Synthesis of Triphenylethylene-Naphthalimide Conjugates as topoisomerase-IIα inhibitor and HSA binder

Article abstract of DOI:10.1002/cmdc.202100034

A series of triphenylethylene-naphthalimide (TPE-naph) conjugates was synthesized by a molecular hybridization technique, and their anticancer activity was evaluated in vitro on 60 human cancer cell lines through their cytotoxicity. The ratios of E and Z isomers were determined on the basis of HPLC methodology and NMR spectroscopy. The structure-activity relationship for anticancer activity was deduced on the basis of the nature and bulkiness of the amine attached to the C-4 position of the naphthalene ring. Experimental and molecular modeling studies of the most active TPE-naph conjugate bearing a morpholinyl group showed that it was able to inhibit topoisomerase-II (TOPO-II) as a possible intracellular target. Moreover, the transportation behavior of TPE-naph conjugate towards human serum albumin (HSA) indicated efficient binding affinity. The steady-state and time-dependent fluorescent results suggested that this conjugate quenched HSA significantly through static as well as dynamic quenching. Thus, this report discloses the scope of triphenylethylene-naphthalimide (TPE-naph) conjugates as efficient anticancer agents.

Full text of DOI:10.1002/cmdc.202100034

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Synthesis of Triphenylethylene-Naphthalimide Conjugates
as topoisomerase-IIα inhibitor and HSA binder
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Sudesh Rani,[a] Vijay Luxami,[a] and Kamaldeep Paul*[a]
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A series of triphenylethylene-naphthalimide (TPE-naph) con-
jugates was synthesized by a molecular hybridization techni-
que, and their anticancer activity was evaluated in vitro on 60
human cancer cell lines through their cytotoxicity. The ratios of
E and Z isomers were determined on the basis of HPLC
methodology and NMR spectroscopy. The structure-activity
relationship for anticancer activity was deduced on the basis of
the nature and bulkiness of the amine attached to the C-4
position of the naphthalene ring. Experimental and molecular
modeling studies of the most active TPE-naph conjugate
bearing a morpholinyl group showed that it was able to inhibit
topoisomerase-II (TOPO-II) as a possible intracellular target.
Moreover, the transportation behavior of TPE-naph conjugate
towards human serum albumin (HSA) indicated efficient bind-
ing affinity. The steady-state and time-dependent fluorescent
results suggested that this conjugate quenched HSA signifi-
cantly through static as well as dynamic quenching. Thus, this
report discloses the scope of triphenylethylene-naphthalimide
(TPE-naph) conjugates as efficient anticancer agents.
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Introduction
such as coumarin (III) and xanthene (IV) manifested significant
anticancer activity (Figure 1).[13,14]
Cancer is a cluster of diseases that accounts for leading cause of
mortality globally.[1] According to cancer reports in 2018, about
17.0 million cancer cases were diagnosed and 9.5 million died
worldwide.[2] Due to prevalence of cancer, development of
potential anti-tumor drugs with optimal physicochemical profile
is of utmost importance.[3] Long term administration of drug is
the main cause of paramount failure in drug discovery and
remains prevalent as challenge for contemporary medicinal
chemistry.[4] Combinational principle or molecular hybridization
is the effective way to develop multi-target drug candidates
with enhanced biological activities.[5] The success of substituted
olefins (such as tamoxifen (I), a marketed drug) and naphthali-
mide (II; such as amonafide, in clinical trials) provoked to
synthesize their conjugate molecules in the area of cancer
therapy.[6,7]
The triphenylethylene (TPE) scaffold viz., tamoxifen, a first-
generation selective estrogen receptor modulator (SERM) owing
the feature of estrogen diethylstilbestrol has emerged as target
motif for the treatment of cancer in pharmacotherapy.[8,9] As an
anticancer agent, it resists tumor formation either by blocking
estrogen receptor or by interacting with factors which impair
DNA replication.[10,11] On account of partial agonistic character
of tamoxifen,[12] extensive efforts have been devoted to endow
new triarylethylene analogues as potent anticancer agents with
increased efficacy and toxicological profile. Adopting the
technique of molecular hybridization, coupling of triphenyl-
ethylene analogue (tamoxifen) with various pharmacophores
On the other hand, naphthalimide is a class of heterocycles,
constituting π-deficient planar aromatic structure and
a
versatile pharmacophore with diverse biological applications in
pharmaceuticals such as anticancer, antibacterial, antiviral and
analgesic.[15,16] Naphthalimides exert antiproliferative activity
due to their ability of intercalate into the base pairs of DNA,
through DNA-groove binding and topoisomerase (TOPO)
inhibition.[17] Naphthalimide analogue such as amonafide, has
reached in phase II clinical trials and displayed promising
anticancer activity.[18] But apparently, clinical development of
this moiety was impeded due to its neurological toxicity, poor
physicochemical property and poor therapeutic index.[19,20] Thus,
researchers have focused on efforts geared toward the mod-
ification of imide position and substituents at C-4 position of
naphthalene ring. Rao et al. have synthesized naphthalimide-
benzothiazole/cinnamide derivatives (V) as active anticancer
agent against lung and colon cancer cell lines as well as
inhibiting the topoisomerase-II activity,[21] whereas Lu et al.
reported a series of C-4 naphthalimide-benzothiazole conju-
gates (VI) with potent inhibitory activity towards murine
melanoma cell lines.[22] These promising anticancer activities of
naphthalimide and triphenylethylene analogues have prompted
us to develop novel structural hybrids evading drug resistance.
To the best of our knowledge, no report on triphenylethylene-
naphthalimide (TPE-naph) conjugate has been explored as
anticancer agents in drug discovery.
In view of these investigations, we designed target hybrid
by reducing the flexibility as depicted in Figure 2, by conjugat-
ing amino triphenylethylene scaffold to 4-substituted naph-
thalic anhydride without any linker. Further, triphenylethylene-
naphthalimide conjugates substituted with various primary and
secondary amines were synthesized and evaluated for their
cytotoxicity against 60 human cancer cell lines. As triphenyl-
ethylene and naphthalimide derivatives are observed as top-
[a] S. Rani, Dr. V. Luxami, Dr. K. Paul
School of Chemistry and Biochemistry
Thapar Institute of Engineering and Technology
Patiala-147004, Punjab (India)
E-mail: kpaul@thapar.edu
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100034
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Figure 1. Marketed drug tamoxifen (I), drug in clinical trials amonafide (II), lead hybrids based on triphenylethylene (III, IV) and naphthalimide (V, VI).
Subsequent treatment of acenaphthene (3) with N-bromosucci-
nimide in DMF at room temperature, furnished intermediate 4
in 97% yield. Further, oxidation of intermediate
4 was
accomplished with refluxing of K2Cr2O7 in acetic acid to afford
6-bromo-1H,3H-benzo[de]isochromene-1,3-dione (5) in 72%
yield. 6-Bromo-2-(4-(1,2-diphenylprop-1-en-yl)phenyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (6) was obtained in quantitative
yield on refluxing of compound 2 to a stirred solution of 5 in
zinc acetate and ethanol for 15 h. Finally, compound 6 was
refluxed with primary or secondary amine in the presence of
potassium carbonate and DMF for 2–3 h to obtain isomeric
products 7–21 in good yields. Attempts were made to separate
E and Z isomers from mixture by column and preparative
chromatography but was unsuccessful. Without separation of E
and Z isomers, these synthesized compounds were character-
ized by NMR and mass spectrometric methods. The structures
of synthesized TPE-naph were assigned on the basis of spectral
data (Figures S1–S34 in the Supporting Information). For
instance, 1H NMR of conjugate 7 showed two characteristic
multiplets (due to mixture of E and Z isomers) of morpholine at
δ 4.04–3.99 and 3.29–3.24 ppm. The signals at δ values 67.0 and
53.5 ppm in 13C NMR further confirmed the assigned structure.
To confirm the ratio and stereochemical assignment of E
and Z configuration of TPE-naphthalimide conjugate 7, re-
versed-phase high performance liquid chromatography (RP-
HPLC) has been performed. Figure S35 represents the HPLC
separation of E and Z isomers of analogue 7 with retention
times 15.77 and 17.42 min, respectively. Further, E/Z ratio of
TPE-naph was determined by 1H NMR from the respective
integral of methyl protons.[24] 1H NMR spectrum of compound 7
(E/Z) exhibited two methyl singlets centered at 2.21 and
2.15 ppm. The singlets at δ values 2.21 and 2.15 ppm are due to
CH3 group, which was assigned E (tR =15.77 min) and Z
configurations (tR =17.42 min), respectively. The signal identities
were established on the basis of retention times of compound
7 obtained from chromatogram. As well the ratios of E and Z
Figure 2. Molecular hybridization of TPE with 4-substituted naphthalic
anhydride.
oisomerase inhibitors,[11,21] therefore herein, the mechanism of
cytotoxicity of most potent TPE-naphthalimide 7 is elucidated
through topoisomerase-II (TOPO-II) inhibition assay. Further-
more, molecular modeling was performed to determine the
interactions between TOPO-II and TPE-naphthalimide which
supports the plausible experimental data. The transportation
behavior against most potent TPE-naph conjugate with human
serum albumin (HSA) has also been explored with spectroscopy
techniques.
