N-substituted 1,2-dihydroquinolines as anticancer agents: Electronic control of redox stability, assessment of antiproliferative effects, and mechanistic insights

Full text of DOI:10.1002/cmdc.201300210

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Redox chemotherapy: Antiproliferative
activities of a series of N-substituted
1,2-dihydroquinolines capable of caus-
ing redox imbalance in cancer cells are
presented. Detailed studies showed that
these derivatives arrest the cell cycle in
the G2/M phase and induce apoptosis
through an intrinsic pathway character-
ized by loss of mitochondrial membrane
potential, DNA fragmentation, cyto-
chrome c release, and activation of
caspases 9 and 3.
N. John Victor, R. Sakthivel,
K. M. Muraleedharan,* D. Karunagaran*
&& – &&
N-Substituted 1,2-Dihydroquinolines
as Anticancer Agents: Electronic
Control of Redox Stability, Assessment
of Antiproliferative Effects, and
Mechanistic Insights
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DOI: 10.1002/cmdc.201300210
N-Substituted 1,2-Dihydroquinolines as Anticancer Agents:
Electronic Control of Redox Stability, Assessment of
Antiproliferative Effects, and Mechanistic Insights
Napoleon John Victor,[a] Ramasamy Sakthivel,[b] Kannoth Manheri Muraleedharan,*[a] and
Devarajan Karunagaran*[b]
Quinolines and quinolones are privileged core structures in
various therapeutic agents, especially those used against infec-
tious diseases. Antimalarial drugs such as chloroquine, meflo-
quine, and premaquine,[1] and antibacterial agents such as ci-
profloxacin, levofloxacin, and norfloxacin,[2] are some of the
well-known examples from these groups. Our laboratory re-
cently reported the synthesis of 4- and 2-quinolones from
Baylis–Hillman acetates and amines through an SN2’!SNAr!
(D3,4–D2,3 shift)!oxidation sequence involving 1,2-dihydroqui-
nolines (DHQs) as key intermediates.[3] Subsequent analyses
showed that these DHQs, though sensitive to light and
oxygen, can gain stability if the aromatic ring is substituted
with electron-withdrawing groups. Because compounds such
as menadione, ascorbic acid, and piperlongumine can elicit an
antiproliferative response by directly or indirectly perturbing
the redox balance in rapidly proliferating cells, we envisaged
that DHQs will also be interesting candidates for further
study.[4] Results from our investigations in this direction are
presented here.
dant machinery of the parasite by generating reactive oxygen
species (ROS) is believed to be responsible for the activity.
Replacement of the C2 and C4 hydrogen atoms by alkyl
groups (as in compound III) or acylation of the ring nitrogen
A literature search revealed that DHQs have not received
significant attention in drug design, likely due to their de-
creased stability. Synthetic DHQ derivatives with 1,2- or 2,2-dis-
ubstitution pattern have been shown to act as anti-inflamma-
tory agents (e.g., compound I)[5] or HIV-1 reverse transcriptase
inhibitors (e.g., compound II).[6] In addition, derivatives of 2,2,4-
trisubstituted DHQs are well known as lipid peroxidation inhib-
itors,[7] glucocorticoid receptor modulators (e.g., compound
III),[8] and antioxidants.[9] Interestingly, esters of 2,2,4-trimethyl-
1,2-dihydroquinoline-6-ols (e.g., compound IV) were found to
be redox-active due to the possibility of quinone imine forma-
tion through electron transfer after deacetylation by trypano-
somal esterases.[10] A number of DHQ derivatives from this
group possess antitrypanosomal activity with IC50 values in the
nanomolar range. Their ability to exert strain on the antioxi-
(as in compound II) is likely to lower the propensity of the
compound to undergo oxidative transformations. The tenden-
cy of 1,4-DHQs to undergo redox transformation in biological
milieu was made use of by Foucout and co-workers in the de-
velopment of a chemical delivery system (CDS) for central
nervous system (CNS)-active agents, such as g-aminobutyric
acid (GABA) and meta-iodobenzylguanidine (V).[11] These com-
pounds, when conjugated to 1,4-DHQ, were able to cross the
blood brain barrier with ease, and the formation of quinolini-
um salt inside the cells led to accumulation of the agent
through an ion trap mechanism. Because cancer cells are more
susceptible than normal cells to redox imbalance, DHQs are at-
tractive candidates for selectively targeting malignant cells.[4]
Synthesis of 1,2-DHQ derivatives 5–7 typically involves for-
mation of the Baylis–Hillman adduct from the appropriate aro-
matic aldehyde and a,b-unsaturated carbonyl compound,[12] its
acetylation to form intermediate 3, and subsequent tandem
SN2’–SNAr reactions using an amine of interest.[3,13] The required
1,2-DHQs were obtained in moderate to good yields
(Scheme 1).
