Synthesis of new naphthyl aceto hydrazone-based metal complexes: Micellar interactions, DNA binding, antimicrobial, and cancer inhibition studies

Article abstract of DOI:10.3390/molecules26041044

In the present study, naphthyl acetohydrazide (HL) ligand was prepared and used for the synthesis of new six amorphous transition metal (Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II)) complexes. All the compounds were characterized by elemental analysis, UV-vis, FT-IR, 1H- and 13C-NMR, and Matrix-Assisted Laser Desorption Ionization (MALDI). The solubilization study was carried out by estimating the interaction between the metal complexes with surfactants viz. sodium stearate (SS) and Cetyltrimethylammonium bromide (CTAB). UV-Visible spectroscopy was employed to determine partitioning and binding parameters, whereas electrical conductivity measurements were employed to estimate critical micellar concentration (CMC), the extent of dissociation, and free energy of micellization. The CT-DNA interaction of synthesized compounds with DNA represents the major groove binding. The synthesized ligand and metal complexes were also tested against bacterial and fungal strains and it has been observed that Cu(II) complex is active against all the strains except Candida albicans, while Cd(II) complex is active against all bacterial and fungal strains except Pseudomonas. Among all compounds, only the Pd(II) complex shows reasonable activity against cervical cancer HeLa cell lines, representing 97% inhibition.

Full text of DOI:10.3390/molecules26041044

molecules
Article
Synthesis of New Naphthyl Aceto Hydrazone-Based Metal
Complexes: Micellar Interactions, DNA Binding, Antimicrobial,
and Cancer Inhibition Studies
Fawad Ahmad 1,† , Muneera D. F. Alkahtani 2,
* * ,
, Muhammad Babar Taj 3,4, ,† , Afnan M. Alnajeebi 5
Seraj Omar Alzahrani 6 , Nouf Abubakr Babteen 5, Walla Alelwani 5, Azzah M. Bannunah 7, Sadia Noor 8
Rabia Ayub 9 , Syed Ahmad Tirmizi 1,* and Heba Alshater 10
,
1
Department of Chemistry, Quaid-e-Azam University Islamabad, Islamabad 44000, Pakistan;
fawadahmadchemist@yahoo.com
Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University,
2
Riyadh 11675, Saudi Arabia
Department of Chemistry, Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
Department of Chemistry, University of Sahiwal, Sahiwal 57000, Pakistan
Department of Biochemistry, Faculty of Science, University of Jeddah, Jeddah 80203, Saudi Arabia;
3
4
5
Amalnajeebi@uj.edu.sa (A.M.A.); Nababteen@uj.edu.sa (N.A.B.); welwani@uj.edu.sa (W.A.)
Department of Chemistry, College of Science, Taibah University, Madinah 42353, Saudi Arabia;
6
szahrani@taibahu.edu.sa
Department of Basic Sciences, Common First Year Deanship, Umm Al-Qura University,
Makkah 21955, Saudi Arabia; ambannunah@uqu.edu.sa
Department of Chemistry, Govt. College for Women University Faisalabad, Faisalabad 38000, Pakistan;
sadiaa613@gmail.com
Arrhenius Laboratory, Department of Organic Chemistry, Stockholm University, Svante Arrhenius Vag 16C,
SE-10691 Stockholm, Sweden; rabiaay1@gmail.com
Department of Forensic Medicine and Clinical Toxicology, Menoufia University, Shbien El-Kom 32511, Egypt;
hebaalshater@yahoo.com
Correspondence: mdf.alkahtani@gmail.com (M.D.F.A.); dr.taj@iub.edu.pk (M.B.T.);
satirmizi_qau@yahoo.com (S.A.T.); Tel.: +92-300-754-2669 (M.B.T.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
7
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Ahmad, F.; Alkahtani,
M.D.F.; Taj, M.B.; Alnajeebi, A.M.;
Alzahrani, S.O.; Babteen, N.A.;
Alelwani, W.; Bannunah, A.M.; Noor,
S.; Ayub, R.; et al. Synthesis of New
Naphthyl Aceto Hydrazone-Based
Metal Complexes: Micellar
8
9
10
*
Interactions, DNA Binding,
†
Antimicrobial, and Cancer Inhibition
Studies. Molecules 2021, 26, 1044.
https://doi.org/10.3390/
Abstract: In the present study, naphthyl acetohydrazide (HL) ligand was prepared and used for
the synthesis of new six amorphous transition metal (Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II))
complexes. All the compounds were characterized by elemental analysis, UV-vis, FT-IR, 1H- and
13C-NMR, and Matrix-Assisted Laser Desorption Ionization (MALDI). The solubilization study was
carried out by estimating the interaction between the metal complexes with surfactants viz. sodium
stearate (SS) and Cetyltrimethylammonium bromide (CTAB). UV-Visible spectroscopy was employed
to determine partitioning and binding parameters, whereas electrical conductivity measurements
were employed to estimate critical micellar concentration (CMC), the extent of dissociation, and free
energy of micellization. The CT-DNA interaction of synthesized compounds with DNA represents
the major groove binding. The synthesized ligand and metal complexes were also tested against
bacterial and fungal strains and it has been observed that Cu(II) complex is active against all the
strains except Candida albicans, while Cd(II) complex is active against all bacterial and fungal strains
except Pseudomonas. Among all compounds, only the Pd(II) complex shows reasonable activity
against cervical cancer HeLa cell lines, representing 97% inhibition.
molecules26041044
Academic Editors: Takashiro Akitsu
and Franco Bisceglie
Received: 31 December 2020
Accepted: 13 February 2021
Published: 17 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: hydrazones; DNA interaction; MALDI; micellar interaction; microbial inhibition; anti-
cancer activity
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Metal-based drugs have a strong therapeutic effect and it has been observed that
incorporation of metals not only increases the bioactivity of bioactive ligands but also
Molecules 2021, 26, 1044. https://doi.org/10.3390/molecules26041044
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1044
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significantly enhances the pharmacological potential of inactive ligands [
1]. Metal coordina-
tion is one of the most effectual tactics to plan long-acting or slow-releasing drugs [
many years, increasing attention has been paid to the synthesis of hydrazone ligands and
their metal-based complexes because of their multipurpose coordination performance [ ],
chelating scope [ ], structural amenability [ ], and several other pharmacological proper-
ties [ ]. Hydrazone with numerous metals has been extensively used as a building block
for a wide range of biological treatments [ ]. Functional diversity of the azomethine
group (C-N-N) in hydrazone enables its use in many fields [ ]. A close examination of
2,3]. For
4
5
6
6
7,8
9
the structure of hydrazone (Figure 1) reveals that it has (i) an imine carbon that has both
nucleophilic and electrophilic characters, (ii) amino-type nitrogen’s and nucleophilic imine,
(iii) configurational isomerism springing from the native nature of C14 N bond, and (iv) an
acidic N–H proton.
Figure 1. The structural and functional diversity of hydrazine scaffold [10].
The interaction of metal complexes with various surfactants has been previously
reported [11–13] and such solubilization phenomenon helps to increase the solubility of
insoluble or sparingly soluble species in aqueous media. The amphiphilic nature of surfac-
tants helps to accommodate the metal complexes inside the micelles. Such interactions of
metal complexes show synergistic interactions of biomembranes and drugs [14–16]. Thus,
the micelles facilitate solubility of less soluble excipients/additives by controlling their
release rates, minimizing degradation, and in turn reducing toxicity. Solubilization or
micellar interaction of bioactive compounds is a crucial and decisive role player in many
processes at biological and industrial levels. The metal complexes partition themselves
between intermolecular bulk aqueous phase and surrounding micellar phase which helps
in solubilization [13].
Our previous work [8] on Dehydroacetic acid (DHA) based hydrazone revealed
that due to its specific nature of two isomeric keto and enol forms, limited attention is
devoted to their metal complexes. There is no literature available on the micellar interaction
and biological activity of dehydroacetic acid-based naphthyl aceto hydrazone ligand and
its metal complexes. Taken together, due to the status of DHA, hydrazine, and metal
incorporated moieties, herein, we designed and synthesized the dehydroacetic acid-based
naphthyl aceto hydrazide ligand and its metal complexes to investigate their micellar
interaction parameters and their pharmaceutical potential, especially against bacterial and
fungal strains.
2. Results and Discussion
The characterization of ligand and metal complexes (Figure 2) is explained as follows.

