FULL PAPER

DOI: 10.1002/ajoc.201300153

Synthesis and Fluorescence Properties of Isatin-Based Spiro Compounds:

Switch off Chemosensing of Copper(II) Ions
Ashis Kundu, Sudipta Pathak, and Animesh Pramanik*[a]
Abstract: A series of isatin-based
spiro compounds 2,7-diamino-2-oxo1,4-dihydrospiro[indoline-3,4-quinoline]-3-carbonitriles have been synthesized by heating mixtures of isatins,
malononitrile, and meta-phenylenediamine to reflux in ethanol. Some of the
compounds have high fluorescence
quantum yields (FF = ca. 0.8) and long
fluorescence lifetimes (t = ca. 9.0 ns)
in the polar solvent acetone. The
value of FF decreases with increasing

polarity of the solvents in the sequence acetone, CH3CN, N,N-dimethylformamide, and MeOH. All of the
compounds fluoresce in a weak basic
medium, such as triethylamine, but do
not emit in acetic acid. Efficient and
selective
fluorescence
quenching
occurs for Cu2 + ions in solution
through complexation, which can be
used for detection of Cu2 + ions in the
presence of several other metal ions.
Preliminary studies show that these

Introduction

isatin-based spiro compounds can be

used as fluorescence probe for bioimaging of human squamous epithelium and peripheral blood mononuclear
(PBM) cells and also for detecting
Cu2 + ions in living cells.

Keywords: chemosensors copper

isatins multicomponent reactions
spiro compounds

Copper, the third most abundant essential trace element in

the human body after iron and zinc, plays a critical role in
many biological processes and is also required by the
human nervous system.[8] However, high levels of copper
ions in living cell leads to neurodegenerative diseases, such
as Alzheimers and Wilsons diseases.[9] Thus, the sensing
and recognition of divalent copper ions have attracted considerable attention in recent years[10] and still there is
a urgent need for development of new fluorescent sensors
for the detection of copper ions, especially inside living
cells.

In recent years, the versatile reactivity of isatin has been exploited extensively in multicomponent reactions for the
synthesis of diverse biologically active heterocyclic compounds.[1] A number of isatin-based spiro compounds are
important pharmacophores and have promising biological
activities, such as antimicrobial,[2a] antifungal[2a,b] and antitubercular[2c] activities. Moreover the spirooxindole systems,
the core structure of many pharmacological agents and natural alkaloids[3, 4] have superior biological activities and,
therefore, have prompted many efforts toward their synthesis.[5, 6] Nevertheless, the search for simple and efficient
methods for the synthesis of new classes of isatin based
spiro compounds is important for exploring their biological,
biophysical, and photophysical properties.
Although isatin-based molecules are biologically important, there have been only a very few reports of isatin
based fluorescent compounds to date.[7] Herein, we report
the development of a new class of isatin-based spiro compounds with excellent fluorescence properties. They also
have diverse kinds of photophysical properties, such as selective chemosensing of Cu2 + ions, pH dependent fluorescence, and significantly different fluorescence quantum
yields and lifetimes depending upon the solvent polarity.

Results and Discussion

Synthesis
During our laboratory efforts on the development of multicomponent reactions for synthesis of biologically important
heterocyclic compounds,[11] we discovered that heating
a mixture of isatin 1, malononitrile 2, and meta-phenylenediamine 3 to reflux in ethanol produces the isatin-based
spiro compound 2,7-diamino-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]-3-carbonitrile 4 a in high yield within
one hour (Scheme 1, Table 1). Under similar reaction conditions, isatins containing 5-halo substituents and N-alkyl/

[a] A. Kundu, S. Pathak, Dr. A. Pramanik

Department of Chemistry, University of Calcutta
92, A. P. C. Road, Kolkata-700009 (India)
Fax: (+ 91) 33-2351-9755
E-mail: animesh_in2001@yahoo.co.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/ajoc.201300153.

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Scheme 1. Synthesis of 2,7-diamino-2-oxo-1,4-dihydrospiro[indoline3,4-quinoline]-3-carbonitriles 4.

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Table 1. Synthesis of compounds 4.

