Exploratory chemistry toward the identification of a new class of multidrug resistance reverters inspired by pervilleine and verapamil models

Article abstract of DOI:10.1021/jm050542x

On the basis of the present knowledge of the substrate recognition site of ABC transporter proteins and inspired by the structures of verapamil and pervilleine A, a new class of Pgp-mediated multidrug resistance (MDR) reverters has been designed and synthesized. The new compounds are flexible molecules carrying one or two basic nitrogen atoms flanked, at properly modulated distance, by two aromatic moieties. Most of the molecules studied possess MDR inhibitory activity on anthracycline-resistant erythroleukemia K 562 cells, showing a potency that is higher than that of the reference compound verapamil and, in a few cases (7, 12, 13, 17, 20, 22, 28), is in the high nanomolar range. These compounds may be useful leads to develop new MDR reverting agents. In fact, the chemical structure of the class is fairly simple and can be implemented in a variety of ways that will allow the synthesis of new compounds that might be useful leads for the development of drugs to control Pgp-dependent MDR.

Full text of DOI:10.1021/jm050542x

7426
J. Med. Chem. 2005, 48, 7426-7436
Exploratory Chemistry toward the Identification of a New Class of Multidrug
Resistance Reverters Inspired by Pervilleine and Verapamil Models
Elisabetta Teodori,*,† Silvia Dei,† Arlette Garnier-Suillerot,‡ Fulvio Gualtieri,† Dina Manetti,† Cecilia Martelli,†
Maria Novella Romanelli,† Serena Scapecchi,† Paiwan Sudwan,‡ and Milena Salerno‡
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze, via U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy, and
Laboratoire BioMoCeTi UMR 7033, Universite´ Paris 13, 74 rue Marcel Cachin, 93017 Bobigny, France
Received June 8, 2005
On the basis of the present knowledge of the substrate recognition site of ABC transporter
proteins and inspired by the structures of verapamil and pervilleine A, a new class of Pgp-
mediated multidrug resistance (MDR) reverters has been designed and synthesized. The new
compounds are flexible molecules carrying one or two basic nitrogen atoms flanked, at properly
modulated distance, by two aromatic moieties. Most of the molecules studied possess MDR
inhibitory activity on anthracycline-resistant erythroleukemia K 562 cells, showing a potency
that is higher than that of the reference compound verapamil and, in a few cases (7, 12, 13,
17, 20, 22, 28), is in the high nanomolar range. These compounds may be useful leads to develop
new MDR reverting agents. In fact, the chemical structure of the class is fairly simple and can
be implemented in a variety of ways that will allow the synthesis of new compounds that might
be useful leads for the development of drugs to control Pgp-dependent MDR.
Introduction
models.12 Very recently, a model that is a hybrid of the
first two mentioned above has been proposed by Loo and
co-workers.13
Drug resistance is the major cause of failure of
chemotherapy. Multidrug resistance (MDR) is a kind
of acquired drug resistance of cancer cells and micro-
organisms to a variety of chemotherapic drugs that
usually are structurally and mechanistically unre-
lated.1,2 MDR can be the result of several biochemical
mechanisms among which the most widely implicated
and studied is that resulting from altered cell membrane
transport.3 This kind of resistance is often referred to
as classical MDR4 and is due to a lower cell concentra-
tion of cytotoxic drugs associated with accelerated efflux
of the chemotherapic, as a consequence of the overex-
pression of a number of proteins that act as extrusion
pumps. Several families of pumps, using a variety of
energy sources, are present in mammals and microor-
ganisms.2,5,6 At the moment, the best-known extrusion
pumps are P-glycoprotein (Pgp) and MRP1 proteins7
that belong to the ABC superfamily of transporters that
use ATP as their energy source. In mammals, besides
cancer cells, these proteins have been found in several
important tissues, such as the blood-brain barrier
(BBB), intestinal epithelium, and hepatic cells, where
they apparently regulate the secretion of lipophilic
molecules and the extrusion of xenobiotics that have
entered the organism,8-10 processing a large variety of
substrates.
The structure of Pgp has so far been resolved only at
low resolution,14,15 whereas the structure of some pumps
of bacterial origin showing homology with Pgp, such as
MsbA, has been obtained at 4.5 (EC-MsbA) and 3.8 Å
resolution (VC-MsbA).16-18 This achievement has cast
new light on their structures and mechanism of action
and has opened the way to the development of homology
models for Pgp19,20 and MRP1.21 From these and many
other reports on the structure of extruding pumps, it
appears that the recognition sites are large, flexible and
rich in amino acids able to give electrostatic, hydropho-
bic, and π-stacking interactions, such as aromatic amino
acids.22-26 For instance, it has been reported that the
volume of the recognition site of Escherichia coli AcbR
pump (that does not belong to the ABC but to the RND
superfamily) is some 5000 Å3 in size and can bind sev-
eral ligands simultaneously.25 Moreover, it has been
shown that ligands with very close structures bind in
quite different modes to the recognition site of the
Staphylococcus aureus QacR repressor protein, which
is considered a model for multidrug recognition.26 At
present, it appears realistic to think that the interacting
sites of these transport proteins are large, polymorphous
drug recognition domains, where a variety of substrates
can be accommodated in
a plurality of binding
The mechanism of action of these extruding proteins
is still controversial. As a matter of fact, a number of
models have been proposed to explain their involvement
in MDR:11 the dominant drug pump concept12 has given
origin to three main models, such as the “aqueous pore”,
the “hydrophobic vacuum cleaner”, and the “flippase”
modes.25,27-29
Inhibition of the functions of Pgp and sister proteins
is considered a realistic approach to circumvent MDR,
and drugs possessing inhibitory properties have been
and are actively being sought.30-33 However, even if
several molecules are being evaluated in clinical trials,
so far, no drug has been approved for therapy.34,35 The
main problem associated with the development of ef-
fective Pgp-mediated MDR inhibitors seems due to poor
specificity, low affinity for the binding site, interference
* Corresponding author. Tel.: ++39 55 4573693. Fax: ++39 55
4573671. E-mail: elisabetta.teodori@unifi.it.
† Universita` di Firenze.
‡ Universite´ Paris 13.
10.1021/jm050542x CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/14/2005

New Class of Multidrug Resistance Reverters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7427
Chart 1
with the physiological role of Pgp and sister proteins,
and, last but not least, interference with the pharma-
cokinetics of the associated chemotherapic.
Nature is a generous source of molecules able to
modulate extruding pumps.31,32 Very recently, the MDR
modulating activity of a new family of tropane alkaloids
named pervilleines has been reported.36-38 To us, since
we have for a long time been engaged in the design and
study of MDR modulators,39-42 the structural similarity
of pervilleines and verapamil was immediately appar-
ent. In fact, the two compounds may be reconciled to a
unique structural scaffold, where a basic linker (L)
tethers two aromatic moieties (H1, H2), as shown in
Figure 1. Building on this idea, we designed a new series
Figure 2. Flexible molecules such as bis- or tris(arylalkyl)-
amines carrying aromatic moieties at different distances can
adapt to the recognition site of Pgp binding with high affinity,
in a variety of binding modes. X and Y represent atoms or
groups of atoms able to establish electrostatic interactions.
such flexible molecules would pay a toll in terms of
entropy; however, we were confident that loss in binding
energy would be compensated by the enthalpic gain
deriving from the fact that flexible molecules can choose,
within the recognition site, the most productive binding
mode. Interestingly, a similar approach, labeled as
“polyvalency”, has recently been used successfully by
Sauna and co-workers,48,49 who synthesized and studied
several homodimers of the natural MDR inhibitor
stipiamide.
