Facile synthesis of indole heterocyclic compounds based micellar nano anti-cancer drugs

Article abstract of DOI:10.1039/C8RA07060A

Facile synthesis of micellar “nano” indole heterocyclic anti-cancer compounds is described. The synthesized compounds (11-23) were characterized by UV-VIS, ¹H NMR, FT-IR and mass spectroscopy. The binding energies of DNA-compound adducts varied

Full text of DOI:10.1039/C8RA07060A

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Facile synthesis of indole heterocyclic compounds
based micellar nano anti-cancer drugs†
ab
Cite this: RSC Adv., 2018, 8, 37905
Sofi Danish Mukhtar,a Ming Fa Hsieh,c Zeid A. Alothmand
*
Imran Ali,
and Abdulrahman Alwarthand
Facile synthesis of micellar “nano” indole heterocyclic anti-cancer compounds is described. The
synthesized compounds (11–23) were characterized by UV-VIS, 1H NMR, FT-IR and mass spectroscopy.
The binding energies of DNA–compound adducts varied from ꢀ20.08 to ꢀ23.85 kJ molꢀ1, and they
were stabilized by hydrophobic interactions and H-bonding. The synthesized compounds enter into
minor grooves of DNA during adduct formation. The DNA binding constant of compounds 11–23 was
1.00 to 2.00 ꢁ 105
M
ꢀ1. The drug-loading efficiency and drug-loading content in their micellar forms
Received 23rd August 2018
Accepted 31st October 2018
were recorded. Compounds 11, 12, 14 and 19 at a micellar concentration of 670 mL mLꢀ1 displayed
excellent anticancer activities against the HepG2/C3A line (25–50%). The potency of nano anticancer
drugs was predicted by drug likeness using Lipinski's “rule of five”. Taken together, compounds 11–23
could be used to treat cancers.
DOI: 10.1039/c8ra07060a
rsc.li/rsc-advances
a broad range of pharmacological properties, including anti-
tumor.20–23 Ahmad et al.24 reported apoptosis of human cancer
1. Introduction
cells using indoles. Indoles have been shown to inhibit the
proliferation of human cancer cells.25–27 Liao et al.28 showed
that 2-aryl-3-substituted indoles derivatives were active against
the breast cancer cell line MCF-7. Treatment with indoles or
their derivatives [e.g., I3C(indole-3-carbinol) and DIM (3,30-
diindolylmethane)] can result in the apoptosis of breast,29
squamous cell carcinoma,30 cholangiocarcinoma,31 colon32
and ovarian tumor cells.33 These indolic heterocycles may be
decisive anti-cancer agents for several human cancer cell
lines.34 Csipo et al.35 studied the chemosensitization of indole
moieties such as I3C and DIM.
Here, we synthesized a heterocyclic series ingrained with
indolic moieties, followed by purication and characterization by
column chromatography and spectroscopy. DNA binding studies
of synthesized molecules were carried out and DNA binding
constants calculated. The synthesized molecules were loaded
onto micellar “nanocarriers”. The activities of the developed
nano anticancer drugs were tested on HepG2(C3A) cell lines.
Finally, Lipinski's rule was applied to these synthesized
compounds to ascertain if they could become anti-cancer drugs.
Molecules such as cisplatin, 5-uorouracil and taxol have been
used to treat cancer but cannot cure cancers, especially for
late-stage disease. Besides, these molecules show various
serious side effects by damaging healthy body cells.1 The most
notorious problems with these drugs are a narrow therapeutic
range, cytotoxicity and poor solubility.2 Moreover, no single
drug can cure different types of cancers.3,4 These challenges
can be addressed by developing “nano” anti-cancer drugs
because they are cell-specic, targeted drugs that show few
side effects.5–15 Hence, different research teams, clinicians and
regulatory authorities are looking for effective nano anti-
cancer drugs.
