Synthesis, anticonvulsant, antioxidant and binding interaction of novel N-substituted methylquinazoline-2,4(1H, 3H)-dione derivatives to bovine serum albumin: A structure-activity relationship study

Article abstract of DOI:10.1016/j.saa.2013.03.064

A novel class of N-substituted glycosmicine derivatives was synthesized, and their anticonvulsant, antioxidant activity and interaction with bovine serum albumin (BSA) were evaluated. The synthesized compounds 4a-j were examined for anticonvulsant activity by maximal electroshock induced seizures (MESs) test and their neurotoxic effects were determined by rotorod test in mice. The structure-activity relationships (SARs) of these compounds were also investigated. Compounds 4d, 4g, 4i and 4j were found to have good protective effect from seizure. The in vitro antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging assay. The interaction between novel N-substituted methylquinazoline-2,4(1H, 3H)-dione (NMQ) and BSA was analyzed by fluorescence and ultraviolet spectroscopy at 304 K under simulative physiological conditions. BSA fluorescence quenched by NMQ is discussed according to the Stern-Volmer equation. The binding constant and binding sites of NMQ with BSA were calculated. According to Forster non-radiation energy transfer theory, the binding distance (r) between NMQ and BSA was calculated.

Full text of DOI:10.1016/j.saa.2013.03.064

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, anticonvulsant, antioxidant and binding interaction of novel
N-substituted methylquinazoline-2,4(1H,3H)-dione derivatives to bovine serum
albumin: A structure–activity relationship study
b
M.K. Prashanth a, M. Madaiah a, H.D. Revanasiddappa a, , B. Veeresh
⇑
a Department of Chemistry, University of Mysore, Manasagangothri, Mysore 570 006, Karnataka, India
b Department of Pharmacology, G. Pullareddy College of Pharmacy, Mehdipatnam, Hyderabad 580 028, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Novel anticonvulsant agents containing essential pharmocophoric elements in their structure.
ꢀ
A new compounds NMQ was
synthesized and characterized by
1H NMR, IR, and mass spectra.
Anticonvulsant and antioxidant
activity were performed for the
prepared compounds.
ꢀ
ꢀ
ꢀ
ꢀ
We explored the interaction of BSA
and NMQ by spectroscopic methods.
The binding constants, binding sites
and binding distance were calculated.
We have studied the structure–
activity relationship to the
synthesized compounds.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2012
Received in revised form 5 November 2012
Accepted 16 March 2013
Available online 25 March 2013
A novel class of N-substituted glycosmicine derivatives was synthesized, and their anticonvulsant,
antioxidant activity and interaction with bovine serum albumin (BSA) were evaluated. The synthesized
compounds 4a–j were examined for anticonvulsant activity by maximal electroshock induced seizures
(MESs) test and their neurotoxic effects were determined by rotorod test in mice. The structure–activity
relationships (SARs) of these compounds were also investigated. Compounds 4d, 4g, 4i and 4j were found
to have good protective effect from seizure. The in vitro antioxidant activity was evaluated by 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging assay. The interaction between novel
N-substituted methylquinazoline-2,4(1H,3H)-dione (NMQ) and BSA was analyzed by fluorescence and
ultraviolet spectroscopy at 304 K under simulative physiological conditions. BSA fluorescence quenched
by NMQ is discussed according to the Stern–Volmer equation. The binding constant and binding sites of
NMQ with BSA were calculated. According to Forster non-radiation energy transfer theory, the binding
distance (r) between NMQ and BSA was calculated.
Keywords:
Glycosmicine
Anticonvulsant
Antioxidant activity
Bovine serum albumin
Fluorescence spectroscopy
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
significant number of effective antiepileptic drugs (AEDs) are
now on the market. Classical AEDs comprise phenobarbital,
Epilepsy is a heterogeneous mixture of disorders characterized
by neuronal hyperexcitability and hypersynchronous neuronal
firing, and it affects up to 1% of the world’s population [1]. The
anticonvulsants are commonly known as antiepileptic drugs, a
phenytoin, carbamazepine and valproic acid [2]. With the available
AEDs on the market, about 70% of people with epilepsy achieve
satisfactory seizure control. But these drugs have severe side
effects like drowsiness, ataxia, gastrointestinal disturbances,
hirsutism and megaloblastic anemia [3–5]. Thus, it is very essential
to search for newer chemical entities for the treatment of epilepsy
with lower toxicity and fewer side effects.
⇑
Corresponding author. Tel.: +91 821 2419669; fax: +91 821 2419363.