In the starting phase of the research, as a proof of
concept, we decided to limit our efforts to molecules
carrying one nitrogen atom tethered by very simple
linkers, such as polyethylene chains, to aromatic moi-
eties. We also explored the potential of ethoxyethylene
linkers, mainly to face the expected problems of solubil-
ity for long hydrocarbon chains, and that of linkers
carrying two nitrogen atoms, such as ethylendiamine
Figure 1.
of molecules with the structures shown in Chart 1. We
reasoned that, given the properties of the Pgp recogni-
tion site described above, flexible molecules carrying a
basic nitrogen flanked, at properly modulated distance,
by two (or three) aromatic moieties would bind with high
affinity as they will be able to find the best accommoda-
tion within the site (Figure 2). Multiple aromatic rings
and at least one protonable nitrogen atom are the main
features of most of the pharmacophores proposed for
Pgp interaction with substrates and inhibitors;43-47 here
we add, as a further critical property, molecular flex-
ibility. Of course, we were aware that, upon binding,

7428 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Teodori et al.
Scheme 1a
a Reagents and conditions: (i) CHCl3, X-(CH2)n-OH (n ) 2-7; X ) Br, Cl); (ii) K2CO3/CH3CN, RNH-(CH2)n-OH (n ) 2-7; R ) H,
CH3); (iii) Ar1COCl, CHCl3; (iv) (t-ButOCO)2O/Et3N; (v) HCOOH/HCHO; (vi) CF3COOH, CH2Cl2. For the meaning of Ar and Ar1, see
Table 1.
Scheme 2a
a Reagents and conditions: (i) CHCl3; (ii) K2CO3/CH3CN, NHCH3(CH2)2O(CH2)2OH52 or NH2(CH2)2O(CH2)2OH; (iii) (t-ButOCO)2O/
Et3N; (iv) Ar1COCl, CHCl3; (v) CF3COOH, CH2Cl2. For the meaning of Ar, Ar1, and R, see Table 1.
and piperazine. The aromatic moieties where chosen
among those present in pervilleines or those that, in our
previous work on verapamil-derived MDR reverters,
gave the best results, such as the anthracene and
fluorene moieties present in compounds MM3641 and
SC11,39 which were active at nanomolar doses. It must
be remarked that, for each series, we did not synthesize
all the compounds that can be obtained by combining
the aromatic fragments shown in Chart 1, as we could
have easily done using a combinatorial chemistry ap-
proach. As a matter of fact, since HTS facilities were
not available, we followed the classic medicinal chem-
istry approach, modulating the choice of the compounds
to synthesize by following biological results.
nopropan-1-ol, 5-aminopentan-1-ol, 6-aminohexan-1-ol
or 3-methylaminopropan-1-ol,50 and 7-aminoheptan-1-
ol51 to give 39-45.
The secondary amines 43-45 were alkylated by
reductive methylation with HCOOH/HCHO to give the
tertiary amines 48-50. The amino function of the
secondary amines 41 and 42 was protected by trans-
formation into the tert-butyl carbamate (t-BOC) to give
compounds 46 and 47. Compounds 1-7, 11-17, 51-
53 were then obtained by reaction of 39, 40, and 46-
50 with the proper acyl chloride. Finally the secondary
amines 8-10 were obtained after acidic cleavage of 51-
53 with CF3COOH.
A similar pathway allowed the synthesis of the
ethoxyethylamino derivatives 18-20 (Scheme 2). By
reaction of 2-(2-chloroethoxy)ethanol with trans-3-(3,4,5-
trimethoxyphenyl)acryloyl chloride or 3,4,5-trimethoxy-
benzoyl chloride, compounds 54 and 55 were obtained,
respectively. The tertiary amines 18 and 20 were
obtained by reaction of 54 with 2-(2-methylamino-
ethoxy)ethanol52 followed by esterification with the
appropriate acyl chloride. The secondary amine 19 was
obtained by reaction of 55 with 2-(2-aminoethoxy)-
ethanol (commercially available) to give 57, which in
turn was reacted with di-tert-butyl dicarbonate to give
58. The protected compound was reacted with trans-3-
(3,4,5-trimethoxyphenyl)acryloyl chloride to give 59 that
Chemistry
The reaction pathways used to synthesize the desired
compounds (1-32) are described in Schemes 1-3 and
their chemical and physical characteristics are reported
in Table 1.
Compounds 1-17 were synthesized as reported in
Scheme 1. The halo esters 33-38, obtained by esteri-
fication of the corresponding haloalkyl alcohol (2-bro-
moethanol, 3-bromopropan-1-ol, 5-chloropentan-1-ol,
6-chlorohexan-1-ol, or 7-bromoheptan-1-ol) with trans-
3-(3,4,5-trimethoxyphenyl)acryloyl chloride or anthracene-
9-carbonyl chloride, were reacted with the commercially
available amino alcohols 2-(methylamino)ethanol, 3-ami-

New Class of Multidrug Resistance Reverters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7429
Scheme 3a
a Reagents and conditions: (i) ArCOCl, CHCl3; (ii) 1-benzylpiperazine or N-t-BOC-piperazine,53 K2CO3/CH3CN; (iii) N-benzyl-N,N′-
dimethylethane-1,2-diamine54 or N-t-BOC-N,N′-dimethylethane-1,2-diammine,55 K2CO3/CH3CN; (iv) Pd/C, HCOONH4, MeOH or CF3COOH,
CH2Cl2; (v) Ar1COOCH2CH2Br (34, 35), K2CO3/ CH3CN; (vi) 3-bromopropan-1-ol, K2CO3/CH3CN; (vii) Ar1COCl, CHCl3. For the meaning
of Ar and Ar1, see Table 1.
after cleavage of the protective group with CF3COOH
gave the desired compound.
of the inhibitor) and 1 (when the amount of pirarubicin
in resistant cells is the same as in sensitive cells), (ii)
R
max, which expresses the efficacy of MDR-modulator
Piperazine and dimethylaminoethane derivatives 21-
32 were synthesized as shown in Scheme 3. Compounds
60, 34, and 35, obtained by esterification of 3-bromopro-
pan-1-ol with the suitable acyl chloride, were reacted
with 1-benzylpiperazine or N-t-BOC-piperazine53 to
obtain compounds 61-63. Cleavage of the protective
group was performed by hydrogenation with HCOONH4,
Pd/C 10% or with CF3COOH to give compounds 64-
66, which were alkylated with the bromo esters 34 or
35 to obtain the desired compounds 21-24. In the case
of 25 and 26, this procedure, gave very poor yields and
therefore a different pathway was followed. Compounds
65 and 66 were alkylated with 3-bromopropan-1-ol to
give 67 and 68 that in turn were esterified with 4-cyano-
4-(3,4-dimethoxyphenyl)-5-methylhexanoyl chloride or
fluorene-9-carbonyl chloride to give 25 and 26, respec-
tively.
and is the maximum increase that can be obtained in
the nuclear concentration of pirarubicin in resistant
cells with a given inhibitor; and (iii) [I]0.5, which
measures the potency of MDR-reverting agent and
represents the concentration of the inhibitor that causes
a half-maximal increase (R ) 0.5) in nuclear concentra-
tion of pirarubicin (see Table 2).
