Multifunctional 1,10-phenanthroline derivative and its metal complexes as an anti-Alzheimer’s agent: structure-based drug design, synthesis, characterization and pharmacological studies

Article abstract of DOI:10.1080/00958972.2020.1852223

N-Heterocycles are structural constituents of pharmaceuticals based on FDA approved therapeutic agents with more than 60% of drugs containing N-heterocycles as building blocks. In the present study, a 1,10-phenanthroline derivative with metal acetates forms metal chelates of the type [M^IIL], where L?=?1,10-phenanthroline derivative and M = Co(II), Zn(II), Cu(II) and Ni(II). They were characterized by analysis, spectra [IR, UV–vis, (¹H and ¹³C) NMR, mass, ESR] and magnetism as well as thermal analysis for the structural elucidation. Spectroscopic and analytical measurements confirmed an octahedral geometry for the Co(II), Zn(II), Cu(II) and Ni(II) chelates. The antimicrobial efficacy of the prepared complexes was assessed against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Aspergillus flavus, Candida albicans and Aspergillus niger. The antimycobacterial efficacy of the 1,10-phenanthroline derivative and its chelates was assessed against H₃₇Rv using Microplate Alamar Blue Assay approach and compared with a standard. The acetylcholinesterase (AChE) inhibitory effect of the ligand was examined to find the therapeutic efficiency of compound in the treatment of neurodegenerative diseases. The synthesized ligand exhibited selective inhibition against AChE and butyryl-cholinesterase with IC₅₀ values of 0.20 and 3.20 μM as compared to standards, rivastigmine and galantamine. Further, the in?vitro anti-inflammatory efficiency of metal chelates was examined with the help of egg albumin method. The α-glucosidase inhibition activity was also carried out for the prepared metal complexes.

Full text of DOI:10.1080/00958972.2020.1852223

Journal of Coordination Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Multifunctional 1,10-phenanthroline derivative
and its metal complexes as an anti-Alzheimer’s
agent: structure-based drug design, synthesis,
characterization and pharmacological studies
E. H. Edinsha Gladis , K. Nagashri , A. Suman & J. Joseph
To cite this article: E. H. Edinsha Gladis , K. Nagashri , A. Suman & J. Joseph (2020):
Multifunctional 1,10-phenanthroline derivative and its metal complexes as an anti-Alzheimer’s
agent: structure-based drug design, synthesis, characterization and pharmacological studies,
Journal of Coordination Chemistry, DOI: 10.1080/00958972.2020.1852223
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2020.1852223
Multifunctional 1,10-phenanthroline derivative
and its metal complexes as an anti-Alzheimer’s agent:
structure-based drug design, synthesis, characterization
and pharmacological studies
E. H. Edinsha Gladisa,b, K. Nagashria, A. Sumana and J. Josephb
aDepartment of Chemistry, Manonmaniam Sundaranar University, Tirunelveli, Tamilnadu, India;
bDepartment of Chemistry, Noorul Islam Centre for Higher Education, Kumaracoil, Tamilnadu, India
ABSTRACT
ARTICLE HISTORY
Received 23 May 2020
Accepted 13 October 2020
N-Heterocycles are structural constituents of pharmaceuticals
based on FDA approved therapeutic agents with more than 60%
of drugs containing N-heterocycles as building blocks. In the pre-
sent study, a 1,10-phenanthroline derivative with metal acetates
forms metal chelates of the type [MIIL], where L ¼ 1,10-phenan-
throline derivative and M ¼ Co(II), Zn(II), Cu(II) and Ni(II). They
were characterized by analysis, spectra [IR, UV–vis, (1H and 13C)
NMR, mass, ESR] and magnetism as well as thermal analysis for
the structural elucidation. Spectroscopic and analytical measure-
ments confirmed an octahedral geometry for the Co(II), Zn(II),
Cu(II) and Ni(II) chelates. The antimicrobial efficacy of the pre-
pared complexes was assessed against Bacillus subtilis, Escherichia
coli, Staphylococcus aureus, Aspergillus flavus, Candida albicans and
Aspergillus niger. The antimycobacterial efficacy of the 1,10-phe-
nanthroline derivative and its chelates was assessed against H37Rv
using Microplate Alamar Blue Assay approach and compared with
a standard. The acetylcholinesterase (AChE) inhibitory effect of
the ligand was examined to find the therapeutic efficiency of
compound in the treatment of neurodegenerative diseases. The
synthesized ligand exhibited selective inhibition against AChE and
butyryl-cholinesterase with IC50 values of 0.20 and 3.20 mM as
compared to standards, rivastigmine and galantamine. Further,
the in vitro anti-inflammatory efficiency of metal chelates was
examined with the help of egg albumin method. The a-glucosi-
dase inhibition activity was also carried out for the prepared
metal complexes.
KEYWORDS
Complexes; inhibition;
efficiency; cytotoxicity;
antimicrobial
CONTACT K. Nagashri
shrik1810@gmail.com
Supplemental data for this article is available online at https://doi.org/10.1080/00958972.2020.1852223
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
E. H. E. GLADIS ET AL.
1. Introduction
The emergence of multidrug resistant pathogens exposed the need for new antimicro-
bial agents with multitarget interactions. The acetylcholine (neurotransmitter) is essen-
tial for the treatment of memory and learning in neurodegenerative diseases, and in
patients with Alzheimer’s disease (AD) with lower level of acetylcholine.
Acetylcholinesterase (AChE) is a neurotransmitter, which modulates the acetylcholine
and other esters of choline and terminates essential brain functions [1]. It was also
proposed to play an imperative role in the aggregation of amyloid aggregates during
the formation of fibrils [2–5]. The primary pharmacological strategies for the control of
AD involve enhancement of cholinergic neurotransmission by reducing the rate of
decomposition of acetylcholine (ACh) at synapses through the use of acetylcholinester-
ase inhibitors (AChEIs) [6–8]. As of 2019, around 70 million people worldwide had AD
and with an exponential growth it is expected to reach 140 million by 2030 [9].
Numerous studies have documented that metal chelators have beneficial effects in the
treatment for AD. Some AChEIs are available for AD treatment and inhibit AChE, such
as galantamine, donepezil, tacrine and rivastigmine. They are effective only for treating
mild to moderate AD [10] and showed a non-selective profile along with limited effi-
ciency, adverse cholinergic peripheral side-effects, poor bioavailability and hepatotox-
icity [11]. Evidence from the literature pointed out that 1,10-phenanthroline and 8-
hydroxyquinoline derivatives, both of which are lipophilic in nature, can counteract
the effect of A-toxicity by inhibiting its accumulation in infected cells by metal chela-
tion in AD mice. Pharmacological studies and the development of N-heterocyclic deriv-
atives (1,10-phenanthroline derivatives) as chemotherapeutics have developed interest
to address these factors and have become a promising area of study. Using aromatic
N-heterocycles as ligands which undergo chelation with the metal ion can enable the

