Synthesis, characterization and in vitro antimicrobial evaluation of new compounds incorporating oxindole nucleus

Article abstract of DOI:10.3109/14756366.2011.576251

New compounds incorporating with the oxindole nucleus were synthesized via the reaction of substituted isatins [5-methyl-, 5-chloro- and 1-hydroxymethyl isatins] with different nucleophiles. The structures of the newly compounds were elucidated on the bas

Full text of DOI:10.3109/14756366.2011.576251

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(4): 599–608
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.576251
RESEARCH ARTICLE
Synthesis, characterization and in vitro antimicrobial
evaluation of new compounds incorporating oxindole nucleus
Nadia G. Kandile1, Howida T. Zaky1, Mansoura I. Mohamed1, Hind M. Ismaeel1, and
Nashwa A. Ahmed2
1Chemistry Department, Faculty of Women, Ain Shams University, Heliopolis, Cairo, Egypt and 2Basic Science
Department, Faculty of Applied Medical Science, October 6th University, 6 October City, Egypt
Abstract
New compounds incorporating with the oxindole nucleus were synthesized via the reaction of substituted isatins
[5-methyl-, 5-chloro- and 1-hydroxymethyl isatins] with different nucleophiles.The structures of the newly compounds
1
were elucidated on the basis of FTIR, H NMR, 13CMR spectral data, GC/MS and chemical analysis. Investigation of
antimicrobial activity of the new compounds was evaluated using broth dilution technique in terms of minimal
inhibitory concentration (MIC) count against four pathogenic bacteria and two pathogenic fungi. Most of the new
compounds are significantly active against bacteria and fungi. MIC showed that compound (4a) possesses higher
effect on Gram-positive bacteria Bacillus cereus than the selected antibacterial agent sulphamethoxazole, whereas
compound (11c) possesses more activity against Gram-negative bacteria Shigella dysenterie.
Keywords: Substituted isatins, antimicrobial, antifungal gents, minimal inhibitory concentration (MIC), oxindoles
Introduction
agents and natural alkaloids such as spiroindole-
thiazolidinones showed good activity against the
pathogens, Rhizoctonia solani, Fusarium oxysporum
and Collectotrichum capsici. Also, spirotryprostatins
have been found to have anti-mitotic properties, and as
such they have become of great interest as anticancer
drugs and also were shown to be active as cell cycle
inhibitor and pteropodine and isopteropodine, which
are heteroyohimbine-type oxindole alkaloid, act as
positive modulators of muscarinic M(1) and 5-HT(2)
Oxindoles have a broad range of pharmacological
actions, being described as anxiogenic and sedative
agents1,2 or antagonists of guanylate cyclase-coupled
atrial natriuretic peptide receptor3. Schiff bases and
Mannich bases of isatin were reported to possess
antibacterial4–6, antifungal7–9, anti-TB10–12, anti-HIV13–15
,
analgesic16 antiviral17 and anticancer18 activities.
Spiro compounds represent an important class
of naturally occurring substances characterized by
interesting biological properties19,20. Furthermore, it
has been reported that sharing of the indole 3-carbon
atom in the formation of spiroindoline derivatives
highly enhances biological activity for some Gram-
positive (Bacillus subtilis and Bacillus megatherium),
Gram-negative (Escherichia coli) and fungi (Aspergillus
niger and Aspergillus oryzae)21–23. e spirooxindole
system is the core structure of many pharmacological
receptors24–28
.
In continuation of our studies on the synthesis and
evaluation of biological activity of heterocycles, we
report herein the efficient synthesis of new biologi-
cally active compounds based on oxindole nucleus and
evaluate its antimicrobial activity using broth dilution
technique in terms of minimal inhibitory concentration
(MIC) count.
Address for Correspondence: Nadia G. Kandile, Chemistry Department, Faculty of Women, Ain Shams University, Heliopolis, 11757, Cairo,
Egypt. Tel. and Fax: +2024157804. E-mail: nadiaghk@yahoo.com
(Received 04 March 2011; revised 24 March 2011; accepted 25 March 2011)
599

600 N. G. Kandile et al.
ethanol (20 mL) containing a few drops of glacial acetic
acid for 8 h. After cooling to room temperature, red nee-
dles obtained were filtered and crystallized from ethanol
to give compound 3.
Materials and methods
Chemistry
All melting points are uncorrected and were determined
on Gallenkamp instrument. Infrared spectra of the new
compounds were measured on Perkin-Elmer spectro-
photometer model 1430 using potassium bromide pel-
3-(1H-Benzoimidazol-2-ylimino)-5-methyl indolin-2-one) (3)
Red crystals, 74% yield, mp 205°C, IR (KBr pellet):
3408–3235 for (NHimidazole, NHoxindole), 1710 for (C=O) and
lets and frequencies are reported in cm−1. e H NMR
1
were measured on Varian genini-300 MHZ spectropho-
tometer and chemical shifts (δ) are in ppm. e mass
spectra (m/z) values were measured on mass spectro-
photometer HP model GC MS-QPL000EX (Shimadzu)
at 70 eV. Elemental analyses were carried out at the
Microanalytical Centre, Cairo University. Antimicrobial
activity evaluation were carried out at the Basic Science
Department, Faculty of Applied Medical Science, October
6th University, October City, Egypt. Explorer Automated
Microwave Synthesis Workstation (CEM) was used for
synthesis of compounds.