Even if binding data with Pgp are not available at
the moment, this test indicates that our compounds
inhibit the Pgp-operated extrusion of the reporter
molecule pyrarubicin, as does the reference molecule
verapamil. The molecules studied lack any detectable
cytotoxicity at the doses used in the test.
Results and Discussion
The results of the MDR-reverting activity of the
compounds synthesized are reported in Table 2. Inspec-
tion of the table indicates that most of the molecules
studied possess MDR-inhibitory activity and show a
potency that is higher than that of the reference
compound verapamil and, in a few cases (7, 12, 13, 17,
20, 22, 28), is in the same high nanomolar range of
MM36, the most potent compound that we have found
in previous studies.41 Overall, the results obtained seem
to confirm that the rationale upon which the present
work is based was sound and that the entropic toll paid
by molecules while binding is compensated by the
enthalpic gain deriving from the fact that such flexible
molecules can chose, within the recognition site, the
most productive binding mode. Therefore, the molecules
of this new class can be taken as leads to design new
potent and selective MDR-reverting agents. In fact,
since the new structures are only loosely related to
verapamil, it can be expected that their members lack
The synthesis of compounds 27-32 was performed
using a similar reaction pathway by reaction of 60, 34,
and 35 with N-benzyl-N,N′-dimethylethane-1,2-diamine54
or N-t-BOC-N,N′-dimethylethane-1,2-diamine,55 as shown
in Scheme 3.
Pharmacological studies
MDR-Reverting Activity. The ability of the exam-
ined compounds to revert MDR was evaluated on
anthracycline-resistant erythroleukemia K 562 cells,
measuring the uptake of THP-adriamycin (pirarubicin)
by the continuous spectrofluorometric signal of the
anthracycline at 590 nm (λex ) 480 nm) after incubation
of the cells, following the protocols reported in previous
papers.39,41,56 MDR-reverting activity is described by (i)
R, which represents the fold increase in the nuclear
concentration of pirarubicin in the presence of the MDR-
reverting agent and varies between 0 (in the absence

7430 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Table 1. Chemical and Physical Characteristics of Compounds 1-32
Teodori et al.
%
yield
no.
structure
R
n
Ar
Ar1
salt/mp (°C)
analysisa
1
2
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
C
C
C
C
C
C
D
D
D
D
D
D
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
2
2
a
a
a
a
a
a
a
a
a
c
a
a
a
a
a
a
a
a
a
a
a
a
c
a
c
a
a
a
c
a
c
a
e
c
b
f
34
52
74
58
96
56
90
64
36
60
65
40
57
36
46
57
43
72
43
61
85
59
31
61
33
59
50
69
69
69
22
59
oxab/50-54c
C29H37NO14
C34H35NO11
C29H37NO14
C26H31NO11
C33H35NO11
C31H41NO14
C36H39NO11
C30H39NO14
C35H37NO11
C37H43ClN2O6
C35H49NO14
C40H47NO11
C39H47NO11
C35H51NO10
C42H51NO11
C39H57NO14
C44H55NO11
C33H45NO16
C32H43NO16
C38H43NO13
C34H46N2O14
C39H44N2O11
C40H38N2O4
C34H46N2O10
C43H51N3O10
C38H44N2O11
C34H48N2O14
C39H46N2O11
C40H40N2O4
C36H50N2O14
C43H53N3O10
C38H46N2O11
oxa/97-99d
3
2
oxa/82-85d
4
2
oxa/103-105d
oxa/134-136d
oxa/96-98c
5
2
g
b
c
b
c
d
b
c
g
b
c
b
c
b
b
c
b
c
c
a
d
g
b
c
c
a
d
g
6
3
7
3
oxa/85-87c
8
3
oxa/172-174c
oxa/156-158c
HCl/50-54e
9
H
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
5
oxa/50-54c
5
oxa/60-65c
5
oxa/66-70c
6
liquid free base
oxa/171-173c
oxa/58-60c
6
7
7
oxa/79-81c
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
oxa/58-60c
oxa/121-123c
oxa/88-90c
CH3
-
oxa/215-220c
oxa/214-217c
free base/160-163
free base/159-161
oxa/223-226c
oxa/207-210c
oxa/50-55c
-
-
-
-
-
-
-
oxa/160-163c
free base/120-122
oxa/159-164c
oxa/160-163c
oxa/156-160c
-
-
-
-
a All compounds have been analyzed for C, H, N after vacuum-drying at a temperature below the melting point; the results obtained
range within (0.4% of the theoretical values. b Oxa ) oxalate. c Recrystallization solvent: ethyl acetate. d Recrystallization solvent: absolute
ethanol. e Recrystallization solvent: absolute ethanol/anhydrous diethyl ether.
any cardiovascular activity. Indeed, this has been
confirmed in the case of 2 and 24, which did not show
any detectable cardiovascular action on heart and aorta
models in the assays that we have previously described41
(data not shown).
In detail, some clear-cut structure-activity relation-
ships can be found by inspection of the results reported
in Table 2. Comparing the compounds of the A series
that have the same aromatic moieties at the ends of the
molecule (2, 7, 12, 15, 17), it is apparent that the length
of the linker is critical: MDR-reverting potency in-
creases with the number of methylenes, reaching the
maximum for n ) 5 (12; [I]0.5 ) 0.10 µM). For n ) 6
there is a drop in potency (15; [I]0.5 ) 0.61 µM) that,
however, is nearly completely recovered for n ) 7 (17;
[I]0.5 ) 0.15 µM). Recently, Sauna and co-workers,48
studying stipiamide homodimers, reported that increas-
ing the length of the spacer between the two monomers
from 11 up to 35 Å, significantly enhanced the capacity
of the dimers to inhibit drug efflux. Our series of
compounds seem to behave much in the same way. In
fact, the extended conformation distance between the
two aromatic moieties of the compounds that carry the
same aromatic moieties ranges from 16.43 to 27.53 Å
(Table 3). Reasonably, in both cases, the distance of the
bound conformations might change considerably, since
the molecules are rather flexible, a property that is at
the base of our design and that Sauna and co-workers
consider critical also for stipiamide dimers.48 Indeed, an
expected consequence of flexibility is that any molecule
of either set may bind in its own way, according to the
length of the linker, much like what has been observed

New Class of Multidrug Resistance Reverters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7431
Table 2. MDR-Reverting Activity of Compounds 1-32
no.
