Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters: Further restriction of molecular flexibility maintains high potency and efficacy

Article abstract of DOI:10.1016/j.bmcl.2010.11.059

Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a-d) that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5- trimethoxyphenyl)acrylic acid present in set 1 was

Full text of DOI:10.1016/j.bmcl.2010.11.059

Bioorganic & Medicinal Chemistry Letters 21 (2011) 106–109
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR)
by N,N-bis(cyclohexanol)amine aryl esters: Further restriction
of molecular flexibility maintains high potency and efficacy
a
a
b
a
a
Cecilia Martelli , Silvia Dei , Catherine Lambert , Dina Manetti , Francesca Orlandi ,
a
a
b
a,
⇑
Maria Novella Romanelli , Serena Scapecchi , Milena Salerno , Elisabetta Teodori
Dipartimento di Scienze Farmaceutiche, Università di Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
a
b
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:
Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a–d)
that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5-tri-
methoxyphenyl)acrylic acid present in set 1 was substituted with 3-(3,4,5-trimethoxyphenyl)propanoic
and 3-(3,4,5-trimethoxyphenyl)propiolic moieties to give sets 2 and 3, respectively. While the introduction
of 3-(3,4,5-trimethoxyphenyl)propanoic moiety resulted in a definite drop in potency and efficacy, esteri-
fication with 3-(3,4,5-trimethoxyphenyl)propiolic acid gave four isomers (3a–d) that maintain high
potency and possess optimal efficacy. These results are discussed in terms of conformational flexibility of
the different sets of compounds.
Received 7 October 2010
Revised 15 November 2010
Accepted 15 November 2010
Available online 20 November 2010
Keywords:
MDR reverters
Pgp inhibitors
N,N-Bis(cyclohexanol)amine aryl esters
Chemosensitizers
Ó 2010 Elsevier Ltd. All rights reserved.
Doxorubicin-resistant erythroleukemia
K562 cells (K562/DOX)
Pirarubicin uptake
Drug transporter proteins are fundamental to the functions of
living organisms, being involved in the absorption, translocation,
secretion, and excretion of a variety of endogenous and xenobiotic
molecules. Because of their physiological role, these proteins can
modulate pharmacokinetic and toxic properties of nutrients and
isms to various chemotherapeutic drugs that usually are structur-
ally and mechanistically unrelated.
5
,6
Even if there is no high-resolution crystal structure of human
Pgp, homology models, build upon the resolved structure of other
7
,8
9,10
related proteins such as Sav 1866 and murine Pgp
integrated
1
drugs.
by the information obtained from other bacterial proteins such as
1
1,12
The ABC superfamily of multidrug transporter proteins is one of
the largest in living organisms and its members are involved in
several fundamental cellular processes.2 ABCB1 (Pgp) is the most
intensively studied member of the family since it has been found
in several important tissues and blood–tissue barriers where, to-
gether with other family members, it seems to regulate the secre-
QacR
polymorphous drug binding domain
cules can accommodate in a plurality of binding modes including
, ion– , hydrogen bonds and hydrophobic interactions.
In principle, modulation of the functions of Pgp and like pro-
suggest that Pgp recognition site is characterized by a
1
3,14
where a variety of mole-
p–p
p
teins would be one way of circumventing the appearance of MDR
3
tion of physiologically important lipophilic molecules and the
in cancer cells. This is the main reason prompting the design and
4
15–19
extrusion of xenobiotics that enter the organism. Overexpression
synthesis of Pgp inhibitors,
although thus far, no drug of this
2
0
of Pgp and related proteins (mainly ABCC1 and ABCG2) that act as
extrusion pumps, can induce classical multidrug resistance (MDR),
a kind of acquired drug resistance of cancer cells and microorgan-
class has been approved for cancer therapy. However other po-
tential uses of these agents are emerging, such as that of enhancing
drug penetration through biologically protective barriers, such as
2
1,22
the blood–brain and blood–cerebrospinal fluid.
Recent evi-
dence indicates that Pgp plays a role in the inhibition of viral infec-
2
3
tivity of the human immunodeficiency virus (HIV). Recently, the
important role of ABC transporter proteins like Pgp in stem cells
has been evidenced. Finally, the increasing interest in the func-
Abbreviations: EDCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimmide hydro-
chloride; DMAP, 4-dimethylaminopyridine; DOX, doxorubicin.
24
⇑
Corresponding author. Tel.: +39 055 4573693; fax: +39 055 4573697.
E-mail address: elisabetta.teodori@unifi.it (E. Teodori).
tions and mechanism of action of Pgp and sister proteins requires
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.059

C. Martelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 106–109
107
the availability of new and potent molecules to be used as pharma-
cological tools.
The synthesis of compounds 1a–d has been previously re-
2
5
ported. Compounds 2a–d and 3a–d were obtained following
the reaction pathways described in Scheme 1, starting from inter-
mediates 4a–d whose synthesis has been previously described as
well as their configuration that was attributed on the basis of the
In the search for efficient MDR reverters, we designed a new
2
5
family of compounds on the basis of the characteristics of the sub-
strate recognition sites of Pgp, MRP1 and similar proteins, discussed
above. Structurally, the compounds designed are N,N-bis(cyclohex-
anol)aminearyl esters formedby a scaffold where a basiclinker teth-
ers two aromatic moieties. Some of these novel derivatives showed
low-nanomolar potency and high efficacy in inhibiting Pgp-depen-
dent nuclear pirarubicin efflux in doxorubicin-resistant erythroleu-
kemia K562 cells as well as rat intestinal mucosa ATPase activity
mostly referable to MRP1 and in increasing the cytotoxicity of doxo-
rubicin towards doxorubicin-resistant erythroleukemia K562
cells.2
1
25 1
H NMR characteristics of the cyclohexane protons.
H NMR
spectra indicate that 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.
Final compounds 2a–d and 3a–d were obtained by reaction of
4a–d with 3-(3,4,5-trimethoxyphenyl)propanoyl chloride or with
2
6
3-(3,4,5-trimethoxyphenyl)propiolic acid
in the presence of
hydrochloride
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
5
(EDCl) and 4-dimethylaminopyridine (DMAP). Chemical and phys-
ical characteristics of the new compounds are reported in
Supplementary data (Table 1).
The scaffold of N,N-bis(cyclohexanol)amine aryl esters is fairly
rigid but the aryl substituents maintain some flexibility and we
decided to evaluate the consequences of further conformational
modulation of the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid
moiety in the most active members of the series, the 1a–d set of
isomers (Chart 1). Therefore we synthesized the 2a–d and 3a–d
sets of isomers where trans-3-(3,4,5-trimethoxyphenyl)acrylic acid
is substituted by 3-(3,4,5-trimethoxyphenyl)propanoic acid or
The 1H NMR data reported in Supplementary data (Table 2)
show that the configurations of 4a–d are maintained in the final
products 2a–d and 3a–d, ruling out any isomerization in the sub-
sequent reactions.
Modulation of pirarubicin uptake. The ability of isomers of sets
1–3 to modulate Pgp action was evaluated on doxorubicin-resis-
tant erythroleukemia K562 cells (K562/DOX) that, as reported in
3
-(3,4,5-trimethoxyphenyl)propiolic acid (Chart 1). Unfortunately,
2
7,28
we were unable to obtain the set of isomers esterified with
cis-3-(3,4,5-trimethoxyphenyl)acrylic acid since the compounds
rapidly isomerize to the more stable trans isomers.
the literature, overexpress only Pgp.
K562 is a human leukemia
cell line established from a patient with chronic myelogeneous leu-
2
9
kemia in blast transformation. K562/DOX cells resistant to doxo-
rubicin express a unique membrane glycoprotein with a molecular
3
0
mass of 180,000 Da. We measured the uptake of THP-adriamycin
pirarubicin) by continuous spectrofluorometric signal of the
(
CH3
OCH3
anthracycline at 590 nm (kex = 480 nm) after incubation of the
H CO
cells, following the protocols reported in previous Letters.3
1–34
N
3
Pgp-blocking activity is described by: (i)
a, which represents the
O
O
Ar
H CO
3
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
O
O
1
2
3
a-d: Ar = A
a-d: Ar = B
a-d: Ar = C
cells is the same as in sensitive cells); (ii) amax, which expresses the
Ar =
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
OCH3
OCH3
OCH3
H CO
H CO
H CO
3
3
3
H CO
H CO
3
H CO
3
causes a half-maximal increase (
tion of pirarubicin (see Table 1).
a = 0.5) in the nuclear concentra-
3
A
B
C
The results are reported in Table 1, together with those of com-
2
5
Chart 1. General structures of designed compounds.
pounds 1a–d, MM36, the most potent compound that we have
CH3
CH3
OCH3
OCH3
N
N
H CO
3
H3CO
O
OH
O
OH
H CO
3
4a, 4b
H3CO
4c, 4d
O
O
a
a
CH3
OCH3
CH3
OCH3
N
H CO
3
H CO
3
N
O
O
O
O
H CO
O
Ar
Ar
3
H CO
3
O
O
O
cis/cis
trans/cis
c
is/trans
trans/trans
Ar
N
Ar
Ar
N
Ar
N
N
B
C
2d
3d
B
C
B
C
2b
3b
B
C
2
c
2
3
a
a
3
c
Scheme 1. Synthesis of compounds 2a–d and 3a–d. Reagents and conditions: (a) ArCOCl, CHCl
3
or ArCOOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(
EDCl), 4-dimethylaminopyridine (DMAP); for the meaning of Ar see Table 1. Compounds 4a–d are described in Ref. 25.