Heterocyclic compounds appear to be most effective
against various cancers.16,17 Around 60% of the medications
used for cancer are based on heterocyclic moieties.18 Among
the various heterocyclic moieties, nitrogen-based compounds
have been found to be effective against different types of
cancers.19 Indoles (Fig. 1) are heterocyclic moieties with
aDepartment of Chemistry, College of Sciences, Taibah University, Al-Medina
Al-Munawara, 41477, Saudi Arabia. E-mail: drimran.chiral@gmail.com;
drimran_ali@yahoo.com
bDepartment of Chemistry, Jamia Millia Islamia (Central University), New Delhi-
110025, India
cDepartment of Biomedical Engineering, Chung Yuan Christian University, 200, Chung
Pei Rd., Chung Li, Taiwan
dDepartment of Chemistry, College of Science, King Saud University, Riyadh 11451,
Kingdom of Saudi Arabia
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra07060a
Fig. 1 Chemical structures of indole-3-carboxaldehyde, indole-2-
carboxaldehyde and indole-3-carboxylic acid.
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Waltham, MA, USA). A UV/VIS spectrometer (T80) from PG
Instruments (Lutterworth, UK) was employed. A DPX300 300
MHz system (Bruker, Billerica, MA, USA) was used for NMR
studies. A micrOTOF-Q II™ ESI-Qq-TOF mass spectrometer
(10262; Bruker) was also used. Melting points were determined
using a system developed by Veego Instruments (REC-22038;
Mumbai). Deionized water was collected by a Milli-Q (Milli-
pore; Bedford, MA, USA) water-purication system. Synthesized
compounds were puried by ash chromatography using
a pump (MP-200), UV detector and computer with CHEETAH
soware.
2. Experimental
2.1 Chemicals and reagents
Sulfuric acid, potassium hydroxide and formaldehyde of AR grade
were purchased from Merck (Mumbai, India), as were
LiChrosolv® chloroform, hexane and methanol. Indole-3-
carboxaldehyde, 4-nitroaniline, 4-uroaniline, 4-bromoaniline, 4-
chloroaniline, 4-methylaniline, 2-methylaniline and Ct-DNA were
purchased from Spectrochem (Mumbai, India). Aluminum silica
gel (60 F254) thin-layer plates were procured from Merck (Darm-
stadt, Germany). The human cancer cell line HepG2/C3A was from
National Taiwan University (Taipei City, Taiwan). MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was ob-
tained from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco's
modied Eagle's medium (DMEM) and antimycotics/antibiotics
were purchased from Gibco (Billings, MT, USA). Fetal bovine
2.3 Wet chemistry
2.3.1 Syntheses of the condensates of indole (3–10). A clear
solution of indole-3-carboxaldehyde (1) (10 mM) was prepared
in ethanol/dioxane (2 : 1, v/v). Equimolar amounts of the cor-
responding aniline derivatives (2) [4-nitroaniline, 4-uroani-
line, 4-bromoaniline, 4-chloroaniline, 4-methylaniline, 2-
methylaniline] were added separately. These were stirred to
obtain clear solutions. A few drops of concentrated H2SO4 were
added with constant heating under reux for 4–6 h. The reac-
tion was determined by thin-layer chromatography (TLC) using
¨
serum (FBS) was obtained from HyClone (Julich, Germany). Poly(3-
caprolactone)-block poly(ethylene glycol)-poly(3-caprolactone),
abbreviated as PCL-PEG-PCL (nanocarrier) was obtained from
Reinste Nano Ventures (New Delhi, India).
2.2 Instrumentation
Fourier transform infrared (FT-IR) spectroscopy was carried out MeOH : CHCl3 (1 : 3, v/v) solvent. Solid products started to
using RXIFT spectrometer (LR 64912C; PerkinElmer, precipitate and were ltered off aer completion of the reaction.
a
Scheme 1 Synthesis of indole derivatives.
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They were re-crystallized in an ethanol and dioxane (2 : 1, v/v) atom interact with the major and minor grooves of DNA.39 The
mixture to obtain pure products. The synthesized compounds synthesized compounds contained the same number of
(3–10) are shown in Scheme 1.