1
1622, 1651 for (C=N) cm−1. H NMR (DMSO, 300 MHz):
δ 2.49 (s, 3H, CH3), 6.71–7.40 (m, 7H, 2Ar-H), 8.78 (s, 1H,
NHimidazole) and 10.90 (s, 1H, NHoxindole) ppm; Anal. Calcd.
for C16H12N4O (276.29), C, 69.55; H, 4.38; N, 20.28; found:
C, 69.25; H, 4.39; N, 20.00; m/z
Preparation of N-(5-methylisoxazol-3-yl)-4-
(5-substituted 2-oxindolin-3-ylideneamino)
benzenesulphonamides (4a,b) and 4-[(1-
hydroxymethyl)-2-oxindolin-3-ylideneamino]-N-(5-
methylisoxazol-3-yl) benzenesulphonamide (4c)
General procedure
Synthesis of 2-(5-substituted 2-oxindolin-3-
ylideneamino) isoindolin-1,3-diones (2a,b)
General procedure
A mixture of 5-substituted isatins or 1-hydroxymethyli-
satin (0.01 mol) 1a–c and 4-amino-N-(5-methyl isoxazol-3
-yl) benzene sulphonamide (0.01 mol) in ethanol (20 mL)
was refluxed for 8 h. After cooling to room temperature,
the solid product obtained was filtered and crystallized
Method (A): A mixture of 5-substituted isatin 1a,b
(0.01 mol) and N-aminophthalimide (0.01 mol) in abso-
lute ethanol (30 mL) was refluxed for 30 min. e solid
product formed during heating was filtered and crystal-
lized from ethanol to give 2a,b.
Method (B): A mixture of 5-substituted isatin 1a,b
(0.01 mol) and N-aminophthalimide (0.01 mol) in a little
amount of ethanol made as a slurry was irradiated with
MW (temperature 130°C, pressure 250 psi , time 1min).
e solid product obtained after cooling was filtered and
from ethanol to give 4a–c
.
N-(5-Methylisoxazol-3-yl)-4-(5-methyl-2-oxindolin-3-
ylideneamino) benzenesulphonamide (4a)
Red crystals, 78% yield, mp 150°C; IR (KBr pellet):
3469–3180 for (NHsulphonamide, NHoxindole), 1718 for (C=O),
1623 for (C=N) and 1362 for SO2 cm−1. 1H NMR (DMSO,
300 MHz): δ 2.47–2.48 (s, 6H, 2CH3), 6.05 (s, 1H, CH),
crystallized from ethanol to give 2a,b
.
6.54–7.40 (m, 7H, 2Ar-H), 10.85 (s, 1H, NHoxindole
)
and 10.88 (s, 1H, NHsulphonamide) ppm; Anal. Calcd. for
C19H16N4O4S (396.41); N, 14.13; S, 8.09; found: N, 13.90;
S, 8.13; m/z 397 (M++1).
2-(5-Methyl-2-oxindolin-3-ylideneamino) isoindoline-1,3-
dione (2a)
Orange crystals, 86% yield, mp 269°C; IR (KBr pellet):
3275 for (NH), 1740–1720 for (C=O1,3dione, C=Ooxindole) and
N-(5-Methylisoxazol-3-yl)-4-(5-chloro-2-oxindolin-3-
ylideneamino) benzene sulphonamide (4b)
1
1603 for (C=N) cm−1. H NMR (DMSO, 300 MHz): δ 2.49
(s, 3H, CH3), 6.80–7.90 (m, 7H, 2Ar-H) and 10.9 (s, 1H,
NH) ppm; Anal. Calcd. for C17H11N3O3 (305.28); C, 66.88;
H, 3.63; N, 13.76; found: C, 66.65; H, 3.77; N, 13.77; m/z
(305) (M+).
Orange crystals, 85% yield, mp 230–232°C; IR (KBr pellet):
3463–3168 for (NHsulphonamide, NHoxindole), 1708 for (C=O),
1617 for (C=N) and 1353 for (SO2) cm−1. Anal. Calcd.
for C18H13ClN4O4S (416.83); C, 51.86; H, 3.14; Cl, 8.51; N,
13.44; S, 7.69; found: C, 52.00; H, 3.00; Cl, 8.81; N, 13.45; S,
7.47; m/z 417 (M+).
2-(5-Chloro-2-oxindolin-3-ylideneamino) isoindoline-1,3-
dione (2b)
Orange crystals, 76.7% yield, mp 279–280°C; IR (KBr pel-
4-[(1-Hydroxymethyl)-2-oxindolin-3-ylideneamino]-N-(5-
methylisoxazol-3-yl) benzene sulphonamide (4c)
let): 3264 for (NH), 1730–1710 for (C=O1,3dione, C=Ooxindole
)
and 1602 for (C=N) cm−1. Anal. Calcd. for C16H8ClN3O3
(325.70); C, 59.00; H, 2.48; Cl, 10.88; N, 12.90; found: C,
58.85; H, 2.87; Cl, 10.85; N, 12.70; m/z 328 (M++3).