[a] N. John Victor,+ Dr. K. M. Muraleedharan
Department of Chemistry
Indian Institute of Technology Madras, Chennai, 600036 (India)
E-mail: mkm@iitm.ac.in
[b] R. Sakthivel,+ Prof. D. Karunagaran
DHQs 5–7 differ in the nature of the substituent on the aro-
matic ring. In order to get an understanding of their cytotoxic
effects, the viability of HeLa, SiHa and SW480 cell lines after in-
cubation with these compounds was studied by MTT assay,
and the results are presented in Table 1. Among these, the 6,7-
difluoro- and 6-nitro-substituted DHQs (5 and 7.1, respectively)
were more potent than the 5,6,7,8-tetrafluoro-substituted de-
Department of Biotechnology
Indian Institute of Technology Madras, Chennai, 600036 (India)
E-mail: karuna@iitm.ac.in
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300210.
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DHQs 7.7, 7.8 and 7.10 were superior in terms of their inhibi-
tory activities. As the oxidation of these DHQs after cellular
uptake is a possibility, we prepared quinolones 8–12, quinoline
13 and quinolinium salts 14–15 to investigate whether such
activation in vivo is responsible for the observed cytotoxicity.[3]
Scheme 1. Synthesis of dihydroquinolines 5, 6, and 7.1–7.10. Reagents and
conditions: a) 1,4-diazabicyclo[2.2.2]octane (DABCO), THF, 278C, 24–48 h;
b) AcCl, Et3N, CH2Cl2, 08C!RT, 1–2 h; c) R5NH2 (4), NMP (N-methyl pyrroli-
done), K2CO3, 278C, 1–12 h. Full protocol details and characterization data
are given in the Experimental Section and Supporting Information.
When assayed against HeLa cells, the IC50 values of these com-
pounds were found to be greater than 100 mm suggesting that
the biological effects from such species, if at all, would be
rather weak; this does not undermine the effect from redox re-
actions involving DHQs in which quinolinium ions could be
a byproduct.
rivative 6. Considering the inhibitory effects and the ease of
synthesis of compound 7.1, a subset of compounds (7.2–7.10)
was then prepared by varying the nature of the N-substitution
and subjected to cytotoxicity evaluation. Interestingly, all of
these compounds showed antiproliferative effects in a concen-
tration-dependent manner and had IC50 values in the range of
5.2–84 mm (Table 1). 1-Phenylethylamine- and serine-based
There are a number of anticancer agents that exert their
effect by inducing apoptosis, a process characterized by
plasma membrane blebbing, externalization of phosphatidyl-
serine, cell shrinkage, nuclear and cytoplasmic condensation,
cytochrome c release, activation of caspases, and DNA frag-
mentation.[14] In order to assess the cellular effects of exposure
to DHQs, the most active compounds (7.10 and 7.7) were sub-
jected to further analysis. Because cleavage of DNA into oligo-
nucleosomal-sized fragments is an indication of apoptosis,
these compounds were first evaluated in a DNA fragmentation
assay according to the reported protocol.[15] Western blot anal-
ysis of lysates from HeLa cells treated with these DHQs clearly
showed DNA laddering, which point toward their ability to
induce apoptosis (Figure 1).
Table 1. Cytotoxic effects of 1,2-dihydroquinoline (DHQ) derivatives
against three human cancer cell lines.[a]
Compd
IC50 [mm]
HeLa
SiHa
SW480
5
6
47.8ꢀ1.0
32.4ꢀ6.1
33.2ꢀ0.4
>100
>100
>100
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
42.4ꢀ4.0
20.3ꢀ0.5
19.6ꢀ1.5
29.7ꢀ0.8
27.4ꢀ1.0
34.4ꢀ2.9
17.6ꢀ3.5
13.9ꢀ0.3
84.0ꢀ2.1
8.5ꢀ0.3
47.3ꢀ1.4
24.1ꢀ4.6
18.0ꢀ0.3
27.1ꢀ1.0
23.0ꢀ0.3
27.3ꢀ2.1
5.2ꢀ0.9
37.3ꢀ2.0
18.8ꢀ0.7
12.6ꢀ0.4
20.4ꢀ0.7
20.5ꢀ0.6
19.0ꢀ1.0
5.7ꢀ0.3
9.0ꢀ0.3
60ꢀ0.8
Apoptosis is accompanied by a loss of mitochondrial mem-
brane potential that can be studied using JC-1, a cationic dye
capable of undergoing a reversible change in fluorescence
emission.[16] High membrane potential promotes aggregation
of the dye inside the mitochondria, where it fluoresces orange.