Entry

R1

R2

Product

t [min]

Yield [%][a]

1
2
3
4
5
6
7
8
9
10
11
12

H
H
H
H
H
Me
Me
Me
Me
Et
Et
Ph

H
F
Cl
Br
I
H
F
Cl
I
H
Cl
H

4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l

60
45
30
30
45
90
75
60
75
120
120
60

91
87
90
81
83
76
71
72
74
75
70
74
Scheme 2. Mechanistic pathway for the formation of compounds 4.

[a] Yield of isolated product.

addition to afford intermediate 6. After that, intermediate

6 undergoes intramolecular cyclization, and subsequent tautomerisation to give the final products 4.

aryl substituents also react smoothly with malononitrile 2

and meta-phenylenediamine 3 to generate corresponding
spiro derivatives 4 bl in moderate-to-good yields (Table 1).
All the products were fully characterized by 1H and
13
C NMR spectroscopy. Determination of the X-ray crystal
structures of compound 4 k (Figure 1) and 4 a (see Figure S1

Photophysical Properties
Solvent-Dependency Studies
The photophysical properties of the synthesized spiro[indoline-3,4-quinoline]-3-carbonitriles 4 were investigated in
a variety of solvents with increasing polarity in the sequence acetone, CH3CN, N,N-dimethylformamide (DMF),
and MeOH (Table 2, Figure 2). For all the compounds, the
emission maxima shifted to the red wavelength region with
increasing solvent polarity. The most bathochromic emission was found in methanol. Concurrently, the fluorescence
quantum yields (FF) decreased with increasing solvent polarity, which may be because of the solvation. The highest
FF values were measured in acetone, and the lowest were
measured in protic polar methanol. The substantial fluorescence quenching of the compounds 4 in the protic solvent
methanol compared with that in CH3CN and DMF may be
attributed to extensive solvation of 4 through intermolecular hydrogen bonding.
Interestingly for compounds 4 fl, in which the N1 atom
of the isatin moiety is either alkylated or arylated (R1), the

Figure 1. ORTEP diagram of 4 k with atom numbering scheme. Thermal

ellipsoids are shown at the 50 % probability (CCDC 903275).

in the Supporting Information) further confirmed the

product formation.[12]
A plausible mechanistic
path way for the formation
of compounds 4 is depicted
in Scheme 2. The process
constitutes a cascade of reactions with an initial Knoevenagel condensation between
isatin 1 and malononitrile 2
to furnish isatylidene malononitrile 5. Subsequently 5
reacts with meta-phenylenediamine 3 in a Michael-type

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Figure 2. a) UV/vis absorption spectra and b) fluorescence emission spectra of representative compound 4 c in
different solvents.

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have significantly lower t values in acetone. Interestingly

crystals of 4 were also fluorescent when observed under
a fluorescence microscope (see Figure S13 in the Supporting information).

Table 2. Spectroscopic data of 4 in different solvents.

4

Solvent

lmax [nm]

lem [nm]

FF[b]

t [ns]

4a

acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH
acetone
CH3CN
DMF
MeOH

418
420
413
427
417
412
428
421
420
414
431
423
418
414
429
422
417
414
430
422
424
420
434
428
417
413
419
408
416
410
412
409
416
406
433
398
423
424
432
424
423
420
433
424
422
419
423
412

509
515
525
530
516
518
529
534
515
519
529
534
516
519
526
533
516
520
528
534
515
521
530
530
518
519
526
530
518
521
528
521
501
502
518
519
516
519
527
528
515
516
528
532
518
519
519
523

0.833
0.389
0.194
0.090
0.618
0.564
0.288
0.096
0.589
0.316
0.433
0.099
0.880
0.326
0.320
0.094
0.791
0.324
0.364
0.112
0.247
0.117
0.115
0.050
0.006
0.004
0.002
0.002
0.013
0.004
0.003
0.003
0.009
0.014
0.002
0.004
0.047
0.015
0.011
0.010
0.150
0.075
0.033
0.024
0.0058
0.0056
0.0041
0.0040

9.04

4b

4c

4d

4e

4f

4g

4h

4i

4j

4k

4l

8.74

Effect of Different Metal Ions

9.35

The UV/vis absorption spectra of representative compound

4 c and also 4 c in presence of 100 equivalents of various
metal perchlorate salts in acetonitrile were measured
(Figure 3). The excess metal cations were used to prove the

9.22

8.85

4.31

3.75

2.41

Figure 3. UV/vis absorption spectra of 4 c in the presence of 100 equivalents of metal ions in acetonitrile; [4 c] = 10 4 m.