nitrogen and sulfur atoms. These heteronuclear compounds
2.3.2 Syntheses N-substituted indoles (11–23). A solution tend to interact with DNA, as shown by docking studies theo-
(15 mL) of indole N-(1H-indol-3-yl methylene)-4-nitroaniline3 retically and by DNA binding experimentally. Also, the cancer-
(10 mM) was prepared in a mixture of ethanol and dioxane causing genes (i.e., oncogenes) are also present on DNA. “DNA
(2 : 1, v/v). Then, formaldehyde (40%; 1.5 mL) and morpholine intercalation” refers to insertion of molecules into DNA bases
(10 mM; 0.087 mL) in ethanol were added to the solution. Then, and cause frameshi mutations. Our synthesized compounds
the mixture was stimulated for 3–4 h and le overnight. The could act as DNA intercalators.
solid was collected by ltration and recrystallized from ethanol
and dioxane (2 : 1, v/v) to yield 11. Compounds 12–23 were
2.5 DNA binding
synthesized by the same procedure used in the preparation of
Interactions of compounds 11–23 were studied with Ct-DNA
compound 3. Compounds having Z-geometry of C]N were
(pH 7.4) in
a solution of deionized water with tris-
formed because of the peak in IR spectra (i.e., only Z/cis can
show a peak in IR spectra). Also, the synthesized compound was
stable in biological conditions. The purity of the synthesized
compounds was ascertained by column chromatography and
ash chromatography.
2.3.3 Encapsulation of synthesized compounds (11–23) in
PCL-PEG-PCL nanocarriers. These compounds (3.0 mg each)
were dissolved in 1.0 mL of a binary solution of THF : DMSO
(hydroxymethyl)-amino methane buffer (Tris, 10ꢀ2 M). Initially,
the concentration of freshly prepared Ct-DNA solution was
determined by UV-VIS absorption spectrophotometry at 260 nm
(3 ¼ 6600 Mꢀ1 cmꢀ1) by knowing its absorbance.40 To carry out
binding experiments, the absorption spectra of freshly prepared
compounds 11–23 at a xed concentration of 1.6 ꢁ 10ꢀ4 M were
taken separately, and then at different concentrations of DNA
(5.15 ꢁ 10ꢀ5, 3.0 ꢁ 10ꢀ5, 1.00 ꢁ 10ꢀ5, 0.9 ꢁ 10ꢀ5 and 0.7 ꢁ
10ꢀ5 M). lmax was recorded and the absorbance of the mixture
(i.e., with each different solution of DNA and compounds) was
also measured. Experiments were repeated ve times. The
intrinsic DNA binding constant (Kb) was resolved using the
Benesi–Hildebrand equation as modied by Wolfe et al.:41
(1 : 1, v/v), separately, as was 20 mg of the polymer (PCL60
-
PEG40-PCL60). The compounds and polymer solutions were
mixed in 3.0 mL of double-distilled water and stirred for 3 h.
The resultant samples were placed in dialysis bags (molecular
weight (MW) cutoff ¼ 8000) and dialyzed for 24 h to obtain
micellar solutions, the formation of which was conrmed by
UV-VIS spectrometry by determining lmax
.
[DNA]/(3a ꢀ 3f) ¼ [DNA]/(3a ꢀ 3f) + 1/K(3b ꢀ 3f)
(1)
2.4 Docking studies
where the absorption coefficients 3a, 3f, and 3b represent Aobs
/
Docking studies were carried out as per standard protocol.36–38 [compound], the extinction coefficient for the molecule, and the
Heterocyclic molecules containing nitrogen, oxygen and sulfur extinction coefficient for molecule in the fully bound form,
Table 1 Docking parameters of compounds 11–23
Binding energy
KJ molꢀ1
Residues involved in hydrophobic