structure
R
n
Ar
Ar1
[I]0.5, µM
Rmax
1
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
C
C
C
C
C
C
D
D
D
D
D
D
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
2
2
a
a
a
a
a
a
a
a
a
c
a
a
a
a
a
a
a
a
a
a
a
a
c
a
c
a
a
a
c
a
c
a
e
c
b
f
6.40 ( 1.6
1.00 ( 0.2
3.10 ( 0.8
3.70 ( 0.9
0.80 ( 0.2
0.60 ( 0.15
0.18 ( 0.05
0.50 ( 0.1
0.21 ( 0.05
2.10 ( 0.5
0.80 ( 0.2
0.10 ( 0.02
0.11 ( 0.03
2.0 ( 0.4
0.80
0.84
0.86
0.86
0.70
0.90
0.78
1
0.74
0.90
0.84
0.80
0.83
0.6
2
3
2
4
2
5
2
g
b
c
b
c
d
b
c
g
b
c
b
c
b
b
c
b
c
c
a
d
g
b
c
c
a
d
g
6
3
7
3
8
3
9
H
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
H
3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
5
5
5
6
6
0.61 ( 0.15
1.0 ( 0.4
0.68
0.55
0.55
0.90
1
7
7
0.15 ( 0.05
4.20 ( 1.0
7.0 ( 2.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CH3
-
0.17 ( 0.04
0.38 ( 0.10
0.16 ( 0.04
2.50 ( 0.5a
0.37 ( 0.10
0.62 ( 0.15
0.23 ( 0.06
1.30 ( 0.3
0.19 ( 0.04
0.80 ( 0.2a
2.60 ( 0.5
0.40 ( 0.1
0.57 ( 0.15
1.60 ( 0.3
0.05 ( 0.01
0.84
0.61
0.90
0.50
0.80
0.65
0.70
0.78
0.70
0.50
0.80
0.74
0.64
0.70
0.70
-
-
-
-
-
-
-
-
-
-
-
verapamil
MM36
a Concentration of the inhibitor that causes a 40% increase in nuclear concentration of pirarubicin (R ) 0.4).
Table 3. Calculated Extended Conformation Distance (d)
17. It is interesting that, a few months ago, Cianchetta
and co-workers57 have proposed that Pgp substrates
interact, among others, with two hydrophobic areas 16.5
Å apart, which seems compatible with the length of
inhibitors such as our compounds and stipiamide ho-
modimers.
between the Centers of the Aromatic Moietiesa
A second important feature of the A series is that also
the nature of the aromatic hydrophobic moieties is
critical. As observed in our previous works,39,41 the
presence of antracene (c) or fluorene (g) moieties is
no.
n
d (Å)
no.
n
d (Å)
2
7
12
2
3
5
16.43
17.49
22.35
15
17
6
7
25.85
27.53
beneficial with respect to the benzene (f) one (2, [I]0.5
)
a The molecules have been drawn in the all-trans configuration
and minimized with Discover (cvff force field) (d < 0.01). A
pseudoatom has been created at the center of aromatic rings.
1.0 µM, and 5, [I]0.5 ) 0.80 µM, versus 4, [I]0.5 ) 3.70
µM), as is the presence of the hydrogen-bond-acceptor
methoxy groups. As a matter of fact, within the A series,
the best combination of aryl moieties is that presenting
antracene (c) [or fluorene (g)] and (3,4,5-trimethoxyphen-
yl)vinyl moieties (a) (2, [I]0.5 ) 1.0 µM; 7, [I]0.5 ) 0.18
for the already mentioned S. aureus QacR repressor
protein.26 If this is true, the difference in potency of 15
with respect to 12 and 17 can be rationalized by
admitting that 15 cannot find, inside the recognition
site, a binding mode as productive as those of 12 and
µM; 9, [I]0.5 ) 0.21 µM; 12, [I]0.5 ) 0.10 µM; 13, [I]0.5
0.11 µM; 17, [I]0.5 ) 0.15 µM).
)

7432 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Teodori et al.
substances obtained were almost pure and used as such for
the next reaction. Their IR and 1H NMR spectra are consistent
with the proposed structures.
A third interesting finding in the A series is that
secondary and tertiary amines present more or less the
same activity (compare 6, [I]0.5 ) 0.60 µM, and 8, [I]0.5
) 0.50 µM, or 7, [I]0.5 ) 0.18 µM, and 9, [I]0.5 ) 0.21
µM).
The spectra of trans-3-(3,4,5-trimethoxyphenyl)acrylic acid
2-bromoethyl ester 33 are reported as an example. IR (Nujol):
1
ν cm-1 1711 (CO). H NMR (CDCl3): δ 7.65 (d, J ) 15.9 Hz,
All these SARs are essentially maintained in the B,
C, and D series. Thus, in the B series, the compound
carrying the antracene (c) and (3,4,5-trimethoxyphenyl)-
vinyl moieties (a) and having a length similar to
compound 12, is the most potent (20, [I]0.5 ) 0.17 µM),
whereas secondary and tertiary amines show similar
activity (18, [I]0.5 ) 4.2 µM; 19, [I]0.5 ) 7.0 µM). Series
C and D are characterized by two ionizable nitrogen
atoms, but SARs found in the one nitrogen series hold
also in this case: the most potent compounds are 22
and 28, both containing the antracene (c) and (3,4,5-
trimethoxyphenyl)vinyl moieties (a) (22, [I]0.5 ) 0.16 µM;
28, [I]0.5 ) 0.19 µM) and showing a length close to that
of 12 (22.28 and 22.71 Å vs 22.35 Å). The results
obtained in all series indicate that the nature of the
linker is not critical, provided it contains at least a
protonable nitrogen atom.
In conclusion, we have designed and studied a new
class of MDR reverters, among which potent and selec-
tive molecules have been found. Even if suggested by
the structure of a drug used for other purposes (vera-
pamil) and of a compound of natural origin (pervilleine
A), the class was mainly designed on the basis of the
information that has recently been collected on the
nature of the recognition site of Pgp and sister proteins.
The general structure of the compounds of the class is
fairly simple and can be implemented in several ways
that will allow the synthesis of a variety of new
compounds that might be useful leads for the develop-
ment of drugs to control Pgp-dependent MDR.
1H, CHdCH), 6.76 (s, 2H, aromatics), 6.38 (d, J ) 15.9 Hz,
1H, CHdCH), 4.52 (t, J ) 6.0 Hz, 2H, CH2O), 3.89 (s, 9H,
3OCH3), 3.65 (t, J ) 6.0 Hz, 2H, CH2Br) ppm.
General Procedure for the Synthesis of Hydroxyami-
no Esters (39-45, 56, 57). The appropriate halo ester (1
mmol) and the suitable aminoalkyl alcohol (1.2 mmol) were
dissolved in 1 mL of anhydrous CH3CN, and K2CO3 (1 mmol)
was added. The mixture was heated at 60 °C for 5-10 h. The
reaction mixture was cooled to room temperature and 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 using CHCl3/MeOH (95:5) (compounds
39, 40, 42, 45, 56) or CH2Cl2/abs EtOH/petroleum ether/NH4-
OH (340:65:60:8) (compounds 41, 43, 44, 57) as the eluting
system. Yields 50-70%. Their IR and 1H NMR spectra are
consistent with the proposed structures.
