1-[4-(2-Aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidin es: A novel series of highly potent revertants of P-glycoprotein associated multidrug resistance

Article abstract of DOI:10.1016/j.bmcl.2008.05.034

The 1-[4-(2-aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidin es 5-8 are a novel cluster of highly potent P-glycoprotein dependent multidrug resistance (MDR) revertants. Using a concentration of 4 μg/mL, these compounds possess 11-43 times the potency of verapamil in reversing MDR in murine L-5178 lymphoma cells transfected with the human MDR1 gene. Structure-activity relationships reveal that the attachment of the N-aroyl group to various 3,5-bis(benzylidene)-4-piperidones is essential for MDR reversal to occur. In terms of potencies, the 1-piperidinyl group is the preferred terminal amine while the 4-methyl and 4-chloro substituents are the optimal groups for placement in the benzylidene aryl rings.

Full text of DOI:10.1016/j.bmcl.2008.05.034

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3484–3487
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-[4-(2-Aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidines:
A novel series of highly potent revertants of P-glycoprotein associated
multidrug resistance
Umashankar Das a, Joseph Molnár b, Zoltán Baráth b, Zsuzsanna Bata c, Jonathan R. Dimmock a,
*
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Sask., Canada S7N 5C9
b Institute of Medical Microbiology and Immunology, University of Szeged, Szeged H-6720, Hungary
c Department of Dermatology, University of Szeged, Szeged H-6720, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The 1-[4-(2-aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidines 5–8 are a novel clus-
ter of highly potent P-glycoprotein dependent multidrug resistance (MDR) revertants. Using a concentra-
tion of 4 lg/mL, these compounds possess 11–43 times the potency of verapamil in reversing MDR in
murine L-5178 lymphoma cells transfected with the human MDR1 gene. Structure–activity relationships
reveal that the attachment of the N-aroyl group to various 3,5-bis(benzylidene)-4-piperidones is essential
for MDR reversal to occur. In terms of potencies, the 1-piperidinyl group is the preferred terminal amine
while the 4-methyl and 4-chloro substituents are the optimal groups for placement in the benzylidene
aryl rings.
Received 14 March 2008
Revised 6 May 2008
Accepted 9 May 2008
Available online 16 May 2008
Keywords:
Multidrug resistance
Structure–activity relationships
Physicochemical constants
N-Aroyl-3,5-bis(benzylidene)-4-piperidones
Ó 2008 Elsevier Ltd. All rights reserved.
The principal objective of our laboratory is finding chemical ap-
proaches to counteract the ravages caused by cancer. A recent
emphasis has been directed to finding compounds which reverse
the vexatious problem of multidrug resistance (MDR).1,2 The study
described herein reveals the remarkable potencies of a novel series
of P-glycoprotein (P-gp) associated MDR revertants namely the 1-
[4-(2-aminoethoxy)phenylcarbonyl]-3,5-benzylidene-4-oxo-
piperidines.
The problem of drug resistance occurs in many tumours and
leads to an increased drug efflux from the neoplasms causing de-
creased intracellular drug concentrations thereby reducing the
effectiveness of anticancer drugs. The mechanism of drug resis-
tance is multifactorial but principally it is due to the overexpres-
gp, inhibiting the efflux of the anticancer drugs from the tumours
and reducing the binding of cytotoxins to P-gp.8,9 In addition these
compounds may reduce P-gp synthesis and/or inhibit MDR gene
expression. A number of MDR modulators have pronounced bioac-
tivities of their own such as verapamil and cyclosporine A,10 which
limit their clinical usefulness while most inhibitors of MDR are
transporter substrates thus requiring high concentrations to over-
come MDR.11,12 To the best knowledge of the authors, these severe
limitations have resulted in there being no clinically available MDR
reversal agents to date and hence such medication is urgently
required.
The design of a novel cluster of MDR revertants was made as
follows. First, the common molecular features of a number of
MDR modulators are their hydrophobicity, having two aryl rings
and atoms capable of hydrogen bonding as well as bearing a posi-
tive charge at neutral pH.13,14 Second, in terms of specific groups to
be incorporated into the candidate MDR revertants, previous inves-
tigations from this laboratory revealed that various compounds
which contain the 1,5-diaryl-3-oxo-1,4-pentadienyl moiety re-
verse MDR.1,2 In addition, the 4-(2-aminoethoxy)phenyl substitu-
ent is present in the established MDR modulators amiodarone
sion of P-gp, which is
a member of the ABC (ATP-binding
cassette) family of transporters.3 P-gp is a 170 kDa membrane pro-
tein which behaves as a drug efflux pump with a very broad spec-
ificity, and appears to act from the intracellular leaflet.4 In humans,
this protein is encoded by the mdr1 and mdr3 genes. While the
MDR3 glycoprotein can bind to some of the substrates and inhibi-
tors of MDR,5 the low rate of this transport process usually makes
it undetectable.
A number of studies revealed the increased potencies of anti-
cancer drugs when administered with MDR revertants.6,7 These
chemosensitizers act in different ways, namely by binding to P-
1
15 and toremifene 2.16,17 These general and specific molecular fea-
tures were incorporated into the design of the candidate MDR rev-
ertants, whose general structure 3 is presented in Figure 1. The
decision was made to evaluate the effect of placing different R
groups in the arylidene aryl rings as well as varying the basic cen-
tre X. The compounds in series 5–9 were prepared by a previously
* Corresponding author. Tel.: +1 306 966 6331; fax: +1 306 966 6377.
E-mail address: jr.dimmock@usask.ca (J.R. Dimmock).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.034

U. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3484–3487
3485
Table 1
CH3
MDR-reversing properties in murine L-5178Y/MDR1 lymphoma cells and logP values
of the compounds in series 4–9 and verapamil
I
CH3
CH3
CH3
CH3
O
O
N
N
FAR valuea
logPb
O
Cl
Compound
O
I
4 lg/ml
40 lg/ml
2
1
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
1.58
3.26
1.25
1.04
45.5
83.7
98.0
63.0
49.5
106
118
77.5
123
179
157
145
48.5
95.8
76.0
63.3
1.37
1.93
1.21
1.03
4.2
2.00
4.03
0.91
0.80
51.6
3.38
4.25
4.71
3.27
4.60
5.50
5.95
4.52
5.35
6.25
6.71
5.27
5.51
6.40
6.86
5.42
4.64
5.34
5.80
4.36
1.37
2.27
2.73
1.30
4.55
The 1,5-diaryl-3-oxo-
1,4-pentadienyl group
O
N
R
R
c
R1
R1
—
O
98.9
3.23
23.8
66.6
99.7
14.1
136
146
133
51.5
63.3
124
6.88
10.9
1.05
114
1.85
1.22
N
O
3
Protonable
nitrogen atom
Atoms capable of
hydrogen bonding
Figure 1. The structures of amidarone 1, toremiphene 2 and the general formula 3
of the candidate MDR revertants prepared in this study. The groups within the
dotted lines in structures 1–3 are the common molecular features of some MDR
revertants.
reported methodology18 and the synthetic chemical route is sum-
marized in Figure 2. Since the 4-piperidones 4a–d possess some of
the molecular features found in series 5–9, their assessment for
MDR reversal properties was undertaken.
9d
Verapamil
d
—
The compounds were assessed for MDR revertant properties
using a literature method.19 This assay employed murine L-5178
lymphoma cells transfected with the human MDR1 gene and in
these cells the levels of P-gp are substantially higher than in the
parental cells.20 The concentrations of rhodamine 123 were mea-
sured in treated and untreated transfected and parental cells and
the ratios of the fluorescence intensities are referred to as fluores-
cence activity ratio (FAR) values. Since MDR is due, inter alia, to an
increase in the efflux of a compound from cells, a FAR value of
greater than 1 indicates that reversal of MDR has taken place. All
of the compounds were assessed using concentrations of 4 and
40 lg/mL and the data are presented in Table 1.
On a few occasions, the FAR values are lower at 40 lg/ml than
when the lower concentration is used. This observation has been
observed previously.1 A possible explanation for this phenomenon
is that when the concentration of a compound is elevated, although
binding to P-gp continues, other behavioural mechanisms are acti-
vated which expedite the extrusion of cellular contents. However
the FAR values using 4 lg/mL will be considered since, with the
exception of 8b and 9b, the higher concentration of compounds
did not lead to substantial increases in the FAR data. The following
observations were made from the MDR reversal experiments. First,
MDR-reversal was displayed by all of the compounds in series 5–8
and the FAR values of these compounds ranged from 11 (5a) to 43
(7b) times that of the established MDR-reversing agent verapamil.
These data clearly indicate that the incorporation of the 4-(2-
a
The fluorescence activity ratio (FAR) values are the ratios of the fluorescent
intensities of rhodamine 123 in treated and untreated murine L-5178Y cells
transfected with human MDR1 gene.
b
The logP values are of the unprotonated molecules and were determined using
a commercial software package.30
c
A concentration of 40 lg/ml of 5b was toxic to the cells.
Limitations of solubility precluded an assessment of verapamil at 40 lg/mL. The
d
FAR value of this compound at 10 lg/mL is 5.61.
aminoethoxy)phenyl carbonyl group into series 4 leading to series
5–8 generated a novel class of potent MDR-reversal agents. In gen-
eral, the quaternary ammonium compounds 9 were inactive in this
bioassay. Second, the optimal basic centre in series 5–8 was con-
sidered and the data presented in Table 1 reveals that the presence
of a 1-piperidino group in series 7 is greatly preferred. Thus the
average FAR values for the compounds in series 5–8 are 72.6,
87.8, 151 and 70.9, respectively. The pKa values of dimethylamine,
diethylamine, piperidine and morpholine are 10.73, 10.84, 11.12
and 8.50, respectively,21 revealing that under the conditions of
the bioassay, all of these compounds exist principally as ions.22
The solvent-accessible surface area (SASA) figures for the proton-
ated dimethylamino, diethylamino, piperidino and morpholino
species are 183.8, 233.0, 234.6 and 224.1, respectively.23 Thus
apart from the smaller SASA figure for the dimethylamino group,
neither the basicity nor the size of the amino group appears to gov-
O
COCl
O
O
i, ii
+
R
N
H
R
R
N
R
HCl
X
O
4a-d
X
O
5a-d: X = N(CH3)2 HCl;
6a-d: X = N(C2H5)2 HCl;
8a-d: X =
7a-d: X =
HCl;
N
HCl;
N
O
I-
iii
9a-d: X = N(CH3) (C2H5)2+
Figure 2. Synthesis of the compounds 5–9. The reaction conditions are as follows: i—CH2Cl2/N(C2H5)3, ii—HCl/ (CH3)2CHOH; iii—a, K2CO3; b, CH3I/CH3COCH3. The nature of
the R groups in series 4–9 are as follows, namely a: R = H; b: R = CH3; c: R = Cl; d: R = NO2.