JOURNAL OF COORDINATION CHEMISTRY
3
targeting of DNA and have greater ability to bind double strands to DNA via different
interaction modes [12–16].
Generally, metal ions play central roles in the structural organization of microorgan-
isms and in the biological functions of various enzymes. Literature evidence high-
lighted that metal complexes showed significant achievements in therapeutic
efficiency. The utilization of metals/metal ions in drug architecture expanded because
of the success of the anticancer agent cisplatin [17, 18]. Structural modification of
organic ligands with metal complexes is used in the treatment of malignant diseases
as therapeutic agents (e.g. Pt, Au and Ru), including many forms of cancer [19, 20].
Inspired by these literature results, ligand synthesis with an N4O2 chromophore
(obtained by mixing 1,10-phenanthroline-2,9-dicarboxaldehyde with 3-aminonaphtha-
len-2-ol) was focused in the present investigations. We underwent formation with the
ions Co(II), Zn(II), Cu(II) and Ni(II) (Scheme 1). With the aid of FTIR, UV–vis, NMR and
electron spin resonance (ESR) spectroscopies, the ligand and metal chelates were char-
acterized. The cytotoxicity was determined in the HeLa cell line and the influence of
different structural moieties on the pharmacological performances is discussed.
Further, the in vitro anti-inflammatory efficiency of metal chelates was performed with
the help of the egg albumin method. The a-glucosidase inhibition activity was carried
out for the prepared metal complexes. The DNA-binding interaction was studied using
electrochemical, electronic absorption titration, viscosity and thermal denatur-
ation approach.
2. Experimental
2.1. Materials and methods
All chemicals, reagents and solvents were purchased from Sigma and utilized for the
present study. With the help of pretreated, silica gel-coated plates, Thin Layer
Chromatography (TLC) was utilized to monitor the progress of the reaction. Using col-
umn chromatography, the proper choice of mesh and size (60–120) of silica gel was
adopted for purification purposes. The C, H, N composition analyses were recorded
with the assistance of an Elemental Analyzer Carlo Erba EA1108. The metal contents
were measured using gravimetric approach, copper, nickel, cobalt and zinc as cuprous
thiocyanate, nickel dimethylglyoximate, cobalt pyridine thiocyanate and zinc ammo-
nium phosphate, respectively. FTIR spectra of ligand and its metal complexes were
recorded using a Shimadzu FTIR Affinity-1 spectrophotometer. The electronic absorp-
tion spectra of 1,10-phenanthroline derivative and its metal complexes were recorded
using a Systronics UV–visible spectrophotometer (200–1000 nm). The nature of proton
1
and anionic coordination sites were ascertained using H NMR spectrum of ligand and
[ZnL] with the help of a BRUKER 400 MHz spectrometer. Chemical shifts are summar-
ized in ppm relative to TMS (tetramethylsilane) as an internal reference. The Fast Atom
Bombardment (FAB) mass spectra show the molecular mass and fragmentation of lig-
and and metal chelates using a JEOL SX 102/DA-6000 mass spectrometer/data system.
The Systronics conductivity bridge was utilized to measure the molar conductance
and magnetic moment was calculated using Guoy’s electronic balance. The electro-
chemical features of metal chelates were examined with CH Instruments, USA (Model

4
E. H. E. GLADIS ET AL.
Scheme 1. Outline for the synthesis of 1,10-phenanthroline derivative and its metal chelates.
CHI 604D) in DMSO (Dimethyl sulfoxide) (n-Bu4NClO4 as supporting electrolyte)
through a three-electrode cell assembly with platinum electrode as working electrode,
standard calomel as reference electrode and platinum as auxiliary electrode. Thermal
analyses of complexes were performed under nitrogen at heating rate of 10 ꢀC minꢁ1
using a Perkin Elmer instrument.
2.2. Preparation of 1,10-phenanthroline derivative
First step: A reaction mixture of 2,9-dimethyl-1,10-phenanthroline hemihydrate (10 mM)
and selenium dioxide (10 mM) was placed in 100 mL of dioxane in a 250-mL round bottom
(RB) flask. The mixture was manually stirred and allowed to reflux in a wax bath for 5 h. The
hot solution was filtered and a yellow-orange product separated from the filtrate.

JOURNAL OF COORDINATION CHEMISTRY
5
Second step: The ethanolic solution of 1,10-phenanthroline-2,9-dicarbaldehyde
(10 mM) was taken in the RB flask and the solution of 3-aminonaphthalen-2-ol (20 mM)
in the molecular ratio 1:2 was added dropwise. The resulting solution was refluxed for
3 h at RT. TLC plate monitored the course of the chemical process. The synthetic route
to achieve the target compound is outlined in Scheme 1.
Ligand (H2L): Mol. formula: C34H22N4O4, MW ¼ 518.5. Yield: 70%; CHN analysis:
Calcd for: N 10.80, C 78.75, H 4.28; Found: N 10.66, C 78.64, H 4.16%; UV k (nm) (e,
Mꢁ1 cmꢁ1): 252 (5250), 344 (4380). FTIR (cmꢁ1): 3320 v(O–H)aromatic; 3086–3070
1
v(C–H)aromatic; 2964, 2880 v(C–H); 1654 v(C ¼ N)azomethine; 1228 v(C–OH)aromatic. H-NMR,
d (ppm): 8.4 (HC ¼ N, 2H, s), 11.6 (-OH, 2H, s), 6.6–7.8 (18H, m). 13C-NMR, d (ppm)
(DMSO-d6): 108.36–130.06 (aromatic carbons), 135.58 (C ¼ N, phenanthroline moiety),
152.18 (C-O, naphthalene moiety), 160.40 (C ¼ N, imine). FAB MS (m/z): 519.
2.3. Preparation of metal chelates
Equimolar hot ethanolic solutions of 1,10-phenanthroline derivative and copper acet-
ate (0.05 M) were placed in a RB flask. At RT, the reacting mixture was stirred and
allowed to precipitate. The solid product was then separated with methanol and hex-
ane and washed repeatedly. The remaining metal complexes were prepared with the
same procedure. The metal chelates were dried in a vacuum desiccator over fused cal-
cium chloride.
[CuL]: Mol. formula: C34H20N4O2Cu. MW ¼ 580. Yield: 74%; CHN: Calcd for: C 70.39,
H 3.47, N 9.66, Cu 10.96; Found: C 70.25, H 3.34, N 9.52, Cu 10.84%. UV–vis, k (nm) (e,
Mꢁ1 cmꢁ1): 240 (5140), 352 (4600), 495 (1150). FTIR (KBr): 3186 v(N–H), 1170 v(C–N),
1632 v(C ¼ N), 550 v(Cu–N), 486 v(Cu–O). FAB mass: 581 m/z [M þ 1]. Magnetic moment
¼ 1.86 BM. Km (S cm2 molꢁ1) ¼ 20.
[NiL]: Mol. formula: C34H20N4O2Ni. MW ¼ 575. Yield: 60%; CHN: Calcd for: C 70.99, H
3.50, N 9.74, Ni 10.20; Found: C 70.85, H 3.38, N 9.62, Ni 10.06%. UV–vis, k (nm) (e,
Mꢁ1 cmꢁ1): 352 (4480), 250 (5000), 550 (210), 730 (175). FTIR (cmꢁ1): 3176 v(N–H),
1164 v(C ꢁ N), 1622 v(C ¼ N), 540 v(Ni–N), 486 v(Ni–O). FAB-MS, m/z: 576 [M þ 1].
meff(BM) ¼ 3.38. Km (S cm2 molꢁ1) ¼ 12.
[CoL]: Mol. formula: C34H20N4O2Co. MW ¼ 575. Yield: 68%; CHN: Calcd for: C 70.96,
H 3.50, N 9.74, Co 10.24. Found: C 70.78, H 3.36, N 9.60, Co 10.18%. UV–vis, k (nm) (e,
Mꢁ1 cmꢁ1): 350 (4450), 500 (320), 650 (240). FTIR (cmꢁ1): 3196 v(N–H), 1150 v(C–N),
1620 v(C ¼ N), 540 v(Co–N), 490 v(Co–O). FAB mass: 576 m/z [M þ 1]. meff(BM) ¼ 4.32.
Km (S cm2 molꢁ1) ¼ 10.
[ZnL]: Mol. formula: C34H20N4O2Zn. MW ¼ 582. Yield: 70%; CHN: Calcd for; C 70.17,
H 3.46, N 9.63, Zn 11.24; Found: C 70.00, H 3.35, N 9.58, Zn 11.14%. UV–vis, k (nm) (e,
Mꢁ1 cmꢁ1): 268 (5100), 380 (4230) nm. FTIR (cmꢁ1): 3178 v(N–H), 1156 v(C–N), 1614
1
v(C ¼ N), 556 v(Zn–N), 482 v(Zn–O). H NMR, d (ppm): 8.2 (HC ¼ N, 2H, s), 6.4–7.8 (18H,
m). FAB mass: 583 m/z [M þ 1]. Km (S cm2 molꢁ1) ¼ 14.
2.4. Pharmacological studies
2.4.1. Antioxidant activity: ferricyanide reducing assay
The reducing power of tested compounds was assessed with the ferricyanide assay.
Different concentrations of tested compounds were mixed with 2.0 mL of phosphate