1
08
C. Martelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 106–109
Table 1
MDR-reverting activity of compounds 1a–d, 2a–d, and 3a–d
CH3
OCH3
N
H CO
3
O
O
Ar
H CO
3
O
O
Ar =
OCH3
OCH3
OCH3
H CO
3
H CO
H CO
3
3
H CO
H CO
3
H CO
3
Figure 1. Overlay of 1d (black) with 2d (dark gray) and 3d (light gray).
3
A
B
C
moment, research is in progress to synthesize metabolically stable
ester bioisosteres.
[I]0.5a
(l
M)
a
max
b
Compound
Ar
The compounds of set 3, with their excellent efficacy are fairly
interesting and, together with those of set 1, have been selected
for further pharmacological studies. Preliminary results show that
the compounds of set 3 display very promising properties that will
be reported in due time.
1
1
1
1
2
2
2
2
3
3
3
3
a (cis/trans)
b (trans/trans)
c (cis/cis)
A
A
A
A
B
B
B
B
C
C
C
C
0.092 ± 0.015c
0.85
0.81
0.80
0.98
0.75
0.76
0.76
0.77
0.98
0.99
1
0.32 ± 0.10c
c
0.03 ± 0.01
d (trans/cis)
a (cis/trans)
b (trans/trans)
c (cis/cis)
d (trans/cis)
a (cis/trans)
b (trans/trans)
c (cis/cis)
0.012 ± 0.001c
0.63 ± 0.2
0.61 ± 0.18
1.10 ± 0.3
0.35 ± 0.1
0.08 ± 0.01
0.07 ± 0.01
0.07 ± 0.01
0.02 ± 0.002
0.05 ± 0.01d
1.60 ± 0.3
Acknowledgment
We are grateful to Professor Fulvio Gualtieri for helpful
discussion.
d (trans/cis)
1
0.70
0.70
MM36
Verapamil
Supplementary data
a
Concentration of the inhibitor that causes a 50% increase in nuclear concen-
tration of pirarubicin ( = 0.5).
Supplementary data (the synthesis of the reported compounds;
chemical and physical characteristics, IR and H NMR spectra and
a
1
b
Efficacy of MDR-modulator and maximum increase that can be obtained in the
nuclear concentration of pirarubicin in resistant cells.
elemental analyses of compounds 2a–d, 3a–d) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.11.059.
c
See Ref. 25.
d
See Ref. 33.
found in previous studies,33 and verapamil used as reference com-
pounds. These data show that reduction of the double bond in the
isomers 1a–d to give 2a–d is detrimental for both potency and effi-
cacy. The most potent isomers are in both sets the trans/cis ones,
but 2d is some 30 times less potent than 1d and, as far as efficacy
is concerned, the compounds of set 2 are definitely less efficacious
than those of set 1. On the contrary, the transformation of the dou-
ble bond of set 1 into the triple bond of set 3 leave nearly un-
changed the potency of the four isomers (the trans/cis still being
the most potent one) but definitely increase the efficacy, so that
all the isomers of set 3, under the conditions of our essay, are able
to completely restore the sensitivity of K562/DOX cells to
pirarubicin.
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