 37 ꢀC. The LCeMS/MS analysis
was carried out using a Varian 1200L triple quadrupole system

S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
411
[17] R.W. Robey, P.R. Massey, L. Amiri-Kordestani, S.E. Bates, ABC transporters:
unvalidated therapeutic targets in cancer and the CNS, Anticancer Agents
Med. Chem. 10 (2010) 625e633.
[18] E. Libby, R. Hromas, Dismounting the MDR horse, Blood 116 (2010)
4037e4038.
[19] N.A. Colabufo, F. Berardi, M. Cantore, M. Contino, C. Inglese, M. Niso,
R. Perrone, Perspectives of P-glycoprotein modulating agents in oncology and
neurodegenerative diseases: pharmaceutical, biological, and diagnostic po-
tentials, J. Med. Chem. 53 (2010) 1883e1897.
[20] D.S. Miller, B. Bauer, A.M. Hartz, Modulation of P-glycoprotein at the blood-
brain barrier: opportunities to improve central nervous system pharmaco-
therapy, Pharmacol. Rev. 60 (2008) 196e209.
[21] D. Kuhnke, G. Jedlitschky, M. Grube, M. Krohn, M. Juker, I. Mosyagin,
I. Cascorbi, L.C. Walker, H.K. Kroemer, R.W. Warzog, S. Vogelgesang, MDR1-P-
Glycoprotein (ABCB1) mediates transport of Alzheimer's amyloid-beta pep-
tides-implications for the mechanism of Abeta clearance at the blood-brain
barrier, Brain Pathol. 17 (2007) 347e353.
[22] A.H. Abuznait, C. Cain, D. Ingram, D. Burk, A. Kaddoumi, Up-regulation of P-
glycoprotein reduces intracellular accumulation of beta amyloid: investiga-
tion of P-glycoprotein as a novel therapeutic target for Alzheimer's disease,
J. Pharm. Pharmacol. 63 (2011) 1111e1118.
equipped by two Prostar 210 pumps, a Prostar 410 autosampler and
an Electrospray Source (ESI) operated in positive ions. The analyt-
ical parameters (chromatographic, MRM and calibration curves) are
reported in the Supplementary material.
6.4.2. Preparation of samples
The samples of 15e17, at concentration of 1
tained at 37 ꢀC in PBS for different times: 0; 30; 60 and 120 min.
After the incubation the samples were added with 300 L of ISTD
mM, were main-
m
solution and centrifuged. The supernatants were transferred in
vials and dried under a gentle stream of nitrogen. The dried sam-
ples were dissolved in 1.5 mL of mQ water:acetonitrile
80:20 þ 10 mM of formic acid solution and the obtained solutions
were analyzed by LCeMS/MS method described in the
Supplementary material.
[23] F.C. Lam, R. Liu, P. Lu, A.B. Shapiro, J.M. Renoir, F.J. Sharom, P.B. Reiner, Beta-
amyloid efflux mediated by P-glycoprotein, J. Neurochem. 76 (2001)
1121e1128.
Acknowledgments
[24] S. Ohtsuki, S. Ito, T. Terasaki, Is P-glycoprotein involved in amyloid-b elimi-
The authors wish to thank Prof. Fulvio Gualtieri for helpful
discussion and Dr. Sara Ulivelli for the excellent assistance in the
cytotoxicity tests. This work was partially supported by ITT (Istituto
Toscano Tumori) DR 7197/2009 and by MIUR (FIRB 2012
RBFR12SOQ1).
nation across the blood-brain barrier in Alzheimer's disease, Clin. Pharmacol.
Ther. 88 (2010) 443e445.
[25] A. Lazarowski, L. Czornyj, F. Lubienieki, E. Girardi, S. Vazquez, C. D'Giano, ABC
transporters during epilepsy and mechanisms underlying multidrug resis-
tance in refractory epilepsy, Epilepsia 48 (2007) 140e149.
[26] E. Teodori, S. Dei, A. Garnier-Suillerot, F. Gualtieri, D. Manetti, C. Martelli,
M.N. Romanelli, S. Scapecchi, P. Sudwan, M. Salerno, Exploratory chemistry
toward the identification of a new class of multidrug resistance reverters
inspired by pervilleine and verapamil models, J. Med. Chem. 48 (2005)
7426e7436.
Appendix A. Supplementary material
[27] C. Martelli, M. Coronnello, S. Dei, D. Manetti, F. Orlandi, S. Scapecchi,
M.N. Romanelli, M. Salerno, E. Mini, E. Teodori, Structure-activity relation-
ships studies in a series of N,N-bis(alkanol)amine aryl esters as P-glycoprotein
(Pgp) dependent multidrug resistance (MDR) inhibitors, J. Med. Chem. 53
(2010) 1755e1762.
