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)

Article abstract of DOI:10.1016/j.bmc.2012.11.019

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure-activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis). The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I] _0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a-d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a-d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells. The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site. In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.

Full text of DOI:10.1016/j.bmc.2012.11.019

Bioorganic & Medicinal Chemistry 21 (2013) 456–465
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
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)
Francesca Orlandi a, Marcella Coronnello b, Cristina Bellucci a, Silvia Dei a, Luca Guandalini a,
Dina Manetti a, Cecilia Martelli a, Maria Novella Romanelli a, Serena Scapecchi a, Milena Salerno c,
Hayette Menif c, Ivan Bello c, Enrico Mini b, Elisabetta Teodori a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università di Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy
c Laboratoire des Acides Nucléiques et Biophotonique, FRE 3207 CNRS, Université Paris 13, UFR SMBH, 74 rue Marcel Cachin, 93017 Bobigny, France
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multi-
drug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and syn-
thesized several analogs by modulation of the two aromatic moieties linked through ester functions to
the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relation-
ships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids,
gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).
The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in
the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump
P-gp, showing potency values ([I]0.5) in the submicromolar and nanomolar range. Of these, compounds
2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a amax close to 1. Selected compounds (2d, 3a,
3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubi-
cin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin
cytotoxicity on K562/DOX cells.
Received 6 September 2012
Revised 7 November 2012
Accepted 12 November 2012
Available online 24 November 2012
Keywords:
MDR reversers
P-gp inhibitors
N,N-Bis(cyclohexanol)amine aryl esters
Chemosensitizers
Doxorubicin-resistant erythroleukemia
K562 cells (K562/DOX)
Pirarubicin uptake
The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of
the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and
rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the
several binding modes possible in the transporter recognition site.
In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,29 are
potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the develop-
ment of MDR-reversal drugs.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
perform an ATP-dependent active outward transport of chemo-
therapeutic drugs.2,3 These proteins such as ABCB1, ABCC subfam-
The development of resistance in cancer cells and microorgan-
isms is the main obstacle to achieving success with chemotherapy.
Frequently, following drug treatment, cells become resistant to a
variety of chemotherapeutic drugs that are even structurally and
mechanistically unrelated, thus resulting in so-called multidrug
resistance (MDR).1 In humans, MDR is associated with the over-
expression of transporter proteins acting as extrusion pumps that
ily and ABCG2, belong to the ABC super family of multidrug
transporters and, in normal conditions, are expressed in cells of
several tissues where they have an important physiological role
regulating the permeability of biological membranes,3 the secre-
tion of physiologically important lipophilic molecules4 and the
extrusion of xenobiotics that enter the organism.5
Among the MDR transporter proteins, ABCB1 (P-gp) is the most
extensively studied and in recent years several biochemical, bio-
physical and bioinformatic tools have been used to study its mech-
anism of action and to explore its structural characteristics.6–8
Even if there is no high-resolution crystal structure of human
P-gp, several homology models have been proposed, based on
Abbreviations: EDCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimmide hydro-
chloride; DMAP, 4-(N,N-dimethylamino)pyridine; DOX, doxorubicin.
⇑
Corresponding author. Tel.: +39 055 4573693; fax: +39 055 4573671.
E-mail address: elisabetta.teodori@unifi.it (E. Teodori).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.019

F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
457
the resolved structure of related bacterial proteins9–15 which have
provided useful information on the P-gp mechanism of action and
its recognition site. An important advancement in the field was the
recently described structure of murine P-gp16 which has 87% se-
quence identity to human ABCB1 transporter and obviously is a
better template for homology models.17,18
Although an atomic structure of human P-gp is still missing,
several important features of its structure are known, in particular
its recognition site that results characterized as a large, polymor-
phous drug binding domain where a variety of molecules can be
When esterified with different acids the N,N-bis(cyclohexa-
nol)amine moiety gives origin to four geometric isomers (cis/trans,
trans/trans, cis/cis and trans/cis) with varying geometry and possi-
bly different biological activity. Some of these novel derivatives
had low nanomolar potency and very high efficacy in inhibiting
P-gp-dependent nuclear pirarubicin efflux in doxorubicin-resistant
erythroleukemia K562 cells (K562/DOX) and rat intestinal mucosa
ATPase activity, and on increasing the cytotoxicity of doxorubicin
towards the K562/DOX cell line.30,31 Among the several sets of
isomers synthesized, those containing the trans-3-(3,4,5-trime-
thoxyphenyl)acrylic acid moiety combined with the 3,4,5-trimeth-
oxybenzoic acid one (compounds 1a–d, Table 1) showed the best
MDR-reversing activity both in terms of potency and efficacy.
Taking into consideration the outstanding potency and efficacy
of compounds 1a–d, we decided to extend the structure–activity
relationships studies in this series of molecules by studying new
analogues characterized by the N,N-bis(cyclohexanol)amine scaf-
fold with suitably modulated aromatic ester portions (Chart 1).
First of all, the trans-3-(3,4,5-trimethoxyphenyl)vinyl moiety
(A) was combined with 4-cyano-4-(3,4-dimethoxyphenyl)-5-
methylpentyl and 9H-fluorene moieties (C and D structures of
Chart 1) that are present in compounds active at nanomolar doses
reported in our previous work on verapamil-derived MDR revers-
ers32,33 to give sets 2 and 3.
Then, to investigate the consequence of geometric modulation of
the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid moiety (A), present
in set 1, analogous compounds where this residue was substituted
by 3-(3,4,5-trimethoxyphenyl)propanoic acid (E) (set 4) or 3-(3,4,5-
trimethoxyphenyl)propiolic acid (F) (set 5) were synthesized. Preli-
minary results of this research have been anticipated in a letter.34
The good activity of compounds containing the 3-(3,4,5-trime-
thoxyphenyl)propiolic acid moiety (5a–d) prompted us to synthe-
size other compounds containing this aromatic residue combined
with the 2,2-bis(4-methoxyphenyl)acetic acid residue and with
the 9H-fluorene-9-carboxylic acid one (sets 6a–d and 7a–d).