Results and Discussion
Triphenylethylene-naphthalimide conjugates were synthesized
via multistep reactions including molecular hybridization
technique (Scheme 1). Initially, McMurry reaction[23] was em-
ployed using the precursors 4-aminobenzophenone (1) and
acetophenone in the presence of Zn and TiCl4 in anhydrous THF
to afford 4-(1,2-diphenylprop-1-en-1-yl)aniline (2) in 60% yield.
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°
Scheme 1. Synthesis of triphenylethylene-naphthalimide conjugates. a) Zn, TiCl4, acetophenone, pyridine, THF, N2, 0 C to reflux, 60% yield; b) N-
bromosuccinimide, DMF, RT, 3 h, 97% yield; c) K2Cr2O7, acetic acid, reflux, 4 h, 72% yield; d) Zn(OAc)2, EtOH, reflux, 15 h, 98% yield; e) HNR1R2, K2CO3, DMF,
reflux, 2–3 h, 60–80% yields.
1
isomers of analogue 7 were calculated from H NMR is same as
that found from HPLC methodology, that is, 1:3.3. Moreover,
the stereochemical assignments of E and Z configurations were
given on the basis of NOESY spectroscopy (Figure S36). It has
been observed that methyl signal at 2.15 ppm (Z isomer)
interacts with the protons of alpha phenyl ring, which are
shielded, that is, at δ value 7.23 ppm. Therefore, it is concluded
that protons of E isomer of TPE-naphthalimide conjugate were
found to be deshielded than Z isomer and this relationship also
holds good for tamoxifen.[25,26]
Anti-proliferative activity
Preliminary, in vitro antiproliferative activity of compounds (E/Z)
7–21 at single dose concentration (10 μM) was evaluated
against 60 human cancer cell lines by National Cancer Institute
(NCI, Bethedsa, USA; Figure S37).[27] Among tested compounds,
derivative 7 possessing morpholine group displayed excellent
percentage growth inhibition against almost all cancer cell
lines. Compound 7 exhibited good cytotoxicity towards non-
small cell lung (HOP-92, GI=78.7%; NCI-H23, GI=78.4%), CNS
(SF-295, GI=71.5%) and ovarian (OVCAR-3, GI=75.1%) cancer
cell lines (Figure 3). It was inferred that compound 7 elicited
improve cytotoxic potency towards NCI-H23 non-small cell
Figure 3. Comparison of anticancer activity between compound 7 and 9
lung), OVCAR-3 (ovarian) and MDA-MB-231 (breast cancer) cell
lines in comparison to amonafide. When morpholinyl group of
analogue 7 was replaced with piperidine moiety, it led to
compound 8 with complete loss of antiproliferative activity
which indicated that increase in lipophilicity reduces cytotox-
icity towards cancer cell lines. Compound 9 (GI=À 9.19%) with
methyl piperazine moiety was found to be potent against SR
against an in vitro panel of a) leukemia, non-small cell lung, colon, CNS
cancer cell lines and b) melanoma, ovarian, renal, prostate, breast cancer cell
lines.
leukemia cancer cell line when compared to amonafide (GI=
91.9%). Analogue 9 also exerted excellent anticancer activity
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towards K-562 (leukemia cancer) cell line with growth inhibition
of 90.8% (Figure 3). It has also been observed that compound
10 substituted with N-ethyl piperazinyl group showed lower
activity when compared to 9 having N-methylpiperazinyl
substituent that also proved that cytotoxicity is dependent
upon lipophilicity. Derivative 11 (N-phenyl piperazine) displayed
weak anticancer activity with average percentage of inhibition
<40% except UO-31 (renal cancer) cell line while derivative
belonging to N-benzyl piperazine (12) did not show any
significant inhibition due to the presence of bulky amine.
On the other hand, compounds 13 (cyclohexyl amine), 15
(N,N-dimethyl ethylene diamine) and 17 (ethanol amine) with
primary amines were least effective against cancer cell lines.
Compound 14 bearing 4-(2-aminoethylene)morpholine was
found to be sensitive towards leukemia (K-562, MOLT-4, RPMI-
8226, SR), non-small cell lung (EKVX), colon (HCT-116, HCT-15),
ovarian (OVCAR-4) and breast (MCF-7, T-47D) cancer cell lines
with GI>50%. Analogues 16 (GI=40.4%) and 18 (GI=45.4%)
with respective butyl amine and allyl amine displayed selectivity
towards UO-31 renal cancer cell line. Among derivatives 19, 20
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Figure 4. Effect of TPE-naph 7 on the relaxation of supercoiled plasmid DNA
topoisomerase-II. Supercoiled DNA (lane b) was incubated with topoisomer-
ase II in the absence (lane a) and presence of analogue 7 at 25 μM (lane d),
50 μM (lane e) and 100 μM (lane f). Etoposide was used as positive control at
100 μM (lane c).
and 21 substituted with aromatic amines, only analogue 20 Human serum albumin studies
with 4-fluoroaniline showed significant activity against leukemia
cancer cell line (K-562, GI=67.9%; SR, GI=63.1%) and colon
cancer cell lines (HCT-15, GI=52.9%; HT-29, GI=61.4%). This
might be attributed to the presence of fluoro at para-position
of aniline. Thus, TPE-naph conjugates with secondary amines
displayed promising percentage growth inhibition than ana-
logues bearing primary amines. It was inferred that in this series
7–21, compounds were found to be more sensitive towards
leukemia and colon cancer cell lines. Moreover, from structure-
activity relationship, it is demonstrated that increasing lip-
ophilicity and bulkiness of substituent attached to C-4 position
of naphthalimide ring resulted in decrease in cytotoxic activity.
Therefore, the nature and size of amine influenced the
anticancer activity of TPE-naph conjugates.
HSA is a major extracellular plasma protein in blood and
featuring specific binding site for transportation and deposition
of drug. The binding behavior of drug towards HSA could
modulate its key pharmacodynamic and pharmacokinetic
properties like efficacy, metabolism and drug distribution.[29]
Various promising drugs due to their high binding affinity
towards HSA were found to be futile. Henceforth, the binding
interaction studies have been done between HSA and the
biological active molecules insights drug-likeliness properties as
well as explore screening of molecules in drug discovery. Thus,
to analyze the interaction between most active TPE-naph 7 and
HSA, in vitro studies were carried out by UV-visible absorption
and fluorescence emission techniques.
Topoisomerase inhibition
UV-visible absorption spectroscopy
Topoisomerase-II plays a key role in the segregation of strands
of dsDNA which leads to replication resulting in proliferation of
cell.[28] Therefore, to evaluate the mechanism of cytotoxicity of
TPE-naph conjugates, we targeted the inhibitory effect of
analogue 7 on topoisomerase-IIα using supercoiled pHOT1
plasmid DNA. Etoposide, a well-known DNA intercalator was
used as a positive control at 100 μM. Figure 4 represents the
inhibitory effect of 7 on the relaxation of plasmid DNA
mediated by TOPO-IIα at three different concentrations, that is,
25, 50 and 100 μM. Compound 7 induces partial inhibitory
activity at 25 μM and complete inhibition at 50 and 100 μM. It
revealed that the inhibiting of TOPO-II by compound 7
suggested a possible mechanism of cytotoxicity through top-
oisomerase-II inhibition.
UV-visible spectroscopy is a reliable tool employed to deter-
mine the structural alterations in protein and to investigate the
formation of complex on drug binding. In this study, absorption
band of HSA at 280 nm (7 μM) was recorded in phosphate
buffer (0.1 M) at pH 7.4. The band at 280 nm corresponds to
aromatic ring of amino acid residues Trp214, Tyr411 and
Phe403 in HSA. On incremental addition of compound 7, the
enhancement in absorption band of HSA was observed with
appearance of a new band at 400 nm (Figure 5a). The maximum
peak of absorption remains unaltered which indicated that
analogue 7 binds with the cavity of HSA through noncovalent
interactions. This is evident by the existence of π-π stacking
interactions between the aromatic rings of analogue 7 and
tryptophan, tyrosine and phenylalanine residues in the binding
cavity of HSA.[30]
In order to evaluate the binding affinity of TPE-naph with
HSA, the binding constant (Kb) was calculated from a plot of 1/
A-Ao versus 1/Q using Benesi-Hildebrand equation (1, SI)[31] and
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Figure 5. Effect of the incremental addition of analogue 7 on a) UV/Vis absorption and b) fluorescence emission of HSA (7 μM) in phosphate buffer (pH 7.4).
the ratio of intercept to slope was found to be equal to 1.4×
104 MÀ 1 (Figure S38).
nearly one that revealed strong binding interaction among HSA
and TPE-naph (Figure S40).