[28] R. Hayeshi, C. Masimirembwa, S. Mukanganyama, A.L. Ungell, The potential
inhibitory effect of antiparasitic drugs and natural products on P-glycoprotein
mediated efflux, Eur. J. Pharm. Sci. 29 (2006) 70e81.
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2014.09.084.
References
ꢁ
ꢁ
[1] R. Perez-Tomas, Multidrug resistance: retrospect and prospects in anti-cancer
drug treatment, Curr. Med. Chem. 13 (2006) 1859e1876.
[29] M.M. Pires, D. Emmert, C.A. Hrycyna, J. Chmielewski, Inhibition of P-glyco-
protein-mediated paclitaxel resistance by reversibly linked quinine homo-
dimers, Mol. Pharmacol. 75 (2009) 92e100.
[2] K. Dano, Active outward transport of daunomycin in resistant Ehrlich ascites
tumor cells, Biochim. Biophys. Acta 323 (1973) 466e483.
[30] F. Orlandi, M. Coronnello, C. Bellucci, S. Dei, L. Guandalini, D. Manetti,
C. Martelli, M.N. Romanelli, S. Scapecchi, M. Salerno, H. Menif, I. Bello, E. Mini,
E. Teodori, New structure-activity relationship studies in a series of N,N-
bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-
mediated multidrug resistance (MDR), Bioorg. Med. Chem. 21 (2013)
456e465.
[3] R.L. Juliano, V. Ling, A surface glycoprotein modulating drug permeability in
Chinese hamster ovary cell mutants, Biochim. Biophys. Acta 455 (1976)
152e162.
[4] A.H. Schinkel, J.W. Jonker, Mammalian drug efflux transporters of the ATP
binding cassette (ABC) family: an overview, Adv. Drug Deliv. Rev. 55 (2003)
3e29.
[31] C. Martelli, S. Dei, C. Lambert, D. Manetti, F. Orlandi, M.N. Romanelli,
S. Scapecchi, M. Salerno, E. Teodori, Inhibition of P-glycoprotein-mediated
multidrug resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters:
further restriction of molecular flexibility maintains high potency and effi-
cacy, Bioorg. Med. Chem. Lett. 21 (2011) 106e109.
[32] J. He, J. Zhao, T. Shen, H. Hidaka, N. Serpone, Photosensitization of colloidal
titania particles by electron. Injection from an excited organic dye-antennae
function, J. Phys. Chem. B 101 (1997) 9027e9034.
[5] M. Dean, Y. Hamon, G. Chimini, The human ATP-binding cassette (ABC)
transporter superfamily, J. Lipid Res. 7 (2001) 1007e1017.
[6] H. Kusuhara, Y. Sugiyama, Active efflux across the blood-brain barrier: role of
the solute carrier family, NeuroRx 2 (2005) 73e85.
[7] R.J. Dawson, K.P. Locher, Structure of a bacterial multidrug ABC transporter,
Nature 443 (2006) 180e185.
[8] S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell,
Y.T. Trinh, Q. Zhang, I.L. Urbatsch, G. Chang, Structure of P-glycoprotein re-
veals a molecular basis for poly-specific drug binding, Science 323 (2009)
1718e1722.
[33] N. Kowanko, Biosynthesis of the Cinchona alkaloids. I. The incorporation of
tryptophan into quinine, JACS 84 (1962) 4919e4921.
[34] J. Boudron, L. Commeiras, P. Barbier, V. Bourgarel-Rey, E. Pasquier,
N. Vanthuyne, J.C. Hubaud, V. Peyrot, J.L. Parrain, Caulerpenyne-colchicine
hybrid: synthesis and biological evaluation, Bioorg. Med. Chem. 14 (2006)
5540e5548.
[35] L.H. Klemm, K.W. Gopinath, G.C. Karaboyas, G.L. Capp, D. Hsu Lee, Synthesis
and spectral studies of some dienic and enynic esters, Tetrahedron 20 (1964)
871e876.
[36] C.B. Lozzio, B.B. Lozzio, Human chronic myelogenous leukemia cell line pos-
itive philadelphia chromosome, Blood 45 (1975) 321e334.
[37] J. Vergote, J.L. Moretti, E.G.E. De Vries, A. Garnier-Suillerot, Comparison of the
kinetics of active efflux of 99mTc-MIBI in cells with P-glycoprotein-mediated
and multidrug-resistance protein-associated multidrug-resistance pheno-
thype, Eur. J. Biochem. 252 (1998) 140e146.