The reversal activity of the studied compounds was evaluated
by the pirarubicin doxorubicin-resistant erythroleukemia K562
cell uptake assay that was used as preliminary screening. The com-
pounds that showed the best results in this test (2d, 3a, 3b, 5a–d)
were further studied by evaluating their doxorubicin cytotoxicity
potentiation (RF) on the K562/DOX cell line.
accommodated in a plurality of binding modes, including
p
À
p,
ion– , hydrogen bonds and hydrophobic interactions. This means
p
that, unlike receptors and enzymes, P-gp and sister proteins lack
pre-existent and specific binding sites for the large variety of
chemicals that they are able to transport. This characteristic makes
it difficult to develop useful general pharmacophores but at the
same time appears fruitful in terms of drug research since it im-
plies that efficient modulators can be found in very different chem-
ical classes, as indeed is the case.19–21
In the past 30 years, P-gp overexpression in cancer cells has
been considered a possible therapeutic target for circumventing
MDR.22 Many P-gp-dependent MDR reversers, that should act by
being co-administrated with chemotherapeutic drugs, have been
identified20,23 and a few have reached clinical trials.24 Neverthe-
less, no drug of this kind has been cleared for the market so far.
Like substrates, even ABC transporter-targeting drugs present a
variety of chemical structures, suggesting that most of them bind
to the large substrate-recognition sites with the same plurality of
binding modes proposed for substrates.
The lack of a high-resolution crystal structure of human P-gp
and sister proteins, as well as their ability to interact with a very
large variety of chemical species, makes it nearly impossible to
use the two classic approaches of medicinal chemistry: structure-
based (including fragment-based drug design, FBDD) and ligand-
based drug design, to discover new drugs. As a consequence, search
for ABC protein inhibitors is, at present, mostly done by extensive
screening of synthetic or natural compounds and by chemical
modulation of those found active.
A general pharmacophore for P-gp interaction could be very
useful to direct the synthesis of more potent and selective revers-
ers. However, structure–activity relationship studies performed
until now have proved to be of limited value in attaining this goal,
because they have identified only some general characteristics
such as the importance of hydrogen bond acceptor groups, aro-
matic or hydrophobic features and the presence of a protonable
nitrogen atom.25–27 As expected from the broad substrate selectiv-
ity of ABC transporters like P-gp, more detailed SARs seem valid
only within a specific chemical class as the case of the model
proposed by Pajeva and Wiese on verapamil-related MDR
reversers.28
2. Chemistry
Compounds 2a–d, 3a–d, 6a–d and 7a–d were obtained follow-
ing the reaction pathways described in Schemes 1 and 2 and their
chemical and physical characteristics are reported in Table S1
(Supplementary data). The synthesis of compounds 4a–d and 5a–
d has been previously reported.34
Key intermediates 11a–d (Scheme 1) were synthesized starting
from the 4-oxocyclohexyl ester 8 obtained by esterification of
4-hydroxycyclohexanone35 with 3-(3,4,5-trimethoxyphenyl)propi-
olic acid36 in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCl) and 4-dimethylaminopyridine
(DMAP). Reductive amination of compound 8 with commercially
available trans-4-aminocyclohexanol gave an approximately 1:1
mixture of cis/trans and trans/trans isomers (a/b) of alcohol 9 (9a/
b). The same reaction with cis-4-aminocyclohexanol37 gave an
approximately 1:1 mixture of cis/cis and trans/cis isomers (c/d) of
9 (9c/d), as it results from 1H NMR spectra. In both cases titanium
(IV) isopropoxide as Lewis acid catalyst and NaBH3CN as reducing
agent were used, according to the Mattson procedure.38
In an ongoing search for potent P-gp-dependent MDR reversers
started a few years ago, we used some of the information available
about the recognition site of P-gp, reasoning that in a large recog-
nition site like that proposed, flexible basic molecules carrying
suitably positioned aryl moieties could easily find the most pro-
ductive interaction, compensating for the entropy toll with enthal-
py gain. The discovery of potent P-gp dependent MDR reversers
among the compounds synthesized showed that this was indeed
the case.29 Then, considering that an excess of rotatable bonds is
usually detrimental to proper pharmacokinetics, we decided to re-
duce the very high flexibility of linear structures and designed and
synthesized a new series of compounds characterized by a N,N-
bis(cyclohexanol)amine scaffold linked to two aromatic moieties
by ester functions.
A chromatographic separation was performed on the mixtures
9a/b and 9c/d, and the pure isomers 9a–d were obtained. Their

458
F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
configuration was attributed on the basis of the 1H NMR character-
istics of the cyclohexane protons.31 1H NMR spectra indicate that
the substituents on the two cyclohexane rings are in a cis/trans
(series a), trans/trans (series b), cis/cis (series c) and trans/cis (series
d) configuration.
The pure secondary aminoalcohols 9a–d were alkylated by
reductive methylation with HCOOH/HCHO to give the correspond-
ing tertiary aminoalcohols 11a–d.
3. Biological studies
3.1. Modulation of pirarubicin uptake
The ability of compounds of sets 2–7 to modulate P-gp action
was evaluated on the K562/DOX cell line. K562 is a human leuke-
mia cell line established from a patient with chronic myelogeneous
leukemia in blast transformation.41 K562/DOX cells resistant to
doxorubicin overexpress a unique membrane glycoprotein, P-
gp.42,43 The uptake of THP-adriamycin (pirarubicin) was measured
by a continuous spectrofluorometric signal of anthracycline at
590 nm (kex = 480 nm) after incubation of the cells, following the
protocols reported in previous papers.32,44 In brief, P-gp-blocking
In the same manner were also obtained aminoalcohols 10a–d,
whose synthesis has been previously described.31
Final compounds 2a–d, 3a–d, 6a–d and 7a–d (Scheme 2)
were obtained by reaction of 10a–d (for 2 and 3 sets) and 11a–d
(for 6 and 7 sets) with 4-cyano-4-(3,4-dimethoxyphenyl)-5-meth-
ylhexanoic acid,39 9H-fluorene-9-carboxylic acid or 2,2-bis-(4-
methoxyphenyl)acetic acid40 in the presence of 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) and 4-
dimethylamino-pyridine (DMAP).