6
E. H. E. GLADIS ET AL.
buffer and 2.0 mL of 1% potassium ferriccyanide and incubated at 40 ꢀC for 20 min. To
this reaction mixture, a solution of 2.0 mL of 10% trichloroacetic acid was added and
centrifuged for 20 min. The top layer was collected and mixed with 2.5 mL of deion-
ized water and 0.5 mL of 0.1% ferric chloride. The absorbance of the resulting solution
was obtained from a spectrometer at 700 nm. Increase in the absorbance of reaction
mixture indicates higher reducing power.
2.4.2. Antituberculosis activity
The anti-mycobacterial activities of the synthesized ligand and its metal complexes
were screened against M. tuberculosis using Microplate Alamar Blue Assay (MABA).
2.4.3. Antimicrobial activities
The in vitro antimicrobial activities of the compounds were tested against bacterial and
fungal species. Prior to the experiment, bacterial and fungal cultures were inoculated in
broth (inoculation medium) and incubated overnight at 35 ꢀC. Inoculation medium con-
taining grown culture was added aseptically to the nutrient medium and mixed thor-
oughly to get uniform distribution. The solution was poured into petri dishes and then
allowed to gel at room temperature. Wells (5 mm in diameter) were cut in the agar plates
using proper sterile tubes. Then, wells were filled to the surface of agar with 0.1 mL of
the test compounds dissolved in DMSO (100 lM mL–1). The plates were allowed to stand
for 1 h to facilitate diffusion of the drug solution. Then, the plates were incubated at
35 ꢀC for 24 h for bacteria and 72h for fungi and the diameter of the inhibition zones
were measured. Minimum inhibitory concentrations (MICs) were determined by using
the serial dilution method. The lowest concentration (lg mL–1) of the compound which
inhibits the growth of bacteria after 24-h incubation at 35 ꢀC, and of fungi after 72-h
incubation was taken as the MIC. The concentration of DMSO-d6 in the medium did not
affect the growth of any of the microorganisms tested.
2.4.4. a-Glucosidase inhibitory assay
The prepared metal chelates were assessed for a-glucosidase inhibitory efficiency with
slightly modified methodology adopted by Pistia Brueggeman and Hollingsworth. The
metal chelates were prepared at different concentrations (20–400 lg mL–1); 100 mL of
phosphate buffer along with enzyme (a-glucosidase) concentration of 10 lL were incu-
bated for 20 min at RT. After incubation time, the inhibitory action was started with
slow addition of pNPG(b-Galactosidase) (substrate) of 10 lL and again incubated for
30 min. The inhibitory action was terminated with drop by drop addition of 50 lL
(0.01 N) Na2CO3 and absorbance was noticed at 405 nm using a multi-well plate
reader. Enzymatic activity was calculated as:
ODBlank ꢁ ODSample =ODBlank ꢂ 100
ð
Þ
The IC50 values were calculated and tabulated for the prepared chelates against
a-glucosidase inhibitory efficiency.
2.4.5. Anticholinesterase activity assays
The inhibitory activities of acetyl- (AChE) and butyryl-cholinesterase (BuChE) of the
synthesized 1,10-phenanthroline derivative was determined according to modified

JOURNAL OF COORDINATION CHEMISTRY
7
Ellman’s method [21]. The inhibitory concentration (IC50) was determined by plotting
of absorbance against inhibition (%) curve. IC50 values were calculated for a given
sample, the concentration at which the sample inhibits half of the maximum biological
response. The substrates, acetylthiocholine iodide and butyrylthiocholine iodide, were
utilized for the measurements. 5,50-Dithio-bis(2-nitrobenzoic) acid (DTNB) was used to
measure anticholinesterase activity. Stock solutions of the 1,10-phenanthroline deriva-
tive, galanthamine and rivastigmine, in DMSO were prepared at 200 mg mL–1. The reac-
tion mixture consists of 100 mL of 0.1 mM phosphate buffer (pH 8.0), 10 mL of sample
solution (1,10-phenanthroline derivative) and 20 mL AChE (2.45 ꢂ 10ꢁ4 U mL–1) (or
3.10 ꢂ 10ꢁ4 U mL–1 BuChE) solution and were mixed and incubated for 15 min at 25 ꢀC.
About 10 mL of DTNB solution was prepared by adding 2.0 mL of pH 7.0 and 4.0 mL of
pH 8.0 phosphate buffers to a mixture of 1.0 mL of 16 mg mL–1 DTNB and
7.5 mg mL–1 NaHCO3 in pH 7.0 phosphate buffers. The reaction was initiated by add-
ition of 10 mL (7.0 mM) acetylthiocholine iodide or 0.79 mM butyrylthiocholine iodide.
The activity was measured by following the yellow color produced as a result of the
thioanion produced by reacting the enzymatic hydrolysis of the substrate with DTNB.
DMSO was used as solvent. The standards galantamine and rivastigmine were chosen
as standards.
2.4.6. Anti-inflammatory study
The prepared ligand and their metal chelates were reacted with diclofenac at 10 lM,
50 lM, 100 lM, 250 lM and 500 lM mixed in double distilled water and phosphate
buffer. The reaction mixture (2 mL) and 1% egg albumin solution (1 mL) in buffer was
kept at 37 ꢀC for 20 min in an incubator. The denaturation of egg albumin was
observed at 40 ꢀC in a water bath for 15 min. The prepared chelates were assessed for
anti-inflammatory efficiency with the help of denaturation of egg albumin protocol. In
this assay, the denaturation induces the change of absorbance at 660 nm with the
help of a UV–vis double beam spectrophotometer; diclofenac was utilized as standard.
The observed outcomes were compared with standard drug/literature resources at dif-
ferent concentrations (10, 50, 100, 250 and 500 lM/mL). The inhibition percentage
(IC50) was arrived using the formula:
% denaturation of egg albumin inhibition ¼ ½ðAt ꢁ AcÞ=Atꢃ ꢂ 100
where At and Ac are absorbance of sample and standard, respectively [22].
2.4.7. DNA-binding studies
2.4.7.1. UV–vis absorption titration studies. Absorption titrations were carried out for
metal(II) complexes with constant concentration (10 mM) while varying the DNA con-
centration from 0 to 10 mM. DNA concentration was determined from its absorbance
with the help of molar extinction coefficient (6600 Mꢁ1 cmꢁ1) at 260 nm. The CT-DNA
stock solution was prepared by diluting DNA in Tris-HCl/NaCl buffer at pH 7.2 (5 mM
Tris-HCl and 50 mM NaCl). The absorption (A260/A280) ratio of DNA solution is 1.8–1.9,
indicating that DNA was sufficiently pure and free of protein. All stock solutions were
stored at 4 ꢀC and used within 3–4 days or freshly prepared. After addition of DNA to
the metal complex, the resulting solution was allowed to equilibrate at RT for 5 min,
ꢄ
and then absorption readings were measured. The changes in the absorbance of n–p