[9] A.B. Ward, P. Szewczyk, V. Grimard, C.W. Lee, L. Martinez, R. Doshi, A. Caya,
M. Villaluz, E. Pardon, C. Cregger, D.J. Swartz, P.G. Falson, I.L. Urbatsch,
C. Govaerts, J. Steyaert, G. Chang, Structures of P-glycoprotein reveal its
conformational flexibility and an epitope on the nucleotide-binding domain,
PNAS 110 (2013) 13386e13391.
[10] I.K. Pajeva, C. Globisch, M. Wiese, Comparison of the inward- and outward-
open homology models and ligand binding of human P-glycoprotein, FEBS J.
23 (2009) 7016e7026.
[11] E. Teodori, S. Dei, S. Scapecchi, F. Gualtieri, The medicinal chemistry of
multidrug resistance (MDR) reversing drugs, Farmaco 57 (2002) 385e415.
[12] T.J. Raub, P-glycoprotein recognition of substrates and circumvention through
rational drug design, Mol. Pharm. 3 (2006) 3e25.
[13] E. Teodori, S. Dei, C. Martelli, S. Scapecchi, F. Gualtieri, The functions and
structure of ABC transporters: implications for the design of new inhibitors of
Pgp and MRP1 to control multidrug resistance (MDR), Curr. Drug Targets 7
(2006) 893e909.
[38] P. Reungpatthanaphong, C. Marbeuf-Gueye, L. Le Moyec, M. Salerno,
A. Garnier-Suillerot, Decrease of P-glycoprotein activity in K562/ADR cells by
MbCD and filipin and lack of effect induced by cholesterol oxidase indicate
that this transporter is not located in rafts, J. Bioenerg. Biomembr. 36 (2004)
533e543.
[14] K. Yang, J. Wu, X. Li, Recent advances in the research of P-glycoprotein in-
hibitors, Biosci. Trends 2 (2008) 137e146.
[39] T. Tsuruo, H. Ida, H. Kawataba, T. Oh-Hara, H. Hamada, T. Utakoji, Character-
istics of resistance to adriamycin in human myelogeneous leukemia K562
resistant to adriamycin and in isolated clones, Jpn. J. Cancer Res. 77 (1986)
682e687.
[15] H.M. Coley, Overcoming multidrug resistance in cancer: clinical studies of P-
glycoprotein inhibitors, Methods Mol. Biol. 596 (2010) 341e358.
[16] S. Modok, H.R. Mellor, R. Callaghan, Modulation of multidrug resistance efflux
pump activity to overcome chemoresistance in cancer, Curr. Opin. Pharmacol.
6 (2006) 350e354.

412
S. Dei et al. / European Journal of Medicinal Chemistry 87 (2014) 398e412
[40] E. Teodori, S. Dei, P. Quidu, R. Budriesi, A. Chiarini, A. Garnier-Suillerot,
F. Gualtieri, D. Manetti, M.N. Romanelli, S. Scapecchi, Design, synthesis, and
in vitro activity of catamphiphilic reverters of multidrug resistance: discovery
of a selective, highly efficacious chemosensitizer with potency in the nano-
molar range, J. Med. Chem. 42 (1999) 1687e1697.
[41] E. Pereira, A. Garnier-Suillerot, Correlation between the short-term mea-
surements of drug accumulation in living cells and the long-term growth
inhibition, Biochem. Pharmacol. 47 (1994) 1851e1857.
G. Sgaragli, E. Teodori, N,N-bis(cyclohexanol)amine aryl esters: a new class of
highly potent transporter-dependent multidrug resistance inhibitors, J. Med.
Chem. 52 (2009) 807e817.
[44] A. Tarcsay, G.M. Keseru, Homology modeling and binding site assessment of
the human P-glycoprotein, Future Med. Chem. 3 (2011) 297e307.
[45] J. Tarasiuk, A. Garnier-Suillerot, Kinetic parameters for the uptake of anthra-
cycline by drug-resistant and drug-sensitive K562 cells, Eur. J. Biochem. 204
(1992) 693e698.
[42] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D.L. Fine,
B.J. Abbott, J.G. Mayo, R.H. Shoemaker, M.R. Boyd, Feasibility of drug screening
with panels of human tumour cell lines using a microculture tetrazolium
assay, Cancer Res. 48 (1988) 589e601.
[46] S. Mankehetkorn, A. Garnier-Suillerot, The ability of verapamil to restore
intracellular accumulation of anthracyclines in multidrug resistant cells
depends on the kinetics of their uptake, Eur. J. Pharmacol. 343 (1998)
313e321.
[43] C. Martelli, D. Alderighi, M. Coronnello, S. Dei, M. Frosini, B. Le Bozec,
D. Manetti, A. Neri, M.N. Romanelli, M. Salerno, S. Scapecchi, E. Mini,