activity is described by: (i) a, which represents the fold increase
in the nuclear concentration of pirarubicin in the presence of the
P-gp modulator and varies between 0 (in the absence of the mod-
ulator) and 1 (when the amount of pirarubicin in resistant cells is
Table 1
MDR-reversing activity of compounds 1–7
CH3
N
O
Ar1
O
Ar2
O
O
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
OCH3
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
CN
H3CO
H3CO
A
C
D
E
OCH3
G
B
F
Ma
amax
b
N
Ar1
Ar2
[I]0.5
l
1a cis/trans
1b trans/trans
1c cis/cis
1d trans/cis
2a cis/trans
2b trans/trans
2c cis/cis
2d trans/cis
3a cis/trans
3b trans/trans
3c cis/cis
3d trans/cis
4a cis/trans
4b trans/trans
4c cis/cis
4d trans/cis
5a cis/trans
5b trans/trans
5c cis/cis
5d trans/cis
6a cis/trans
6b trans/trans
6c cis/cis
6d trans/cis
7a cis/trans
7b trans/trans
7c cis/cis
7d trans/cis
Verapamil
MM36
B
B
B
B
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
F
A
A
A
A
C
C
C
C
D
D
D
D
E
E
E
E
F
F
F
F
0.092 0.015c
0.32 0.10c
0.03 0.01c
0.012 0.001c
0.24 0.04
0.14 0.02
0.15 0.03
0.08 0.03
0.08 0.01
0.075 0.01
0.18 0.04
0.15 0.03
0.63 0.2d
0.61 0.18d
1.10 0.3d
0.35 0.1d
0.08 0.01d
0.07 0.01d
0.07 0.01d
0.02 0.01d
0.13 0.03
0.14 0.04
0.12 0.04
0.02 0.001
0.12 0.04
0.50 0.07
0.05 0.01
0.29 0.08
1.60 0.3
0.85 0.03
0.81 0.03
0.80 0.02
0.98 0.03
0.88 0.03
0.99 0.01
0.99 0.01
0.89 0.05
0.81 0.03
0.94 0.04
0.90 0.03
0.99 0.01
0.75 0.03
0.76 0.03
0.76 0.05
0.77 0.02
0.98 0.02
0.99 0.01
0.99 0.01
0.99 0.01
0.76 0.03
0.74 0.02
0.94 0.04
0.99 0.01
0.96 0.04
0.77 0.02
0.89 0.05
0.91 0.06
0.70 0.07
0.70 0.06
G
G
G
G
D
D
D
D
F
F
F
F
F
F
F
0.05 0.01e
a
b
c
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. 31.
See Ref. 34.
See Ref. 32.
a
= 0.5).
d
e

F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
459
CH3
N
O
Ar1
O
Ar2
1-7
O
O
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
OCH3
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
CN
H3CO
H3CO
E
A
C
D
OCH3
B
F
G
Chart 1. General structure of designed compounds.
OCH3
H3CO
OH
O
i
H3CO
O
8
O
O
ii,iv
iii, iv
OCH3
OCH3
H3CO
H
H
H3CO
N
N
H3CO
O
H3CO
O
OH
OH
O
O
9a cis/ trans
9c cis/ cis
9b trans/ trans
9d trans/ cis
v
v
OCH3
OCH3
CH3
N
H3CO
H3CO
CH3
N
H3CO
H3CO
O
O
O
OH
OH
O
11a cis/ trans
11c cis/ cis
11b trans/ trans
11d trans/ cis
Scheme 1. Synthesis of key intermediates 11a–d. Reagents and conditions: (i) anhydrous CH2Cl2, 3-(3,4,5-trimetoxyphenyl)propiolic acid,36 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCl), 4-dimethylaminopyridine (DMAP); (ii) trans-4-aminocyclohexanol,37 (i-PrO)4Ti, NaBH3CN; (iii) cis-4-aminocyclohexanol, (i-PrO)4Ti,
NaBH3CN; (iv) chromatographic separation; (v) HCOOH/HCHO.
the same as in sensitive cells); (ii)
a
max, which expresses the effi-
phenotype. The reversal effects on the MDR phenotype of selected
compounds (2d, 3a, 3b, 5a–d) were investigated on K562/DOX
cells. Preliminarily, the compounds were evaluated for their intrin-
sic cytotoxicity on both K562 and K562/DOX cell lines by the MTT
cacy of the P-gp modulator and is the maximum increase that
can be obtained in the nuclear concentration of pirarubicin in resis-
tant cells with a given compound; and (iii) [I]0.5, which measures
the potency of the modulator and represents the concentration
that causes a half-maximal increase (
centration of pirarubicin (see Table 1).
This test indicates that the compounds inhibit the P-gp-oper-
ated extrusion of the reporter molecule pirarubicin, as does the ref-
erence molecule verapamil.
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide)
a
= 0.5) in the nuclear con-
assay,45 then the MDR-reversal activity of studied compounds
was tested using the same method in the presence of doxorubicin.
Based on the values of [I]0.5, two concentrations were chosen, one
very close to the [I]0.5 value (0.1
lM) and the other ten times high-
er (1 M). Verapamil was used as reference compound.
l
The IC50 doxorubicin values resulting in 50% inhibition of cell
growth compared to untreated control were calculated from plot-
ted results. The reversal-fold (RF) values, a measure of MDR rever-
sal, were calculated by dividing the doxorubicin IC50 value on
K562/DOX cells in the absence of modulators by the doxorubicin
IC50 values in the same cells in the presence of modulators (see
Table 2).
3.2. Cytotoxicity test and MDR reversal
The ability of a compound to enhance the growth inhibitory ef-
fects of doxorubicin in cancer cell lines showing MDR due to P-gp
overexpression, has also been proved useful in the quantification
and characterization of MDR reversal by modulators of the MDR

460
F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
CH3
N
CH3
N
O
Ar1
OH
O
OH
Ar1
Ar1
N
O
Ar1
N
O
10c, 10d
11c, 11d
A
A
10a, 10b
11a, 11b
F
F
i
i
CH3
N
CH3
N
O
O
Ar1
O
O
Ar2
Ar1
O
Ar2
O
O
O
cis/ cis
cis/ trans
trans/ trans
trans/ cis
Ar1
Ar2
N
N
N
Ar1 Ar2
N
Ar1
Ar1 Ar2
Ar2
C
D
G
D
A
A
F
C
D
G
D
A
A
F
C
D
G
D
A
A
F
C
D
G
D
2c
3c
6c
7c
A
A
F
2d
3d
6d
7d
2a
3a
6a
7a
2b
3b
6b
7b
F
F
F
F
Ar1, Ar2:
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
CN
A
C
F
D
G
OCH3
Scheme 2. Synthesis of final compounds 2a–d, 3a–d, 6a–d, and 7a–d. The synthesis of compounds 10a–d is similar to that of compounds 11a–d and described in Ref. 31.