Orange crystals, 90% yield, mp 215°C; IR (KBr pellet):
3417–3318 for (NHsulphamethoxazole, OH), 1732 for (C=O),
1610 for (C=N) and 1362 for (SO2) cm−1. 1H NMR (DMSO,
300 MHz): δ 2.50 (s, 3H, CH3), 5.13 (s, 2H, CH2 of CH2OH),
6.08 (s, 1H, CH), 6.09 (s, 1H, OH), 6.72–7.70 (m, 8H,
2Ar-H) and 11.01 (s, 1H, NHsulphonamide) ppm; Anal. Calcd.
for C19H16N4O5S (412.41); C, 55.33; H, 3.91; N, 13.58; S,
7.77; found: C, 55.61; H, 4.22; N, 13.45; S, 7.75; m/z 411
(M+−1).
Preparation of 3-(1H-benzoimidazol-2-ylimino)-5-
methyl-indolin-2-one (3)
General procedure
A mixture of 5-methylisatin 1a (0.01 mol) and 1H-ben-
zoimidazol-2-amine (0.01 mol) was refluxed in absolute
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of new compounds incorporating oxindole nucleus 601
Preparation of 2-[(5-substituted 2-oxindolin-3-
ylidene) hydrazono] carbonyl-N-propylbenzamides
(6a,b)
Preparation of 3-[2-(1-phenyl-2-oxoethyl) hydrazono]-
5-substituted indolin-2-ones (9b,c)
General procedure
A mixture of 8b,c (0.01 mol) and phenacyl chloride
(0.01 mol) in absolute ethanol (20 mL) containing a few
drops of triethylamine was refluxed for 6 h. e reaction
mixture was concentrated under reduced pressure. e
oily residue obtained was triturated with petroleum ether
40–60°C and then crystallized from petroleum ether
General procedure
A mixture of 1a,b (0.01 mol) and n-propylamine (0.01 mol)
in absolute ethanol (30 mL) was stirred at room tempera-
ture for 24 h. e solid product obtained was filtered off
and crystallized from benzene to give 6a,b
.
60–80°C to give 9b,c
.
2-[5-Methyl-2-oxindolin-3-ylidene) hydrazono] carbonyl-N-
propylbenzamide (6a)
3-2(1-Phenyl-2-oxoethyl) hydrazono]-5-chloroindolin-2-one
(9b)
Yellow crystals, 74% yield, mp 237–238°C; IR (KBr pellet):
3170–3136 for (NHoxindole, NHamide), 1696–1692 for (C=O)
Brown crystals, 50% yield, mp 236–238°C; IR (KBr pel-
let): 3375–3180 for (NHhydrazono, NHoxindole), 1699–1659 for
(C=Oacetophenone, C=Ooxindole) and 1616 for (C=N) cm−1. Anal.
Calcd. for C16H12ClN3O2 (313.74); Cl, 11.30; N, 13.39;
found: Cl, 11.79; N, 13.39.
1
and 1605 for (C=N) cm−1. H NMR (DMSO, 300 MHz); δ
2.27 (s, 3H, CH3), 2.49 (t, 3H, CH3), 3.40 (t, 2H, CH2), 3.51
(m, 2H, CH2), 6.70–7.90 (m, 7H, 2Ar-H), 8.30, 8.31 (s, 2H,
2NHamide) and 10.30 (s, 1H, NHoxindole) ppm. Anal. Calcd.
for C20H20N4O3 (364.39); C, 65.92; H, 5.53;N, 15.38; found;
C, 66.15; H, 5.75; N, 15.49; m/z 362 (M+−2).
3-[2-(1-Phenyl-2-oxoethyl) hydrazono]-5H-indolin-2-one (9c)
Brown crystals, 54% yield, mp 98°; IR (KBr pellet): 3391–
2-[5-Chloro-2-oxindolin-3-yilidene) hydrazono] carbonyl-N-
propylbenzamide (6b)
3221 for (NHhydrazono, NHoxindole), 1723–1687 for (C=Oacetophenone
,
C=Ooxindole) and 1619 for (C=N) cm−1. 1H NMR (DMSO,
300MHz): δ 4.79 (s, 2H, CH2), 6.80–7.99 (m, 9H, 2Ar-H),
9.70 (s, 1H, NHhydrazono) and 10.86 (s, 1H, NHoxindole) ppm.
Anal. Calcd. for C16H13N3O2 (279.29); C, 68.81; H, 4.69; N,
15.05; found: C, 68.93; H, 4.69; N, 15.16 m/z 278(M+−1).
Yellow crystals, 76.7% yield, mp 227°C; IR (KBr pellet):
3170–3140 for (NHoxindole, NHamide), 1690–1685 for (C=O)
and 1605 for (C=N) cm−1. Anal. Calcd. for C19H17ClN4O3
(384.81); C, 59.30; H, 4.45; Cl, 9.21; N, 14.56; found: C,
59.61; H, 4.51; Cl, 9.30; N, 14.32; m/z 385 (M++1).
Preparation of 3-[2-(2-chloro-1-phenylethylidene)
hydrazono] indolin-2-one (10)
General procedure
A mixture of 8c (0.01 mol) and phenacyl chloride
(0.01 mol) in HCl (2 M, 20 mL) was refluxed for 3 h. e
solid product separated while hot was filtered and crys-
tallized from benzene to give 10.
