2
Journal of Chemistry
apparatus (Loughborough, England) and are uncorrected.
2.2.5. (p-Tolyl)isoindoline-1,3-dione 5. Yield: 93%; m.p.
102°C; 1H NMR (500 MHz, CDCl3) δH 7.34 (s, 4H), 7.80 (dd,
2H, J � 3.0 & 8.5 Hz), 7.96 (dd, 2H, J � 3.0 & 8.5 Hz), 2.43 (s,
3H); 13C NMR (125 MHz, CDCl3) δC 167.4 × 2, 138.2, 134.3 × 2,
131.8 × 2, 129.8 × 2, 129.0, 126.4 × 2, 123.7 × 2, 21.2. +ESIMS
m/z C15H11NO2 238.
1
*e H and 13C NMR spectra were preceded on a Bruker
¨
AMX500 spectrometer (Bruker BioSpin, Fallanden,
Switzerland) in deuterated solvent with tetramethylsilane
(TMS) as an internal standard. Chemical shifts are shown in
ppm (δ), and coupling constants (J) are presented in Hertz.
ESI mass experiment was done on Agilent Triple Quadrupole
6410 QQQ LC-mass spectrometer with ESI ion source (gas
temperature 350°C, nebulizer pressure 60 psi, and flow rate of
gas 10 L/min) operating in the both scan ionization modes
with direct infusion method in the presence of solvent
methanol/water (1 :1 v/v) with flow rate 0.2 mL/min. *in
layer chromatography (TLC) was performed on precoated
TLC plates (silica gel F254, Merck, Germany); the UV de-
tection was done at low wavelength 254 nm and sprayed with
ceric sulfate in 10% H2SO4 (heating at 120°C).
2.2.6. (o-Tolyl)isoindoline-1,3-dione 6. Yield: 95%; m.p.
103°C; 1H NMR (500 MHz, CDCl3) δH 7.23 (d, 1H,
J � 7.5 Hz), 7.35 (m, 1H),7.40 (s, 2H), 7.81 (dd, 2H, J � 3.0 &
8.5 Hz), 7.99 (dd, 2H, J � 3.0 & 8.5 Hz), 2.24 (s, 3H);
13C NMR (125 MHz, CDCl3) δC 167.3 × 2, 136.5, 134.3 × 2,
132.0, 131.1, 130.6 × 2, 129.4, 128.7, 126.9 123.7 × 2,
18.0. +ESIMS m/z C15H11NO2 238.253, found 1088.578.
2.2.7. N-(2-Chloro-4-methoxyphenyl)isoindoline-1,3-dione 7.
Yield: 94%; m.p. 110°C; 1H NMR (500 MHz, CD3OD +
CDCl3) δH 7.44 (brs, 1H), 7.42 (brs, 1H), 7.06 (brs, 1H), 7.80
(brs, 2H), 7.91 (brs, 2H), 3.91 (s, 3H, OCH3); 13C NMR
(125 MHz, CD3OD + CDCl3) δC 171.5 × 2,158.8, 138.7,
132.4 × 2, 130.2, 128.3, 126.5, 115.9 × 2, 135.4 × 2, 127.7 × 2,
60.0. +ESIMS m/z C15H10ClNO3 288.
2.2. General Procedure for the Synthesis of Phthalimides
through L-Proline-Catalyzed Reaction. *e phthalic acid
(1.0 mmol), aryl amine (1.0 mmol), and L-proline (0.5 mmol)
were sequentially added in ethanol (5.0 mL) into a rb (round
bottom) flask to form a solution. *e resultant mixture was
stirred at 30°C room temperature for six hours. After
completion of the reaction, the reaction mixture was then
diluted with EtOAc ethyl acetate and washed with water. *e
organic phase was dried, filtered, and concentrated to give
the pure phthalimide product. Purity of the products was
checked by TLC.
2.3. Biological Assays
2.3.1. Inhibition of Albumin Denaturation Assay.
Anti-inflammatory potential as membrane stabilization of
the products 1–7 was evaluated by modified method [16].