Reagents and conditions: (i) anhydrous CH2Cl2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl), 4-dimethylaminopyridine (DMAP), Ar2COOH (4-
cyano-4-(3,4-dimethoxyphenyl)-5-methylhexanoic acid,39 9H-fluorene-9-carboxylic acid or 2,2-bis(4-methoxyphenyl)acetic acid.40
4. Results
4.1. Modulation of pirarubicin uptake.
Table 2
Effects of MDR-reversing agents 2d, 3a, 3b, 5a–d in comparison to 1a–d on
doxorubicin cytotoxicity in K562 and K562/DOX cells
The results obtained in doxorubicin-resistant erythroleukemia
K562 cells are reported in Table 1, together with those of MM36
Compounds
K562 cells
IC50
M)a
K562/DOX cells
M)a
(Fig. 1, the most potent compound that we have discovered in pre-
vious studies32) and verapamil (the gold standard of P-gp inhibi-
tion) used as reference compounds.
The new compounds, except compound 4c, show potency val-
ues ([I]0.5) in the submicromolar and nanomolar range; among
them, compounds 2b–c, 3d, 5a–d and 6d show excellent efficacy
with an amax close to 1. They are therefore potent and efficacious
modulators of the P-gp extrusion pump.
(l
IC50
(l
RFb
DOXOc
0.03 0.002
0.05 0.011
0.05 0.010
0.04 0.015
0.05 0.009
0.05 0.007
0.04 0.010
0.013
0.04 0.020
0.03 0.017
0.02 0.004
0.02 0.004
0.14 0.020
0.07 0.016
0.09 0.020
0.07 0.020
0.04 0.040
0.04 0.010
0.06 0.010
0.06 0.040
0.09 0.030
0.05 0.006
2.84 0.047
0.37 0.130
0.31 0.030
0.29 0.060
0.14 0.030
2.99 0.200
0.99 0.200
0.28 0.080
1.3 0.060
DOXO + 1a (1
DOXO + 1b (1
DOXO + 1c (1
DOXO + 1d (1
DOXO
l
l
l
l
M)c
M)c
M)c
M)c
7.7
9.2
9.8
20.3
DOXO + 2d (0.1
DOXO + 2d (1
DOXO + 3a (0.1
DOXO + 3a (1
DOXO + 3b (0.1
DOXO + 3b (1
DOXO + 5a (0.1
DOXO + 5a (1
DOXO + 5b (0.1
DOXO + 5b (1
DOXO + 5c (0.1
DOXO + 5c (1
DOXO + 5d (0.1
DOXO + 5d (1
lM)
3.0
10.7
2.3
5.0
2.0
2.9
1.6
1.5
0.6
1.9
2.3
4.5
2.0
8.5
1.2
4.7
l
M)
lM)
lM)
0.6 0.100
4.2. Cytotoxicity test and MDR reversal
lM)
1.56 0.410
1.06 0.270
1.85 0.300
1.98 0.200
4.8 0.600
l
M)
The reversal effects (RF) of compounds on the MDR phenotype
lM)
lM)
have been investigated in K562/DOX cells at 0.1 and 1.0
centrations. The results are reported in Table 2 together with those
of the reference compound verapamil tested at 2.0 and 20 M and
of compounds 1a–d.31 Compounds 2d, 3a, 3b, 5a–d and verapamil
tested at 1.0 M without doxorubicin had no cytotoxic effects on
K562 and K562/DOX cells (data not shown), therefore, we tested
all compounds at 0.1 and 1.0 M concentrations in the presence
of doxorubicin. The lowest concentration (0.1 M) of compounds
lM-con-
lM)
l
M)
1.54 0.070
1.3 0.100
l
lM)
lM)
0.67 0.030d
1.47 0.070
0.35 0.053d
2.51 0.160
0.63 0.060
lM)
l
l
M)
DOXO + verapamil (2
DOXO + verapamil (20
l
l
M)
M)
l
l
a
Mean SE of at least three determinations or mean of two determinations
performed with quadruplicate cultures at each drug concentration tested and
measured as described in Section 6.
Reversal fold of MDR was determined by dividing the doxorubicin IC50 values
on K562/DOX cells in the absence of modulators by those in the presence of
modulators.
From Ref. 31. Note that in that paper the DOXO IC50 value on K562 and K562/
DOX cells was slightly different from that obtained in the present tests.
p <0.001 versus control doxorubicin-treated. Where not specified the IC50 value
is not significant compared to control cells (K562 and K562/DOX cells treated with
doxorubicin only).
moderately reversed the resistance of K562/DOX cells with RF val-
ues ranging between 1.6 and 3.0; at this concentration, compound
5b had no reversal effect (RF = 0.6). Comparing the RF value ob-
tained at both concentrations, the compounds 2d, 3a, 5b, 5c and
b
5d at a higher tested concentration (1.0
increase in RF (RF = 10.7, 5.0, 1.9, 4.5, 8.5, respectively) compared
to the lower concentration (0.1 M) (RF = 3.0, 2.3, 0.6, 2.3, 2.0,
lM) caused a 2- to 4-fold
c
l
d
respectively). Compounds 3b and 5a instead showed the same RF
values at both concentrations.

F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
461
H3CO
H3CO
O
OMe
OMe
CN
H
N
N
N
OMe H
O
N
H
MM36
GF120918 (Elacridar)
O
CH3
O
HO
CH3
CH3
N
CH3
N
O
O
H
N
H3C
N
N
N
H
O
CH3
CH3
O
O
O
O
CH3
N
CH3
O
O
NH
N
N
N
H
O
CH3
CH3
CSA (Cyclosporin A)
Figure 1. MM3632, GF12091820 (Elacridar) and CSA19 (Cyclosporin A) structures.