3.01

specific binding of Cu2 + ions and the lack of binding ability

of other cations. Clearly no significant changes in lmax
values were detected when 100 equivalents of Na + , Mn2 + ,
Co2 + , Ni2 + , Zn2 + , Cd2 + , Hg2 + , and Pb2 + ions were added
to the solution of 4 c in acetonitrile. However, on addition
of CuACHTUNGRE(ClO4)2, the two absorption peaks at 294 and 415 nm
were found to shift to 367 and 500 nm respectively. This significant red shift clearly indicates specific binding of Cu2 +
ions with 4 c to form a complex.
As fluorescence emission spectroscopy is more sensitive
than UV/vis spectroscopy toward small changes, fluorescence spectroscopy experiments were also performed to
assess the selectivity of 4 c towards various metal ions.
Figure 4 shows the fluorescence response of 4 c in acetonitrile upon the addition of different metal cations. The emission spectra of free 4 c in acetonitrile has only one band at
519 nm. The fluorescence intensity of the band at 519 nm
decreased substantially with the addition of Cu2 + ions,
which indicates fluorescence quenching. However, no appreciable changes were detected upon addition of other
metal cations, such as Zn2 + , Co2 + , Ni2 + , Mn2 + , Cd2 + , Hg2 + ,
and Pb2 + . Only a small extent of fluorescence quenching
was observed on addition of Na + ion, which was not detectable in UV/vis spectroscopy. Binding of the ligand 4 c with
Cu2 + ions was accompanied by a change in color of the solution from light green to brown, which was easily observa-

4.55

4.13

4.61

[b] Determined with reference to coumarin 153 in ethanol (FF = 0.54).[13]

FF values were significantly lower in all of the solvents. On

the other hand, the FF values of compounds 4 be with 5halo R2 substituents F, Cl, Br, and I, respectively, were not
significantly different compared with 4 a. Probably the replacement of N1 H bonds with N1 C bonds in the N-alkylated/arylated compounds 4 fl causes more nonradiative
decay resulting in lower FF values. As compounds 4 a
e have high FF values in acetone, the fluorescence lifetimes
(t) were measured in the same solvent. The results show
that 4 ae also have long fluorescence lifetimes in acetone
(Table 2). On the other hand, the N-alkylated/arylated compounds 4 fl, which have low FF values in acetone, also

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Animesh Pramanik et al.

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Figure 6. The selectivity of 4 c (10 mm) in the presence of various metal

ions in acetonitrile. The grey bars represent the emission intensity of 4 c
in the presence of other metal ions (1.0 mm). The black bars represent
the emission intensity that occurs upon the subsequent addition of Cu2 +
ions (1.0 mm) to the above solution (lem = 519 nm).

Figure 4. Fluorescence emission spectra of 4 c in the presence of different metal ions in CH3CN. [4 c] = 10 mm.

ble by the naked-eye (Figure 5 a). This response was selective for Cu2 + ions. The addition of ten equivalents of other
cations resulted no appreciable changes in color under visible as well as UV light (Figure 5 b). The fluorescence
quenching manifested by a change in color upon addition
of Cu2 + ions was also observed with all other compounds 4.

Figure 5. Changes of a solution of 4 c in acetonitrile (10.0 mm) in the

presence of ten equivalents of each cation under a) visible light and
b) UV light.

Figure 7. UV/vis absorption of compound 4 c (10 4 m) upon addition of

increasing amount of Cu2 + ions (1, 3, 5, 7, 10, 14, 20, 27, 34, 42, and
50 mm) in acetonitrile. Arrows indicate the absorbance that increased
and decreased during the titration experiments.