At the same time, cells with low potential contain monomeric
JC-1 in the cytoplasm, where it fluoresces green. During our
studies, the dye was found localized in the mitochondria of
DMSO-treated (control) HeLa cells (Figure 2a), where it formed
orange fluorescent aggregates. However, there was a notable
7.5ꢀ0.2
72.5ꢀ3.5
6.8ꢀ1.8
5.2ꢀ0.1
[a] Cells were treated with various concentrations of test compound and
incubated for 72 h; DMSO was used as the vehicle-only control; cell via-
bility was determined by MTT assay, and the IC50 values were calculated
from the results. Data represent the meanꢀSD of three independent ex-
periments performed in triplicate.
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Figure 1. DNA fragmentation induced in HeLa cells by DMSO (control), Taxol
(25 nm), 7.7 (25 mm) and 7.10 (25 mm).
Figure 3. Western blots obtained after exposing HeLa cells to Taxol (12 nm),
7.7 (25 mm), and 7.10 (25 mm). The following effects were observed: a) cyto-
chrome c release into cytosol, b) activation of cleaved caspase-9, and c) acti-
vation of cleaved caspase-3.
loss of mitochondrial membrane potential after 24 h treatment
of the cells with Taxol and 7.10, as indicated by green fluores-
cence (Figure 2b,c respectively).
only 8.8% apoptotic cells were seen in vehicle-only control
cells (Figure 4b). Similarly, there were 26.8% of G2/M-phase
cells compared with 13.1% in vehicle-only control cells (Fig-
ure 4c). These data were determined to be statistically signifi-
cant (p<0.01) suggesting that 7.10 and related compounds
induce G2/M phase cell-cycle
Caspases are a family of cytosolic cysteine proteases in-
volved in the initiation and execution of apoptosis.[17] In mito-
chondrion-dependent apoptosis, cytochrome c is released into
the cytosol where it binds with Apaf1 and procaspase-9 form-
arrest.
To investigate the possibility
of
DHQ-induced
apoptosis
through the formation of ROS,
a cell-based assay using 2’-7’-di-
chlorodihydrofluorescein diace-
tate (DCFH-DA) was carried
out.[20] Intracellular ROS were de-
tected using the redox-sensitive
Figure 2. Fluorescence microscopic images of HeLa cells stained with JC-1 after 24 h exposure to a) DMSO,
b) Taxol (12 nm), and c) 7.10 (25 mm).
fluorescent
dye
DCFH-DA,
a membrane permeable fluoro-
genic reagent that measures the
ing an apoptosome complex, which then gives caspase-9; sub-
sequent activation of downstream effectors such as caspase-3
triggers apoptosis.[18] In order to see whether DHQs operate
through a mitochondrion-mediated mechanism, HeLa cells
were incubated with 7.7 (25 mm) or 7.10 (25 mm) for 48 h and
analyzed for cytochrome c, cleaved caspase-9, and cleaved cas-
pase-3 expression by Western blot. Results indicate that these
compounds trigger release of cytochrome c, and cleaved cas-
pases 9 and 3, which confirmed that these DHQs induce apop-
tosis via a mitochondrial pathway (Figure 3).
ROS activity within a cell. After diffusion into the cell, DCFH-DA
is deacetylated by cellular esterases to a non-fluorescent com-
pound, which undergoes oxidation by ROS to the highly fluo-
rescent derivative 2’,7’-dichlorofluorescein (DCF). The produc-
tion of ROS was determined by measuring the intensity of the
fluorescence (Figure 5a). Relative DCF fluorescence intensities
were quantitated from the representative images of cells treat-
ed with 7.7 (42.23ꢀ0.004) and 7.10 (32.45ꢀ0.78) and com-
pared with the control group (6.069ꢀ0.03) (Figure 5b). A sig-
nificant increase in intracellular ROS was observed in cells sub-
jected to oxidative stress injury caused by DHQs 7.7 and 7.10
but not in the control cells. To determine whether intracellular
ROS increment originates directly from the compound or indi-
rectly after its effect on mitochondria, the DCFH-DA assay was
carried out with varying incubation times (i.e., 15 min, 30 min,
1 h, 6 h, and 12 h). The results indicate that oxidative stress in-
duced by DHQs is very rapid, and hence the generated ROS is
likely due to a direct effect (for details, see the Supporting In-
formation).