Preparation of N-(5-substituted 2-oxindolin-3-
ylidene)-2-(piperidin-1-ylcarbonyl) benzohydrazides
(7a,b)
General procedure
A mixture of 1a,b (0.01 mol) and piperidine (0.01 mol) in
absolute ethanol (30 mL) was stirred at room tempera-
ture for 24 h. e solid product obtained was filtered off
3-[2-(2-Chloro-1-phenylethylidene) hydrazono] indolin-2-one
(10)
and crystallized from benzene to give 7a,b
.
Violet crystals, 53% yield, mp 300°C; IR (KBr pellet): 3227
for (NH), 1699 for (C=O) and 1618, 1614 for (C=N) cm−1.
1H NMR (DMSO, 300MHz): δ 5.10 (s, 2H, CH2), 6.82–7.60
(m, 9H, 2Ar-H) and 10.66 (s, 1H, NH) ppm. Anal. Calcd. for
C16H12ClN3O (297.74); C, 64.54; H, 4.06; Cl, 11.91; N, 14.11;
found: C, 64.72; H, 4.30; N, Cl, 12.11, 14.61, m/z 297 (M+).
N-(5-Methyl-2-oxindolin-3-ylidene)-2-(piperidin-1-ylcarbonyl)
benzohydrazide (7a)
Yellow crystals, 65% yield, mp 240°C; IR (KBr pellet):
3190–3166 for (NHoxindole, NHamide), 1701 for (C=O) and
1617 for (C=N) cm−1. 1H NMR (DMSO, 300MHz): δ 2.49 (s,
3H, CH3), 3.30 (m, 6H, 3CH2), 3.51 (t, 4H, 2CH2), 6.90–7.90
(m, 7H, 2Ar-H), 8.29 (s, 1H, NHamide) and 10.30 (s, 1H,
NHoxindole) ppm. Anal. Calcd. for C22H22N4O3 (390.43); C,
67.68; H, 5.68; N, 14.35; found: C, 67.36; H, 5.39; N, 14.09;
m/z 390 (M+).
Preparation of 3-[2-(4-oxopentan-2-ylidene)
hydrazono]-5-substituted indolin-2-ones (11a–c
)
General procedure
A mixture of 8a–c (0.01 mol) and acetylacetone (0.01 mol)
was refluxed in absolute ethanol (20 mL) for 5 h. After
cooling to room temperature, the solid product obtained
N-(5-Chloro-2-oxindolin-3-ylidene)-2-(piperidin-1-ylcarbonyl)
benzohydrazide (7b)
was filtered and crystallized from ethanol to give 11a–c
.
Yellow crystals, 64% yield, mp 236–238°C; IR (KBr pel-
let): 3182–3150 for (NHoxindole, NHamide), 1691 for (C=O)
and 1606 for (C=N) cm−1. Anal. Calcd. for C21H19ClN4O3
(410.85); C, 61.39; H, 4.66; Cl, 8.63; N, 13.64; found: C,
61.59; H, 4.56; Cl, 8.77; N, 13.21; m/z 412 (M++2).
3-[2-(4-Oxopentan-2-ylidene) hydrazono]-5-methyl-indolin-
2-one (11a)
Orange crystals, 79% yield, mp 260–262°C; IR (KBr pellet):
3180 for (NH), 1699 for (C=O) and 1590 for (C=N) cm−1;
© 2012 Informa UK, Ltd.

602 N. G. Kandile et al.
Anal. Calcd. for C14H15N3O2 (257.28) C, 65.35; H, 5.88; N,
16.33; found: C, 65.14; H, 6.12; N, 16.28; m/z 257(M+).
Preparation of spirocyclic compound (13)
General procedure
Method (A): A mixture of compound 12a (0.01 mol) and
thiosemicarbazide (0.01 mol) in absolute ethanol was
refluxed for 6 h, the solid product after cooling was fil-
tered and crystallized from ethanol to give 13.
Method (B): A mixture of compound 12a (0.01 mol)
and thiosemicarbazide (0.01 mol) in a little amount of
ethanol made as a slurry was irradiated with MW (tem-
perature 130°C, pressure 250, time for 1 min).e solid
product obtained after cooling was filtered and crystal-
lized from ethanol to give 13.
3-[2-(4-Oxopentan-2-ylidene) hydrazono]-5-chloroindolin-2-
one (11b)
Orange crystals, 73% yield, mp 280–282°C; IR (KBr pel-
let): 3139 for (NH), 1701 for (C=O) and 1583 for (C=N)
cm−1. Anal. Calcd. for C13H12ClN3O2 (277.70): C, 56.22; H,
4.36; Cl, 12.77; N, 15.13; found: C, 56.02; H, 4.70; Cl, 12.74;
N, 15.02; m/z 277(M+).