Competition Experiments
Competition experiments in which Cu2 + ions were added to
various solutions of 4 c containing one of the different
metal ions Zn2 + , Ni2 + , Cd2 + , Mn2 + , Co2 + , Na + , Hg2 + , and
Pb2 + in acetonitrile indicated that the recognition of Cu2 +
ions by 4 c was not significantly affected by the presence of
these other cations (Figure 6). Therefore, the intensity of
the fluorescence band of the complex at 519 nm can be
used to monitor the presence of Cu2 + ions alone as well as
Cu2 + ions in presence of other metal ions.

two isosbestic points were found at 393 nm and 307 nm

during the course of the titration, which indicates the formation of a Cu2 + complex through two intermediates.[14]
Further UV studies in the 7001000 nm range with addition
of 0.5, 1.0, 1.5, 2.0, and 2.5 equivalents of 4 c to solution of
Cu2 + ions (10 2 m) showed significant differences between
the UV spectra of free 4 c and 4 c in the presence of Cu2 +
ions, which confirms the formation of a complex (see Figure S14 in the Supporting Information). The association
constant for complexation of 4 a with Cu2 + ions in acetonitrile was determined as 7.377  103 m 1 by using the Benesi
Hildebrand equation, which indicates the formation of
a weak complex (see Figure S15 in the Supporting Information). Additionally, NMR titration was carried out by gradually adding Cu2 + ions to a [D6]DMSO solution of 4 c. The

Complexation with Cu2 + Ions

The changes in the absorption spectra of 4 c in acetonitrile
(10 4 m) on gradual addition of CuACHTUNGRE(ClO4)2 are shown in
Figure 7. On increasing the concentration of Cu2 + ions
from 1.0 mm to 50.0 mm, the absorption intensities of the
bands centered at 413 and 294 nm decreased gradually. Simultaneously, two new bands appeared at about 382 and
500 nm with increased intensities. In the absorption spectra,

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Animesh Pramanik et al.

Figure 9. Fluorescence emission spectra of 4 cCu2 + complex with gradual addition of PBS solution.

acidified with acetic acid (10 4 m), quenching of the fluorescence band at 530 nm was detected (Figure 10 b). On the
other hand, all of the compounds 4 fluoresce significantly
in a weak basic medium, such as triethylamine (see Figure S16 in the Supporting Information).

Figure 8. Change in 1H NMR spectra of 4 c (c = 5  10 2 m) upon addition

of Cu2 + ions (0.5, 1, 2, and 3 equiv.) in [D6]DMSO.

result showed that the peak

intensity of the NH and NH2
groups, which are probable
binding sites for Cu2 + ions,
decreased significantly with
increasing concentration of
Cu2 + ions (Figure 8). This
result also indicates the formation of weak complex between 4 c and Cu2 + ions.
Further, the formation of
complex between 4 c and
Cu2 + ion was proved unam- Figure 10. a) UV/vis spectra of 4 c (10 4 m) in methanol with gradual addition of acetic acid (10 2 m) and
biguously when phosphate b) fluorescence spectra of 4 c (10.0 mm) in methanol with gradual addition of acetic acid (10 4 m).
buffer solution (PBS) was
added to a solution of 4 cCu2 + complex in DMF. On
Staining of Living Cells
gradual addition of PBS, the fluorescence intensity of the
Fluorescent molecular probes play a crucial role in bioresultant solution increased substantially without any
imaging studies. Fluorescent dyes that are easily compatible
change in lem (Figure 9). This may be attributed to the
with biological systems are very useful in the field of physbreaking of 4 c-Cu2 + complex in presence of PBS for the
iological research.[15, 16] In a preliminary cell imaging experiformation of stable copper hydrogenphosphate in the solution causing the release of ligand 4 c in the medium.
ment, we chose compound 4 c as the fluorescent probe,
which was tested in bioimaging of human squamous epithelium and peripheral blood mononuclear (PBM) cells. IniEffect of Acid and Base
tially the cells were incubated in phosphate-buffered saline
(PBS) solution. Then a solution of 4 c (0.1 mm) in dimethyl
In the spectrophotometric study, when a methanolic solusulfoxide (DMSO)/H2O (1:10 v/v) was added to the cells at
tion of 4 c (10 4 m) was gradually acidified with acetic acid
(10 2 m), the absorption band maximum at 418 nm steadily
room temperature. Interestingly, we observed that 4 c can
stain both types of cells instantaneously (Figure 11). Furdecreased and at the same time a new band also appeared
thermore, we examined the effect of Cu2 + ions on the
at 508 nm (Figure 10 a). Similarly, in a fluorimetric titration,
when a methanolic solution of 4 c (10.0 mm) was gradually
stained cells. In this experiment saline water was used in-

