A New Class of 1-Aryl-5,6-dihydropyrrolo[2,1-a]isoquinoline Derivatives as Reversers of P-Glycoprotein-Mediated Multidrug Resistance in Tumor Cells

Article abstract of DOI:10.1002/cmdc.201800177

A number of aza-heterocyclic compounds, which share the 5,6-dihydropyrrolo[2,1-a]isoquinoline (DHPIQ) scaffold with members of the lamellarin alkaloid family, were synthesized and evaluated for their ability to reverse in vitro multidrug resistance in cancer cells through inhibition of P-glycoprotein (P-gp) and/or multidrug-resistance-associated protein 1. Most of the investigated DHPIQ compounds proved to be selective P-gp modulators, and the most potent modulator, 8,9-diethoxy-1-(3,4-diethoxyphenyl)-3-(furan-2-yl)-5,6-dihydropyrrolo[2,1-a]isoquinoline-2-carbaldehyde, attained sub-micromolar inhibitory potency (IC₅₀: 0.19 μm). Schiff bases prepared by the condensation of some 1-aryl-DHPIQ aldehydes with p-aminophenol also proved to be of some interest, and one of them, 4-((1-(4-fluorophenyl)-5,6-dihydro-8,9-dimethoxypyrrolo[2,1-a]isoquinolin-2-yl)methyleneamino)phenol, had an IC₅₀ value of 1.01 μm. In drug combination assays in multidrug-resistant cells, some DHPIQ compounds, at nontoxic concentrations, significantly increased the cytotoxicity of doxorubicin in a concentration-dependent manner. Studies of structure–activity relationships and investigation of the chemical stability of Schiff bases provided physicochemical information useful for molecular optimization of lamellarin-like cytotoxic drugs active toward chemoresistant tumors as well as nontoxic reversers of P-gp-mediated multidrug resistance in tumor cells.

Full text of DOI:10.1002/cmdc.201800177

Accepted Article
Title: A new class of 1-aryl-5,6-dihydropyrrolo[2,1-a]isoquinoline
derivatives as reversers of P-glycoprotein-mediated multidrug
resistance in tumor cells
Authors: Cosimo Damiano Altomare, Alisa A. Nevskaya, Maria D.
Matveeva, Tatiana N. Borisova, Mauro Niso, Nicola Antonio
Colabufo, Angelina Boccarelli, Rosa Purgatorio, Modesto de
Candia, Saverio Cellamare, and Leonid G. Voskressensky
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800177
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800177
A Journal of
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10.1002/cmdc.201800177
ChemMedChem
FULL PAPER
A new class of 1-aryl-5,6-dihydropyrrolo[2,1-a]isoquinoline
derivatives as reversers of P-glycoprotein-mediated multidrug
resistance in tumor cells
Alisa A. Nevskaya,[a] Maria D. Matveeva,[a] Tatiana N. Borisova,[a] Mauro Niso,[b] Nicola A. Colabufo,[b]
Angelina Boccarelli,[c] Rosa Purgatorio,[b] Modesto de Candia,[b] Saverio Cellamare,[b] Leonid G.
Voskressensky,[a] and Cosimo D. Altomare*[b]
[a]
Mrs. A. A. Nevskaya, Mrs. M. D. Matveeva, Prof. T. N. Borisova, Prof. L. G. Voskressensky, Organic Chemistry Department, Peoples’ Friendship University
of Russia, 6 Miklukho-Maklaya St., Moscow 117198 (Russia)
[b]
Dr. M. Niso, Prof. N. A. Colabufo, Dr. R. Purgatorio, Dr. M. de Candia, Prof. S. Cellamare, Prof. C. D. Altomare, Department of Pharmacy-Drug Sciences,
University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari (Italy)
E-mail: cosimodamiano.altomare@uniba.it
[c]
Dr. A. Boccarelli, Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro, Piazza Giulio Cesare 11, 70124 Bari (Italy)
Supporting Information (chemical stability tests of compounds 14 and 15) for this article is given via a link at the end of the document.
Abstract: A number of aza-heterocyclic compounds, which share
with members of the lamellarin alkaloids’ family the 5,6-
in dose-dependent manner the cytotoxicity of doxorubicin (DXR),
daunorubicin and vinblastine in multidrug-resistant cells, with a
potency as MDR modulator higher than that of verapamil (VRP),
dihydropyrrolo[2,1-a]isoquinoline
(DHPIQ)
scaffold,
were
synthesized and evaluated for their ability to reverse in vitro
multidrug resistance (MDR) in cancer cells, through inhibition of P-
glycoprotein (P-gp) and/or multidrug-resistance-associated protein-1
(MRP-1). Most of the investigated DHPIQs proved to be selective P-
gp modulators, and the most potent one, 8,9-diethoxy-1-(3,4-
diethoxyphenyl)-3-(furan-2-yl)-5,6-dihydropyrrolo[2,1-a]isoquinoline-
2-carbaldehyde (4), attained submicromolar inhibition potencies (IC50
0.19 µM). Schiff bases prepared by condensation of some 1-aryl-
DHPIQ aldehydes with p-aminophenol also proved to be of some
interest, and one of them, 4-((1-(4-fluorophenyl)-5,6-dihydro-8,9-
dimethoxypyrrolo[2,1-a]isoquinolin-2-yl)methyleneamino)phenol (15),
displayed IC50 of 1.01 µM. In drug combination assays in multidrug-
resistant cells, some DHPIQ compounds, at non-toxic doses,
a
first-generation P-glycoprotein (P-gp) inhibitor.[2] Besides
lamellarins, numerous natural alkaloids have been discovered
as potent inhibitors of P-gp efflux pump and other related pumps
responsible for the development of MDR.[3-5]
Nevertheless, intrinsic or acquired MDR still remains a major
hurdle to achieve success with the conventional chemotherapy
in cancer patients.[6] Several mechanisms underlie MDR, which
include enhanced drug efflux, increased DNA repair, reduced
apoptosis and altered drug metabolism.[7-9] MDR in human tumor
tissues is mostly related to the overexpression of the ATP-
binding cassette (ABC) transporters,[10] which are encoded in
humans by 49 genes.
significantly increased the cytotoxicity of doxorubicin in
a
concentration-dependent manner. Structure-activity relationship
studies and investigation of the chemical stability of the Schiff bases
provided physicochemical information useful for molecular
optimization of lamellarin-like cytotoxic drugs active toward
chemoresistant tumors as well as non-toxic reversers of P-gp-
mediated MDR in tumor cells.
Introduction
Figure 1. Structures of natural (lamellarin I) and synthetic (1-3) compounds
containing 1-aryl-5,6-dihydropyrrolo[2,1-a]isoquinoline as scaffold.