*e reaction mixture consists of test solution (1 mg/ml) and
1% aq. solution of bovine albumin fraction. *e pH of the
reaction mixture was adjusted by using small amount of
HCl. *e reaction mixture was incubated for 20 min at 37°C
and then heated for 20 min at 51°C. After cooling of the
tubes, the turbidity was measured at 660 nm. Aspirin was
used as a standard. *e experiment was repeated thrice, and
the percent inhibition of protein denaturation was calculated
as follows:
2.2.1. N-(4-Methoxyphenyl)isoindoline-1,3-dione 1. Yield:
94%; m.p. 107°C; 1H NMR (500 MHz, CDCl3) δH 7.04 (d, 1H,
J � 8.5 Hz), 7.35 (d, 2H, J � 8.5 Hz), 7.80 (m, 2H), 7.96 (m, 2H),
3.88 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3) δC
167.5 × 2,159.2, 134.3 × 2, 131.8 × 2,127.9 × 2, 124.2, 123.6 × 2,
114.5 × 2, 55.5. ESIMS m/z C15H11NO3 254.
2.2.2. N-(Phenyl)isoindoline-1,3-dione 2. Yield: 93%; m.p.
101°C; 1H NMR (500 MHz, CDCl3) δH 7.43 (m, 3H), 7.52 (d,
2H, J � 8.0 Hz), 7.83 (dd, 2H, J � 3.0 & 8.5 Hz), 7.99 (dd, 2H,
J � 3.0 & 8.5 Hz); 13C NMR (125 MHz, CDCl3) δC 167.3 × 2,
134.4 × 2, 131.7 × 2, 129.1 × 2, 128.1, 126.6 × 2, 123.7 × 3.
ESIMS m/z C14H9NO2 224.
Abs control − Abs
ꢀ
ꢁ
test
% inhibition �
× 100,
(1)
ꢂ
ꢃ
Abs control
where Abs control is absorbance without any sample and
Abstest is the absorbance of test.
2.3.2. Membrane Stabilization Assay (Heat-Induced Hemolysis).
Anti-inflammatory potential as membrane stabilization of
the products 1–7 was evaluated by reported method [17].
Red blood cells suspension was prepared as follows: freshly
collected human whole blood (10 ml) was transferred to the
anticoagulant-containing centrifuge tubes. *e tubes were
centrifuged for 10 min at 3000 rpm and then washed thrice
with normal saline. *e blood volume was measured and
reconstituted as 10% v/v suspension of normal saline. *e
reaction mixture (2 ml) was prepared with 1 ml of test
sample (1 mg/ml) solution and 1 ml of 10% red blood cells
suspension. Aspirin was used as a standard drug. In the
control test tube, saline was added instead of test sample.
*e reaction mixtures were incubated for 30 min at 56°C on
2.2.3. N-(4-Chlorophenyl)isoindoline-1,3-dione 3. Yield:
92%; m.p.105°C; 1H NMR (500 MHz, CDCl3) δH 7.43 (d, 2H,
J � 8.5 Hz), 7.51 (d, 2H, J � 8.5 Hz H), 7.82 (dd, 2H, J � 3.0 &
8.0 Hz), 7.98 (dd, 2H, J � 3.0 & 8.0 Hz); 13C NMR (125 MHz,
CDCl3) δC 167.0 × 2, 134.5 × 2, 133.8, 131.6 × 2, 130.2,
129.3 × 2, 127.6 × 2, 123.8 × 2. ESIMS m/z C14H8ClNO4 258.
2.2.4. N-(4-nitro)isoindoline-1,3-dione 4. Yield: 95%; m.p.
108.0°C; 1H NMR (500 MHz, CD3OD + CDCl3) δH 7.42 (m,
2H), 7.56 (d, 2H, m), 7.80 (dd, 2H, m), 7.91 (dd, 2H, m); 13
C
NMR (125 MHz, CD3OD + CDCl3) δC 167.3 × 2, 134.4 × 2,
131.7 × 2, 129.1 × 2, 128.1 × 2, 126.6 × 2, 123.7 × 2. ESIMS m/z
C14H8N2O4 268.