3-[2-(4-Oxopentan-2-ylidene) hydrazono]-5H-indolin-2-one
(11c)
Orange crystals, 76% yield, mp 229–230°C; IR (KBr pellet):
3206 for (NH), 1689 for (C=O) and 1625, 1590 for (C=N)
cm−1. 1H NMR (DMSO, 300 MHz): δ 2.07 (s, 3H, CH3), 2.20
(s, 3H, CH3), 5.51 (s, 2H, CH2), 6.87–7.46 (m, 4H, Ar-H)
and 10.98 (s, 1H, NH) ppm. Anal. Calcd. for C13H13N3O2
(243.26); C, 64.19; H, 5.39; N, 17.27; found: C, 63.94; H,
5.64; N, 17.16, m/z 243(M+).
Spiro compound (13)
Yellow crystals, 78% yield, mp 216°C; IR (KBr pellet):
3395–3225 for (OH, NH), 3216–3172 for (NH of NH2),
1690 for (C=O) and 1592 for (C=N) cm−1. 1H NMR
(DMSO, 300 MHz) δ 2.25 (s, 3H, CH3), 3.38 (s, 3H,
OCH3), 6.70–7.17 (m, 6H, 2Ar-H), 7.70 (s, 1H, CH of
CH=N), 8.68 (s, 1H, NHtriazole), 9.42, 9.46 (s, 2H, NH2),
10.15 (s, 1H, OH) and 10.54 (s, 1H, NHoxindole) ppm.
13CMR (DMSO) δ 162.9, 136.4 130.1, 129.8, 128.8, 127.5,
126.4, 123.4, 122.2, 120.7, 117.9, 109.6, 109.2, 40.3, 40.0,
39.4, 20.7. Anal. Calcd. for C18H18N6O3 (366.37); C, 59.01;
H, 4.95; N, 22.94; found: C, 59.25; H, 4.74; N, 22.63; m/z
368 (M++2).
Preparation of 3-[2-(4-hydroxy-3-
methoxybenzylidene) hydrazono]-5-substituted
indolin-2-ones (12a–c
)
General procedure
A mixture of 8a–c (0.01 mol) and vanillin (0.01 mol) was
refluxed in absolute ethanol (20 mL) for 5 h. e solid
product after cooling was filtered and crystallized from
ethanol to give 12a–c
.
Biological activity evaluation
In vitro antimicrobial activity measurement
Primary screening
3-[2-(4-Hydroxy-3-methoxybenzylidene) hydrazono]-5-
methyl-indolin-2-one (12a)
Most of the newly synthesized compounds were
screened for their antibacterial and antifungal activities
using the agar well diffusion technique29. e microor-
ganisms (reference and clinical isolates) used include
Gram-negative E. coli (ATCC-25922) and Shigella dys-
enterie, Gram-positive Staphylococcus aureus (ATCC-
25923) and Bacillus cereus, fungi Aspergillus flavus and
Candida albicans (ATCC 10231). For the antibacterial
assay, a standard inoculum (105 CFU/mL) was distrib-
uted on the surface of sterile nutrient agar plates by a
sterile glass spreader, whereas for the antifungal assay
a loopful of a particular fungal strain was transferred
to 3 mL saline to get a suspension of the corresponding
species; 0.1 mL of the spore suspension was distributed
on the surface of sterile Sabouraud dextrose agar plates.
Six millimetre diameter wells were punched in the agar
media and filled with 100 µL (500 µg/mL in DMSO) of
the tested chemical compounds previously sterilized
through 0.45 sterile membrane filter30. e plates were
kept at room temperature for 1 h and then incubated at
37°C for 24 h for bacteria and 30°C for 4 days for fungi.
e antimicrobial activities were evaluated by measur-
ing the inhibition zone diameters. Commercial antibi-
otic discs were used as positive reference standard to
determine the sensitivity of the strains (Table 1).
Orange crystals, 85% yield, mp 270°C; IR (KBr pellet): 3400–
3204 for (OH, NH), 1719 for (C=O) and 1615 for (C=N).
Anal. Calcd. for C17H15N3O3 (309.31); C, 66.01; H, 4.89; N,
13.58; found: C, 65.90; H, 5.22; N, 13.47, m/z 311(M+2).
3-[2-(4-Hydroxy-3-methoxybenzylidene) hydrazono]-5-
chloroindolin-2-one (12b)
Orange crystals, 64% yield, mp 290°C; IR (KBr pellet):
3380–3185 for (OH, NH), 1713 for (C=O) and 1613 for
1
(C=N) cm−1. H NMR (DMSO, 300 MHz): δ 3.87 (s, 3H,
OCH3), 6.82–8.10 (m, 6H, 2Ar-H), 8.62 (s, 1H, CH of
CH=N), 10.10 (s, 1H, OH) and 10.91 (s, 1H, NH) ppm.
Anal. Calcd. for C16H12ClN3O3 (329.73); C, 58.28; H, 3.67;
Cl, 10.75; N, 12.74; found: C, 58.34; H, 3.82; Cl, 10.58; N,
12.56; m/z 329(M+2).
3-[2-(4-Hydroxy-3-methoxybenzylidene) hydrazono]-5H-
indolin-2-one (12c)
Orange crystals, 79% yield, mp 278°C; IR (KBr pellet):
3436–3251 for (OH, NH), 1728 for (C=O) and 1619 for
1
(C=N) cm−1. H NMR (DMSO, 300 MHz): δ 3.92 (s, 3H,
OCH3), 6.78–7.90 (m, 7H, 2Ar-H), 8.58 (s, 1H, CH of
CH=N), 10.13 (s, 1H, OH) and 10.68 (s, 1H, NH) ppm.