The heterocyclic system 5,6-dihydropyrrolo[2,1-a]isoquinoline
(DHPIQ), bearing an aryl group at 1-position, is the aza-
heterocyclic scaffold of a group of marine alkaloids, i.e.,
lamellarins, which is a family of more than thirty polyaromatic
compounds endowed with several biological activities, including
anticancer activity.[1] Some members of the lamellarin family
showed inhibition of HIV-1 integrase and human topoisomerase
I, along with other effects on nuclear proteins. Some of these
alkaloids showed cytotoxicity against tumor cells in vitro,
whereas other members, at non-toxic doses, proved to be
efficacious in reversing MDR, thereby increasing the
Among the ABC transporters, three are mainly associated with
MDR, namely P-gp (ABCB1), the multidrug-resistance-
associated protein-1 (MRP1, ABCC1), and the breast cancer
resistance protein (BCRP, ABCG2).[11-14] P-gp is overexpressed
in many cancer cells under chemotherapeutic treatment; it
exports a variety of chemotherapeutic agents outside of the
cancer cells, decreasing intracellular drug accumulation.[15] The
overexpression of the efflux pump MRP1 is responsible for MDR
to many chemotherapeutics (e.g., doxorubicin, vincristine,
cisplatin, methotrexate).[16] BCRP, the most recently identified
antiproliferative
activity
of
conventional
anti-tumor
chemotherapeutic agents in multidrug-resistant cells. In
particular, lamellarin I (Figure 1) proved to significantly increase
1
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10.1002/cmdc.201800177
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ABC transporter, is expressed in several hematological and solid
tumors together with P-gp.[17,18]
Scheme 1. Synthesis of 1-aryl-DHPIQ congeners and adducts
The P-gp-mediated MDR reversal activity shown by lamellarin I
prompted us to investigate the activity as P-gp modulators of
recently synthesized 2-carbaldehyde of 1-aryl-DHPIQ (1-3,
Figure 1).[19] These compounds were efficiently synthesized
through a domino reaction between 1-aroyl-substituted 3,4-
dihydroisoquinolines and α,β-unsaturated aldehydes, in the
absence of catalyst under microwave irradiation. In the same
study, some of the synthesized compounds were screened for
the antiproliferative/cytotoxic activity on a panel of human cancer
cell lines, including HCT116 (colon carcinoma cells), showing
low growth inhibition potencies. The group at 3-position of the
DHPIQ nucleus was the main structural variant explored, and
compound 1 (R5 = H), with IC50 of about 12 M, proved to be a
more potent cytotoxic than 2 (R5 = Me) and 3 (R5 = Ph), which
showed IC50s of about 67 and 183 M, respectively.
In this study, a number of previously and newly synthesized
DHPIQ derivatives were tested for their ability of modulating the
activity of P-gp and MRP1 efflux pumps, through Calcein-AM
transport assays in MDCK-MDR1 and MDCK-MRP1 cells. With
the aim of investigating the ability to sensitize MDCK-MDR1
cells, some DHPIQ derivatives were screened for their cytotoxic
effects in drug combination assays with DXR. Structure-activity
relationships were examined and the stability of some imino
derivatives of 1-aryl-DHPIQ toward chemical hydrolysis was
evaluated.
on some group replacements (4, 8) and on a number of carbonyl
adducts, namely oxime (9), thiosemicarbazone (10), and Schiff
bases (11-15) prepared by condensation of a number of DHPIQ
aldehydes with para-aminophenol (PAP).
The synthesis of 2-CHO DHPIQ compounds (1-7) was
accomplished through a domino-reaction of 1-aroyl substituted
3,4-dihydroisoquinolines with α,β-unsaturated aldehydes, such
as acrolein, сrotonaldehyde, cinnamaldehyde and 3-(furan-2-
yl)acrylaldehyde. The 2-NO2 derivative 8 was prepared through
a
similar reaction between drotaveraldine and 2-
nitrovinylbenzene. The aldehyde adducts, namely oxime 9 and
thiosemicarbazone 10, were prepared by condensation of
compound
1
with hydroxylamine and thiosemicarbazide,
respectively. The aldehyde compounds 1, 3, 5-7 were
condensed with PAP to afford the Schiff bases 11-15. The
secondary amino derivative 16 was prepared through
hydrogenation of the C=N double bond of 11, using NaBH3CN
as the reducing agent.
Biological studies
The P-gp inhibition potency of the DHPIQ compounds was
assessed by measuring the transport inhibition of Calcein-AM,
as a profluorescent P-gp substrate, in MDCK-MDR1 cell line
overexpressing P-gp.[20] The activity of the same compounds
was evaluated in MDCK-MRP1 cells overexpressing MRP1.
MC18[21] and verapamil (VRP)[21], as selective inhibitors of P-gp
and MRP1 efflux pumps, respectively, were used as positive
controls (Figure 2).
Results and Discussion
Chemistry
The synthesis of compounds 1-3 and 5-7 has been reported
earlier.[19] Most of them (2, 3, 4 and 6) were screened in this
study in order to explore the effects on the biological activity of
the substituents R1 and R2 (MeO, EtO), R3 and R4 (OEt, F), and
R5 (Me, Ph). To extend the structure-activity relationship (SAR)
study on these lamellarin-like compounds, new analogs and
derivatives were synthesized according to Scheme 1, focusing
Figure 2. Structures of the selective inhibitors of the efflux pumps P-gp
(MC18) and MRP1 (verapamil) used as positive controls.
2
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The inhibition potencies (IC50s) are reported in Table 1. With the
only exception of two less soluble compounds 8 and 13, which
showed low activity at the maximum test concentration (50 M),
the DHPIQ derivatives showed high-to-moderate inhibitory
potency on P-gp (IC50 from 0.19 to 11.2 M). Most of the test
compounds proved to be quite selective towards P-gp compared
to MRP1; in two cases, namely 6 and the respective Schiff base
14, a reversed or nil selectivity was observed. Compared to the
reference compound MC18, several DHPIQ derivatives proved
to be two-to-six times more potent as P-gp inhibitors. As for
MRP1, compounds 6 and 15 proved to be slightly more potent
than VRP.
at 10 and 25 M concentrations. Compound 6 did not show any
own cytotoxicity up to 25 M concentration, but did not even
show any significant reversal of the DXR resistance (a low
potentiating effect on DXR was observed only at 25 M), which
reflects the lower P-gp inhibition potency (IC50 = 10.7 M).
Table 2. In vitro inhibition potency (IC50) toward tumor cell growth.
IC50 ± SEM (µM)[a]
Cmpd
HepG2
HCT116
ND
3
> 100
4
> 100
ND
Compounds 3, 4, 6, 12, 15 and 16 were also evaluated for their
ability to restore the cytotoxicity of DXR in MDCK-MDR1 cells,
as a consequence of the P-gp inhibition. Figure 3 shows the
effects of the test compounds at three concentrations (1, 10 and
25 M) on the cytotoxicity of 10 M DXR in multidrug-resistant
cells. As shown in Figure 3, MDCK-MDR1 cells displayed
resistance to DXR, but they were sensitized when co-incubated
with the test DHPIQ compounds; their potentiating effects
appeared related to the P-gp inhibition potencies. Indeed,
compounds 3 and 4, which inhibited P-gp with a submicromolar
potency (IC50s 0.24 and 0.19 M, respectively), significantly
reversed the resistance of tumor cells to DXR in a concentration-
dependent manner, showing significant potentiating effects (3 >
4) at concentration as low as 1 M with no own cytotoxicity even
6
55.2 ± 7.2
> 100
41.3 ± 6.8
24.7 ± 1.5
ND
11
12
> 100
14
51.7 ± 5.8
5.74 ± 1.06
> 100
26.9 ± 2.8
ND
15
16
17.5 ± 0.3
17.2 ± 0.5
0.30 ± 0.08
PAP[b]
DXR[c]
92.0 ± 11
5.68 ± 1.30
[a] Each IC50 value is the mean ± SEM of two independent experiments
performed in triplicate; HepG2: Human liver cancer cell line; HCT116: Human
colon carcinoma cell line; ND = not determined. [b] Para-aminophenol. [c]
Doxorubicin.
Table 1. Inhibition potency (IC50) of 1-aryl-DHPIQ derivatives toward P-gp and MPR1 drug efflux pumps.