Residues involved in H-bonding (bond length) interactions
Compound
No. of H-bonds
3
11
ꢀ20.50
A: DT08H:Unk0:O of nitro group (3.55)
B: DG010H : Unk0:N of C]N group (3.43)
C: DC015H:Unko:N of morpholine group (3.48)
A: DG014H:Unk0:N of C]N group (3.40)
B: DG010H:Unk0:O of nitro group (3.39)
Dt8, Dc9, Dg10, Dg14 & Dc15
12
13
ꢀ21.34
ꢀ22.59
2
2
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
A: DG014H:Unk0:O of morpholine group (3.36) Dt8, Dc9, Dg10, Dg14, Dc15 & Dg16
B: DG010H:Unk0:N of C]N group (3.42)
14
15
16
ꢀ22.59
ꢀ23.85
ꢀ20.50
1
1
2
A: DG010H:Unk0:Nof morpholine group (3.51)
A: DG010H:Unk0:Nof morpholine group (3.49)
A: DC013H:Unk0:O of methoxy group (3.39)
B: DG014H:Unko:O of morpholine group (3.44)
A: DG010H:Unk0:N of C]N group (3.32)
A: DG014H:Unk0:N of C]N group (3.43)
B: DG010H:Unk0:N of indole group (3.40)
A: DG010H:Unk0:N of C]N group (3.42)
B: DC013H : Unk0:O of nitro group (3.24)
A: DG010H : Unk0:N of C]N group (3.35)
B: DG014H:Unk0:N of indole group (3.46)
A: DC09H:Unk0:N of indole group (3.57)
A: DC09H:Unk0:N of indole group (3.58)
A: DC014H:Unk0:N of C]N group (3.41)
B: DG010H:Unk0:N of indole group (3.28)
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
Dt8, Dc9, Dg10, Dg14 & Dc15
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
17
18
ꢀ20.92
ꢀ21.34
1
2
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
19
20
ꢀ20.08
ꢀ20.92
2
2
Dt8, Dc9, Dg10, Dc13, Dg14 & Dc15
Dt8, Dc9, Dg10, Dc13, Dg14 & Dc15
21
22
23
ꢀ20.92
ꢀ21.34
ꢀ20.92
1
1
2
Dt8, Dc9, Dg10, Dc13, Dg14, Dc15 & Dg16
Dt8, Dc9, Dg10, Dc13, Dg14 & Dc15
Dt8, Dc9, Dg10, Dc13, Dg14 & Dc15
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respectively. The Kb for dissimilar compounds was ascertained
by division of the slopes and intercepts of the plots of [DNA]/(3a
ꢀ 3f) vs. [DNA].
2.6 Determination of drug loading
2.6.1 Drug-loaded micelles. Synthesized molecules (0.2
mg) were loaded onto micelles and measured by UV-VIS spec-
trophotometry. DMF (4.0 mL) and H2O (0.9 mL) were intro-
duced into 0.1 mL of a drug-loaded micellar solution. Due to
ultrasonic vibration for 10 min, micelles were broken up and
the molecules (11–23) dissolved in the solutions. The charac-
teristic absorbance of the molecules was recorded at 320 nm
and compared with a standard curve generated from a solvent
mixture of DMF/H2O in 4 : 1 (v/v) ratio. Two main concepts were
utilized to determine molecule loading: drug-loading efficiency
(DLE) (also known as entrapment efficiency [EE]), and drug-
loading content (DLC). DLE is given by:42
ꢀ
ꢁ
weight of loaded drug
weight of polymer
DLC ¼
ꢁ 100%
(2)
DLE/EE is given by:
ꢀ
ꢁ
weight of loaded drug
weight of drug in feed
DLE=EE ¼
ꢁ 100%
(3)
2.6.2 Anticancer assays. In vitro anticancer proles of the
nano drugs (11–23) were determined reiteratively with a murine
microglial cell line (HepG2/C3A) by a cell viability (MTT) assay.43
The principle of the MTT assay is to remove succinic acid from
the mitochondria of living cells under the action of hydrogenase
and cytochrome C. The tetrazolium ring opens to form purple
crystals, which are eluted and have an absorbance peak at
570 nm. The experimental method involved four steps. First,
HepG2/C3A cells were seeded in 96-well dishes at 1 ꢁ 104/well.