The spectra of trans-3-(3,4,5-trimethoxyphenyl)acrylic acid
2-[(2-hydroxyethyl)methylamino]ethyl ester (39) are reported
1
as an example. IR (neat): ν cm-1 3410 (OH), 1709 (CO). H
NMR (CDCl3): δ 7.63 (d, J ) 16.1 Hz, 1H, CHdCH), 6.78 (s,
2H, aromatics), 6.36 (d, J ) 16.1 Hz, 1H, CHdCH), 4.33 (t, J
) 5.5 Hz, 2H, CH2O), 3.90 (s, 6H, 2OCH3), 3.88 (s, 3H, OCH3),
3.61 (t, J ) 5.1 Hz, 2H, CH2OH), 2.80 (t, J ) 5.1 Hz, 2H,
CH2N), 2.65 (t, J ) 5.5 Hz, 2H, CH2N), 2.37 (s, 3H, NCH3)
ppm.
trans-3-(3,4,5-Trimethoxyphenyl)acrylic Acid 3-(tert-
Butoxycarbonylbutylamino)propyl Ester (46). To a solu-
tion of 0.1 g (0.28 mmol) of 41 in 5 mL of THF cooled to 0 °C
were added 80 mg (0.36 mmol) of di-tert-butyl dicarbonate and
0.08 mL of triethylamine. The mixture was maintained at
room temperature for 6 h and then concentrated in vacuo. The
residue was dissolved in CH2Cl2 and the organic layer was
washed with water. After drying with Na2SO4, the solvent was
removed under reduced pressure. Compound 46 was obtained
as a light yellow oil (120 mg, 95% yield). IR (neat): ν cm-1
Experimental Section
1
3448 (OH), 1708 (CO). H NMR (CDCl3): δ 7.60 (d, J ) 16.1
Chemistry. All melting points were taken on a Bu¨chi
apparatus and are uncorrected. Infrared spectra were recorded
with a Perkin-Elmer 681 or a Perkin-Elmer Spectrum RX I
FT-IR spectrophotometer in Nujol mull for solids and neat for
liquids. NMR spectra were recorded on a Gemini 200 or a
Bruker Avance 400 spectrometer. Chromatographic separa-
tions were performed on a silica gel column by gravity
chromatography (Kieselgel 40, 0.063-0.200 mm; Merck) or
flash chromatography (Kieselgel 40, 0.040-0.063 mm; Merck).
Yields are given after purification, unless otherwise stated.
Where analyses are indicated by symbols, the analytical
results are within (0.4% of the theoretical values. We have
chosen to perform and report only the combustion analyses of
final compounds. The identity and purity of the intermediates
was ascertained through IR, NMR, and TLC chromatography.
Compounds were named following IUPAC rules as applied by
Beilstein-Institut AutoNom (version 2.1), a software for sys-
tematic names in organic chemistry. When reactions were
performed in anhydrous conditions, the mixtures were main-
tained under nitrogen.
General Procedure for the Synthesis of Esters (33-
38, 54, 55, 60). A 1-mmol portion of the appropriate carboxylic
acid (commercially available) was transformed into the acyl
chloride by reaction with SOCl2 (2 mL), in 5 mL of anhydrous
C6H6, at 60 °C for 4-8 h. The reaction mixture was cooled to
room temperature and the solvent was removed under reduced
pressure. The acyl chloride obtained was dissolved in CHCl3
(free of EtOH) and the suitable alcohol (1 equiv) was added.
The reaction mixture was heated to 60 °C for 4-8 h, cooled to
room temperature, and treated with CH2Cl2, and the organic
layer washed with 10% NaOH solution. After drying with Na2-
SO4, the solvent was removed under reduced pressure. The
Hz, 1H, CHdCH), 6.75 (s, 2H, aromatics), 6.33 (d, J ) 16.1
Hz, 1H, CHdCH), 4.23 (t, J ) 5.8 Hz, 2H, CH2O), 3.88 (s, 6H,
2OCH3), 3.87 (s, 3H, OCH3), 3.57-3.25 (m, 6H, 2CH2N, CH2-
OH), 1.98-1.52 (m, 5H, 2CH2 and OH), 1.46 (s, 9H, 3CH3)
ppm.
Compounds 47 and 58 were obtained in the same way from
the corresponding secondary amines 42 and 57, respectively.
Their IR and 1H NMR spectra are consistent with the proposed
structures.
trans-3-(3,4,5-Trimethoxyphenyl)acrylic Acid 5-[(5-Hy-
droxypentyl)methylamino]pentyl Ester (48). To a solution
of 0.8 g (1.95 mmol) of 43 in 25 mL of anhydrous ethanol were
added 24 mL of HCOOH and 24 mL of HCHO. The mixture
was heated to 80 °C for 7 h and concentrated in vacuo. The
residue was then dissolved in CH2Cl2 and the organic layer
was washed with a saturated solution of Na2CO3 and with
water. After drying with Na2SO4, the solvent was removed
under reduced pressure. The crude product was then purified
by flash chromatography using CH2Cl2/abs EtOH/ethyl ether/
petroleum ether/NH4OH 360:180:360:900:9.9 as the eluting
system. Compound 48 was obtained as an oil (0.53 g, 64%
yield). IR (neat): ν cm-1 3404 (OH), 1710 (CO). 1H NMR
(CDCl3): δ 7.55 (d, J ) 16.1 Hz, 1H, CHdCH), 6.72 (s, 2H,
aromatics), 6.31 (d, J ) 16.1 Hz, 1H, CHdCH), 4.16 (t, J )
6.2 Hz, 2H, CH2O), 3.85 (s, 6H, 2OCH3), 3.83 (s, 3H, OCH3),
3.59 (t, J ) 6.1 Hz, 2H, CH2OH), 2.62 (s, 1H, OH), 2.31-2.27
(m, 4H, 2CH2N), 2.16 (s, 3H, NCH3), 1.76-1.22 (m, 12H, 6CH2)
ppm.
Compounds 49 and 50 were obtained in the same way
1
starting from 44 and 45, respectively. Their IR and H NMR
spectra are consistent with the proposed structures.

New Class of Multidrug Resistance Reverters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7433
2,3,4-Trimethoxybenzoic Acid 2-(N-Methyl-[2-[trans-
3-(3,4,5-trimethoxyphenyl)acryloyloxy]ethyl]amino)eth-
yl Ester (1). Following the general procedure described above
for the general synthesis of esters, the acyl chloride, obtained
from 2,3,4-trimethoxybenzoic acid (375 mg, 1.76 mmol), was
allowed to react with 39 (300 mg, 0.88 mmol). The crude
product was then purified by flash chromatography using CH2-
Cl2/abs EtOH/ethyl ether/petroleum ether/NH4OH 360:180:
360:900:9.9 as the eluting system.
mmol). The mixture was heated at 60 °C for 6 h, cooled to room
temperature, and treated with CH2Cl2. The organic layer was
washed with 10% NaOH solution, and after drying with Na2-
SO4, the solvent was removed under reduced pressure and the
residue purified by flash chromatography using CH2Cl2/abs
EtOH/ethyl ether/petroleum ether/NH4OH 360:180:360:900:
9.9 as the eluting system. Title compound (0.45 g, 70% yield)
was obtained as an oil. IR (neat): ν cm-1 1714 (CO). 1H NMR
(CDCl3): δ 7.29-7.25 (m, 7H, aromatics), 4.33 (t, J ) 6.6 Hz,
2H, CH2O), 3.87 (s, 9H, 3OCH3), 3.48 (s, 2H, CH2Ph), 2.55-
2.43 (m, 10H, 5CH2N), 2.03-1.89 (m, 2H, CH2) ppm.