IC50 ± SEM (µM)[a]
MRP1
Cmpd
R1/R2
R3
R4
R5
X
P-gp
2
OEt
OEt
OEt
OMe
OMe
OEt
OEt
OEt
OEt
OMe
OMe
OMe
OEt
OEt
OEt
OEt
H
OEt
OEt
OEt
F
Me
Ph
2-Furyl
H
CHO
0.43 ± 0.07
0.24 ± 0.05
0.19 ± 0.02
10.7 ± 1.0
40%[b]
9.34 ± 1.0
>100
3
CHO
4
CHO
22.8 ± 3.4
4.24 ± 0.7
ND
6
CHO
8
H
Cl
Ph
H
NO2
9
OEt
OEt
OEt
OEt
H
OEt
OEt
OEt
OEt
Cl
CHNOH
1.28 ± 0.05
0.24 ± 0.03
11.2 ± 1.3
1.91 ± 0.7
29%[b]
8.96 ± 0.90
> 100
10
H
CHNNHC(S)NH2
CHNC6H4-4-OH
CHNC6H4-4-OH
CHNC6H4-4-OH
CHNC6H4-4-OH
CHNC6H4-4-OH
CH2NC6H4-4-OH
11
H
51.4 ± 4.3
6.68 ± 1.0
ND
12
Ph
H
13
14
H
F
H
10.2 ± 1.1
1.01 ± 0.2
0.71 ± 0.09
1.20 ± 0.30
9.93 ± 1.2
2.23 ± 0.4
7.37 ± 0.8
15
H
F
Ph
H
16
OEt
OEt
MC18[c]
VRP[d]
4.53 ± 0.5
[a] Each IC50 value is the mean ± SEM of two independent experiments performed in triplicate; ND = not determined. [b] Average % inhibition at 50 M
concentration (maximum concentration tested; solubility limit). [c] MC18, P-gp-selective positive control. [d] Verapamil, MRP1-selective positive control.
3
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The Schiff base 12 (IC50 = 1.91 M) was per se not cytotoxic,
because of different nitrogen basicity and lipophilicity. The 2-NO2
DHPIQ analog (8) showed lower aqueous solubility and activity
compared to the aldehyde derivatives. For P-gp modulation, no
noteworthy advantage came from the aldehyde adducts.
However, three derivatives (10, 15 and 16) showed potency
higher than the positive control MC18. The MDR reversal effects
of the examined compounds, as assessed in co-incubation
assays with DXR in a multidrug resistance cell model (Figure 3),
appeared to be reasonably related to their P-gp inhibition
potency, whereas the Schiff base 15 showed moderate
cytotoxicity in both MDCK-MDR1 and HepG2 cell lines.
but reversed the resistance to DXR in
a concentration-
dependent manner, showing potentiating effects slightly lower
than those observed with 3. In contrast, 15 (IC50 = 1.01 M)
showed either own cytotoxicity and sensitizing effects toward
DXR at 10 and 25 M. Finally, the amino derivative 16 (IC50
=
0.71 M) showed a low but significant cytotoxicity at the highest
concentration, but at non-toxic concentrations (1 and 10 M)
potentiated the effects of DXR in a concentration-dependent
manner.
The cytotoxicity of these and two other compounds (11 and
14), as well as PAP as a possible toxic product [23] of the Schiff
bases’ hydrolysis, was evaluated on two human tumor cell lines,
namely HepG2 (human liver cancer cells) and HCT116 (human
colon carcinoma cells). The IC50 values are summarized in Table
2. HepG2 cell lines are widely used, not only to evaluate toxic
effects of a variety of chemicals and drugs, but also in
genotoxicity testing, as these cells express metabolizing
enzymes required for activation of DNA-reactive carcinogens.[24]
The HCT116 cells have been chosen for this screening because
they express high levels of glutathione S-transferases (GST),
which catalyze glutathione (GSH) conjugation of many different
cytotoxic agents, making the compounds easily eliminable, with
consequent MDR.[25]
As shown in Table 2, both tumor cell lines showed low
sensitivity toward the test compounds. The Schiff base 15 (IC50
= 5.74 M) turned out to be that with the highest cytotoxicity on
the HepG2 cells, whereas the other compounds displayed IC50s
> 50 M. The cytotoxicity of 15 does not appear to depend upon
the hydrolytic release of PAP, which in turn inhibited the HepG2
cell growth with IC50 value of 92 M. However, the Schiff base
15 proved to be the most cytotoxic toward MDCK-MDR1 (Figure
3) and HepG2 (Table 2) cell lines.
cmpd 4
cmpd 3
100
80
60
40
20
0
100
80
60
40
20
0
***
**
***
***
***
***
1M
10M
25M
1M
10M 25M
cmpd 6
cmpd 12
100
80
60
40
20
0
100
80
60
40
20
0
**
**
***
***
1M
10M
25M
1M
10M 25M
cmpd 15
cmpd 16
100
80
60
40
20
0
100
80
60
40
20
0
##
***
##
The HCT116 cell line showed a sensitivity toward the test
DHPIQ compounds slightly higher than the HepG2 cell line,
spanning a range of IC50 values from 17 to 40 M. The aldehyde
###
***
***
***
***
6
was found to be about 1.5-fold less toxic than the
1M
10M 25M
1M
10M 25M
corresponding Schiff base 14, which in turn showed a potency
comparable to PAP in inhibiting the growth of human colon
carcinoma cells. Moreover, the Schiff base 11 (IC50 = 24.7 M)
resulted about 1.5-fold less toxic than the corresponding amino
derivative 16, which in turn showed a potency superimposable to
that of PAP (IC50s 17.5 and 17.2 M, respectively). This was
more evident with HCT116 cells than with HepG2 cells. It
remains to prove that, at least in colon carcinoma cells, the
amino derivative 16 may undergo oxidation (dehydrogenation)
and subsequent hydrolysis of the imine derivative to release
PAP in cell.
DXR (10 µM)
cmpd alone
cmpd + DXR (10 µM)
Figure 3. Dose-dependent effects on the in vitro growth of multidrug-resistant
MDCK-MDR1 cells by 10 µM doxorubicin (DXR) alone or in the presence of 1,
10 and 25 µM concentrations of compounds 3, 4, 6, 12, 15 and 16. Cell
survival is represented as % of control cell growth in cultures containing no
drug and test compounds. Each bar represents the mean ± SEM of two
experiments in triplicate; one-way ANOVA followed by Bonferroni’s post-hoc
comparison test: ** P < 0.01, *** P < 0.001 vs. DXR alone; ## P < 0.01, ### P
< 0.001 vs. respective test compound at the lowest concentration (1 µM).
Structure-activity relationships
Hydrolytic stability studies
Within the limits of the examined molecular space, some clues in
SARs can be deduced from the P-gp inhibition data, which may
have utility in optimization studies. Lipophilicity of the R1-R5
substituents do increase the P-gp inhibition potency; indeed: (i)
EtO groups proved to be more favorable than OMe, F, Cl as R1-
R4 substituents; (ii) the lipophilic phenyl group (3) and its
bioisosteric replacement 2-furyl (4), as R5 substituents, improved
the P-gp interaction of the DHPIQ derivatives compared to the
methyl-substituted (2) or unsubstituted compounds (e.g., 12 >
11; 15 > 14); (iii) the amino derivative (16) turned out to be a
more potent inhibitor than the parent Schiff base (11), likely
Compounds 11-15 are Schiff bases with PAP, which is known
for being cytotoxic in some tissues, due to its ability to trigger
formation of reactive oxygen species (ROS).[23] To understand if
the activity of these Schiff bases might be related to the release
of PAP, we carried out a hydrolytic stability study on compounds
14 and 15. These Schiff bases differ one from each other for R5
(H and Ph in 14 and 15, respectively), which could affect the rate
of hydrolysis of the imine linkage.
The reversed phase (RP) HPLC proved to be unsuitable as
analytical method for monitoring their hydrolytic stability.
Compounds 14 and 15, which are hydrophobic weak bases (with
4
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10.1002/cmdc.201800177
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calculated pKa of the imine group about 4.5), could be reliably
analyzed using aqueous mobile phases at acidic pH values (3 
4.5) to which the imino derivatives are not enough stable during
the chromatographic analysis. Indeed, as shown by 1H NMR
data (Supporting Information), the Schiff bases 14 (ca. 80%
hydrolyzed after 30 min) and 15 (ca. 40% hydrolyzed after 15
min) underwent rapid degradation in DCl/DMSO-d6 solution. In
this study, also UV spectrophotometry proved to be of limited
applicability (Supporting Information).
progresses from about 28% at 1 h to 48% at 2.5 h and 64% at
22 h.