The RF data indicate that all the N,N-bis(cyclohexanol)amine
We will discuss the results obtained in this ranking test in three
ways: (i) by analyzing the behavior of the isomers in each set; (ii)
by comparing each set with the others; (iii) by examining the
whole library of compounds.
aryl esters tested were moderately active at a 0.1
tion with the exception of compound 5b. Their activity was sub-
stantially increased at 1.0 M with the exception of compounds
3b and 5a. All compounds were more potent than the reference
drug verapamil at 2.0 M. Compounds 2d and 5d were the most
lM concentra-
l
l
(i) In five out of the seven sets the most active isomer is the
trans/cis one (1d, 2d, 4d, 5d, 6d); in set 3 the two isomers
3a and 3b, that present cis/trans and trans/trans geometry
respectively, have nearly identical potency. Isomer 7c pre-
senting cis/cis geometry is the most potent of set 7. These
results would suggest that, among the compounds of this
class, trans/cis geometry31 is the most suitable to interact
with the P-gp recognition site. However, with the exception
of set 1, where the most active isomer 1d is about 27 times
effective, exhibiting a similar behavior to that of isomers 1a–c
(RF = 7.7, 9.2 and 9.8, respectively). They were, however, less effec-
tive than the 1d isomer (RF = 20.3).
4.3. Chemical stability
In order to evaluate the chemical stability of our compounds
during the biological tests, a selection of derivatives were main-
tained under the same conditions as the cytotoxicity test (37 °C
for 3 days in the cell culture medium described in the Section 6)
in four different concentrations (10À3 to 10À6 M). The stability of
the compounds was verified by TLC and by LC–ESI-MS (Supple-
mentary data); both analytical methods showed that the tested
compounds are stable under these conditions. however, at the
moment, no information is available on their metabolic stability.
Studies are in progress to evaluate the metabolic profile of these
compounds.
more potent than 1b ([I]0.5 = 0.012 and 0.32
tively) and of set 7 where 7c is approximately 10 times more
potent than 7b ([I]0.5 = 0.05 and 0.50 M, respectively), the
lM, respec-
l
differences in potency are generally small (3- to 5-fold)
which makes it risky to generalize about the interaction
geometry. As far as efficacy is concerned, the situation is
nearly the same as only in sets 1, 4, 5, 6 maximum efficacy (amax
)
corresponds to the most potent isomer of each set.
(ii) A comparison of the different sets confirms that the N,N-
bis(cyclohexanol)amine scaffold, when esterified with suit-
able aromatic acids, is definitely efficient in establishing
productive interactions with the P-gp recognition site.31,46
Activity was maintained with only modest variation in set
1 when the trans-2-(3,4,5-trimethoxyphenyl)vinyl moiety
(A) was combined, to give sets 2 and 3, with 4-cyano-4-
(3,4-dimethoxyphenyl)-5-methylpentyl (C) and 9H-fluo-
rene (D) moieties that characterized early MDR reversers
active at nanomolar doses.31,32 Results obtained with sets
4 and 5, designed to evaluate the consequences of modula-
tion of the geometry imposed by the double bond of trans-
2-(3,4,5-trimethoxyphenyl)vinyl moiety present in set 1,
were particularly interesting. Clearly, the reduction of the
double bond (set 4) and the consequent increase of confor-
mational freedom, and of the related entropy toll, are det-
rimental to both affinity and efficacy. On the contrary,
further restriction of the conformational freedom of the
5. Discussion
In the search for potent and efficient reversers of P-gp-depen-
dent multidrug resistance, we synthesized a series of compounds
characterized by an N,N-bis(cyclohexanol)amine scaffold linked to
two different aromatic moieties through ester functions. This scaf-
fold gives origin to four geometric isomers (cis/trans, trans/trans,
cis/cis and trans/cis), resulting in a library of 24 pure isomers (sets
2–7). All compounds were screened in the preliminary test through
the evaluation of pirarubicin uptake on doxorubicin-resistant eryth-
roleukemia K562 cells (K562/DOX) as a measure of their ability to
reverse P-gp extruding action. In Table 1, where the results of this
assay are reported, we have included the data of the four isomers
of the previously studied set 1.30,31 Accordingly we will take into
consideration the isomers of this set, that are the most interesting
compounds of the class, in the following discussion.

462
F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
molecule, obtained through the introduction of the triple
by the cells. These kinetics have a role not only in the cytotoxicity
of the drug towards the resistant cells but also in the ability of
inhibitors to restore intracellular incorporation to a level compara-
ble with that observed in parent cells.48 Moreover, metabolic pro-
cesses on doxorubicin, during the long lasting cytotoxicity
experiment (72 h) can alter the cytotoxic response of the
cells and require higher concentrations of Pgp modulators. Studies
are in progress to evaluate the metabolic profile of these
compounds.
bond (set 5) maintained the potency, smoothening the dif-
ferences among the four isomers, and significantly
increased efficacy. In both cases trans/cis isomers (1d and
5d) are the most potent ones, suggesting that sets 1 and
5 bind in very similar binding sites.
Unfortunately, the set carrying the cis-2-(3,4,5-trimethoxy-
phenyl)vinyl moiety, where the double bond of set 1 would
be in the cis geometry, could not be isolated for biological
testing, since the final products isomerize to the more sta-
ble trans isomers (set 1), which precludes a more complete
analysis of the geometric requests of the binding space.-
Since 9H-fluorene-9-carboxylic acid can be considered a
restricted conformational analogue of 2,2-bis(4-methoxy-
phenyl)acetic acid, sets 6 and 7 were synthesized following
the good activity of the compounds of set 5, to collect
information on the geometric requirements at both ends
of the molecules. Further conformational restriction of set
6 to set 7 had a different effect on the activity of the iso-
Neri et al.49 have extended the study of P-gp modulation activ-
ity of sets 1 and 5 to the inhibition of rhodamine123 (R123) efflux
in human MDR1-gene transfected mouse T-lymphoma L5178 cells.
All the compounds were active at low concentration, 1c, 1d, 5c and
5d being active at nanomolar doses. These compounds were more
potent than reference drugs GF12091820 (Elacridar) and CSA19
(Cyclosporin A), two of the most potent P-gp modulators studied.
Their structure is reported in Figure 1.