Anal. Calcd. for C16H13N3O3 (295.29); C, 65.08; H, 4.44; N,
14.23; found: C, 64.81; H, 4.47; N, 14.29; m/z 297 (M+).
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of new compounds incorporating oxindole nucleus 603
required to arrest the microbial growth was regarded as
MICs and are given in Table 2.
Determination of MIC of the synthesized compounds
Compounds inhibiting the growth of one or more of the
above microorganisms were further tested for their MIC
and were determined by broth dilution technique31. e
nutrient broth and the yeast extract broth media, which
contained 1 mL of different concentrations of the tested
compounds (5, 10, 15, 20, 25 µg/mL), were inoculated
with the microbial strains; the bacterial cultures were
incubated for 24 h at 37°C, whereas the fungal ones
were incubated at 30°C for 48 h; the growth was moni-
tored spectrophotometrically. e lowest concentration
Results and discussion
Chemistry
We investigated earlier the reaction of isatin,
N-aminophthalimide or pyridazines as key start-
ing materials for the synthesis of biologically active
compounds32–40
. In the present work, we studied
the reaction of 5-substituted isatins and 1-hydroxy
Table 1. Antimicrobial screening results of the tested compounds.
Inhibition zone diameter (mm)
Compounds (50 µg/mL)
E. coli
21
23
27
23
32
20
21
17
25
23
21
20
24
22
19
S. dysenterie
S. aureus
20
B. cereus
21
A. flavus
19
C. albicans
2a
20
25
27
23
30
19
23
20
25
28
28
25
27
25
20
20
20
21
22
21
19
18
18
19
21
20
19
23
23
3
4a
22
23
19
31
25
19
4b
31
32
20
5
6a
29
25
19
16
20
17
6b
17
23
18
7b
19
19
16
9b
26
22
18
11a
21
19
20
11b
18
19
18
11c
18
18
18
12a
23
23
21
12b
25
19
22
13
15
18
15
a
17
a
Sulphamethoxazole (10 µg/mL)
Fluconazole (10 µg/mL)
aNot tested.
38
a
35
a
30
a
38
a
25
27
Table 2. MIC (µg/mL) results of the tested compounds.
MIC values (µg/mL)
Compounds
E. coli
10
10
5
S. dysenterie
S. aureus
B. cereus
A. flavus
15
C. albicans
2a
10
10
5
10
10
5
10
10
1
15
15
15
5
3
4a
15
15
4b
5
10
5
10
5
5
10
5
6a
5
10
15
15
10
10
10
10
15
10
10
10
10
10
5
10
10
15
10
10
10
15
10
10
10
15
15
10
15
5
15
15
15
15
10
10
15
10
10
10
10
6b
20
15
15
15
10
10
15
10
10
7b
20
9b
20
11a
10
11b
5
10
11c
1
10
12a
10
10
15
15
12b
15
13
15
a
10
a
Sulphamethoxazole (10 µg/mL)
Fluconazole (10 µg/mL)
2.5
a
2.5
a
2.5
a
2.5
a
2.5
2.5
© 2012 Informa UK, Ltd.

604 N. G. Kandile et al.
methylisatin (1a–c) with different amines, such as
ylideneamino) isoindolin-1,3-diones (2a,b) were reacted
with benzylamine, they underwent ring cleavage to give
N,N′-dibenzylphthalamide (5), as expected (mp, mixed
melting point with authentic sample)33,34, in addition
to 3-hydrazonoindolin-2-ones (8a,b) as by-product. e
structure of (8a,b) were confirmed by mp (mixed melt-
ing point with authentic sample)42–44. However, reaction
of (2a,b) with n-propylamine and piperidine took place
smoothly via ring opening of the phthalide moiety to
give the corresponding 2-[(5-substituted 2-oxindolin-
3-ylidene) hydrazino] carbonyl-N-propylbenzamides
(6a,b) and N-(5-substituted 2-oxindolin-3-ylidene)-2-
(piperidin-1-ylcarbonyl) benzohydrazides (7a,b), respec-
tively. e new compounds (6a,b and 7a,b) have been
characterized by standard procedures such as elemental
analysis, IR and NMR spectroscopy and MS, which con-
formed their structure (cf. experimental part). Scheme 2
illustrates the synthetic pathway for target compounds
(5, 6 and 7).
