Synthesis, structural and antioxidant studies of some novel N-ethyl phthalimide esters

Article abstract of DOI:10.1371/journal.pone.0119440

A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.

Full text of DOI:10.1371/journal.pone.0119440

RESEARCH ARTICLE
Synthesis, Structural and Antioxidant Studies
of Some Novel N-Ethyl Phthalimide Esters
C. S. Chidan Kumar1,2☯*, Wan-Sin Loh1☯, Siddegowda Chandraju3, Yip-Foo Win4,
Weng Kang Tan5, Ching Kheng Quah1*, Hoong-Kun Fun1,6
1 X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang, Malaysia, 2 Department
of Chemistry, Alva’s Institute of Engineering & Technology, Mijar, Karnataka, India, 3 Department of Sugar
Technology & Chemistry, University of Mysore, Sir M.V. PG Center, Tubinakere, Karnataka, India,
4 Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Perak Campus,
Jalan Universiti, Bandar Barat, Perak, Malaysia, 5 Klinik Kesihatan Batu Kawa, Jalan Stapok Utara/ Utara,
Kuching, Sarawak, Malaysia, 6 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia
☯ These authors contributed equally to this work.
* ckquah@usm.my (CKQ); chidankumar@gmail.com (CSCK)
Abstract
OPEN ACCESS
Citation: Chidan Kumar CS, Loh W-S, Chandraju S,
Win Y-F, Tan WK, Quah CK, et al. (2015) Synthesis,
Structural and Antioxidant Studies of Some Novel N-
Ethyl Phthalimide Esters. PLoS ONE 10(3):
A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spec-
troscopic studies. Further, the molecular structure of majority of compounds were analysed
by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of
substituents on the crystal stability and molecular packing. All the synthesized compounds
were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and
CUPRAC methods. Few of them have shown good antioxidant activity.
e0119440. doi:10.1371/journal.pone.0119440
Academic Editor: Hans-Joachim Lehmler, The
University of Iowa, UNITED STATES
Received: September 26, 2014
Accepted: January 11, 2015
Published: March 5, 2015
Introduction
Copyright: © 2015 Chidan Kumar et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
The chemistry of heterocyclic compounds is one of the most complex branches of organic
chemistry. Heterocyclic compounds play vital role in biological processes and the researchers
are trying to understand the chemistry of heterocyclic compounds in order to improve the
quality of daily life [1]. Major fractions of organic compounds isolated from nature are com-
prised of nitrogen heterocycles. Numerous lines of evidence suggest that heterocyclic com-
pounds used as analgesic, anti-inflammatory and anti-migraine agents can be potent regulators
of the nitroxidative stress and targeting free nitrogen and oxygen radicals is a very promising
strategy for future pain management [2]. The structural diversity and biological importance of
nitrogen containing heterocycles have made them attractive targets for synthesis over many
years. Constructing highly functionalized heterocyclic compounds would seem to be essential
and significant. The 5-membered N-heterocycles are of exceptional interest in the pharmaceu-
tical industry, as they appear in the core structure of several drugs. Among heterocyclic scaf-
folds, phthalimides are of particular biological interest and have been reported as herbicides,
insecticides, antipsychotics and anti-inflammatory agents. Generally, in organic synthesis, they
are used as starting materials and intermediates for the synthesis of variety of bioactive com-
pounds. The use of phthalimides as primary amine protecting groups is extensively
Data Availability Statement: Crystallographic data
for compounds 4(a-n) (excluding 4d, 4h and 4m)
have been deposited at the Cambridge
Crystallographic Data Centre (Table1). Copies of the
data can be obtained free of charge on application to
the CCDC, 12 Union Road, Cambridge CB2 IEZ, UK.
Fax: +44-(0)1223-336033 or E-Mail: deposit@ccdc.
cam.ac.uk
Funding: CSCK thanks Universiti Sains Malaysia
(USM) for a postdoctoral research fellowship. WSL
thanks Malaysian Government for MyBrain15
(MyPhD) scholarship. CKQ thanks Malaysian
Government and USM for Fundamental Research
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Grant Scheme (FRGS) (203/PFIZIK/6711411). The
authors extend their appreciation to The Deanship of
Scientific Research at King Saud University for the
funding the work through the research group project
No. RGP VPP-207. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
documented in the chemical literature, especially for α–amino acids. Substituted phthalimides
are used predominantly as chiral building blocks in organic synthesis and can be used as key
intermediates in the preparation of bio-active compounds i.e. antibacterial, analgesic, antifun-
gal, virucidal, plant growth regulator and also in dye industry. In view of their significant roles
in biological activities, such as anti-inflamatary [3], hypolipidemic [4], analgesic [5] and other
biomedical activities [6], therefore, development of new and efficient methodologies for these
bioactive compounds is important. Research accounts in the field of organic chemistry and
synthesis of five-membered nitrogen heterocyclic compounds (both aromatic and non-
aromatic) as well as natural products with such heterocyclic systems, is still an open challenge.
Encouraged with the above findings, herein we are reporting the synthesis, structural and
antioxidant studies of several novel phthalimide ester derivatives by introducing ester group to
the main core structure. In the present study, the spacer of one carbon atom distance was intro-
duced to connect the N-terminius of the phthalimide with ester group and to investigate their
antioxidant properties.
Competing Interests: The authors have declared
that no competing interests exist.
Materials and Methods
The reagents and solvents for the synthesis were obtained from the Aldrich Chemical Co., and
were used without additional purification. The purity of each compound was confirmed by
thin layer chromatography using Merck silica gel 60 F254-coated aluminium plates. Open cap-
illary method was employed to determine the melting points and were found uncorrected.
The infrared spectra were recorded using a Perkin-Elmer System 2000 FTIR Spectropho-
tometer as KBr disc from 4000–400 cm-1. The spectra for 1H, 13C and 1H-13C HMQC NMR
were recorded on a JEOL JNM-ECX 400 FT-NMR Spectrometer using deuterated CDCl3 as
the solvent and tetramethylsilane, TMS as the internal standard. Elemental analyses (CHN)
were carried out on a Perkin Elmer Series II, 2400 analyzer. X-ray analysis was done using
Apex II Duo CCDC diffractometer. The data were processed with SAINT and absorption cor-
rection was done using SADABS [7]. The structures were solved by direct method using the
program SHELXTL [8], and were refined by full-matrix lowest squares technique on F2 using
anisotropic displacement parameters. The non-hydrogen atoms were refined anisotropically.
In these compounds, all the H atoms were calculated geometrically with isotropic displacement
parameters set to 1.2 (1.5 for methyl groups) times the equivalent isotropic U values of the par-
ent carbon atoms. The overlay structures were drawn using Olex2 software [9]. Crystallograph-
ic data for compounds 4(a-n) (excluding 4d, 4h and 4m) have been deposited at the
Cambridge Crystallographic Data Centre. S1 Supporting Information contains the checkCIF
report of these deposited compounds. Copies of the data can be obtained free of charge on ap-
plication to the CCDC, 12 Union Road, Cambridge CB2 IEZ, UK. Fax: +44-(0)1223–336033 or
E-Mail: deposit@ccdc.cam.ac.uk.
Synthesis
The systematic procedure followed for the synthesis of target compounds is depicted in Fig. 1.