Molecules 2021, 26, 1044
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Figure 2. Synthesis of naphthyl hydrazone ligand and their metal (M) derivatives.
2.1. FT-IR Spectroscopy
The FT-IR spectra of synthesized ligands represent its formation by indicating the
peak at 3275 cm−1 due to N-H stretching frequency followed by a weak signal of hydrogen-

Molecules 2021, 26, 1044
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bonded O-H at 2977 cm−1. The C=O stretching frequency appeared at 1702 cm−1 and a
sharp signal at 1654 cm−1 depicts hydrazone based immine C=N. Finally, two peaks at
1511 and 1466 cm−1 reflect aromatic stretching frequencies [17]. The comparative structural
elucidation of novel metal (Co, Ni, Cu, Zn, Pd, Cd) complexes of synthesized ligand depict
the differences in the FT-IR spectra. The FT-IR spectrum of Co(II) complex showed a weak
stretching band of hydrogen-bonded O-H at 3142 cm−1 and C-H stretching appeared at
3015 cm−1. The variation in the cases of C=O and C=N stretching bands at 1693 and
1619 cm−1 indicates the formation of a complex. Besides these, the new peaks at 489 cm−1
and 568 cm−1 indicate the Co-N and Co-O bands, respectively [18]. In the FT-IR spectrum of
paramagnetic Ni(II) complex N-H stretching band appears with a broad peak at 3437 cm−1
followed by C=O and C=N stretch at 1676 and 1619 cm−1 respectively. The two bands at
−1
−1
440 cm and 570 cm characterize Ni-N and Ni-O respectively [18]. The Cu(II) complex,
due to its paramagnetic nature, was characterized only by FT-IR spectroscopy where O-H
stretching frequency shifted from 3275 to 3208 cm−1 followed by more shifting of stretching
frequency bands of C=O and C=N at 1702, 1684 cm−1 to 1680 and 1654 cm−1 respectively
as compared to the ligand. The two bands at 538 cm−1 and 480 cm−1 represent Cu-N
and Cu-O respectively [19]. FT-IR spectrum of the Zn(II) complex showed an absence of
O-H stretching frequency with appearing of N-H band at 3147 cm−1 followed by the clear
difference of C=O and C=N stretching frequencies at 1692, 1625 cm−1, respectively The new
Zn-N and Zn-O bands appear at 488 cm−1 and 527 cm−1, respectively [19]. FT-IR spectrum
of the Pd(II) diamagnetic complex showed an absence of O-H stretching frequency with the
appearance of N-H band at 3310 cm−1 followed by C=O and C=N stretching frequencies
at 1689, 1563 cm−1, respectively. The new Pd-O and Pd-N bands appear at 492 cm−1 and
437 cm−1, respectively [20]. The FT-IR spectrum of Cd(II) complex displayed the absence
−1
of O-H stretching band and showed the weak band of hydrogen-bonded N-H at 3142 cm
,
followed the appearance of C-H stretch at 2962 cm−1. This may be due to the tautomerism
in complex structure as depicted in Figure 1. The C=O and immine C=N st−re1tch appeared
−1
−1
at 1676 and 1624 cm respectively. The new bands at 482 cm and 434 cm successively
represent Cd-N and Cd-O, respectively [21,22] as shown in Table 1.
Table 1. Infra-Red absorption frequency of compounds.
−1
Infra-Red Bands (Stretching’s in cm
)
Compounds
υ(N-H) υ(O-H) υ(C=O) υ(C=N)
υ(arom.)
υ(M-O)
υ(M-N)
Ligand
Co(L)2
Ni(L)2
Cu(L)2
Zn(L)2
Pd(L)2
Cd(L)2
3275(w) 2997(w) 1702(s)
1654(s)
1619(s)
1619(s)
1654(s)
1625(s)
1563(s)
1624(s)
1511,1466
1449–1428
1455–1428
1459–1428
1456–1442
1437–1425
1452–1427
-
-
-
3142(w) 1693(s)
1676(s)
3208(w) 1680(s)
568(m)
570(m)
538(m)
527(m)
492(m)
434(m)
489(m)
440(m)
480(m)
488(m)
437(m)
482(m)
3437(w)
-
-
3147(w)
3310(w)
3142(w)
-
-
-
1692(s)
1689(s)
1676(s)
2.2. Multi-Nuclear (1H, 13C) NMR and MALDI Analysis
The results of 1H-NMR spectrum of a ligand, when recorded in DMSO-d6, confirmed
its structure. For example, the presence of various respective shifts, singlet of hydroxyl
proton at 16.14 ppm and singlet of NH proton bonded to carbonyl group give a chemical
shift at 11.54 ppm. The seven naphthyl aromatic protons appeared as multiplets in the
region between 8.10–7.47 ppm. An olefinic singlet due to one proton was observed at a
chemical shift of 5.85 ppm. Moreover, two methylene protons with a singlet of azomethine
and doublet olefinic methyl protons appear at 4.16, 2.10, 2.56, 2.50 ppm (J = 2.51) respec-
tively. Further investigation of the structure via 13C-NMR illustrates that azomethine and
olefin linked carbon nuclei of CH3 appeared at 17.24 and 19.71 ppm. Active methylene
signals at 37.77 ppm and the two carbonyl carbon nuclei in the ligand showed a couple of
signals at 168.33 and 168.53 ppm, respectively. The naphthyl carbon nuclei appeared in the
region between 124.52–133.82 ppm and pyran ring carbon nuclei are characterized at 95.10