8
E. H. E. GLADIS ET AL.
transition band was monitored after each addition of CT-DNA solution. The intrinsic
binding constant (Kb, equation given in the Supplementary Information) was calcu-
lated using the absorbance and extinction coefficient values.
2.4.7.2. Viscosity study. Viscosity experiments were carried out using Ostwald’s viscom-
eter. In this experiment, DNA concentration was kept constant and concentration of
complex solution increased gradually from 0 to 100 mM. The flow times were measured
with a stop-watch and each sample was measured in triplicate for the same complex
solution to get an average flow time. To study the effect of DNA binding on the vis-
cosity of the complex, the results were presented as (g/go)1/3 versus [complex]/[DNA],
where g and go are the viscosity of CT-DNA solutions in the presence and absence of
the complex, respectively. Viscosity values were calculated from the experimental flow
time of CT-DNA containing solutions corrected from the flow time of buffer alone.
2.4.7.3. Thermal denaturation. Thermal denaturation investigations were performed
with a Shimadzu double beam spectrophotometer equipped with a temperature-con-
trolling programmer (0.1 ꢀC). The absorbance at 260 nm was continuously monitored
for solutions of CT-DNA (50 lM) in the absence and presence of the complexes
(10 lM). The temperature of the solution was increased by 1 ꢀC minꢁ1. The measure-
ments were carried out in 10 mM Tris/HCl buffer, pH 7. The DNA-melting is observed
when double-stranded DNA molecules are heated and separate into two single
strands. It occurs due to disruption in intermolecular forces such as p-stacking and
hydrogen bonding interactions between DNA base pairs.
3. Results and discussion
Preparation of the desired 1,10-phenanthroline derivative started with 2,9-dimethyl-
1,10-phenanthroline in two steps. The first synthetic step involved the reaction of 2,9-
dimethyl-1,10-phenanthroline with selenium dioxide in dioxane at room temperature
with stirring for 5 h to obtain the desired 1,10-phenanthroline-2,9-dicarbaldehyde. It
was then condensed with 3-aminonaphthalen-2-ol in hot ethanolic solution for 3 h
and refluxed to obtain the ligand (H2L). Metal acetate(s) were reacted to achieve the
desired complexes for medicinal applications. Elemental composition, molecular mass
and conductivity values suggested molecular formulas of metal chelate with excellent
matching with theoretical results. With the assistance of spectral measurements, ther-
mal and magnetic behavior, structural elucidation was attempted and supported the
proposed structural formulas. In water or in non-polar organic solvents, the complexes
were insoluble, but partly soluble in DMSO or DMF. Numerous attempts, such as crys-
tallization using solvent mixtures and low temperatures, failed for the growth of single
crystals appropriate for X-ray crystallography. The analytical, spectroscopic and mag-
netic data, however, permitted assigning possible structures of the synthe-
sized complexes.
By elemental analyses, the stoichiometry and formulation of the Schiff base ligand
(H2L) and its metal complexes were confirmed. In the complexes, the metal-to-ligand
ratio was 1:1 from carbon, hydrogen, nitrogen and metal content(s). The elemental

JOURNAL OF COORDINATION CHEMISTRY
9
analyses of ligand and complexes showed strong agreement with the structure pro-
posed. The molar conductivity values of metal chelates (0.001 M) in DMSO with 1,10-
phenanthroline derivative (10–20 mho molꢁ1 cm2) suggest non-electrolytic behavior
[23] and removal of counter ion from complex formation as acetic acid.
3.1. FTIR spectra
The FTIR spectra of ligand and its copper complex are shown in Supplementary
Information Figures S1 and S2. The IR spectra of the free ligand exhibited a broad
band at 3380 cmꢁ1 corresponding to phenolic -OH which disappeared in IR spectra of
metal complexes, indicating coordination through phenolic hydroxyl group. The shift
of ꢀ(C–O) at 1208 cmꢁ1 to a lower frequency suggests weakening of ꢀ(C–O) and for-
mation of stronger M–O bond. Another important band in the IR spectra of the ligand
at 1654 cmꢁ1 due to C ¼ N group stretching vibration was shifted by 10–20 cmꢁ1
upon complexation, implying its coordination via azomethine nitrogen [24]. The shift
of ꢀ(C ¼ N) vibration in all complexes to a lower frequency suggests that the nitrogen
of the ring contributes to complexation. The lower ꢀ(C ¼ N) frequency indicates stron-
ger M–N bonding. Furthermore, the formation of complexes was also ascertained by
the presence of medium intensity bands at 576–578 cmꢁ1 and 468–470 cmꢁ1, which
were assigned to v(M-O) and v(M-N), respectively. Therefore, from IR spectra, it is con-
cluded that H2L is a bi-negative hexadentate ligand with N,O-donor sites coordinating
to the metal ions via the azomethine N and deprotonated phenolic–O atoms.
1
3.2. H NMR spectra
The 1H NMR spectra of 1,10-phenanthroline derivative (Supplementary Information
Figure S3) H2L showed a signal at 11.10 ppm (singlet, 2H), which was the phenolic
–OH protons. Upon addition of D2O, the OH signal disappeared. The singlet at
8.3 ppm was assigned to azomethine (–CH ¼ N–) proton. The spectrum also contains
multiplets at 6.84–7.86 ppm which were assigned to aromatic protons (both 1,10-phe-
nanthroline and phenol moieties). In comparing the spectrum of ligand and its Zn(II)
complex, the OH proton signal of ligand disappeared in the Zn(II) complex
(Supplementary Information Figure S4). The vanishing of –OH signal indicated depro-
tonation of the hydroxyl group of ligand and confirmed the bonding of oxygen to the
metal ion (C–O–M), consistent with FTIR observations. 1H NMR spectra provide evi-
dence for the presence of either one or two azomethine groups. In the Zn(II) complex,
the position of azomethine signal was shifted upfield at 8.34 ppm in comparison with
that of the free ligand, inferring coordination through the azomethine nitrogen.
3.3. 13C NMR spectra
The 13C NMR spectrum of H2L shows the signals corresponding to aromatic carbons at
108.26–135.58 ppm. The peaks at 160.40 and 152.18 ppm correspond to carbons
(C ¼ N)
and (C-O)phenolic, respectively. In the case of Zn(II) complex, the above
carbon signals were changed to 158.62 and 149.50 ppm, demonstrating coordination
azomethine