e present study was extended to synthesize new
biologically active oxindole derivatives using 3-hy-
drazono-5-substituted indolin-2-ones (8a–c) as start-
ing materials. us, reaction of (8a–c) with phenacyl
chloride was investigated at various pH values. When
isatin-3-hydrazones (8b,c) reacted with phenacyl chlo-
ride in boiling ethanol and in the presence of catalytic
amount of triethylamine afforded 3-[2-(1-phenyl-2-
oxoethyl) hydrazono]-5-substituted indolin-2-ones
N-aminophthalimide,
1H-benzodimidazol-2-amine
and sulphamethoxazole. It was reported by Popp41 that
the reaction of N-aminophthalimide with isatin and
5-chloro isatin gave the corresponding ylideneamino
derivatives. In the present study, we reported the
microwave assists reaction of 5-methyl, 5-chloro isatins
(1a,b) with N-aminophthalimide to give 2-(5-substituted
2-oxindolin-3-ylideneamino)
isoindolin-1,3-diones
(2a,b). e structures of (2a,b) were confirmed by ele-
1
mental analysis: FTIR, H NMR and MS (cf. Materials
and methods). However, refluxing 5-methyl isatin (1a)
with 3-(1H-benzoimidazol-2-amine) in ethanol led to
the formation of 3-(1H-benzoimidazol-2-ylimino)-5-
methyl indolin-2-one (3). Sulphamethoxazole reacted
with (1a–c) in refluxing absolute ethanol to afford N-(5-
methylisoxazol-3-yl)-4-(5-substituted 2-oxindolin-3
ylideneamino) benzenesulphonamides (4a,b) and 4-(1-
hydroxymethyl)-2-oxindolin-3-ylideneamino)-N-(5-
methylisoxazol-3-yl) benzenesulphonamide (4c). e
target compounds (2–4) were prepared as depicted in
Scheme 1.
IncontinuationofourinterestinN-aminophthalimide
chemistry, with a view directed towards preparing bio-
logically active compounds33,35, in the present article,
we studied the reaction of 2-(5-substituted 2-oxindo-
lin-3-ylideneamino) isoindolin-1,3-diones (2a,b) with
different amines. When 2-(5-substituted 2-oxindolin-3-
O
N
N
R
O
i
O
N
H
(2a,b
)
N
O
N
H
N
R
H3C
ii
O
O
N
R'
N
H
(1a-c
)
(3)
R
R'
O
S
O
a = CH3
b = Cl
c = H
H
H
NH
N
CH2OH
N
R
iii
O
CH3
O
N
R'
(4a-c
)
Scheme 1. Synthesis of the target compounds 2a,b, 3 and 4a–c. (i) N-Aminophthalimide, (ii) 1H-benzoimidazol-2-amine and (iii) 4-amino-
N-(5-methylisoxazol-3-yl) benzenesulphonamide.
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of new compounds incorporating oxindole nucleus 605
CONHCH2Ph
i
CONHCH2Ph
(5)
O
O
N
NH
O
O
N
N
HN
O
R
R
ii
CH3
O
N
H
N
H
(2a,b
R
)
(6a,b
)
a = CH3
b = Cl
NH
O
N
R
O
O
iii
N
N
H
(7a,b
)
Scheme 2. Synthetic pathway for target compounds 5, 6a,b and 7a,b. (i) Benzylamine, (ii) n-propylamine and (iii) piperidine.
(9b,c), on the other hand, it has been found that isatin-3
-hydrazone (8c) behaved differently on adding 2 M HCl
to the reaction condition, the product was formulated
as 3-[2-(2-chloro-1-phenylethylidene) hydrazono]
indolin-2-one (10). Moreover, the reaction of isatin-3
-hydrazones (8a–c) with dicarbonyl compound such as
acetylacetone in boiling ethanol afforded 3-[2-(4-oxo-
pentan-2-ylidene) hydrazono]-5-substituted indolin-
Biological evaluation (in vitro antimicrobial
measurement)
Most of the synthesized compounds were tested for
their in vitro antimicrobial activity by the broth dilution
technique in terms of MIC. e MICs of the compounds
against six pathogenic microbial species are present in
Table 2. e study also included the activity of reference
compounds sulphamethoxazole as antibacterial agent
and fluconazole as antifungal agent. From the data
of primary antimicrobial screening followed by MICs
count in Tables 1 and 2, the following conclusion can be
drawn:
2-ones (11a–c). Reaction of isatin-3-hydrazones (8a–c
)
with 3-methoxy-4-hydroxybenzaldehyde in boiling
ethyl alcohol it afforded 3-[2-(4-hydroxy-3-methoxy
benzylidene) hydrazono]-5-substituted indolin-2-ones
(12a–c).
Finally, spirocyclic compound (13) was synthesized
via spiro annelation reaction of 3[2-(4-hydroxy-3-meth-
1. E. coli: Compounds N,N′-dibenzylphthalamide (5)
and
N-(5-methylisoxazol-3-yl)-4-(5-substituted
2-oxindolin-3-ylideneamino)
benzenesulphon-
oxybenzylidene)
hydrazono]-5-methyl-indolin-2-one
amides (4a,b) are the most active compounds compa-
rable with other tested compounds.
2. S. dysenterie: Compound 3-[2-(4-oxopentan-2
(12a) with thiosemicarbazide. e spiro compound was
achieved either by the usual thermal method or by using
microwave irradiation. Scheme 3 illustrates the synthetic
pathway for target compounds (9–13).
-ylidene)
hydrazono]-5H-indolin-2-one
(11c)
is the most potent compound and showed sig-
nificant activity comparable with the standard
sulphamethoxazole.
e proposed mechanism for the synthesis of the spi-
rocyclo derivative (13) is described in Scheme 4, and it
involves a nucleophilic attack of the amino electrons of
thiosemicarbazide at the electrophilic indolyl carbon,
accompanied by the migration of a hydrogen atom to
form an intermediate thiol derivative, followed by cycl-
ization and desulphurization with the loss of H2S to give
the suggested spirocyclic compound (13).