Molecules 2021, 26, 1044
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and 105.96 ppm. Finally, azomethine carbon nuclei appeared at 181.60 ppm. The structure
of ligand was further supported and confirmed by MALDI spectrum where m/z = 351
represents the molecular ion peak which is also acting like base peak based on its stability.
The formation of the Zn(II) complex was further supported by 1H NMR in which the
major difference is the disappearance of OH proton and shifting of NH singlet to downfield
at 5.51 ppm. Besides these, there is a clear shift of an olefinic proton from 5.85 to 4.17 ppm
in comparison with the ligand. Similarly, the 13C-NMR spectrum of the complex is entirely
different from the ligand, for example, carbon nuclei of CH3 appear at 19.39 and 20.18
ppm, azomethine carbon nuclei displayed a signal at 170.47 ppm while the carbonyl carbon
appeared at 179.21 ppm. The structure was further confirmed by MALDI spectrum where
m/z = 762.00 is a molecular ion peak while after fragmentation m/z = 413.54 represents the
base peak.
Similarly, the 1H-NMR of Pd(II) complex supported its formation by the disappear-
ance of OH proton and appearance of NH singlet at 5.92–5.85 ppm. Moreover, olefinic
protons gave two signals at 4.15 to 4.08 ppm. The CH2 (methylene) protons appeared
at 3.83 ppm. Similarly, the 13C-NMR spectrum of the complex is entirely different from
the ligand regarding carbon nuclei of CH3 split up into four signals appearing between
19.72–17.23 ppm. Azomethine carbon nuclei gave a signal at 174.33 ppm while the car-
bonyl carbon appeared at 181.61 ppm. Finally, olefinic carbon nuclei presented its peak at
95.10 ppm. Moreover, the formation of the complex was further confirmed by MALDI spec-
trum where m/z = 805.34 is a molecular ion peak while after fragmentation m/z = 413.54
represents the base peak which is maybe due to the removal of Pd(L).
The 1H NMR of Cd(II) complex showed the naphthyl protons between 8.07–7.39 and
NH protons connected with C=O gave a shift of 5.54 ppm. The olefinic proton appeared at
4.12 ppm with the addition of CH2 protons and CH3 protons between 3.35 ppm and 2.01
ppm in the spectrum. The structural elucidation was further supported by 13C-NMR in
which carbon of CH3 appeared at 20.63 and 19.44 ppm. Pyran ring carbon nuclei appear
at 108.18 ppm closer to naphthyl carbon region between 124–133.76 ppm. Azomethine
carbon nuclei showed a peak at 159.91 ppm. The carbonyl carbon appeared at 170.34 ppm.
Finally, the structure was confirmed by MALDI, where m/z = 813 is behaving dually as a
molecular ion peak as well as base peak due to its good stability.
The Co(II), Ni(II), and Cu(II) complexes were only characterized by FT-IR and MALDI
spectrometry due to their paramagnetic nature. The structure of Co(II), Ni(II), and Cu(II)
complexes were confirmed by MALDI spectrometer with an instrumental error m/z =
±
2.
The Co(II) complex was also confirmed by MALDI spectra where m/z = 758.26 indicates
the molecular ion peak and after the fragmentation of one ligand, the complex indicates its
base peak at m/z = 409.9 with good stability. In the case of Cu(II) complex, the molecular
ion peak appeared at m/z = 762.45 with less stability and a base peak at m/z = 413.18
which mainly represents the detachment of one ligand from the complex after a possible
fragmentation. The MALDI spectrum of Ni(II) complex showed a molecular ion peak at
m/z = 758.26 with good stability while a base peak appeared at m/z = 407.31.
2.3. UV-Vis Studies of Ligand and Metal Complexes
UV-vis absorption spectra of ligand and metal complexes separately were studied
by taking a varied concentration of synthesized compounds. The recorded UV-vis spec-
tra represent a good illustration for comparative data between non-conjugated ligands
and conjugated ligands. The synthesized ligand of concentration, (0.009 g/10 mM),
(
0.23 mL/2.3 mL DMSO) showed an intense absorption band
π
–
π* with
λ
max
value of
325 nm The observed higher shift in value was due to two main factors, (1) The resonance
and slight conjugation present only on one terminal of synthesized ligands, (2) It may
be due to pyran ring involved in resonance and keto-enol tautomerism, and one more
band at lower frequency was due to n–
and azomethine linkage as a charge transfer band of a wavelength having
294 nm (A = 1.026). Besides these, there were some other bands with a lower value of
π* arising by non-bonding electrons of carbonyl
λmax value at

Molecules 2021, 26, 1044
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λmax. There is a clear difference in the shift between the ligand and synthesized metal
complexes, which mainly corresponds to L
of the Co(II) complex, (0.019 g/10 mM), (0.22 mL/2.3 mL DMSO) vary in comparison to
a free ligand and displayed values at 322 nm (A = 1.137) and 282 nm (A = 1.416).
→
M charge transfer bands. The spectral data
λ
max
The d-d transitions in these cases were too weak to be observed. Similarly, spectra of
Ni(II) having concentration, (0.018 g/10 mM), (0.12 mL/2.3 mL DMSO) was presented
by one intense and one weak band and showed
λ
max
values of 337 nm (A = 1.123) and
283 nm (A = 1.138), respectively. The electronic spectra of Cu(II) complex having concen-
tration, (0.019 g/10 mM), (0.205 mL/2.3 mL DMSO) represented a broad absorption band
at 337 nm (A = 1.067), which is due to LMCT and only difference of 12 nm in the shift was
observed in comparison to free ligand. The electronic spectra of Pd(II) complex having
concentration, (0.019 g/10 mM), (0.14 mL/2.3 mL DMSO) showed an absorption band
having λmax at 330 nm (A = 1.106), which could be assigned as charge transfer transition.
As Zn(II) complex having concentration, (0.019 g/10 mM), (0.2 mL/2.3 mL DMSO) and
Cd(II) complex having concentration, (0.020 g/10 mM), (0.2 mL/2.3 mL DMSO) complexes
were diamagnetic in nature, so in these cases d-d transitions were forbidden. For such
types of complexes, the electronic transition occurred only because of ligand, but there are
observable shifts in
λ
max
values at 341 nm (A = 1.226) and 359 nm (A = 1.131) respectively
from free ligand, which indicates co-ordination (Figure S1 and Table S1).
2.4. Micellar Interaction of Metal Complexes
2.4.1. UV-Vis Spectroscopic Measurements
The partition and binding attributes and their corresponding trends were estimated
from the spectroscopic analysis of complex molecules with the surfactants. The maximum
absorbance of the synthesized complexes was recorded in DMSO and water, initially due
to less solubility in pure water (Table 1) and thereafter these values were selected for the
subsequent micellar study. The bathochromic shift was observed to various extents in all
cases which verified the interaction of complexes with surfactants due to the predominant
host-guest type association. Higher magnitude of maximum absorbance in the case of
CTAB indicated deep penetration within micelles compared to SS. The involvement of
structural attributes of all complexes changed with a molar concentration of surfactants
from pre-micellar to the micellar region (critical micellar concentration CMC); however, in
the post-micellar region, the microenvironment became steadier and no further change in
absorbance was observed.
The differential spectroscopy provides a comprehensive interpretation of the presence
and extent of interactions of surfactants and additives, drugs, or metal complex molecules.
The similar trends in simple and differential absorption were seen and the gradual increase
in absorbance until CMC was observed due to the inclusion of metal complex molecules
in micellar media of SS and CTAB (Figures 3 and 4). The partition and binding trends
were calculated using expressions given in Equations (1)–(5) (see experimental section) and
results are tabulated in Table 2.
Figure 3. Plots of simple and differential absorbance vs. sodium stearate (SS) concentration.