10
E. H. E. GLADIS ET AL.
Figure 1. ESR spectrum of [CuL] at 77 K.
between zinc and azomethine nitrogen and (C-O)phenolic [25]. The NMR spectra thus
1
authenticate the vibrational frequencies from the IR. The observations of H and 13C
NMR spectra support the ligand and its Zn(II) complex structures.
3.4. Mass spectra
FAB mass spectra confirmed the molecular weight of ligand and its metal chelates. The
ligand mass spectrum conforms to the suggested formula (molecular ion m/z 503,
Supplementary Information Figure S5). The molecular mass peak in [CuL] was witnessed
at m/z 581 (Supplementary Information Figure S6) which confirms that metal complex is
monomeric. In addition, mass spectra showed different peaks reflecting successive deg-
radation of the complexes, an indication of fragment stability. Therefore, the molecular
mass of complexes and molar conductivity values suggested [ML] metal complexes.
3.5. ESR spectra
The ESR spectra of Cu(II) complex are recorded at 77 K in DMSO (Figure 1), and g and A
were determined. The observed g values for copper chelate of 2.265 suggest a mix of
jj
covalent and ionic character in the metal ligand bond. In order to measure the degree of
distortion of the copper(II) complex, we calculated the factor (f ¼ g /A ), which is known
jj jj
to be an empirical index of tetrahedral distortion [26] and is found to be 146.4, character-
istic of mild to moderate distortion [27], indicating the existence of substantial distortion
of the dihedral angle in the xy-plane. The results are consistent with distorted octahedral
geometry around the copper site and supports coordination through azomethine nitro-
gen, which forces the structure to remain in a nearly octahedral geometry [28].
3.6. Electronic absorption spectra
The electronic absorption spectrum of ligand showed bands at 252 nm
(e ¼ 5250 Mꢁ1 cmꢁ1) and 344 nm (e ¼ 4380 Mꢁ1 cmꢁ1), which are recognized to be p–p
ꢄ
ꢄ
and n–p transitions. In the spectra of metal complexes, these transitions shifted to lower

JOURNAL OF COORDINATION CHEMISTRY
11
wavelength and some significant changes are connected with donor atoms that are
involved in the bonding between metal and ligand. The electronic absorption spectrum of
Co(II) complex exhibited bands at 650 nm (e ¼ 240 Mꢁ1 cmꢁ1
) and 500 nm
(e ¼ 320 Mꢁ1 cmꢁ1) corresponding to 4T1g! T2g(F) and 4T1g! A2g(F) transitions. Its mag-
netic moment value of 4.34 BM suggested octahedral geometry of the Co(II) complex. The
electronic spectra of copper(II) complex exhibited bands at 240 nm (e ¼ 5140 Mꢁ1 cmꢁ1),
352 nm (e ¼ 4600 Mꢁ1 cmꢁ1) and 495 nm (e ¼ 1150 Mꢁ1 cmꢁ1) which may be assigned to
4
4
2
2
2
2
2
2
the transitions T1g! T2g(F), T1g! A2g(F) and T1g! T1g(P), respectively. Based on the
absorption bands, an octahedral geometry was assigned for the Cu(II) complex. The mag-
netic value of 1.92 BM further supports the octahedral geometry of the Cu(II) complex. The
UV–vis spectra of the Ni(II) complex showed charge-transfer transition in the absorption
region 550 nm (e ¼ 210 Mꢁ1 cmꢁ1) and 700 nm (e ¼ 175 Mꢁ1 cmꢁ1). The observed transi-
tions 3A2g(F)-3T2g(F) and 3A2g(F)-3T1g(P) are dependable with well-defined octahedral con-
figuration around ligand structural core. The [NiL] also showed a magnetic value of 3.28
BM, indicating distorted octahedral geometry.
3.7. Stability study
With the assistance of a UV–vis spectrophotometer, the stability of prepared metal
chelates in DMSO was monitored. The complexes are unchanging, preserving spectral
appearance with minor deviations after 24 h. The stability of metal chelates in
Phosphate Buffered Saline (PBS) buffer was also assessed under biological conditions.
All complexes are stable up to 24 h and express only minor variations in spectral
appearance after a long period (after 24 h) due to ligand under physiological condi-
tions undergoing hydrolysis. The stability of [ZnL] is also studied with NMR spectrum.
3.8. Metal-chelating properties of ligand
Experimental data on the role of metals (Cu, Fe, Zn, etc.) involved in aggregation with
Ab protein induces neurotoxicity with the formation of various reactive oxygen species
in the brain. Several experimental techniques indicated that these metals are capable
of binding with Ab protein, enhancing its aggregation and leading to biochemical
damages observed in AD brain. To reduce the neurotoxicity based on the above
observations, the exclusion of metals from the brain (infected parts) is an emerging
beneficial approach for AD. In the current investigations, the complexation ability of
ligand with biometals such as Cu, Ni, Co and Zn was studied by a UV–vis spectropho-
tometer. The electronic absorption spectral values of ligand were remarkably shifted in
combination with the exposure of metal acetates and suggested complex formation
with ligand. Therefore, the synthesized ligand may behave as metal chelator to
remove metal ions from solution.
3.9. Thermogravimetric analysis
The thermogravimetric profile shows lattice water or coordinated water molecules,
anions, molecular fragments after decomposition of ligand and metal oxide as residue

12
E. H. E. GLADIS ET AL.
Table 1. MIC values of ligand, metal salts and its metal complexes against different microbial spe-
cies (mg/mL).
Compound/Standard
E. coli
S. aureus
B. subtilis
C. albicans
A. niger
A. flavus
Ligand, H2L
[CuL]
[NiL]
92
30
46
86
26
52
90
22
40
98
20
36
82
18
44
80
25
38
[CoL]
40
30
38
35
40
42
[ZnL]
28
35
46
30
38
30
Copper acetate
Nickel acetate
Cobalt acetate
Zinc acetate
Sreptomycin
Nystatin
102
106
128
116
8
110
110
144
110
12
108
116
130
108
6
98
100
132
140
120
–
112
125
122
104
–
120
136
114
–
–
–
–
14
10
15
[29]. The thermogravimetric profile of the metal complexes was recorded up to 800 ꢀC
with the heating rate of 10 ꢀC min–1 under nitrogen.
For [CuL], no decomposition below 200 ꢀC indicates there are no water molecules
present in the complex. At 260–400 ꢀC, the first decomposition was detected which
partially corresponds to decomposition of the ligand center. There is a small deviation
from the straight line, suggesting the second decomposition with complete loss of
organic moiety at 340–468 ꢀC and copper oxide (CuO) was observed along with min-
imal amount of ash. Identical thermogravimetric characteristics were observed for the
other complexes. Thermal stability is important in optoelectronic applications (for
OLEDs it is important to provide material stability without decomposition at high tem-
perature). Materials with high Ti values (600 ꢀC) are essential for device fabrications
with improved performance. The prepared metal chelates exhibited high thermal sta-
bility values (Ti) and may be suitable for optoelectronics applications [30]. The
observed decomposition shows excellent agreement with the proposed structural for-
mulas of the metal chelates as depicted in Scheme 1.
3.10. Antimicrobial activity
The World Health Organization (WHO) has concerns due to increased antimicrobial
resistance. Metal-based pharmaceuticals represent a promising approach to new drugs
with various action mechanisms. The goal of destroying hydrophobic interactions of
bacterial plasma membranes, consisting of a phospholipid bilayer similar to that of
eukaryotic cells, involves amphiphilic metal complexes. The synthesized ligand and its
metal complexes were screened against microbial species Bacillus subtilis, Escherichia
coli, Aspergillus flavus, Staphylococcus aureus, Aspergillus niger and Candida albicans.
The growth inhibition of microbial species is expressed in terms of MIC and summar-
ized in Table 1. The experimental findings suggested that the prepared copper chelate
inhibits microbial growth better than other chelates, 1,10-phenanthroline derivative
and standards under the same experimental conditions due to its chelation via the
action sequence of RNA/DNA and protein synthesis inhibitors. The observed findings
suggested better efficiency of the prepared metal chelates against Gram-positive bac-
terial strain than Gram-negative bacteria due to their difference in the composition of
the cell membranes.