Cells were allowed to attach for 24 h and washed several times
with phosphate-buffered saline (PBS) and serum-free medium
containing micellar solution-encapsulated drugs. Second, the
old medium was removed at the end of incubation time, and
fresh water containing 0.5 mg mLꢀ1 of MTT added. Third, the
medium was cultured for 4 h, the culture medium removed and
100 mL of DMSO was added to the ELISA Reader. Crystals were
dissolved by shaking for 5 min, and the absorbance at 570 nm
measured. Finally, cell cytotoxicity was calculated using the
following equation:
Abs ð570 nmÞsample
Cell cytotoxicity ¼
ꢁ 100%
Abs ð570 nmÞcontrol
Fig. 2 (a–c) Absorption spectra of compounds 11, 21 and 23 in the
absence and presence of different Ct-DNA concentrations. Arrows
indicate the decrease in absorbance upon addition of decreasing
concentrations of DNA. Inset: plots of [DNA]/3b vs. [DNA] for the
absorption titration of Ct-DNA with compounds. DNA concentrations
are (1) 5.15 ꢁ 10ꢀ5, (2) 3.0 ꢁ 10ꢀ5, (3) 1.00 ꢁ 10ꢀ5, (4) 0.9 ꢁ 10ꢀ5, and
3. Results and discussion
Finally, 13 compounds (11–23) were synthesized (Scheme 1) and
puried by ash chromatography. The melting points of
synthesized compounds were recorded. The synthesized
compounds were characterized by UV-VIS, FT-IR, mass and
NMR spectroscopy. Their structural data are described in ESI.†
(5) 0.7 ꢁ 10ꢀ5
.
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Table 2 Wavelength shifts, %hypochromism and binding constants of compounds 11–23
lmax
Compound
lmax (free)
(bound to DNA)
Change
%hypochromisma
Kbb (Mꢀ1
)
11
12
13
14
15
16
17
18
19
20
21
22
23
300 nm
261 nm
261 nm
255 nm
255 nm
256 nm
261 nm
262 nm
260 nm
264 nm
260 nm
258 nm
259 nm
260 nm
262 nm
260 nm
257 nm
255 nm
255 nm
258 nm
264 nm
260 nm
258 nm
261 nm
256 nm
260 nm
40
1
1
2
0
1
3
2
0
6
1
2
1
39.29
0.72
0.22
9.11
0.34
3.28
6.69
4.53
0.46
0.41
4.17
5.89
5.03
2.0 ꢁ 105
1.0 ꢁ 105
1.0 ꢁ 105
1.125 ꢁ 105
1.125 ꢁ 105
1.111 ꢁ 105
1.125 ꢁ 105
1.111 ꢁ 105
1.0 ꢁ 105
1.125 ꢁ 105
1.0 ꢁ 105
1.25 ꢁ 105
1.111 ꢁ 105
a
b
% hypochromicity (H%) ¼ [(Af ꢀ Ab)/Af] ꢁ 100, where Af and Ab represent the absorbance of free and bound compounds. Binding constants.
3.1 Docking studies
Table 3 DLC and DLE values of compounds 11–23
To study the in silico properties of compounds 11–23 with DNA,
theoretical DNA docking studies were carried out. B-DNA is
most common form used due to its wide and deep major
grooves, narrow and deep minor grooves, and its pattern of base
pairing between the two DNA strands. It gives different
hydrogen bond donor/acceptor forms in the major and minor
grooves, respectively.
The DNA docking of compounds 11–23 was done with DNA
dodecamers d(CGCGAATTCGCG)2 having a (PDB ID: 1BNA) le.
The DNA docking results are shown in Fig. SI1–5 (ESI)† and
Table 1. Almost all compounds adjusted themselves in a co-
crystalized ligand-like appearance in the vicinities of DNA.
The binding affinities of compounds 11–23 followed the
order 15 > 14 z 13 > 18 z 22 z 12 > 23 z 17 z 20 z 21 > 16 z
11 > 19.
Compound
DLC
DLE/EE
11
12
13
14
15
16
17
18
19
20
21
22
23
5.42%
2.44%
4.52%
3.54%
0.58%
3.30%
0.33%
4.10%
1.44%
4.87%
3.53%
4.79%
3.17%
54.2%
24.4%
45.2%
35.4%
5.8%
33.0%
3.28%
41.0%
14.4%
48.7%
35.3%
47.9%
31.7%
Fig. 3 Percentage viabilities of HepG2/C3A cells upon treatment with compounds 11–23 at micellar concentrations of 6.7, 67, 134, 335 and 670
mL mLꢀ1
.
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Fig. 4 Percentage viabilities of HepG2/C3A cells upon treatment with compounds 11–23 at micellar concentrations of 0.1, 1.0, 10, 50 and 100 mg
mLꢀ1
.
molecular attraction,44 and more repulsion from phosphate
groups in the backbone. Also, twisting can lead to more
hydrogen bonding. It can be seen in Fig. SI1† that, during the
molecular interaction of compound 11 with DNA, it favored the
minor grooves of DNA with ꢀ20.50 kJ molꢀ1 of binding energy.
