N,N-bis(cyclohexanol)amine aryl esters: A new class of highly potent transporter-dependent multidrug resistance inhibitors

Article abstract of DOI:10.1021/jm8012745

A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol) amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 × 10^-9 and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 μM concentration.

Full text of DOI:10.1021/jm8012745

J. Med. Chem. 2009, 52, 807–817
807
N,N-bis(Cyclohexanol)amine Aryl Esters: A New Class of Highly Potent Transporter-Dependent
Multidrug Resistance Inhibitors
Cecilia Martelli,† Daniela Alderighi,‡ Marcella Coronnello,§ Silvia Dei,† Maria Frosini,‡ Be´ne´dicte Le Bozec,| Dina Manetti,†
Annalisa Neri,‡ Maria Novella Romanelli,† Milena Salerno,| Serena Scapecchi,† Enrico Mini,§ Giampietro Sgaragli,‡ and
Elisabetta Teodori*,†
Dipartimento di Scienze Farmaceutiche, UniVersita` di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy, Dipartimento di
Neuroscienze, Sezione di Farmacologia, Fisiologia e Tossicologia, UniVersita` di Siena, Via A. Moro 2, 53100 Siena, Italy, Dipartimento di
Farmacologia Preclinica e Clinica, UniVersita` di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy, Laboratoire de Biophysique Mole´culaire,
Cellulaire et Tissulaire (BioMoCeTi), UMR CNRS 7033, UMPC UniVersité Paris 6 and UniVersite´ Paris 13, 74 rue Marcel Cachin,
93017 Bobigny, France
ReceiVed October 8, 2008
A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed
on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical
isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant
erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3)
were studied further evaluating their action on the ATPase activity present in rat small intestine membrane
vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on
the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound
3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin
extrusion at low nanomolar concentration, inhibited ATPase activity at 5 × 10-9 and increased the cytotoxicity
of doxorubicin with a reversal fold (RF) of 36.4 at 3 µM concentration.
Introduction
through π-π, ion-π, hydrogen bond, and hydrophobic
interactions.15-17
Multidrug resistance (MDR) is a kind of acquired drug
resistance of cancer cells and microorganisms to a variety of
chemotherapic drugs that usually are structurally and mecha-
nistically unrelated.1 Multidrug resistance may originate from
several biochemical mechanisms; classical MDR2 is due to a
lower cell concentration of cytotoxic drugs associated with
accelerated efflux of the chemotherapeutic agent as a conse-
quence of the overexpression of proteins such as ABCB1 (Pgp)
and ABCC1 (MRP1)3 that act as extrusion pumps. These
proteins belong to the ABC superfamily of transporters that use
ATP as energy source, but several families of similar pumps
that use a variety of energy sources are present in mammals
and microorganisms.1,4 In mammals, these proteins have been
found in several important tissues besides cancer cells and
blood-tissue barriers where they apparently regulate the secre-
tion of physiologically important lipophilic molecules5 and the
extrusion of xenobiotics that enter the organism.6
Direct information on the structure of Pgp and MRP1 is
scarce,7,8 but resolution of the structure of homologous bacterial
transporters9-11 has opened the way to the development of
homology models12-14 and provided many useful details on the
structure of the recognition site of ABC transporters. All
information collected so far points to the existence of a large,
polymorphous drug recognition domain, where a variety of
molecules can be accommodated in a plurality of binding modes
Inhibition of the functions of Pgp, MRP1, and sister proteins
is considered a suitable approach to circumvent MDR and drugs
possessing inhibitory properties have been and are actively being
sought,18-22 even if so far, no drug has been approved for
therapy.23,24 An emerging potential use of these agents may be
to increase drug penetration through biologically important
protective barriers, such as the blood-brain and blood-cerebro-
spinal fluid barriers.25 The main problems associated with the
development of effective Pgp-mediated MDR inhibitors seem
due to poor specificity, low potency, interference with physi-
ological functions and, as a consequence, interference with the
pharmacokinetics of the chemotherapeutic drug used.
Recently, we have described a new family of MDR reverters,
endowed with fairly good potency, designed on the basis of a
new concept arising from the emerging picture of the substrate
recognition sites of Pgp, MRP1, and sister proteins.26 In brief,
we guessed that flexible molecules carrying a basic nitrogen
flanked, at suitable distances, by two (or three) aromatic
moieties, would accommodate in the recognition cavity choosing
the most productive binding modes and therefore would interact
with high affinity with the protein. The good potencies of most
of the compounds synthesized and studied confirmed our
prediction that the entropy toll paid had been compensated by
the enthalpy gain due to the fact that flexible molecules can
optimize their interaction within the recognition site.
* To whom correspondence should be addressed. Phone: ++39 055
4573693. Fax: + +39 055 4573671. E-mail: elisabetta.teodori@unifi.it.
Building on the results obtained and having in mind possible
therapeutic applications, we have decided to verify the conse-
quences of a partial reduction of the very high flexibility of our
compounds. In fact, it has empirically been shown that a high
number of rotatable bonds is detrimental for good absorption
and drug-likeness,27,28 while it is well-known that flexibility does
not favor selectivity. Toward this end, we designed a new series
of molecules where the N,N-bis-arylalkylamine moiety of pre-
†
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze.
‡
Dipartimento di Neuroscienze, Sezione di Farmacologia, Fisiologia e
Tossicologia, Universita` di Siena.
§
Dipartimento di Farmacologia Preclinica e Clinica, Universita` di
Firenze.
|
Laboratoire de Biophysique Mole´culaire, Cellulaire et Tissulaire
(BioMoCeTi), UMR CNRS 7033, UMPC Universite` Paris 6 and Universite´
Paris 13.
10.1021/jm8012745 CCC: $40.75
2009 American Chemical Society
Published on Web 01/13/2009

808 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Chart 1. General Structures of Designed Compounds
Martelli et al.
and 15a-d were obtained. Their configuration was attributed
on the basis of the 1H NMR characteristics of the cyclohexane
protons H1, H4 and H1′, H4′ as exemplified in Table S2
(Supporting Information) for compounds 14, 17, 3, and 4. In
most cases, it was possible to extract the Jaa and sometimes the
Jae constants; when the signal does not allow extraction of the
coupling constants, the half-height amplitude of the signal
(w/2)37-39 allows confident attribution of the equatorial or axial
nature of the protons. Moreover the chemical shift of the signal
is diagnostic, because axial protons resonate at higher fields,
while the corresponding equatorial ones are deshielded. For
example, the isomers 14a and 14c present the H1 proton with
equatorial characteristics (δ ) 5.12 ppm, w/2 ) 15.6 Hz and δ
) 5.15 ppm, w/2 ) 11.8 Hz, respectively), while isomers 14b
and 14d present the H1 proton with axial characteristics (δ )
4.92 ppm, Jaa ) 10.8 Hz, Jae ) 4.4 Hz and δ ) 4.93 ppm, Jaa
) 10.8 Hz Jae ) 4.4 Hz, respectively). The isomers 14a and
14b present the H1′ proton with axial characteristics (δ ) 3.60
vious series (general structure I) was substituted with that of
N,N-bis-cyclohexylamine (general structure II) (Chart 1). The
aromatic moieties selected were those that gave the best results
in the linear series.