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Experimental Section
All chemicals were purchased commercially from SigmaAldrich and
Spectrochem companies and used without further purification. Absorption and fluorescence spectra of all compounds were recorded on a Hitachi U-3510 spectrophotometer and PerkinElmer LS55 fluorescence
spectrometer at 25 8C. 1H (300 MHz) and 13C (75 MHz) NMR spectra
were recorded in [D6]DMSO solution with a Bruker 300 MHz instrument. Elemental analyses (C, H, and N) were performed with a Perkin
Elmer 240C elemental analyzer. HRMS was performed with a QTOF I
(quadrupole-hexapole-TOF) mass spectrometer. Fluorescence microscopic images of cell staining were captured on a BD Pathway 855 instrument. The X-ray diffraction data for crystallized compounds were
collected with MoKa radiation at 296 K on a Bruker APEX-II CCD
system. The crystals were positioned at 50 mm from the CCD. Frames
were measured with a counting time of 5 s. Data analysis was carried
out with the Bruker APEX2 and Bruker SAINT program. The structures were solved by direct methods with the Shelxs97 program.[17]
CCDC 903274
(4 a)
and
CCDC 903275 (4 k) contain the
supplementary
crystallographic
data for this paper.[12] These data
can be obtained free of charge
from The Cambridge Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/
cif.

Figure 11. Images of stained a) human squamous epithelium cells and

b) PBM cells under a fluorescence microscope.

stead of PBS solution, as PBS breaks the 4 cCu2 + complex.

At first, human squamous epithelium cells were stained
with 4 c in saline water (Figure 12 a) and then the stained

Figure 12. Images of human squamous epithelium cells. a) Normal stained cells and after addition of
b) 0.25 equiv.; c) 0.5 equiv. and d) 1.0 equiv. of Cu2 + ions under a fluorescence microscope after 15 mins.

A mixture of isatin (147 mg,

1.0 mmol), malononitrile (100 mg,
1.5 mmol), and meta-phenylenediamine (108 mg, 1.0 mmol) in ethanol (10 mL) was heated to reflux for
one hour. After completion of the reaction (monitored by TLC), the solvent was removed under vacuum to obtain the solid products, which
were purified by repeated crystallization from EtOH.

cells were incubated with Cu2 + ions (0.25, 0.5, and

1.0 equivalents) for 15 mins. Fluorescence quenching was
observed inside the cells under a fluorescence microscope
(Figure 12 bd). The preliminary result of this in vitro study
indicates that compound 4 c is cell-permeable and can be
used to detect copper ions in living cells.

2,7-Diamino-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]-3carbonitrile (4 a)
Yellow solid (276 mg, 91 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 9.34 (s, 1 H), 9.21 (s, 1 H), 7.76 (d, J = 9 Hz, 1 H), 7.69
(d, J = 7.8 Hz, 1 H), 7.41 (t, J = 7.5 Hz, 1 H), 7.267.17 (m, 2 H), 7.10 (d,
J = 9 Hz, 1 H), 6.82 (s, 1 H), 6.26 ppm (bs, 2 H); 13C NMR (75 MHz,
[D6]DMSO): d = 161.6, 152.7, 152.1, 148.5, 140.4, 131.8, 130.6, 126.2,
125.1, 124.5, 121.7, 120.5, 117.5, 116.5, 105.0, 52.7, 51.5 ppm; IR (KBr):
n = 3320, 2225, 1706 cm 1; Elemental analysis: calcd (%) for C17H13N5O:
C 67.32, H 4.32, N 23.09; found: C 67.16, H 4.26, N 23.02.