Compound 11 formed three hydrogen bonds with the residues
(thymine, guanine and cytosine) of DNA with bond lengths of
Table 4 IC50 values of drugs in micelles in the HepG2/C3A line
Compounds
IC50 (mg mLꢀ1
)
11
12
13
14
16
17
18
19
20
22
23
3.85
2.03
3.30
1.91
5.28
3.27
3.66
1.63
4.29
˚
3.55, 3.43 and 3.48 A, respectively. With regard to hydrophobic
interactions, the nitrogen-containing nucleobases Dt8, Dc9,
Dg10, Dg14 and Dc15 were involved. Similarly, compound 19
preferred the minor grooves of DNA and formed two hydrogen
bonds with the residues of DNA with a binding affinity of
ꢀ20.08 kJ molꢀ1 (ESI†). The residues involved in hydrogen
bonding were DG'10H and DC'13H. Besides these interactions,
compound 19 (ESI†) also interacted with the nitrogen-
containing nucleobases of DNA hydrophobically in a two-
dimensional network. The residues involved in these hydro-
phobic interactions were Dt8, Dc9, Dg10, Dc13, Dg14 and Dc15.
It is not useful to discuss the details of docking studies for all
compounds as all the compounds interacted with DNA in
a similar fashion.
18.7
7.28
The number of hydrophobic interactions and H-bonds of
compounds 11–23 with DNA are given in Table 1. The hydrogen
bonds formed in the DNA-compound adducts were one for 14,
15, 17, 21 and 22, two for 12, 13, 16, 18, 19 and 20, and three for
11 compounds, respectively. During the interaction with DNA,
compounds 11–23 adjusted themselves so that their substituted
indole, morpholine and N-methylpiperazine rings were within
the minor grooves of DNA mostly, whereas Z-geometry C]N
moieties attached to benzene rings were mainly in the major
grooves of DNA. Also, 1–3 H-bonds were formed by this spatial
orientation. The residues of DNA involved in H-bonding were
guanine, adenine, cytosine and thymine bases (DG'14H,
DG'10H, DC'9H, DC'13H, DC'15H, DT'8H) with the amine group
of the indole ring, morpholine and N-methylpiperazine rings.
Also, cyano and methoxy groups were attached to benzene rings.
One hydrogen bond was attached to guanine and cytosine
bases. This twisting of rings might have been because of the GC-
rich region with a large positive potential responsible for
3.2 DNA binding
Interactive study of newly synthesized heterocyclic moieties
with DNA is crucial for estimation of their anticancer activity,
and to shed light on a viable mechanism of their action. Hence,
DNA binding is considered to be the most essential experi-
mental step to measure the activity of anticancer drugs because
most anticancer drugs target DNA specically.45 In general,
there are two modes of binding of a drug with DNA: non-
covalent or covalent. In covalent binding, a labile ligand is
replaced with a nitrogen atom of a DNA base, such as N7 of
guanine. In non-covalent binding, there are “electrostatic”,
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Table 5 Lipinski's ‘rule-of-five’ data for compounds 11–23
Mol log S
Mol PSA
(A2)
Compounds
Mol. wt
No. of HBA
No. of HBD
Mol log P
[log(moles per L)]/(mg Lꢀ1
)
Mol vol (A3)
11
12
13
14
15
16
17
18
19
20
21
22
23
365.16
397.08
337.16
349.18
333.18
349.18
333.18
353.13
378.19
350.19
362.21
346.22
366.16
5
3
3
4
3
4
3
3
5
3
4
3
3
1
0
0
0
0
0
0
0
1
0
0
0
0
2.74
4.00
3.42
3.12
3.43
3.24
3.55
3.86
2.80
3.47
3.17
3.60
3.92
ꢀ5.78(0.61)
ꢀ6.43(0.15)
ꢀ5.80(0.53)
ꢀ5.24(2.02)
ꢀ5.71(0.65)
ꢀ5.54(1.00)
ꢀ5.74(0.61)
ꢀ6.24(0.20)
ꢀ5.39(1.56)
ꢀ5.41(1.37)
ꢀ4.84(5.18)
ꢀ5.35(1.56)
ꢀ5.85(0.52)
61.69
23.43
23.43
30.37
22.73
30.98
23.43
23.43
57.26
19.00
25.93
19.00
19.00
352.50
344.16
328.22
354.43
342.94
354.14
343.24
339.50
377.68
353.40
379.61
368.42
364.68
“groove” and “intercalative” types of interactions. Bath- conrmed the outer-groove binding of DNA with the compound
ochromic and hypochromic shis revealed the intercalative with low hyperchromism and negligible bathochromism. The
binding of compounds with DNA. Intercalation enhanced Kb was calculated for compounds 11–23 and ranged from 1.0 to