E-mail address: hdrevanasiddappa@yahoo.com (H.D. Revanasiddappa).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.03.064

M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
325
In recent years, there has been an increased interest in the
application of antioxidants to medical treatment as information
is constantly gathered linking the development of human diseases
to oxidative stress. However, oxygen-derived free radicals result-
ing from uncontrolled production are hostile and damaging to cells
and their functions, and thus play an important role in pathogene-
sis of some diseases, such as cancer, cardiovascular diseases, ath-
erosclerosis and inflammation [6]. Owing to these facts, there is a
constant need for searching new and effective therapeutic agents.
It is known that drug–protein interactions greatly influence the
absorption, distribution, metabolism, and excretion properties of
drugs [7]. The serum albumin, one of the most abundant proteins,
plays an important role in the transport and deposition of a variety
of endogenous and exogenous ligands in blood [8]. The most pop-
ularly studied albumins are bovine serum albumin (BSA) and hu-
man serum albumin (HSA). BSA and HSA display approximately
80% sequence homology [9,10]. BSA has two tryptophan residues
that possess intrinsic fluorescence [11]. For its medical importance
and ready availability, BSA was usually selected for studying drug–
protein interaction in vitro.
All other reagents were of analytical reagent grade, and double
distilled water was used during the experiment.
Optical measurements
Elemental analysis (C, H, N) was determined using a Carlo-Erba
1160 elemental analyzer. IR spectra were recorded on a JASCO
FTIR-8400 spectrophotometer using Nujol mulls. The 1H NMR
spectra were recorded on a Varian AC 400 spectrometer instru-
ment in CDCl3 using TMS as the internal standard. Low resolution
mass spectra were obtained on a Varian 1200L model mass spec-
trometer (solvent: CH3OH). Melting points were determined with
a Buchi 530 melting point apparatus in open capillaries and are
uncorrected. Compound purity was checked by thin layer chroma-
tography (TLC) on precoated silica gel plates (Merck, Kieselgel 60
F254, layer thickness 0.25 mm). The fluorescence measurements
were performed on a fluorophotometer (Varioskan Flash 4.00.53)
and the UV–vis absorption spectra were recorded with an UV–vis
spectrophotometer (Systronics 118, India).
The quinazolinedione moiety is an important scaffold embed-
ded in a variety of natural alkaloids [12,13]. In recent decades, qui-
nazoline-2,4(1H,3H)-diones have drawn the attention of chemists
and medicinal chemists because of their various biological activi-
ties for use as anticonvulsants [14], antibacterial [15], psychoseda-
tive [16] and antihypertensive or hypotensive compounds [17]. As
such, quinazoline-2,4(1H,3H)-dione derivatives have been widely
used as key structures in the production of medicinal drugs
[18,19].
Synthesis
Synthesis of glycosmicine (2)
Sodium cyanate (28 mmol) in water (25 mL) was added to a
solution of N-methyl anthranilic acid (20 mmol) and acetic acid
(0.2 mL) in water (50 mL) with stirring. When the temperature of
the reaction mixture reached 40 °C, NaOH was added in portions
till the reaction temperature reach to 75 °C. Stirring was continued
without cooling for 4 h, after which the crystals were filtered off
and dissolved in boiling water (50 mL). The solution was acidified
with 50% H2SO4 to pH 1–2. The precipitated crystals were filtered
off, washed with water and recrystallized from 50% acetic acid to
give compound 2 (2.92 g, 84%); m p 278 °C [24].
Natural products are always special sources for the discovery of
potential drugs with novel structures and varying biological activity.
The genus Glycosmis (Rutaceae) is such animportant medicinalplant
and has an extensive application in the ayurvedic system of medi-
cine. Glycosmis arborea an Indian medicinal plant is used locally
against fever, liver complaints and certain other diseases [20]. A
wide array of secondary compounds has been isolated from G. arbo-
rea including carbazole, acridone, furoquinoline, quinoline and qui-
nazoline [21]. They possess a variety of biological effects; viz the
acridone alkaloids from G. arborea including atalaphyllidine, 5-hy-
droxy-N-methylseverifoline, atalaphyllinine, and des-N-methyln-
oracrony-cine showed potent antiproliferative activity against
tumor cell lines [22]. Along with acridones, monomeric alkaloids
belonging to the carbazole, quinazoline, furoquinoline and quino-
lone exhibited significant anti-tumor-promoting activity on Ep-
stein-Barr virus early antigen activation [23]. Glycosmicine, one
derivative of quinazolinedione has been isolated from theG. arborea.
Based on the above literature review and considering the wide
applications of quinazolinedione molecule in medicinal chemistry
an attempt has been made to synthesize different substituted
amine derivatives containing glycosmicine moiety as antiepileptic
and antioxidant agents. In addition, the interaction between the
NMQ and BSA has been investigated using fluorescence and UV–
vis absorption spectroscopy.