The scaffold chosen gives origin to four geometrical isomers
(three when the two aryl groups are identical) of quite different
shape that somehow represent restricted conformation analogues
of the parent linear compounds. According to the frozen
analogue approach,29 they can provide further insight on the
characteristics of the recognition site.
ppm, Jaa ) 10.6 Hz, Jae ) 4.0 Hz and δ ) 3.62 ppm, Jaa
)
10.6 Hz, Jae ) 4.0 Hz, respectively) while isomers 14c and 14d
present the H1′ proton with equatorial characteristics (δ ) 3.94
ppm, w/2 ) 10.9 Hz for both isomers). Furthermore, the protons
of the two carbon atoms carrying the amine function have axial
characteristics in every isomer of 14 (see Table S2, Supporting
Information), indicating that the two cyclohexane rings are in
a cis/trans (14a), trans/trans (14b), cis/cis (14c), and trans/cis
Preliminary results of this research have been published.30
In the present paper, the set of studied compounds has been
extended to evaluate SARs. The new compounds were synthe-
sized and tested either as single isomers (compounds 3-8) or
as a 1:1 mixture of cis/trans (a) + trans/trans (b) isomers
(compounds 9 and 10). Mixtures 9a/b and 10a/b were not
separated into the isomers because their MDR-reversing activity
was less interesting than that of compounds 3-8, a fact which
suggested skipping the time-consuming preparation of the single
isomers.
A variety of assays are available to test the modulating activity
of MDR reverters, in particular those acting on Pgp.31-33
Pirarubicin erythroleukemia K562 cell uptake assay was used
as a preliminary screening. The pharmacological profile of the
compounds that showed the best results in this assay, was further
studied evaluating their action on the ATPase activity present
in rat small intestine membrane vesicles (3a-d) and doxorubicin
cytotoxicity potentiation (RF) on doxorubicin-resistant eryth-
roleukemia K562 cells (3a-d and 4a-d).
1
(14d) configuration. Therefore, H NMR data show that 14a
and 14c are frozen in a conformation having the bulky
substituent in position 1 forced into an axial position, while
14b and 14d have the same group in the more relaxed equatorial
position.
Reductive methylation of 14a-d, 15a-d, and 16a/b with
HCOOH/HCHO gave the corresponding tertiary amines 17a-d,
18a-d, and 19a/b. The amino function of the secondary amine
mixture 14a/b was protected by transformation into the t-
butylcarbamate (t-BOCa) to give the mixture 20a/b, which was
then reacted with trans-3-(3,4,5-trimethoxyphenyl)acryloyl chlo-
ride to give 21a/b, which, in turn, was transformed into 10a/b
by acidic cleavage with CF3COOH. Final compounds 3a-d,
4a-d, 5a-d, 6a-d, 7a-c, 8a-c, and mixture 9a/b were then
obtained by reaction of 17a-d, 18a-d, and mixture 19a/b with
the proper acyl chloride.
The 1H NMR data, reported in Table S2 (Supporting
Information), show that the configurations of 14a-d are
maintained in compounds 17a-d and in the final products
3a-d, 4a-d, ruling out any isomerization in the subsequent
reactions. The same behavior is detectable for the intermediates
15a-d, 18a-d, and final compounds 5a-d, 6a-d, 7a-c, and
8a-c.
Chemistry
The reaction pathways used to synthesize the desired com-
pounds (3-10) are described in Scheme 1; their chemical and
physical characteristics are reported in Table S1 (Supporting
Information).
The 4-oxocyclohexyl esters 11-13 were obtained by esteri-
fication of 4-hydroxycyclohexanone34 with 3,4,5-trimethoxy-
benzoyl chloride, trans-3-(3,4,5-trimethoxyphenyl)acryloyl chlo-
ride, or anthracene-9-carbonyl chloride, respectively. Reductive
amination of compounds 11-13 with trans-4-aminocyclohex-
anol gave compounds 14a/b, 15a/b, and 16a/b, while the same
reaction of compounds 11 and 12 with cis-4-aminocyclohex-
anol35 gave compounds 14c/d and 15c/d; in both cases,
titanium(IV) isopropoxide as Lewis acid catalyst and NaBH3CN
as reducing agent were used, according to the Mattson proce-
dure.36 The secondary amines obtained were an approximately
1:1 mixture of cis/trans and trans/trans isomers (a/b) or cis/cis
and trans/cis isomers (c/d), respectively, as results from 1H NMR
spectra.
The information on preferred conformations obtained from
1H NMR was confirmed by molecular modeling (Figure 1)
where isomers 3a-d are shown in their lowest energy confor-
mation with the 3,4,5-trimethoxybenzoate group in axial posi-
tion. The lowest energy conformations of the set of isomers
4-8 are practically identical.
Pharmacological Studies
Modulation of Pirarubucin Uptake. The ability of com-
pounds 3-10 to modulate Pgp action was evaluated on
doxorubicin-resistant erythroleukemia K562 cells (K562/DOX)
that, as reported in the literature, overexpress only Pgp.40,41 As
a Abbreviations: RF, reversal fold; MTT, 3-(4,5-dimethylthiazolyl-2)-
2,5-diphenyltetrazolium bromide; abs EtOH, absolute ethanol; (i-PrO)4Ti,
titanium (IV) isopropoxide; t-BOC, t-butylcarbamate.
A chromatographic separation was performed on the amines
14a/b, 14c/d, 15a/b, and 15c/d and the pure isomers 14a-d

N,N-bis(Cyclohexanol)amine Aryl Esters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 809
a
Scheme 1. Synthesis of Compounds 3-10
a Reagents and conditions: (i) CHCl3, Ar1COCl (3,4,5-trimethoxybenzoyl chloride, trans-3-(3,4,5-trimethoxyphenyl)acryloyl chloride, anthracene-9-carbonyl
chloride; (ii) trans-4-aminocyclohexanol, (i-PrO)4Ti, NaBH3CN; (iii) cis-4-aminocyclohexanol, (i-PrO)4Ti, NaBH3CN; (iv) chromatographic separation; (v)
HCOOH/HCHO; (vi) (t-ButOCO)2O/Et3N; (vii) Ar2COCl, CHCl3; (viii) CF3COOH, CH2Cl2; for the meaning of Ar1 and Ar2, see Table 1.
a matter of fact, K562 is a human leukemia cell line, established
from a patient with a chronic myelogeneous leukemia in blast
transformation.42 K562/DOX cells resistant to doxorubicin
express a unique membrane glycoprotein with a molecular mass
of 180000 Da.43 We measured the uptake of THP-adriamycin
(pirarubicin) by continuous spectrofluorometric signal of the
anthracycline at 590 nm (λex ) 480 nm) after incubation of the
cells, following the protocols reported in previous papers.38,44-46
Pgp-blocking activity is described by (i) R, which represents
the fold increase in the nuclear concentration of pirarubicin in
the presence of the Pgp inhibitor and varies between 0 (in the
absence of the inhibitor) and 1 (when the amount of pirarubicin
in resistant cells is the same as in sensitive cells); (ii) Rmax, which
expresses the efficacy of the Pgp inhibitor and is the maximum
increase that can be obtained in the nuclear concentration of
pirarubicin in resistant cells with a given compound; and (iii)
[I]0.5, which measures the potency of the inhibitor and represents
the concentration that causes a half-maximal increase (R ) 0.5)
in nuclear concentration of pirarubicin (see Table 1).
Even if binding experiments on Pgp were not performed, this
test indicates that the compounds inhibit the Pgp-operated
extrusion of the reporter molecule pirarubicin, as does the
reference molecule verapamil. The molecules studied lack any
detectable cytotoxicity at the doses used in the test.
