Isomeric N,N-bis(cyclohexanol)amine aryl esters: The discovery of a new class of highly potent P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors

Article abstract of DOI:10.1021/jm0614432

A new series of P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine scaffold have been designed, following the frozen analog approach. With respect to the parent flexible molecules, the new compounds sh

Full text of DOI:10.1021/jm0614432

J. Med. Chem. 2007, 50, 599-602
599
Chart 1. General Structures of Designed Compounds
Isomeric N,N-Bis(cyclohexanol)amine Aryl
Esters: The Discovery of a New Class of Highly
Potent P-Glycoprotein (Pgp)-dependent
Multidrug Resistance (MDR) Inhibitors
Elisabetta Teodori,*,† Cecilia Martelli,† Milena Salerno,‡
Nacira Darghal,‡ Silvia Dei,† Arlette Garnier-Suillerot,‡
Fulvio Gualtieri,† Dina Manetti,† Serena Scapecchi,† and
Maria Novella Romanelli†
Dipartimento di Scienze Farmaceutiche, UniVersita` di Firenze, Via
U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy, and Laboratoire
BioMoCeTi UMR 7033, UniVersite´ Paris 13, 74 rue Marcel Cachin,
93017 Bobigny, France
if, so far, no drug has been approved for therapy.17 Furthermore,
a potential use of these agents may be to increase drug
penetration to biologically important protective barriers, such
as the blood-brain and blood-cerebrospinal fluid.18 The main
problems associated with the development of effective Pgp-
mediated MDR inhibitors seem due to poor specificity, low
potency, interference with physiological functions, and, as a
consequence, interference with the pharmacokinetics of the
chemotherapic used.
Very recently, we have described a new family of MDR
reverters, endowed with fairly good potency, designed on the
basis of a new concept. In brief, given the properties of the
Pgp recognition site described above, 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
because flexible molecules can optimize their interaction within
the recognition site.19
We are still building on this idea and further studies are in
progress. At the same time, having in mind possible therapeutic
applications, we decided to verify the consequences 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,20,21
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 a previous series (1,19 2)
was substituted with N,N-bis-cyclohexylamine (3, 4) (Chart 1).
The aromatic moieties selected were those that gave the best
results in the linear series. The scaffold chosen may give origin
to four geometrical isomers of quite different shape that
somehow represent restricted conformation analogs of the parent
linear compounds 1 and 2. According to the frozen analog
approach,22 they can provide further insight on the characteristics
of the recognition site. In the present paper, we describe a set
of such derivatives that are fairly potent inhibitors of Pgp-
dependent MDR, reaching unprecedented low-nanomolar activ-
ity.
ReceiVed December 18, 2006
Abstract: A new series of P-glycoprotein (Pgp)-dependent multidrug
resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine
scaffold have been designed, following the frozen analog approach.
With respect to the parent flexible molecules, the new compounds show
improved potency and efficacy. Among them, compound 1d, on
anthracycline-resistant erythroleukemia K562 cells, is able to completely
reverse Pgp-dependent MDR at low nanomolar concentration.
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 drug as a conse-
quence of the overexpression of proteins such as ABCB1 (P-
glycoprotein, Pgp) and ABCC1 (multidrug resistance associate
protein 1, 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 pumps that use a variety
of energy sources are present in mammals and microorgan-
isms.1,4 In mammals, besides cancer cells, these proteins have
been found in several important tissues and blood-tissue
barriers where they apparently regulate the secretion of lipophilic
molecules and the extrusion of xenobiotics that enter the
organism.5
Direct information on the structure of Pgp and MRP1 is
scarce,6,7 but resolution of the structure of homologous bacterial
transporters8 has opened the way to the development of
homology models9,10 and provided many useful details on the
structure of the recognition site of ABC transporters. According
to these findings, the recognition sites appear to be large,
flexible, and rich in amino acids able to establish a variety of
interactions with substrates, in particular π-π, ion-π, hydrogen
bond, and hydrophobic interactions.11,12 All information col-
lected 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.13
Inhibition of the functions of Pgp and sister proteins is
considered a suitable approach to circumvent MDR and drugs
possessing inhibitory properties have been and are actively being
sought (reviewed by Teodori,14 Avendano,15 and Robert16), even
The reaction pathway used to synthesize the desired com-
pounds (3a-d and 4a-d) is presented in Scheme 1, and their
chemical and physical characteristics are reported in Table S1
(Supporting Information).
The 4-oxocyclohexyl ester 5 was obtained by esterification
of 4-hydroxycyclohexanone23 with 3,4,5-trimethoxybenzoyl
chloride. Compounds 6 were then obtained by reductive
amination of compound 5 with trans-4-aminocyclohexanol or
* To whom correspondence should be addressed. Phone: 39 055
4573693. Fax: 39 055 4573671. E-mail: elisabetta.teodori@unifi.it.
† Universita` di Firenze.
‡ Universite´ Paris 13.
10.1021/jm0614432 CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/26/2007