Ha
t0
Ha
Ha
Hb
Hb
1h
t0
Ha
Hb
24h
Hc
Hd
1h
Figure 5. 1H NMR spectra at 500 MHz of the Schiff base 15 and its hydrolysis
product 7 in D2O/DMSO-d6 solutions. The proton peaks labeled as Ha and Hb
were monitored during the solvolysis reaction.
2.5 h
As shown in Figure 6, the hydrolysis rate of compound 14 in
D2O/DMSO-d6 solution is much greater than that of 15. The
higher stability of 15 (t1/2 >> 24 h) is clearly due to the steric
shield of the 3-phenyl group to the nucleophilic addition of water
to the imine linkage. According to the 1H NMR data, the
hydrolysis of 14 in D2O/DMSO-d6 solution followed a pseudo-
first-order kinetics with an experimental apparent rate constant
(kobs) of 0.207 h-1 and t1/2 of 3.34 h.
1.
Figure 4. 1H NMR spectra at 500 MHz of the Schiff base 14 and the aldehyde
product 6 in D2O/DMSO-d6 solutions. The proton peaks labeled as Ha, Hb, Hc
and Hd were monitored during the solvolysis reaction. Signals indicating PAP
appeared in the range of 6.42-6.47 ppm (dd, 4Har).
In contrast, 1H NMR (500 MHz) provided a means for
monitoring the course of hydrolytic reaction of the examined
Schiff bases. Characteristic proton peaks of the starting
compounds (14 and 15) and the respective reaction products (6
and 7, respectively, and PAP) appeared in distinct regions of the
spectra and the signals could be quantitatively related to each
other by integration. The stability of 14 and 15 was monitored in
D2O/DMSO-d6 solution at room temperature. Typical 1H NMR
spectra of the Schiff base 14 and hydrolyzed samples at three
times (0, 1 and 2.5 h) in D2O/DMSO-d6 solution (1:15, v/v) are
shown in Figure 4. Variations in the AUCs of the proton peaks
(singlets) at 8.03 (Ha) and 7.53 ppm (Hb) in the Schiff base 14,
and 9.48 (Hc) and 7.66 ppm (Hd) in the aldehyde product 6, as
well as the appearance of a double doublet (dd) related to the
aromatic protons of PAP (6.42-6.47 ppm), were monitored
during the reaction course. After 30 min, two new peaks
appeared as singlets at 9.48 (Hc) and 7.66 (Hd) ppm, clearly
indicating the formation of the hydrolysis product 6. After about 1
h, new peaks (6.47-6.42 ppm, dd, 4H) assigned to the aromatic
protons of PAP appeared in solution. Calculation of area-under-
the-curve (AUC) ratios related to the above proton peaks proved
the occurrence of hydrolytic conversion of 14 into 6, which
60
14
40
20
15
0
0
1
2
3
Time / h
Figure 6. Solvolysis reaction course of the Schiff bases 14 and 15 in
D2O/DMSO-d6 solution at room temperature, as determined by 1H NMR
spectrometry (500 MHz), during the first 3 hs of observation. Data points
represent average values ± SEMs from two experiments in triplicate.
Similar results were obtained by simultaneously monitoring
the disappearance of the Schiff base 14 and the appearance of
the two hydrolysis products 6 and PAP in a buffered solution at
pH 7.4 and fixed ionic strength (50 mM PBS, 0.15 M KCl, and
0.5% DMSO as the co-solvent) at room temperature, through
5
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10.1002/cmdc.201800177
ChemMedChem
FULL PAPER
UV spectrophotometric analysis of the ternary mixture
(Supporting Information). As shown in Figure 7, the Schiff base
highlighted the role of lipophilicity of the different substituents in
increasing the biological potency of the studied lamellarin-like
compounds.
14 underwent a pseudo first-order kinetics hydrolysis (r2
0.9953) with kobs of 0.214 h-1 and t1/2 of 3.21 h.
=
Overall, this work provides evidence in support of the DHPIQ
heterocyclic nucleus as a molecular scaffold for building up
promising non-cytotoxic modulators of the MDR phenotype, able
to potentiate the cytotoxic activity of other antitumor drugs, as
well as new anticancer agents active on resistant cells. The
synthesis of additional well-designed DHPIQ derivatives, their
biological testing and further SAR studies may help disclosing of
novel agents combining higher cytotoxic activity on tumor cells
and more potent modulating activity on MDR tumor cells.
Although carried out in remote conditions compared to those
recurring in tumor cells, the results from these stability studies
combined with the above biological findings may help in
understanding the behavior of the examined Schiff bases.
0.0
-0.5
-1.0
-1.5
Experimental Section
Chemistry
0
1
2
3
4
5
6
Materials and general procedures: All reagents and solvents were
purchased from Merck, J.T. Baker, or Sigma-Aldrich Chemical Co. and
used without further purification. 1H and 13C NMR spectra were recorded
in CDCl3 or [D6]DMSO solutions at 25 °C, with a 600 MHz NMR
spectrometer; peak positions are given in parts per million (δ) referenced
to the appropriate solvent residual peak. Mass spectra were recorded
with an LCMS-8040 Triple quadrupole liquid chromatograph-mass
spectrometer from Shimadzu.
Time / h
Figure 7. Pseudo-first-order plot for hydrolysis of compound 14 at 50 M
concentration in phosphate buffer (pH 7.4, 0.15 M KCl) and room temperature.
The progress of the hydrolytic reaction was monitored by UV
spectrophotometry. Data points represent the average values ± SEMs of two
experimental measurements in triplicate.
The synthesis procedures of compounds 1-3 and 5-7 were recently
described;[19] compounds 4 and 8-16 were synthesized according to the
following procedures.
The activity of compound 15, which turned out to be highly
stable at neutral (cytosolic) pH, may be attributed to the intact
molecule. High stability toward the hydrolytic reaction at neutral
pH could be predicted for compound 12 (R5 = Ph). The potent P-
gp inhibition, as well as the intrinsic cytotoxicity against MDCK-
MDR1 cells and HepG2 cells, could be predominantly ascribed
to the whole molecule 15, and not to its hydrolytic products. In
contrast, the Schiff base 14 (a less potent P-gp inhibitor) proved
to be quite unstable in buffered solution at pH 7.4 (t1/2 about 3.2
h), which suggests that its low cytotoxicity against HepG2 and
HCT116 cells may be due to the combined effects of itself and
the hydrolytic products 6 and PAP.
1-(3,4-Diethoxyphenyl)-8,9-diethoxy-3-(furan-2-yl)-5,6-
dihydropyrrolo[2,1-a]isoquinoline-2-carbaldehyde (4): 3-(Furan-2-
yl)acrylaldehyde (71 mg, 0.58 mmol) was added to a solution of (6,7-
diethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)(3,4-diethoxyphenyl)
methanone (197 mg, 0.48 mmol) in trifluoroethanol (6 mL). The mixture
was refluxed for 16 h, and TLC (sorbfil, EtOAc/hexane 2:1) monitored the
reaction progress. The solvent was removed under vacuum; the residue
was crystallized from EtOH to afford compound 4 as a beige powder (76
mg, 31%): mp 125-128 оС; 1H NMR (600 MHz, CDCl3,): δ = 1.17 (t, 3H, J
= 7.0 Hz, О-CH2-CH3), 1.39 (t, 3H, J = 7.0 Hz, О-CH2-CH3), 1.42 (t, 3H, J
= 7.0 Hz, О-CH2-CH3), 1.46 (t, 3H, J = 7.0 Hz, О-CH2-CH3), 2.98 (t, 2H, J
= 6.6 Hz, 6-CH2), 3.57 (q, 2H, J = 7.0 Hz, О-CH2-CH3), 4.02-4.08 (m, 4H,
О-CH2-CH3), 4.13 (q, 2H, J = 7.0 Hz, О-CH2-CH3), 4.21 (t, 2H, J = 6.6
Hz, 5-CH2), 6.58 (dd, 1H, J = 1.6, 3.3 Hz, CH-fur), 6.59 (s, 1H, 7-H), 6.68
(s, 1H, 10-H), 6.90 (d, 1H, J = 3.3 Hz, CH-fur), 6.92-6.94 (m, 3H, CH-Ar),
7.60 (br.s., 1H, CH-fur), 9.77 (s, 1H, CHO); 13C NMR (150 MHz, DMSO-
d6): δ = 14.6, 14.9 (4C), 29.0, 43.2, 64.7, 64.6, 64.7 (2C), 109.9, 111.6,
112.8, 113.8, 114.0, 116.2, 120.9, 122.0, 123.2, 124.9, 127.1, 128.3,
128.6, 143.5, 143.7, 147.3, 147.6, 148.2, 148.9, 186.3; MS (LCMS) m/z =
516 [M+Н]+; Anal. calcd for C31H33NO6: C, 72.21; H, 6.45; N, 2.72, found:
C 73.0, H 6.58, N 2.90.