Synthesis of 3-(3-chloropropanoyl)-1-methylquinazoline-2,4(1H,3H)-
dione (3)
Compound 2 (6 mmol) was refluxed with 3-chloro propionyl
chloride (6.2 mmol) in dry benzene (25 mL) and in the presence
of triethylamine (6.2 mmol). Then the reaction mixture was stirred
at room temperature for about 8 h. After completion of the reaction
(TLC), the reaction mixture was quenched in ice cold water and ex-
tracted with dichloromethane. The organic layer was washed with
5% NaHCO3 and dried over Na2SO4 and concentrated in vacuo to
give the light brown product.
Yield: (1.39 g, 87%), m.p.: 257 °C. Anal. calc. for C12H11ClN2O3: C,
54.05; H, 4.16; N, 10.50. found: C, 54.01; H, 4.15; N, 10.63. 1H NMR
(300 MHz, CDCl3) d: 2.73 (t, 2H, COACH2), 3.62 (t, 2H, CH2Cl), 3.78
(s, 3H, NACH3), 7.37–7.88 (m, 4H, ArAH). IR (Nujol, cmꢁ1): 3234.5
(NAH), 3053–2829.5 (Ar CAH), 2971.0–3026.1 (CH2). 1706, 1675,
1635 (C@O).
Synthesis of 3-(3-((substitutedphenyl)amino)propanoyl)-1-
methylquinazoline-2,4 (1H,3H)-dione derivatives (4a–j)
Experimental
The substituted amines (2 mmol) and triethylamine were taken
in THF (20 mL) and anhydrous potassium carbonate was added
(600 mg) to it. A solution of 3 (2 mmol), in THF (10 mL) was added
drop wise with stirring for 30 min. The reaction mixture was re-
fluxed for 10 h. After completion of the reaction (TLC), the reaction
mixture was then desolventized in rotavapor and the compound is
extracted in dichloromethane. The dichloromethane layer was
washed with water and dried over anhydrous Na2SO4 and further
desolventization in rotary evaporator to get the desired compound.
Structure verification data for novel compounds are documented in
Supplementary File.
General
All the chemicals and solvents were of AR grade. Solvents were
used as supplied by commercial sources without any further puri-
fication. BSA (essentially fatty acid free) was purchased from Sigma
Aldrich Bangalore, and stored in refrigerator at 4.0 °C. BSA solution
was prepared in the Tris–HCl buffer solution (0.05 mol Lꢁ1 Tris,
0.15 mol Lꢁ1 NaCl, pH 7.4) and it was kept in the dark at 304 K.
The compounds were prepared as a stock solutions using DMF.

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M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
Pharmacology
Animals
Superoxide scavenging assay
Superoxide dismutase activity was measured as described by
reported method [27]. The assay is based on the reduction of
nitroblue tetrazolium (NBT) by superoxide ions generated by the
xanthine–xanthine oxidase (X–XO) system. The reaction system
contained 0.2 mmol xanthine and 0.6 mmol NBT in 0.1 mol
phosphate buffer (pH 7.8). The tested compounds were dissolved
in methanol. The reaction was started by addition of XO
(0.07 U mLꢁ1), an activity which allowed to yield the absorbance
change between 0.03 and 0.04 per minute at 560 nm. The extent
of NBT reduction was followed spectrophotometrically by measur-
ing the increase of the absorbance at 560 nm. The IC50 of each com-
pound was defined as the concentration which inhibited 50% of the
NBT reduction by superoxide anion radical produced in the X–XO
system.
The anticonvulsant activity was evaluated by maximal electro-
shock seizure (MES) test and neurotoxicity screening. The albino
mice (18–20 g) were procured from National Institute of Nutrition,
Hyderabad and they were used in this study. The animals were
kept in individual cages for 1 week to acclimatize for the labora-
tory conditions. They were allowed to free access of water and
food.
All the experimental procedures were carried out in accordance
with Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEAs) guidelines. The study was re-
viewed and approved by the Institutional Animal Ethics Commit-
tee, G Pulla Reddy College of Pharmacy, Hyderabad, India.
Measurement of binding parameters
Maximal electroshock seizure model (MES)
In this study, seizures were induced in mice by delivering elec-
tro shock of 150 mA for 0.2 s by means of a convulsiometer
For each of the four active compounds (4a, 4e, 4i and 4j), the bind-
ing parameters with BSA molecules were measured by fluorescence
spectroscopy. A solution (2.5 mL) containing 1.00 ꢂ 10ꢁ5 mol Lꢁ1
BSA was titrated by successive additions of 1.00 ꢂ 10ꢁ3 mol Lꢁ1
NMQ stock solution and the concentration of NMQ varied from 0
to 2.40 ꢂ 10ꢁ5 mol Lꢁ1. Titrations were performed manually using
micro-injector. Fluorescence spectra were measured in the range
of 280–500 nm at the excitation wavelength of 280 nm. The fluores-
cence spectra were performed at 304 K.
through
a pair of ear clip electrodes. The test compounds
(100 mg/kg) were administered by oral route in the form of solu-
tion (The compounds were dissolved in 1% sodium carboxymethyl
cellulose), 30 min before the maximal electroshock seizure test.