Effects on ATPase Activity. ATP binding and hydrolysis
are critical for efflux pump function, although it is still a matter
of debate whether ATP hydrolysis is required for driving the
substrate across the membrane or for resetting the transporter
to the starting position or for both.47,48 Nevertheless, studies of
the effects of MDR modulators on ATPase activity of the ABC
transporter family are considered useful to get insight into the
action mechanisms of these drugs. The effects of some of the

810 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Table 1. MDR-Reverting Activity of Compounds 3-10
Martelli et al.
c
compd
structurea
R
Ar1
Ar2
[I]0.5 µMb
Rmax
1
2
I
I
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
A
A
A
B
B
B
C
A
B
C
B
B
B
B
C
C
C
C
C
C
C
C
D
D
D
D
A
A
A
B
B
B
C
B
0.60 ( 0.15d
0.28 ( 0.05e
0.092 ( 0.015
0.32 ( 0.10
0.03 ( 0.01
0.012 ( 0.001
0.30 ( 0.06
0.30 ( 0.06
0.18 ( 0.03
0.16 ( 0.06
0.14 ( 0.02
0.15 ( 0.01
0.17 ( 0.08f
0.16 ( 0.07f
0.19 ( 0.03f
0.12 ( 0.06f
1.19 ( 0.4
0.90
0.71
0.85
0.81
0.80
0.98
0.95
0.88
0.79
0.78
0.67
0.60
0.38
0.40
0.39
0.46
0.52
0.44
0.61
0.73
0.98
0.36
0.47
0.44
0.50
0.70
0.70
0.70
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)
8a (cis/trans)
8b (trans/trans)
8c (cis/cis)
9a/b (cis/trans + trans/trans)
10a/b (cis/trans + trans/trans)
verapamil
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
0.13 ( 0.08f
0.13 ( 0.06
0.35 ( 0.11
2.0 ( 0.6
0.12 ( 0.04f
0.11 ( 0.03f
1.13 ( 0.4f
0.70 ( 0.14g
3.30 ( 0.06
1.60 ( 0.3
MM36
0.05 ( 0.01h
a See Chart 1. b Concentration of the inhibitor that causes a 50% increase in nuclear concentration of pirarubicin (R ) 0.5). c Efficacy of MDR-modulator:
maximum increase that can be obtained in the nuclear concentration of pirarubicin in resistant cells. d See ref 26. e See ref 30. f Concentration of the inhibitor
that causes a 30% increase in nuclear concentration of pirarubicin (R ) 0.3). g Concentration of the inhibitor that causes a 40% increase in nuclear concentration
of pirarubicin (R ) 0.4). h See ref 46.
synthesized compounds on pump ATPase activity of brush
border plasma membrane vesicles, prepared from homogenates
of rat small intestine mucosa, were evaluated.49 In plasma
membrane vesicles, as shown in Figure S1 (Supporting Informa-
tion), MRP1, MRP2, and Pgp were present in detectable
amounts. On the assumption of the existence of multiple binding
sites on Pgp and sister proteins, both basal ATPase activity and
ATPase activity stimulated by the substrate verapamil were
assayed in the absence or in the presence of MDR-inhibitors.
Pgp.40,41 Test compounds were evaluated for their intrinsic
cytotoxicity and MDR-reversal activity in vitro using the MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)
assay.50
The IC50 drug concentrations resulting in a 50% reduction in
the conversion of MTT in drug-treated cells compared to
untreated control cells were calculated from plotted results. The
potency of MDR reversal was expressed by the reversal-fold
(RF) values, obtained by dividing the doxorubicin IC50 values
of K562 and K562/DOX cells in the absence of modulators by
those in the same cells in the presence of modulators (see Table
2). All results are presented as mean ( SE, and statistical
analysis was performed using one-way Anova test and Bon-
ferroni’s multiple comparison test (GraphPad Prism software,
Inc. CA).
Effects on Doxorubicin Cytotoxicity. The assessment of the
ability of a compound to enhance the growth inhibitory effects
of doxorubicin in tumor cell lines showing MDR due to Pgp
overexpression has also been proved useful in the quantification
and characterization of MDR reversal by modulators of the
MDR phenotype. Comparisons are made between the effects
on the sensitivity to doxorubicin in doxorubicin-resistant cells
in the presence and in the absence of an inhibitor.
Results
The reversal effects of compounds 3a-d and 4a-d on the
MDR phenotype were investigated on doxorubicin-resistant
erythroleukemia K562 cells (K562/DOX) overexpressing only
Modulation of Pirarubucin Uptake. The results obtained
on doxorubicin-resistant erythroleukemia K562 cells are reported
in Table 1, together with those of two parent linear compounds

N,N-bis(Cyclohexanol)amine Aryl Esters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 811
Figure 1. Lowest-energy conformations of 3a, 3b, 3c, and 3d. The compounds were built within Spartan ’04 (wave function) and minimized using
semiempirical methods (AM1). A conformational analysis was performed starting from the cyclohexane rings built in the chair conformation and
by rotating each flexible bond. The groups on the trans-substituted cyclohexane ring have been calculated only in the diequatorial position. For 3a
and 3c, both cis isomers (i.e., those having the ester and amino groups in axial/equatorial position) have been calculated: it was found that lower
energy is always associated with the conformer showing the trimethoxybenzoate group axial and the methylcyclohexylamino group equatorial, with
∆E values changing according to the different conformation on the rest of the molecules (2-5 kcal/mol). Fairly similar results were obtained with
the isomers of compounds 4-8.
Table 2. Effects of Studied MDR-Reversing Agents on Doxorubicin Cytotoxicity in K562 Cells and K562/DOX Cellsa
K562
K562/DOX
compd
IC50 (µM)b
RFc
IC50 (µM)b
RFc
DOX
0.032 ( 0.002
nd
0.046 ( 0.011
0.069 ( 0.013
nd
0.048 ( 0.01
0.038 ( 0.013
nd
0.045 ( 0.015
0.046 ( 0.013
nd
0.050 ( 0.009
0.037 ( 0.008
nd
0.027 ( 0.005
0.019
1.0
nd
0.7
0.5
nd
0.7
0.8
nd
0.7
0.7
nd
0.6
0.9
nd
1.2
1.7
nd
0.7
1.4
nd
1.3
4.6
nd
2.84 ( 0.047
2.77 ( 0.24
0.37 ( 0.13e
0.29 ( 0.06e
1.26 ( 0.082f
0.31 ( 0.03e
0.21 ( 0.03e
1.39 ( 0.13
0.29 ( 0.06e
0.16 ( 0.02e
0.77 ( 0.16e
0.14 ( 0.03e
1.0
1.0
7.7
9.8
2.3
9.2
13.5
2.0
9.8
17.8
3.7
20.3
36.4
1.3
5.0
11.4
1.2
8.4
13.5
5.5
16.7
20.3
1.6
14.2
20.3
3.4
DOX + 3a (0.092 µM)d
DOX + 3a (1 µM)
DOX + 3a (3 µM)
DOX + 3b (0.32 µM)d
DOX + 3b (1 µM)
DOX + 3b (3 µM)
DOX + 3c (0.03 µM)d
DOX + 3c (1 µM)
DOX + 3c (3 µM)
DOX + 3d (0.01 µM)d
DOX + 3d (1 µM)
DOX + 3d (3 µM)
DOX + 4a (0.3 µM)d
DOX + 4a (1 µM)
0.078 ( 0.006e
2.23 ( 0.29
0.57 ( 0.06e
0.25 ( 0.044e
2.33 ( 0.3e
DOX + 4a (3 µM)
DOX + 4b (0.3 µM)d
DOX + 4b (1 µM)
DOX + 4b (3 µM)
DOX + 4c (0.18 µM)d
DOX + 4c (1 µM)
nd
0.048 ( 0.001
0.023
nd
0.025 ( 0.001
0.0069
nd
0.028 ( 0.008
0.011
nd
0.048 ( 0.008
0.058 ( 0.004
0.34 ( 0.065e
0.21 ( 0.036e
0.52 ( 0.093e
0.17 ( 0.032e
0.14 ( 0.032e
1.75 ( 0.18e
0.2 ( 0.014e
0.14 ( 0.036e
0.84f
DOX + 4c (3 µM)
DOX + 4d (0.16 µM)d
DOX + 4d (1 µM)
DOX + 4d (3 µM)
DOX + verapamil (1.6 µM)d
DOX + verapamil (1 µM)
DOX + verapamil (3 µM)
1.1
2.9
nd
0.7
0.6
1.7 ( 0.09e
1.7
4.2
0.68 ( 0.05e
a nd ) not determined. b 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 Experimental section. c Reversal fold of MDR was determined by dividing the IC50 values of doxorubicin
of K562 and K562/DOX cells in the absence of modulators by those in the presence of modulators. d [I]0.5 value which measures the potency of the inhibitor
and represents the concentration that causes a half-maximal increase (R ) 0.5) in nuclear concentration of pirarubicin. e p < 0.001 of IC50 of treated
K562/DOX cells vs control cells (K562/DOX cells treated with doxorubicin only). Where not specified the IC50 values of treated K562 and K562/DOX cells
are not significantly different compared to control cells (K562 and K562/DOX cells treated with doxorubicin only). f p < 0.01 of IC50 of treated K562/DOX
cells vs control cells (K562/DOX cells treated with doxorubicin only). Where not specified the IC50 values of treated K562 and K562/DOX cells are not
significantly different compared to control cells (K562 and K562/DOX cells treated with doxorubicin only).
