Potent acetylcholinesterase inhibitors: Synthesis, biological assay and docking study of nitro acridone derivatives

Article abstract of DOI:10.1016/j.jphotobiol.2016.05.028

The reaction of o-halobenzoic acid with aniline derivatives and their subsequent cyclization reaction yielded the acridone derivatives. The series of nitro acridone derivatives were prepared by Ullmann condensation in presence of copper as catalyst and we

Full text of DOI:10.1016/j.jphotobiol.2016.05.028

Potent Acetylcholinesterase Inhibitors: Synthesis, Biological Assay and
Docking Study of Nitro Acridone Derivatives
Mehtab Parveen, Afroz Aslam, Shahab A.A. Nami, Ali Mohammed
Malla, Mahboob Alam, Dong-Ung Lee, Sumbul Rehman, P.S. Pereira Silva,
M. Ramos Silva
PII:
DOI:
Reference:
S1011-1344(16)30205-6
doi: 10.1016/j.jphotobiol.2016.05.028
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25 March 2016
15 May 2016
30 May 2016
Please cite this article as: Mehtab Parveen, Afroz Aslam, Shahab A.A. Nami, Ali
Mohammed Malla, Mahboob Alam, Dong-Ung Lee, Sumbul Rehman, P.S. Pereira
Silva, M. Ramos Silva, Potent Acetylcholinesterase Inhibitors: Synthesis, Biolog-
ical Assay and Docking Study of Nitro Acridone Derivatives,
10.1016/j.jphotobiol.2016.05.028
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ACCEPTED MANUSCRIPT
Potent Acetylcholinesterase Inhibitors: Synthesis, Biological Assay and Docking Study of
Nitro Acridone Derivatives
Mehtab Parveen*,1, Afroz Aslam1, Shahab A. A. Nami2, Ali Mohammed Malla1, Mahboob
Alam3, Dong-Ung Lee3, Sumbul Rehman4, P. S. Pereira Silva5, M. Ramos Silva5
1Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
2Department of Kulliyat, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh
202002, India
3Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
4Department of Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh
202002, India
5CEMDRX, Physics Department, University of Coimbra, P-3004-516 Coimbra, Portugal.
*Email: mehtab.organic2009@gmail.com
Tel: +91 09897179498
Abstract
The reaction of o-halobenzoic acid with aniline derivatives and their subsequent
cyclization reaction yielded the acridone derivatives. The series of nitro acridone derivatives
were prepared by Ullmann condensation in presence of copper as catalyst and were
1
13
characterized by FTIR, H, C NMR and Mass spectra. The structure of 5-nitro-(2-phenyl
amino) benzoic acid (4) was confirmed by X-ray crystallography and was found to crystallize
in P21/c space group. The in vitro efficacy of the compounds for their acetylcholinesterase
(AChE) and antimicrobial inhibitory activities have been evaluated against the standard drugs
Ampicillin and Gentamicin against gram positive and gram negative bacteria. 1,7-
dinitroacridone was found to be the most potent AChE inhibitor (IC50 = 0.22 µM). Moreover,
the compounds have been screened for their antioxidant activity using the DPPH assay. Also,
docking study results were found to be in good agreement with the results obtained through
in vitro experiments. The docking study further predicted possible binding conformation.
Keywords: Acridone; Acetylcholinesterase; Antioxidant activity, Docking study.
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1.
Introduction
Chemical modification of bioactive components is one of the most commonly employed
approaches in drug discovery research because of its promising results [1]. The occurrence of
heterocyclic derivatives in bioactive natural products and pharmaceuticals has made them
significant synthetic targets. Acridones are an exclusive kind of heterocycle, a subclass of
acridines with a basic structure consisting of 9(10H)-acridone which is present in a large
number of natural products and synthetic compounds that are known as multi-targeted agents
with biomedical perspective. Acridone-based derivatives were first pursued for their
antimicrobial activity against bacteria, parasites and fungi [2,3]. It is an important organic
compound bearing tricyclic nitrogen containing ring and was first used in 19th century against
malaria [4]. Acridone consists of two benzene rings fused together having a keto group and a
nitrogen atom at 9th and 10th position, respectively, resulting in a planner structure. This
chemical scaffold bears a wide range of biological activity including anti-cancer [5], anti-
malarial [6], anti-viral [7,8] and modulation of multi-drug resistance (MDR) [9-11]. On the
molecular level, the planner structure of acridone molecule facilitates its interaction with
nucleotides leading to intercalation with DNA and RNA strands. Interaction of DNA with
various biologically important organic molecules has been extensively reviewed in the
literature [12-14]. A series of 1-amino thioacridones were designed as DNA intercalating
agents with covalent bond formation potential [15]. The intercalation of acridone molecule
with DNA and RNA is primarily based on its π-π stacking interactions with base pairs of
double-stranded nucleic acids. These interactions of acridine analogs have also been
confirmed by X-ray crystallography revealing ring overlap between acridine ring and base
pairs of nucleic acid [16]. The biological consequence of these interactions is the disruption
of DNA functions in the cells, serving as a prerequisite for effective anti-cancer activity of
acridine analogs [17]. Moreover, acridone molecule also possesses hydrophilic and lipophilic
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balance which facilitates its transverse through biological membranes to reach into the
nucleus, exerting their action [18].