Conclusions
In this study, some synthetic lamellarin-like compounds were
identified as potent P-gp inhibitors having potential for the
treatment of multidrug-resistant tumors. A number of previously
and
newly
synthesized
1-aryl-5,6-dihydropyrrolo[2,1-
a]isoquinoline (DHPIQ) derivatives were prepared and assayed
for their ability to modulate P-gp and MRP1-mediated MDR in
suitable tumor cell models. The majority of them proved to be in
vitro inhibitors of P-gp with selectivity over the MRP1 efflux
pump, and some compounds attained submicromolar P-gp
inhibition potency. Studies of the MDR-reversal activity of
differently substituted 1-aryl-DHPIQ compounds, carried out in
cells exhibiting MDR (MDCK-MDR1 cell line), provided in vitro
proof that, at non-toxic concentrations, in case of 2-CHO
derivatives (e.g., 3), the cytotoxicity of DXR increased
significantly. Among the aldehyde adducts, the Schiff base 15,
which should be hydrolytically stable at the cell pH, turned out to
be itself moderately cytotoxic toward human HCT116 cells (IC50
5.7 M) and MDCK-MDR1 cells, as well as able to potentiate the
cytotoxic activity of DXR. Structure-activity relationship analysis
1-(4-Chlorophenyl)-8,9-dimethoxy-2-nitro-3-phenyl-5,6-
dihydropyrrolo[2,1-a]isoquinoline (8): 2-Nitrovinylbenzene (173 mg,
1.2 mmol) was added to a solution of (4-chlorophenyl)(6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinolin-1-yl)methanone (383 mg, 1.2 mmol) in
trifluoroethanol (10 mL). The mixture was refluxed for 24 h, and the
reaction progress was monitored by TLC (sorbfil, EtOAc/hexane 2:1).
The solvent was removed under vacuum, and the residue was
crystallized from ethyl acetate to afford compound 8 as a yellow powder
(300 mg, 56%): mp 225-226 оС; 1H NMR (600 MHz, CDCl3): δ = 2.96 (t,
2H, J = 6.1 Hz, 6-CH2), 3.37 (s, 3H, OCH3), 3.84-3.91 (m, 5H, OCH3, 5-
CH2), 6.35 (s, 1H, 7-H), 6.69 (s, 1H, 10-H), 7.40-7.47 (m, 6H, CH-Ar),
7.49-7.54 (m, 3H, CH-Ar); 13C NMR (150 MHz, DMSO-d6): δ = 28.7, 42.5,
55.0, 55.9, 56.0, 107.4, 111.0, 114.6, 119.9, 125.1, 126.2, 128.6 (2C),
128.8, 129.1, 129.3, 130.2 (2C), 132.1 (2C), 132.5, 132.8, 133.0, 133.6,
147.7, 148.2; MS (LCMS): m/z
C26H21ClN2O4: C 67.75, H 4.59, N 6.08; found: C 67.88, H 4.71, N 6.25.
=
461 [M+Н]+; Anal. calcd for
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800177
ChemMedChem
FULL PAPER
1-(3,4-Diethoxyphenyl)-8,9-diethoxy-5,6-dihydropyrrolo[2,1-
121.3 (2C), 121.6, 122.0 (3C), 123.5, 125.3, 126.1, 127.9, 144.9, 146.8,
147.9, 148.7, 152.3, 155.6; MS (LCMS) m/z = 541 [M+Н]+; Anal. calcd for
C33H36N2O5: C, 73.31, H, 6.71, N, 5.18, found: C, 73.42, H, 6.90, N, 4.86.
a]isoquinoline-2-carbaldehyde
oxime
(9):
Hydroxylamine
hydrochloride (10 mg, 0.22 mmol) was added to a solution of 1-(3,4-
diethoxyphenyl)-8,9-diethoxy-5,6-dihydropyrrolo[2,1-a]isoquinoline-2-
carbaldehyde (1, 70 mg, 0.16 mmol) in EtOH (4 mL). The mixture was
refluxed for 32 h, and the reaction progress was monitored by TLC
(sorbfil, EtOAc/hexane 2:3). The solvent was removed under vacuum,
and water (3 mL) was added to the resulting residue and extracted with
Et2O (3×8 mL). The organic layers were combined and dried over
MgSO4. The solvent was removed under vacuum, and the residue was
recrystallized from EtOAc–hexane to afford compound 9 as a yellow solid
4-(((1-(3,4-Diethoxyphenyl)-8,9-diethoxy-3-phenyl-5,6-
dihydropyrrolo[2,1-a]isoquinolin-2-yl)methylene)amino)phenol (12):
о
White solid (210 mg, 52%): mp 202-204 С; 1H NMR (600 MHz, DMSO-
d6): δ = 1.03 (t, 3Н, J = 7.0 Hz, O-CH2-CH3), 1.23 (t, 3Н, J = 7.0 Hz, O-
CH2-CH3), 1.26 (t, 3Н, J = 7.0 Hz, O-CH2-CH3), 1.31 (t, 3Н, J = 7.0 Hz,
O-CH2-CH3), 2.89 (t, 2Н, J = 6.1 Hz, 6-СH2), 3.45 (q, 2Н, J = 7.0 Hz, O-
CH2-CH3), 3.89 (t, 2Н, J = 6.1 Hz, 5-СH2), 3.94-3.97 (m, 4Н, O-CH2-CH3),
4.02 (q, 2Н, J = 7.0 Hz, O-CH2-CH3), 6.45 (s, 1Н, 7-H), 6.55-6.61 (m, 4Н,
СН-Ar), 6.82 (s, 1Н, 10-H), 6.83-6.84 (m, 1Н, СН-Ar), 6.94-6.95 (m, 2Н,
СН-Ar), 7.39-7.42 (m, 1Н, СН-Ar), 7.47 (t, 2Н, J = 7.2 Hz, СН-Ar), 7.51 (t,
2Н, J = 7.2 Hz, СН-Ar), 8.04 (s, 1H, CH=N), 9.15 (s, 1H, OH); 13C NMR
(150 MHz, DMSO-d6): δ = 14.9 (4C), 28.6, 31.0, 64.9, 65.0, 65.09, 65.8,
112.1, 113.0, 113.7, 115.1 (2C), 116.2, 117.5, 118.9 (2C), 119.3, 120.4,
122.2 (3C), 124.3, 124.5, 124.8, 126.7, 128.0 (2C), 131.0, 133.5, 137.6
(2C), 142.0, 146.8, 149.0, 151.9, 165.7; MS (LCMS) m/z = 617 [M+Н]+;
Anal. calcd for C39H40N2O5: C, 75.95; H, 6.54; N, 4.54, found: C, 76.13, H
6.70, N 4.63.