To determine the spontaneity for formation of complex
between conjugate 7 and HSA, change in free energy has been
calculated that was found to be À 8.4 kcal/mol by employing
Fluorescence spectroscopy
following equation 1.[36] The negative value of G demonstrates
~
Fluorescence spectroscopy is a sophisticated technique which
explores the binding interactions of drug and protein. Under
physiological conditions, the fluorescence spectrum of HSA of 7
in phosphate buffer (pH 7.4) was recorded with an excitation
wavelength of 280 nm. The fluorescence spectrum of HSA
spontaneity and existence of hydrophobic interaction among
HSA:TPE-naph.
DG ¼ À RTlnKb
(1)
exhibited an emission band at 340 nm due to tryptophan Life-time fluorescence technique
residues. With incremental addition of compound 7, the
emission band of HSA at 340 nm was consistently quenched
which revealed some changes around Trp214 of HSA (Fig-
ure 5b). The λmax of emission of HSA displayed hypsochromic
shift by 5 nm (from 340 to 335 nm). This indicated that
analogue 7 interacts with the hydrophobic cavity of HSA.
The quenching of HSA emission by TPE-naph is either due to
static quenching or dynamic quenching mechanism. In order to
have better understanding of quenching mechanism, time-
resolved fluorescence experiment was carried out.[37] Figure 6
The quenching of emission band of HSA upon incremental
addition of 7 is the result of either dynamic or static quenching.
Further, the Stern-Volmer equation (S2) was employed to
evaluate Stern-Volmer constant, Ksv and the bimolecular
collision constant, Kq which explicate the binding mode of 7 to
HSA.[32] (Figure S39). From the linear plot of regression curve of
Fo/F versus [Q], Ksv and Kq were calculated to be 6.8×104 MÀ 1
and 1.6×1013 MÀ 1 sÀ 1, respectively. As the bimolecular collision
constant (Kq) for HSA:TPE-naph system is greater than diffusion
limiting quenching in water,[33] that is, 2×1010 MÀ 1 sÀ 1 which
suggested the nature of quenching in the present case is static,
that is, formation of analogue 7 and HSA complex in ground
state.[34] Further, the binding constant and the number of
binding sites have been determined using a modified Stern-
Volmer equation (S3) from the plot of log[(Fo-F)/F] versus log[Q]
which give Kb and n as intercept and slope, respectively.[35] The
binding constant of compound 7 toward HSA was calculated to
be 1.4×104 MÀ 1 and the number of binding sites per HSA was
Figure 6. Fluorescence decay profile of HSA on addition of various concen-
trations of 7 in phosphate buffer (pH 7.4).
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Table 1. Fluorescence decay profile of HSA and compound 7.
1
2
System
Conc.
τ1 [ns]
τ2 [ns]
τ3 [ns]
α1
α2
α3
τav
χ2
3
4
5
6
HSA
HSA-7
3.64
3.65
4.19
3.47
6.97
7.47
7.94
7.83
1.17
0.51
0.64
0.43
0.26
0.35
0.42
0.32
0.70
0.60
0.45
0.53
0.02
0.04
0.12
0.13
5.06
4.80
4.10
2.11
1.01
1.11
1.39
1.23
1:2
1:3
1:5
7
8
9
represents the life-time decay profile of HSA (7 μM) in the
absence and presence of different concentrations of compound
7. The average lifetime triexponential values were calculated
using the equation 2. Upon addition of 14 μM of analogue 7,
the life-time of HSA decrease is marginal (5.06 to 4.80 ns). As
depicted from Table 1, at higher concentration (21–35 μM) of
conjugate 7, reduction in lifetime of HSA is more pronounced. It
revealed that other phenomena of quenching may also exist
i.e. dynamic quenching. While the steady state fluorescence
experiment demonstrate the possibility of static quenching.
From above results, it was inferred that both the static as well
as dynamic quenching might be responsible for the interaction
of analogue 7 to HSA.
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tav ¼ a1t1 þ a2t2 þ a3t3
(2)
Molecular docking studies
Figure 7. 3D docked structures of compound 7. a) E isomer, b) Z isomer and
c) etoposide with topoisomerase-II-DNA complex (PDB ID: 5GWK).
To better understand the inhibition behavior of E and Z isomers
of TPE-naph analogue 7 towards TOPO-II-DNA complex (PDB ID:
5GWK), docking studies have been performed using AutoDock
vina 4.0 software.[38] From docking studies, the binding energy
scores E and Z isomers for analogue 7 and reference drug Conclusion
etoposide were calculated to be À 11.3, À 11.3 and À 9.8 kcal/
mol, respectively. It was observed that E and Z isomers of
conjugate 7 exhibited better binding affinity than reference
drug etoposide while these binds only with amino acid residues
of TOPO-II. It demonstrates the existence of strong intermolec-
ular forces between the ligands and TOPO-II. Figure 7 repre-
sents the 3D docked structures of compound 7 (E isomer, a; Z
isomer, b) and etoposide with TOPO-II-DNA complex. From the
binding pattern, it was inferred that E isomer of 7 interacts with
the active sites of TOPO-II-DNA complex via THR618 (d=2.23 Å)
through conventional-hydrogen bonding; TYR805, LYS611,
LYS614 and PHE807 residues through alkyl-alkyl and π-alkyl
hydrophobic interactions while Z-isomer of analogue 7 showed
unconventional hydrogen bonding (Asp541, Tyr805) and hydro-
phobic interactions, (Gly617, Thr618, Lys614 and Phe807). On
the other hand, etoposide binds with TOPO-II complex through
Asn770 (d=2.38 Å) and Gln773 (d=2.52 Å) residues by conven-
tional hydrogen bondings; Met766 through hydrophobic inter-
actions; and sugar backbone of DNA through DT-5 (d=2.82 Å),
DC-4 (d=3.75 Å), DA-6 (d=2.95 Å), DG-5 (d=3.10 Å). The
results of docking studies are consistent with our experimental
data and proved that TPE-naphthalimide conjugate acts as
TOPO-II inhibitor.
A novel series of triphenylethylene-naphthalimide conjugates
has been synthesized by substituting different amines at the C-
4 position of naphthalimide ring and evaluated for their
cytotoxicity towards 60 human cancer cell lines. Preliminary
results indicated that TPE-naph conjugates were found to be
more sensitive towards leukemia and colon subpanels at one-
dose concentration (10 μM). Among synthesized analogues,
only compound
7 bearing morpholinyl group exhibited
excellent broad-spectrum activity. It was also observed that an
increase in the lipophilicity of the substituent lowers the
cytotoxicity of TPE-naph hybrids towards cancer cell lines. More
importantly, it is concluded that TPE-naph derivatives substi-
tuted with secondary amines exhibited better cytotoxicity.
Thus, SAR studies revealed that the nature and bulkiness of
substituent play key roles in antiproliferative activity of TPE-
naph. The most active compound, 7, significantly inhibited the
activity of TOPO-II at 50 μM concentration. This suggested the
plausible mechanism of cytotoxicity through TOPO-II inhibition;
also supported by docking results. The docking results analyzed
the preferential binding sites of TPE-conjugate 7 with the
TOPO-II-DNA complex. It was observed that the oxygen atoms
of naphthalimide and phenyl rings of triphenylethylene inter-
acts only with TOPO-II through hydrogen bonding and π-π
stacking hydrophobic forces with binding affinity À 11.3 kcal/
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5-Bromo-1,2-dihydroacenaphthylene (4):[39] To a suspension of
acenaphthene (3 g, 19.4 mmol) in DMF, a solution of N-bromosucci-
nimide (4.15 g, 23.35 mmol) in 30 mL of DMF was added slowly at
room temperature. The solution was stirred vigorously for 3 h. The
reaction was quenched by adding 300 mL of cold water into
solution, and brown colored precipitates were obtained on
filtration. The crude was recrystallized to obtain pure 5-bromo-1,2-
mol. Unlike etoposide, TPE-naph conjugate 7 interacts only with
TOPO-II. Thus, it indicated TPE-naph conjugate act as TOPO-II
inhibitor. Moreover, the interaction of 7 with HSA indicated the
binding constant in order of 104 MÀ 1 which is the evident of
drug-likeness properties of these analogues. Thus, the present
manuscript highlights the approaches employed to design and
synthesize new molecular framework (triphenylethylene-naph-
thalimide conjugates) as therapeutic template to target TOPO-
IIα for anticancer activity. These results extended a new route
to design and develop efficient drug candidates as anticancer
agent.