Acridone is highly fluorescent and stable against photo degradation, oxidation, and
heat [19,20]. It is a small molecule with no charge. Several acridone derivatives have been
used as fluorescent labels for peptides [21], amino acids [22], antibodies [23] and substrates
for catalysis [24]. A series of acridone linked with nitro group have been developed as
potential anticancer agent, among which nitracrine appeared to be very potent cytotoxin and
4-nitroacridone was found to be the best mycophenolic acid.
There are several methods for the synthesis of acridone including the Bernthsen
reaction [25] which is one of the oldest methods for the synthesis of acridones. The most well
known methodologies for the synthesis of acridone are reported by Meyer [26], Koller [27],
besides several new methods have also been cited in the literature [28-33]. In 1937,
Tanasescu and Suciu [34] described a synthetic approach to acridones through rearrangement
of benzo[c]isoxazoles (anthranil) in the presence of concentrated Sulphuric acid containing
nitrous acid as catalyst. Keeping in view of these and in continuation of our research on
biologically active molecules, we hereby report the synthesis and characterization of some
acridone derivatives using Ullmann condensation [35] between substituted o-halobenzoic
acid and aniline derivatives followed by Friedel-Crafts acylation. The compounds (8-10) have
also been screened for their plausible in vivo antimicrobial and antioxidant activities. The
synthesized compounds (8-10) were further subjected for their docking studies to explore the
binding pattern of the compounds.
2.
Result and discussion
2.1. Chemistry
The substrate N-phenylanthranilic acids (4-6) used as starting compounds (Scheme 1) were
prepared through condensation of substituted o-halobenzoic acid with an excess of the
appropriate substituted aromatic amine. To this mixture POCl3 was added and refluxed for 1
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h and stirred for another 2 h in presence of 1 M HCl to obtain the final substituted acridone
derivatives (8-10). Its purity has been ascertained on the basis of TLC. Moreover, the
1
13
precursor has been subjected to spectral (IR, H NMR, C NMR and MS studies) analysis.
The compounds (8-10) have been obtained in good yield. The selected IR bands of the
synthesized compound provide useful information regarding the structure of the acridone
derivatives. The strong absorption band at 1635 (8), 1632 (9) and 1620 (10) cm-1 may be
assigned to characteristic cross conjugated (C=O) group while the closely spaced band
observed at 1545, 1337 (8), 1532, 1350 (9) and 1535, 1337 (10) may be ascribed to the nitro
1
group implying the formation of acridone derivatives (8-10). In the H NMR spectrum, the
assignments of the signals are based on the chemical shift and intensity pattern. The spectra
of the compounds (8-10) exhibited a singlet at δ 12.35 (8), 12.31 (9) and 12.85 (10)
integrating for N-H protons. A sharp singlet at δ 8.42 and 8.56 ppm (8 and 10) has been
accredited to H-1 and H-8. This prominent downfield shift of H-1 and H-8 protons is
supposed to be due to their hydrogen bonding interaction with the neighbouring carbonyl
group, which causes them to resonate at elevated δ values.
13
The C NMR signals are found to be in corroboration with the proposed structure of the
compound (6-8). The compounds exhibited carbonyl carbon (C=O) signal 169.49 (8), 176.14
(9) and 170.25 ppm (10) respectively. A group of signals resonating in between 151.83-
111.32 (8), 148.88-110.20 (9) and 149.80-113.65 ppm (10) are assigned to aromatic ring
carbons. However, compounds (8-10) showed characteristic molecular ion peaks, MS (ESI)
at m/z: 240 (8), 240 (9) and 285 (10) respectively which were in good concurrence with the
proposed structures.
2.2. Crystal structure
Single crystals appropriate for analysis were obtained by slow evaporation of methanol at
room temperature. X-ray crystallographic analysis of the compound (4) reveals that it
crystallizes in the monoclinic crystal system with space group P 21/c. The asymmetric unit of
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the compound (4) has been shown in Fig. 1.The asymmetric unit showed that molecule
crystallize as its sodium salt with three molecules of hydration. The hydrated sodium atom
appears to bind with oxygen atom of the nitro group via electrostatic interaction. This type of
interaction has been previously reported by Yazicilar et.al. [36]. Pertinent crystallographic
data for compound (4) has been summarized in Table 1. It can be seen from the three
dimensional (3D) framework of compound (4) that it is stabilized via an intricate array of
hydrogen bonding. The three water molecules play an important role in stabilizing 3-D
structure, forming extensive hydrogen bonding between carboxylic oxygen and water
molecules (COO....H-O) as depicted in Fig. 2.