о
(24 mg, 33%): mp 156-158 С; 1H NMR (600 MHz, DMSO-d6): δ = 1.17
(m, 3H, J = 7.0 Hz, О-CH2-CH3), 1.35-1.45 (m, 6H, О-CH2-CH3), 1.47 (m,
3H, J = 7.0 Hz, О-CH2-CH3), 3.02 (m, 2H, J = 6.6 Hz, 6-CH2), 3.59 (q,
2H, J = 7.0 Hz, О-CH2-CH3), 3.98 - 4.07 (m, 4H, О-CH2-CH3, 5-CH2),
4.08 - 4.19 (m, 4H, О-CH2-CH3), 6.61 (s, 1H, 7-H), 6.68 (s, 1H, 10-H),
6.85 (s, 1H, СH-Ar), 6.86 (d, 1H, J = 8.3 Hz, СH-Ar), 6.95 (d, 1H, J = 8.3
Hz, СH-Ar), 7.24 (s, 1H, 3-Н), 7.92 (s, 1H, CH=NOH); 13C NMR (150
MHz, DMSO-d6): δ = 14.6, 14.9 (3C), 29.2, 45.1, 63.8, 64.6, 64.7, 65.0,
109.2, 113.3, 114.0, 115.9, 117.4, 119.7, 121.5, 123.1 , 124.0, 127.5,
128.7, 139.5, 147.1, 147.3, 147.8, 149.1, 177; MS (LCMS) m/z = 465
[M+Н]+; Anal. calcd for C27H32N2O5: C 69.81, H 6.94, N 6.03, found: C
69.70, H 6.81, N 6.22.
4-(((1-(4-Chlorophenyl)-8,9-dimethoxy-5,6-dihydropyrrolo[2,1-
a]isoquinolin-2-yl)methylene)amino)phenol (13): Beige solid (110 mg,
43 %): mp 281-283 оС; 1H NMR (600 MHz, DMSO-d6): δ = 3.05 (t, 2H, J
= 6.3 Hz, 6-CH2), 3.41 (s, 3H, O-CH3), 3.87 (s, 3H, O-CH3), 4.13 (t, 2H, J
= 6.3, 5-CH2), 6.51 (s, 1H, 7-H), 6.71 (s, 1H, 10-H), 6.77 (d, 2H, J = 8.6
Hz, C6H4-OH), 7.02 (d, 2H, J = 8.6 Hz, C6H4-OH), 7.42 (dd, 4H, J = 5.7,
8.5 Hz, С6Н4-4-Cl), 7.58 (s, 1H, 3-H), 8.11 (s, 1H, CH=N), 9.15 (s, 1H,
OН); 13C NMR (150 MHz, DMSO-d6): δ = 21.7, 28.9, 40.6, 44.7, 55.1,
56.1 (2C), 107.7, 112.8, 116.14, 119.5, 121.1, 121.3, 122.1 (3C), 122.5,
125.6, 126.5, 129.0, 132.2, 133.2, 134.5, 144.7, 147.6, 147.8, 151.7; MS
(LCMS) m/z = 457 [M+Н]+; Anal. calcd for C27H23ClN2O3: C, 70.66; H,
5.05; Cl, 7.73; N, 6.10, found: C 70.52, H 5.25, N 6.21.
2-((1-(3,4-Diethoxyphenyl)-8,9-diethoxy-5,6-dihydropyrrolo[2,1-
a]isoquinolin-2-yl)methylene)hydrazinecarbothioamide
(10):
Thiosemicarbazide (17 mg, 0.19 mmol) was added to a solution of
compound 1 (70 mg, 0.16 mmol) in EtOH (4 mL). The mixture was
refluxed for 8 h, and the reaction progress was monitored by TLC (sorbfil,
EtOAc/hexane 2:3). The solvent was removed under vacuum, and the
residue was crystallized from EtOH to afford compound 10 as a yellow
solid (206 mg, 88%): mp 174-176 оС; 1H NMR (600 MHz, CDCl3): δ =
1.15 (t, 3H, J = 6.9 Hz, О-CH2-CH3), 1.36-1.43 (m, 6H, О-CH2-CH3), 1.46
(t, 3H, J = 6.9 Hz, О-CH2-CH3), 3.00 (t, 2H, J = 6.2 Hz, 6-CH2), 3.57 (q,
2H, J = 6.9 Hz, О-CH2-CH3), 3.97-4.03 (m, 2H, О-CH2-CH3), 4.03-4.09
(m, 4H, О-CH2-CH3, 5-CH2), 4,12 (q, 2H, J = 6.9 Hz, О-CH2-CH3), 5.99
(br.s, 1H, NH), 6.60 (s, 1H, 10-H), 6.61 (br.s., 1H, NH), 6.67 (s, 1H, 7-H),
6.84-6.89 (m, 2H, СH-Ar), 6.92 (d, 1H, J = 8.1 Hz, СH-Ar), 7.06 (s, 1H, 3-
Н), 7.60 (s, 1H, 10-H), 7.97 (s, 1H, CH=N); 13C NMR (150 MHz, DMSO-
d6): δ = 14.6, 14.9 (2C), 15.0, 29.2, 29.3, 45.2 (2C), 63.9, 64.6, 64.7, 64.9
(2C), 109.2, 116.1, 118.7, 123.4 (2C), 124.1 (2C), 126.0, 126.9, 127.3,
147.2, 147.3, 148.1, 149.1 (2C); MS (LCMS) m/z = 523 [M+Н]+; Anal.
calcd for C28H34N4O4S: 64.34%, H 6.56%, N 10.72%, found: C 64.1, H
6.36, N 10.54.
4-(((1-(4-Fluorophenyl)-8,9-dimethoxy-5,6-dihydropyrrolo[2,1-
a]isoquinolin-2-yl)methylene)amino)phenol (14): Beige solid (290 mg,
65 %): mp 168-170 оС; 1H NMR (600 MHz, DMSO-d6): δ = 2.99 (t, 2H, J
= 5.8 Hz, 6-СН2), 3.26 (s, 3H, ОСН3), 3.74 (s, 3H, ОСН3), 4.13 (t, 2H, J =
5.8 Hz, 5-СН2), 6.41 (s, 1H, 7-Н), 6.70 (d, 2Н, J = 8.7 Hz, C6H4-OH), 6.87
(d, 2Н, J = 8.7 Hz, C6H4-OH), 6.91 (s, 1H, 10-Н), 7.29 (d, 2Н, J = 8.7 Hz,
С6Н4-4-F), 7.43 (d, 2Н, J = 8.7 Hz, С6Н4-4-F), 7.56 (s, 1H, 3-Н), 8.04 (s,
1H, CН=N), 9.28 (s, 1H, OН); 13C NMR (150 MHz, DMSO-d6): δ = 28.9,
44.8, 55.1, 56.07, 107.5, 112.7, 115.9 (d, J = 21.7, 2C), 116.0, 116.1,
119.8, 121.3, 121.4, 122.1 (2C), 125.4, 126.4, 131.8 (d, J = 131.8, 1C),
133.3 (d, J = 7.2, 2C), 144.7, 147.5, 147.6, 147.7, 151.8, 155.7, 161.9 (d,
J
=
244.2, 1C); MS (LCMS) m/z = 443 [M+Н]+; Anal. calcd for
Synthesis of Shiff bases with para-aminophenol (11-15)
C27H23FN2O3: C, 73.29; H, 5.24; N, 6.33, found: C 73.61, H 5.00, N 6.11.
Para-aminophenol (PAP, 1.0 mmol) was added in a flask with a solution
of the corresponding aldehyde derivative 1, 3, 5-7 (1.0 mmol) in absolute
alcohol (MeOH for synthesis of 11 from 1 and 12 from 3, EtOH for 13-15
from 5-7). The reaction was carried out in the presence of glacial acetic
acid (0.01 mmol) and MgSO4 as a water-removal agent (2.0 mmol). The
mixture was stirred and heated under reflux; the reaction progress was
monitored by TLC (alufol, EtOAc/hexane 2:1). After cooling, the residue
was filtered off and washed once with MeOH (2 mL) to afford compounds
11 and 12. Isolation of 13-15 was obtained by removing solvent under
vacuum and recrystallizing the residues from EtOAc/hexane.