Molecules 2021, 26, 1044
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Figure 4. Plots of simple and differential absorbance vs. Cetyltrimethylammonium bromide (CTAB)
concentration.
Table 2. Partitioning and binding parameters of M(II) complexes in micellar media.
Kc × 103
Kx × 103
Kb
∆Gp
(kJ/mol)
∆Gb
(kJ/mol)
Surfactant
Complex
(dm3/mol)
(dm3/mol)
(dm3/mol)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Pd(II)
Cd(II)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Pd(II)
Cd(II)
3.54
1.76
1.81
2.36
7.96
6.28
1.52
22.8
6.6
197
97.6
100
131
442
349
84.2
1270
367
102
396
323
567
225
−30.2
−28.46
−28.53
−29.19
−32.2
−31.61
−28.09
−34.81
−31.74
−28.56
−31.93
−31.42
−15.71
−13.41
−11.79
−14.13
−15.31
−16.23
−19.17
−23.98
−21.41
−18.27
−15.91
−21.1
SS
117
300
125
700
2300
16000
56700
1600
617
CTAB
1.83
7.13
5.81
5000
2.4.2. Electrical Conductivity Measurements
The change in electrical conductivity is a useful indication of interaction between
surfactant and complex molecules. The plots in Figure 5 are representing the electrical
conductivity against the concentrations of SS and CTAB at 298 K. The involvement of both
surfactants enhanced the numerical value of the CMC compared to the pure surfactants at
ambient temperature. Significant variation in conductivity was observed after attaining
the CMC of the aqueous solutions of complexes in the presence of surfactant. The point
of intersection in the conductivity/concentration plots in pre-micellar and post-micellar
regions gave us the value of CMC as shown in Figure 5 for SS and CTAB.

Molecules 2021, 26, 1044
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Figure 5. Plots of electrical conductivity vs. concentration of surfactants.
Micellization is a complicated process and there is a critical relationship between
surfactant’s concentration and formation of micelles. The real mechanistic details of
this process are still under debate. The negative values of the free energy and change of
micellization (
process. Higher negative values of
∆
G◦ m) of both micellar media revealed that this was a spontaneous and stable
∆
G◦ m showed the higher solubilization of complexes
in the micelles of CTAB in comparison to SS. From the increase in the CMC seen in the
experimental outcomes, values were observed, and complexes interacted with both ionic
surfactants differently. Moreover, the electrostatic forces and the nature of metal decided
their positions in corresponding micellar systems. The micellar and thermodynamic
attributes are presented in Table 3.
Table 3. Micellar attributes of the metal complex in surfactants at 298 K.
Complex
SS
CTAB
CMC
(mM)
∆G◦
(kJ mol
CMC
(mM)
∆G◦
m
−1
m
−1
β
β
)
(kJ mol
−42.56
−46.21
−45.41
−40.18
−44.58
−47.99
)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Pd(II)
Cd(II)
4.48
4.73
4.28
4.59
4.85
4.26
−40.31
−41.89
−34.75
−37.30
−39.19
−34.44
0.195
0.274
0.519
0.399
0.533
0.307
1.04
1.11
1.2
0.276
0.422
0.294
0.485
0.229
0.317
1.24
1.26
0.99
2.4.3. Comparative Interaction of Metal Complexes in Both Surfactants
Micellization or solubilization is a dynamic process and it allows excipient/additive
molecules to reside for different periods in the micellar interior and its outer surface; thus,
we witnessed random or fluctuating absorbance values. The degree of solubilization of
the complexes in micellar media and their plausible loci in micellar regions are directly
influenced by the affected temperature, chemical, structural nature of complex molecules,
the concentration of surfactants and metal complexes. Thus, the movement and mechanism
of solubilization were governed by partitioning of metal complexes between micellar and
aqueous phases.
The higher value of Kx of Ni(II) in CTAB revealed its higher tendency to transfer
towards micellar media than SS and is positioned near the peripheral region of micelles.

Molecules 2021, 26, 1044
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The value of partition coefficient Kx (22.8
higher as compared to SS micelles (1.76
and 28.46) confirmed the stability and spontaneity of the system. The negative values of
×
103 dm3/mol) in the micelles of CTAB was
×
103 dm3/mol). The negative value of
∆
Gp ( 34.81
−
−
energies of binding and portioning indicated that the interaction of metal complexes in the
micellar media was spontaneous and higher the value of Kx, the more was solubilization.
The values of all complexes in both micellar media (as summarized in Table 3) indicates
the higher solubilization of complexes in the micelles of CTAB as compared to SS.
Figure 6 shows how all metal complexes were positioned in different zones of micellar
media depending on their portioning tendencies. The partitioning study shows that
complexes were distributed between aqueous and micellar medium depending on their
interaction with the micelles. The structure breaking effect of metal complexes increased
the values of CMC in all cases in comparison to pure surfactants (SS 4 mM and CTAB
0.9 mM). The values of CMC of SS shifted to 4.56 mM for Co(II) complex; 4.69 mM for
Ni(II) complex; 4.34 mM for Cu(II) complex; 4.62 mM for Zn(II); 4.8 mM for Pd(II) complex,
and 4.19 mM for Cd(II) complex. Whereas in case of CTAB, the values of CMC shifted
to 1.03 mM for Co(II) complex; 1.09 mM for Ni(II) complex; 1.17 mM for Cu(II) complex;
1.19 mM for Zn(II) complex; 1.23 mM for Pd(II) complex, and 0.995 mM for Cd(II) complex
as determined by UV-vis absorbance vs. concentration plots. The values of CMC were
obtained from the intersectional point absorbance-concentration plot in the pre-micellar
and post-micellar regions. The continuously rising simple differential absorption with the
increasing concentrations of SS and CTAB was attributed to incessant and uninterrupted
incorporation of complex molecules in micelles. The values of CMC obtained from both
techniques were in close agreement. The slight difference in CMC values represents a range
of concentration and separate working principles of both techniques.
Figure 6. Partitioning behaviour of M(II) complexes in the micelles of SS and CTAB.

