Synthesis, structural analysis and application of a series of solid-state fluorochromes - Aryl hydrazones of 4-hydrazino-N-hexyl-1,8- naphthalimide

Article abstract of DOI:10.1016/j.tet.2012.10.093

The development of red solid-state fluorochromes is important for different applications. The influence of the electronic effects of substituents on the chemical shifts in the 1H NMR spectra and solid-state fluorescent properties of aryl hydrazones of 4-hydrazino-N-hexyl-1,8-naphthalimide is evaluated. The main fragmentation pathway of hydrazones is determined using electrospray ionization mass spectrometry and high resolution MS/MS. A possible application of these fluorochromes for the in situ imaging of enzyme activities is presented.

Full text of DOI:10.1016/j.tet.2012.10.093

Tetrahedron 69 (2013) 712e721
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, structural analysis and application of a series of solid-state
fluorochromesdaryl hydrazones of 4-hydrazino-N-hexyl-1,8-
naphthalimide
,
b
c
c
Ivaylo P. Ivanova , Mashenka B. Dimitrova , Donka N. Tasheva , Diana V. Cheshmedzhieva ,
*
Valentin S. Lozanov d, Sonia V. Ilieva c
a Faculty of Biology, Sofia University, 1164 Sofia, Bulgaria
b Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
c Faculty of Chemistry and Pharmacy, Sofia University, 1164 Sofia, Bulgaria
d Department of Medical Chemistry and Biochemistry, Medical University of Sofia, 1431 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2012
Received in revised form 12 October 2012
Accepted 29 October 2012
Available online 10 November 2012
The development of red solid-state fluorochromes is important for different applications. The influence
of the electronic effects of substituents on the chemical shifts in the 1H NMR spectra and solid-state
fluorescent properties of aryl hydrazones of 4-hydrazino-N-hexyl-1,8-naphthalimide is evaluated. The
main fragmentation pathway of hydrazones is determined using electrospray ionization mass spec-
trometry and high resolution MS/MS. A possible application of these fluorochromes for the in situ im-
aging of enzyme activities is presented.
Keywords:
1,8-Naphthalimide
Hydrazone
Ó 2012 Elsevier Ltd. All rights reserved.
Chemical shift
Hammett correlation
Mass spectrometry
Solid-state fluorescence
Enzyme activity imaging
1. Introduction
electronic and fluorescence spectra for application as materials for
organic light-emitting diodes (OLEDs). The authors have mentioned
1,8-Naphthalimides have been widely studied due to their pho-
tophysical properties. These are one of the most important building
blocks for the synthesis of efficient fluorescent materials showing
strong fluorescence, large quantum yields and sizable Stokes shifts.
Naphthalimides are widely used as colourants in the polymer
industry,1,2 laser dyes,1,3,4 chemosensors5,6 and fluorescence probes
for biomedical purposes, including fluorescence cell makers7 and
anti-cancer agents.8 Efficient solid-state emission of organic mate-
rials is essential for optoelectronic devices, and the molecular de-
sign and development of novel fluorochromes that emit visible light
in the solid state with high efficiency are strongly desired for dif-
ferent applications.9 Though many organic compounds show red
fluorescence in solution, fewer compounds fluoresce in the solid
state due to intermolecular interactions in the condensed states.10
Gan and co-workers11,12 have synthesized several aryl hydrazones
of 4-hydrazino-N-butyl-1,8-naphthalimide and studied their
that materials for green and blue OLEDs are available, whereas red
fluorescent materials of high efficiency are not common. Photo-
physical properties of the other synthesized hydrazones of
1,8-naphthalimide have been investigated,13,14 including those co-
valently bound to single-wall carbon nanotubes15 and components
of copolymers.16e18 Naphthalimide hydrazones based on vanillin19
and salicylaldehyde20 were designed and synthesized as highly
selective fluorescent probes for Agþ and Cu2þ, respectively.
Correlation analysis, the study of the relationships between the
parameters known as substituent constants and various directly
measurable quantities, plays an important role in physical organic
chemistry for the description of the electronic properties of mole-
cules. Free energy relationships have been observed for a number of
spectroscopic properties,21 including mass spectrometry.21,22 Cor-
relation analyses have also been applied to demonstrate that de-
pendencies exist between substituent effect and chemical shifts in
nuclear magnetic resonance spectroscopy.23,24 So, the established
relationships are of substantial value for the prediction of NMR
properties.25
* Corresponding author. Tel.: þ359 2 8167 214; e-mail address: ivayloi@abv.bg
(I.P. Ivanov).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.10.093