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9
°
dihydroacenaphthylene (4) in 97% yield (mp: 54–56 C).
4-Bromo-1,8-naphthalic anhydride (5):[40] Potassium dichromate
(6.31 g, 21.45 mmol) was added slowly to a solution of 5-bromo
°
acenaphthene 4 (1 g, 4.2 mmol) in glacial acetic acid at 0 C. The
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mixture was stirred for 10 min at room temperature and then
allowed to reflux for 4 h. The cooled reaction mixture was poured
into 500 mL of water and filtered to provide green colored
precipitates. The excess chromium salt left in residue was removed
by washing with boiled water. After washing, white colored solid
Experimental Section
°
(5) was obtained in 72% yield (mp: 221–224 C).
Reagents and conditions
6-Bromo-2-(4-(1,2-diphenylprop-1-en-1-yl)phenyl)-1H-benzo[de]
isoq uinoline-1,3(2H)-dione (6): To a two necked round-bottom
flask, 4-bromo-1,8-naphthalic anhydride (5; 1 g, 3.61 mmol) and
zinc acetate (0.95 g, 4.3 mmol) were dispersed in 15 mL of ethanol
under the inert atmosphere. To the stirred reaction mixture, 4-(1,2-
diphenylprop-1-en-1-yl)aniline (2) (0.52 g, 1.81 mmol) was added
and heated to reflux for 15 h. After completion of reaction, the hot
filtration of the suspension was carried out and repeatedly washed
with ethanol. The crude was purified by column chromatography
(hexane/chloroform 8:2) to obtain a yellow colored pure product
All the chemicals and reagents were commercially available from
Spectrochem, Aldrich (Sigma-Aldrich), TCI and used without further
purification. Analytical TLC was performed using spectrochem silica
gel (GF-254) pre-coated plates and visualized TLC under UV light or
by iodine indicator. 1H NMR spectra were carried out on Jeol
spectrometer 400 MHz and Bruker À 500 MHz while 13C NMR spectra
were recorded on Jeol spectrometer 100 MHz, using deuterated
solvents (CDCl3 and [D6]DMSO). Using TMS as an internal reference,
chemical shifts (δ) and coupling constants (J) were recorded in ppm
and Hz, respectively. Melting points were determined on equi-
ptronics melting point apparatus and were uncorrected. UV/Vis and
fluorescence studies were accomplished on Shimadzu and Varian
Cary Eclipse fluorescence spectrophotometer, respectively. The
time fluorescence studies were carried out using deltaflexTM
spectrometer. Mass spectra of synthesized compounds were
observed at water Micromass-Q-T of Micro.
1
(6) in 98% yield; E/Z 1:2; H NMR (400 MHz, CDCl3): δ=8.67–8.64
(m, 1H, ArH), 8.62–8.59 (m, 2H, ArH), 8.44–8.42 (d, J=7.88 Hz, 1H,
ArH), 8.37–8.35 (d, J=7.88 Hz, 1H, ArH), 8.14–8.12 (d, J=7.92 Hz,
1H, ArH), 8.10–8.08 (d, J=7.92 Hz, 1H, ArH), 7.97–7.89 (m, 2H, ArH),
7.43–7.37 (m, 4H, ArH), 7.31–7.29 (d, J=7.96 Hz, 4H, ArH), 7.23–7.22
(m, 2H, ArH), 7.16–7.10 (m, 4H, ArH), 7.08–7.02 (m, 4H, ArH), 6.97–
6.95 (m, 3H, ArH), 2.21 (s, 3H, CH3), 2.13 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3 +[D6]DMSO): δ=163.7, 163.6, 163.5, 143.7, 143.6,
143.5, 143.2, 143.1, 142.7, 138.4, 138.3, 136.6, 136.5, 133.7, 133.6,
132.8, 132.3, 132.2, 131.5, 131.4, 131.3, 131.2, 130.8, 130.7, 130.6,
130.5, 130.0, 129.9, 129.3, 129.2, 128.5, 128.4, 128.3, 128.1, 127.9,
127.7, 127.6, 126.8, 126.6, 123.3, 123.2, 122.4, 122.3 (ArC), 23.6 (CH3),
23.5 (CH3); ESI MS: m/z 544.0 (M+H)+.
Synthesis
4-(1,2-Diphenylprop-1-en-1-yl)aniline (2): Titanium tetrachloride
(2.77 mL, 25.30 mmol) was added gradually to a suspension of Zn
(3.24 g, 50.75 mmol) in dry THF (40 mL) under the atmosphere of
°
6-Amino subsituted-2-(4-(1,2-diphenylprop-1-en-1-yl)phenyl)-1H-
nitrogen at 0 C. Subsequently, the resulting black mixture was
allowed to reflux for 3 h to produce titanium reagent. After
benzo[de]isoquinoline-1,3(2H)-diones (7–21):
A mixture of 6-
°
bromo-2-(4-(1,2-diphenylprop-1-en-1-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (6) (0.2 g, 0.37 mmol) and corresponding primary or
secondary amine (1.1 mmol) was refluxed for 2–3 h in the presence
of potassium carbonate (0.06 g, 0.44 mmol) in 10 mL of DMF. The
reaction mixture was allowed to cool and poured in water (50 mL)
to obtain precipitates. Then, the mixture was filtered off to get
corresponding solid crude product of 6-amino subsituted-2-(4-(1,2-
diphenylprop-1-en-1-yl)-1H-benzo[de]isoquin oline-1,3(2H)-diones
(7–21) which was purified by column chromatography over silica
gel using hexane and chloroform as eluents.
refluxing, the obtained titanium reagent was cooled to 0 C and
charged with pyridine (1.02 mL, 12.7 mmol). Then, a solution of
acetophenone (0.59 mL, 5.07 mmol) and 4-amino benzophenone
(1 g, 5.07 mmol) in THF was added dropwise to reaction mixture at
°
0 C and stirred for 30 min. The reaction mixture was heated to
reflux for 2.5 h. The completion of reaction was monitored by TLC.
On completion, 10% aq. soln. of K2CO3 was poured into the cooled
reaction mixture and stirred vigorously for 10 min. On stirring, a
black mass was separated out that removed by vacuum filtration.
The crude product was extracted from the filtrate with ethyl
acetate. The extracted organic layer was dried over Na2SO4 and the
solvent was evaporated. Further, the resulting brownish-yellow oil
was purified by column chromatography (hexane/ethylacetate
97:3) to provide a desired cross-coupled product (2) in diastero-
2-(4-(1,2-Diphenylprop-1-en-1yl)phenyl)-6-morpholino-1H-benzo
[de] isoq uinoline-1,3(2 H)-dione (7): Yellow solid, E/Z 1:3.3; Yield
1
°
77%; mp: 236–240 C; H NMR (400 MHz, CDCl3): δ=8.63–8.61 (dd,
2
3
1H, J=7.28 Hz, J=0.92 Hz, ArH), 8.58–8.55 (m, 1H, ArH), 8.51–8.49
(d, 1H, J=8.00 Hz, ArH), 8.48–8.45 (m, 1H, ArH), 8.44–8.41 (m, 1H,
ArH), 7.76–7.74 (m, 1H, ArH), 7.72–7.68 (m, 1H, ArH), 7.41–7.34 (m,
3H, ArH), 7.30–7.25 (m, 4H, ArH), 7.23–7.21 (m, 5H, ArH), 7.16–7.11
(m, 2H, ArH), 7.03–7.00 (m, 3H, ArH), 6.95–6.92 (m, 3H, ArH), 4.04–
3.99 (m, 5H, morph-OCH2), 3.29–3.24 (m, 5H, morph-NCH2), 2.21 (s,
1H, CH3), 2.15 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ=164.7,
164.5, 164.2, 164.1, 156.0, 155.9, 144.0, 143.7, 143.3, 143.1, 142.8,
138.7, 138.6, 136.5, 136.4, 133.1, 133.0, 132.9, 131.7, 131.5, 131.1,
meric isomers E/Z 1:6; yield 60%; mp. 142–144 C; 1H NMR
°
(400 MHz, CDCl3): δ=7.35–7.32 (m, 2H, ArH), 7.26–7.23 (m, 3H, ArH),
7.17 (brs, 3H, ArH), 7.13–7.09 (m, 2H, ArH), 7.04–6.96 (m, 1H, ArH),
6.91–6.88 (m, 1H, ArH), 6.67–6.65 (m, 2H, ArH), 6.36–6.33 (m, 2H,
ArH), 3.48 (s, 2H, NH2), 2.18 (s, 3H, CH3), 2.10 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ=144.6, 144.2, 144.1, 144.0, 139.1, 134.1, 134.0,
133.6, 132.2, 130.1, 129.5, 129.4, 128.1, 127.9, 127.8, 126.5, 126.0,
114.3 (ArC), 23.4 (CH3); ESIMS: m/z 286.2 (M+H)+.