1
26 and 230 (Structure I, Chart 1) and of MM36, the most potent
Except for the mixture 10a/b, the new molecules are all able
to modulate the activity of Pgp, being more potent than the
standard reference compound verapamil. Their potencies range
compound that we have found in previous studies,46 and
verapamil used as reference compounds.

812 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Martelli et al.
Figure 2. Effects of 3c and 3d on basal (A,C) and verapamil-stimulated (B,D) ATPase activity. Under control conditions (no drug added, CTRL)
basal ATPase activity was considered to be 100%. *p < 0.05; **p < 0.01 one sample t test vs CTRL (n ) 3-5). #p < 0.05; ##p < 0.01 one sample
t test vs verapamil (n ) 3-5).
from low nanomolar (compounds 3a, 3c, 3d) to micromolar
values (6c, 7c, 8c), the remaining compounds being in the high
nanomolar range. Some compounds (3d, 4a, 7c) show nearly
complete reversal of MDR (Rmax close to 1), while a few show
very low Rmax (<0.5) and seem unable to achieve a significant
control of MDR. In these cases (5c, 5d, 6a, 6b, 6d, 8a, 8b, 8c,
9a/b), the [I]0.5 could not be evaluated and potency was
measured at the lower value of R reported in Table 1.
Confirming previously found structure-activity relationships,26
N-methyl derivatives seem more active than the corresponding
secondary amines as it can be seen comparing 3a and 3b with
10a/b ([I]0.5 ) 0.092 µM, and 0.32 µM and 3.30 µM,
respectively).
Effects on ATPase Activity. The effect of isomers 3a-d
on ATPase activity was evaluated. In the assay protocol used,
ATPase activity was fully inhibited by low micromolar con-
centrations of Na3VO4, thus indicating that it was totally
ascribable to Pgp or MRPs.51 Verapamil stimulated the basal
ATPase activity, thus behaving like a substrate for the pumps.
As reported in panel A of Figure 2, compound 3c gave a
typical bell-shaped concentration-activation curve with 5 ×
10-8 M maximum effective concentration, thus behaving like a
Pgp ATPase substrate. Moreover, this isomer showed a bimodal
effect on 30 µM verapamil-stimulated ATPase activity (Figure
2, panel B). In fact, this activity was stimulated at concentrations
e2.5 × 10-8 M, while it was inhibited at higher concentrations.
Compound 3d inhibited, with a typical bell-shaped concentra-
tion-inhibition curve, the basal ATPase activity, maximal
inhibition (about 55%) being achieved at 5 × 10-9 M concen-
tration (Figure 2, panel C). At higher concentrations, the
inhibition was reversed up to values comparable to those of
control preparations. Moreover, at concentrations e5 × 10-9
M, 3d fully antagonized the portion of ATPase activity
stimulated by verapamil (Figure 2, panel D), thus indicating a
clear antagonism toward verapamil activation. Compounds 3a
and 3b did not exert significant effects (data not shown).
Effects on Doxorubicin Cytotoxicity. The ability of com-
pounds 3a-d and 4a-d to reverse drug resistance of K562/
DOX cells was examined in comparison to verapamil.
Preliminarily, it was checked whether compounds 3a-d,
4a-d and verapamil alone exerted cytotoxic effects on K562
and K562/DOX cells. In both cell lines, isomers of compounds
3 and 4 and verapamil had an intrinsic toxicity not exceeding
20% at all concentrations tested ([I]0.5, 1 µM and 3 µM) with
the exception of isomers 4c and 4d, which presented a 35%
cell growth inhibition at 3 µM concentration (data not shown).
The effects on the reversion of doxorubicin resistance were
examined after the cells were incubated at 37 °C for 72 h at
[I]0.5 concentration of each compound, which represents the
concentration that causes a half-maximal increase in nuclear
concentration of pirarubicin and at 1 and 3 µM concentrations.
Results are reported in Table 2.
At the [I]0.5 concentration, isomer 3a did not display any drug
resistance reversal activity in K562/DOX cells. At the same
concentration, isomers 3b and 3c significantly reverted doxo-
rubicin resistance in these cells by a factor (RF) of about 2.
The most relevant and significant effect was obtained with
isomer 3d, which reverted doxorubicin resistance in K562/DOX
cells by a RF of 3.7. At the higher concentrations (1 and 3 µM)
all isomers (3a-d) were able to markedly reverse doxorubicin
resistance (RF values ranging from 7.7 to 36.4). Also at these
concentrations isomer 3d was the most active, isomers 3b and
3c displayed intermediate activity, and isomer 3a was the least
active.
Isomers 4a and 4b at the [I]0.5 dose did not reverse dox-
orubicin resistance in K562/DOX cells showing RF values
slightly above 1. At higher concentrations (1 and 3 µM), they
exerted marked reversal effects on drug-induced cell growth
inhibition (RF values ranging from 5.0 to 13.5). At [I]0.5, 4c
was a potent resistance reversal agent in this cell line (RF value
5.5), while isomer 4d displayed borderline reversal activity (RF
value 1.6). At the higher concentrations tested, isomers 4c and

N,N-bis(Cyclohexanol)amine Aryl Esters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 813
4d increased significantly cell growth inhibitory effects induced
by doxorubicin and RF values ranging from 14.2 to 20.3 were
observed. While at 1 µM, these interactions may be attributed
to specific modulatory effects on Pgp function, at 3 µM, a direct
cytotoxic effect of studied isomers may have affected experi-
mental results.
When comparing reversal effects of the new compounds and
verapamil at [I]0.5, similar reversal activities were observed for
isomers 3d, 4c, and verapamil. At higher concentrations (1 and
3 µM), all isomers were clearly more effective in enhancing
the cell growth inhibitory effects of doxorubicin as compared
to equimolar verapamil concentrations.
When the effects of the new compounds were examined on
parental doxorubicin sensitive K562 cells at the concentrations
of 1 and 3 µM, statistically not significant differences in IC50
were observed for isomers 3a-d (RF values ranging from 0.5
to 0.9). When isomers 4a-d were used in combination with
doxorubicin, increased cytotoxic effects were usually observed.