The title compound (160 mg, 34% yield) was obtained as an
1
oil. IR (neat): ν cm-1 1714 (CO). H NMR (CDCl3): δ 7.63-
4-[3-[trans-3-(3,4,5-Trimethoxyphenyl)acryloyloxy]pro-
py]piperazine-1-carboxylic Acid tert-Butyl Ester (62).
Following the procedure described for compound 61, starting
from 34 (1.16 g, 3.2 mmol) and 1-BOC-piperazine53 (0.74 g,
4.0 mmol), compound 62 (1.41 g, 94% yield) was obtained as a
7.55 (m, 2H, CHdCH and aromatics), 6.73 (s, 2H, aromatics),
6.69 (d, J ) 10.9 Hz, 1H, aromatics), 6.37 (d, J ) 16.1 Hz, 1H,
CHdCH), 4.37 (t, J ) 5.6 Hz, 2H, CH2O), 4.31 (t, J ) 5.8 Hz,
2H, CH2O), 3.92 (s, 3H, OCH3), 3.90-3.84 (m, 15H, 5OCH3),
2.87-2.80 (m, 4H, 2 CH2N), 2.43 (s, 3H, NCH3) ppm. The oily
product was transformed into the oxalate that was recrystal-
lized from ethyl acetate. Mp: 50-54 °C. Anal. (C29H37NO14)
C, H, N.
Compounds 2-7, 11-18, and 20 were obtained in the same
way by reaction of the corresponding alcohol with the suitable
acyl chloride. Compounds 2-7, 11-13, 15-18, and 20 were
transformed into the oxalate and recrystallized from the
solvent reported in Table 1. Their chemical and physical
characteristics are reported in Table 1. IR and 1H NMR spectra
are reported in the Supporting Information.
1
yellow oil. IR (neat): ν cm-1 1714 (CO), 1693 (CO). H NMR
(CDCl3): δ 7.49 (d, J ) 15.9 Hz, 1H, CHdCH), 6.65 (s, 2H,
aromatics), 6.24 (d, J ) 15.9 Hz, 1H, CHdCH), 4.16 (t, J )
6.4 Hz, 2H, CH2O), 3.78 (s, 6H, 2OCH3), 3.76 (s, 3H, OCH3),
3.33 (t, J ) 4.9 Hz, 4H, 2CH2N), 2.37 (t, J ) 7.0 Hz, 2H, CH2N),
2.32-2.29 (m, 4H, 2CH2N), 1.82-1.77 (m, 2H, CH2), 1.35 (s,
9H, 3CH3) ppm.
Compound 63 was obtained in the same way from 35. Its
IR and 1H NMR spectra are consistent with the proposed
structure.
3,4,5-Trimethoxybenzoic Acid 3-[[2-(Benzylmethylami-
no)ethyl]methylamino]propyl Ester (69). Following the
procedure described for compound 61, starting from 60 (0.29
g, 0.87 mmol) and N-benzyl-N,N′-dimethylethane-1,2-di-
amine54 (0.18 g, 1.0 mmol), compound 69 (0.19 g, 51% yield)
3,4,5-Trimethoxybenzoic Acid 3-(N-tert-Butoxycarbon-
yl[3-[trans-3-(3,4,5-trimethoxyphenyl)acryloyloxy]propyl]-
amino)propyl Ester (51). Following the procedure described
for compound 1, starting from compound 46 (120 mg, 0.26
mmol) and 3,4,5-trimethoxybenzoyl chloride (120 mg, 0.52
mmol), compound 51 (180 mg, 36% yield) was obtained as a
1
was obtained as an oil. IR (neat) ν cm-1 1714 (CO). H NMR
1
light yellow oil. IR (neat): ν cm-1 1714 (CO), 1693 (CO). H
(CDCl3): δ 7.29-7.24 (m, 7H, aromatics), 4.33 (t, J ) 6.1 Hz,
2H, CH2O), 3.90 (s, 9H, 3OCH3), 3.50 (s, 2H, CH2Ph), 2.53-
2.46 (m, 6H, 3CH2N), 2.23 (s, 3H, NCH3), 2.20 (s, 3H, NCH3),
1.96-1.89 (m, 2H, CH2) ppm.
NMR (CDCl3): δ 7.60 (d, J ) 16.0 Hz, 1H, CHdCH), 7.29 (s,
2H, aromatics), 6.75 (s, 2H, aromatics), 6.34 (d, J ) 16.0 Hz,
1H, CHdCH), 4.34 (t, J ) 6.2 Hz, 2H, CH2O), 4.22 (t, J ) 6.2
Hz, 2H, CH2O), 3.90 (s, 12H, 4OCH3), 3.88 (s, 6H, 2OCH3),
3.46-3.31 (m, 4H, 2CH2N), 2.16-1.94 (m, 4H, 2CH2), 1.45 (s,
9H, 3CH3) ppm.
Compounds 52, 53, and 59 were obtained in the same way
from the corresponding alcohol with the suitable acyl chloride.
Their IR and 1H NMR spectra are consistent with the proposed
structures.
trans-3-(3,4,5-Trimethoxyphenyl)acrylic Acid 3-[[2-
(tert-Butoxycarbonylmethylamino)ethyl]methylamino]-
propyl Ester (70). Following the procedure described for
compound 61, starting from 34 (1.5 g, 4.2 mmol) and 1-BOC-
N,N′-dimethylethane-1,2-diamine55 (0.74 mg, 4.2 mmol), com-
pound 70 (1.0 g, 53% yield) was obtained as a brown oil. IR
(neat): ν cm-1 1714 (CO), 1693 (CO). 1H NMR (CDCl3): δ 7.50
(d, J ) 15.9 Hz, 1H, CHdCH), 6.67 (s, 2H, aromatics), 6.26
(d, J ) 15.9 Hz, 1H, CHdCH), 4.15 (t, J ) 6.4 Hz, 2H, CH2O),
3.79 (s, 6H, 2OCH3), 3.77 (s, 3H, OCH3), 3.28-3.15 (m, 2H,
CH2N), 2.78 (s, 3H, NCH3), 2.43-2.40 (m, 4H, 2CH2N), 2.18
(s, 3H, NCH3), 1.80-1.74 (m, 2H, CH2), 1.36 (s, 9H, 3CH3) ppm.
Compound 71 was obtained in the same way from 35. Its
IR and 1H NMR spectra are consistent with the proposed
structure.
3,4,5-Trimethoxybenzoic Acid 3-(piperazin-1-yl)propyl
Ester (64). To 0.42 g (1.0 mmol) of 61 dissolved in 5 mL of
anhydrous MeOH were added 0.21 g of Pd/C 10% and 0.31 g
(4.9 mmol) of HCOONH4. The mixture was refluxed for 5 h
and then filtered, and the solvent was removed under reduced
pressure. The residue was made alkaline with NaHCO3,
extracted with CH2Cl2, and dried. After removal of the solvent
under reduced pressure, the residue was purified by flash
chromatography using CH2Cl2/abs EtOH/petroleum ether/NH4-
OH (340:65:60:8). Compound 64 (160 mg, 48% yield) was
obtained as an oil. IR (neat): ν cm-1 3340 (NH), 1713 (CO).