4-(((1-(4-Fluorophenyl)-8,9-dimethoxy-3-phenyl-5,6-
dihydropyrrolo[2,1-a]isoquinolin-2-yl)methylene)amino)phenol (15):
Beige solid (250 mg, 69 %): mp 309-311 оС; 1H NMR (600 MHz, DMSO-
d6): δ = 2.92 (t, 2H, J = 6.2 Hz, 6-СН2), 3.20 (s, 3H, О-СН3), 3.70 (s, 3H,
ОСН3), 3.92 (t, 2H, J = 6.2 Hz, 5-СН2), 6.33 (s, 1H, 7-Н), 6.59-6.60 (m,
4H, CH-Ar), 6.87 (s, 1H, 10-Н), 7.20 (t, 2H, J = 8.7 Hz, СН-Ar), 7.41 (dd,
2H, J = 6.0, 8.7 Hz, СН-Ar), 7.43-7.45 (m, 1Н, СН-Ar), 7.48 - 7.50 (m,
2Н, СН-Ar), 7.51-7.52 (m, 2H, CH-Ar), 8.07 (s, 1H, CН=N), 9.15 (s, 1H,
OН); 13C NMR (150 MHz, DMSO-d6): δ = 14.6, 21.3, 28.9, 42.58, 54.9,
56.0, 60.3, 108.2, 112,4, 115.3 (d, J=21.7, 1C), 116.0, 118.6, 118.9,
121.2, 121.82, 126.27, 127.2, 128.8, 128.9, 130.4, 131.5 (d, J = 7.2, 2C),
133.1 (d, J = 2.9, 2C), 133.58, 136.5, 145.0, 147.5, 152.0, 155.5, 161.7
(d, J = 242.8, 1C), 162.5, 170.9; MS (LCMS) m/z = 519 [M+Н]+; Anal.
calcd for C33H27FN2O3: C 76.43, H 5.25, N 5.40, found: C 76.63, H 5.35,
N 5.58.
4-(((1-(3,4-Diethoxyphenyl)-8,9-diethoxy-5,6-dihydropyrrolo[2,1-
a]isoquinolin-2-yl)methylene)amino)phenol (11): White powder (250
mg, 53%): mp 216-218 оС; 1H NMR (600 MHz, DMSO-d6): δ = 1.03 (t,
3Н, J = 7.0 Hz, O-CH2-CH3), 1.23 (t, 3Н, J = 7.0 Hz, O-CH2-CH3), 1.26 (t,
3Н, J = 7.0 Hz, O-CH2-CH3), 1.30 (t, 3Н, J = 7.0 Hz, O-CH2-CH3), 2.93 (t,
2Н, J = 6.4 Hz, 6-СH2), 3.48 (q, 2Н, J = 7.0 Hz, O-CH2-CH3), 3.92-3.96
(m, 4Н, O-CH2-CH3), 4.03 (q, 2Н, J = 7.0 Hz, O-CH2-CH3), 4.08 (t, 2Н, J
= 6.4 Hz, 5-СH2), 6.49 (s, 1Н, 7-H), 6.66 (d, 2Н, J = 8.5 Hz, C6H4-OH),
6.82-6.84 (m, 3Н, СН-Ar, 10-H), 6.88 (d, 1Н, J = 2.1 Hz, CH-Ar), 7.00 (d,
2H, J = 8.5 Hz, C6H4-OH), 7.48 (s, 1H, 3-H), 8.00 (s, 1H, CH=N), 9.23 (s,
1H, OH); 13C NMR (150 MHz, DMSO-d6): δ = 15.0, 15.2, 15.3 (2C), 28.9,
44.8, 63.7, 64.3 (2C), 64.4, 64.5, 109.2, 114.0, 114.4, 116.2 (2C), 116.4,
4-(((1-(3,4-Diethoxyphenyl)-8,9-diethoxy-5,6-dihydropyrrolo[2,1-
a]isoquinolin-2-yl)methyl)amino)phenol (16): NaBH3CN (70 mg, 1.11
mmol) was added to a solution of the compound 11 (200 mg, 0.37 mmol)
in MeOH (15 mL). The reaction was carried out in the presence of glacial
acetic acid (1 drop). The resulting solution was stirred at room
temperature for 5 h; the reaction progress was monitored by TLC (sorbfil,
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800177
ChemMedChem
FULL PAPER
EtOAc/hexane 1:1). The solvent was removed under vacuum, and glacial
acetic acid was added to the residue up to pH 7. The resulting solution
was extracted with EtOAc (4×9 mL). The organic layers were combined
and dried over MgSO4. The solvent was removed under vacuum, and the
residue was recrystallized from EtOAc/hexane to afford the amine
product 16 as a beige solid (122 mg, 61%): mp 194-196 оС; 1H NMR
(600 MHz, DMSO-d6): δ = 1.17 (t, 3Н, J = 6.8 Hz, O-CH2-CH3), 1.32 (t,
3H, J = 6.8 Hz, O-CH2-CH3), 1.41 (t, 3Н, J = 6.8 Hz, O-CH2-CH3), 1.44 (t,
3H, J = 6.8 Hz, O-CH2-CH3), 2.97 (t, 2Н, J = 6.1 Hz, 6-СH2), 3.59 (q, 2H,
J = 6.8 Hz, O-CH2-CH3), 3.92 (q, 2H, J = 6.8 Hz O-CH2-CH3), 3.98-3.99
(m, 4Н, 5-СН2, СН2-NH), 4.05 (q, 2H, J = 6.8 Hz, O-CH2-CH3), 4.10 (q,
2H, J = 6.8 Hz, O-CH2-CH3), 6.47 (d, 2Н, J = 8.6 Hz, C6H4-OH), 6.64 (d,
2H, J = 8.6 Hz, C6H4-OH), 6.65-6.70 (m, 3H, CH-Ar, 7-H, 10-H), 6.88 (d,
1Н, J = 8.1 Hz, CH-Ar), 6.92 (d, 1Н, J = 8.1 Hz, CH-Ar), 6.98 (s, 1Н, 3-
H); 13C NMR (150 MHz, DMSO-d6): δ = 15.7, 19.1, 29.0, 55.0, 56.1 (2C),
56.6, 65.4, 108.3, 112.4, 115.2, 115.3, 116.0, 118.6, 118.9, 121.3, 121.8,
126.3, 127.2, 128.8, 130.4, 131.5, 133.0, 133.5, 133.6, 136.5, 145.0,
147.5, 147.8, 151.9, 155.5, 160.9, 162.5; MS (LCMS) m/z = 543 [M+Н]+;
Anal. calcd for C33H38N2O5: C 73.04, H 7.06, N 5.16, found C 73.28, H
7.24, N 5.33.
and in combination with 10 μM doxorubicin. In all the experiments, the
solvents (EtOH, DMSO) were added in each control to evaluate a
possible solvent cytotoxicity. After the established incubation time, 0.5
mg/mL MTT was added to each well and after 3 h incubation at 37 °C the
supernatant was removed. The formazan crystals were solubilized using
100 μL of DMSO and the absorbance values at 570 and 630 nm were
determined on the microplate reader Victor 3.
Antiproliferative assay in HepG2 cells: The antiproliferative assay was
performed in HepG2 cells at 48 h.[28,29] On day 1, 10,000 cells/well were
seeded into 96-well plates in a volume of 100 μL. On day 2, the test
compounds, each at different concentrations ranging from 0.1 to 100
μM), were added. In all the experiments, the solvents (EtOH, DMSO)
were added in each control to evaluate a possible solvent cytotoxicity.
After the established incubation time with test compound (48 h), 0.5
mg/mL MTT was added to each well and after 3 h incubation at 37 °C the
supernatant was removed. The formazan crystals were solubilized using
100 μL of DMSO and the absorbance values at 570 and 630 nm were
measured on the microplate reader Victor 3.