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ChemMedChem
131.0, 130.5, 130.4, 130.3, 129.4, 128.5, 128.3, 128.2, 127.9, 127.6,
127.5, 126.7, 126.6, 126.3, 126.2, 126.1, 126.0, 123.5, 117.2, 115.2,
115.1 (ArC), 67.0 (morph-OCH2), 53.5 (morph-NCH2), 23.6 (CH3), 22.9
(CH3); HRMS (TOF MS): m/z calcd C37H30N2O3 551.2256, found
551.2230.
136.5, 132.9, 131.7, 131.5, 131.1, 131.0, 130.3, 130.2, 129.4, 129.3,
128.2, 128.1, 127.9, 127.7, 126.7, 126.6, 126.4, 126.3, 126.1, 126.0,
125.9, 123.6, 123.5, 120.5, 117.2, 117.1, 116.6, 116.5, 115.2 (ArC),
53.2 (pip-NCH2), 53.1 (pip-NCH2), 49.7 (pip-NCH2), 49.6 (pip-NCH2),
23.7 (CH3), 23.6 (CH3); HRMS (TOF MS): m/z calcd C43H35N3O2
626.2729, found 626.2709.
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5
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7
8
9
2-(4-(1,2-Diphenylprop-1-en-1yl)phenyl)-6-(piperidin-1-yl)-1H-
benzo [de]isoquinoline-1,3(2H)-dione (8): Yellow solid, E/Z 1:2.2;
6-(4-Benzylpiperazin-1-yl)-2-(4-(1,2-diphenylprop-1-en-1-yl)
phenyl)-1H-benzo[de]isoq uinoline-1,3(2H)-dione (12): Yellow sol-
1
°
Yield 72%; mp: 235–238 C; H NMR (400 MHz, CDCl3): δ=8.61–8.59
1
°
(m, 1H, ArH), 8.54–8.51 (m, 1H, ArH), 8.47–8.45 (d, J=7.28 Hz, 1H,
ArH), 8.44–8.42 (m, 1H, ArH), 8.40–8.38 (m, 1H, ArH), 7.41–7.33 (m,
3H, ArH), 7.30–7.26 (m, 4H, ArH), 7.22–7.17 (m, 5H, ArH), 7.15–7.08
(m, 4H, ArH), 7.03–6.99 (m, 3H, ArH), 6.95–6.92 (m, 3H, ArH), 3.28–
3.20 (m, 6H, pip-NCH2), 1.92–1.84 (m, 6H, pip-CH2), 1.76–1.70 (m, 3H,
pip-CH2); 13C NMR (100 MHz, CDCl3): δ=163.9, 163.7, 163.4, 163.2,
156.7, 156.6, 143.0, 142.7, 142.5, 142.3, 142.0, 141.8, 137.7, 137.6,
135.4, 132.2, 132.0, 130.7, 130.5, 130.4, 130.1, 130.0, 129.4, 129.3,
128.4, 127.4, 127.2, 127.1, 126.9, 126.7, 126.5, 125.7, 125.6, 125.4,
125.0, 124.5, 124.4, 122.3, 114.9, 113.9, 113.8 (ArC), 53.6 (pip-NCH2),
28.7 (pip-CH2), 25.3 (pip-CH2), 23.4 (CH3), 22.6 (CH3); HRMS (TOF MS):
m/z calcd C38H32N2O2 549.2464, found 549.2438.
id, E/Z 1:1.4; Yield 75%; mp: 246–249 C; H NMR (400 MHz, CDCl3)
δ 8.62–8.60 (d, J=7.2 Hz, 1H, ArH), 8.55–8.53 (d, J=7.80 Hz, 2H,
ArH), 8.49–8.40 (m, 3H, ArH), 7.72–7.64 (m, 2H, ArH), 7.41–7.32 (m,
11H, ArH), 7.31–7.22 (m, 10H, ArH), 7.18–7.14 (m, 5H, ArH), 7.11–
7.07 (m, 2H, ArH), 7.05–7.00 (m, 5H, ArH), 6.95–6.92 (m, 4H, ArH),
3.67 (s, 2H, benzyl-CH2), 3.65 (s, 2H, benzyl-CH2), 3.32–3.29 (m, 7H,
pip-NCH2), 2.78 (brs, 7H, pip-NCH2), 2.22 (s, 3H, CH3), 2.15 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3): δ=164.8, 164.6, 164.3, 164.1, 144.1,
144.0, 143.7, 143.6, 143.3, 143.1, 142.8, 140.0, 138.8, 138.7, 138.6,
138.0, 136.4, 133.1, 133.0, 132.9, 131.8, 131.7, 131.6, 131.4, 131.1,
130.9, 130.7, 130.6, 130.3, 130.2, 129.4, 129.3, 128.5, 128.4, 128.2,
128.1, 127.9, 127.7, 127.5, 127.4, 126.7, 126.6, 1263, 126.2, 125.9,
125.8, 125.8, 123.4 (ArC), 65.4 (benzyl CH2), 65.3 (benzyl CH2), 53.2
(pip-CH2), 53.1 (pip-CH2), 53.0 (pip-CH2), 23.7 (CH3), 23.6 (CH3); HRMS
(TOF MS): m/z calcd C44H37N3O2 640.2886, found 640.2860.
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2-(4-(1,2-Diphenylprop-1-en-1-yl)phenyl)-6-(4-methylpiperazin-1-
yl)-1H-benzo[de]isoq uinoline-1,3(2H)-dione (9): Yellow solid, E/Z
1:3.5; yield 78%; mp: 239–242 C; 1H NMR (400 MHz, CDCl3): δ=
°
8.62–8.60 (m, 1H,ArH), 8.56–8.54 (m, 1H, ArH), 8.49–8.47 (d, J=
8.08 Hz, 1H, ArH), 8.46–8.44 (m, 1H, ArH), 8.42–8.40 (m, 1H, ArH),
7.74–7.72 (m, 1H, ArH) 7.70–7.66 (m, 1H, ArH), 7.41–7.33 (m, 3H,
ArH), 7.30–7.25 (m, 4H, ArH), 7.22–7.20 (m, 5H, ArH), 7.16–7.07 (m,
3H, ArH), 7.03–7.00 (m, 3H, ArH), 6.95–6.92 (m, 3H, ArH), 3.32–3.30
(m, 5H, pip-NCH2), 2.74–2.73 (m, 5H, pip-NCH2), 2.44 (s, 1H, pip-
NCH3), 2.42 (s, 3H, pip-NCH3), 2.21 (s, 1H, CH3), 2.15 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3 +[D6]DMSO): δ=164.4, 163.9, 156.4, 156.3,
143.6, 143.2, 142.9, 138.4, 136.4, 131.3, 131.2, 130.8, 130.1, 130.0,
129.2, 128.3, 128.1, 127.8, 126.8, 126.6, 126.1, 125.7, 123.3, 123.2,
116.4, 116.3, 115.0 (ArC), 55.0 (pip-NCH2), 53.0 (pip-NCH2), 52.9 (pip-
NCH2), 46.2 (NCH3), 46.1 (NCH3), 23.6 (CH3); HRMS (TOF MS): m/z
calcd C38H33N3O2 564.2573, found 564.2546.