I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
713
Fluorescence-based imaging of enzyme activity in vitro and
2.2. Effect of phenyl substituents on the proton chemical
shift
in vivo promises many applications from elucidation of the enzyme
function and mechanism, to better disease detection and mon-
itoring.26e28 The possibility for application of 1,8-naphthalimide
hydrazones for fluorescent histochemical localization of enzy-
mesdmarkers for malignant or other diseases has been explored.
Members of our group have developed methods for fluorescent
histochemical localization of dipeptidyl peptidase IV, tripeptidyl
peptidase I29 and gamma-glutamyl transpeptidase.30 The enzyme
hydrolyzes the corresponding synthetic substrate to yield free 4-
hydrazino-N-hexyl-1,8-naphthalimide, which couples simulta-
neously with piperonal and gives a water-insoluble fluorescent
hydrazone precipitating on the sites of enzyme activity and mark-
ing them.
According to the principle of substituent-induced changes in the
chemical shift (SCS), the variations in the 1H NMR spectra of
hydrazones 3ae3g were correlated using different linear free en-
ergy relationship (LFER) models, based on the mono and dual
substituent parameters (sp,
sþp , F, R and Rþ). The protons chemical
shifts (dH), which correlate with the Hammett type substituent
constants are shown in Table 1. Good linear correlation (n¼7,
Table 1
NMR chemical shifts (dH) for two types of hydrogen atoms in the series of p-
substituted hydrazones 3ae3g and Hammett substituent constants correlating with
the respective chemical shifts
In the present work, a series of substituted aryl hydrazones of 4-
hydrazino-N-hexyl-1,8-naphthalimide were synthesized and char-
acterized by nuclear magnetic resonance and mass spectrometry.
The effects of substituents at the benzene ring on the 1H NMR
chemical shifts have been studied using models, based on mono
and dual substituent parameter relationship. Electrospray ioniza-
tion mass spectrometry (ESI-MS) and high resolution MS/MS have
been used in order to explore the general and specific fragmenta-
tion of the compounds studied. The substituent effect on the solid-
state fluorescence properties has been rationalized using Hammett
type correlation, and the possibilities for application of the series of
hydrazones to develop new techniques for fluorescent imaging of
enzyme activity have been explored.
Hammett substituent constantsb
a
Compound
X
dH (ppm)
NH
H-5
sp
spþ
R
3a
3b
3c
3d
3e
3f
N(CH3)2
11.25
11.39
11.46
11.52
11.54
11.72
11.82
7.65
7.70
7.74
7.77
7.73
7.78
7.84
ꢁ0.83
ꢁ0.27
ꢁ0.17
0.00
ꢁ1.70
ꢁ0.78
ꢁ0.31
0.00
ꢁ0.98
ꢁ0.56
ꢁ0.18
0.00
OCH3
CH3
H
Cl
CN
0.23
0.66
0.11
0.66
ꢁ0.19
0.15
3g
NO2
0.78
0.79
0.13
a
SolventdCD3SOCD3.
b
Taken from Ref. 33.
r¼0.9865) between the chemical shift of the amine hydrogen (NH)
and sp substituent constants was observed for this series (Fig. 1).
The slope
r
is 0.34ꢀ0.02. A similar dependence ( ¼0.65ꢀ0.03) was
r
2. Results and discussion
2.1. Synthesis
observed in the series of N-phenylhydrazones of p-substituted
benzaldehydes.32 In these compounds the sensitivity to the elec-
tronic effects of substituents is double that of the established for N-
hexyl-1,8-naphthalimide hydrazones (3ae3g).
The most common method for the preparation of N-substituted
hydrazones is the reaction of aldehydes or ketones with N-alkyl or
N-aryl hydrazines.31 Usually the reaction proceeds in ethanol so-
lution under reflux for a few hours. N-Aryl hydrazones and espe-
cially those substituted with electron-withdrawing groups are
stable to acids and can be prepared under acidic conditions. In
a previous work29 we described the synthesis of the piperonal
hydrazone of 4-hydrazino-N-hexyl-1,8-naphthalimide in the pres-
ence of acetic acid. In the present study this approach is success-
fully applied for the preparation of a series of substituted aryl
hydrazones of N-hexyl-1,8-naphthalimide (Scheme 1).
The observed LFER in the two series of hydrazones shows that
shielding/deshielding of the NH hydrogen atoms is determined
from the nitrogen atom shielding/deshielding caused by the elec-
tronic effect of the aromatic ring substituents. The excellent linear
correlation (r¼0.9957) obtained between dH and M06/6-31þG(d)
computed Hirshfeld charges on the amine nitrogen for N-methyl
analogues of 3ae3g14 also supports this assumption (Fig. S1 in the
Supplementary data).
Hammett correlations of 15N NMR chemical shifts of the amine
and imine type nitrogen atoms (dN) with sp constants in the series
Scheme 1. Synthesis of hydrazones 3aeh.
The reactions between aldehydes 1ae1h and 4-hydrazino-1,8-
naphthalimide (2) were carried out under reflux in ethanol solu-
tion in the presence of acetic acid for several minutes. The reaction
time depends on the electronic effects of the substituents. The re-
action between p-nitrobenzaldehyde and 2 was carried out for
5 min, whereas the interaction with p-dimethylaminobenzalde-
hyde was completed in 15 min. Hydrazones 3be3h precipitated on
cooling; compound 3g crystallized partially during the reaction. For
the isolation of hydrazone 3a a few drops of water were added to
the solution and the resulting solid was filtered. The crude crys-
talline compounds were purified by column chromatography.
of N-phenyl34 and N-alkyl-N-(2-hydroxycyclohexyl)35 hydrazones
of p-substituted benzaldehydes have also been determined. In both
cases
r is negative. This indicates that electron-withdrawing (EW)
substituents cause deshielding of the nitrogen atoms, whereas
electron-donating (ED) substituents cause shielding effects. The
resonance structure 4 has the main contribution34e36 to the elec-
tron density distribution in hydrazones (Scheme 2). Electron-
withdrawing substituents increase the stability of this struc-
ture35,37,38 by field and resonance (form 5) effects. In contrast, ED
substituents increase the stability of resonance forms 6 and 7
(Scheme 2). To estimate the influence of the field and resonance