The animals were observed closely for 2 min. The percentage of
inhibition of seizure relative to control was recorded and calcu-
lated [25]. Phenytoin (100 mg/kg, p.o) was used as a standard drug.
The UV–vis absorption spectra of NMQ solution at 1.00 ꢂ
10ꢁ5 mol Lꢁ1 concentration were measured in the range of 200–
500 nm at 304 K.
Neurotoxicity screening
The minimal motor impairment was measured in mice by roto-
rod test. The mice were trained to stay on the accelerating rotorod
that rotates at 10 revolutions per min. Trained animals were given
an ip injection of the test compounds at dose of 100 mg/kg. Neuro-
toxicity was indicated by the inability of the animal to maintain
equilibrium on the rod for at least one min in each of the four trials.
Results and discussion
Chemistry
In view of the above findings, it was considered of interest to
undertake the synthesis of N-substituted glycosmicine derivatives,
hoping that these compounds might possess certain anticonvul-
sant and antioxidant activity. The syntheses of the compounds
4a–j were accomplished according to the reaction sequence illus-
trated in Scheme 1. The structures of the synthesized compounds
were deduced on the basis of 1H NMR, IR and mass spectra. The
composition of all the compounds was obtained by elemental anal-
ysis. In addition, the chemical shift and multiplicity patterns corre-
lated well with the proposed structures. The elemental analysis
data showed good agreement between the experimentally deter-
mined values and the theoretically calculated values.
The naturally occurring plant derived 1-methylquinazoline-
2,4(1H,3H)-dione (glycosmicine) 2 was made by cyclization of
N-methyl-anthranilic acid 1 with sodium cyanate in basic media.
Synthesis of 3-(3-chloropropanoyl)-1-methylquinazoline-2,4(1H,
3H)-dione 3 as a key intermediate was achieved by the N-acylation
of glycosmicine2 with3-chloropropionyl chloride in presenceoftri-
ethylamine. The proton spectral data agree with respect to the num-
ber of protons and their chemical shifts with the proposed
structures. The proton spectral data of the intermediate, glycosmi-
cine 2 showed resonance at 8.57 ppm (s, 1H, NH), but it was not
observed in compound 3. The two characteristic peaks at 2.73 and
3.62 ppm due to ACH2 were observed in compound 3. IR spectrum
revealed that the presence of three carbonyl groups at 1706, 1675
and 1635 cmꢁ1. This confirmed the N-acylation of glycosmicine 2.
Finally, 3-(3-chloropropanoyl)-1-methylquinazoline-2,4(1H,3H)-
dione 3 was allowed to base condensation on treatment with differ-
ent substituted amines in THF and afforded the compounds 4a–j in
good yields (see Supporting Information File 1 for full experimental
Statistical analysis
The experimental data were analyzed by one way analysis of
variance (ANOVA) followed by Dunnet test to compare the differ-
ence between the groups.
Antioxidant activity
DPPH free radical scavenging activity
The capacity to scavenge the stable free radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH) was monitored according to the Blois meth-
od [26]. The test samples (10–100 lL) were mixed with 1 mL of
DPPH (0.1 mmol) solution and filled up with methanol to a final
volume of 4 mL. Absorbance of the resulting solution was mea-
sured at 517 nm in a visible spectrophotometer (Model 166, Sys-
tronics, India). The free radical scavenging rate of the reaction
solution was calculated as a percentage (%) of DPPH decoloration
using the equation:
Ið%Þ ¼ ðAblank ꢁ Asample=AblankÞ ꢂ 100
here Ablank is the absorbance of the control reaction mixture
excluding the test compounds and Asample is the absorbance of
the test compounds. Radical scavenging potential was expressed
as IC50 value, which represents the sample concentration at which
50% of the DPPH radicals scavenged. Tests were carried out in trip-
licate and the results were expressed as mean values standard
deviations.