6-(Cyclohexylamino)-2-(4-(1,2-diphenylprop-1-en-1-yl)phenyl)-1H-
benzo[de]iso quinoline-1,3(2H)-dione (13): Yellow solid, E/Z 1:1.4;
1
°
Yield 71%; mp: 238–242 C; H NMR (400 MHz, CDCl3): δ=8.63–8.61
(d, J=7.24 Hz, 1H, ArH), 8.57–8.55 (d, J=7.16 Hz, 1H, ArH), 8.50–
8.42 (m, 2H, ArH), 8.13–8.07 (m, 1H, ArH), 7.70–7.59 (m, 2H, ArH),
7.41–7.34 (m, 4H, ArH), 7.31–7.28 (m, 5H, ArH), 7.23–7.22 (m, 5H,
ArH), 7.17–7.07 (m, 6H, ArH), 7.05–7.01 (m, 5H, ArH), 6.96–6.94 (m,
4H, ArH), 3.64 (brs, 2H, cyclohexyl-NH), 3.14 (s, 1H, cyclohexyl-CH),
3.11 (s, 1H, cyclohex yl-CH), 2.23 (s, 3H, CH3), 2.16 (s, 3H, CH3), 1.88–
1.85 (m, 3H, cyclohexyl-CH2), 1.76–1.72 (m, 2H, cyclohexyl-CH2),
1.42–1.33 (m, 7H, cyclohexyl-CH2), 1.29–1.28 (m, 2H, cyclohexyl-
CH2), 1.25–1.21 (m, 6H, cyclohexyl-CH2); 13C NMR (100 MHz, CDCl3):
δ=165.0, 164.8, 164.3, 164.2, 148.7, 148.6, 144.1, 143.7, 143.4, 143.3,
143.0, 142.8, 138.8, 138.7, 136.4, 136.3, 134.9, 134.8, 133.6, 131.7,
131.6, 131.5, 131.2, 131.1, 130.9, 130.6, 130.5, 130.4, 130.3, 129.4,
128.5, 128.3, 128.2, 128.1, 127.9, 127.7, 127.6, 127.5, 126.8, 126.7,
126.6, 126.5, 126.2, 126.1, 125.9, 124.7, 124.6, 123.4, 123.3, 120.3,
120.2 (ArC), 51.8 (cyclohexyl-CH), 32.8 (cyclohexyl-CH2), 25.7
(cyclohexyl-CH2), 24.9 (cyclohexyl-CH2), 23.7 (CH3), 23.6 (CH3); HRMS
(TOF MS): m/z calcd C39H34N2O2 563.2620, found 563.2623.
2-(4-(1,2-Diphenylprop-1-en-1-yl)phenyl)-6-(4-ethylpiperazin-1-
yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10): Yellow solid, E/Z
1:3.6; yield 74%; mp: 240–243 C; 1H NMR (400 MHz, CDCl3): δ=
°
8.62–8.57 (m, 1H, ArH), 8.55–8.53 (m, 1H, ArH), 8.49–8.46 (m, 1H,
ArH), 8.44–8.40 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 7.77–7.71 (m,
1H, ArH), 7.69–7.65 (m, 1H, ArH), 7.40–7.33 (m, 3H, ArH), 7.30–7.26
(m, 4H, ArH), 7.22–7.19 (m, 5H, ArH), 7.15–7.09 (m, 2H, ArH), 7.04–
6.99 (m, 3H, ArH), 6.98–6.92 (m, 3H, ArH), 3.31 (brs, 4H, pip-NCH2),
2.77 (brs, 4H, pip-NCH2), 2.60–2.53 (m, 3H, NCH2), 2.21 (s, 1H, CH3),
2.14 (s, 3H, CH3), 1.19–1.14 (m, 4H, NCH3); 13C NMR (100 MHz, CDCl3
+[D6]DMSO): δ=164.5, 163.9, 156.3, 143.8, 143.6, 143.5, 143.3,
143.2, 142.9, 138.5, 138.4, 138.3, 136.5, 136.4, 133.2, 132.8, 131.5,
131.4, 131.3, 131.2, 130.9, 130.8, 130.6, 130.1, 130.0, 129.3, 129.2,
128.1, 127.9, 127.7, 127.5, 126.7, 126.5, 126.1, 125.7, 123.1, 116.3,
114.9 (ArC), 53.0 (pip-NCH2), 52.9 (pip-NCH2), 52.7 (ethyl-NCH2), 52.3
(pip-NCH2), 23.6 (CH3), 23.5 (CH3), 12.0 (ethyl-CH3); HRMS (TOF MS):
m/z calcd C39H35N3O2 578.2729, found 578.2800.
2-(4-(1,2-Diphenylprop-1-en-1-yl)-6-((2-morpholinoethyl)amino)-
1H-benzo[de]isoquino line-1,3(2H)-dione (14): Yellow solid, E/Z
1
°
1:1; yield 62%; mp: 249–252 C; H NMR (500 MHz, CDCl3): δ=8.65–
8.64 (d, J=7.20 Hz, 1H, ArH), 8.59–8.57 (d, J=7.25 Hz, 1H, ArH),
8.52–8.50 (d, J=8.3 Hz, 1H, ArH), 8.46–8.44 (d, J=8.35 Hz, 2H, ArH),
8.18–8.12 (m, 2H, ArH), 7.71–7.64 (m, 2H, ArH), 7.41–7.35 (m, 4H,
ArH), 7.32–7.28 (m, 5H, ArH), 7.23–7.20 (m, 4H, ArH), 7.17–7.10 (m,
6H, ArH), 7.04–7.00 (m, 5H, ArH), 6.96–6.95 (m, 4H, ArH), 6.37 (brs,
1H, NH), 6.33 (brs, 1H, NH), 3.79 (brs, 8H, morph-OCH2), 3.47–3.40
(m, 4H, ethyl-NHCH2), 2.86–2.80 (m, 4H, ethyl-CH2), 2.59 (brs, 8H,
morph-NCH2), 2.23 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): δ=165.0, 164.8, 149.7, 149.6, 144.1, 143.7, 143.3, 142.8,
138.8, 138.7, 136.4, 136.3, 135.0, 134.8, 134.3, 133.5, 131.6, 131.5,
131.2, 131.1, 131.0, 130.9, 130.3, 130.2, 129.4, 128.5, 128.2, 128.1,
127.9, 127.7, 127.5, 126.7, 126.6, 126.2, 125.9, 125.2, 125.1, 125.0,
123.4, 123.3, 120.6, 120.5, 120.1, 110.3 (ArC), 67.1 (morph-OC), 67.0
(morph-OC), 56.0 (aliphatic-NC), 55.9 (aliphatic-NC), 53.2 (ethyl-NC),
53.1 (ethyl-NC), 23.7 (CH3), 23.6 (CH3); HRMS (TOF MS): m/z calcd
C39H35N3O3 594.2678, found 594.2650.
2-(4-(1,2-Diphenylprop-1-en-1-yl)phenyl)-6-(4-phenylpiperazin-1-
yl)-1H-benzo[de]iso quinoline-1,3(2H)-dione (11): Yellow solid, E/Z
1
°
1:1; Yield 79%; mp: 245–248 C; H NMR (500 MHz, CDCl3): δ=8.66–
8.64 (d, J=7.25 Hz, 1H, ArH), 8.60–8.58 (m, 2H, ArH), 8.54–8.52 (d,
J=7.85 Hz, 2H, ArH), 8.50–8.48 (d, J=8.40 Hz, 1H, ArH), 7.78–7.71
(m, 2H, ArH), 7.43–7.41 (d, J=8.35 Hz, 2H, ArH), 7.39–7.28 (m, 15H,
ArH), 7.24–7.21 (m, 4H, ArH), 7.17–7.16 (m, 4H, ArH), 7.13–7.10 (m,
1H, ArH), 7.06–7.03 (m, 8H, ArH), 6.96–6.92 (m, 6H, ArH), 3.55–3.50
(m, 8H, pip-NCH2), 3.48–3.44 (m, 8H, pip-NCH2), 2.23 (s, 3H, CH3),
2.17 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ=164.7, 164.6, 164.3,
164.1, 156.1, 156.0, 151.1, 143.7, 143.6, 143.3, 143.1, 142.8, 138.6,
6-(2-(Dimethylamino)ethylamino)-2-(4-(1,2-diphenylprop-en-1-yl)
phe nyl)-1H-benzo[de] isoquinoline-1,3(2H)-dione (15): Yellow
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solid, E/Z 1:1.3; Yield 68%; mp: 233–235 C; 1H NMR (500 MHz,
CDCl3): δ=8.61–8.59 (d, J=7.15 Hz, 1H, ArH), 8.54–8.53 (d, J=
7.25 Hz, 1H, ArH), 8.50–8.48 (d, J=8.30 Hz 1H, ArH), 8.44–8.42 (d,
J=8.30 Hz, 1H, ArH), 8.30–8.25 (m, 2H, ArH), 7.63–7.56 (m, 2H, ArH),
7.40–7.35 (m, 4H, ArH), 7.31–7.28 (m, 5H, ArH), 7.23–7.20 (m, 5H,
ArH), 7.17–7.09 (m, 5H, ArH), 7.05–7.01 (m, 4H, ArH), 6.96–6.93 (m,
3H, ArH), 3.91–3.88 (m, 4H, ethyl-NCH2), 3.83–3.80 (m, 4H, ethyl-
NCH2), 3.43 (s, 4H, CH3), 3.41 (s, 6H, CH3), 2.23 (s, 3H, CH3), 2.16 (s,
3H, CH3); 13C NMR (100 MHz, CDCl3): δ=165.1, 164.9, 164.5, 164.4,
149.9, 149.8, 144.0, 143.7, 143.4, 143.3, 142.9, 142.8, 138.8, 138.6,
136.4, 134.8, 134.7, 134.4, 133.6, 131.6, 131.5, 131.1, 130.8, 130.3,
130.2, 129.3, 128.5, 128.2, 128.1, 127.9, 127.8, 127.5, 126.7, 126.6,
126.2, 125.9, 125.2, 125.1, 122.8, 120.9, 110.0 (ArC), 59.0 (ethyl-
NHCH2), 45.0 (ethyl-NCH2), 44.8 (ethyl-NCH2), 40.1 (NCH3) 40.0
(NCH3), 23.7 (CH3), 23.6 (CH3); HRMS (TOF MS): m/z calcd C37H33N3O2
552.2573, found 552.2545.