20.0 g (0.085 mol) of indane (1) was taken in 500 mL round-bottomed flask, and then added
with 200 mL of acetic acid and potassium dichromate (76.7 g, 0.257 mol). The reaction mixture
was refluxed for 3 h at 120°C. The solvent was removed in vacuum, the chromium salt was re-
moved from the residue using boiling water and the white crude product was recrystallized
from glacial acetic acid to give white needle precipitates (1a). In a 250 mL round bottom flask,
powdered phthalic anhydride (1a; 15 g) was mixed with aqueous ammonia (11 mL) and fitted
with a wide air condenser. The reaction mixture was heated at reflux temperature until a ho-
mogeneous melt is formed. All the water gets evaporated during the first hour of the reaction
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 1. Synthesis of (1,3‐dioxo‐2,3‐dihydro‐1H‐isoindol‐2‐yl)methyl (substituted) benzoates 4(a-n).
doi:10.1371/journal.pone.0119440.g001
time. The flask was shaken occasionally, the heating was continued and any material which
sublimes into condenser was push down with a glass rod. After the completion of reaction, the
contents were allowed to cool, dried and recrystallized to get a fine powder of 2 (95%).
The synthesized compound (2) (10.0 g, 0.036 mol) was dispersed in a solution of potassium
hydroxide in ethanol (100 mL). The solution was refluxed for 4 h; the liquor was then cooled
and the product (2a) was filtered, washed successively with water and dried in vacuum. 0.3 mol
of potassium phthalimide (2a) was reacted with dibromomethane (10 mol) in 30 mL of DMF.
The reaction mixture was stirred for 4 h at 70°C. The liquor was then cooled and the product
was filtered, washed with water and dried in vacuum. The precipitate was recrystallized in etha-
nol to obtain the pure compound (3).
Synthesis of (1,3‐dioxo‐2,3‐dihydro‐1H‐isoindol‐2‐yl)methyl (substituted)benzoates
4(a-n). The vacuum dried intermediate 3 (0.002 mol) was then reacted with mono- and di-
substituted benzoic acids (0.003 mol) separately in presence of anhydrous potassium carbonate
(0.003 mol) in DMF (10 mL). The reaction mixture was stirred at room temperature for about
2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
reaction mixture was poured into ice-cold water and allowed stir for 10 min. The solid obtained
was filtered and washed successively with distilled water and recrystallized from ethanol to ob-
tain 4(a–n).
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl benzoate (4a). Isolated yield 78%; Sol-
vent for growing crystals: Acetone; M. P.: 149–151°C; FTIR as KBr disc (cm-1): ν (C-H) aro-
matic 3062, 3049, 3024; ν(C = O) 1782, 1726; ν(C = C) 1448, ν(C-N) 1298, ν(C-O) 1279. 1H-
NMR (ppm) (CDCl3): δ: aromatic protons 7.40 (t, 5.9 Hz, 2H); 7.54 (t, 7.3 Hz, 1H); 7.79 (q, 2.3
Hz, 2H); 7.94 (q, 2.8 Hz, 2H); 8.01 (d, 6.9 Hz, 2H); CH2 5.97 (s, 2H). 13C-NMR (ppm)
(CDCl3): δ: aromatic carbons 124.10, 128.49, 129.19, 129.98, 131.86, 133.53, 134.78; CH2 61.36,
COO 165.50, C = O 166.86. Anal. Calc. for C16H11NO4: C, 68.32; H, 3.94; N, 4.98. Found: C,
68.29; H, 3.97; N, 4.99. CCDC No.: 1019340.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 2-chlorobenzoate (4b). Isolated yield
74%; Solvent for growing crystals: Mixture of acetone and ethanol (1:1 v/v); M. P.: 161–163°C;
FTIR as KBr disc (cm-1): ν(C-H) aromatic 3099, 3043; ν(C-H) aliphatic 2970, ν(C = O) 1782,
1735; ν(C = C) 1591, 1470; ν(C-N) 1298, ν(C-O) 1244, ν(C-Cl) 1038. 1H-NMR (ppm)
(CDCl3): δ: aromatic protons 7.27 (t, 5.9 Hz, 1H); 7.41 (q, 6.4 Hz, 2H); 7.78 (q, 2.3 Hz, 2H);
7.82 (s, 1H); 7.94 (q, 2.3 Hz, 2H); CH2 5.96 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic car-
bons 124.14, 126.65, 128.86, 131.29, 131.83, 131.88, 133.16, 134.30 134.81; CH2 61.75, COO
164.38, C = O 166.76. Anal. Calc. for C16H10ClNO4: C, 60.87; H, 3.19; N, 4.44. Found: C, 60.85;
H, 3.17; N, 4.43. CCDC No.: 1019275.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 3-chlorobenzoate (4c). Isolated yield
81%; Solvent for growing crystals: Mixture of acetone and ethanol (1:1 v/v); M. P.: 157–159°C;
FTIR as KBr disc (cm-1): ν(C-H) aromatic 3103, 3091, 3073, 3047, 3033; ν(C-H) aliphatic 2975,
ν(C = O) 1782, 1728; ν(C = C) 1597, 1470; ν(C-N) 1312, ν(C-O) 1251, ν(C-Cl) 1071. 1H-NMR
(ppm) (CDCl3): δ: aromatic protons 7.35 (t, 7.8 Hz, 1H); 7.51 (d, 8.3 Hz, 1H); 7.79 (q, 2.3 Hz,
2H); 7.89 (d, 7.8 Hz, 1H); 7.94 (q, 2.3 Hz, 2H); 7.97 (t, 1.8 Hz, 1H); CH2 5.96 (s, 2H). 13C-NMR
(ppm) (CDCl3): δ: aromatic carbons 124.16, 128.12, 129.84, 129.95, 130.92, 131.82, 133.57,
134.66 134.85; CH2 61.59, COO 164.32, C = O 166.78. Anal. Calc. for C16H10ClNO4: C, 60.87;
H, 3.19; N, 4.44. Found: 60.84; H, 3.32; N, 4.42. CCDC No.: 1019341.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 4-chlorobenzoate (4d). Isolated yield
79%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 145–
147°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3101, 3072, 3044; ν(C-H) aliphatic 2968,
ν(C = O) 1785, 1735; ν(C = C) 1594, ν(C-N) 1306, ν(C-O) 1270, ν(C-Cl) 1095. 1H-NMR
(ppm) (CDCl3): δ: aromatic protons 7.38 (d, 8.7 Hz, 2H); 7.79 (q, 2.3 Hz, 2H); 7.94 (q, 2.3 Hz,
4H); CH2 5.95 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 124.14, 127.64, 128.87,
131.36, 131.82, 134.83, 140.05; CH2 61.47, COO 164.65, C = O 166.81. Anal. Calc. for
C16H10ClNO4: C, 60.87; H, 3.19; N, 4.44. Found: C, 60.84; H, 3.21; N, 4.41.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 2,4-dichlorobenzoate (4e). Isolated
yield 76%; Solvent for growing crystals: Acetone; M. P.: 134–136°C; FTIR as KBr disc (cm-1):
ν(C-H) aromatic 3093, 3067, 3046; ν(C-H) aliphatic 2977, ν(C = O) 1783, 1727; ν(C = C) 1586,
1470; ν(C-N) 1311, ν(C-O) 1277, ν(C-Cl) 1046. 1H-NMR (ppm) (CDCl3): δ: aromatic protons
7.25 (qn, 1.8 Hz, 1H); 7.44 (d, 1.8 Hz, 1H); 7.78–7.82 (m, 3H); 7.94 (q, 2.3 Hz, 2H); CH2 5.95
(s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 124.17, 127.10, 131.25, 131.79, 132.96,
134.87, 135.54, 139.04; CH2 61.83, COO 163.46, C = O 166.71. Anal. Calc. for C16H9Cl2NO4:
C, 54.88; H, 2.59; N, 4.00. Found: C, 54.85; H, 2.61; N, 3.98. CCDC No.: 1019342.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 2-methylbenzoate (4f). Isolated yield
80%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 144–
146°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3101, 3045, 3029; ν(C-H) aliphatic 2971,
2924; ν(C = O) 1783, 1732; ν(C = C) 1602, 1459; ν(C-N) 1315, ν(C-O) 1241. 1H-NMR (ppm)
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
(CDCl3): δ: aromatic protons 7.19 (q, 7.7 Hz, 2H); 7.38 (t, 7.3 Hz, 1H); 7.78 (q, 2.3 Hz, 2H);
7.87 (d, 7.8 Hz, 1H); 7.94 (q, 2.7 Hz, 2H); CH3 2.58 (s, 3H); CH2 5.94 (s, 2H). 13C-NMR (ppm)
(CDCl3): δ: aromatic carbons 124.07, 125.79, 128.44, 131.00, 131.82, 131.87, 132.57, 134.73,
140.86; CH3 21.84, CH2 61.23, COO 166.25, C = O 166.88. Anal. Calc. for C17H13NO4: C,
69.15; H, 4.44; N, 4.74. Found: C, 69.12; H, 4.46; N, 4.71. CCDC No.: 1019276.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 3-methylbenzoate (4g). Isolated yield
74%; Solvent for growing crystals: Acetone; M. P.: 148–150°C; FTIR as KBr disc (cm-1): ν(C-H)
aromatic 3060, ν(C-H) aliphatic 2965, 2923; ν(C = O) 1777, 1725; ν(C = C) 1610, 1459; ν(C-N)
1311, ν(C-O) 1275. 1H-NMR (ppm) (CDCl3): δ: aromatic protons 7.28 (t, 7.8 Hz, 1H); 7.34 (d,
7.4 Hz, 1H); 7.78 (q, 2.3 Hz, 2H); 7.82 (s, 2H); 7.94 (q, 2.7 Hz, 2H); CH3 2.34 (s, 3H); CH2 5.96
(s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 124.08, 127.15, 128.37, 129.11, 130.44,
131.89, 134.29, 134.74, 138.30; CH3 21.29, CH2 61.33, COO 165.69, C = O 166.87. Anal. Calc.