M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
327
O
NH CH3
NH
i
COOH
N
O
CH3
1
2
O
N
O
N
O
O
O
N
O
O
NH
ii
R
N
Cl
iii
N
N
H
O
CH3
CH3
CH3
2
3
4a-j
Br
NO2
HO
CH3
Cl
g, R =
d, R =
a,
R =
HO
O2N
h,
R =
OH
e, R =
f, R =
b, R =
c, R =
i, R =
j, R =
OCH3
NH2
Cl
OH
F
Scheme 1. Reagents and conditions: (i) sodium cyanate, NaOH, 4 h; (ii) 3-Chloro propionyl chloride, TEA, benzene, 8 h; (iii) substituted amine (a–j), THF, reflux, 10 h.
data). TLC was run throughout the experiment to optimize the reac-
tion for purity and completion. The IR spectra of compounds 4a–c
showed broad phenolic stretching at 3220–3500 cmꢁ1 and the
appearance of strong absorption band at 3300–3380 cmꢁ1 is due
to the stretching vibration of NAH bond formation in the synthe-
sized compounds. The 1H NMR spectra of compounds 4a–j showed
singlet at d 5.62–8.86 which revealed the presence of N–H proton
and singlet at d 3.71–3.83 due to NACH3 group. The signal due to phe-
nolicOH inallthe analogues appeared assinglet atd 9.02–9.66. The 1H
NMR spectrum of compound 4g showed singlet of ACH3 at 1.48 ppm.
Compound 4f showed singlet at 5.72 ppm due to ANH2 group. The
aromatic cluster of compound also supports the synthesis of com-
pounds. Mass spectra of all newly synthesized compounds showed
M + 1 peak, in agreement with their molecular formula.
these compounds to prevent the seizure spread. The most active
of these compounds were 4d, 4g, 4i and 4j which showed 63.82–
71.98% protection and among these 4i and 4j were recorded 70%
of protection. The compound 4h accessible MES effect by 50%,
while compounds 4b, 4c and 4e revealed 30–42% protection. In
the neurotoxicity screening, the compounds were administrated
by oral route at 100 mg/kg in mice. The tested compounds did
not showed neurotoxicity at 0.5 and 1 h. The results showed com-
pounds having 2-hydroxy 4a, 2-nitro 4-methoxy 4e and 4-amine 4f
substituents showed 25% neurotoxicity compared to the standard
at 2 h of oral administration (Table 2).
Antioxidant activity
The in vitro antioxidant activity for compounds 4a–j was deter-
mined by the use of the DPPH test and the results are given in Table
3. The stable free radical DPPH is a useful reagent to investigate the
scavenger properties of phenols, catechols and anilines. This assay
evaluates the ability of the substance under examination to donate
hydrogen atoms or electron donating (conceivable) and quench the
visible absorption at 517 nm of the nitrogen radical of DPPH [31].
There are many reports describing the antiradical effect of phenolic
compounds by DPPH assay [32,33].
Antioxidants are defined as substances that even at low concen-
tration significantly delay or prevent oxidation of easily oxidizable
substrates. It can be seen, from Table 3, compounds 4a, 4b, 4c, 4e
and 4f present the highest scavenging activity on DPPH radical,
whereas the 4d and 4g present moderate and 4h, 4i and 4j present
very low scavenging activity on DPPH radical. The results showed
significantly higher activity for compounds 4c and 4f. The activity
of 4c and 4f is remarkably higher than that of naturally occurring
Pharmacology
The new derivatives obtained from the reaction sequence were
injected intraperitoneally into mice and evaluated in the maximal
electroshock (MES) and neurotoxicity screens, using doses of
100 mg/kg at four different time intervals (0.5, 1, 2 and 4 h). The
pre-clinical discovery and development of new chemical agents
for the treatment of epilepsy are based mainly on the use of pre-
dictable animal models, from which the MES seizure model remain
the ‘‘gold standards’’ in early stages of discovery of new AEDs and
have gained an appreciable degree of predictability [28,29]. The
MES test is associated with the electrical induction of the seizure.
The neurotoxicity of the compounds was primarily determined in
the rotorod method [30]. The characteristic feature of this series
is the presence of the propionyl group at third position and the dif-
ferent substituents on phenyl ring.
Compounds 4b, 4c, 4d, 4e, 4g, 4h, 4i and 4j exhibited anticon-
vulsant activity against MES-induced seizure at the dose of
100 mg/kg (Table 1). It was the indicative of the good ability of
glycosmicine 2 (21.7 0.38 lg/mL) and other compounds, the
main reason might be that the free hydroxyl or amine group is at
the position 4 [34,35]. The wide variations in free radical scavenging

328
M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
Table 1
Superoxide effects are involved in many pathological processes,
such as inflammation, cancer and aging [37]. The enzymatic super-
oxide anion radical was generated by the xanthine/xanthine
oxidase reaction system [38]. The production of superoxide was
estimated by the nitroblue tetrazolium method. Table 1 shows
the IC50 values of individual compounds by superoxide radical
scavenging assay ranging from 10.8 0.13 to 59.0 0.09 lM.
According to the results, the relative IC50 of 4c was about five times
higher than that of 4j and about two times higher than glycosmi-
cine 2 (23.5 0.19 lg/mL). Compound 4c has hydroxyl group at
Anticonvulsant activity of the newly synthesized compounds in the maximal
electroshock seizure test.