JOURNAL OF COORDINATION CHEMISTRY
13
The biological activity of a given molecule is affected by many factors, including
lipophilia, resonance, chemical structure and thermodynamic stability [31–35]. Many
Schiff bases and their metal complexes exhibit a range of cytotoxic activities, with
small changes in chemical composition, often leading to vast differences in selectivity
and activity [36, 37]. Schiff base complexation with metal ions reduces the polarity of
metal ions upon chelation [38]. This results in enhancement of the metal lipophilic
characteristics, allowing for the blockage of cellular enzymatic activity [39].
Coordination also decreases the metal ion’s polarity, mainly due to the partial sharing
of its positive charge with the donor groups [40]. The compounds showed more activ-
ity against Gram-positive bacteria than toward Gram-negative bacteria. This may be
attributed to the difference in the composition of the cell membrane, where Gram-
negative bacteria’s outer membrane will minimize the damage from the synthesized
compounds, playing a preventive role in the antibacterial effect of the compounds.
Compounds with an imine group, conjugated planar moiety and metal ion are suit-
able for anti-TB agents and may also serve as modulators to disrupt bacterial metabol-
ism by inhibiting enzymes and inhibiting the DNA-function by binding with the active
DNA-sites through hydrogen bonding, hydrophobic interaction and electrostatic inter-
actions. Furthermore, there is no toxic nature of metal complexes as compared to
metal salts, owing to its liphophilic 1,10-phenanthroline and naphthalene moieties
which enhance the efficacy.
3.11. Ferric reducing antioxidant power
In this assay, the yellow color of the test solution changed into different shades of
color depending on the reducing power of each compound/complex. The reducing
power of a compound serves as a significant indicator of its potential antioxidant
activity. The antioxidant efficiency was monitored at 700 nm and standard (BHT) at
various concentrations with higher absorbance compared to that of the tested com-
plexes. The reducing power of the complexes increased with increase in concentration.
Metal complexes showed much better activity than the ligand and metal precursors.
The antioxidant values are expressed in IC50 (mM) values. The antioxidant efficiency of
tested samples is 0.17 0.02 ([CuL]), 0.28 0.10 ([NiL]), 0.26 0.20 ([CoL]), 0.18 0.30
([ZnL]), 0.52 0.25 (ligand) and 0.30 0.05 mM (BHT), respectively. Among the metal
complexes studied, the [CuL] exhibited higher antioxidant power due to its redox
potential and organic moieties (1,10-phenanthroline and naphthalene).
3.12. Anti-tubercular activity
Compared with 1,10-phenanthroline derivative and reference compound (INH), the
metal complexes displayed enhanced in vitro anti-tuberculosis activity against
Mycobacterium tuberculosis H37Rv. Comparing the efficacy of antituberculosis, the Cu(II)
complex found higher potency with a MIC value of 0.54 mM (also a higher binding
interaction with DNA), while other metal chelates displayed slightly lower efficiency
(0.80 ([ZnL]), 0.90 ([NiL]) and 0.75 ([CoL]) mM). The greater potency of [CuL] against M.
tuberculosis stems from the ability of 1,10-phenanthroline derivative to form stable

14
E. H. E. GLADIS ET AL.
lipophilic chelates with Cu2þ, which can be transmitted across membrane into myco-
bacterial cells. Results show that compounds with imine group, conjugated planar
moiety and metal ion show strong anti-TB activity.
In pharmaceutical laboratories/industries, logP is used to arrive at the characteristic
activity of chemotherapy agents in the human body. Lipophilia is a major factor that
influences absorption, body distribution, penetration through essential membranes
and biological barriers, metabolism and excretion (ADME properties). The logP of a
compound intended for oral administration should be <5 according to “Lipinski’s rule
of 5” (developed at Pfizer). In the present analysis, the chelate logP were 1.20 ([CuL]),
1.86 ([ZnL]), 2.12 ([NiL]) and 2.30 ([CoL]). The copper chelate is an excellent candidate
for therapeutic applications; targeting the central nervous system (CNS) would have a
value of logP around 2 (ideal value is 1.35–1.8).
The “Briggs Rule of 3” indicates to be bioavailable (among other parameters) for an
agrochemical and should have a value of logP < 3. The [CuL] will, in the present case,
be used for agricultural purposes without harming plants and enriching the
productivity.
3.13. DNA-interaction studies
One of the possible mechanisms responsible for cytotoxicity is the interaction of metal
complexes with DNA (along with a host of other targets that occur in living cells).
There are two types of binding metal complexes to DNA: covalent [41] and non-cova-
lent (which involves electrostatic interactions, groove binding and base pair intercal-
ation) [42]. An elucidation of the DNA–metal complex binding mode versus biological
activity relationship offers important evidence to new DNA-oriented metal complexes
with an enhanced therapeutic potential.
3.13.1. Electrochemical studies
Cyclic voltammetric investigations on the Cu2þ complex with 1,10-phenanthroline
derivative displayed a quasi-reversible one-electron redox process involving the Cu2þ
/
Cuþ couple at Epa ¼ 0.05 and Epc ¼ ꢁ0.20 V within potential scan range of 1.0 to
–1.0 V [40]. With the addition of CT-DNA to the metal complex (Figure 2), a significant
decrease in both anodic and cathodic peak current, a positive shift in E1/2 and a differ-
ence in peak potential (Ep) suggested slow diffusion at the electrode surface due to
the binding of copper chelate to the electrode surface via intercalation of phenyl rings
and electrostatic interactions between the negatively charged phosphate backbone
and positively charged copper ion [42, 43]. Metal complex displayed substantial cyto-
toxicity activity against the cell line of HeLa.
3.13.2. Electronic absorption titration
Using electronic absorption spectroscopy, the interactions between metal complexes
and DNA were established. DNA is a therapeutic target molecule which binds with
metal complexes through intercalation due to a strong stacking interaction between
an aromatic chromophore and DNA-base pairs. The electronic absorption spectra of
[CuL] in the absence and presence of DNA are shown in Figure 3 and Table 2. In the

JOURNAL OF COORDINATION CHEMISTRY
15
Figure 2. Cyclic voltammogram of [CuL] in the absence (red color) and presence of DNA
(green color).
Figure 3. Electronic absorption spectrum of [CuL] in the absence (black color) and presence of
DNA (colored lines) [Complex] ¼ 10 mM; [DNA] ¼ 10 mM, 20 mM, 30 mM, 40 mM; 50 mM.
ꢄ
absence of DNA, [CuL] showed a band at 344 nm which corresponds to p!p transi-
tion. This absorption band experienced both hypochromism and bathochromism upon
addition of DNA, indicating that the complex binds to CT-DNA through intercalation.
The intrinsic binding constant (Kb) of [CuL] with DNA was determined based on the