for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.13; H, 4.46; N, 4.72. CCDC No.:
1019277.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 4-methylbenzoate (4h). Isolated
yield 80%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 139–
141°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3101, 3031; ν(C-H) aliphatic 2974, 2923;
ν(C = O) 1782, 1727; ν(C = C) 1611, 1448; ν(C-N) 1307, ν(C-O) 1272. 1H-NMR (ppm)
(CDCl3): δ: aromatic protons 7.19 (d, 7.8 Hz, 2H); 7.78 (q 2.3 Hz, 2H); 7.89 (d, 8.3 Hz, 2H);
7.93 (q, 2.3 Hz, 2H); CH3 2.37 (s, 3H); CH2 5.95 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic
carbons 124.07, 126.45, 129.19, 130.01, 131.89, 134.72, 144.30; CH3 21.78, CH2 61.23, COO
165.56, C = O 166.89. Anal. Calc. for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.11;
H, 4.45; N, 4.73.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 2-methoxybenzoate (4i). Isolated
yield 71%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 144–
146°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3072, 3026; ν(C-H) aliphatic 2981, 2949,
2844; ν(C = O) 1786, 1742, 1721; ν(C = C) 1598, 1492; ν(C-N) 1331, ν(C-O) 1237. 1H-NMR
(ppm) (CDCl3): δ: aromatic protons 6.89 (s, 1H); 6.93 (t, 8.2 Hz, 1H); 7.77 (q, 2.3 Hz, 2H); 7.44
(t, 6.9 Hz, 1H); 7.75 (d, 2.8 Hz, 1H); 7.93 (q, 2.3 Hz, 2H); OCH3 3.86 (s, 3H); CH2 5.92 (s, 2H).
13C-NMR (ppm) (CDCl3): δ: aromatic carbons 112.12, 118.81, 120.11, 124.02, 131.93, 132.03,
134.21, 134.68, 159.72; OCH3 56.06, CH2 61.39, COO 164.71, C = O 166.89. Anal. Calc. for
C17H13NO5: C, 65.59; H, 4.21; N, 4.50. Found: C, 65.57; H, 4.24; N, 4.49. CCDC No.: 1019343.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 3-methoxybenzoate (4j). Isolated
yield 78%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 133–
135°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3084, 3028; ν(C-H) aliphatic 2977, 2945,
2840; ν(C = O) 1780, 1736; ν(C = C) 1602, 1465; ν(C-N) 1323, ν(C-O) 1273. 1H-NMR (ppm)
(CDCl3): δ: aromatic protons 7.08 (dd, 2.8 Hz, 0.92Hz, 1H); 7.29 (t, 7.8 Hz, 1H); 7.51 (s, 1H);
7.59 (d, 7.8 Hz, 1H); 7.78 (q, 2.3 Hz, 2H); 7.93 (q, 2.3 Hz, 2H); OCH3 3.80 (s, 3H); CH2 5.95
(s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 114.28, 120.11, 122.40, 124.09, 129.52,
130.45, 131.85, 134.77, 159.60; OCH3 55.56, CH2 61.40, COO 165.39, C = O 166.84. Anal. Calc.
for C17H13NO5: C, 65.59; H, 4.21; N, 4.50. Found: C, 65.57; H, 4.24; N, 4.48. CCDC No.:
1019278.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 4-methoxybenzoate (4k). Isolated
yield 82%; Solvent for growing crystals: Acetone; M. P.: 130–133°C; FTIR as KBr disc (cm-1):
ν(C-H) aromatic 3097, 3046; ν(C-H) aliphatic 2964, 2945, 2845; ν(C = O) 1782, 1728; ν(C = C)
1607, 1471; ν(C-N) 1305, ν(C-O) 1258. 1H-NMR (ppm) (CDCl3): δ: aromatic protons 6.86 (d,
8.7 Hz, 2H); 7.77 (q, 2.3 Hz, 2H); 7.93 (q, 2.3 Hz, 2H); 7.96 (d, 11.0 Hz, 2H); OCH3 3.86 (s,
3H); CH2 5.93 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 113.73, 121.55, 124.07,
131.89, 132.09, 134.72, 163.83; OCH3 55.54, CH2 61.13, COO 165.20, C = O 166.90. Anal. Calc.
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
for C17H13NO5: C, 65.59; H, 4.21; N, 4.50. Found: C, 65.56; H, 4.23; N, 4.47. CCDC No.:
1019279.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 2-nitrobenzoate (4l). Isolated yield
79%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 152–
154°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3110, 3099, 3045; ν(C-H) aliphatic 2974,
ν(C = O) 1784, 1733; ν(C = C) 1603, 1472; ν(NO2)1540, ν(C-N) 1315, ν(C-O) 1273. 1H-NMR
(ppm) (CDCl3): δ: aromatic protons 7.59–7.68 (m, 2H); 7.73 (d, 5.6 Hz, 1.8 Hz, 1H); 7.78 (q,
2.3 Hz, 2H); 7.90 (d, 1.4 Hz, 1H); 7.94 (q, 2.3 Hz, 2H); CH2 5.97 (s, 2H). 13C-NMR (ppm)
(CDCl3): δ: aromatic carbons 124.14, 126.93, 130.01, 131.76, 132.13, 133.16, 134.48, 134.82,
148.00; CH2 61.83, COO 164.35, C = O 166.57. Anal. Calc. for C16H10N2O6: C, 58.90; H, 3.09;
N, 8.59. Found: C, 58.97; H, 3.12; N, 8.57. CCDC No.: 1019280.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 3-nitrobenzoate (4m). Isolated
yield 73%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 157–
159°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3103, 3083; ν(C-H) aliphatic 2974, ν(C = O)
1785, 1725; ν(C = C) 1616, 1468; ν(NO2)1529, ν(C-N) 1314, ν(C-O) 1259. 1H-NMR (ppm)
(CDCl3): δ: aromatic protons 7.64 (t, 8.2 Hz, 1H); 7.81 (q, 2.3 Hz, 2H); 7.96 (q, 2.3 Hz, 2H);
8.35 (d, 7.8 Hz, 1H); 8.41 (d, 8.2 Hz, 1H); 8.81 (s, 1H); CH2 6.01 (s, 2H). 13C-NMR (ppm)
(CDCl3): δ: aromatic carbons 124.25, 124.91, 127.97, 129.82, 130.99, 131.79, 134.94, 135.65,
148.34; CH2 61.99, COO 163.49, C = O 166.73. Anal. Calc. for C16H10N2O6: C, 58.90; H, 3.09;
N, 8.59. Found: C, 58.97; H, 3.11; N, 8.55.