Molecules 2021, 26, 1044
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2.5. DNA Interaction Study
The ligand displayed the two prominent
sition. With the successive addition of 0.1 mL (0.9 µM) DNA in a complex solution of
λ
max at 325 nm which indicates π→π* tran-
(
0.23 mL in 2.3 mL DMSO), only the intercalative modes were obtained due to hydro-
gen bonding. The
λ
max
at 325 nm leads to blue shift (hypsochromic) of 5.5 nm from
325 nm→319.5 nm with a decrease in absorbance from 1.083
intercalating or groove mode with DNA. The binding constant, as well as Gibb’s free
energy ( G = RTlnK), was also calculated. The cobalt and nickel complexes exhibited
→
0.877 and it confirmed
∆
λ
max
towards lower wavelength, at 318.5 nm and 341 nm from 322 nm and 345 nm respectively,
with an average difference of 3.25 nm and this indicates a negligible binding with DNA.
Cu(II) complex without DNA gives its
λmax at 337 nm comparative to ligand hydrazone
on successive addition of 0.1–0.9 mL DNA and represented a shift of
λmax up to 328.5 nm
with a difference of only 8.5 nm which confirms the interaction via intercalative mode. The
Zn(II) complex displayed entirely different behavior in interaction studies with DNA. Free
complex prepared by dissolving 0.2 mL in 2.2 mL DMSO to get absorbance close to 1, as
with all other complexes, represents
higher
max with the decrease in absorbance after the first three additions from 0.1→0.3 mL
Furthermore, shifted back to 342 nm after two more additions. The continual increase
λmax at 341.5 nm. The redshift was observed toward
λ
.
λ
max
of the next five additions leads to
λ
max
of 345 nm, which is a shift of only 4.5 nm. This
behavior of the Zn(II) complex may be due to weak intercalative mode of interaction with
DNA. This could be due to the coupling of the * orbital of the intercalators with the
orbital of the base pairs which decrease the * transition probabilities and consequently
π
π
π-π
lead to hypochromism [23]. Pd(II) complex with a minute difference displayed the same
results as Zn(II) complex with a blue shift of only 4.5 nm. Cd(II) complex interacted DNA
by showing an irregular trend with a redshift from 359
→
363 nm (Figures S2–S7). The
difference in activity of metal complexes is due to the different nature of metals present in
it. It may also be attributed to the purity of the compound. The interaction mood of DNA
with ligand and zinc complex is illustrated in Figure 7.
Figure 7.
(a) DNA Binding study of ligand (b) DNA Binding study of Zn(II) complex. (a–k, represent addition of DNA
from 0.1 mL to 1.1 mL).
2.6. Anti-Bacterial and Anti-Fungal Studies
It has been observed that Hydrazone based ligand, Co(II) and Pd(II) complexes
only show zone of inhibition (mm/100
µL) against Pseudomonas and Candida albicans
respectively. Ni(II) and Zn(II) complexes are found to be inactive against all bacterial and

Molecules 2021, 26, 1044
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fungal strains. Cu(II) complex is active against all the strains except candida albicans
while Cd(II) complex is active against all bacterial and fungal strains except pseudomonas
(
Table 4). The activity of Cu(II) complex could be due to the redox nature of Cu2+ ions [24].
Table 4. Anti-bacterial activity of the ligand and its M(II) complexes expressed as the diameter (mm)
of the inhibition zone.
Tested
Compound
E. coli
Pseudomonas
S. aureus
Listeria
C. albicans
Ligand
-
-
5
5
-
-
-
-
-
-
-
Co(II) complex
Ni(II) complex
Cu(II) complex
Zn(II) complex
Pd(II) complex
Cd(II) complex
-
-
-
-
10
-
5
-
10
-
10
-
-
-
-
-
-
-
5
9
9
-
12
15
- inactive.
2.7. MTT Based Cell Viability
The cell viability test was performed in the form of triplicate. From the series of com-
plexes examined herein only Cu(II) and Pd(II) complexes were found to be active against
cervical HeLa cell lines in comparison with standard Doxorubicin (positive control). The
comparative results (Table 5) represent the activity of the ligand and the M(II) complexes.
The activity of Cu(II) and Pd(II) hydrazone complexes could be due to the presence of Cu(II)
and Pd(II) metals. Further research is needed to investigate the mechanism of inhibition of
Cu(II) and Pd(II) complexes.
Table 5. Anti-cancer studies against Hela cancer cell lines.
Tested Compound
Ligand
Conc. (mg/mL)
30 µM
% Inhibition
26
28
41
87
28
97
31
95
Co(II) complex
Ni(II) complex
Cu(II) complex
Zn(II) complex
Pd(II) complex
Cd(II) complex
Doxorubicin
30 µM
30 µM
30 µM
30 µM
30 µM
30 µM
30 µM
3. Materials and Methods
All the chemicals used for synthesis were of analytical grade. Metal salts were pur-
chased from Merck. Dehydroacetic acid (DHA) was obtained from Aldrich. Ethanol,
Methanol, THF, and DMSO were used as it without further purification and were pur-
chased from Sigma Aldrich. The progress of the reaction was monitored through TLC by
using a combination of hexane-ethyl acetate (7:3) solvents. Melting points were determined
on Gallonkamp melting point apparatus using open capillaries and were uncorrected.
The solution conductivity measurements were performed by dissolving the complexes in
DMSO with 10−3 M concentration and their molar conductivities were measured at room
temperature. Infrared spectra were recorded on Jasco FT-IR-420 spectrophotometer. The
multinuclear (1H and 13C) NMR were recorded on Bruker AM-250 spectrometers in DMSO
solution using TMS as an internal standard. UV-vis absorption spectra and DNA binding