They were modest at 1 µM and more pronounced, albeit not
significant, at 3 µM.
If the atomic structure of Pgp were available, we could check
the soundness of this hypothesis and improve the affinity of
the compounds of the series. Unfortunately, only a few homo-
logy models12-14 developed after the structure of homologous
bacterial transporters and not very useful for this purpose, are
known. Therefore, we had to rely on the old trial and error
approach, which, so far, has yielded disappointing results. For
instance, the attempt to improve the fitting of set 5 by restoring
the conformational freedom of the two phenyl rings of an-
thracene (set 6) afforded nearly less potent and less efficacious
compounds. Disappointing results were also obtained with the
series of compounds substituted with identical aromatic moieties
(sets 7 and 8).
Pharmacological Profiling of Most Potent Compounds.
The ATPase modulation of the isomers of set 3 was evaluated:
the test confirmed the activity and the high potency revealed
by the pirarubicin uptake assay. Also, in this test, the isomers
behave in a quite different way: compounds 3a and 3b did not
exert significant effects; compound 3c gave a typical bell-shaped
activation curve with a maximum at 5 × 10-8 M, while 3d
inhibited the basal ATPase activity, maximal inhibition (about
55%) being achieved at a 5 × 10-9 M concentration. When
ATPase activity was stimulated with 30 µM verapamil, com-
pound 3c increased enzymatic activity up to 2.5 × 10-8 M but
inhibited it at higher concentrations, whereas compound 3d fully
antagonized verapamil-stimulated enzymatic activity at 5 × 10-9
M concentration. These results show that of the four isomers
tested, 3c behaves like a substrate while 3d behaves like an
inhibitor/substrate of ATPase activity.33
Discussion
Modulation of Pirarubicin Uptake and SARs. As stated
above, we have used pirarubicin uptake in doxorubicin-resistant
erythroleukemia K562 cells as a screening assay to evaluate
the interaction of our compounds with Pgp; as a consequence,
we will rely on the results obtained in this test to draw some
structure-activity relationships for the whole series. The results
reported in Table 1 show that, as it is usual when applying the
frozen analogue approach, the compounds of the series show a
quite different pharmacological behavior that spans from the
excellent activity of the isomers of set 3 to the poor performance
of those of set 8. Very likely, this is the consequence of two
concurrent properties: isomeric geometry and the nature of the
aryl moieties. In each isomer of the series, the scaffold is fairly
rigid and the aryl moieties are forced into discrete positions
with limited conformational freedom, something that may favor
or disfavor the affinity for the binding site depending on the
nature of the aromatic moieties.
The results of cytotoxicity tests demonstrated that isomers
3d, 4c, and 4d significantly enhanced doxorubicin sensitivity
of K562/DOX cells. While intrinsic cytotoxicity of isomer 3d
was limited (<20%), isomers 4c and 4d at high concentration
(3 µM) induced cell growth inhibition of both K562/DOX and
K562 cells (about 35%). This suggests that the effects of isomers
3d on doxorubicin cytotoxicity may be specifically mediated
by Pgp interactions in an overexpressing cell phenotype, while
those of 4c and 4d may be partially not specific. Lack of isomer
3d effects on the sensitivity to doxorubicin of the sensitive K562
cell line also suggests specificity.
The best combination of these properties is apparently that
of compound 3d, the most potent and efficacious member of
the series, that combines a trans/cis geometry with 3,4,5-
trimethoxybenzoic and trans-3,4,5-trimethoxycinnamic aryl
moieties. The other isomers of the set (3a, 3b, 3c) are also potent
and efficacious modulators of Pgp, being more active than the
corresponding linear compound 1,26 the differences in their
potency and efficacy reflecting their different geometry. A
similar situation is found in the set of isomers 4: even in this
case the trans/cis isomer (4d) is the most active, yet some ten
times less than 3d. In this case, restriction of conformational
freedom does not pay, and the four isomers of 4 maintain the
activity of the corresponding linear compound 2.30 Clearly,
because the isomers of set 4 have the same geometrical features
of those of set 3, these differences have to be ascribed to the
anthracene moiety. Apparently, as the conformational freedom
is reduced, the nature of the aromatic moieties gains importance,
a consequence that is in accord with our original hypothesis
that flexible molecules can more easily find the most productive
interaction with the recognition site. As a matter of fact, the
anthracene moiety (set 4 and 5) that produced very effective
Pgp inhibitors in flexible molecules (see compound 2, but also
the analogous compounds reported in the previous exploratory
work26) appears to be less effective when inserted in less flexible
structures.
As far as the selectivity of our compounds against different
types of ABC transporters is concerned, the results of intestine
vesicles would suggest that they may be active also on MRP1
and sister proteins. On these bases, a novel ongoing investigation
has been started and the results will be disclosed in due time.
In conclusion, applying the frozen analogue approach to a
series of very flexible MDR reverters26 has lead us to identify
a new series of drugs that show very interesting MDR-reverting
properties. Among them, compound 3d, which consistently
shows low nanomolar potency and high efficacy in all the tests
used, appears as a new pharmacological tool for Pgp studies
and a promising lead for the development of potent, efficient,
and safe MDR reverters.
Experimental Section
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 spectropho-
1
tometer. H NMR spectra were recorded on Bruker Avance 400
spectrometer. Chromatographic separations were performed on a
silicagelcolumnbyflashchromatography(Kieselgel40,0.040-0.063
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

814 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Martelli et al.
compounds. The identity and purity of the intermediates was
ascertained through IR, 1H NMR, and TLC chromatography. When
reactions were performed in anhydrous conditions, the mixtures
were maintained under nitrogen.
Compounds were named following IUPAC rules as applied by
Beilstein-Institut AutoNom 2000 4.01.305, a software for systematic
names in organic chemistry.
2.60 (t, J ) 10.8 Hz, 1H, NCH), 2.15-1.13 (m, 18H, 8CH2, OH
and NH) ppm.
The two isomers 15a and 15b were obtained in the same way
by chromatographic separation of the approximately 50/50 mixture
of cis/trans and trans/trans isomers 15a/b (1.08 g).
cis/trans 15a. Yield 350 mg as a colorless oil; Rf ) 0.38. IR
(neat): ν 3350 (OH e NH); 1708 (CO) cm-1. 1H NMR (CDCl3): δ
7.58 (d, 1H, CHdCH, J ) 16.0 Hz), 6.75 (s, 2H, aromatics), 6.32
(d, 1H, CHdCH, J ) 16.0 Hz), 5.08 (bs, 1H, CHO), 3.88 (s, 6H,
2OCH3), 3.86 (s, 3H, OCH3), 3.63-3.53 (m, 1H, CHOH),
2.75-2.65 (m, 1H, NCH), 2.63-2.54 (m, 1H, NCH), 2.01-1.90
(m, 6H, 3CH2), 1.77-1.69 (m, 2H, CH2), 1.63-1.48 (m, 4H, 2CH2),
1.36-1.23 (m, 2H, CH2), 1.21-1.06 (m, 2H, CH2) ppm.
trans/trans 15b. Yield 200 mg as a white solid (mp: 168-170
°C); Rf ) 0.32. IR (neat): ν 3350 (OH e NH); 1708 (CO) cm-1. 1H
NMR (CDCl3): δ 7.57 (d, 1H, CHdCH, J ) 16.0 Hz), 6.75 (s,
2H, aromatics), 6.32 (d, 1H, CHdCH, J ) 16.0 Hz), 4.82 (tt, 1H,
CHO, J ) 10.8 Hz, J ) 4.4 Hz), 3.87 (s, 9H, 3OCH3), 3.62 (tt,
1H, CHO, J ) 10.8 Hz, J ) 4.0 Hz), 2.69-2.59 (m, 2H, 2NCH),
2.14-1.93 (m, 8H, 4CH2), 1.52-1.40 (m, 2H, CH2), 1.39-1.13
(m, 6H, 3CH2) ppm.