2.3. Acetylcholinesterase Inhibition (AChEI) Docking Studies
The molecular docking studies were carried out to support the experimental AChE inhibition
results. The synthesized molecules were subjected to docking study using PatchDock,
Discovery studio 4.0 Client and iGEMDOCK softwares to predict the binding mode of
compounds towards target enzyme (PDB:3NM8). The docking results and their scoring
functions presented crucial information regarding the orientation of the compounds and the
strength of the non-covalent interaction (binding affinity) between ligand and receptor in the
binding cavities. On the basis of docking simulations, the strong binding affinity of
compound 10 with AChE can be explained on the basis of hydrogen bonding/secondary
interactions as well as orientation and electronic features of the substituents towards the
active site of the target enzyme Fig. 3. The hydrogen bonds interacted with amino acids of
the enzyme are in good agreement with the predicted binding affinities obtained by molecular
docking studies as verified by AChE inhibition activity data where compound 10 was found
to be most potent AChE inhibitor with IC50 value of 0.22 µM in close proximity to standard
drug Tacrine with IC50 value of 0.20 µM (Table 2). The improved acetylcholinesterase
inhibition of compound 10 in comparison to the other products 8 and 9 can be explained on
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the basis of mode of interaction among the substituents at the molecular skeleton and amino
acids of enzyme acting as receptor. The two nitro groups at 1 and 7-position of acridinone
moiety could interact with THR31, LYS89 and SER 88 of the enzyme’s amino acids through
hydrogen bonds. The amino acids of enzyme around the cavity can have a contact with
ligands making the long distance secondary bonds. In case of compound 8 and 9, the nitro
group of compound 9 at position 1 makes intermolecular hydrogen bond with adjacent keto
group of the molecule, while nitro group of compound 8 is not in proper orientation in order
to make effective bonds with amino acids. Therefore, the available site of ligand either
engaged in the formation of secondary bond within the molecule or not in proper orientation
for making additional non classical bonds. However, compound 10 is believed to have a
better pharmacological activity among other derivatives 8 and 9. The increased activity of
compound 10 among the three compounds could be explained on the basis of obtained
docking score, a representative of the binding affinity of ligand to receptor. The binding score
of compounds 10, 9 and 8 obtained are -89.12, -88.66 and -81.04 (kcal/mol), respectively
(Fig. 4). The mentioned docking score clearly depicts that binding energy of compound 10 (-
89.12) is higher than that of 8 and 9 implying stronger interactions between compound 10 and
amino residues of target enzyme. Therefore, compound 10 is an effective blocker for AChE.
Additionally, the skeleton of acridinone also plays an important role in stabilizing the ligand-
receptor complex by pi (π)-interaction with amino acids of the target enzyme as shown in
Fig. 5. The π- π interactions between aromatic rings of the compound with amino acids of the
protein further stabilize the orientation of the molecule into cavity of receptor. From the
combined table and chart shown in Fig. 4 it is evident that compound 10 showed an effective
activity against AChE and revealed better affinity to receptor and showed maximum docking
score as it was buried well inside the cavity of the target enzyme. Ligand map (Fig. 6) was
used to explain the in-depth interactions between the docked molecule and active site of the
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protein. Number of non-covalent interactions such as hydrophobic, hydrogen bonding and
van der Waals forces and their pattern were matched with the help of diagrams automatically
generated by MMV software. Ligand map gives a set of divergent intuitive future and are
also helpful in knowing the secondary interactions pattern after docking. Moreover,
geometrical descriptors of compounds 8, 9 and 10 were also used to show the extent of the
surface interaction and can be applied for predicting pharmacological and toxicological
activities of molecules on the basis of structure activity relationship (Fig. 7). The values of
descriptors obtained using ChemAxon software were employed to establish the idea of active
compound among the studied compounds. In general, more projection area implies more
interaction to the receptors as shown in Table 3.
2.4. Antimicrobial activity
The in vitro antimicrobial activity of compounds (8-10) were studied against Staphylococcus
aureus (ATCC 29213), S. epidermedis (MTCC 435), Streptococcus mutans (ATCC 25175),
S. pyrogenes (MTCC 435), S.viridans, Corynebacterium diphtheria, (gram-positive bacterial
strains) E. coli (ATCC 25922), P. aeruginosa (MTCC 424) and Proteus vulgaris (MTCC
426) (gram-negative bacterial strains) by disc diffusion method. The antibacterial activity of
each compound was compared with Gentamicin and Ampicillin drug. The disc poured in
DMSO was used as negative control. The antibacterial data obtained has been reported in
Tables 4 and 5 and the values are reported in terms of the zone of inhibition in millimetre
(mm). Among the synthesized compounds, compound (10) was found to be active against all
the Gram positive bacterial strains except Streptococcus pyrogenes. It can be inferred from
the data reported in Table 4, that compound (10) displayed potential inhibition zones 18 and
14 mm against Staphylococcus aureus and S. viridians bacterial strains. All the synthesized
compounds (8-10) were found to be more active against Gram positive as compared to Gram
negative bacterial strains (Table 5). Compound (9) displayed highest activity against Proteus
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vulgaris with zone of inhibition 16 mm in vicinity to standard drug Gentamicin with zone of
inhibition 18 mm for the same bacterial strain.