(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl 4-nitrobenzoate (4n). Isolated yield
83%; Solvent for growing crystals: Mixture of acetone and methanol (1:1 v/v); M. P.: 161–
163°C; FTIR as KBr disc (cm-1): ν(C-H) aromatic 3113, 3083, 3055; ν(C = O) 1784, 1740;
ν(C = C) 1606, 1467; ν(NO2)1527, ν(C-N) 1324, ν(C-O) 1276. 1H-NMR (ppm) (CDCl3): δ: ar-
omatic protons 7.81 (q, 2.3 Hz, 2H); 7.95 (q, 2.3 Hz, 2H); 8.18 (d, 8.7 Hz, 2H); 8.25 (d, 8.7 Hz,
2H); CH2 6.00 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: aromatic carbons 123.66, 124.24, 131.13,
131.75, 134.57, 134.96, 150.87; CH2 61.94, COO 163.66, C = O 166.71.Anal. Calc. for
C16H10N2O6: C, 58.90; H, 3.09; N, 8.59. Found: C, 58.98; H, 3.11; N, 8.58. CCDC No.: 1019344.
Antioxidant evaluation
2,2’-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. All the test samples in
addition to the standard antioxidant butylated hydroxytoluene (BHT) on DPPH radical scav-
enging was estimated according to the method reported [10]. Methanolic solution of the sam-
ples (10, 25, 50, 100, 200 and 500 μg/mL for samples; 0–5 μg/mL for BHT) in 200 μL aliquot
was mixed with 100 mM tris-HCl buffer (800 μL, pH 7.4) and then added 1 mL of 500 μM
DPPH in methanol (final concentration of 250 μM). The mixture was vigorously shaken and
incubated in the dark at room temperature for 20 min. A DPPH blank solution (control) was
prepared as above without the sample, and methanol was used for the baseline correction. The
absorbance of the test solutions were measured spectro-photometrically at 517 nm. The DPPH
radical scavenging activities were calculated using the equation:
DPPH radical scavenging activity ð%Þ ¼ ½ðAc ꢀ AsÞ=Acꢁx 100
where, Ac is the absorbance of the control and As is the absorbance of the test samples. The in-
hibition concentration of the samples for 50% (IC50) DPPH radical scavenging was also calcu-
lated. Results were expressed as mean of the three determinations.
Ferric ion-reducing antioxidant power (FRAP) assay. All the synthesized compounds
were evaluated for ferric reducing antioxidant property as described earlier by Oyaizu [11].
The theory behind this method is the reduction of ferric (Fe3+) to ferrous (Fe2+), which is
PLOS ONE | DOI:10.1371/journal.pone.0119440 March 5, 2015
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
accomplished in presence of antioxidants. Samples 4(a-n) with the concentration of 10 μg/mL
were mixed with equal volume of 0.2M phosphate buffer (pH 6.6) and 1% potassium ferricya-
nide and the mixture were incubated for 20 min at 50°C. Later the mixture was acidified with
2.5 mL of 10% trichloroacetic acid and centrifuged at 3000 rpm for about 15 min. The upper
supernatant liquid was diluted with distilled water and 0.1% ferric chloride was added. The ab-
sorbance of this solution was measured at 700 nm. The increase in absorbance is directly pro-
portional to the reducing ability of the tested samples. The control was prepared as above
without the sample.
Cupric ion-reducing antioxidant capacity (CUPRAC) assay. The compounds were also
tested for their cupric ion reducing property by the method reported [12]. CUPRAC is a widely
applicable method for evaluating the antioxidant property of the substance. A mixture of
CuCl2 (1 mL, 0.01 M) solution, ethanolic neocuproine (1 mL, 0.0075 M) and ammonium ace-
tate (1 mL, 1 M) were dissolved and added 1 mL of test samples (10–50 μg/mL) along with
0.1 mL of distilled water. After 30 min of incubation, the mixture was measured at 450 nm
against the blank solution. Control is prepared as above without the sample.
Statistical analysis
All the assay measurement were performed in triplicate (n = 3) and are expressed as mean of
the three determinations. The amount of compound required to inhibit DPPH free radicals by
50% (IC50) was graphically estimated using linear regression algorithm. Statistical significance
was evaluated employing t-test and P < 0.05 which were considered to be significant.
Results and Discussion
Chemistry
The detailed synthetic routes adopted for the synthesis of the derivatives 4(a-n) is depicted in
Fig. 1. The intermediate 3 was synthesized according to the procedure reported [13, 14]. The
derivatives 4(a-n) was obtained by nucleophilic substitutions by benzoic acid derivatives, as
per the reported procedure [15]. The target products 4(a-n) were confirmed by analytical and
spectral studies. Further, the compounds 4(a-n) (except 4d, 4h & 4m) were also characterized
by single crystal X-ray diffraction studies. All the synthesized compounds 4(a-n) were evaluat-
ed for their in-vitro antioxidant properties.
The IR spectra of the phthalimide esters 4(a-n) showed the absorption bands above 3000 cm-1
indicating unsaturation or the presence of C-H (benzene and isoindoline-3,5-dione) groups
whereas the methylene group,-CH2, as well as methyl group,-CH3- revealed the asymmetric and
symmetric C-H stretching frequencies near ꢂ2970 and ꢂ2840 cm-1 respectively [16]. Based on
the infrared spectra studies, compounds 4(a-n) also revealed the present of ν(C = C) and ν(C-N)
bands which are usually found for benzene and isoindoline-3,5-dione groups. In addition, the
4(a-n) compounds revealed two distinct ν(C = O) bands in the range of 1786–1725 cm-1; the
ν(C = O) band with the higher wavelength number are attributed to the C = O of isoindoline-
3,5-dione group and the ν(C = O) band with the lower wavelength number are attributed to the
C = O of carboxylate anion [17–20]. The only exceptional cases are compounds 4(b-e) revealed
the present of ν(C-Cl) centering at ꢂ1071 cm-1 and compounds 4(l-n) revealed the present of
ν(NO2) centering at ꢂ1530 cm-1 respectively. The 1H NMR spectra of the phthalimide esters
4(a-n) produced similarities to each other with the presence of-CH2- protons centering around
δꢂ 5.96 ppm and revealed two well resolved sets of quartets centering around δꢂ 7.78 and
7.94 ppm with the integration values of 2:2, ascribed to the-CH- protons of isoindoline-3,5-dione
group [19,20]. In addition, the benzene protons revealed different sets of multiplicity and integra-
tion values due to different position of substitution and these protons signals are located in the
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
downfield region in the 1H NMR spectra. The only exceptional and predictable observation was
the occurrence of the-CH3 and-OCH3 protons signals of compounds 4(f-k) in the up-field re-
gion. Based on the integration values, the number of protons in compounds 4(a-n) is in accor-
dance with the number of protons proposed. 13C NMR spectrum of the phthalimide esters
4(a-n) showed three distinct sets of carbon signals. In the downfield region, both δ(C = O) and
δ(COO) signals are located at δꢂ 166.85 ppm and δꢂ 165.00 ppm respectively, whereas the-
CH2- carbon signals are located in the up-field region centering around δꢂ 61.50 ppm [16–20].
The only exceptional and predictable observation was the occurrence of-CH3 and-OCH3 carbon
signals of compounds 4(f-h) and 4(i-k) in the up-field region of 13C NMR spectra respectively.