Molecules 2021, 26, 1044
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studies were recorded on UV-vis Spectrophotometer Shimadzu UV-1700. Mass spectra
were recorded by using low mass MALDI standard mixture under 6000 m/z Ultra Flex
Analysis placing 1
µL spot of DHB (2,4-dihydroxybenzoic acid) as matrix and 1 µL of the
standard mixture in acetonitrile.
3.1. General Procedure for the Synthesis of (E)-N0-(1-(2-Hydroxy-6-Methyl-4-oxo-4H-Pyran-3-yl
Ethylidene)-2-(Naphthalen-1-yl)Acetohydrazide Ligand (HL)
(i) Synthesis of methyl 2-(naphthalen-1-yl)acetate
First, the mixture of 2-(naphthalen-1-yl) acetic acid (5 g, 0.03 moles), 50 mL methanol
and a catalyst (3 mL conc. H2SO4) was refluxed for twelve hours to yield a methyl 2-
(naphthalen-1-yl)acetate. The basic workup was followed by extraction with ethyl acetate
and further purification through column chromatography which results in the successful
separation of powdered product.
(ii) Synthesis of 2-(naphthalen-1-yl)acetohydrazide
The ethanolic (20 mL) mixture of methyl 2-(naphthalen-1-yl)acetate and hydrazine
hydrate (1:1) was refluxed for six hours and then poured into ice. The formed fluffy solid
was washed with water and ethanol to give 90% of 2-(naphthalen-1-yl)acetohydrazide.
(iii) Synthesis of (E)-N0-(1-(2-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl ethylidene)-2-
(naphthalen-1-yl)acetohydrazide ligand (HL)
Finally, the ethanolic solution of 2-(naphthalen-1-yl)acetohydrazide and an ethanolic
solution of Dehydroacetic acid, DHA (1:1 ratio) were refluxed for four hours in the presence
of five drops of glacial acetic acid as a catalyst. (E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-
pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)acetohydrazide (HL) appeared in the form of
◦
fluffy shiny white ppt. (Figure 1) Yield: 83%; m.p. 199–203 C. Anal. Calcd. for C20H18N2O4
(Mw = 350.4 gmol−1). C, 68.56; H, 5.18; N, 8.00%. Found: C, 67.90; H, 5.55; N, 7.81%. FTIR
(KBr, cm−1): 3275 (O-H str.), 2997 (O-H str.), 1702 (C=O str.), 1654 (C=N str.), 1511, 1466
1
(aromatic str.) H-NMR (DMSO-d6, 300 MHz,
δ
/ppm): 16.14 (1H, s, OH), 11.54 (1H, s, NH),
8.10–7.47 (7H, s, naphthyl), 5.85 (1H, s, olefinic), 4.16 (2H, s, CH2), 2.56 (3H, s, CH3 olefinic),
2.10 (3H, s, CH3 azomethine); 13C-NMR (DMSO-d6, 75 MHz,
δ
/ppm): 181.60, 168.33, 163.62,
162.75, 133.82–124.52, 105.96, 95.10, 37.77, 19.71, 17.24. λmax (nm): 325. m/z: 351
3.2. Synthesis of Metal Complexes
To an ethanolic solution (5 mL) containing 2.0 mmol of HL, an ethanolic solution
(
5 mL) of metal acetates or nitrates (1.0 mmol) were added dropwise with constant stirring.
The triethylamine was added to facilitate the binding of metal with co-ordination site of
ligand and stirred at room temperature for about 3h. The precipitated metal complex
was filtered off and washed with hot ethanol, petroleum ether, and finally air-dried to
form amorphous solid (Figure 3). Unfortunately, no single crystals were obtained, and
characterization is reduced to elemental analysis, UV-vis, FT-IR, multinuclear (1H and 13C)
NMR, and MALDI mass spectrometry.
(iv) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl) ethylidene)-2-(naphthalen-2-
yl)acetohydrazide)2Co.
Pale orange solid. Yield: 61%; m.p. >350 ◦C. Anal. Calcd. for C40H34N4CoO8 (Mw
= 757.67 gmol−1). C, 63.41; H, 4.52; N, 7.39%. Found: C, 62.47; H, 4.38; N, 6.85%. FT-IR
(KBr, cm−1): 3142 (O-H str.), 3015 (C-H str.), 1693 (C=O olefinic str.), 1619 (C=N), 1428, 1410
(aromatic str.), 568 (Co-N), 457 (Co-O). λmax (nm): 322. m/z: 758.26
(v) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)
acetohydrazide)2Ni.
Orange solid. Yield: 61%; m.p. 321 ◦C. Anal. Calcd. for C40H34N4NiO8 (Mw = 757.5
gmol−1). C, 63.43; H, 4.52; N, 7.40 %. Found: C, 63.40; H, 4.55; N, 7.30 %. FT-IR (KBr, cm−1):
3437 (N-H str.), 3035 (C-H str.), 1676 (C=O olefinic str.), 1619 (C=N), 1455, 1428 (aromatic
str.), 456 (Co-N), 429 (Ni-O). λmax (nm): 345. m/z: 758