2.5. Antioxidant study
The synthesized acridone derivatives (8-10) were subjected to free radical scavenging activity
by DPPH method. This model of scavenging activity by DPPH radical is extensively applied
to evaluate antioxidant activity conveniently as compared with other methods. The odd
electron in the DPPH free radical gives a strong absorption band at 517 nm, which is purple
in color. This property makes it suitable for spectrometric studies. The DPPH assay has often
been used to estimate the antiradical activity of a given antioxidant. The free radical
scavenging capabilities of the compounds were measured in term of hydrogen donating of
free radical scavenging ability after adding methanolic solution of DPPH to the sample
solution having different concentrations. The extent of decolourizing is indicative of
antioxidant behaviour of a particular compound. Ascorbic acid was used as the reference
compound. All the tests were performed in triplicate. The compound (10) showed the highest
IC50 value (27.80 µ/mL) followed by compounds (8) and (9) respectively and results are
reported in Table 6.
3.
Experimental
3.1. Synthesis and characterization
Melting points were determined on a Kofler apparatus and are uncorrected. Elemental
analysis (C, H, N) were conducted using Carlo Erba analyzer model 1108. The IR spectra
were recorded with Shimadzu IR-408 Perkin-Elmer 1800 (FTIR) and its values are given in
cm-1. 1H NMR and 13C NMR spectra were run in DMSO-d6 on a Bruker Avance-II 400 MHz
instrument with TMS as internal standard. Chemical shifts are reported in ppm (δ) relative to
the TMS. Mass spectra were recorded on a JEOL D-300 mass spectrometer. Thin layer
chromatography (TLC) plates were coated with silica gel G and exposed to iodine vapours to
check the homogeneity as well as the progress of reaction. Sodium sulfate or magnesium
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sulfate (anhydrous) was used as a drying agent. All the reagents were purchased from Sigma-
Aldich (India) which were of analytical grade and used without further purification.
3.2. General procedure for the synthesis of substituted N-phenylanthranilic acid
derivatives (4-6)
The mixture of substituted o-halobenzoic acid (1) (0.04 mol) and substituted aniline (2) (0.08
mol) were refluxed in DMF in presence of anhydrous sodium carbonate (1 mol%) and copper
powder (3 mol%) for 4 hrs. After completion of the reaction as evident from thin layer
chromatography (TLC), the reaction mixture was cooled to room temperature and then
slowly added with shaking to a HCI and water (1:1) solution (100 mL). The reaction mixture
was left to stand overnight. The solid precipitated out was filtered, washed with boiling water
and then crystallized with appropriate solvent to yield substituted N-phenylanthranilic acid
(4-6) Scheme 1.
4.2.1 5-nitro-(2-phenyl amino) benzoic acid (4)
Crystallized from methanol-acetone as an orange-yellow color solid; Yield: 90%, m.p. >300
oC. Anal.Calc. for C13H10N2O4; C, 60.47; H, 3.90; N, 10.85; O, 24.78; found: C, 60.52; H,
4.72; N, 8.24; O, 24.76. IR (KBr) cm-1: 3067 (N-H), 1590-1433 (C=Caromatic), 1523-1329
(NO2), 1618 (C=O, acid carbonyl) 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.58 (s, 1H, N-
H, D2O-exchangeable), 8.82 (s, 1H, COOH), 8.81 (s, 1H, H-1), 8.03 (d, 1H, H-3), 7.40 (dd,
13
2H, H5-H9), 7.26 (dd, 2H, H6-H8) 7.15 (d, 1H, H-4), 7.11 (m, 1H, H-7). C NMR (100
MHz, DMSO-d6, δ, ppm): 169.49 (C-1), 151.83 (C-13), 139.51 (C-6), 136.14 (C-3), 129.54
(C-4), 128.58 (C-7, C-9), 126.90 (C-2), 124.08 (C-9), 122.24 (C-7, C-11), 111.32 (C-5, C-
12). MS (ESI): (m/z) 334.26 [M+H]+• (C13H15N2NaO7).
4.2.2. 2-(3-nitrophenyl) amino benzoic acid (5)
o
Crystallized from methanol-acetone as orange red color solid; Yield: 85%, m.p. 260-265 C.