From the 13C NMR spectra study, the carbon signals of benzene and isoindoline-3,5-dione
groups were found in the range of 112.12–163.82 ppm [16–20]. The carbons signals centering at
δꢂ 124.10, 131.80 and 134.80 ppm were attributed to isoindoline-3,5-dione groups and the re-
maining signals were attributed to benzene carbons.
X-ray crystallography
All the compounds were colourless and data were collected using MoKα radiation (λ = 0.71073
Å). The crystal structures of 4(a-n) (excluding 4d, 4h and 4m) are depicted in Fig. 2. Tables 1
Fig 2. ORTEP diagram of compounds 4a to 4n (excluding 4d, 4h and 4m). Compounds 4b, 4f, 4g, 4j, 4k
and 4n are drawn at 50% ellipsoids for non-hydrogen atoms and the remaining compounds are drawn at 30%
ellipsoids for non-hydrogen atoms.
doi:10.1371/journal.pone.0119440.g002
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Table 1. Crystal data and parameters for structure refinement of 4a, 4b, 4c, 4e and 4f.
Compound
4a
4b
4c
4e
4f
CCDC number
1019340
C16H11NO4
281.26
1019275
C16H10ClNO4
315.70
1019341
C16H10ClNO4
315.70
1019342
C16H9Cl2NO4
350.14
1019276
C17H13NO4
295.28
Molecular formula
Molecular weight
Crystal system
Space group
a (Å)
Monoclinic
P21/c
Triclinic
Tetragonal
I41/a
Monoclinic
P21/c
Triclinic
P-1
P-1
10.839 (1)
4.823 (1)
26.129 (3)
90
7.052 (2)
7.604 (2)
13.666 (6)
99.403 (1)
98.632 (1)
107.496 (3)
673.89 (4)
2
13.8967 (6)
13.8967 (6)
30.436 (3)
90
14.340 (3)
13.951 (2)
7.6331 (13)
90
7.3517 (5)
7.6033 (5)
13.658 (1)
98.867 (2)
99.506 (3)
110.979 (1)
683.93 (8)
2
b (Å)
c (Å)
α (°)
β (°)
106.372 (4)
90
90
103.005 (3)
90
γ (°)
V (Å3)
90
1310.6 (3)
4
5877.8 (8)
16
1487.9 (4)
4
Z
D
calc (g cm− 3
Crystal dimensions (mm)
μ (mm− 1
min/Tmax
)
1.425
1.556
1.427
1.563
1.434
0.56 × 0.17 × 0.12
0.10
0.55 × 0.41 × 0.08
0.30
0.43 × 0.33 × 0.16
0.28
0.57 × 0.19 × 0.09
0.46
0.54× 0.40× 0.39
0.10
)
T
0.944/0.988
14810
0.8577/0.9829
24638
0.890/0.956
63100
0.781/0.962
16105
0.946 /0.961
26359
Reflections measured
Ranges/indices (h, k, l)
− 13, 15; − 6, 6; − 36,
− 10, 9; − 10, 10; − 19,
− 10, 9; − 10, 10; − 19,
− 19, 15; − 19, 17; − 10,
− 10, 10; − 10, 10; − 19,
36
19
19
10
19
θ limit (°)
2.9–29.4
2.9–30.5
4129
2.5–24.6
4351
2.9–22.0
3964
3.0–30.4
4183
Unique reflections
3822
Observed reflections (I> 2σ 2514
3332
2731
1900
3359
(I))
Paramters
190
199
199
257
200
Goodness of fit on F2
1.04
1.05
1.02
0.99
1.07
R1, wR2 [I ꢄ 2σ(I)]
0.043, 0.121
0.065, 0.188
0.046, 0.153
0.049, 0.199
0.050, 0.141
doi:10.1371/journal.pone.0119440.t001
& 2 give the crystallographic data and parameters for structure refinement. Table 3 lists the hy-
drogen bond geometries of these compounds. The dihedral angles and the torsion angles
formed between the phthalimide and the benzene ring systems are shown in Table 4. Four out
of the eleven compounds (4a, 4e, 4g and 4k) were crystallized in monoclinic system with space
group P21/c. Whereas 4b, 4f, 4j and 4l crystallized in triclinic system with space group P-1 and
the compounds 4i, 4n and 4c crystallized in orthorhombic and tetragonal systems with space
groups Pbca, Pbcn and I41/a, respectively.
Compound 4a with no substitution on the benzene ring is illustrated in Fig. 2(4a). The
phthalimide (N1/C1—C8) and the benzene (C11—C16) rings are almost perpendicular to each
other with the dihedral angle of 87.00 (6)° and the torsion angle between C10—O3—C9—N1
being-176.16 (11)° (Table 4). Compound 4a is considered as a parental skeleton for the com-
parison of compounds with varrying substitutions on the benzene ring. In the crystal structure
(Fig. 3), molecules are linked into dimers via intermolecular C3—H3AꢃꢃꢃO1 hydrogen bonds
(Table 3), forming R22ð10Þring motif [20] and are further connected into sheets parallel to bc-
plane by intermolecular C9—H9AꢃꢃꢃO4 hydrogen bonds (Table 3). Compounds 4b and 4c
exist with one—chloro substituent at—ortho and—para positions of the benzene rings (Fig. 2
(4b & 4c)), respectively, whereas compound 4e (Fig. 2(4e)) differs by having—dichloro substit-
uents at–ortho and—meta positions of the benzene ring. The dichloro-benzene ring in
PLOS ONE | DOI:10.1371/journal.pone.0119440 March 5, 2015
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Table 2. Crystal data and parameters for structure refinement of 4g, 4i, 4j, 4k, 4l and 4n.
Compound
4g
4i
4j
4k
4l
4n
CCDC number
1019277
C17H13NO4
295.28
1019343
C17H13NO5
311.28
1019278
C17H13NO5
311.28
1019279
C17H13NO5
311.28
1019280
C16H10N2O6
326.26
1019344
C16H10N2O6
326.26
Molecular formula
Molecular weight
Crystal system
Space group
a (Å)
Monoclinic
P21/c
Orthorhombic
Pbca
Triclinic
Monoclinic
P21/c
Triclinic
Orthorhombic
Pbcn
P-1
P-1
4.3984 (2)
13.9960 (8)
22.487 (1)
90
12.4243 (8)
7.1597 (5)
32.917 (2)
90
8.4216 (5)
8.4471 (6)
10.8739 (7)
80.311 (2)
86.382 (2)
66.304 (2)
698.22 (8)
2
14.2125 (8)
13.0603 (8)
7.9266 (4)
90
7.5556 (5)
7.6118 (4)
13.6551 (8)
99.132 (2)
97.068 (2)
113.204 (2)
697.59 (7)
2
13.298 (2)
7.549 (1)
29.095 (3)
90
b (Å)
c (Å)
α (°)
β (°)
97.622 (2)
90
90
100.672 (2)
90
90
γ (°)
V (Å3)
90
90
1372.07 (12)
4
2928.1 (3)
8
1445.88 (14)
4
2920.6 (7)
8
Z
D
calc (g cm− 3
)
1.429
1.412
1.481
1.430
1.553
1.484
Crystal dimensions
(mm)
0.70 × 0.07 ×
0.06
0.52 × 0.46 × 0.18
0.46 × 0.40 × 0.16
0.57 × 0.35 × 0.11
0.56× 0.25× 0.16
0.70 × 0.07 × 0.06
μ (mm− 1
)
0.10
0.11
0.11
0.11
0.12
0.12
T
min/Tmax
0.931/0.994
0.947/0.981
32340
0.951/0.983
18754
0.942/0.988
38131
0.935 /0.981
34125
0.930/0.991
17655
Reflections measured 72106
Ranges/indices (h, k,
l)
− 6, 6; − 20, 20;
− 17, 17; − 9, 10;
− 46, 46
− 11, 12; − 12, 12;
− 15, 15
− 20, 20; − 18, 18;
− 10, 11
10, 10; − 10, 10;
− 19, 19
− 15, 18; − 10, 10;
− 40, 40
− 32, 32
2.3–24.3
4235
θ limit (°)
2.5–28.6
4405
2.6–30.5
4282
2.9–30.5
4446
3.0–30.5
4279
3.1–21.6
4242
Unique reflections
Observed reflections
(I> 2σ(I))
2400
3155
3198
2696
3327
1707
Parameters
200
209
209
209
217
217
Goodness of fit on F2
1.00
1.03
1.02
1.00
1.04
0.93
R1, wR2 [I ꢄ 2σ(I)]
0.061, 0.130
0.045, 0.138
0.068, 0.193
0.075, 0.211
0.047, 0.130
0.057, 0.213
doi:10.1371/journal.pone.0119440.t002
compound 4e is disordered over two positions with the occupancy ratio of 0.791 (2): 0.219 (2).