strong stacking interactions between aromatic chromophores 2.0 ꢁ 105 Mꢀ1, thereby conrming that the synthesized
and the base pairs of DNA.46 Also, the electrostatic binding of compounds interacted strongly with DNA. Using Microso
a compound with DNA decreased the p / p* transition energy Excel™, regression analysis was carried out for DNA binding
because the p* orbital of the ligand interacted with DNA base studies. The correlation coefficient (R2) was found to be 0.7468–
pairs. This led to a lower hypochromic effect with no or negli- 0.9555%. The order of Kb for compounds 11–23 was almost
gible bathochromic shi.47 However, covalent bonding led to identical. Finally, we summarized that nearly all the synthesized
bathochromism and hyperchromism due to breaking of the heterocyclic compounds interacted with Ct-DNA through minor
DNA structure when a compound interacted with DNA cova- grooves, as noted by other scholars.51–53 Overall, the order of
lently. A compound coordinating with DNA through the N7 binding of differently substituted compounds with DNA was
position of guanine was indicated by a bathochromic shi. nitro > halogen > methoxy > methyl groups. The binding
Binding with the outside groove of DNA had a minor or no effect constants,
and, rarely, some hyperchromism was shown by groove binding compounds 11–23 are given in Table 2. Hence, DNA binding
in the UV-VIS spectrum48,49
studies were comparable with DNA docking studies. Therefore,
% hypochromism and wavelength shis of
The purity of DNA in the Ct-DNA stock solution was shown compounds 11–23 acted as DNA-binding agents.
by A260/A280 $ 1.80, which indicated a sufficiently DNA protein-
free nature.50 With the addition of DNA, slight band shiing was
3.3 DLC and DLE of drugs encapsulated in micelles
observed at 200–350 nm. The concentration of the stock solu-
Drug-loaded micelles were prepared by dialysis. All the drugs
loaded in micelles showed good results of DLC and DLE (Table
tion of DNA was determined experimentally by utilizing 3 ¼
6600 Mꢀ1 cmꢀ1 at 260 nm. Additional solutions of DNA (3.0, 1.0,
3). DLC ranged from 0.33% to 5.42%. Higher DLC was shown by
0.9 and 0.7 ꢁ 10ꢀ4 M) were made from a stock solution (5.15 ꢁ
compound 11 and lower by compound 17. DLE ranged from
10ꢀ5 M). The concentration of the compounds was xed at 1.6 ꢁ
3.28% to 54.2%. Compound 11 showed highest DLE and the
10ꢀ4 M. With each different concentration of DNA solution,
lowest was shown by compound 17. DLE was relatively good but
showing no clear pattern. During dialysis, no precipitate or
aggregate was observed. DLC and EE data predicted drug
loading into nanocarriers. Also, the encapsulation experiment
absorption spectra were recorded.
The DNA binding spectra of the most effective compounds,
11, 21 and 23, are shown in Fig. 2a–c, respectively. Changes such
as hypochromism (low to moderate) and slight shiing of bands
done with PCL-PEG-PCL nanocarrier showed its efficiency as
indicated the interaction of compounds with DNA bands via
a nanodrug. Micelle formation is shown in ESI.†
electrostatic mode due to a decrease of the p / p* transition
energy because the p* orbital of the intercalated ligand coupled
with the orbital of the base pairs. Graphs of compounds 20 and
3.4 Anticancer proles of nano drugs
The prepared nano drugs were tested for anticancer activities in
terms of % viability on the HepG2/C3A line. The viability of
HepG2C3A cells aer exposure to compounds 11–23 at varying
micellar concentrations (6.7,67, 134, 335, and 670 mL mLꢀ1) are
given in Fig. 3 and 4. Fig. 3 reveals that the indole derivatives
11–23 at a micellar concentration of 670 mL mLꢀ1 led to
viabilities of HepG2/C3A cells of 25, 35, 28, 48, 32, 30, 39, 50, 40,
17 (ESI†) showed that hyperchromism was because these
compounds were covalently bonded with DNA, which was due
to breakage of the DNA structure.