3486
U. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3484–3487
ern the magnitude of the FAR values. However the biodata gener-
ated suggest that development of analogs of series 7 should take
place in which the terminal basic group is a heterocycle containing
one nitrogen atom such as hexamethyleneimine or heptamethyle-
neimine. Third, the substituents in the arylidene aryl rings were
examined in terms of MDR-reversing potencies. In each of the ser-
ies 5–8, greater potencies were noted with the compounds bearing
4-methyl and 4-chloro substitutents. This observation may have
been due to the p and molecular refractivity (MR) values of these
groups. The p constants for hydrogen, methyl, chloro and nitro
group are 0.00, 0.56, 0.71 and À0.28, respectively,24 indicating
the greater hydrophobicity of the methyl and chloro substituents.
The MR figures of hydrogen, methyl, chloro and nitro groups are
1.03, 5.65, 6.03 and 7.36, respectively,24 suggesting that an optimal
MR value may have been reached while smaller and larger MR fig-
ures detract from MDR-reversing properties. Thus groups which
are lipophilic and have MR values of approximately 5–6 should
be placed in the arylidene aryl rings, for example, the trifluoro-
methyl substituent has p and MR values of 0.88 and 5.02,
respectively.24
A number of studies revealed the high lipophilicity of various
MDR-reversal agents25,26; for example, the calculated logP values
of a number of 1,4-dihydropyridines which reverse MDR were in
the region of 5.1–7.5.27 The calculated logP values of the com-
pounds in series 4–9 and verapamil are presented in Table 1. The
average logP figures for the compounds in series 4–9 are 3.90,
5.14, 5.90, 6.05, 4.99 and 1.92, respectively. Thus the compounds
displaying significant capabilities in reversing MDR, namely series
5–8, have logP values in the region of 5–6. On the other hand, the
compounds in series 4 and 9, which are virtually bereft of anti-
MDR properties, have lower logP values. However, linear and log-
arithmic plots between the FAR values and the logP figures of the
compounds in series 5–8 did not reveal any correlation (p > 0.05).
The potential of these compounds as MDR revertants will be en-
hanced if bioactivity is displayed at lower concentrations than
4 lg/ml and reversal of MDR occurs in a different species and an-
other neoplastic disease. Consequently, the most potent compound
identified in this study, namely 7b along with its analog which is
bereft of a N-acyl group viz. 4b was evaluated further. These results
are presented in Table 2. A concentration of 0.4 lg/mL of 7b but not
4b demonstrated MDR-revertant properties using L5178/MDR1
cells. In addition, 7b reverses MDR in Colo 320/MDR1 cells while
4b displays marginal potencies. These data confirm the relative
potencies of 4b and 7b and in particular emphasize the potential
of 7b as a MDR revertant.
The MDR modulators are believed to bind to the transmem-
brane domains of P-gp which leads to inhibition of ABC transport-
ers due to the induced conformational changes.25 The functionally
active conformation of P-gp depends on the integrity of the mem-
brane bilayer in which P-gp is embedded. Due to the existence of
12 transmembrane domains of P-gp, the co-crystallization of
MDR inhibitors and P-gp is not possible thereby precluding direct
evidence of ligand binding.
A final consideration which demonstrates the importance of
this seminal work is the very recent disclosure of the excellent tol-
erability of the compounds in series 4–8.29 Thus doses of up to and
including 300 mg/kg of each of these compounds did not induce
lethalities in a short-term toxicity study in mice. A few compounds
were examined at lower doses in rats which also did not induce
mortalities. However, most of the compounds in series 9 causing
deaths in some of the mice and bearing in mind the lack of MDR
revertant properties in general, suggest that formation of further
quaternary ammonium analogs of 9 is not pursued.
In conclusion, this study has identified a novel class of P-gp
associated MDR-reversal agents 5–8. These compounds demon-
strate high potencies which far exceed that of a reference drug
verapamil. Optimal structural fragments in terms of potencies
are the 1-piperidinyl group as the terminal base and 4-methyl
and 4-chloro substituents are present in the arylidene aryl rings.
Series 4 has the general molecular features of various MDR rever-
tants, as well as possessing the 1,5-diaryl-3-oxo-1,4-pentadienyl
group. However, the absence of the 4-(2-aminoethoxy)- phenylcar-
bonyl group attached to the piperidyl nitrogen atom resulted in
compounds displaying a lack of MDR reversal. Development of
one or more of these molecules may produce a single drug candi-
date to treat P-gp mediated MDR cancers and structural modifica-
tions can be undertaken in the future in order to increase potencies
still further. In this regard, various guidelines for amplifying the
project have been obtained based on the physicochemical proper-
ties of these molecules.
Acknowledgement
The authors thank the Canadian Institutes of Health Research
for an operating Grant to J.R. Dimmock. Support was also provided
by the Szeged Foundation of Cancer Research, Hungary to J.
Molnár.
References and notes
A further issue to be resolved is whether the N-acylpiperidones
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were assayed for cytocidal activity towards L5178Y/MDR1 cells.28
The ID50 figures for 6a–c are 3.27, 1.56 and 1.53 lg/mL, respec-
tively, while for 7a–d, the relevant values are 3.28, 2.83, 4.17 and
2.15 lg/mL, respectively. Verapamil has an ID50 figure of 42 lg/
mL. This observation emphasizes further the importance of devel-
oping these cytotoxins, which possess MDR-revertant properties.
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Table 2
Evaluation of 4b and 7b for MDR-revertant properties using murine L5178Y/MDR1
and human colo320/MDR1 cells
Compound
FAR value
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L5178Y/MDR1 cells
colo320/MDR1 cells
0.4 lg/mLa 4 lg/mLb
2.42
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a
Verapamil is inactive using this concentration.
The FAR value of verapamil is 3.84 using a concentration of 10 lg/mL.
b