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(vi) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)
acetohydrazide)2Cu.
◦
Light green solid. Yield: 65%; m.p. 330 C. Anal. Calcd. for C40H34N4CuO8
(
Mw = 762.28 gmol−1). C, 62.86; H, 4.75; N, 7.33%. Found: C, 62.94; H, 4.69; N, 7.35%.
FT-IR (KBr, cm−1): 3208 (O-H str.), 3048 (C-H str.), 1680 (C=O str.), 1654 (C=N), 1459, 1428
(aromatic str.), 480 (Cu-N), 427 (Cu-O). λmax (nm): 337. m/z: 762.4
(vii) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)
acetohydrazide)2Zn.
White solid. Yield: 84%; m.p. >350 ◦C. Anal. Calcd. for C40H34N4ZnO8 (Mw = 762.1
gmol−1). C, 62.88; H, 4.79; N, 7.33%. Found: C, 62.90; H, 4.70; N, 7.45%. FT-IR (KBr, cm−1):
3147 (N-H str.), 2961 (C-H str.), 1692 (C=O str.), 1625 (C=N), 1456, 1442 (aromatic str.),
488 (Co-N), 454 (Co-O). 1H-NMR (DMSO-d6, 300 MHz,
δ
/ppm): 7.99–7.25 (14H, m, 2
NH), 4.17 (2H, s, olefinic), 4.12–3.86 (4H, m, 2 CH2), 3.37–250.
CH3 olefinic), 2.43–2.01 (6H, s, 2
CH3 azomethine); 13C-NMR (DMSO-d6,
/ppm): 179.21, 170.47, 164.60, 162.72, 133.75-124.29, 107.60, 96.85, 20.18, 19.39.
max (nm): 337. m/z: 762
×
naphthyl), 5.51 (2H, s, 2
(6H, m, 2
75 MHz
×
×
×
×
,
δ
λ
(viii) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)
acetohydrazide)2Pd.
◦
Pale yellow solid. Yield: 74%; m.p. 320 C. Anal. Calcd. for C40H34N4PdO8
(
Mw = 805.5 gmol−1). C, 59.67; H, 4.26; N, 6.96%. Found: C, 60.57; H, 4.30; N, 7.25%. FT-IR
(KBr, cm−1): 3310 (N-H str.), 3182 (C-H str.), 1689(C=O str.), 1563(C=N), 1437,1346(aro-
matic str.), 492(Co-N), 437(Co-O). 1H-NMR (DMSO-d6, 300 MHz, δ/ppm): 8.22-7.43 (14H,
m, 2
CH2), 2.73–2.55 (6H, m, 2
(DMSO-d6, 75 MHz, /ppm): 181.61, 174.33, 168.34, 133.82, 131.71–124.51, 105.98, 95.10,
×
naphthyl), 5.85–5.92 (2H, s, 2
×
NH), 4.15–4.08 (2H, s, olefinic), 3.83 (4H, m, 2
×
×
CH3 olefinic), 2.10–2.04 (6H, s, 2
×
CH3 azomethine); 13C-NMR
δ
33.64, 19.72, 17.23. λmax (nm): 330. m/z: 805
(ix) ((E)-N’-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)ethylidene)-2-(naphthalen-2-yl)
acetohydrazide)2Cd.
White solid. Yield: 55%; m.p. >350 ◦C. Anal. Calcd. for C40H34N4CdO8 (Mw = 812.5
gmol−1). C, 59.23; H, 4.23; N, 6.91%. Found: C, 60.47; H, 4.28; N, 6.85%. FT-IR (KBr, cm−1):
3142 (N-H str.), 2962 (C-H str.), 1676 (C=O str.), 1624 (C=N), 1427, 1452 (aromatic str.),
482 (Co-N), 434 (Co-O). 1H-NMR (DMSO-d6, 300 MHz, x
δ
/ppm): 8.07–7.39 (14H, m, 2
NH), 4.12 (2H, s, olefinic), 3.35 (4H, m, 2 CH2), 2.50–2.49 (6H,
CH3 olefinic), 2.32–2.01 (6H, s, 2
CH3 azomethine); 13C NMR (DMSO-d6, 75 MHz,
/ppm): 170.34, 159.91, 133.76–124.50, 108.18, 41.32-39.14, 20.63, 19.44. (nm): 360.
×
naphthyl), 5.54 (2H, s, 2
×
×
m, 2
×
×
δ
λ
max
m/z: 813
3.3. Matrix-Assisted Laser Desorption Ionization (MALDI) Protocol
The mass analysis of compounds was carried out by MALDI instrument (standard
mixture under 6000 m/z Ultra Flex Analysis. UCLA, USA.), The matrix consisting of DHB
(2,4-dihydroxybenzoic acid) in 100 µL of 50% Acetonitrile in water was prepared in a small
sample vial and mixed well on the rotary mixer. MALDI steel plate containing 300 spots
was first washed with water followed by washing with EtOH to get rid of any traces of
residual impurities. A volume of 1 µL of the matrix was spread on every multiple circular
spot then a non-calculated little quantity of ligand and metal complexes were added on
them with the help of a pipette, and on these spots, another layer of the matrix was added
to make a sandwich. It was air-dried for 3 min, the plate was inserted in the instrument,
and data were recorded after taking multiple flashes on centroid spots to get maximum
fragmented peaks by using low mass MALDI standard mixture under 6000 m/z Ultra Flex
Analysis. The acquisition of data was in the form of a graph between relative abundance
and m/z.

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3.4. Micellar Interaction of Metal Complexes
Since the complexes were not completely soluble in pure water therefore their inter-
action was studied initially in a mixture of DMSO and distilled water (1:1). The slight
change in the maximum absorbance of metal complexes was observed with redshifts,
which indicated interaction and solubilization in micellar media. Both surfactants showed
interaction to different extends. The comparative table of both surfactants’ interaction with
complexes is presented in Table 4.
3.4.1. Preparation of Stock Solutions and Dilution Method
The stock solutions of all metal complexes (1 mM) were prepared in distilled de-
ionized water and DMSO (1:1) and these primary solutions were used subsequently
for the preparation of secondary solutions by dilution method with surfactant solutions
for spectroscopic measurements. The secondary solutions were prepared by dissolv-
ing different amounts of surfactants from pre-micellar to post-micellar concentrations
(
2.5–6.5 mM for SS and 0.1–2.0 mM for CTAB). The stock solutions were diluted to hold
the Lambert-Beer’s law.
3.4.2. UV-Vis Spectroscopic Measurements
UV-vis absorbance values (simple and differential) were measured for the complexes
at 298 K maintaining
±
0.5K accuracy control in UV-vis spectrophotometer (Shimadzu,
UV-1700, Kyoto, Japan). In the case of simple absorbance distilled water was taken as a
reference standard while for differential spectroscopic analysis metal complex solution was
taken in reference cell and the aqueous solutions of metal complexes in the presence of
surfactants were taken in the sample cell. All the samples were homogenized and rested
for overnight to attain equilibrium before measurements.
3.4.3. Calculation of Partitioning and Binding Parameters
The partitioning and binding parameters were calculated from the spectroscopic data
using the Equations (1)–(5) as given below.
(i) Partitioning coefficient (Kx)
1
∆A
1
1
ꢀ
ꢁ
=
+
(1)
(2)
◦
KC∆A∞ Ca + Csm
∆A∞
Kx = Kcnw
The differential absorbance (∆A) helped to calculate binding constant Kc which was
then used to calculate portioning Kx (Equations (1) and (2)).
(ii) Binding constant (Kb)
CSCa
∆A
CS
∆el
1
=
+
(3)
Kb∆εl
(iii) Standard change in free energy of partition (∆Gp)
∆Gp = −RTlnKx
(iv) Standard change in free energy of partition (∆Gb)
(4)
(5)
∆Gb = −RTlnKb
In the above equations, Kc represents partition constant; Ca is the concentration
of dye;
∆A∞ stands for differential absorbance at infinite dilution. The relationship
Csm = Cs − CMCo; represents CMC of the surfactants in water as CMCo, while Cs is the