600 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Letters
Scheme 1a
a Reagents and conditions: (i) CHCl3, 3,4,5-trimethoxybenzoyl chloride; (ii) trans-4-aminocyclohexanol, (iPrO)4Ti, NaBH3CN; (iii) cis-4-aminocyclohexanol,
(iPrO)4Ti, NaBH3CN; (iv) chromatographic separation; (v) HCOOH/HCHO; (vi) Ar2COCl, CHCl3. For the meaning of Ar1and Ar2, see Table 1.
Table 1. 1H NMR Signals at 400 MHz
axial
H1
equatorial
H1
axial
H1′
equatorial
compd
6a
R
Y
H
H1′
H4
H4′
H
δ ) 5.12a
δ ) 3.60a
Jaa ) 10.6b
Jae ) 4.0b
δ ) 3.62a
Jaa ) 10.6b
Jae ) 4.0b
δ ) 2.71a,c
Jaa ) 9.6b
Jae ) 3.2b
δ ) 2.66a,c
Jaa ) 11.2b
δ ) 2.59a,c
Jaa ) 10.8b
Jae ) 3.6b
δ ) 2.60a,c
Jaa ) 10.8b
cis/trans
w/2 ) 15.6b
6b
H
H
δ ) 4.92a
Jaa ) 10.8b
Jae ) 4.4b
trans/trans
6c
cis/cis
6d
H
H
H
H
δ ) 5.15a
δ ) 3.94a
δ ) 2.81-2.70a,d
w/2 ) 11.8b
w/2 ) 10.9b
δ ) 3.94a
δ ) 4.93a
Jaa ) 10.8b
Jae ) 4.4b
δ ) 2.80-2.72a,d
trans/cis
w/2 ) 10.9b
7a
cis/trans
7b
trans/trans
7c
cis/cis
7d
trans/cis
3a
cis/trans
CH3
CH3
CH3
CH3
CH3
H
H
H
H
B
δ ) 5.18a
δ ) 3.61-3.51a
w/2 ) 26.7b
δ ) 2.69-2.51a,d
δ ) 2.70-2.50a,d
δ ) 2.79-2.62a,d
δ ) 2.77-2.56a,d
δ ) 2.70-2.52a,d
w/2 ) 13.3b
δ ) 4.95-4.78a
w/2 ) 26.4b
δ ) 3.61-3.55a
w/2 ) 25.7b
δ ) 5.19a
δ ) 3.96a
w/2 ) 12.1b
w/2 ) 10.9b
δ ) 3.97a
δ ) 4.93-4.81a
w/2 ) 25.5b
w/2 ) 11.1b
δ ) 5.21a
δ ) 4.80a
Jaa ) 10.2b
Jae ) 4.4b
δ ) 4.80a
Jaa ) 10.2b
Jae ) 4.4b
w/2 ) 10.2b
3b
CH3
B
δ ) 4.92a
Jaa ) 9.2b
Jae ) 4.4b
δ ) 2.75-2.52a,d
trans/trans
3c
cis/cis
3d
trans/cis
4a
cis/trans
4b
CH3
CH3
CH3
CH3
B
B
C
C
δ ) 5.22a
w/2 ) 9.1b
δ ) 5.11a
w/2 ) 10.7b
δ ) 5.11a
w/2 ) 9.6b
δ ) 2.85-2.58a,d
δ ) 2.88-2.58a,d
δ ) 2.76-2.55a,d
δ ) 2.75-2.68a,d
δ ) 4.95-4.85a
w/2 ) 21.6b
δ ) 5.18a
δ ) 5.38-5.26a
w/2 ) 24.1b
w/2 ) 11.2b
δ ) 4.92a
Jaa ) 10.4b
Jae ) 4.4b
δ ) 5.38-5.25a
w/2 ) 26.4b
trans/trans
4c
cis/cis
4d
CH3
CH3
C
C
δ ) 5.17a
δ ) 5.60a
w/2 ) 9.1b
δ ) 5.60a
w/2 ) 11.4b
δ ) 2.76-2.58a,d
δ ) 2.76-2.58a,d
w/2 ) 10.8b
δ ) 4.91-4.82a
w/2 ) 25.4b
trans/cis
a Parts per million. b Hertz. c The attribution of H4 and H4′ signal can be interchanged. d Superimposed signals.
cis-4-aminocyclohexanol,24 using titanium(IV) isopropoxide as
Lewis acid catalyst and NaBH3CN as reducing agent, according
to the Mattson procedure.25 The secondary amines obtained are
an approximately 1:1 mixture of cis/trans and trans/trans
isomers or cis/cis and trans/cis isomers, respectively, as results
from H NMR spectra.
A chromatographic separation was performed on the amines
6, and the pure isomers 6a, 6b, 6c, and 6d were obtained. Their
configuration was attributed on the basis of the 1H NMR
characteristics of the cyclohexane protons at C1, C4 and C1′,
C4′ (Table 1). 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)26-28 allows confident attribution of their
equatorial or axial nature. Moreover the chemical shift of the
signal is diagnostic, because axial protons resonate at higher
1