3,4,5-Trimethoxybenzoic Acid 4-Oxocyclohexyl Ester (11). 3,4,5-
Trimethoxybenzoic acid (710 mg, 3.34 mmol) was transformed into
the acyl chloride by reaction with SOCl2 (2 equiv) in 5 mL CHCl3
(free of EtOH) at 60 °C for 4 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 4-hydroxycyclohexanone34 (0.23 g, 2.00 mmol) was
added. The reaction mixture was heated to 60 °C for 8 h and then
cooled to room temperature, treated with CH2Cl2, and the organic
layer washed with 10% NaOH solution. 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 (420 mg, 68% yield)
1
was obtained. IR (neat): ν 1712 (CO), 1694 (CO) cm-1. H NMR
The two isomers 14c and 14d were obtained in the same way
by chromatographic separation of the approximately 50/50 mixture
of cis/cis and trans/cis isomers 14c/d (0.72 g).
(CDCl3): δ 7.25 (s, 2H, aromatics); 5.41-5.32 (m, 1H, CHO); 3.85
(s, 9H, 3OCH3); 2.57-2.53 (m, 2H, CH2); 2.51-2.40 (m, 2H, CH2);
2.21-2.16 (m, 4H, 2CH2) ppm.
cis/cis 14c. Yield 200 mg as a light-yellow oil; Rf ) 0.52. IR
(neat): ν 3350 (OH and NH); 1723 (CO) cm-1. 1H NMR (CDCl3):
δ 7.31 (s, 2H, aromatics), 5.15 (bs, 1H, CHO), 3.94 (bs, 1H,
CHOH), 3.90 (m, 9H, 3OCH3), 2.81-2.70 (m, 2H, 2NCH),
2.06-1.98 (m, 2H, CH2), 1.83-1.51 (m, 14H, 7CH2) ppm.
trans/cis 14d. Yield 150 mg as a light-yellow oil; Rf ) 0.45. IR
(neat): ν 3350 (OH and NH); 1723 (CO) cm-1. 1H NMR (CDCl3):
δ 7.27 (s, 2H, aromatics), 4.93 (tt, J ) 10.8 Hz, J ) 4.4 Hz, 1H,
CHO), 3.94 (bs, 1H, CHOH), 3.90 (m, 9H, 3OCH3), 2.80-2.72
(m, 2H, 2NCH), 2.18-2.11 (m, 2H, CH2), 2.08-2.01 (m, 2H, CH2),
1.83-1.34 (m, 12H, 6CH2) ppm.
Compounds 12 and 13 were obtained in the same way from
trans-3-(3,4,5-trimethoxyphenyl)-acryloyl chloride or anthracene-
9-carbonyl chloride, respectively. Their IR and H NMR spectra
1
are consistent with the proposed structures.
3,4,5-Trimethoxybenzoic Acid 4-(4-Hydroxycyclohexylamino)cy-
clohexyl Ester (14a/b). A mixture of compound 11 (420 mg, 1.36
mmol), trans-4-aminocyclohexanol (160 mg, 1.36 mmol), and
titanium(IV) isopropoxide (0.44 mL, 1.36 mmol) was stirred at room
temperature. After 3 h, absolute ethanol (2.5 mL) and NaBH3CN
(170 mg, 1.79 mmol) were added and the solution was stirred for
20 h. Water (10 mL) was added, the organic solvent was removed
under reduced pressure, to 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
CHCl3/MeOH (9:1) as eluting system. The title compound (410
mg, 74% yield) as a mixture of cis/trans and trans/trans isomers
was obtained as a white solid; mp: 45-47 °C. IR (neat): ν 3350
(OH and NH); 1723 (CO) cm-1. 1H NMR (CDCl3): δ 7.27 (s, 2H,
aromatics); 5.12 (bs, 0.5H, CHO); 4.92 (tt, J ) 10.8 Hz, J ) 4.4
Hz, 0.5H, CHO); 3.87 (s, 9H, 3OCH3); 3.70-3.48 (m, 1H, CHOH);
2.73-2.49 (m, 2H, 2NCH); 2.20-1.01 (m, 18H, 8CH2, OH and
NH) ppm.
The two isomers 15c and 15d, were obtained in the same way
by chromatographic separation of the approximately 50/50 mixture
of cis/cis and trans/cis isomers 15c/d (1.70 g).
cis/cis 15c. Yield 500 mg as a light-pink oil; Rf ) 0.38. IR (neat):
1
ν 3350 (OH e NH); 1708 (CO) cm-1. H NMR (CDCl3): δ 7.61
(d, 1H, CHdCH, J ) 16.0 Hz), 6.78 (s, 2H, aromatics), 6.33 (d,
1H, CHdCH, J ) 16.0 Hz), 5.11-5.07 (m, 1H, CHO), 3.92-3.88
(m, 7H, 2OCH3 + CHO), 3.86 (s, 3H, OCH3), 2.82-2.70 (m, 2H,
2NCH), 2.02-1.92 (m, 2H, CH2), 1.81-1.51 (m, 14H, 7CH2) ppm.
trans/cis 15d. Yield 280 mg as a light-pink oil; Rf ) 0.33. IR
(neat): ν 3350 (OH e NH); 1708 (CO) cm-1. 1H NMR (CDCl3): δ
7.56 (d, 1H, CHdCH, J ) 16.0 Hz), 6.74 (s, 2H, aromatics), 6.31
(d, 1H, CHdCH, J ) 16.0 Hz), 4.82 (tt, 1H, CHO, J ) 10.8 Hz,
J ) 4.4 Hz), 3.95-3.89 (m, 1H, CHO), 3.88 (s, 6H, 2OCH3), 3.86
(s, 3H, OCH3), 2.76-2.64 (m, 2H, 2NCH), 2.14-2.06 (m, 2H,
CH2), 2.05-1.95 (m, 2H, CH2), 1.80-1.25 (m, 12H, 6CH2) ppm.
cis/trans-3,4,5-TrimethoxybenzoicAcid4-[(4-Hydroxycyclohexyl)-
methylamino]cyclohexyl Ester (17a). To a solution of 410 mg (1.0
mmol) of 14a in 1 mL of absolute ethanol, 0.66 mL of HCOOH
and 0.38 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 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 eluting system. Compound 17a was obtained as an oil (250
mg, 59% yield). IR (neat): ν 3370 (OH); 1714 (CO) cm-1. 1H NMR
(CDCl3): δ. 7.31 (s, 2H, aromatics), 5.18 (bs, 1H, CHO), 3.90 (s,
9H, 3OCH3), 3.61-3.51 (m, 1H, CHOH), 2.69-2.51 (m, 2H,
2NCH), 2.26 (s, 3H, NCH3), 2.20-1.18 (m, 17H, 8CH2, and OH)
ppm.
Compounds 15a/b and 16a/b were obtained in the same way
from 12 and 13, respectively. Compounds 14c/d and 15c/d were
obtained in the same way from 11 and 12, respectively, using cis-
4-aminocyclohexanol.35 Their IR and 1H NMR spectra are consis-
tent with the proposed structures.
cis/trans-3,4,5-Trimethoxybenzoic Acid 4-(4-Hydroxycyclohexy-
lamino)cyclohexyl Ester (14a) and trans/trans-3,4,5-Trimethoxy-
benzoic Acid 4-(4-Hydroxycyclohexylamino)cyclohexyl Ester (14b).