1H NMR (CDCl3): δ 7.24 (s, 2H, aromatics), 4.33 (t, J ) 6.1
Hz, 2H, CH2O), 3.86 (s, 9H, 3OCH3), 2.88-2.84 (m, 4H,
2CH2N), 2.48-2.40 (m, 6H, 3CH2N), 2.00-1.89 (m, 3H, CH2
and NH) ppm.
3,4,5-Trimethoxybenzoic Acid 3-[3-[trans-3-(3,4,5-Tri-
methoxyphenyl)acryloyloxy]propyl]amino)propyl Ester
(8). To a solution of 120 mg (0.18 mmol) of 51 in 2 mL of CH2-
Cl2 was added 0.4 mL of CF3COOH under vigorous stirring.
After 3 h at room temperature, the solution was concentrated
in vacuo. The residue was then dissolved in ethyl acetate and
the organic layer was washed with a saturated solution of
NaHCO3. After drying with Na2SO4, the solvent was removed
under reduced pressure. Then 60 mg (64% yield) of the pure
title compound was obtained. IR (neat): ν cm-1 3580 (NH),
1711 (CO). 1H NMR (CDCl3): δ 7.60 (d, J ) 16.0 Hz, 1H, CHd
CH), 7.29 (s, 2H, aromatics), 6.75 (s, 2H, aromatics), 6.33 (d,
J ) 16.0 Hz 1H, CHdCH), 4.40 (t, J ) 6.6 Hz, 2H, CH2O),
4.29 (t, J ) 6.2 Hz, 2H, CH2O), 3.90 (s, 12H, 4OCH3), 3.88 (s,
6H, 2OCH3), 2.82-2.73 (m, 4H, 2CH2N), 2.01-1.87 (m, 4H,
2CH2), 1.65 (bs, 1H, NH) ppm. The oily product was trans-
formed into the oxalate that was recrystallized from ethyl
acetate. Mp: 172-174 °C. Anal. (C30H39NO14) C, H, N.
Compounds 9, 10, and 19 were obtained in the same way
by reaction of the corresponding N-tert-butoxycarbonylamino
ester. Compounds 9 and 19 were transformed into the oxalate
and recrystallized from ethyl acetate, and compound 10 was
transformed into the hydrochloride by treating the free base
with HCl/abs EtOH and recrystallizing from EtOH/anhydrous
diethyl ether. Their chemical and physical characteristics are
Compound 72 was obtained in the same way from 69. Its
IR and 1H NMR spectra are consistent with the proposed
structure.
trans 3-(3,4,5-Trimethoxyphenyl)acrylic Acid 3-(pip-
erazin-1-yl)propyl Ester (65). To 1.41 g (3.03 mmol) of 62
dissolved in 5 mL of CH2Cl2 was added 6.06 mL of trifluoro-
acetic acid. After 2 h at room temperature, the solvent and
the acid in excess were removed under reduced pressure. The
1
reported in Table 1. IR and H NMR spectra are reported in
the Supporting Information.
3,4,5-Trimethoxybenzoic Acid 3-(4-Benzylpiperazin-1-
yl)propyl Ester (61). To a solution of 0.5 g (1.5 mmol) of
compound 60 in 1 mL of anhydrous CH3CN were added
1-benzylpiperazine (0.41 mL, 2.3 mmol) and K2CO3 (0.2 g, 1.5

7434 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Teodori et al.
dark green oil obtained was dissolved in CH2Cl2 and washed
with 10% NaOH solution. After drying of the organic phase
with Na2SO4, the solvent was removed under reduced pressure.
Compound 65 (1.0 g, 91% yield) was obtained as a yellow oil.
1H NMR (CDCl3): δ 8.55 (s, 1H, aromatics), 8.05-8.03 (m, 4H,
aromatics), 7.57-7.49 (m, 4H, aromatics), 6.94-6.79 (m, 3H,
aromatics), 4.66 (t, J ) 6.0 Hz, 2H, CH2O), 4.08-3.93 (m, 2H,
CH2O), 3.90 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 2.84-2.45 (m,
12H, 6CH2N), 2.22-1.95 (m, 5H, 2CH2, CH(CH3)2), 1.88-1.65
(m, 4H, 2CH2) 1.22 (d, J ) 6.2 Hz, 3H, CHCH3), 0.80 (d, J )
6.2 Hz, 3H, CHCH3) ppm. The oily product was transformed
into the oxalate that was recrystallized from ethyl acetate.
Mp: 223-226 °C. Anal. (C43H51N3O10) C, H, N.
Compound 31 was obtained in the same way from 76 and
transformed into the oxalate that was recrystallized from ethyl
acetate. Its chemical and physical characteristics are reported
in Table 1 and IR and 1H NMR spectra are reported in the
Supporting Information.
9H-Fluorene-9-carboxylic Acid 3-(4-{3-[trans-3-(3,4,5-
Trimethoxyphenyl)acryloyloxy]propyl}piperazin-1-yl)-
propyl Ester (26). Following the procedure described for 25,
starting from 67 (70 mg, 0.16 mmol) and the acyl chloride
obtained from 70 mg (0.33 mmol) of fluorencarboxylic acid and
0.5 mL (0.66 mmol) of SOCl2 in dry C6H6, compound 26 (60
mg, 59% yield) was obtained as an oil. IR (neat): ν cm-1 1713
(CO). 1H NMR (CDCl3): δ 7.76 (d, J ) 7.5 Hz, 2H, aromatics),
7.66 (d, J ) 7.5 Hz, 2H, aromatics), 7.61 (d, J ) 15.8 Hz, 1H,
CHdCH), 7.43 (t, J ) 7.5 Hz, 2H, aromatics), 7.34 (t, J ) 7.5
Hz, 2H, aromatics), 6.77 (s, 2H, aromatics), 6.36 (d, J ) 15.8
Hz, 1H, CHdCH), 4.87 (s, 1H, CH), 4.27 (t, J ) 6.4 Hz, 2H,
CH2O), 4.21 (t, J ) 6.4 Hz, 2H, CH2O), 3.89 (s, 6H, 2OCH3),
3.88 (s, 3H, OCH3), 2.50-2.46 (m, 8H, 4CH2N), 2.38-2.34 (m,
4H, 2CH2N), 1.95-1.88 (m, 2H, CH2), 1.87-1.80 (m, 2H, CH2)
ppm. The oily product was transformed into the oxalate that
was recrystallized from ethyl acetate. Mp: 207-210 °C. Anal.
(C38H44N2O11) C, H, N.
Compound 32 was obtained in the same way from 75 and
transformed into the oxalate that was recrystallized from ethyl
acetate. Its chemical and physical characteristics are reported
in Table 1 and IR and 1H NMR spectra are reported in the
Supporting Information.
Pharmacology. Drugs and Chemicals. Purified piraru-
bicin was provided by Laboratoire Roger Bellon. Concentra-
tions were determined by diluting stock solutions to approxi-
mately 10-5 M and using ꢀ480 ) 11 500 M-1 cm-1. 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 7.25.