The same two sets were also tested on the ATPase activity of hu-
man P-gp enriched intestinal Spodoptera frugiperda membranes
mers. The most potent isomer of set
([I]0.5 = 0.02 M trans/cis geometry) while the most potent
isomer of set 7 is 7c ([I]0.5 = 0.05 M cis/cis geometry). In
this case reduction of conformational freedom slightly
reduces the potency while maintaining good efficacy and
seemingly forcing 7c to change binding mode.
6
is 6d
(Sf9), both in the absence and presence of 0.1 lM epirubicin. Com-
l
pounds 1c, 1d, 5c and 5d potently inhibited epirubicin-stimulated
ATPase activity. Based on the results of their experiments, the
authors suggest that this class of P-gp modulators might act as
transport substrates.49
In conclusion, we have expanded SARs of a new class of po-
tent N,N-bis(cyclohexanol)amine aryl esters which act as revers-
ers of P-gp-mediated MDR by synthesizing several new
compounds. Even if the potency of the parent compounds 1c
and 1d,31 remained unmatched, we have identified two other
molecules (5c and 5d) that, like 1c and 1d,31 are potent and
highly efficacious modulators of P-gp-dependent MDR and ap-
pear to be very promising leads for the development of MDR-
reversal drugs.
l
(iii) Almost all compounds in Table 1 are very potent and effica-
cious reversers of P-gp-dependent-MDR. In terms of binding
modes, the results presented above seem to indicate that the
rather rigid N,N-bis(cyclohexanol)amine scaffold plays a
major role, while the flexible arylester moieties produce
quantitatively different effects on potency and efficacy.
These results can be explained by proposing that the N,N-
bis(cyclohexanol)amine scaffold provides much of the bind-
ing energy of the members of this class, while arylester moi-
eties provide the fine tuning. As proposed at the very
beginning of this research, it is likely that compounds of this
series, that are quite different from most of known MDR
modulators,22,24 exploit at best the possibility, within the
large and polymorphous recognition site, of adopting the
most efficient binding modes within the recognition site in
a mutual molding (induced fit) of their chemical and physi-
cal characteristics.29,47
6. Experimental section
6.1. Chemistry
All melting points were taken on a Büchi apparatus and are
uncorrected. Infrared spectra were recorded with a Perkin–Elmer
681 or a Perkin–Elmer Spectrum RX I FT-IR spectrophotometer.
1H NMR spectra were recorded on Bruker Avance 400 spectrome-
ter. Chromatographic separations were performed on a silica gel
column by flash chromatography (Kieselgel 40, 0.040–0.063 mm;
Merck). Yields are given after purification, unless stated otherwise.
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, 1H NMR and TLC chromatography. When reactions were per-
formed in anhydrous conditions, the mixtures were maintained
under nitrogen.
The MDR reversal effects of selected compounds of the library
(2d, 3a, 3b, 5a–d) on the MDR phenotype were also investigated
using the cytotoxicity test on doxorubicin-resistant erythroleuke-
mia K562 cells (K562/DOX) overexpressing only Pgp and the re-
sults are reported in Table 2, together with those of previously
reported compounds 1a–d.31
As expected, all compounds, with the exception of 5b, were
fairly effective in restoring the sensitivity of K562/DOX cells to
doxorubicin since, at both 0.1 and 1.0
lM concentrations they
are more effective than verapamil at 2 and 20
lM. However, corre-
Compounds were named following IUPAC rules as applied by
Beilstein-Institut AutoNom 2000 4.01.305, a software for system-
atic names in organic chemistry.
lation with the results of the pirarubicin test is modest. Actually,
the two most potent members of sets 1 and 5 (1d and 5d) in the
pirarubicin test are the most potent also in the doxorubicin test.
However their RF values (20.3 and 8.5) do not seem closely related
to potency and efficacy in the pirarubicin test ([I]0.5 = 0.012,
6.2. 3-(3,4,5-Trimethoxyphenyl)propiolic acid 4-oxocyclohexyl
ester (8)
a
max = 0.98; [I]0.5 = 0.02,
a
max = 0.99). Compare also 2d (RF = 10.7,
max = 0.99).
[I]0.5 = 0.08, max = 0.89), and 5c (RF = 4.5, [I]0.5 = 0.07,
a
a
To a solution of 4-hydroxycyclohexanone35 (725 mg, 6.36 mmol)
in dry CH2Cl2 cooled to 0 °C were added in this sequence:
3-(3,4,5-trimethoxyphenyl)propiolic acid36 (2.25 g, 9.53 mmol),
4-dimethylaminopyridine (DMAP) (621 mg, 5.10 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl)
(2.19 g, 11.0 mmol). The reaction mixture was stirred at 0 °C for
It is difficult to indicate the reasons for these inconsistencies be-
tween the two tests. However, we must consider that two anthra-
cyclines with different rates of cellular uptake, pirarubicin faster
than doxorubicin, are used. Pirarubicin and doxorubicin intracellu-
lar concentrations depend on the kinetics of P-glycoprotein
mediated efflux of the drugs but also on the kinetics of their uptake

F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
463
1 h and then was kept at room temperature for 48 h. The mixture
6.4.1. cis/cis 9c
was treated with CH2Cl2 and the organic layer washed with water
and then with a saturated solution of NaHCO3. After drying with
Na2SO4, the solvent was removed under reduced pressure and
the residue purified by flash chromatography using cyclohexane/
ethyl acetate (7:3) as eluting system. The title compound
(658 mg, 34% yield) was obtained as a colourless oil. 1H NMR
(CDCl3): d 6.81 (s, 2H, aromatic); 5.29–5.27 (m, 1H, CHO); 3.84 (s,
3H, OCH3); 3.82 (s, 6H, OCH3); 2.67–2.59 (m, 2H, CH2); 2.43–2.37
(m, 2H, CH2); 2.24–2.11 (m, 4H, 2CH2) ppm.
Hundred and twenty milligram as a light yellow oil; IR (neat): m
3390 (OH and NH); 1706 (CO) cmÀ1. 1H NMR (CDCl3): d 6.86 (s, 2H,
aromatic); 5.12 (br s, 1H, CHO); 3.97 (br s, 1H, CHOH); 3.86 (s, 3H,
OCH3); 3.85 (s, 6H, 2OCH3); 2.98–2.72 (m, 2H, 2NCH); 2.07–2.01
(m, 2H, CH2); 1.92–1.51 (m, 14H, 7CH2) ppm.