16
E. H. E. GLADIS ET AL.
Table 2. Effect of CT-DNA on the absorbance bands and binding constant of the complexes.
kmax (nm)
Complex
Free
Bound
Dk (nm)
% Hypochromism
Kb (ꢂ105 M–1
)
[CuL]
[NiL]
[CoL]
[ZnL]
344
320
335
350
349
324
341
355
5
4
6
5
15.30
10.20
12.40
14.10
4.11
3.62
3.85
2.04
absorbance and molar extinction coefficient. In order to compare the binding
strengths of the metal complexes to CT-DNA, the Kb obtained for [CuL], [NiL], [CoL]
and [ZnL] were 4.11, 3.62, 3.85 and 2.04 ꢂ 105 Mꢁ1, respectively, and suggested that
ꢄ
[CuL] has good binding affinities due to p!p stacking interactions between the com-
plex and the DNA-base pairs [37, 39, 40]. The binding strength of copper(II) complex
to DNA is stronger than that of ethidium bromide (EB) to DNA (Kb ¼ 3.3 ꢂ 105 Mꢁ1
)
due to distorted octahedral geometry. The [CuL] has higher binding constant which
correlates with it having the lowest IC50 value among the complexes performed. The
greater efficacy of [CuL] against cancer cells arises from its ability to block the
enzymes that bind to the nitrogenous bases of DNA or RNA.
3.13.3. Viscosity measurements
The viscosity experiment is an important method for evaluating the binding of metal
complexes with DNA. Viscosity measurements were performed to establish the interac-
tions between the complex and DNA through intercalation or electrostatic or groove-
binding mode. In the absence of crystallographic structural data, hydrodynamic meas-
urements, based on changes in length (viscosity and sedimentation), are considered to
be important binding information. The relative viscosity changes indicate the binding
affinity of complexes with DNA. A classical mode of intercalation means the helix of
DNA must be lengthened and the viscosity of DNA increased. Lower viscosity changes
are due to electrostatic or groove surface binding. Ethidium bromide, a well-known
DNA intercalator, greatly increases the relative viscosity by lengthening the double
helix of DNA by intercalation. Upon addition of metal complexes to DNA (Figure 4), an
increase in the viscosity was noticed. The observed increase in viscosity indicated that
the complexes bind through intercalation, consistent with electronic spectral data. The
above experimental observations indicated that [CuL] has higher DNA-binding affinity
than other metal complexes. The interaction of these complexes with DNA is mainly
by intercalation of the planar 1,10-phenanthroline ligand into the DNA-base pairs, pro-
ducing the strong p–p stacking interaction.
3.13.4. DNA melting studies
The binding affinity of metal complexes into the double DNA-helix is known to signifi-
cantly increase the melting temperature of CT DNA, at which the double helix dena-
tures into single-stranded CT DNA. The interaction of complexes with CT DNA through
intercalation was ascertained on the basis of melting temperature. The melting tem-
perature of metal complexes with CT DNA was monitored at the absorbance at
260 nm from 30 to 90 ꢀC (Figure 5). The melting curves of CT DNA in the absence and
presence of the copper(II) complex are illustrated in Figure 5 and Table 3. The Tm

JOURNAL OF COORDINATION CHEMISTRY
17
Figure 4. Effect of metal(II) complexes on the relative viscosity of CT-DNA (pH ¼ 7.4). Acetonitrile
as solvent: [DNA] ¼ 10 mM; 30 ꢀC; [Complex] ¼ 10 mM.
Figure 5. Plots of absorbance versus temperature (ꢀC) for the melting of CT DNA in the presence
and absence of metal complexes. [DNA] ¼ 10 mM; [Complex] ¼ 10 mM.
value for the free CT DNA is 65 ꢀC and Tm value in the presence of [CuL] is 75 ꢀC. A
small change in the DNA melting temperature or denaturation temperature (DTm) on
addition of the complex to CT DNA was observed. The extinction coefficient of DNA at
260 nm in the double helical form is much less than that in the single-stranded form;
hence, the melting of the helix leads to an increase in the absorption at this wave-
length. Thus, the helix to coil transition temperature can be determined by monitoring
the absorbance of DNA bases at 260 nm as a function of temperature (Tm). The desta-
bilization of DNA-structure occurred due to the p-stacking interaction between com-
plex and the base pairs of DNA. Metal coordination and the organic structural core
(1,10-phenanthroline and naphthalene) can greatly influence the structure, conform-
ation, stability and affinity of nucleic acids. The intercalation of complexes into the

18
E. H. E. GLADIS ET AL.
Table 3. Melting temperature in the absence (DNA alone) and in the
presence of different metal complexes.
S. No
Metal complexes
Tm (ꢀC)
1
2
3
4
5
Absence of metal complex
70
80
72
75
76
[CuL]
[CoL]
[NiL]
[ZnL]
double helix is known to significantly increase the helix melting temperature, at which
the double helix denatures into single-stranded DNA [26, 28]. A similar trend was also
observed for all other metal complexes (Figure 5).
3.14. Cytotoxicity activity
After 24 h of incubation, the cytotoxic activities of the complexes and free ligand were
evaluated on the human cancer cell line HeLa. It depends on their ability to bind DNA
and damage its structure resulting in impairment of its function which is followed by
inhibition of the replication and transcription process and eventually cell death. The
results were analyzed using curves of cell viability and values of IC50. The values for
IC50 have been calculated using the MTT assay, which tests the activities of mitochon-
drial dehydrogenase in living cells. At 24-h incubation, the [CuL] exhibited better activ-
ity than cisplatin. There is a substantial increase in anti-proliferative activity due to the
synergetic effect of combining Cu(II) and a conjugated ligand system, which is further
strengthened by the presence of phen coordinated to the metal core, significantly
increasing cytototoxicity, particularly in the HeLa cell line, with a decrease of more
than 20 times in IC50. Although the compound concentration rises, the viability of the
cells decreases (Figure 6); the viability of cells incubated without any compound was
considered to be 100%. Increasing the complex concentration increases the adsorption
of the complex on cell membranes, leading to increased penetration, encouraging
binding to DNA bases, and inducing successful cell killing activity. [CuL] showed a
cytotoxic profile based on concentration in HeLa. The Cu(II) complex (IC50 ¼ 0.20 mM)
showed greater cytotoxicity than its parent ligand (0.90 mM) and other complexes (IC50
¼ 0.32 mM ([ZnL]), 0.38 mM ([NiL]), 0.44 mM ([CoL])).
3.15. a-Glucosidase inhibition
The study on a-glucosidase inhibition for the complexes is described in Table 4,
reflecting the IC50 values. The chelation of various metal acetates produced a percep-
tible increase in the inhibitory potential of a-glucosidase and demonstrated the vital
function of metal(II) center during enzyme inhibition. This research predicts concentra-
tion-dependent inhibition of a-glucosidase induced by the complexes. Among the
complexes tested, the copper(II) complex was a potent a-glucosidase inhibitor (IC50
0.20 lM), approximately 200 times more active than deoxynojirimycin (DNJ) (IC50
300 lM) standard a-glucosidase inhibitor. The active site residues of a-glucosidases
(imidazole and carboxy groups) interact with metal(II) ion by soft electron donors such
as imidazole nitrogen, and imparts enzyme inhibition. The a-glucosidase inhibition is
¼
¼