129.9, 129.4, 128.5, 128.2, 128.1, 127.9, 127.7, 127.5, 126.7, 126.6,
126.4, 126.3, 126.2, 125.9, 125.0, 124.9, 124.8, 123.4, 123.3, 123.1,
120.5, 120.4, 120.3, 118.1, 118.0, 117.1, 110.7, 110.6 (ArC), 46.2 (allyl-
NC), 46.1 (allyl-NC), 23.7 (CH3), 23.6 (CH3); HRMS (TOF MS): m/z calcd
C36H28N2O2 521.2151, found 521.2177.
°
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6-(Benzylamino)-2-(4-(1,2-diphenylprop-1-en-yl)phenyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (19): Yellow solid, E/Z 1:1.2; Yield
1
°
65%; mp: 244–248 C; H NMR (400 MHz, CDCl3): δ=8.65–8.62 (d,
J=7.30 Hz, 1H, ArH), 8.59–8.57 (d, J=7.25 Hz, 1H, ArH), 8.51–8.49
(d, J=8.35 Hz, 1H, ArH), 8.45–8.41 (d, J=8.35 Hz, 1H, ArH), 8.18–
8.12 (m, 2H, ArH), 7.69–7.62 (m, 2H, ArH), 7.43–7.35 (m, 13H, ArH),
7.31–7.28 (m, 5H, ArH), 7.23–7.20 (m, 4H, ArH), 7.17–7.11 (m, 5H,
ArH), 7.03–7.00 (m, 4H, ArH), 6.96–6.92 (m, 3H, ArH), 6.83–6.77 (m,
2H, ArH), 5.62 (brs, 1H, NH), 5.55 (brs, 1H, NH), 4.65–4.61 (m, 4H,
benzyl-NCH2), 2.23 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (100 Hz,
CDCl3): δ=165.0, 164.8, 164.4, 164.2, 144.0, 143.7, 143.4, 143.3,
142.9, 142.8, 138.8, 138.6, 137.1, 136.4, 134.9, 134.7, 134.2, 133.4,
131.6, 131.5, 131.1, 130.9, 130.2, 129.4, 129.2, 129.1, 128.2, 128.1,
127.9, 127.6, 127.5, 126.7, 126.6, 126.5, 126.3, 125.9, 125.1, 125.0,
123.3, 120.5, 120.4, 110.7 (ArC), 48.0 (benzyl-CH2), 47.9 (benzyl-CH2),
23.7 (CH3), 23.6 (CH3); HRMS (TOF MS): m/z calcd C40H30N2O2
571.2307, found 571.2381.
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6-(Butylamino)-2-(4-(1,2-diphenylprop-1-en-yl)phenyl)-1H-benzo
[de] isoq uinoline-1,3(2H)-dione (16): Yellow solid, E/Z 1:1; Yield
60%; mp: 245–248 C; H NMR (500 MHz, CDCl3): δ=8.64–8.62 (m,
1
°
J=7.20 Hz, 1H, ArH), 8.59–8.56 (m, 1H, ArH), 8.52–8.49 (m, 1H, ArH),
8.48–8.44 (m, 1H, ArH), 8.14–8.06 (m, 2H, ArH), 7.72–7.59 (m, 2H,
ArH), 7.41–7.35 (m, 3H, ArH), 7.31–7.28 (m, 5H, ArH), 7.23–7.23 (m,
5H, ArH), 7.17–7.08 (m, 6H, ArH), 7.06–7.02 (m, 4H, ArH), 6.96–6.92
(m, 3H, ArH), 6.78–6.72 (m, 2H, ArH), 4.16 (brs, 2H, NH), 3.45–3.41
(m, 4H, butyl-NCH2), 2.23 (s, 3H, CH3), 2.16 (s, 3H, CH3), 1.86–1.77 (m,
4H, butyl-CH2), 1.73–1.67 (m, 4H, butyl-CH2), 0.89–0.86 (m, 4H, butyl-
CH2); 13C NMR (100 MHz, CDCl3): δ=165.0, 164.9, 164.5, 164.4, 164.3,
164.2, 150.0, 149.9, 149.8, 144.2, 144.1, 143.8, 143.7, 143.4, 143.3,
142.9, 142.8, 138.9, 138.8, 138.7, 138.6, 136.4, 136.3, 136.2, 135.1,
135.0, 134.9, 134.4, 134.3, 133.6, 133.5, 131.6, 131.1, 130.9, 130.3,
130.2, 129.4, 128.5, 128.3, 128.2, 128.1, 127.9, 127.7, 127.5, 126.7,
126.6, 126.4, 126.3, 125.9, 124.9, 124.8, 124.7, 124.6, 123.4, 123.3,
120.3, 120.2, 110.1, 110.0 (ArC), 43.5 (butyl-NC), 43.4 (butyl-NC), 31.1
(butyl-C), 31.0 (butyl-C), 23.7 (CH3), 23.6 (CH3), 20.5 (butyl-C), 20.4
(butyl-C), 14.0 (butyl-C), 13.9 (butyl-C); HRMS (TOF MS): m/z calcd
C37H32N2O2 537.2464, found 537.2438.
2-(4-(1,2-Diphenylprop-1-en-1-yl)phenyl)-6-((4-fluorophenyl)
amino)-1H-benzo[de]is oquinoline-1,3(2H)-dione (20): Yellow solid,
1
°
E/Z 1:4.8; Yield 61%; mp: 232–234 C; H NMR (400 MHz, CDCl3): δ=
8.70–8.58 (m, 2H, ArH), 8.45–8.38 (m, 1H, ArH), 8.09–8.03 (m, 1H,
ArH), 7.90–7.82 (m, 1H, ArH), 7.40–7.27 (m, 7H, ArH), 7.24–7.21 (m,
3H, ArH), 7.16–7.10 (m, 5H, ArH), 7.05–6.93 (m, 6H, ArH), 6.66–6.63
(m, 2H, ArH), 6.35–6.33 (d, J=8.40 Hz, 1H, ArH), 3.13 (brs, 1H, NH),
3.10 (brs, 1H, NH), 2.21 (s, 1H, CH3), 2.16 (s, 1H, CH3); 13C NMR
(100 MHz, CDCl3): δ=163.9, 163.8, 163.7, 155.8, 144.9, 144.6, 144.5,
144.2, 144.1, 143.7, 143.6, 143.4, 143.3, 139.3, 139.1, 138.5, 136.7,
136.6, 134.7, 134.0, 132.6, 132.4, 132.0, 131.8, 131.8, 131.7, 131.6,
131.3, 131.2, 131.0, 130.2, 130.1, 129.5, 129.4, 129.3, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 126.8, 126.7, 126.6,
126.4, 126.0, 125.9, 125.7, 114.7, 114.3 (ArC), 23.7 (CH3), 23.6 (CH3);
HRMS (TOF MS): m/z calcd C39H27FN2O2 575.2057, found 575.2051.
2-(4-(1,2-Diphenylprop-1-en-yl)phenyl)-6-((2-hydroxyethyl)
amino)-1H-benzo[de]isoquinoline-1,3(2H)-dione (17): Yellow solid,
E/Z 1:3; Yield 63%; mp: 232–235 C; H NMR (500 MHz, CDCl3): δ=
2-(4-(1,2-Diphenylprop-1-en-1-yl)phenyl)-6-(pyrimidin-2-ylami-
1
°
no)-1H-benzo[de]isoquinoline-1,3(2H)-dione (21): Yellow solid, E/Z
8.63–8.43 (m, 3H, ArH), 8.18–8.15 (m, 1H, ArH), 7.70–7.58 (m, 2H,
ArH), 7.46–7.35 (m, 3H, ArH), 7.32–7.28 (m, 4H, ArH), 7.23–7.20 (m,
4H, ArH), 7.17–7.10 (m, 3H, ArH), 7.04–7.02 (m, 3H, ArH), 6.96–6.93
(m, 2H, ArH), 6.79–6.71 (m, 1H, ArH), 5.79 (brs, 2H, OH), 4.45 (brs,
1H, NH), 4.05 (brs, 2H, ethyl-OCH2), 3.97 (brs, 1H, ethyl-OCH2), 3.58
(brs, 3H, ethyl-NCH2), 2.23 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ=165.0, 164.8, 164.4, 164.3, 144.9, 144.8, 144.6,
144.5, 144.2, 144.1, 143.8, 143.7, 143.3, 143.0, 142.8, 132.0, 131.6,
131.2, 131.0, 130.2, 130.1, 129.4, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 127.3, 126.7, 126.6, 126.4, 126.0, 123.0, 120.5, 114.7, 114.3,
110.4, 110.3, 104.6, 104.5 (ArC), 60.5 (CH2OH), 60.4 (CH2OH), 45.4
(NCH2), 45.3 (NCH2), 23.5 (CH3), 23.4 (CH3); HRMS (TOF MS): m/z
calcd C35H28N2O3 525.2100, found 525.2173.