Flash chromatography of 240 mg of compound 14a/b, as an
approximately 50/50 mixture of cis/trans and trans/trans isomers,
afforded the two isomers cis/trans and trans/trans (14a and 14b)
using CHCl3/MeOH/NH3 (90:10:0.5) as eluting system.
cis/trans 14a. Yield 90 mg as a colorless oil; Rf ) 0.50. IR (neat):
ν 3350 (OH and NH); 1723 (CO) cm-1. 1H NMR (CDCl3): δ 7.27
(s, 2H, aromatics), 5.12 (bs, 1H, CHO), 3.91 (s, 9H, 3OCH3), 3.60
(tt, J ) 10.6 Hz, J ) 4.0 Hz, 1H, CHOH), 2.71 (tt, J ) 9.6 Hz, J
) 3.2 Hz, 1H, NCH), 2.59 (tt, J ) 10.8 Hz, J ) 3.6 Hz, 1H, NCH),
2.06-1.10 (m, 18H, 8CH2, OH and NH) ppm.
trans/trans 14b. Yield 70 mg as a white solid (mp: 127-128
°C); Rf ) 0.43. IR (neat): ν 3350 (OH and NH); 1723 (CO) cm-1
.
Compounds 17b, 17c, and 17d were obtained in the same way
from 14b, 14c, and 14d, respectively.
1H NMR (CDCl3): δ 7.27 (s, 2H, aromatics), 4.92 (tt, J ) 10.8 Hz,
J ) 4.4 Hz, 1H, CHO), 3.90 (s, 9H, 3OCH3), 3.62 (tt, J ) 10.6
Hz, J ) 4.0 Hz, 1H, CHOH), 2.66 (t, J ) 11.2 Hz, 1H, NCH),
1
trans/trans 17b. IR (neat): ν 3370 (OH); 1714 (CO) cm-1. H
NMR (CDCl3): δ 7.28 (s, 2H, aromatics), 4.95-4.78 (m, 1H, CHO),

N,N-bis(Cyclohexanol)amine Aryl Esters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 815
3.91 (s, 9H, 3OCH3), 3.61-3.55 (m, 1H, CHOH), 2.70-2.50 (m,
2H, 2NCH), 2.27 (s, 3H, NCH3), 2.20-1.18 (m, 17H, 8CH2, and
OH) ppm.
3OCH3), 3.90 (s, 9H, 3OCH3), 2.70-2.52 (m, 2H, 2NCH), 2.31
(s, 3H, NCH3), 2.13-1.20 (m, 16H, 8CH2) ppm. The oily product
was transformed into the hydrochloride and recrystallized from
absolute ethanol/anhydrous diethylether; mp: 147-150 °C. Anal.
(C35H48ClNO10) C, H, N.
Compounds 3b-d, 4a-d, 5a-d, 6a-d, 7a-c, 8a-c, 9a/b, and
21a/b were obtained in the same way by reaction of the corre-
sponding alcohol with the suitable acyl chloride. Compounds 3c,
3d, 5a-d, 6a-d, 7a, and 8a-c were transformed into the oxalate,
compounds 3b, 4a-d, 7b-c, and 9a/b were transformed into the
hydrochloride and recrystallized from the solvent reported in Table
1. Their chemical and physical characteristics are reported in Table
S1, and IR and 1H NMR spectra are reported in Table S3
(Supporting Information).
1
cis/cis 17c. IR (neat): ν 3370 (OH); 1714 (CO) cm-1. H NMR
(CDCl3): δ 7.32 (s, 2H, aromatics), 5.19 (bs, 1H, CHO), 3.96 (bs,
1H, CHOH), 3.90 (s, 9H, 3OCH3), 2.79-2.62 (m, 2H, 2NCH), 2.33
(s, 3H, NCH3), 2.18-2.05 (m, 2H, CH2), 1.83-1.51 (m, 15H, 7CH2,
and OH) ppm.
trans/cis 17d. IR (neat): ν 3370 (OH); 1714 (CO) cm-1. 1H NMR
(CDCl3): δ 7.26 (s, 2H, aromatics), 4.93-4.81 (m, 1H, CHO), 3.97
(bs, 1H, CHOH), 3.89 (s, 9H, 3OCH3), 2.77-2.56 (m, 2H, 2NCH),
2.31 (s, 3H, NCH3), 2.21-2.13 (m, 2H, CH2), 1.98-1.49 (m, 15H,
7CH2, and OH) ppm.
Compounds 18a, 18b, 18c, and 18d were obtained in the same
way from 15a, 15b, 15c, and 15d, respectively.
3,4,5-Trimethoxybenzoic Acid 4-{4-[3-(3,4,5-Trimethoxyphenyl)-
acryloyloxy]cyclohexylamino}cyclohexyl Ester (10a/b). To a solu-
tion of 110 mg (0.16 mmol) of 21a/b in 0.32 mL of CH2Cl2, 0.32
mL of CF3COOH were added under vigorous stirring. After 2 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; 90
mg (90% yield) of the pure title compound were obtained. IR (neat):
1
cis/trans 18a. H NMR (CDCl3): δ 7.61 (d, 1H, CHdCH, J )
16.0 Hz), 6.77 (s, 2H, aromatics), 6.35 (d, 1H, CHdCH, J ) 16.0
Hz), 5.12-5.08 (m, 1H, CHO), 3.90 (s, 6H, 2OCH3), 3.88 (s, 3H,
OCH3), 3.57 (tt, 1H, CHOH, J ) 10.8 Hz, J ) 4.0 Hz), 2.64-2.55
(m, 2H, 2NCH), 2.30 (s, 3H, NCH3), 2.09-2.00 (m, 4H, 2CH2),
1.89-1.30 (m, 12H, 6CH2) ppm.
trans/trans 18b. 1H NMR (CDCl3): δ 7.56 (d, 1H, CHdCH, J )
16.0 Hz), 6.73 (s, 2H, aromatics), 6.31 (d, 1H, CHdCH, J ) 16.0
Hz), 4.76 (tt, 1H, CHO, J ) 10.8 Hz, J ) 4.4 Hz), 3.88 (s, 6H, 2
OCH3), 3.86 (s, 3H, OCH3), 3.55 (tt, 1H, CHOH, J ) 10.8 Hz, J
) 4.0 Hz), 2.60-2.46 (m, 2H, 2NCH), 2.22 (s, 3H, NCH3),
2.12-1.96 (m, 4H, 2CH2), 1.89-1.77 (m, 4H, 2CH2), 1.52-1.22
(m, 8H, 4CH2) ppm.
1
ν 3520 (NH), 1706 (CO) cm-1. H NMR (CDCl3): δ 7.59 (d, J )
16.0 Hz, 1H, CHdCH), 7.29 (s, 2H, aromatics), 6.76 (s, 2H,
aromatics), 6.33 (d, J ) 16.0 Hz 1H, CHdCH), 5.17 (bs, 0.5H,
CHO), 4.96-4.84 (m, 0.5H, CHO), 4.73-4.81 (m, 1H, CHO), 3.90
(s, 18H, 6OCH3), 2.71-2.58 (m, 2H, 2NCH), 2.15-1.07 (m, 17H,
8CH2, NH) ppm.
cis/cis 18c. 1H NMR (CDCl3): δ 7.61 (d, 1H, CHdCH, J ) 16.0
Hz), 6.77 (s, 2H, aromatics), 6.34 (d, 1H, CHdCH, J ) 16.0 Hz),
5.12-5.07 (m, 1H, CHO), 4.00-3.95 (m, 1H, CHOH), 3.88 (s,
6H, 2 OCH3), 3.85 (s, 3H, OCH3), 2.75-2.60 (m, 2H, 2NCH), 2.32
(s, 3H, NCH3), 2.80-2.00 (m, 2H, CH2), 1.90-1.69 (m, 8H, 4CH2),
1.63-1.48 (m, 6H, 3CH2) ppm.