In the crystal structure of 4b (Fig. 4a), intermolecular C3—H3AꢃꢃꢃO1 and C6—H6AꢃꢃꢃO2 hy-
drogen bonds (Table 3) link the molecules to form their respective dimers with R22ð10Þring mo-
tifs [20]. In compounds 4c and 4e, the adjacent molecules are connected to form R22ð10Þring
motifs [20] through intermolecular C3—H3AꢃꢃꢃO2 and C6—H6AꢃꢃꢃO1 hydrogen bonds
(Table 3, Figs. 5 & 6a). Further, the molecules in compounds 4b and 4e are linked into sheets
via intermolecular C15—H15AꢃꢃꢃO4 hydrogen bonds (Table 3) extending parallel to ab and
bc-plane for 4b and 4e, respectively (Fig. 4b & Fig. 6b). The crystal packing of 4b is further con-
solidated by πꢃꢃꢃπ interactions between the five-membered ring (N1/C1/C2/C7/C8; centroid
Cg1) and benzene ring (C2—C7; centroid Cg2), where the separations of centroids of
Cg1ꢃꢃꢃCg1, Cg1ꢃꢃꢃCg2 and Cg2ꢃꢃꢃCg2 being 3.4860 (13), 3.6022 (13) and 3.5996 (12) Å, respec-
tively. Whereas, a three-dimensional network involving the ring motifs was produced by the
C14—H14AꢃꢃꢃO4 and C16—H16AꢃꢃꢃO2 intermolecular hydrogen bonds in compound 4c
(Fig. 6).
The dihedral angles between the phthalimide and the benzene rings (Table 4) of 4b, 4c and
4e indicate that, they lie perpendicular to each other in the range of 85.05 (8) to 89.34° and the
C10—O3—C9—N1 torsion angles fall within the range of 170.31 (14) to-179.63 (13)°. Fig. 7
PLOS ONE | DOI:10.1371/journal.pone.0119440 March 5, 2015
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Table 3. Hydrogen bond geometries for the compounds 4a-4n (excluding 4d, 4h and 4m).
D–H ꢃ ꢃ ꢃA
d(D–H) (Å)
d(H ꢃ ꢃ ꢃA) (Å)
d(Dꢃ ꢃ ꢃA) (Å)
Angle (D–H ꢃ ꢃ ꢃA) (°)
4a
C3—H3Aꢃ ꢃ ꢃ O1i
C9—H9A ꢃ ꢃ ꢃ O4ii
4b
0.93
0.97
2.46
2.59
3.354 (2)
3.300 (2)
162
130
C3—H3Aꢃ ꢃ ꢃ O1iii
C6—H6A ꢃ ꢃ ꢃ O2iv
C15—H15A ꢃ ꢃ ꢃ O4v
4c
0.93
0.93
0.93
2.51
2.42
2.37
3.382(3)
3.270(3)
3.290(3)
157
151
169
C6—H6A ꢃ ꢃ ꢃ O1vi
C3—H3Aꢃ ꢃ ꢃ O2vii
C14—H14A ꢃ ꢃ ꢃ O4viii
C16—H16A ꢃ ꢃ ꢃ O2ix
4e
0.93
0.93
0.93
0.93
2.40
2.37
2.36
2.56
3.317(2)
3.289(2)
3.111(3)
3.437(2)
167
168
137
157
C3—H3Aꢃ ꢃ ꢃ O2x
C6—H6A ꢃ ꢃ ꢃ O1xi
C15—H15Aꢃ ꢃ ꢃ O4xii
4f
0.93
0.93
0.93
2.44
2.46
2.44
3.349 (4)
3.379 (3)
3.334 (6)
166
168
162
C3—H3Aꢃ ꢃ ꢃ O1xiii
C6—H6Aꢃ ꢃ ꢃ O2xiv
C15—H15Aꢃ ꢃ ꢃ O4xv
4g
0.93
0.93
0.93
2.36
2.42
2.39
3.256 (2)
3.332 (2)
3.300 (2)
163
168
168
C3—H3Aꢃ ꢃ ꢃ O1xvi
4j
0.93
2.49
3.393 (2)
164
C3—H3Aꢃ ꢃ ꢃ O4xiv
C17—H17Aꢃ ꢃ ꢃ O2 xvii
4k
0.93
0.96
2.55
2.50
3.287 (2)
3.340 (2)
136
146
C4—H4Aꢃ ꢃ ꢃ O5xviii
C15—H15Aꢃ ꢃ ꢃ O4xii
4l
0.93
0.93
2.50
2.56
3.220 (3)
3.491 (3)
134
176
C3—H3Aꢃ ꢃ ꢃ O1xix
C5—H5Aꢃ ꢃ ꢃ O6xx
C6—H6Aꢃ ꢃ ꢃ O2xxi
C15—H15A ꢃ ꢃ ꢃ O4xxii
4n
0.93
0.93
0.93
0.93
2.56
2.58
2.41
2.35
3.470 (2)
3.467 (2)
3.304 (2)
3.262 (2)
166
159
161
168
C12—H12A ꢃ ꢃ ꢃ O6v
0.93
2.45
3.363 (4)
167
(Continued)
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Table 3. (Continued)
D–H ꢃ ꢃ ꢃA
d(D–H) (Å)
d(H ꢃ ꢃ ꢃA) (Å)
2.36
d(Dꢃ ꢃ ꢃA) (Å)
3.274 (4)
Angle (D–H ꢃ ꢃ ꢃA) (°)
166
C15—H15A ꢃ ꢃ ꢃ O4xv
0.93
Symmetry codes:
(i) − x, − y+3, − z+2;
(ii) − x, y+1/2, − z+3/2;
(iii) − x+1, − y+1, − z+1;
(iv) − x− 1, − y, − z+1;
(v) x, y+1, z;
(vi) x− 1/2, y, − z+1/2;
(vii) x+1/2, y, − z+1/2;
(viii) x, y− 1/2, − z;
(ix) − x+1, − y+1/2, z;
(x) − x+1, y+1/2, − z− 1/2;
(xi) − x+1, y− 1/2, − z− 1/2;
(xii) x, y, z+1;
(xiii) − x+3, − y+1, − z+1;
(xiv) − x+1, − y, − z+1;
(xv) x, y− 1, z;
(xvi) − x− 1, − y+1, − z+2;
(xvii) x+1, y− 1, z;
(xviii) x− 1, − y+3/2, z− 1/2;
(xix)-x,-y,-z+1;
(xx) x, y+1, z+1;
(xxi)-x+1,-y+2,-z+1;
(xxii) x-1, y, z.
doi:10.1371/journal.pone.0119440.t003
shows the overlaid molecules over all non-H atoms, calculated using the phthalimide moiety
with the r.m.s values of 0.095 Å for 4a/4b, 0.141 Å for 4a/4c and 0.350 Å for 4a/4e.