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 601
Table 2. MDR-Reverting Activity of Compounds 3a-d and 4a-d
bodimmide hydrochloride (EDAC) as the coupling reagent (see
Supporting Information).
The information obtained from 1H NMR on preferred
conformations was confirmed by molecular modeling (Figure
S1, see Supporting Information) where compounds 3a-d are
shown in their lowest energy conformation with the 3,4,5-
trimethoxybenzoate group in axial position. The lowest energy
conformations of the set of isomers 4 are practically identical.
The ability of compounds 3a-d and 4a-d to revert MDR
was evaluated on anthracycline-resistant erythroleukemia K562
cells, measuring the uptake of pirarubicin by continuous
spectrofluorometric signal of the anthracycline at 590 nm (λex
) 480 nm) after incubation of the cells, following previously
reported protocols.27,29,30 MDR-reverting activity is described
by (i) R, which represents the fold increase in the nuclear
concentration of pirarubicin in the presence of the MDR-
reverting agent and varies between 0 (in the absence 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 MDR-modulator and is the maximum
increase that can be obtained in the nuclear concentration of
pirarubicin in resistant cells with a given inhibitor; and (iii) [I]0.5,
which measures the potency of the MDR-reverting agent and
represents the concentration of the inhibitor that causes a half-
maximal increase (R ) 0.5) in nuclear concentration of
pirarubicin (see Table 2). Even if binding data on Pgp are not
available at the moment, this test indicates that active com-
pounds inhibit the Pgp-operated extrusion of the reporter
molecule pirarubicin, as does the reference molecule verapamil.
The molecules studied in this work did not show any detectable
antiproliferative activity at the doses used in the test.
b
compd
3a
cis/trans
3b
trans/trans
3c
cis/cis
3d
trans/cis
4a
cis/trans
4b
trans/trans
4c
cis/cis
4d
structure
II
Ar1
a
Ar2
b
[I]0.5 (µM)a
Rmax
0.09 ( 0.015
0.85
0.81
0.80
0.98
0.95
0.88
0.79
0.78
II
II
II
II
II
II
II
a
a
a
a
a
a
a
b
b
b
c
c
c
c
0.32 ( 0.10
0.03 ( 0.01
0.01 ( 0.001
0.30 ( 0.06
0.30 ( 0.06
0.18 ( 0.03
0.16 ( 0.04
trans/cis
1
2
verapamil
MM36
I
I
a
a
b
c
0.60 ( 0.15c
0.28 ( 0.05
1.60 ( 0.03
0.05 ( 0.01d
0.90
0.71
0.70
0.70
a Concentration of the inhibitor that causes a 50% increase in nuclear
concentration of pirarubicin (R ) 0.5). b Efficacy of MDR modulator and
maximum increase that can be obtained in the nuclear concentration of
pirarubicin in resistant cells. c See ref 19. d See ref 30.
The results obtained on anthracycline-resistant erythroleuke-
mia K562 cells are reported in Table 2, together with those of
the parent linear compounds 119 and 2 (Chart 1) and of MM3630
and verapamil used as reference compounds.
fields, while the corresponding equatorial ones are de-
shielded.
The isomers 6a and 6c present the C1 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 6b and 6d