Conclusions
In conclusion, a simple and efficient one-pot method for
the synthesis of a new class of highly fluorescent isatinbased spiro compounds 2,7-diamino-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]-3-carbonitriles has been developed by heating a mixture of isatins, malononitrile, and
meta-phenylenediamine to reflux in ethanol. Some of the
compounds have high fluorescence quantum yields and
long fluorescence lifetimes in polar solvents like acetone.
The FF value decreases with increasing polarity of the solvents, which may be attributed to extensive solvation of the
compounds in polar solvents. Furthermore, the compounds
fluoresce significantly in weak basic medium, such as triethylamine, but do not emit in acetic acid. The efficient and
selective fluorescence quenching for Cu2 + ions can be used
for detecting Cu2 + ions in the presence of several other
metal ions. Preliminary studies show that these isatin-based
spiro compounds can be used as fluorescent probes for bioimaging of human squamous epithelium and PBM cells,
and also for detecting copper ions in living cells.

Asian J. Org. Chem. 2013, 2, 869 876

Preparation of 2,7-Diamino-2-oxo1,4-dihydrospiro[indoline-3,4quinoline]-3-carbonitrile (4 a)

2,7-Diamino-5-fluoro-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]3-carbonitrile (4 b)
Yellow solid (279 mg, 87 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 9.41 (s, 1 H), 9.29 (s, 1 H), 7.81 (d, J = 9 Hz, 1 H), 7.66
(dd, J1 = 9.6 Hz, J2 = 2.7 Hz, 1 H), 7.36- 7.23 (m, 2 H), 7.16 (dd, J1 = 9 Hz,
J2 = 2.1 Hz, 1 H), 6.88(d, J=1.8 Hz, 1 H), 6.36 ppm (bs, 2 H); 13C NMR
(75 MHz, [D6]DMSO) [F coupled 13C spectra]: d 161.3, 159.9, 156.8,
152.6, 151.5, 148.4, 136.5, 126.5, 126.4, 126.0, 123.1, 120.3, 119.7, 117.5,
117.4, 117.2 117.1, 116.9, 115.9, 104.6, 56.1, 54.5 ppm; IR (KBr): n =
3364, 2233, 1694 cm 1; Elemental analysis: calcd (%) for C17H12N5OF: C
63.55, H 3.76, N 21.80; found: C 63.38, H 3.69, N 21.71.
2,7-Diamino-5-chloro-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]3-carbonitrile (4 c).
Yellow solid (304 mg, 90 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 9.44 (s, 1 H), 9.31 (s, 1 H), 7.797.72 (m, 2 H), 7.45 (dd,
J1 = 8.7 Hz, J2 = 2.4 Hz, 1 H), 7.16 (d, J = 8.7 Hz, 1 H), 7.09 (dd, J1 = 9 Hz,
J2 = 2.1 Hz, 1 H), 6.79 (d, J = 1.8 Hz, 1 H), 6.32 ppm (bs, 2 H); 13C NMR

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(75 MHz, [D6]DMSO): d = 160.9, 152.6, 151.3, 148.4, 139.0, 130.5, 129.8,
128.0, 126.4, 126.0, 122.9, 120.3, 117.3, 115.8, 104.5, 52.2, 51.7 ppm; IR
(KBr): n = 3360, 2227, 1697 cm 1; (M + H) + : calculated 338.0809, found:
337.9980; Elemental analysis: calcd (%) for C17H12N5OCl: C 60.45, H
3.58, N 20.73; found: C 60.26, H 3.50, N 20.64.

111.1, 109.1, 107.1, 99.6, 85.9, 52.1, 51.6, 26.2 ppm; IR (KBr): n = 3370,
2165, 1681 cm 1; Elemental analysis: calcd (%) for C18H14N5OI: C 48.78,
H 3.18, N 15.80; found: C 48.58, H 3.11, N 15.72.
2,7-Diamino-1-ethyl-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]-3carbonitrile (4 j).

2,7-Diamino-5-bromo-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]3-carbonitrile (4 d).

White solid (248 mg, 75 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 8.58 (s, 1 H), 7.08- 7.02 (m, 1 H), 6.86- 6.79 (m, 3 H),
5.82- 5.68 (m, 3 H), 5.45 (bs, 2 H), 4.89 (bs, 2 H), 3.47 (q, J = 6.9 Hz, 2 H),
0.93 ppm (t, J = 6.9 Hz, 3 H); 13C NMR (75 MHz, [D6]DMSO): d = 178.5,
153.3, 148.7, 141.8, 137.0, 136.7, 128.2, 127.0, 124.6, 122.7, 120.9, 109.0,
108.4, 108.0, 99.6, 52.8, 51.3, 34.2, 12.6 ppm; IR (KBr): n = 3350, 2172,
1664 cm 1; Elemental analysis: calcd (%) for C19H17N5O: C 68.87, H
5.17, N 21.13; found: C 68.66, H 5.10, N 21.02.