JOURNAL OF COORDINATION CHEMISTRY
19
Figure 6. A plot of % cell viability vs. concentration of metal complexes.
Table 4. a-Glucosidase inhibition activity of metal(II) complexes, metal precur-
sors and standard.
S. No
Complex/standard
IC50 (mM)
1
2
3
4
5
6
7
8
9
[CuL]
[NiL]
[CoL]
0.20 0.02
0.86 0.5
0.70 0.2
0.55 0.4
0.84 0.01
1.20 0.04
0.90 0.02
0.94 0.02
300 0.5
[ZnL]
Copper acetate
Nickel acetate
Cobalt acetate
Zinc acetate
DNJ
modulated by the existence and location of the substituents in the Cu(II) complexes.
The inhibitory values suggest that [CuL] occupies the active a-glucosidase sites more
easily than the normal, DNJ. [CuL] can also undergo hydrogen bonding with the
enzyme and modulate the conformation changes to make it easier to inhibit a-glucosi-
dase. Hence, the redox properties and molecular scaffolds make [CuL] an efficient
inhibitor in the inhibition of a-glucosidase.
3.16. Anti-inflammatory efficiency
The purchase and utility of animals for pharmacological research work is complicated
due to ethical issues. The present work was concentrated on protein denaturation
methodology for in vitro anti-inflammatory efficiency of metal chelates with the help
of the egg albumin method. The molecules may inhibit protein denaturation and
thereby improve the anti-inflammatory process. Among the prepared metal com-
plexes, copper complex (IC50 ¼ 40 mM) exhibited greater inhibitory efficacy than other
complexes (IC50 ¼ 78–90 mM) due to the existence of redox and chemical molecular
moiety as compared to diclofenac (IC50 ¼ 50 mM). The upward rising of absorbance of
chelates with respect to control showed stabilization of protein and thereby inhibited
the denaturation process. Among the chelates, the copper complex is active at lower

20
E. H. E. GLADIS ET AL.
Table 5. In vitro inhibition IC50 values (lM) and selectivity index of compound and standards for
AChE and BuChe.
IC50 (mM)
Selectivity
index ¼ IC50(BuChE)
IC50(AChE)
/
Compound structure
Ligand (H2L)
AChe
BuChe
0.20 0.16
3.2 0.20
16
Galantamine
2.41 0.10
17.30 0.15
7.21
Rivastigmine
3.01 0.2
0.30 0.1
0.10
concentration as compared to the standard. Therefore, the biochemical interpretations
suggested that the synthesized metal complexes possessed anti-inflammatory effi-
ciency. Furthermore, clinical trials are essential to ascertain the mechanism of anti-
inflammatory actions.
3.17. Cholinesterase inhibitory activity
Cholinesterase inhibitors (ChEIs) are perspective drugs that can enhance cholinergic
activity to boost memory, efficiency, minimize psychiatric and behavioral disorders.
The inhibitory activities on AChE and BuChE of the synthesized 1,10-phenanthroline
derivative were determined using galantamine and rivastigmine as the reference
compounds by the modified Ellman approach. Some inhibitors of AChE, such as
donepezil, rivastigmine and galantamine, are commonly used to treat AD cognitive
problems. The IC50 values for inhibitions of AChE and BuChE are summarized in
Table 5. Similar to galantamine (IC50 ¼ 2.41 lM), the strongly conjugated 1,10-phe-
nanthroline derivative displayed the best inhibition against AChE with an IC50 value
of 0.20 lM and rivastigmine (IC50 ¼ 3.01 lM). The IC50 values obtained against BuChE

JOURNAL OF COORDINATION CHEMISTRY
21
demonstrated that 1,10-phenanthroline derivatives act as a selective inhibitor of
AChE as opposed to galantamine. Phenolic compounds interact with residues of
amino acids that characterize the active site of AChE through a hydrogen bond,
hydrophobic interaction and a p–p interaction. Because of greater binding ability,
several hydroxyl groups in the phenolic compound are thought to improve AChE
inhibitory action. The aromatic center (conjugation) illuminates the fused aromatic
nuclei with higher inhibitory AChE potencies than their corresponding molecules
with just one or two fused ring systems. The best inhibitors behave as potent inhibi-
tors of AChE with fused aromatic characteristics. The azomethine moiety also pro-
motes tight bonding to the active site of the AChE and commendably inhibits
hydrolysis of choline substrates. Consequently, the main requirement for anti-AChE
activity may be the azomethine, naphthyl and phenolic moiety or lipophilic moieties
on the conjugated aromatic center. This research finding may provide opportunities
for researchers to develop new effective inhibitors of ChEs by adequately modulating
the pattern of substitution and also some pharmacophores from the perspective of
multifunctional anti-AD agents. Based on selectivity index, the prepared ligand (H2L)
might provide selective inhibition toward BuChE with respect to AChE. More clinical
trials, however, are required to clarify the effectiveness of the 1,10-phenanthroline
derivative in AD management.
3.18. Comparative study
Different metal(II) complexes show varied abilities as chemotherapeutic agents. The
experimental observations reveal that [CuL] showed higher activity. The biological
activities were influenced by the nature of ligands, central metal ion and geometry.
The present investigations were focused on different biological activities of metal com-
plexes with highly conjugated ligand system comprised of 1,10-phenanthroline and
naphathalene moieties.
Some related literature information are: Ali et al. [44] synthesized Co(II), Cu(II), Ni(II)
and Zn(II) complexes of a Schiff base containing o-hydroxyl group. Among the com-
plexes, [ZnL] was more effective with 83.60% and 88.52% inhibition for A549 and
H1299 lung cancer lines at 20 mM concentration. Singh et al. [45] indicated that
copper(II) complexes can cleave DNA through oxidative and hydrolytic pathways, cell
apoptosis via intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway
due to excessive production of ROS and hence, are found more active than Ni and Pt
complexes. The TSC ligand and its complexes have been studied for their possible bio-
logical activity including antibacterial and antifungal activity by Refat et al. [46].
ꢀ
Mendez-Arriaga et al. [47] synthesized a series of triazolopyrimidine transition metal
complexes by the reaction between 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp)
with M(NO3)2ꢅxH2O (M ¼ Cu(II), Co(II), Ni(II) and Zn(II)). The in vitro antiparasitic activity
of these complexes against four Leishmania spp. strands and their toxicity against
macrophage host cells has also been studied. Researchers have concluded that metal
ion along with pharmacophore are responsible for the pharmacological activities.
In the present investigations, there are three essential structural moieties present in
the molecular system (Scheme 1). First, the naphthalene moiety is an essential

22
E. H. E. GLADIS ET AL.
building block for drug molecules with unique or selective target and induces thera-
peutic efficiency. Second, the metal ion is essential for redox characteristics and modu-
lates pharmacological activities. Third, the whole designed ligand system, 1,10-
phenanthroline derivative includes planarity and conjugation manifested higher
pharmacological activities as compared to standards. The distorted geometry of che-
lates, the structural ligand core and the copper have improved biological activities as
compared to literature data.
4. Conclusion
The present investigation shows the preparation of metal complexes with Schiff base
ligand (1,10-phenanthroline-2,9-dicarboxaldehyde condensation with 2-aminophenol).
The 1,10-phenanthroline derivative demonstrated the highest inhibition of AChE with
an IC50 value of 0.20 lM, which was 10 times higher than that of galantamine (IC50
¼
2.41 lM) and rivastigmine (IC50 ¼ 3.01 lM). The selectivity index of ligand toward
AChE is 16-fold as compared with galantamine and rivastigmine, respectively. Among
the metal complexes, the Cu(II) complex showed an effective inhibition activity of
a-glucosidase compared to the standard inhibitor (DNJ) and may be significant for
antidiabetic and other related disorders.
Acknowledgement
We thank the Head, Department of Chemistry, Manonmaniam Sundaranar University for provid-
ing the necessary facilities and the Chancellor, Noorul Islam Centre for Higher Education. This
research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
Disclosure statement
No potential conflict of interest was reported by the authors.
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