6.4.2. trans/cis 9d
Eighty milligram as a light yellow oil; IR (neat):
m 3390 (OH and
NH); 1706 (CO) cmÀ1 1H NMR (CDCl3): d 6.83 (s, 2H, aromatic);
.
4.88–4.83 (m, 1H, CHO); 3.93 (br s, 1H, CHOH); 3.87 (s, 3H,
OCH3); 3.84 (s, 6H, 2OCH3); 2.81–2.69 (m, 2H, 2NCH); 2.18–2.11
(m, 2H, CH2); 2.08–2.01 (m, 2H, CH2); 1.83–1.30 (m, 12H, 6CH2)
ppm.
6.3. cis/trans-3-(3,4,5-Trimethoxyphenyl)propiolic acid
4-(4-hydroxycyclohexylamino)cyclo-hexyl ester (9a) and
trans/trans-3-(3,4,5-trimethoxyphenyl)propiolic acid
4-(4-hydroxy-cyclohexylamino)cyclohexyl ester (9b)
6.5. cis/trans-3-(3,4,5-Trimethoxyphenyl)propiolic acid
4-[(4-hydroxycyclohexyl)methylamino] cyclohexyl ester (11a)
A mixture of compound 8 (658 mg, 2.14 mmol), trans-4-amino-
cyclohexanol (292 mg, 2.54 mmol) and titanium (IV) isopropoxide
(1.28 mL, 4.28 mmol) was stirred at room temperature. After 3 h,
absolute ethanol (2.0 mL) and NaBH3CN (266 mg, 4.28 mmol) were
added and the solution was stirred for 20 h. Water (10 mL) was
added, the organic solvent was removed under reduced pressure
then the mixture was treated with CH2Cl2 and the organic layer
washed with a saturated solution of NaHCO3 After drying with
Na2SO4, the solvent was removed under reduced pressure and
the residue purified by flash chromatography using CH2Cl2/
To a solution of 80 mg (0.19 mmol) of 9a in 1 mL of absolute
ethanol, 0.12 mL of HCOOH and 0.03 mL of HCHO were added.
The mixture was heated to 80 °C for 4 h and concentrated in vacuo.
The residue was then dissolved in CH2Cl2 and the organic layer was
washed with a saturated solution of NaHCO3 and with water. After
drying with Na2SO4, the solvent was removed under reduced pres-
sure. Compound 11a was obtained as an oil (76.4 mg, 94% yield). IR
(neat):
m .
3360 (OH); 1708 (CO) cmÀ1 1H NMR (CDCl3): d 6.84 (s,
2H, aromatic); 5.11 (br s, 1H, CHO); 3.88 (s, 3H, OCH3); 3.86 (s,
6H, 2OCH3); 3.61-3.51 (m, 1H, CHOH); 2.69-2.59 (m, 2H, 2NCH);
2.31 (s, 3H, NCH3); 2.10-1.18 (m, 17H, 8CH2, and OH) ppm.
Compounds 11b, 11c and 11d were obtained in the same way
from 9b, 9c and 9d respectively.
MeOH/NH4OH (95:5:0.5) as eluting system. Compound
9
(370 mg, 40% yield) as a approximately 50/50 mixture of cis/trans
and trans/trans isomers, was obtained as a white solid. Mp: 67–
69 °C. IR (neat):
m .
3392 (OH and NH); 1705 (CO) cmÀ1 1H NMR
(CDCl3): d 6.82 (s, 2H, aromatic); 5.10 (br s, 0.5H, CHO); 4.83 (m,
0.5H, CHO); 3.88 (s, 3H, OCH3); 3.85 (s, 6H, OCH3); 3.60–3.52 (m,
1H, CHOH); 2.72–2.50 (m, 2H, 2NCH); 2.20–1.00 (m, 18H, 8CH2,
OH and NH) ppm.
Flash chromatography of 370 mg of the mixture 9a/b, using
CH2Cl2/MeOH/NH3 (97:3:0.3) as eluting system afforded the two
isomers cis/trans and trans/trans (9a and 9b).
6.5.1. trans/trans 11b
IR (neat):
m
3360 (OH); 1708 (CO) cmÀ1. 1H NMR (CDCl3): d 6.81
(s, 2H, aromatic); 4.81–4.72 (m, 1H, CHO); 3.85 (s, 3H, OCH3); 3.83
(s, 6H, 2OCH3); 3.60–3.50 (m, 1H, CHOH); 2.68–2.50 (m, 2H,
2NCH); 2.22 (s, 3H, NCH3); 2.18–1.20 (m, 17H, 8CH2, and OH) ppm.
6.5.2. cis/cis 11c
6.3.1. cis/trans 9a
IR (neat):
m
3360 (OH); 1708 (CO) cmÀ1. 1H NMR (CDCl3): d 6.85
Hundred and thirty six milligram as a colourless oil; IR (neat):
m
(s, 2H, aromatic); 5.11 (br s, 1H, CHO); 4.05 (br s, 1H, CHOH); 3.92
(s, 3H, OCH3); 3.90 (s, 6H, 2OCH3); 2.71–2.61 (m, 2H, 2NCH); 2.32
(s, 3H, NCH3); 2.10–2.01 (m, 2H, CH2); 1.83–1.50 (m, 15H, 7CH2,
and OH) ppm.
3390 (OH and NH); 1706 (CO) cmÀ1. 1H NMR (CDCl3): d 6.82 (s, 2H,
aromatic); 5.09 (br s, 1H, CHO); 3.86 (s, 3H, OCH3); 3.85 (s, 6H,
2OCH3); 3.59 (m, 1H, CHOH); 2.71–2.51 (m, 2H, 2NCH); 2.08–
1.10 (m, 18H, 8CH2, OH and NH) ppm.
6.5.3. trans/cis 11d
6.3.2. trans/trans 9b
IR (neat):
m
3360 (OH); 1708 (CO) cmÀ1. 1H NMR (CDCl3): d 6.82
Hundred milligram as a colourless oil; IR (neat):
m 3390 (OH and
(s, 2H, aromatic); 4.83–4.74 (m, 1H, CHO); 3.97 (br s, 1H, CHOH);
3.86 (s, 3H, OCH3); 3.84 (s, 6H, 2OCH3); 2.73–2.52 (m, 2H,
2NCH); 2.28 (s, 3H, NCH3); 2.19–2.08 (m, 2H, CH2); 1.92–1.42 (m,
15H, 7CH2, and OH) ppm.