3. S. aureus: Compounds N,N′-dibenzylphthalamide
(5) and N-(5-methylisoxazol-3-yl)-4-(5-methyl-2-
oxindolin-3-ylideneamino) benzenesulphonamide
(4a) showed the highest activity against S. aureus as
well as against E. coli strains.
© 2012 Informa UK, Ltd.

606 N. G. Kandile et al.
O
Ph
(9b,c)
NH
O
N
R
EtOH
TEA
N
H
i
Ph
Cl
N
O
2M HCl
N
N
(10)
H
CH3
NH2
O
N
ii
O
N
R
N
H
N
N
CH3
R
O
(8a-c)
R, a = CH3
b = Cl
H
(11 )
a-c
c = H
OCH3
OH
N
N
iii
R
O
(12a-c)
iv
N
EtOH
H
NH2
N
N
HN
N
H3C
O
N
OCH3
H
OH
(13)
Scheme 3. Synthetic pathway for target compounds 9b,c, 10, 11a–c, 12a,b and 13. (i) Phenacyl chloride, (ii) acetylacetone, (iii) 4-hydroxy-3-
methoxybenzaldehyde (vanillin) and (iv) thiosemicarbazide.
4. B. cereus: Compound N-(5-ethylisoxazol-3-yl)-4-
Conclusion
(5-methyl-2-oxindolin-3-ylideneamino) benzene-
In this study, we reported a convenient route for the
sulphonamide (4a) was the most active compound
synthesis of some new compounds incorporating with
as with species (1 and 3) and more active than the
the oxindole nucleus starting from substituted isatins
[5-(methyl, chloro) and 1-hydroxymethyl isatins] and
standard sulphamethoxazole.
5. A. flavus: All the tested compounds nearly have the
investigated their antimicrobial and antifungal activities.
same activity.
e in vitro evaluation of their antimicrobial against sev-
6. C. albicans: Compounds N-(5-methylisoxazol-3-yl)-
eral pathogenic bacterial and fungal strains revealed that
4-(5-chloro-2-oxindolin-3-ylideneamino) benzene
compounds N-(5-methylisoxazol-3-yl)-4-(5-substituted
sulphonamide (4b) and 2-[5-methyl-2-oxindolin-3-
2-oxindolin-3-ylideneamino)
(4a,b) are more active comparable with other synthesized
oxindole derivatives against bacterial strains. is higher
benzenesulphonamides
ylidene) hydrazono] carbonyl-N-propylbenzamide
(6a) were the most potent as antifungal comparable
with other tested compounds.
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of new compounds incorporating oxindole nucleus 607
S
HN
NH2
NH2
OCH3
OH
+
SH2
N
NH2
N
N
N
O
H3C
H3C
N
NH
O
N
OCH3
OH
N
H
H
(12a)
- H2S
N
NH2
N
HN
H3C
N
O
N
H
OCH3
OH
(13)
Scheme 4. Mechanism for the synthesis of target compound 13.
6. Varma RS, Nobles WL. Antiviral, antibacterial, and
antifungal activities of isatin N-Mannich bases. J Pharm Sci
1975;64:881–882.
7. PandeyaSN,SriramD,NathG,DeClercqE.Synthesis,antibacterial,
antifungal and anti-HIV activity of Schiff and Mannich bases of
isatin with N-[6-chlorobenzothiazol-2-yl]thiosemicarbazide.
Indian J Pharm Sci 1999;61:358–361.
8. PandeyaSN,SriramD,NathG,DeClercqE.Synthesis,antibacterial,
antifungal and anti-HIV evaluation of norfloxacin Mannich bases.
Sci Pharm 1999;67:103–111.
9. Pandeya SN, Sriram D, Nath G, De Clercq E. Synthesis,
antibacterial, antifungal and anti-HIV evaluation of Schiff
and Mannich bases of isatin derivatives with 3-amino-2-
methylmercapto quinazolin-4(3H)-one. Pharm Acta Helv
1999;74:11–17.
activity may be attributed to benzenesulphonamide moi-
ety. e data show that derivative (4b) with the chlorine
atom at C-5 position in the oxindole nucleus was more
potent as antifungal agent than the (4a) derivative with the
methyl group. Compound (5) showed against all tested
bacterial strains exerted excellent antibacterial activity. e
data of MICs count also indicated that compounds (11c)
and (4a) showed significant activity against S. dysenterie
and B. cereus bacterial species, respectively, comparable
with standard sulphamethoxazole and may serve as useful
lead compounds in search for potent antibacterial agent.
Declaration of interest
10. Sriram D, Yogeeswari P, Gopal G. Synthesis, anti-HIV and
antitubercular activities of lamivudine prodrugs. Eur J Med Chem
2005;40:1373–1376.
11. Patole J, Sandbhor U, Padhye S, Deobagkar DN, Anson CE, Powell
A. Structural chemistry and in vitro antitubercular activity of
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against Mycobacterium smegmatis. Bioorg Med Chem Lett
2003;13:51–55.
eauthorsreportnoconflictofinterest. eauthorsalone
are responsible for the content and writing of the article.
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