Molecules 2021, 26, 1044
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concentration of surfactants. In Equation (2), nw shows the number of moles of H2O. The
experimental values of these parameters are presented in the results section (Table 6).
Table 6. Shift in maximum absorbance of M(II) complexes in aqueous and micellar media.
λ
(nm)
max
Complex
DMSO:H2O (1:1)
SS System
332
CTAB System
Co(II)
Ni(II)
Cu(II)
Zn(II)
Pd(II)
Cd(II)
327
351
347
333
339
373
341
368
365
348
355
387
357
352
339
343
378
1 mM of each metal complex was used to prepare stock solutions.
3.4.4. Electrical Conductivity Measurements
Electrical conductivity (EC) data were measured on Hanna Conductivity meter (HI
99301, Hanna Instruments Inc. Highland Industrial Park 584 Park East Drive Woonsocket,
RI 02895 USA) in 0.01–199.9 mS range (
±
0.5%
±
2 accuracy control). The electrode calibra-
tion was ensured with potassium chloride over the desired micellar concentration range.
The conductivities of all complexes in the presence surfactants were measured at 298 K
temperature.
3.4.5. Parameters Calculated by Conductivity Data
(i) Critical Micellar Concentration (CMC)
The concentration of surfactants at which at their process of micellization initiates
is designated as critical micelle concentration (CMC). Micellization is a dynamic and
complicated process so the measurement of exact and accurate micellar concentration is
difficult because its value is estimated from a very narrow concentration range between
pre-micellar and post-micellar regions. In micelle formation, the surfactants undergo a
sudden change in their physicochemical properties, including conductivity.
(ii) The Degree of Dissociation (β)
The degree of dissociation was determined from the ratio of slopes of the conductivity–
concentration plots in the pre-and post-micellar region (Equation (6)).
S2 (Post−micellar slope)
S1 (Pre−micellar slope)
β =
(6)
(iii) Free Energy of Micellization (∆G◦
)
m
∆G◦ = (2 − β)RT ln X
(7)
m
CMC
In Equation (7), R = Universal gas constant (8.314 J mol−1K−1); T = absolute tem-
perature; XCMC = critical mole fraction of surfactants at their respective CMCs. The
experimental values of these parameters are presented in the results section (Table 4).
3.5. DNA Binding Activity Studies
UV-vis spectrophotometer (Shimadzu, UV-1700, Kyoto, Japan) was used to study
the binding interaction of ligand, metal complexes, and the DNA under the range of
250–780 nm. The concentration of CT-DNA was determined spectrophotometrically by
−1
using an extinction coefficient of 6600 M−1 cm at 260 nm. DNA interaction of free ligand
and metal complexes were studied individually against CT-DNA having a 0.66 µM solution.

Molecules 2021, 26, 1044
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The solution of CT-DNA was stirred for about 24 h by maintaining the temperature at
◦
4 C. The purity of CT-DNA free from protein was confirmed by an observed peak at
260 nm and by taking the ratio of absorbance on
λ
max
on 260 and 280 nm, which was
1.82. Concentrated stock solutions of the ligand and complexes were prepared in DMSO.
The was determined by successive addition of 0.1 mL of DNA with the help of a
λ
max
micropipette in the sample solution.
3.6. Anti-Bacterial and Anti-Fungal Protocol
The 0.02 g of sample was dissolved in 1 mL of DMSO. Freshly prepared cultures of E.
coli, S. aureus, Listeria, Pseudomonas, and Candida albicans were taken. Their suspensions
were made by inoculating single colony of each organism into 10 mL of saline solution
(0.9% NaCl). Muller Hinton Agar (MHA) was used as a growth medium for bacterial
growth and Sabouraud Dextrose Agar was used for fungal growth. The culture was spread
onto the Petri plates and digging of wells were carried out. Afterwards, those wells were
being sealed by adding 0.5 mL of liquefied MHA into the wells and they were kept for
a few minutes for drying. Then, the pre-prepared solution of compounds was poured
(100 uL) into the wells against each microorganism. Thereafter, the Petri plates were kept
overnight in an incubator. The plates were observed the very next day for results in terms
of zones of inhibition.
3.7. MTT Based Cell Viability Assay
Cytotoxic activity of the ligand and its metal complexes was assessed in 96-well flat-
bottomed microplates using the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2, 5-diphenyl-
tetrazolium bromide) colorimetric assay culturing HeLa cells, Cervical Cancer [25].
4. Conclusions
New ligands based on 2-(naphthalene-1-yl) acetic acid (NAA) hydrazone and metal
complexes were successfully synthesized and characterized by FT-IR, 1H and 13C-NMRs,
MALDI, and UV-visible spectroscopy. The synthesized complexes were evaluated for their
solubilization in ionic surfactants viz. sodium stearate (sodium salt of stearic acid, SS)
and Cetyltrimethylammonium bromide (CTAB). UV-Visible spectroscopy was employed
to determine partitioning and binding parameters, whereas electrical conductivity mea-
surements were employed to estimate critical micellar concentration (CMC), the extent of
dissociation, and free energy of micellization. The complexes showed greater solubilization
on the cationic micelles of CTAB as compared to SS owing to the electrostatic interactions
of positively charged head group of CTAB and the negatively charged hydroxyl groups
in the complexes. The experimental results showed an increase in the CMC values, and
complexes interacted differently with both ionic surfactants. Finally, the electrostatic forces
together with the nature of metal decided their positions in corresponding micellar systems.
SS-DNA interaction studies with ligand as well as metal complexes individually displayed
promising results by representing good values of binding constants and Gibbs free energy.
The maximum binding was observed for the Cu(II) complex. All the complexes exhibit
intercalation. Furthermore, anti-bacterial and anti-fungal activity results showed that only
Cu(II) and Cd(II) complexes were active approximately against all strains. The HeLa cervi-
cal cancer cell lines result represented that Cu(II) and Pd(II) complexes are extremely active
compared to the standard dose Doxorubicin. The performance and application of these
drugs in various fields regarding their activity could be a positive turn in pharmaceuticals
and from an industrial point of view.
Supplementary Materials: The comparative UV-vis analysis of ligand and metal complexes is given
in Figure S1. The DNA binding spectra are given in Figures S2–S7. Selected NMR and FT-IR and
MALDI spectra of ligands and complexes are reported in Figures S7–S28, while selected electronic
spectral analysis of compounds is given in Table S1.

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Author Contributions: Conceptualization, supervision and editing, M.B.T.; methodology and writ-
ing, M.B.T., F.A.; software, R.A., S.O.A. and H.A.; biological analysis, W.A., A.M.A.; Investigation,
N.A.B., A.M.B., S.N.; review and supervision, S.A.T.; review and editing, M.D.F.A. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at Princess Nourah Bint
Abdulrahman University though the Fast-Track Research Funding Program Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful to The Islamia University Bahawalpur, Pakistan and
University of Jeddah, Saudi Arabia for providing basic lab facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.
References
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