NH); 1706 (CO) cmÀ1 1H NMR (CDCl3): d 6.83 (s, 2H, aromatic);
.
4.85 (m, 1H, CHO); 3.88 (s, 3H, OCH3); 3.85 (s, 6H, 2OCH3); 3.62
(m, 1H, CHOH); 2.70–2.52 (m, 2H, 2NCH); 2.12–1.10 (m, 18H,
8CH2, OH and NH) ppm.
6.6. cis/trans-9H-Fluorene-9-carboxylic acid 4-(methyl{4-[3-
(3,4,5-trimethoxyphenyl)propyno-yl-oxy]cyclohexyl}amino)-
cyclohexyl ester (7a)
6.4. cis/cis-3-(3,4,5-Trimethoxyphenyl)propiolic acid
4-(4-hydroxycyclohexylamino)cyclohexyl ester (9c) and
trans/cis-3-(3,4,5-trimethoxyphenyl)propiolic acid
4-(4-hydroxycyclohexyl-amino)cyclohexyl ester (9d)
Following the procedure described for compound 8, to a solu-
tion of 11a (76.4 mg, 0.17 mmol) in dry CH2Cl2 cooled at 0° C were
added in this sequence: 9H-fluorene-9-carboxylic acid (54.1 mg,
An approximately 50/50 mixture of cis/cis and trans/cis isomers
9c/d was obtained in the same way as 9a/b starting from 8 and
using cis-4-aminocyclohexanol.37 Its IR and 1H NMR spectra are
consistent with the proposed structure. The chromatographic sep-
aration of 0.25 g of the mixture afforded the two isomers 9c and
9d.
0.26 mmol),
4-dimethylaminopyridine
(DMAP)
(16.7 mg,
0.14 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCl) (59.2 mg, 0.31 mmol). The reaction mixture
was stirred at 0 °C for 1 h and then was kept at room temperature
for 48 h. The mixture was treated with CH2Cl2 and the organic

464
F. Orlandi et al. / Bioorg. Med. Chem. 21 (2013) 456–465
layer washed with water and then with a satured solution of NaH-
CO3. After drying with Na2SO4, the solvent was removed under re-
duced pressure and the residue purified by flash chromatography
using CH2Cl2/MeOH/NH4OH (97:3:0.3) as eluting system. The title
6.9. 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 (kex = 480 nm)
according to the previously described method.50
To evaluate the reverting activity of test compounds, the cells,
in exponential growth phase (3–5 Â 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. The MTT working solution
compound (54 mg, 49% yield) was obtained as an oil. IR (neat):
m
1708 (CO) cmÀ1 1H NMR (CDCl3): d 7.76 (d, J = 7.6 Hz, 2H, aro-
.
matic); 7.64 (d, J = 7.6 Hz, 2H, aromatic); 7.41 (t, J = 7.6 Hz, 2H, aro-
matic); 7.32 (t, J = 7.6 Hz, 2H, aromatic); 6.85 (s, 2H, aromatic);
5.11 (br s, 1H, CHO); 4.82 (s, 1H, aliphatic); 4.75 (m, 1H, CHO);
3.88 (s, 3H, OCH3); 3.86 (s, 6H, 2OCH3); 2.76–2.52 (m, 2H,
2NCH); 2.29 (s, 3H, NCH3); 2.13–1.38 (m, 16H, 8CH2) ppm. 13C
NMR (CDCl3): d 170.27 (C); 153.67 (C); 153.16 (C); 141.43 (C);
140.74 (C); 128.08 (CH aromatic); 127.31 (CH aromatic); 125.54
(CH aromatic); 120.04 (CH aromatic); 114.32 (C); 110.38 (CH aro-
matic); 86.33 (C); 80.29 (C); 73.53 (CHO); 71.28 (CHO); 61.03
(OCH3); 58.60 (NCH); 56.27 (OCH3); 53.53 (CH aliphatic); 32.75
(NCH3); 30.70 (CH2); 29.01 (CH2); 26.71 (CH2) ppm. The oily prod-
uct was transformed into the hydrochloride and recrystallized
from absolute ethanol/anhydrous diethylether. Mp: 140–143 °C.
Anal. (C39H44ClNO7) C, H, N.
(50 lL) was added and plates were further incubated for 3 h. Fol-
lowing 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 lL of DMSO; the absorbance of the solution was then read
on an automated plate reader at a wavelength of 540 nm.
All results are presented as means SE and statistical analysis
was performed using the one-way Anova test and Bonferroni’s
multiple comparison test (GraphPad Prism software, Inc. CA, USA.
Compounds 7b, 7c and 7d were obtained in the same way from
11b, 11c and 11d respectively. Compounds 6a–d were obtained in
the same way from 11a–d using 2,2-bis(4-methoxyphenyl)acetic
acid40 and compounds 2a–d and 3a–d were obtained in the same
way from 10a–d31 using 4-cyano-4-(3,4-dimethoxyphenyl)-5-
methylhexanoic acid39 or 9H-fluorene-9-carboxylic acid, respec-
tively. All compounds were transformed into the hydrochloride.
Their chemical and physical characteristics are reported in
Table S1; IR and 1H and 13C NMR spectra are reported in Tables
S2 and S3 (Supplementary data).
Acknowledgments
The authors wish to thank Professor Fulvio Gualtieri for helpful
discussion and Dr. Gianluca Bartolucci for the LC–ESI-MS analyses.
This work was partially supported by the ITT (Istituto Toscano
Tumori).
Supplementary data
Supplementary data (chemical and physical characteristics, IR,
1H NMR and 13C NMR spectra, elemental analyses of compounds
2a–d, 3a–d, 6a–d and 7a–d and LC–ESI-MS) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.11.019.
6.7. Biology
6.7.1. Cell lines and cultures
The K562 cell line is a highly undifferentiated erythroleukemia
originally derived from a patient with chronic myelogenous leuke-
mia.41 The K562 leukemia cells and the P-gp expressing K562/DOX
cells were obtained from Professor J.P. Marie (Hopital Hotel-Dieu,
Paris, France). These cells were cultured following the previously
reported protocol.31
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