Yellow solid (310 mg, 81 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 9.50 (bs, 1 H), 9.37 (bs, 1 H), 7.967.64 (m, 3 H), 7.17
(bs, 2 H), 6.86 (bs, 1 H), 6.38 ppm (bs, 2 H); 13C NMR (75 MHz,
[D6]DMSO): d = 161.3, 152.6, 151.4, 148.6, 139.4, 133.4, 132.7, 126.5,
126.0, 123.1, 120.3, 119.2, 117.0, 115.8, 104.2, 52.9, 51.4 ppm; IR (KBr):
n = 3214, 2221, 1722 cm 1; Elemental analysis: calcd (%) for
C17H12N5OBr: C 53.42, H 3.16, N 18.32; found: C 53.25, H 3.10, N 18.24.

2,7-Diamino-5-chloro-1-ethyl-2-oxo-1,4-dihydrospiro[indoline-3,4quinoline]-3-carbonitrile (4 k).

2,7-Diamino-5-iodo-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]-3carbonitrile (4 e).

Grey solid (256 mg, 70 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 8.86 (s, 1 H), 7.33 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1 H), 7.13
(d, J = 8.4 Hz, 1 H), 7.01 (d, J = 1.8 Hz, 1 H), 6.04- 5.93 (m, 3 H), 5.75 (bs,
2 H), 5.18 (bs, 2 H), 3.69 (q, J = 7.2 Hz, 2 H), 1.13 ppm (t, J = 6.9 Hz,
3 H); 13C NMR (75 MHz, [D6]DMSO): d = 178.2, 153.3, 148.9, 140.8,
139.1, 136.7, 128.2, 127.0, 126.7, 124.5, 120.7, 110.1, 109.2, 107.1, 99.7,
52.2, 51.7, 34.4, 12.5 ppm; IR (KBr): n = 3355, 2167, 1668 cm 1; HRMS
(ESI): m/z: calcd: 366.1122 [M+H] + ; found: 366.0698. Elemental analysis: calcd (%) for C19H16N5OCl: C 62.38, H 4.41, N 19.14; found: C
62.21, H 4.33, N 19.05.

Yellow solid (356 mg, 83 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 9.46 (s, 1 H), 9.34 (s, 1 H), 8.06 (d, J = 1.8 Hz, 1 H),
7.817.76 (m, 2 H), 7.16 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1 H), 7.01 (d, J =
8.4 Hz, 1 H), 6.85 (d, J = 1.8 Hz, 1 H), 6.35 ppm (bs, 2 H); 13C NMR
(75 MHz, [D6]DMSO): d = 160.8, 152.5, 151.3, 148.3, 139.8, 139.2, 138.4,
126.9, 126.0, 123.2, 120.2, 119.2, 117.2, 115.8, 104.5, 52.1, 51.6 ppm; IR
(KBr): n = 3212, 2221, 1713 cm 1; Elemental analysis: calcd (%) for
C17H12N5OI: C 47.57, H 2.82, N 16.32; found: C 47.42, H 2.74, N 16.23.
2,7-Diamino-1-methyl-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]3-carbonitrile (4 f).

2,7-Diamino-1-phenyl-2-oxo-1,4-dihydrospiro[indoline-3,4-quinoline]3-carbonitrile (4 l).

Grey solid (241 mg, 76 % yield); m.p. 244246 8C; H NMR (300 MHz,
[D6]DMSO): d = 8.75 (s, 1 H), 7.267.21 (m, 1 H), 7.00- 6.97 (m, 3 H),
5.98 (s, 1 H), 5.94- 5.86 (m, 2 H), 5.76- 5.72 (m, 2 H), 4.97 (bs, 2 H),
3.08 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d = 178.9, 153.4,
149.1, 148.7, 143.0, 136.7, 136.6, 127.2, 124.4, 122.9, 121.1, 109.0, 108.4,
107.9, 99.6, 52.6, 51.5, 26.2 ppm; IR (KBr): n = 3361, 2148, 1695 cm 1; Elemental analysis: calcd (%) for C18H15N5O: C 68.13, H 4.76, N 22.07;
found: C 67.95, H 4.69, N 22.01.