present the C1 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 6a and 6b present the
C1′ proton with axial characteristics (δ ) 3.60 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 6c and 6d present the C1′
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 charac-
teristics in every isomer of 6 (see Table 1); this indicates that
the two cyclohexane rings are in cis/trans (6a), trans/trans (6b),
The new molecules are all potent and efficacious inhibitors
of MDR and, in general, present improved potency and efficacy
with respect to the parent flexible compounds. However, some
interesting differences can be observed between the two sets of
isomers. The four isomers of 3 are all more potent than the
flexible parent compound 1. As far as isomers 4 are concerned,
the improvement with respect to the flexible compound 2 is
less impressive and, actually, regards only two isomers.
However, the trend of potencies is very much the same in the
two sets, where the isomers with cis/cis and trans/cis config-
uration are the most potent, indicating that such configurations
allow optimal binding to the recognition site. 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), which produced very effective Pgp inhibitors in flexible
molecules (see compound 2, but also the analogous compounds
reported in the previous exploratory work19), appears to be less
effective than the trans-3,4,5-trimethoxycinnamic one (set 3)
in rigidified structures.
1
cis/cis (6c), and trans/cis (6d) configurations. Therefore, H
NMR data show that 6a and 6c are frozen in a conformation
having the bulky susbstituent in position 1 forced into an axial
position, while 6b and 6d have the same group in the more
relaxed equatorial position as shown in Table 1.
Reductive methylation of 6a-6d with HCOOH/HCHO gave
the corresponding tertiary amines 7a-7d. Final compounds
3a-d and 4a-d were then obtained by reaction of 7a-d with
1
the proper acyl chloride. The H NMR data reported in Table
1 show that the configurations of 6a-d are maintained in
compounds 7a-d and then in the final products 3a-d and 4a-
d, ruling out any isomerization in the subsequent reactions.
Compound 2 (Chart 1) was synthesized for comparative
purposes by reaction of anthracene-9-carboxylic acid 3-(3-
hydroxypropyl-methylamino)propyl ester19 with 3,4,5-trimethoxy-
benzoic acid using 1-(3-dimethylamino-propyl)-3-ethylcar-
Therefore, modulation of molecular flexibility and a careful
choice of the aromatic moieties can give compounds possessing
very interesting Pgp inhibitory properties. As a matter of fact,
compound 3d, which shows low nanomolar potency ([I]0.5
)
0.01 µM) and is able to completely reverse Pgp-dependent MDR
(R ) 0.98) in anthracycline-resistant erythroleukemia K562
cells, is a very interesting compound that deserves further

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JM0614432