Treatment
E/Fa
% Protection
4a
4b
4c
4d
4e
4f
4g
4h
5.17
3.76
3.11
1.95
3.19
5.01
1.69
2.48
1.58
1.51
2.73
1.27
5.39
04.08
30.24b
42.30b
63.82c
40.80b
07.05
68.64c
53.98b
70.68c
71.98c
49.35b
76.43
–
para position, so it showed relatively higher superoxide radical
scavenging than other phenolics examined.
4i
4j
Glycosmicine
Standard
Control
Structure–activity relationship
The most common structural elements of the older generation
clinically active anticonvulsants can be defined as a nitrogen het-
ero-atomic system bearing one or two phenyl rings and at least
one carbonyl group. The hypothesis suggests the presence of an
aryl hydrophobic binding site, hydrogen bonding domain, an elec-
tron donor group and another hydrophobic–hydrophilic site con-
trolling the pharmacokinetic properties of the anticonvulsant. In
the present series of compounds, the active compounds possess
all the requirements essential for anticonvulsant activity as pro-
posed by Dimmock and others [39]. Thus, our new proposed phar-
macophore model includes all the above factors important for
bioactivity (Supplementary Fig. 1).
Quinazolinones are a class of drugs which function as sedative-
hypnotics; the sedative-hypnotic (neurotoxic) properties of qui-
nazolinones are well documented [40]. Initial structure–activity
relationship can be drawn for the synthesized analogues. The
results of bioevaluation led to an understanding of the structure–
activity relationship of these compounds. All the compounds
4a–j exhibited promising anticonvulsant activity. SAR studies indi-
cated that different substitution on the aromatic ring, exerted
varied anticonvulsant activity.
Values are expressed as mean SE. n = 6 animals in each group.
a
Extension/flexion.
p < 0.05.
p < 0.01 when compared to control.
b
c
Table 2
Neurotoxicity screening of the compounds.
Treatment
Neurotoxicity screen
2 h
4 h
4a
4b
4c
4d
4e
4f
4g
4h
1/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
1/4
1/4
0/4
1/4
0/4
0/4
1/4
0/4
4i
4j
Glycosmicine
Standard
It was observed that compound 4a has hydroxyl group at
ortho position and 4f has amine group at para position as sub-
stituents showed least activity while compounds (4i and 4j)
substituted with 4-chloro and 4-fluoro at para position of the
phenyl ring exhibited the maximum percent protection against
seizures induced by MES. Compounds (4b and 4c) having elec-
tron donating hydroxyl group at 3rd and 4th position of phenyl
ring causes decreased in activity. On the other hand, replace-
ment of the fluoro moiety in 4h, with amine in 4f, chloro in
4h and nitro moiety in 4d and 4e substitutions reduced the
biological activity. This emphasizes that the hydrophobic and
lipophilic domains in the molecule are responsible for the potent
anticonvulsant activity. Interestingly, the simple phenyl ring
with fluoro substitution in para-position exhibited the most
potent activity and did not exhibit neurotoxicity at highest
administered dose. It may be proved that substitution of a small
lipophilic group like fluorine at the para-position of the phenyl
ring of this type of compounds resulted in increased activity.
From the SAR studies, it reveals that, the substitution of electron
donating or electron withdrawing groups to phenylaminopropa-
noyl at N-terminal in glycosmicine ring plays key role in the
anticonvulsant activity.
Table 3
Antioxidant activity of the synthesized compounds.
Compounds
IC50
(l
g/mL)a
DPPH
Superoxide
4a
4b
4c
4d
4e
4f
4g
4h
4i
18.4 0.17
19.6 0.24
13.9 0.04
23.9 0.12
17.9 0.31
16.4 0.37
27.5 0.27
41.8 0.04
43.2 0.26
47.2 0.16
21.7 0.38
12.6 0.43
Ntd
13.8 0.19
17.1 0.16
10.8 0.13
27.7 0.22
23.7 0.14
17.3 0.22
30.4 0.10
43.8 0.19
55.3 0.17
59.0 0.09
23.5 0.19
Ntd
4j
2
AAb
BHAc
13.4 0.29
a
b
c
Average of three determinations.
Ascorbic acid.
Butylated hydroxyanisole.
Not tested.
d
The SAR studies indicate that the electron donating hydroxyl
and methoxy goups in 4d and methoxy group in 4f are more likely
to have higher antioxidant activity. The presence of hydroxyl
groups on the phenyl ring greatly influenced antioxidant activity
as observed for all of the compounds of this study. By introducing
different substituents in various positions of the aromatic ring, it
was shown that 4-hydroxy, 4-amine and 2-nitro-4-methoxy
substituted compounds were the most active compounds. Modifi-
activities may be due to the variations in the proton–electron
transfer by the compounds due to difference in their structures
and stability [36]. An insight to the structure–activity relationship
gives an idea that activity generally increases with number and
strength of electron donating groups.