Anal.Calc. for C13H10N2O4; C, 60.47; H, 3.90; N, 10.85; found: C, 60.52; H, 4.72; N, 8.24; O,
42.76. IR (KBr) cm-1: 3191-3079 (N-H), 1582-1455 (C=Caromatic), 1529-1357 (NO2), 1604
9

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1
(C=O, acid carbonyl) H NMR (400 MHz, DMSO-d6, δ, ppm): 12.23 (s,1H, N-H, D2O-
exchangeable), 8.76 (s, 1H, COOH), 7.99 (d, 1H, H-4), 7.97 (d, 1H, H-5), 7.89 (d, 1H, H-1),
13
7.65 (d, 1H, H-7), 7.18-7.51 (m, 2H, H-2, H-3), 7.47 (m, 1H, H-6). C NMR (400 MHz,
DMSO-d6, δ, ppm): 168.65 (C-1), 148.70 (C-9, C-13), 141.51 (C-10), 132.05 (C-2), 130.56
(C-7), 129.45 (C-4), 128.50 (C-6), 123.67 (C-3), 121.76 (C-8), 118.68 (C-12), 113.93 (C-5),
108.64 (C-11). MS (ESI): (m/z) 258.23 [M+H]+• (C13H10N2O4).
4.2.3. 5-nitro-(2-(3-nitrophenyl) amino) benzoic acid (6)
o
Crystallized from methanol-acetone as dark green color solid; Yield: 87%, m.p. 290-295 C.
Anal.Calc. for C13H9N3O6; C, 51.49; H, 2.99; N, 13.86; O, 31.66; found: C, 60.52; H, 4.72;
N, 8.24; O, 31.64. IR (KBr) cm-1: 3277-3091 (N-H), 1535-1440 (C=Caromatic), 1526-1342
(NO2), 1678 (C=O, acid carbonyl) 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.75 (s, 1H, N-
H, D2O-exchangeable), 8.75 (s, 1H, COOH), 8.59 (s, 1H, H-1), 8.37-7.28 (dd, 1H, H-3, H-4),
13
8.06 (d, 1H, H-5), 7.73 (d, 1H, H-7), 7.69 (s, 1H, H-9), 7.63 (m, 1H, H-6). C NMR (400
MHz, DMSO-d6, δ, ppm): 168.35 (C-1), 151.07 (C-13) 148.58 (C-7), 139.65 (C-11), 137.64
(C-3), 130.79 (C-4, C-9), 129.41 (C-2), 126.70 (C-10), 119.46 (C-5), 117.59 (C-8), 113.58
(C-6), 112.36 (C-12). MS (ESI): (m/z) 303.05 [M+H]+• (C13H9N3O6).
3.3. General procedure for the cyclization of N-phenylanthranilic acid derivatives to
substituted acridones (8-10)
Substituted N-phenylanthranilic acid (4-6) (7.77 mmol) synthesised in the first step were
dissolved in POCl3 (50 mL) and refluxed for 1 h. After completion of the reaction as evident
from TLC, the excess of POCl3 was recovered via vacuum distillation and the solid 9-chloro
intermediate product (7) was obtained. To a flask containing the 9-chloro intermediate was
added 30 mL of 1 M HCl. The mixture was mildly heated over a heating mantle with stirring
for 2 h. The reaction mixture was cooled and the product was extracted with
dichloromethane, dried over magnesium sulfate and concentrated. The crude product
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obtained was washed with suitable solvents, filtered, dried and crystallized from appropriate
solvents to afford pure target products (8-10) respectively as shown in Scheme 1.
O
R1
R2
OH
H
R1
COOH
X
Na2CO3, CuI
DMF, Reflux
N
H2N
R2
R1 R2
X
R1
4 NO2 H
5 H NO2
6 NO2 NO2
R2
1 NO2
2 H
H
Cl
NO2 Br
3 NO2 NO2 Cl
O
O
O
R2
R2
POCl3, Reflux
R1
Cl
H
OH
H
R1
1M HCl
stirring 2 hrs
N
N
N
H
R2
R2
4-6
7
R1
R2
H
8 NO2
9 H
NO2
NO2
10 NO2
Scheme 1 Synthetic route for the preparation of acridone analogs
3.3.1. 2-nitroacridinone (8)
o
Crystallized from methanol-acetone as yellow color solid; Yield: 90%, m.p. >300 C.
Anal.Calc. for C13H8N2O3; C, 65.00; H, 3.36; N, 11.66; O, 19.98; found: C, 59.98; H, 3.34; N,
11.64; O, 19.96. IR (KBr) cm-1: 3272-3022 (N-H), 1582-1477 (C=Caromatic), 1545-1337
1
(NO2), 1635 (C=O) H NMR (400 MHz, DMSO-d6, δ, ppm): 12.35 (s, 1H, N-H, D2O-
exchangeable), 8.90 (d, 1H, H-5), 8.41 (d, 1H, H-3), 8.19 (d, 1H, H-1), 7.70 (d, 1H, H-4),
11

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7.62 (m, 1H, H-8), 7.56 (m, 1H, H-6), 7.34 (m, 1H, H-7). 13C NMR (400 MHz, DMSO-d6, δ,
ppm): 176.36 (C-9), 144.40 (C-13), 140.60 (C-12), 140.48 (C-2), 134.51 (C-6), 127.33 (C-3),
126.02 (C-8), 122.96 (C-1), 122.83 (C-7), 120.68 (C-11), 119.04 (C-14), 118.84 (C-4),
117.95 (C-5). MS (ESI): (m/z) 240.05 [M + H]+• (C13H8N2O3).