The oily product was transformed into the hydrochloride, which
was recrystallized from absolute ethanol/anhydrous diethylether;
mp: 192-194 °C. Anal. (C34H46ClNO10) C, H, N.
Pharmacology. Cell Lines and Cultures. The K562 cell line
is a highly undifferentiated erythroleukemia originally derived from
a patient with chronic myelogenous leukemia.42 The K562 leukemia
cells and the P-gp expressing K562/DOX cells were obtained from
Prof. J. P. Marie (Hopital Hotel-Dieu, Paris, France). These cells
were cultured in RPMI 1640 medium with GlutaMAX-I (GIBCO)
medium supplemented with 10% fetal calf serum (FCS) (GIBCO)
at 37 °C in a humidified incubator with 5% CO2. To maintain the
resistance, every month, resistant cells were cultured for three days
with 400 nM doxorubicin. The cell line was then used, one week
latter, during three weeks. Cultures initiated at a density of 105
cells/mL grew exponentially to about 106 cells/mL in 3 days. For
the spectrofluorometric assays, in order 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. Cultured cells were counted with a
Coulter counter before use. The viability of the cells, tested by
Trypan Blue exclusion, was always greater than 95%.
1
trans/cis 18d. H NMR (CDCl3): δ 7.56 (d, 1H, CHdCH, J )
16.0 Hz), 6.73 (s, 2H, aromatics), 6.31 (d, 1H, CHdCH, J ) 16.0
Hz), 4.77 (tt, 1H, CHO, J ) 10.8 Hz, J ) 4.4 Hz), 3.97-3.93 (m,
1H, CHOH), 3.87 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 2.69-2.51
(m, 2H, 2NCH), 2.28 (s, 3H, NCH3), 2.24-2.15 (m, 2H, CH2),
1.90-1.71 (m, 6H, 3CH2), 1.60-1.39 (m, 8H, 4CH2) ppm.
Compound 19a/b was obtained in the same way from 16a/b. Its
IR and 1H NMR spectra are consistent with the proposed structures.
3,4,5-Trimethoxybenzoic Acid 4-[tert-Butoxycarbonyl-(4-hy-
droxycyclohexyl)amino]cyclohexyl Ester (20a/b). To a solution of
260 mg (0.64 mmol) of 14a/b in 15 mL of THF cooled to 0 °C,
140 mg (0.64 mmol) of di-tert-butyldicarbonate, and 0.09 mL of
triethylamine were added. The mixture was maintained at room
temperature for 10 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. The crude product was then purified by flash
chromatography using CH2Cl2/abs EtOH/petroleum ether/NH4OH
340:65:60:8 as eluting system. Compound 20a/b (270 mg, 86%
yield) was obtained as an oil. IR (neat): ν 3370 (OH), 1706 (CO)
Modulation of Pirarubucin Uptake. Drugs and Chemicals.
Purified pirarubicin was provided by Laboratoire Roger Bellon
(France). Concentrations were determined by diluting stock solu-
tions to approximately 10-5 M and using ε480 ) 11500 M-1 cm-1
.
1
cm-1. H NMR (CDCl3): δ 7.28 (s, 1H, aromatic), 7.29 (s, 1H,
Stock solutions were prepared just before use. Buffer solutions were
HEPES buffer containing 5 mM HEPES, 132 mM NaCl, 3.5 mM
CaCl2, 5 mM glucose, at pH 7.3.
aromatic), 5.21 (bs, 0.5H, CHO), 4.99-4.78 (m, 0.5H, CHO), 3.89
(s, 9H, 3OCH3), 3.61-3.48 (m, 1H, CHOH), 2.28-1.25 (m, 28H,
8CH2, 3CH3, 2NCH and OH) ppm.
Cellular Drug Accumulation. The uptake of pirarubicin in cells
was followed by monitoring the decrease in the fluorescence signal
at 590 nm (λex ) 480 nm) according to the previously described
method.52 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 con-
ducted 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.
cis/trans-3,4,5-Trimethoxybenzoic Acid 4-(Methyl{4-[3-(3,4,5-
trimethoxyphenyl)acryloyloxy]cyclohexyl}amino)cyclohexyl Ester
(3a). Following the procedure described for compound 11, the acyl
chloride obtained from trans-3-(3,4,5-trimethoxyphenyl)acryloyl
acid (100 mg, 0.43 mmol) was allowed to react with 17a (120 mg,
0.28 mmol) for 3 h. 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 eluting system.
The title compound (43 mg, 30% yield) was obtained as an oil.
1
IR (neat): ν 1713 (CO) cm-1. H NMR (CDCl3): δ 7.59 (d, J )
15.7 Hz, 1H, CHdCH), 7.34 (s, 2H, aromatics), 6.76 (s, 2H,
aromatics), 6.34 (d, J ) 15.7 Hz, 1H, CHdCH), 5.21 (bs, 1H,
CHO), 4.80 (tt, J ) 10.2 Hz, J ) 4.4 Hz, 1H, CHO), 3.93 (s, 9H,
ATPase Activity. Western-Blot Analysis of Intestinal
Plasma Membrane Vesicles. Plasma membrane vesicles were
prepared from homogenates of rat small intestine mucosa by

816 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Martelli et al.
differential centrifugation.49 Samples of membrane vesicles in
loading buffer (1 part of XT reducing agent 20× and 5 parts of
XT sample buffer 4×, Bio-Rad), were boiled for 1 min, loaded
onto SDS-polyacrylamide electrophoresis gel (Criterion XT, Bio-
Rad), and separated by molecular size. Protein concentration was
determined by Bradford assay. The gels were then electroblotted
onto nitrocellulose membranes (Trans-Blot Transfer-Medium, Bio-
Rad). The surface of the membrane was blocked in TBS solution
(Tris-buffered saline and 0,1% Tween 20) containing 5% nonfat
milk. Primary antibodies were purchased from Alexis (C219: mouse
monoclonal antibody to P-glycoprotein, A23: rabbit polyclonal
antibody to Mrp1, M2III-6: mouse monoclonal antibody to MRP2).
Antibodies were diluted in 1% nonfat milk-TBS buffer and
incubated overnight at 4 °C. Goat antimouse IgG-HRP and goat
antirabbit IgG-HRP (Santa Cruz Biotechnology) were used as
secondary antibodies. Proteins were revealed for colorimetric
evaluation (Opti-4CN Substrate Kit, Bio-Rad).
ATPase activity was measured spectrophotometrically at 37 °C
using an ATP-regenerating system (pyruvate kinase and phospho-
enolpyruvate), coupled to lactate dehydrogenase and NADH, by
monitoring NADH absorbance decay with time at 340 nm.53,54
Sodium azide, EGTA, and ouabain were added to the reaction
medium to inhibit, respectively, H+-, Ca2+-, and Na/K-ATPases.
NADH absorbance decay with time curve followed a polynomial
equation and ATPase activity was calculated by the curve tangent
at 1300 s.
spectra, and elemental analyses of compounds 3-10. This material
is available free of charge via the Internet at http://pubs.acs.org.
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characteristics of compounds 3-10, 1H NMR signals at 400 MHz
1
of compounds 14a-d, 17a-d, 3a-d, 4a-d, IR and H NMR

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