Subsequently, flow cytometry was performed to evaluate
the effect of DHQs on the cell cycle.[19] HeLa cells were treated
with 7.10 (12.5 mm) for 24 h, fixed, stained by propidium
iodide (PI) and analyzed using DMSO and Taxol (12 nm) as neg-
ative and positive controls, respectively (Figure 4). Compound
7.10 caused depletion of cells in the G0/G1 phase with a con-
comitant accumulation of cells in the G2/M phase indicating
a G2/M arrest (Figure 4a). An increase was also observed in the
apoptotic fraction; the S phase, however, seemed unaffected
relative to the DMSO-treated control cells. Treatment of cells
for 24 h with 7.10 resulted in 26% apoptotic cells whereas
Although cancer cells depend on ROS-based signaling for
hyperproliferation, they are very sensitive to fluctuations in
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Figure 4. Flow cytometric analysis of HeLa cells. a) Cell-cycle histograms of HeLa cells treated with DMSO, Taxol (12 nm), and 7.10 (12.5 mm). Statistical analysis
of b) percentage of apoptotic cells, and c) percentage of G2/M phase cells. Data represent the mean ꢀSEM of three independent experiments performed in
triplicate.
Figure 5. Dihydroquinolines (DHQs) induce ROS accumulation in HeLa Cells. a) Representative fluorescent photomicrographs showing relative (rel.) 2’,7’-di-
chlorofluorescein (DCF) fluorescence in cells treated with DMSO (control), 7.7 (25 mm), 7.10 (25 mm), and tert-butyl hydroperoxide (t-BHP) (100 mm). b) Quantifi-
cation of reactive oxygen species (ROS) production as determined by DCF fluorescence. b) Bar graphs presenting the ROS accumulation data. Values represent
the mean ꢀSEM of n=2 independent experiments performed in triplicate; *** p <0.001 vs. control.
redox status. For this reason, compounds capable of inducing
redox imbalance can potentially target such cells. While reac-
tivity and interaction with multiple targets might be consid-
ered a drawback for drug development, if good selectivity can
be achieved, these same features could help overcome resist-
ance, which is a major concern for many anticancer drugs in
clinical use. Active interest in the area of redox chemotherapy
is evident from drug candidates such as 2-methoxyestradiol
(phase II), disulfiram (phase II), motexafin gadolinium (phase III),
and b-lapachone (phase II), which have all reached advanced
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stages of clinical development.[4a,21] The results presented here
clearly show that 1,2-DHQs induce oxidative stress in HeLa
cells. As normal cells are known to be less susceptible to small
fluctuations in ROS levels due to the presence of antioxidant
defense mechanisms, the compounds discussed here can be
considered as leads for the development of new drug candi-
dates. The ready availability of starting materials, ease of syn-
thesis, and possibility of structural diversification for structure–
activity relationship studies are all advantages of this class of
compounds. Efforts to discover highly potent compounds in
this class, preferably with IC50 values in the low nanomolar
range, and investigations to identify the specific targets, if any,
of these compounds are currently underway.
15.5 ppm; IR (neat): n˜ =1708, 1601, 1574, 1499, 1436, 1324, 1289,
1233, 1165, 1106 cmꢁ1
;
HRMS-ESI: m/z [M+K]+ calcd for
C19H18N2O4K: 377.0904, found: 377.0897.
(S)-Methyl
1-(3-hydroxy-1-methoxy-1-oxopropan-2-yl)-6-nitro-
1,2-dihydroquinoline-3-carboxylate (7.10): Reaction of the appro-
priate Baylis–Hillman acetate 3 (R1 =R3 =R4 =H, R2 = NO2, X=Cl)
(200 mg, 0.64 mmol) for 12 h according to the procedure described
above yielded 7.10 as a yellow solid (176 mg, 82%): Rf =0.26
(EtOAc/hexanes, 1:1); mp: 134–1378C; 1H NMR (400 MHz, CDCl3):
d=8.02 (dd, J=9.2, 2.8 Hz, 1H, ArH), 7.92 (d, J=2.4 Hz, 1H, ArH),
7.35 (s, 1H, –C4H), 6.52 (d, J=9.2 Hz, 1H, ArH), 4.55–4.48 (m, 2H,
N–CHaHb/N-CH), 4.34 (d, J=15.2 Hz, 1H, N–CHaHb), 4.25 (dd, J=
11.6, 5.6 Hz, 1H, CHaHbOH), 4.09 (dd, J=11.6, 7.2 Hz, 1H, CHaHbOH),
3.83 (s, 3H, –OCH3), 3.81 (s, 3H, –OCH3), 2.22 ppm (brs, 1H, –OH);
13C NMR (100 MHz, CDCl3): d=169.9, 165.1, 151.0, 139.0, 133.8,
127.9, 125.8, 123.5, 120.7, 110.3, 62.6, 59.8, 53.0, 52.3, 45.6 ppm; IR
(KBr): n˜ =3457, 2954, 1740, 1721, 1640, 1573, 1495, 1434, 1324,
1298, 1221, 1174, 1088, 1010, 929, 817, 742 cmꢁ1; HRMS-ESI: m/z
[M+Na]+ calcd for C15H16N2O7Na: 359.0855, found 359.0866.