3.3.2. 1-nitroacridinone (9)
o
Crystallized from methanol-acetone as brown color solid; Yield: 87%, m.p. 270-275 C.
Anal.Calc. for C13H8N2O3; C, 60.47; H, 3.90; N, 10.85; O, 19.98; found: C, 60.52; H, 4.72;
N, 8.24; O, 19.96. IR (KBr) cm-1: 3241-3096 (N-H), 1545-1468 (C=Caromatic), 1532-1350
1
(NO2), 1632 (C=O) H NMR (400 MHz, DMSO-d6, δ, ppm):12.31 (s, 1H, N-H, D2O-
exchangeable), 8.65 (d, 1H, H-2), 8.26 (d, 1H, H-4), 7.90 (d, 1H, H-5), 7.72 (m, 1H, H-3),
13
7.61 (d, 1H, H-8), 7.45-7.29 (m, 2H, H-6, H-7). C NMR (400 MHz, DMSO-d6, δ, ppm):
176.16 (C-9), 148.88 (C-1), 141.53 (C-11), 140.48 (C-13), 134.42 (C-3), 133.28 (C-6),
126.07 (C-8), 125.73 (C-4), 123.31 (C-12), 122.25 (C-7), 120.93 (C-14), 117.67 (C-5),
115.10 (C-2). MS (ESI): (m/z) 240.05 [M+H]+• (C13H8N2O3).
3.3.3. 1,7-dinitroacridinone (10)
o
Crystallized from methanol-acetone as dark green color solid; Yield: 85%, m.p. 294-296 C.
Anal.Calc. for C13H7N3O5; C, 54.74; H, 2.47; N, 14.73; O, 28.05; found: C, 54.72; H, 2.45;
N, 14.71; O, 2.03, IR (KBr) cm-1: 3273-3026 (N-H), 1525-1470 (C=Caromatic), 1535-1337
1
(NO2), 1620 (C=O) H NMR (400 MHz, DMSO-d6, δ, ppm): 12.85 (s, 1H, N-H, D2O-
exchangeable), 8.90 (d, 1H, H-2), 8.56 (m, 1H, H-8), 8.75 (d, 1H, H-6), 7.97 (m, 1H, H-3),
7.82 (d, 1H, H-5), 7.64 (d,1H, H-4). 13C NMR (400 MHz, DMSO-d6, δ, ppm): 176.20 (C-9),
149.86 (C-1), 147.10 (C-12), 144.23 (C-14), 141.34 (C-7), 135.20 (C-3), 128.45 (C-6),
124.92 (C-4), 123.23 (C-8), 122.40 (C-11), 119.90 (C-13), 118.45 (C-5), 115.21 (C-2). MS
(ESI): (m/z) 285.04 [M+H]+• (C13H7N3O5).
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3.4. Crystal structure determination of compound (4)
Single crystal X-ray data of compound (4) was collected at 100 K on a Bruker SMART
APEX CCD diffractometer using graphite monochromated MoKα radiation (λ = 0.71073Å).
The linear absorption coefficients, scattering factors for the atoms and the anomalous
dispersion corrections were taken from the International Tables for X-ray Crystallography
[37].The data integration and reduction were carried out with SAINT software [38]. An
empirical absorption correction was applied to the collected reflections with SADABS, and
the space group was determined using XPREP [39]. The structure was solved by direct
methods using SHELXTL-97 and refined on F2 by full-matrix least-squares using the
SHELXL-97 [40] program package. All non-hydrogen atoms were refined anisotropically.
Pertinent crystallographic data for compound (4) is summarized in Table 1.
3.5. Molecular Docking
The three-dimensional crystal structure of receptor was taken from Protein Data Bank (pdb).