Compounds 4f and 4g (Fig. 2(4f & 4g)) consist one methyl substituent at—ortho and—
meta positions, respectively. In the crystal structure of 4f (Fig. 8a), dimers were formed by in-
termolecular C3—H3AꢃꢃꢃO1 and C6—H6AꢃꢃꢃO2 hydrogen bonds (Table 3) at an alternate
fashion, giving R22ð10Þring motifs [20]. These set of ring motifs were then further linked into
planes through intermolecular C15—H15AꢃꢃꢃO4 hydrogen bonds (Table 3) parallel to the ab-
plane (Fig. 8b) and πꢃꢃꢃπ interactions stabilized the crystal structure with the separation of cen-
troid-centroid, Cg1ꢃꢃꢃCg1, Cg1ꢃꢃꢃCg2 and Cg2ꢃꢃꢃCg2 being 3.4930 (8), 3.5902 (8) and 3.6003 (8)
Å. Intermolecular C3—H3AꢃꢃꢃO1 hydrogen bonds (Table 3) joined the molecules in 4g into di-
mers as depicted in Fig. 9, forming graph set notation of R22ð10Þring motifs [21]. The conforma-
tion of both the compounds are very analogous to each other with the dihedral angles between
the rings being 86.06 (6) and 80.76 (8)° and their C10—O3—C9—N1 torsion angles are-169.03
(9) and-177.95 (15)°, respectively (Table 4). The analogous conformations is best visualized by
the overlay (Fig. 10) of 4a, 4f and 4g, calculated using phthalimide ring system, with the r.m.s
values of 0.094 Å for 4a/4f and 0.156 Å for 4a/4g.
Compounds 4i, 4j and 4k (Fig. 2(4i-4k)) contain-methoxy substitution at—ortho,—meta
and—para positions on the benzene rings, respectively. The 79.86 (7) and 73.31 (7)° dihedral
angles between phthalimide and benzene rings for compounds 4i and 4j respectively, suggest
that, they have slightly different conformation from the parental skeletal 4a. This is confirmed
by the C10—O3—C9—N1 torsion angles being-86.50 (14) and-91.92 (15)°, respectively for 4i
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Table 4. Dihedral angles and torsion angles formed between the phthalimide and benzene ring
systems.
Compound
Dihedral angle (°)
Torsion angle of C10—O3—C9—N1 (°)
4a
4b
4c
4e
4f
87.00 (6)
-176.16(11)
170.31(14)
-179.63(13)
-175.81(19)
-169.03(9)
-177.95(15)
-86.50(14)
-91.92(15)
-174.63(17)
-171.84(10)
166.2(2)
86.39 (9)
85.05 (8)
Part A 89.34; Part B 87.71
86.06 (6)
4g
4i
80.76 (8)
79.86 (7)
4j
73.31 (7)
4k
4l
83.32 (8)
86.71 (6)
4n
83.91 (10)
doi:10.1371/journal.pone.0119440.t004
and 4j. While, the respective parameters for compound 4k are 83.32 (8) and-174.63 (17)°. This
conformation difference of the compounds 4i and 4j compared with 4a and 4k can be clearly
viewed in Fig. 11, which shows the overlays of all non-H atoms of 4a/4i, 4a/4j and 4a/4k, calcu-
lated using the phthalimide moiety where their H-atoms were excluded, with the r.m.s values
of 0.823, 0.895 and 0.154 Å, respectively. There is no significant intermolecular hydrogen bond
observed in 4i. However, πꢃꢃꢃπ interactions are observed to stabilize the crystal structure, in-
volving the phthalimide ring system and the phenyl ring, with Cg1ꢃꢃꢃCg2 being 3.6764 (10) Å
and Cg3ꢃꢃꢃCg3 being 3.6449 (9) Å, where Cg3 is the centroid of C11—C17. Fig. 12 illustrates
the molecular packing of 4j. Intermolecular C3—H3AꢃꢃꢃO4 hydrogen bond (Table 3) links the
adjacent molecules in 4j to form dimers, giving R22ð10Þring motifs [21]. These set of dimers
were then connected into columns along [–110] by intermolecular C17—H17AꢃꢃꢃO2 hydrogen
bond (Table 3). The crystal structure of 4k is depicted in Fig. 13. The molecules in 4k are linked
into chains along the a-axis by intermolecular C4—H4AꢃꢃꢃO5 hydrogen bonds (Table 3) and
Fig 3. Crystal structure of 4a, showing the sheet parallel to bc-plane, viewed along the b-axis.
doi:10.1371/journal.pone.0119440.g003
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 4. Crystal structure of 4b, showing the formation of sheet parallel to ab-plane.
doi:10.1371/journal.pone.0119440.g004
Fig 5. Crystal structure of 4c, showing the three-dimensional network, viewed along the b-axis.
doi:10.1371/journal.pone.0119440.g005
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 6. Crystal structure of 4e, showing the ring motifs which lead to the formation of sheet parallel to bc-plane.
doi:10.1371/journal.pone.0119440.g006
are further connected into sheets parallel to ac-plane by intermolecular C15—H15AꢃꢃꢃO4 hy-
drogen bonds (Table 3).
The molecular structures of compounds with nitro-substituent at—ortho and—para posi-
tions on the benzene rings, 4l and 4n are shown in Fig. 2(4l & 4n). The dihedral angles formed
between the ring members are 86.71 (6)° for 4l and 83.91 (10)° for 4n. The C10—O3—C9—N1
torsion angle of 4l is-171.84 (10)°, whereas the respective parameter for 4n is 166.2 (2)°. These
two compounds exist with the similar conformation as indicated by the overlay structures in
Fig. 14. The overlays of all non-H atoms of 4a/4l and 4a/4n, calculated using the phthalimide
Fig 7. Overlay of all non-H atoms in compounds 4a, 4b, 4c and 4e, calculated using the phthalimide
moiety.
doi:10.1371/journal.pone.0119440.g007
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 8. Crystal structure of 4f, showing the dimers which are further linked into sheet parallel to ab-plane.
doi:10.1371/journal.pone.0119440.g008
Fig 9. Crystal structure of 4g, showing the formation of dimers stacked along the a-axis.
doi:10.1371/journal.pone.0119440.g009
Fig 10. Overlay of all non-H atoms in compounds 4a, 4f and 4g, calculated using the phthalimide moiety.
doi:10.1371/journal.pone.0119440.g010
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 11. Overlay of all non-H atoms in compounds 4a, 4i, 4j and 4k, calculated using the phthalimide
moiety.
doi:10.1371/journal.pone.0119440.g011
moiety where their H-atoms and nitro-substituent were excluded, with the r.m.s values of
0.117 and 0.096 Å, respectively. In the crystal structure of 4l, intermolecular C3—H3AꢃꢃꢃO1
and C6—H6AꢃꢃꢃO2 hydrogen bonds link the adjacent molecules to form their respective di-
mers (Fig. 15a) alternately, with R22ð10Þring motifs [21]. These set of dimers were then further
connected into sheets parallel to ab-plane via intermolecular C5—H5AꢃꢃꢃO6 and C15—
H15AꢃꢃꢃO4 hydrogen bonds as shown in Fig. 15b. The crystal structure of 4n is shown in
Fig. 16. Intermolecular C12—H12AꢃꢃꢃO6 and C15—H15AꢃꢃꢃO4 hydrogen bonds link the
neighbouring molecules into chains along the b-axis, forming R22ð10Þring motifs [21]. Both 4l
and 4n are observed to have πꢃꢃꢃπ interactions in their crystal packing. In compound 4l,
Cg1ꢃꢃꢃCg2 and Cg2ꢃꢃꢃCg2 with the separations of centroid-centroid being 3.6443 (10) and
3.6076 (9) Å, respectively, consolidated the crystal packing. Whereas in compound 4n, only
Cg1ꢃꢃꢃCg2 with the separation of 3.5571 (17) Å is responsible for its crystal packing.