U. Das et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3484–3487
3487
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activity of 3a–c and 4a–d towards L5178Y/MDR1 was accomplished by a
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experiments. In brief, cells were incubated with varying concentrations of the
compounds at 37 °C for 48 h and the ID50 values were determined by the MTT
method..
solution of the test compound (2 mg/ml, 10 ll) in dimethylsulfoxide was added
to aliquots of the cell suspension and incubated at room temperature for 10
min. Then 10 ll of rhodamine 123 in dimethylsulfoxide was added so that its
final concentration was 5.2 lM and the cells were incubated for a further 20
min at 37 °C. The cells were washed twice and resuspended in PBS (pH 7.4)
after which the fluorescence was measured using a Beckton Dickinson FACScan
instrument. The fluorescence of the cells was measured in treated MDR cells
(F1), untreated MDR cells (F2), treated parental cells (F3) and untreated
parental cells (F4) and the FAR values were obtained from the following
equation, viz. F1/ F2 over F3/ F4. The colo320/MDR1 cells were obtained from
the ATCC (ATCC #CCL-220.1). The colo 320 assay was undertaken in a similar
manner except that prior to flow cytometry, the cells which form a monolayer
were treated with 0.25% trypsin solution for 2–3 min in order to produce a
suspension of individual cells..
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