1:1.5; Yield 67%; mp: 232–235 C; 1H NMR (500 MHz, CDCl3): δ=
°
8.71–8.42 (m, 5H, ArH), 7.80–7.76 (m, 3H, ArH), 7.45–7.29 (m, 9H,
ArH), 7.21–7.12 (m, 6H, ArH), 7.10–7.05 (m, 5H, ArH), 7.03–7.01 (m,
5H, ArH), 6.95–6.92 (m, 3H, ArH), 2.21 (s, 2H, CH3), 2.15 (s, 3H, CH3);
13C NMR (100 Hz, CDCl3 +[D6]DMSO): δ=164.9, 164.8, 164.3, 164.2,
160.8, 143.6, 143.3, 143.2, 142.8, 142.7, 138.5, 136.3, 134.2, 134.1,
133.4, 131.8, 131.5, 131.1, 130.7, 130.1, 129.7, 129.6, 129.3, 128.4,
128.2, 128.0, 127.8, 127.7, 127.4, 126.6, 126.5, 126.2, 125.9, 125.3,
125.2, 123.1, 123.0, 122.3, 113.4, 110.2, 110.1 (ArC), 23.6 (CH3), 23.5
(CH3); HRMS (TOF MS): m/z calcd C37H26N4O2 559.2056, found
559.2050.
HPLC chromatography
6-(Allylamino)-2-(4-(1,2-diphenylprop-1-en-1yl)phenyl)-1H-benzo
[d e]iso quinoline-1,3 (2H)-dione (18): Yellow solid, E/Z 1:1.8; Yield
72%; mp: 230–234 C; H NMR (500 MHz, CDCl3): δ=8.65–8.57 (m,
To analyze the ratio of E and Z isomers of the TPE-naphthalimide
conjugate, reversed-phase (RP) HPLC has been performed using
Ultimate 3000 HPLC (ThermoFisher Scientific) system, equipped
with C-18 column (250×4.6 mm, 5 mm) and UV/Vis detector. The
wavelength used was 400 nm and the mobile phase was comprised
of 0.01 M tetrabutylammoniumhydrogensulfate/acetonitrile (30:70
v/v) with flow rate 1.0 mL/min. The stock solution of E/Z of TPE-
naphthalimide conjugate derivative, that is, 7 (1 mM) was prepared
in acetonitrile and protected from direct light. The stock solution
was then diluted to produce a solution of compound in acetonitrile
(2.3×10À 4 M). First, the prepared sample was filtrated using
1
°
2H, ArH), 8.52–8.44 (m, 2H, ArH), 8.18–8.11 (m, 2H, ArH), 7.70–7.63
(m, 2H, ArH), 7.41–7.35 (m, 4H, ArH), 7.31–7.28 (m, 5H, ArH), 7.23 (m,
3H, ArH), 7.17–7.16 (m, 3H, ArH), 7.03–7.02 (m, 3H, ArH), 6.96–6.93
(m, 2H, ArH), 6.79–6.74 (m, 2H, ArH), 6.04 (brs, 2H, NH), 5.45–5.37
(m, 2H, allyl-CH), 5.35–5.27 (m, 2H, allyl-CH2), 5.19–5.17 (d, J=
10.15 Hz, 1H, allyl-CH2), 4.80–4.73 (m, 1H, allyl-CH2), 4.11–4.02 (m,
4H, allyl-NCH2), 2.23 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ=165.0, 164.8, 164.5, 164.4, 164.2, 163.9, 144.1,
143.7, 143.4, 143.3, 142.9, 142.8, 138.8, 138.6, 136.4, 134.8, 134.7,
134.6, 134.2, 133.5, 133.0, 131.6, 131.5, 131.3, 131.1, 130.9, 130.2,
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0.22 μm cellulose filtrate. Then, 20 μL of the filtered sample was
injected into the column with an injection loop.
Acknowledgments
1
2
KP thanks the CSIR, New Delhi [(02(0310)/17/EMR-II] and SERB,
New Delhi (CRG/2018/002159) for financial support. The authors
express their gratitude to NCI, NIH for antiproliferative activity.
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In vitro antitumor activity
National Cancer Institute, USA selected the TPE-naph conjugates on
account of computer modeling studies. Further, these newly
synthesized derivatives were tested for their anticancer activity
against 60 human cancer cell lines at 10 μM concentration by a
standard procedure.
Conflict of Interest
The authors declare no conflict of interest.
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Topoisomerase-II inhibitory assay
Keywords: anticancer activity
naphthalimide · topoisomerase · triphenylethylene
· human serum albumin ·
Relaxation of supercoiled plasmid DNA by TOPO-IIα was assayed in
20 μL of buffer (0.5 M Tris·HCl (pH 8.0), 1.5 M NaCl, 100 mM MgCl2,
20 mM ATP, 5 mM dithiothreitol, 300 μg/mL BSA) containing drug
solution, 4 U of DNA topoisomerase-IIα and 500 ng of supercoiled
pHOT1 plasmid DNA and incubated at 37 C for 30 min. Reactions
were terminated by the addition of 2 μL of a solution containing
°
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10% sodium dodecyl sulfate (SDS) and 0.5 mg/mL of proteinase K
°
and incubated at 37 C for 30 min. Then, 1μL of loading buffer (50%
glycerol and 0.25% bromophenol blue) was added. The samples
were electrophoresed in 1% agarose gel with TAE buffer (100 mL of
10 stock solution-4.8 g of tris base, 1.14 mL acetic acid, and 0.37 g
EDTA, pH 8.1) for 4 h, then the DNA was stained with ethidium
bromide and photographed under UV illuminator.
Time-resolved fluorescence measurement
Time-resolved fluorescence decay measurements were carried out
by modulator time fluorescence system on deltaflex spectrometer.
A pulsed diode (λmax =280 nm) was used as the excitation source,
and emission was monitored at respective emission wavelengths.
The data was analyzed by using Horiba EZ software attached with
the system.
Molecular modeling
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Molecular docking studies were employed to authenticate the
interaction between ligand and TOPO-II-DNA complex using
software AutoDock 4.0. The three-dimensional X-ray structure of
topoisomerase-II-DNA-etopoiside ternary complex was retrieved
from the protein data bank (PDB ID: 5GWK). Etoposide was
excluded from ternary complex to perform the docking. To setup
ligand TOPO-II interaction, AutoDock tools 1.5.6rc3 was accom-
plished to remove the water molecules, add the polar hydrogen
atoms. As well gasteiger charges were computed and hydrogen
atoms which are nonpolar were merged to carbon atoms. The 3D
structures of the molecules were kept in PDB format with aid of
Gaussian 09 W program. Using ADT package (version 1.5.6rc3), the
PDB file was further modified for partial charges. The charges of the
nonpolar hydrogen atoms have been assigned to the atom to
which hydrogen is attached and file was saved as Pdbqt file. The
docking put files were generate by using the AutoDockTools
program. A cubic grid dimension 60 Å×60 Å×60 Å with the grid
points along the x, y and z axes and a grid spacing of 0.375 Å was
used. The optimized cluster was classified with minimum energy
level in the best suitable conformation of TOPO-II-DNA-ligand
modeled structure. The molecular structures were analyzed using
Discovery studio.
3303–3364.
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Synthesis of Triphenylethylene-
Naphthalimide Conjugates as top-
oisomerase-IIα inhibitor and HSA
binder
Anticancer amines: Triphenylethylne-
naphthalimide hybrids were synthe-
sized and screened for activity against
60 human cancer cell lines. The
compound with a morpholine group
exhibited broad-spectrum anticancer
activity at À 11.3 μM and was found to
be more cytotoxic than amonafide
against four cancer cell lines. It
displayed inhibitory activity against
topoisomerase-IIα and remarkable
binding affinity towards HSA.
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