The overall conformations of the reported structures are described focused onto the dihe-
dral and torsion angles formed between the phthalimide (N1/C1—C8) and the benzene
(C11—C16) ring systems. The molecular structures of the compounds 4a-n (excluding 4d, 4h
and 4m) were overlaid as shown in Fig. 17, excluding H atoms and substituents at the benzene
rings. From the overlay diagram, it is clear that, the molecular conformations of majority of the
compounds are almost similar to each other. Whereas the compounds 4i and 4j with-methoxy
Fig 12. Crystal structure of 4j, showing the column formed along [–110] via intermolecular C—HꢃꢃꢃO hydrogen bonds.
doi:10.1371/journal.pone.0119440.g012
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 13. Crystal structure of 4k, showing the sheet formed parallel to ac-plane via intermolecular C—HꢃꢃꢃO
hydrogen bonds.
doi:10.1371/journal.pone.0119440.g013
substituent at the—ortho and—meta position on the benzene rings are very much deviated
from the parental skeletal (compound 4a).
Antioxidant Activity
The synthesized compounds were screened for their antioxidant potential by employing the in-
vitro assay such as DPPH free radical scavenging assay. The synthesized compounds were fur-
ther tested for their reducing power ability. Ferric ion reducing antioxidant power (FRAP)
assay and cupric ion reducing antioxidant capacity (CUPRAC) were measured using butylated
hydroxytoluene as the standard.
Fig 14. Overlay of all non-H atoms in compounds 4a, 4l and 4n, calculated using the phthalimide moiety.
doi:10.1371/journal.pone.0119440.g014
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 15. Crystal structure of 4l, showing the dimers which are further connected to form sheet parallel to ab-
plane.
doi:10.1371/journal.pone.0119440.g015
DPPH Radical Scavenging Assay
The DPPH radical scavenging test is a standard and widely used assay for in vitro antioxidant
capacity of compounds and it is based on their ability of scavenging of stable 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH) [10]. The results of in vitro antioxidant activity (IC50 values) of
the synthesized compounds in comparison with the reference antioxidant BHT are depicted in
Fig 16. Crystal structure of 4n, showing the ring motifs which are linked into chain along b-axis via intermolecular C—HꢃꢃꢃO hydrogen bonds.
doi:10.1371/journal.pone.0119440.g016
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 17. Overlay of all molecular structures, excluding H atoms and substituents at benzene rings
where 4i and 4j are highlighted in blue and pink, respectively.
doi:10.1371/journal.pone.0119440.g017
Fig. 18. From the results, it is noticed that these compounds exhibit mild to good antioxidant
activity. At the start with concentrations of 10–50 μg/mL, no significant change in the radical
scavenging was noticed. However, the radical scavenging ability increased with increasing con-
centration of the samples (100–500 μg/mL). The IC50 values indicate that the compounds 4i, 4j
and 4k with—methoxy substitution at the—o,—m &—p position, respectively, on the phenyl
ring showed good free radical scavenging activity. Among those, compound 4k with a p-meth-
oxy substituent was more potent in free radical scavenging compared to the other test com-
pounds. The compounds 4(f-h), bearing—methyl substitutions displayed moderate scavenging
activity. The IC50 value for the compound 4a with no substitution is in between to the 4(i-k)
and 4(f-g) compounds. The compounds 4b, 4c, 4d and 4e bearing electronegative—chloro sub-
stitution were mild in their activity, while the compounds 4(l–n) with electron withdrawing—
nitro substitutions respectively displayed very mild scavenging activity. Among them, com-
pound 4n exhibited the lowest activity. The biological data suggested that the substituents on
the phenyl group had a profound effect on the antioxidant activity in the order of NO2 < Cl <
H < CH3 < OCH3, which is consistent with those reported earlier [22].
Fig 18. IC50 (concentration required for 50% inhibition) values for DPPH radical scavenging activities
of the compounds 4(a–n) in comparison with the standard antioxidant BHT. Lower IC50 values indicate
higher radical scavenging activity.
doi:10.1371/journal.pone.0119440.g018
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Fig 19. Ferric and cupric ion reducing power abilities of the tested samples at 10 μg concentration measured at 700 nm. Values are expressed as
absorbance; high absorbance indicates high reducing power
doi:10.1371/journal.pone.0119440.g019
FRAP Assay
In general, the antioxidant activity of a substance is directly correlated to its reducing ability.
Standard assays like FRAP provide a reliable method to verify the antioxidant ability of a sub-
stance. Ferric reducing antioxidant properties for the synthesized compound were evaluated by
the method as described earlier by Oyaizu [11]. Substances having reduction potential react
with potassium ferricyanide forming potassium ferrocyanide. The formed potassium ferrocya-
nide further reacts with FeCl3 to form an intense Prussian blue complex which has a maximum
absorbance at 700 nm. The complex formed is directly proportional to the reducing capacity of
the test sample. An increase in absorbance is equal to the reducing power of the sample. Results
are depicted in Fig. 19 and from the analysis, it is clear that the compounds 4i, 4j, 4k with—
methoxy substitution showed good cupric reducing ability, while the compounds 4a with no
substitution, 4(f-h) with—methyl substitutions were moderate, and the compounds 4b, 4c, 4d,
4e, 4l, 4m and 4n bearing—chloro and—nitro substitutions showed the lowest reducing ability
among the series. This was in agreement with the similar reported structures [22].
CUPRAC Assay
The cupric ion reducing properties of the synthesized compounds were evaluated by the meth-
od reported earlier [12]. In this assay a sample under evaluation effectively reduces Cu2+ to
Cu+, changing the characteristic ion absorption. The reduced Cu+ ion combines with the chro-
mogenic reagent neocuproine forming a stable 2:1 complex which has a maximum absorption
at 450 nm. This method operates at pH 7. Results, shown in Fig. 19, indicate that the majority
of these test compounds have good reducing ability. These compounds displayed 40% less re-
ducing power compared to the standard. Compounds 4i, 4j, 4k showed good cupric reducing
ability, while the compounds 4a, 4f, 4g and 4h were moderate, and the compounds 4b, 4c, 4d,
4e, 4l, 4m and 4n showed the lowest reducing ability among the series. The similar trend of in-
creasing antioxidant activity was also observed in the related structures [22].
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Synthesis, Structural and Antioxidant of N-Ethyl Phthalimide Esters
Conclusion
Herein we describe the efficient synthesis of N-ethyl phthalimide esters 4(a-n). The synthesized
compounds were confirmed from their spectral data. The conformations of the compounds 4
(a-n) (excluding 4d, 4h and 4m) were confirmed by single crystal X-ray diffraction analysis.
The molecular conformations of the reported compounds were compared among each other
and found that, the compounds 4i and 4j deviate from the rest. The X-ray data also revealed
the importance of intermolecular and other minor interactions contributing for the crystal
structure stability and molecular packing, induced by varying substituent(s) on the phenyl
ring. The synthesized compounds were evaluated for their in vitro antioxidant activities.
Among the series, compounds 4i, 4j and 4k with—methoxy substitution at—ortho,-meta
and—para positions respectively displayed good antioxidant properties. However, the incorpo-
ration of electron releasing or electron withdrawing groups on the phthalimide ring system
may result in better activities.
Supporting Information
S1 Supporting Information. CheckCIF report of compounds 4(a-n) (excluding 4d, 4h and
4m).
(PDF)
Author Contributions
Conceived and designed the experiments: CSCK. Performed the experiments: CSCK WSL. An-
alyzed the data: CSCK WSL SC YFW. Contributed reagents/materials/analysis tools: CKQ
HKF. Wrote the paper: CSCK WSL SC YFW WKT CKQ HKF.
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