In conclusion, we have discovered a novel series of 1,2-dihy-
droquinoline (DHQ) derivatives with promising anticancer ac-
tivities. Detailed biochemical studies showed that they are ca-
pable of inducing apoptosis as evidenced by DNA fragmenta-
tion, disruption of mitochondrial membrane potential, cyto-
chrome c release, and activation of caspase-9 and caspase-3.
Their ability to induce oxidative stress in cancer cells was un-
equivocally confirmed by DCFH-DA fluorescence assay. Flow
cytometric analysis of HeLa cells treated with a representative
compound (7.10) revealed that these DHQs cause cell-cycle
arrest in the G2/M phase. Taken together, these observations
point toward the operation of an intrinsic pathway of apopto-
sis in the presence of these compounds. Detailed structure–ac-
tivity relationship studies and efforts to gain an understanding
of their biological effects at a molecular level are currently in
progress.
Acknowledgements
Financial support of this work by the Council of Scientific & In-
dustrial Research (CSIR), India (01(2121)/07/EMR-II) and the
Indian Ministry of Science and Technology through the Depart-
ment of Science and Technology (DST) (SR/S1/OC-13/2007) and
the Department of Biotechnology (DBT) (BT/01/COE/07/04) is
gratefully acknowledged N.J.V. and R.S. thank the CSIR for re-
search fellowships.
Keywords: apoptosis · cytotoxic agents · dihydroquinolines ·
G2/M cell-cycle arrest · reactive oxygen species
Experimental Section
[1] M. Foley, L. Tilley, Pharmacol. Ther. 1998, 79, 55–87.
General procedure for the preparation of dihydroquinolines: A
stirred solution of the Baylis–Hillman acetate (1 equiv) in N-methyl
pyrrolidone (6 mLmmolꢁ1) under a nitrogen atmosphere was treat-
ed with K2CO3 (1.5 equiv). The appropriate amine (2 equiv) was
then added to the reaction mixture, and stirring was continued at
room temperature for 1–12 h. After completion (determined by
TLC), water was added until the solution became turbid, and the
mixture was stirred for an additional 30 min. The precipitated prod-
uct was isolated by filtration, washed with water and dried in
vacuo. This near-pure material was then purified further to remove
trace impurities by column chromatography (EtOAc/hexanes; gra-
dient elution). Isolated yields of dihydroquinolines ranged from
50–85%. Spectral data of representative examples (7.7 and 7.10)
are given below, and that of other compounds are given in the
Supporting Information.
[2] L. A. Mitscher, Chem. Rev. 2005, 105, 559–592.
[3] J. V. Napoleon, M. K. Manheri, Synthesis 2011, 3379–3388.
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(R)-Methyl 6-nitro-1-(1-phenylethyl)-1,2-dihydroquinoline-3-car-
boxylate (7.7): Reaction of the appropriate Baylis–Hillman acetate
3 (R1 =R3 =R4 =H, R2 = NO2, X=Cl) (200 mg, 0.64 mmol) for 5 h ac-
cording to the procedure described above yielded 7.7 as a yellow
gummy solid (162 mg, 75%): Rf =0.43 (EtOAc/hexanes, 1:4);
1H NMR (400 MHz, CDCl3): d=8.00 (dd, J=9.2, 2.4 Hz, 1H, ArH),
7.90 (d, J=2.8 Hz, 1H, ArH), 7.41–7.29 (m, 6H, ArH, –C4H), 6.64 (d,
J=9.2 Hz, 1H, ArH), 5.20 (q, J=6.8 Hz, 1H, N–CH), 4.35 (d, J=
16.0 Hz, 1H, N–CHaHb), 4.14 (d, J=16.0 Hz, 1H, N–CHaHb), 3.76 (s,
3H, OCH3), 1.69 ppm (d, J=6.8 Hz, 3H, CH3–CH); 13C NMR
(100 MHz, CDCl3): d=165.2, 151.2, 139.0, 137.6, 134.1, 129.1 (2C),
128.4, 128.1, 127.0 (2C), 126.0, 122.8, 119.6, 110.0, 55.8, 52.1, 43.3,
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Received: May 12, 2013
Revised: July 12, 2013
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