M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
329
A
B
500
a
a
BSA+4f
500
400
300
200
100
0
BSA+4c
400
300
200
100
g
g
0
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
C
D
500
400
300
200
100
0
500
400
300
200
100
0
a
BSA+4i
a
g
BSA+4j
g
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Fig. 1. Fluorescence quenching spectra of BSA at different concentrations of NMQ. c(BSA) = 1.00 ꢂ 10ꢁ5 mol Lꢁ1 ; c(NMQ)/(ꢂ 10ꢁ5 mol Lꢁ1) (4c, 4f, 4i, 4j): 0, 0.40, 0.80, 1.20,
1.60, 2.00, 2.40 from a to g respectively.
cation of the relative position of the hydroxy moiety from para to
ortho and meta resulted in a clear decrease in antioxidant activity
(4c, 4a and 4b). The difference in radical scavenging activity of the
4a, 4b and 4c were due to the difference in the stability of the oxy-
gen centered radical formed in these compounds. As in the case of
compound 4a, the radical is stabilized by an intramolecular hydro-
gen bond. Additionally, there is a considerable difference between
4b and 4c, probably due to different electronic effects of the OH
groups. The better activity of compound 4c having hydroxyl group
at p-position in the aromatic ring is due to high electron releasing
properties (positive mesomeric effect is higher than negative
inductive effect) and this activates the aromatic ring and stabiliz-
ing the corresponding radical. Differently, in the m-position, the
OH group acts as an electron withdrawing group, providing a
destabilizing effect [41,42].
As can be deduced from the structure–activity relationship, po-
sition of the groups on the molecule affects the electron donating
effect and the scavenging potency of the molecules [43]. When
the electron donating groups were replaced by an electron with-
drawing group like chloro or fluoro group of phenyl ring resulted
in lower the activity. For example, the 3-chloro in 4h, 4-chloro in
4i and 4-fluoro in 4j substituent derivatives were less potent than
4a, 4b, 4c and 4d. The above SAR results suggest that the nature
and position of functional groups are always related to the favor-
able antioxidant activity.
The mechanism of fluorescence quenching of BSA by compounds
The fluorescence quenching of bioactive NMQ derivatives (4c,
4f, 4i and 4j) and BSA are shown in Fig. 1. The fluorescence quench-
ing spectra of solutions containing BSA in fixed concentration were
recorded in the presence of increasing amount of NMQ. As shown
in Fig. 1, BSA exhibits a characteristic fluorescence spectrum with a
maximum at 370 nm with excitation wavelength of 280 nm. The
fluorescence intensity of BSA decreased regularly with increasing
concentration of NMQ but there is no significant emission wave-
length shift which suggested that the NMQ interact with BSA and
quenches its intrinsic fluorescence without altering the environ-
ment in the vicinity of the chromophore tryptophan residues.
In order to clarify the fluorescence quenching mechanism, the
fluorescence quenching data are analyzed by the Stern–Volmer
equation [44]. Eq. (1) can be applied to determine KSV by linear
regression from the Stern–Volmer plot of F0/F against [NMQ]
(Fig. 2). Where F0 and F are the relative fluorescence intensities
of BSA in the absence of and in the presence of quencher, dynamic
and static quenching can be distinguished by the quenching con-
stant Kq. KSV
,
s0 and [Q] are the Stern–Volmer dynamic quenching
constants, average life-time without quencher (
s
0 = 10ꢁ8 s) [45]
and the concentration of the quencher, respectively.
F0=F ¼ 1 þ Ks ½Qꢃ ¼ 1 þ Kqs0½Qꢃ
ð1Þ
m

330
M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
2.1
A
B
BSA-4c
1.8
1.5
1.2
0.9
0.6
0.3
BSA-4f
2.1
1.8
1.5
1.2
0
3
6
9
12
0
3
6
9
12
[Q] x 10-5mol/L
[Q] x 10-5mol/L
3.5
3.0
2.5
2.0
1.5
1.0
C
D
1.8
1.5
1.2
BSA-4j
BSA-4i
0.0
0.6
1.2
1.8
2.4
0
2
4
6
8
[Q] x 10-5mol/L
[Q] x 10-5mol/L
Fig. 2. Stern–Volemr plots F0/F against [NMQ].
Table 4
The Stern–Volmer plots at 310 K are shown in Fig. 2. From Fig. 2,
the values of KSv and Kq were obtained and are listed in Table 4. The
observed values of Kq was larger than the maximum scattering col-
lision quenching constant 2.0 ꢂ 1010 L molꢁ1 sꢁ1, which suggest
that the fluorescence quenching mechanism between NMQ and
BSA may be a static quenching [46].