Graphs of compounds 12, 13, 15, 16, 18 and 19 (ESI†) showed
no change in UV-VIS spectra when the different DNA concen-
trations were added to solutions of the compounds. This
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showed IC50 values in the range 1.63–18.70 mg mLꢀ1 at low
concentrations. Half of the cells were dead and compound 22
showed a low IC50. Morphologies of HepG2 cells before and
aer treatment with nanodrugs are shown in Fig. 5a and b: the
nano drugs were effective because all cells died aer treatment
(Fig. 5b).
3.5 Drug likeness
In general, the time required to synthesize promising
compounds and utilize them is long because their toxicity,
pharmacokinetic properties and effects on metabolism must be
tested. This leads to considerable burdens on the research-and-
development budget of a pharmaceutical company. A number
of scientists and researchers are involved to nd answers of the
above questions. Lipinski's “rule of ve” can be used to identify
some of the important properties of compounds considered
theoretically to be drugs. That is, molecules must have good
membrane permeability, MW < 400, log P < 5, number of H-
bond donors < 5 and number of H-bond acceptors < 10. For
analyses of small drug-like molecules, a lter of log D > 0 and < 3
increases the chances of a compound displaying good intestinal
permeability. Moreover, a compound that has >10 hydrogen
bond acceptors and 5 hydrogen-bond donors shows poor
permeation. Therefore, this rule was applied to compounds 11–
23 synthesized according to the procedure described by Veber
et al.54 As seen in Table 5, compounds 11–23 followed Lipinski's
rule of ve.
4. Conclusions
Thirteen compounds (11–23) were synthesized with good yield
and loaded onto micellar nanocarriers. DNA-binding studies
indicated that compound–DNA adducts were stabilized mainly
by electrostatic attractions, van der Waals forces and hydrogen
bonding. The minor grooves of DNA were the areas through
which the synthesized compounds interacted with DNA.
Hypochromism indicated that the synthesized compounds
bound with DNA through electrostatic attraction and interca-
lations Kb values (1.0 to 2.0 ꢁ 105 Mꢀ1) indicated the strong
binding affinities of compounds 11–23. Good percentages of
DLC and DLE were achieved. Cell viability was 25–50% at 670 mL
mLꢀ1, which indicated excellent anticancer activities. IC50
values were 1.63–18.70 mg mLꢀ1. Compounds 11–23 followed
Lipinski's rule of ve. Compounds 11–23 could be used to treat
cancers.
Fig. 5 Morphology of HepG2 cells (a) before treatment and (b) after
treatment with nano drugs.
45 and 38%. Thus, HepG2/C3A cells displayed low viability
upon treatment with heterocyclic moieties 11, 12, 14, 20 and 19,
which indicated the good anticancer potential of these synthe-
sized heterocyclic compounds. Fig. 4 shows all the indole
moiety derivatives (11–23) caused low viability of HepG2/C3A
cells at a micellar concentration of 100 mg mLꢀ1. Also,
compound 23 showed low viability at micellar concentrations of
10 and 50 mg mLꢀ1. Therefore, these synthesized compounds
interacted differently and had different mechanisms with
different cell targets. Also, the half-maximal inhibitory
concentration (IC50) was calculated for the micelles formed for
compounds 11–23 (Table 4). Compounds 12, 14, 17 and 19
Conflicts of interest
There is no conict of interest for this manuscript.
Acknowledgements
The authors extend their appreciation to the International
Scientic Partnership Program ISPP at King Saud University for
funding this research work (ISPP # 0037).
37912 | RSC Adv., 2018, 8, 37905–37914
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