The retrieved protein (PDB: 3NM8) was loaded in the Molegro virtual docker (MVD) [41]
with the removal of all water molecules. The standard Molegro algorithm was utilized for
rendering the missing charges, protonations states and assigning of polar hydrogens to the
receptor for achieving reliable potential binding sites in the receptor. All the compounds were
drawn using ChemDraw Ultra version 12.0 and structures were analyzed using Chem Draw
Ultra3D software. These structures were energetically minimized using MM2 force field with
RMS gradient set to 0.0001 and finally saved as pdb file for docking process. PatchDock
[42], Discovery studio 4.0 Client [43] and iGEMDOCK [44] softwares were used to sort out
best molecular docking poses of ligand (compound)-receptor interactions, perform
visualization of docked ligands and illustration of basic features of the docked interface and
compute energy calculation of docked ligands, respectively. In all cases, the program’s
default parameters were used. The scoring function obtained by GEMDOCK [44], which is
based on a piece-wise linear potential. GEMDOCK generates and calculates the different
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interactions involved between receptor and molecules, i.e., electrostatic, hydrogen and van
der Waals interactions. The PARS [45], a web server, was used to predict the cavities on the
receptor for the successful docking at binding sites. These locations may cause a regulatory
effect upon binding of the receptor when interacts with the guest.
3.6. Biological activity
The biological activity profile of the synthesized compounds (8-10) as acetylcholinesterase
(AChE) inhibitor was assayed in comparison with Tacrine as a reference drug. The retrieved
protein (PDB: 2JEG) used for this purpose was improved by using import and preparation
option of MVD software and missing bond order, hybridization state, angle and flexibility for
achieving reliable potential binding site in receptor. The energy minimized ligands
(synthesized compounds) were drawn with Chem Draw Ultra (2D and 3D). Discovery studio
3.5 [46] and MVD [47] software were used to perform molecular docking, energy profile of
ligand-receptor interaction independently.
3.7. In vitro acetylcholinesterase inhibition activity
The in vitro inhibition of acetylcholinesterase (AChE) activity of the compounds (8-10) was
carried out spectrophotometrically by Ellaman’s coupled enzyme assay method using Tecrine
as reference drug [48]. Electric ell AChE (Type-VI-S, EC 3.1.1.7) was used as the enzyme
source, acetylthiocholine iodide as substrate and 5,5’-dithio-bis (2-nitrobenzoic) acid
(DTNB) was used for the anti-cholinesterase activity determination. In this procedure, the
assay solution was composed of 0.1mL of each sample (1 mg/mL in methanol), 0.02 mL of
substrate (75 mM acetylthiocholine iodide in H2O) and 0.1 mL of Ellman reagent (10 mM
DTNB and 17.85 mM sodium bicarbonate in sodium phosphate buffer solution, pH 7.0). The
reaction mixture was incubated for 15 minutes at 25 ºC, 25 µL of enzyme solution containing
0.28 U/mL (commercial acetylcholinesterase) was added to above mixture with 3.0 mL of
sodium phosphate buffer and then incubated for 5 min at 25 ºC. The resulting solutions were
placed in a spectrophotometer. For non-enzymatic reaction, the assays were carried out with a
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blank containing all the components except acetylcholineaterase. The difference in
absorbance at 412 nm for sample and control was taken as an inhibition rate (%). The
percentage of enzyme inhibition was calculated using the following formula.
% inhibition = E − S/S  100
where E is the activity of enzyme without test sample and S is the activity of enzyme
with test sample. Tacrine was used as a standard inhibitor. The experiments were done in
triplicate and the results were expressed as an average values.
3.8. Antimicrobial activity
The in vitro antibacterial activity was carried out against nine bacterial strains (six gram
positive and three gram negative). The strains so selected for the study are Staphylococcus
aureus (ATCC 29213), S. epidermedis (MTCC 435), Streptococcus mutans (ATCC 25175),
S. pyrogenes (MTCC 435), S. viridans, Corynebacterium diphtheria, (gram-positive bacterial
strains) E. coli (ATCC 25922), Pseudomonas aeruginosa (MTCC 424) and Proteus vulgaris
(MTCC 426) (gram-negative bacterial strains). These strains were screened for estimation of
antibacterial activities of synthesized compounds. The antimicrobial activity was evaluated
by Disc diffusion method [49,50]. Standard inoculums (1×107 to 2×107) c.f.u mL-1 (0.5
mcFarland standards) was introduced on to the surface of sterile agar plates and sterile glass
spreader was used for even distribution of the inoculums. Every trial compound (1 mg) was
dissolved in 100 µL DMSO to prepare stock solution and from stock solution diverse
concentration 10, 20, 25, 50 and 100 µg/µL of every trial compound were prepared. After
that the compounds of diverse concentration were poured over disk plate. The discs
measuring 6 mm in diameter were prepared from Whatmann no. 1 filter paper and sterilized
by dry heat at 140 ºC for 1 h. The sterile discs previously soaked in a known concentration of
the test compounds were placed in nutrient agar medium. Solvent and growth controls were
also kept. Gentamicin was used as positive control. While the disc poured in DMSO was used
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as negative control. The plates were inverted and incubated for 24 h at 37 ºC. The susceptibil-
ity was assessed on the basis of diameter of zone of inhibition against different strains of
bacteria. Inhibition zones were measured and compared with standard drug. Table 2-3.