Cell Lines and Cultures. K 562 is a human leukemia cell
line.59 Cells resistant to doxorubicin were obtained by continu-
ous exposure to increasing doxorubicin concentrations and
were maintained in medium containing doxorubicin (400 nM)
until 1-4 weeks before experiments. This subline expresses a
unique membrane glycoprotein with a molecular mass of
180 000 Da.60 Doxorubicin-sensitive and -resistant erythro-
leukemia K 562 cells were grown in suspension in RPMI 1640
(Sigma) medium supplemented with L-glutamine and 10% FCS
at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
Cultures, initiated at a density of 105 cells/mL grew exponen-
tially to 8-10 × 105 cells/mL in 3 days. For the spectrofluo-
rometric assays, to have cells in the exponential growth phase,
culture was initiated at 5 × 105 cells/mL, and cells were used
24 h later, when the culture had grown to about 8-10 × 105
cells/mL. Cell viability was assessed by trypan blue exclusion.
The cell number was determined by Coulter counter analysis.
Cellular Drug Accumulation. The uptake of pirarubicin
cells was followed by monitoring the decrease in the fluores-
cence signal at 590 nm (λex ) 480 nm) according to the
previously described method.61 Using this method it is possible
to accurately quantify the kinetics of the drug uptake by the
cells and the amount of anthracycline intercalated between
the base pairs in the nucleus at the steady state, as incubation
of the cells with the drug proceeds without compromising cell
viability. All experiments were conducted in 1 cm quartz
cuvettes containing 2 mL of buffer at 37 °C. We checked that
tested compounds did not affect the fluorescence of pirarubicin.
1
IR (neat): ν cm-1 3340 (NH), 1731 (CO). H NMR (CDCl3): δ
7.55 (d, J ) 15.9 Hz, 1H, CHdCH), 6.71 (s, 2H, aromatics),
6.30 (d, J ) 15.9 Hz, 1H, CHdCH), 4.21 (t, J ) 6.4 Hz, 2H,
CH2O), 3.84 (s, 6H, 2OCH3), 3.83 (s, 3H, OCH3), 2.87-2.83
(m, 4H, 2CH2N), 2.43-2.39 (m, 6H, 3CH2N), 2.09 (bs, 1H, NH),
1.88-1.84 (m, 2H, CH2) ppm.
Compounds 66, 73, 74, were obtained in the same way from
63, 70, and 71, respectively. Their IR and 1H NMR spectra
are consistent with the proposed structures.
3,4,5-Trimethoxybenzoic Acid 3-(4-{3-[trans-3-(3,4,5-
Trimethoxyphenyl)acryloyloxy]propyl}piperazin-1-yl)-
propyl Ester (21). Following the procedure described for 61,
starting from 64 (135 mg, 0.40 mmol) and 34 (140 mg, 0.40
mmol), compound 21 (210 mg, 85% yield) was obtained as a
light yellow oil. IR (neat): ν cm-1 1713 (CO). 1H NMR
(CDCl3): δ 7.61 (d, J ) 15.7 Hz, 1H, CHdCH), 7.30 (s, 2H,
aromatics), 6.76 (s, 2H, aromatics), 6.35 (d, J ) 15.7 Hz, 1H,
CHdCH), 4.38 (t, J ) 6.1 Hz, 2H, CH2O), 4.27 (t, J ) 6.1 Hz,
2H, CH2O), 3.90 (s, 12H, 4OCH3), 3.88 (s, 6H, 2OCH3), 2.65-
2.38 (m, 12H, 6CH2N), 2.18-1.88 (m, 4H, 2CH2) ppm. The oily
product was transformed into the oxalate that was recrystal-
lized from ethyl acetate. Mp: 215-220 °C. Anal. (C34H46N2O14)
C, H, N.
Compounds 22-24 and 27-30 were obtained in the same
way. In particular, compound 22 was generated from 65 and
35, 23 from 66 and 35, 24 from 65 and 34, 27 from 72 and 34,
28 from 74 and 34, 29 from 74 and 35, and 30 from 73 and
34. Compounds 22, 27, 28, and 30 were transformed into the
oxalate and recrystallized from ethyl acetate. Their chemical
and physical characteristics are reported in Table 1. IR and
1H NMR spectra are reported in the Supporting Information.
trans-3-(3,4,5-Trimethoxyphenyl)acrylic Acid 3-[4-(3-
hydroxypropyl)piperazin-1-yl]propyl Ester (67). To a
solution of 0.4 g (1.1 mmol) of compound 65 in 1 mL of
anhydrous CH3CN were added 3-bromo-1-propanol (0.096 mL,
1.1 mmol) and K2CO3 (0.15 g, 1.1 mmol). The mixture was
heated at 60°C for 4 h. The reaction mixture was cooled to
room temperature and 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 was purified by flash chromatography using CH2-
Cl2/abs EtOH/ethyl ether/petroleum ether/NH4OH (360:180:
360:900:9.9) as eluting system. Title compound (70 mg, 15%
yield) was obtained as an oil. IR (neat): ν cm-1 3340 (NH),
1731 (CO). 1H NMR (CDCl3): δ 7.59 (d, J ) 15.8 Hz, 1H, CHd
CH), 6.75 (s, 2H, aromatics), 6.33 (d, J ) 15.8 Hz, 1H, CHd
CH), 4.25 (t, J ) 6.4 Hz, 2H, CH2O), 3.89 (s, 6H, 2OCH3), 3.88
(s, 3H, OCH3), 3.80 (t, J ) 5.0 Hz, 2H, CH2OH), 2.62-2.47
(m, 13H, 6CH2N and OH), 1.93-1.86 (m, 2H, CH2), 1.74-1.70
(m, 2H, CH2) ppm.
Compounds 68, 75, and 76 were obtained in the same way
from 66, 73, and 74, respectively. Their IR and 1H NMR
spectra are consistent with the proposed structures.
Anthracene-9-carboxylic Acid 3-(4-{3-[4-Cyano-4-(3,4-
dimethoxyphenyl)-5-methylhexanoyloxy]propyl}piper-
azin-1-yl)propyl Ester (25). 4-Cyano-4-(3,4-dimethoxyphenyl)-
5-methylhexanoic acid58 (110 mg, 0.36 mmol) was transformed
into the acyl chloride by reaction with oxalyl chloride (0.06
mL, 0.72 mmol) in 5 mL of anhydrous C6H6, at 60 °C for 12 h.
The reaction mixture was cooled to room temperature and the
solvent removed under reduced pressure. The acyl chloride
obtained was dissolved in CHCl3 (EtOH-free), and compound
68 (150 mg, 0.36 mmol) was added. The reaction mixture was
heated at 60 °C for 12 h, cooled to room temperature, and
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 using CHCl3/MeOH/AcOH (85:10:0.5)
as the eluting system. Title compound (80 mg, 33% yield) was
obtained as an oil. IR (neat): ν cm-1 2230 (CN), 1715 (CO).
Supporting Information Available: IR and 1H NMR
spectra and elemental analysis results of compounds 1-32.

New Class of Multidrug Resistance Reverters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7435
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http://pubs.acs.org.
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