The quenching constants of BSA by compounds.
Compound
Ksv (L molꢁ1
)
Kq (L molꢁ1 sꢁ1
)
Ra
4c
4f
4i
4j
2.32 ꢂ 104
9.43 ꢂ 103
9.92 ꢂ 104
7.90 ꢂ 104
2.32 ꢂ 1012
9.43 ꢂ 1011
9.92 ꢂ 1012
7.90 ꢂ 1012
0.9973
0.9994
0.9986
0.9972
a
Binding constant and binding site
Linear quotient.
The binding parameters were then obtained according to the
following equation:
Table 5
The binding constants and the number of binding sites of compounds with BSA.
logðF0 ꢁ FÞ=F ¼ log Ka þ n log½Qꢃ
ð2Þ
Compound
Ka (L molꢁ1
)
n
R
When small molecules bind independently to a set of equiva-
lent sites on a macro-molecule, the equilibrium between free and
bound molecules is given by the equation [47]. Fluorescence inten-
sity data can also be used to obtain the binding constant (Ka) and
the number of binding sites (n).
4c
4f
4i
4j
3.14 ꢂ 104
1.60 ꢂ 104
5.14 ꢂ 105
1.46 ꢂ 105
1.03
1.06
1.17
1.27
0.9981
0.9987
0.9991
0.9994
The plot of log[(F0 ꢁ F)/F] versus log[NMQ] is as shown in Fig. 2
and Table 5 gives the corresponding calculated results. The corre-
lation coefficients (R) are larger than 0.995, indicating that
assumptions underlying the derivation of Eq. (2) are reasonable,
and the curves have good linearity [48]. It showed that the value
of n almost equals to 1 indicated that there is one class of binding
site for these four NMQ to BSA molecule.
Table 6
The distance parameters between compounds and BSA.
Compound
J (cm3 L molꢁ1
)
R0 (nm)
E
R (nm)
4c
4f
4i
4j
4.97 ꢂ 10ꢁ14
1.67 ꢂ 10ꢁ14
1.06 ꢂ 10ꢁ14
2.35 ꢂ 10ꢁ14
2.28
2.68
2.49
2.81
0.31
0.07
0.06
0.28
2.79
4.12
3.75
3.04
The substitutions on the phenyl ring could enhance the binding
affinity of BSA and NMQ. As shown in Table 5, the binding

M.K. Prashanth et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 324–332
331
constants of the interaction between them increased in the
following order: 4c < 4f < 4i < 4j, which indicates that the presence
of fluorine atom and chlorine groups strengthen the interaction of
NMQ with BSA molecules. The binding affinity of these two
compounds may be affected by the polarity of substituent groups.
The stronger polarity of substituent group which strengthened its
binding affinity. In this work, the molecular size and polarity plays
a significant role in the binding between NMQ and BSA.
The interaction between drug molecule NMQ and BSA was
investigated by different spectral methods. The experimental re-
sults demonstrated that the intrinsic fluorescence of BSA was
quenched via dynamic quenching mechanism. The large binding
constant values suggest that NMQ binds to the high affinity bind-
ing sites of albumins. The biological significance of this work is evi-
dent since albumin serves as a carrier molecule for multiple drugs
and the interaction of NMQ with albumin was not characterized
earlier.
Energy transfer from BSA to compound
Acknowledgements
The distance between the buried BSA and the interacted NMQ
can be estimated by Forster’s non-radiative energy transfer theory
and the overlapping of fluorescence spectra of BSA with absorption
spectra of NMQ are depicted in S Fig. 2 (Supplementary file).
According to Forster’s resonance energy transfer theory (FRET),
the effective energy transfer from donor to acceptor can happen
under the following conditions: (a) the donor can produce fluores-
cence, (b) fluorescence emission spectrum of the donor and
absorption spectrum of the acceptor have enough overlap, and
(c) the distance between the donor and the acceptor is shorter than
7 nm.
The authors are thankful to the University of Mysore, Mysore
for providing this project. The authors are grateful to the MHRD
and UGC, New Delhi for the funding through Institution of Excel-
lence Scheme. The authors are grateful to G Pullareddy College of
Pharmacy, Mehdipatnam, Hyderabad, India for providing facilities
to carry out the anticonvulsant activity. One of the author M. K.
Prashanth thankful to the University of Mysore, Mysore for the
award of research fellowship.
Appendix A. Supplementary material
The efficiency of energy transfer in biochemistry can be used to
evaluate the distance between the donor and the fluorophores in
the protein [49]. Using FRET, The energy transfer efficiency E and
the distance between the acceptor and donor r can be defined as
the following equations [50]:
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.03.064.
E ¼ 1 ꢁ ðF=F0Þ ¼ R60=R06 þ r6
ð3Þ
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