Grey solid (314 mg, 74 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 8.86 (s, 1 H), 7.607.55 (m, 2 H), 7.477.39 (m. 3 H),
7.21 (t, J = 7.2 Hz, 1 H), 7.13- 7.05 (m, 2 H), 6.47 (d, J = 7.8 Hz, 1 H), 6.19
(d. J = 8.1 Hz, 1 H), 6.216.18 (m, 2 H), 5.75 (bs, 2 H), 5.16 ppm (bs, 2 H);
13
C NMR (75 MHz, [D6]DMSO): d = 178.8, 153.6, 149.3, 142.9, 137.1,
136.9, 135.0, 130.1, 128.7, 128.5, 127.8, 127.1, 125.4, 124.0, 121.3, 109.6,
109.2, 108.2, 100.1, 53.4, 52.2 ppm; IR (KBr): n = 3361, 2180, 1668 cm 1;
Elemental analysis: calcd (%) for C23H17N5O: C 72.81, H 4.52, N 18.46;
found: C 72.65, H 4.45, N 18.38.

2,7-diamino-5-fluoro-1-methyl-2-oxo-1,4-dihydrospiro[indoline-3,4quinoline]-3-carbonitrile (4 g).
Grey solid (238 mg, 71 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 8.74 (s, 1 H), 7.07- 6.93 (m, 2 H), 6.79 (dd, J1 = 7.8 Hz,
J2 = 2.1 Hz, 1 H), 5.955.84 (m, 3 H), 5.63 (bs, 2 H), 5.05 (bs, 2 H),
3.03 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO) [F coupled 13C spectra]: d = 178.7, 160.6, 157.4, 153.4, 148.9, 139.2, 138.3, 138.2, 136.7, 127.1,
120.8, 114.7, 114.4, 112.2, 111.8, 109.4, 109.3, 109.1, 107.3, 99.7, 52.2, 52.0,
26.3 ppm; IR (KBr): n = 3352, 2179, 1688 cm 1; Elemental analysis: calcd
(%) for C18H14N5OF: C 64.47, H 4.21, N 20.88; found: C 64.28, H 4.16,
N 20.79.

Acknowledgements
A.K. and S.P. thank the University Grant Commission (UGC) and the
Council of Scientific and Industrial Research (CSIR), New Delhi, India
respectively for offering them Senior Research Fellowships (SRF). The
financial assistance of CSIR, New Delhi is gratefully acknowledged
(Major Research Project, No. 02ACHTUNGRE(0007)/11/EMR-II). The authors thank
Prof. Ashutosh Ghosh and Dr. Debabrata Mandal for useful discussions.
Crystallography was performed at the DST-FIST, India-funded single
crystal diffractometer facility at the Department of Chemistry, University of Calcutta. The instrumental facility of the Centre for Research in
Nanoscience & Nanotechnology (CRNN), University of Calcutta is also
acknowledged.

2,7-diamino-5-chloro-1-methyl-2-oxo-1,4-dihydrospiro[indoline-3,4quinoline]-3-carbonitrile (4 h).
Yellow solid (253 mg, 72 % yield); m.p. > 300 8C; 1H NMR (300 MHz,
[D6]DMSO): d = 8.88 (s, 1 H), 7.34 (d, J = 7.2 Hz, 1 H), 7.09- 7.02
(m,2 H), 6.055.96 (m,3 H), 5.76 (bs, 2 H), 5.17 (bs, 2 H), 3.14 ppm (s,
3 H); 13C NMR (75 MHz, [D6]DMSO): d = 178.9, 153.8, 149.3, 142.3,
139.0, 137.1, 128.6, 127.5, 127.3, 124.7, 121.2, 110.4, 109.5, 107.5, 100.1,
52.5, 52.2, 26.7 ppm; IR (KBr): n = 3302, 2179, 1669 cm 1; Elemental
analysis: calcd (%) for C18H14N5OCl: C 61.46, H 4.01, N 19.91; found: C
61.27, H 3.94, N 19.85.

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Received: August 21, 2013

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