Antiproliferative assay in HCT116 cells: The growth inhibitory activities of
the test compounds were evaluated by using the sulforhodamine-B
(SRB) assay.[30] Cells were seeded into 96-well microtiter plates in 100
µL of the appropriate culture medium at plating densities of 50,000
cell/mL. After seeding, microtiter plates were incubated at 37 °C for 24 h
prior to addition of the test compounds. After 24 h, several samples of
each cell line were fixed in situ with cold trichloroacetic acid (TCA), to
obtain a measure of the cell population at the time of compound addition.
The test compounds were freshly dissolved in culture medium and
stepwise diluted to the desired final concentrations. After the addition of
different compound concentrations, the plates were further incubated at
37 °C for 72 h. Cells were fixed in situ by the gentle addition of 50 µL of
cold 50% (w/v) TCA (final concentration, 10%) and incubated for 1 h at
4 °C. The supernatant was discarded, and the plates were washed four
times with tap water and air-dried. Sulforhodamine-B solution (100 µL) at
0.4% (w/v) in 1% acetic acid was added to each well, and the plates were
incubated for 30 min at room temperature. After staining, unbound dye
was removed by washing five times with 1% acetic acid and the plates
were air-dried. Bound stain was then solubilized with 10 mM trizma base
and the absorbance was read on an automatic plate reader at 515 nm.
The compound concentration able to inhibit cell growth by 50% (IC50)
was then calculated from semi-logarithmic dose–response plots.
Biology
Materials: CulturePlate 96/wells plates were purchased from PerkinElmer
Life and Analytical Sciences (Boston, MA, USA). Calcein-AM,
doxorubicin
and
MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazoliumbromide) were purchased from Sigma-Aldrich-RBI
s.r.l. (Milan, Italy). Cell culture medium and reagents were purchased
from EuroClone (Milan, Italy) and Sigma-Aldrich.
Cell cultures: MDCK-MDR1 and MDCK-MRP1 are a gift of Prof. P. Borst,
NKI-AVL Institute, Amsterdam, Netherlands. HepG2 tumor cell line was
purchased from ICLC (Genova, Italy). The HCT-116 tumor cell line was
obtained from the National Cancer Institute, Biological testing Branch
(Frederick, MD, USA). MDCK-MDR1, MDCK-MRP1 were grown in
DMEM high glucose supplemented with 10% fetal bovine serum (FBS), 2
mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, in a
humidified incubator at 37 °C with a 5 % CO2 atmosphere. HepG2 was
grown in MEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL
penicillin, 100 g/mL streptomycin, 1% NEAA, in a humidified incubator
at 37 °C with a 5 % CO2 atmosphere. The HCT-116 tumor cell line was
maintained in the logarithmic phase at 37 °C in 5% CO2 humidified air in
RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine,
penicillin (100 U/mL), and streptomycin (0.1 mg/mL).
Statistical analysis: Data were analyzed by one-way ANOVA for repeated
measures followed by post-hoc Bonferroni’s multiple comparison test.
Results are expressed as mean ± SD of at 2-3 independent experiments
in triplicates. Statistical significance was accepted at a level of P < 0.05.
Calcein-AM assays: These experiments were carried out according to a
previously reported procedure.[25] MDCK-MDR1 or MDCK-MRP1 cell line
(50,000 cells per well) was seeded into black CulturePlate 96/wells plate
with 100 µL medium and allowed to become confluent overnight. Test
compounds (100 L), at different concentrations ranging from 0.1 to 100
M, were solubilized in culture medium and added to each well. The
96/wells plate was incubated at 37 °C for 30 min. Calcein-AM in PBS
(100 L) was added to each well to yield a final concentration of 2.5 M,
and the plate was incubated for 30 min. The plate was washed 3 times
with 100 mL ice cold PBS. Saline buffer (100 L) was added to each well
and the plate was read by a PerkinElmer Victor3 spectrofluorimeter at
excitation and emission wavelengths of 485 nm and 535 nm,
respectively. Under these conditions, Calcein cell accumulation in the
absence and in the presence of tested compounds was evaluated, and a
fluorescence basal level was estimated by untreated cells. In treated
wells, the increase of fluorescence with respect to the basal level was
measured. IC50 values were determined by fitting the fluorescence
increase percentage versus log[dose].
Stability tests by 1H NMR
Deuterium oxide (D2O), deuterium chloride (DCl), DMSO-d6, NaH2PO4,
Na2HPO4 and KCl were all purchased from Sigma-Aldrich (Milan, Italy).
The Schiff base derivatives 14 and 15 were monitored during one day for
chemical stability by 1H-NMR at 500 MHz. Spectra were recorded on
Agilent Spectrometer Technologies (Agilent Technologies Italia S.pA.,
Cernusco sul Naviglio, Milan, Italy). Each compound was studied at one
concentration at room temperature in two different mixtures of deuterated
solvents: a) 50 µL of D2O in 750 µL DMSO-d6, and b) 15 µL of DCl in 750
µL DMSO-d6. The 1H-NMR spectra for the starting materials and the
decomposition products were compared to subsequent spectra at various
time points. The formation of new proton peaks over time indicates
instability of the starting Schiff base derivative. Each stability test was
performed in duplicate.
Hydrolysis kinetics of compound 14 in 50 mM phosphate buffer
solution (0.15 M KCl, pH 7.4) was monitored by UV spectrophotometry
(Supporting Information).
Antiproliferative assay in MDCK-MDR1 cells: The co-administration
assay with doxorubicin was performed in MDCK-MDR1 cells at 72 h.[26,27]
On day 1, 10,000 cells/well were seeded into 96-well plates in a volume
of 100 μL. On day 2, the test compounds, each in three concentrations
(1, 10, and 25 μM), were added. On day 3, the medium was removed
and the test compounds, each in three concentrations, were added alone
Acknowledgements
8
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10.1002/cmdc.201800177
ChemMedChem
FULL PAPER
Berardi, A. Azzariti, N.A. Colabufo, S. Cellamare, J. Med. Chem. 2014,
57, 6403-6418.
The publication has been prepared with the support of the
“RUDN University Program 5-100” and by the Russian
Foundation for Basic Research (project No. 17-53-560020). The
Italian Authors acknowledge financial support from the
University of Bari Aldo Moro (Italy).
[27] M. Niso, C. Abate, M. Contino, S. Ferorelli, A. Azzariti, R. Perrone, N.A.
Colabufo, F. Berardi, ChemMedChem 2013, 8, 2026-2035.
[28] M.L. Pati, M. Niso, D. Spitzer, F. Berardi, M. Contino, C. Riganti, W.G.
Hawkins, C. Abate, Eur. J. Med. Chem. 2018, 144, 359-371.
[29] M. Niso, C. Abate, M. Contino, S. Ferorelli, A. Azzariti, R. Perrone, N.A.
Colabufo, F. Berardi, ChemMedChem 2013, 8, 2026-2035.
[30] K.T. Papazisis, G.D. Geromichalos, K.A. Dimitriadis, A.H. Kortsaris,
Optimization of the sulforhodamine B colorimetric assay. Immunol.
Methods 1997, 208, 151-158.
Conflict of interest
The authors declare no conflict of interest.
Keywords: efflux transporters – multidrug resistance – P-
glycoprotein
relationships
-
pyrrolo[2,1-a]isoquinoline
-
structure-activity
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9
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FULL PAPER
Entry for the Table of Contents
15
100
80
60
40
20
0
DXR (10 µM)
cmpd alone
cmpd + DXR (10 µM)
##
###
***
***
1M
10M
25M
Effects on grow th of MDCK-MDR1 cells
Fighting against multidrug resistance: Newly synthesized analogs of marine alkaloids were discovered as reversers of multidrug
resistance (MDR) in cancer cells. Among them, some aldehyde derivatives (3) and Schiff bases (15) proved to be in vitro potent
inhibitors P-glycoprotein (P-gp) and/or multidrug-resistance-associated protein-1 (MRP-1), showing effectiveness in increasing the
activity of doxorubicin (DXR) in chemoresistant cancer cells.
10
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