3.9. Antioxidant Studies
The acridone derivatives (8-10) were tested for their antioxidant property by 1,1-
diphenylpicrylhydrazyl (DPPH) method [51-53]. In this procedure stock drug solution (1
mg/mL) was diluted to final concentration of 2, 4, 6, 8, 10 and 12 in methanol. Methanolic
DPPH solution (1 mL, 0.3 mmol) was added to 3.0 mL of drug solution of different
concentrations. The tube was kept at an ambient temperature for 30 min and the absorbance
was measured at 517 nm by UV VIS spectrophotometer. The scavenging activity was
calculated by following formula:
% inhibition = [AControl - ASample) / AControl] x 100
where AControl is the absorbance of the L-ascorbic acid (Standard) and ASample is the
absorbance of different compounds. The methanolic DPPH solution (1 mL, 0.3 mM) was
used as control. The inhibitory concentration (IC50) value represents the concentration
required to exhibit 50% antioxidant activity. The IC50 values were calculated by the linear
regression of plots where the abscissa represents the concentration of the compounds
(µg/mL). Explicitly, IC50 is the average percentage of antioxidant activity. Results in the form
of percent inhibition are tabulated in the Table 4. The experiments were done in triplicate.
4.
Conclusions
We have successfully prepared acridone derivatives (8-10) starting from o-halobenzoic acid
and substituted aniline derivatives by Ullmann condensation followed by cyclization of the
ring. The simple method, mild conditions, high yields and particularly environmental
friendliness make this procedure very attractive. All the compounds showed considerable
antimicrobial activity against different strains of bacteria. The compounds also showed good
antioxidant activity by the DPPH method.
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Acknowledgments
The authors are thankful to the Chairman, Department of Chemistry, A.M.U., Aligarh for
providing the necessary research facilities and the UGC is also gratefully acknowledged for
providing the research fellowship to one of the co-authors (AA).
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Fig.1. X-ray crystallographic structure of compound (4).
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Fig.2. Hydrogen bonding network in compound (4) with the H-bond depicted as dashed lines.
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Fig.3. The strong binding affinity of compounds (8-10) with AChE explained on the basis of
hydrogen bonding/secondary interactions as well as orientation and electronic features of the
substituents towards the active site of the target enzyme.
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Fig.4. Estimated binding affinities of synthesized compounds (8-10) based on docked poses
within active site of target enzyme (PDB: 4ey4)
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8
9
10
Fig.5. π-π interaction of compounds (8-10) with amino acid residues of the target enzyme.
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8
9
10
Fig.6. Ligand map of the synthesized compounds showing interactions between the docked
molecules and active site region of the protein.
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Fig. 7. Geometrical structure of compounds 8-10 showing Van der Waals volume and length
perpendicular to the maximum and minimum area. Green, yellow circle represents the radius
of the circumscribed circle of the minimal and maximal projection of the current conformer,
while, green and yellow arrow for min z and max z length of the conformer perpendicular to
its minimal and its maximal projection area, respectively.
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Table 1 Crystallographic data and structure refinement of compound (4)
Compound
Empirical formula
Formula wt.
4
C13H15N2NaO7
334.26
Temperature
Wavelength
293 (2)
0. 71073
Crystal system
Monoclinic
Space group
a, Å
P21/c
20.0793(6)
10.4674(3)
7.2945(2)
90
b, Å
c, Å
α (°)
β (°)
99.018(2)
90
γ (°)
U, Å3
1514.19(8)
4
Z
ρ calc Mg/m3
μ, mm-1
F(000)
1.466
0.143
696
Refl. Collected
Independent refl.
GOF
3363
2870
1.122
Final R indices
[I>2σ(I)]
R indices
(all data)
aR1= 0.0359
WR2= 0.1011( 3359)
R1= 0.0348( 2870)
WR2= 0.1123
b
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Table 2 Quantitative estimation of acetylcholinesterase inhibition activity of compounds (8-
10) by modified Ellaman’s coupled enzyme assay method using tacrine as reference (n=3).
O
R2
R1
N
H
Compound
Nature of Substituents
IC50 (µM)a ± SEMb for hAChEc
inhibition
R1= NO2, R2= NO2
R1= H, R2= NO2
R1= NO2, R= H
0.22±0.01
0.31±0.01
0.45±0.01
10
9
8
Standard
(Tacrine)
0.20±0.01
aIC50 values represent the concentration of inhibitor required to decrease enzyme
activity by 50%
bSEM = standard error of the mean
hAChE = Human recombinant AChE from human serum
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Table 3 Geometrical descriptors data of synthesized compounds (8-10)
Compound
8
9
10
Descriptors
Minimal projection area
27.06
75.06
4.38
28.69
73.01
4.23
30.19
81.02
4.36
Maximal projection area
Minimal projection radius
Maximal projection radius
van der Waals Volume
6.48
6.02
6.49
189.57
4.32
189.80
4.96
212
Length perpendicular to the max area
Length perpendicular to the min area
5.15
12.94
11.54
12.98
29