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I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
Fig. 1. Plot of the NH hydrogen NMR chemical shifts for the series of p-substituted
hydrazones 3ae3g in CD3SOCD3 versus Hammett substituent constants sp
Fig. 2. Plot of the NH hydrogen NMR chemical shifts for the series of p-substituted
hydrazones 3ae3g in CD3SOCD3 versus Hammett substituent constants spþ
.
.
a break at sþp ¼0.00 (Fig. S2 in Supplementary data). The de-
pendence from sþp constants is an indicator for a direct conjugation
between the substituent and the nitrogen atom in the hydrazones.
EW substituents stabilize34 the resonance form 8, whereas ED
substituents stabilize38 the resonance form 9 (Scheme 2). By using
a dual substituent parameter model (Eq. 1) for the first segment of
Fig. 2, where R substituent parameters are replaced by Rþ param-
eters, the following values were obtained: rF¼0.146ꢀ0.048 and
rþR ¼0.156ꢀ0.008 (n¼4, r¼0.999). These results indicate that the
resonance forms 6 and 9 have equal contribution to the molecular
electron density distribution. The polar effects of EW substituents
Scheme 2. Resonance structures of p-substituted hydrazones 3ae3g involving
p-substituents and the benzylidene hydrazine moiety.
are larger and practically identical
(rF¼0.36ꢀ0.10 and
rþR ¼0.38ꢀ0.13; n¼3, r¼0.982) and the contributions of the reso-
nance forms 4 and 8 are also identical. In 1-(4-nitrobenzylidene)-2-
phenylhydrazine the resonance structure29 8 has a significant
contribution. The absorption spectra of N-hexyl-1,8-naphthalimide
hydrazones with EW substituents in acetonitrile showed a hyp-
sochromic shift.14 However, the nitro-substituted compound 3g
showed a bathochromic shift of 13 nm. This effect might be due to
the formation of a quinone type structure with a significant con-
tribution to the resonance structure 8. The resonance stabilization
44548 allows free rotation around the C]N bond and leads to
the rapid conversion between isomeric forms.37 This free rotation
could explain the fact that in the 1H NMR spectrum of 3g the signals
are broad and without expressed multiplicity unlike the other
hydrazones in the series.
To determine whether the formation of biphasic Hammett cor-
relation is typical for hydrazones, the data for dN of the amine ni-
trogen atoms in N-phenyl-34 and N-alkyl-(2-hydroxycyclohexyl)35
hydrazones of p-substituted benzaldehydes were used. The corre-
lations for both series are also biphasic concave upwards with
a break at sþp ¼0.00 (Fig. S3 in Supplementary data). The comparison
of the Hammett type correlations in different series of hydrazones
shows that the electron effects of the two types of substituents are
typical for this class of compounds.
effects on dH(NH) a model based on the dual substituent parameter
(SwaineLupton type equation) has been used for regression anal-
ysis in the following form (Eq. 1):
SCS ¼ rFF þ rRR þ h
(1)
In Eq. 1 F and R are substituent parameters reflecting the field
and resonance electronic effects, respectively, rF and rR indicate the
sensitivity of the chemical shift to the field and resonance elec-
tronic effects of the aromatic ring substituents. Good correlation
(n¼7, r¼0.987) in the series 3ae3g was observed; the parameters
rF¼0.33ꢀ0.07 and rR¼0.35ꢀ0.04 were obtained. Therefore, the
influence of the field and resonance electronic effects are equal and
the contribution of the resonance forms 4 and 5 is equivalent. The
same is valid also for the resonance forms 6 and 7.
Neuvonen et al.35 studied the electronic structures of a series of
imines and hydrazones by 13C and 15N NMR spectroscopy. The
authors carried out statistical analysis of the Hammett type cor-
relations and established fair linear correlation between chemical
shifts of the amine type hydrogen and sþp constants of the sub-
stituents. However, the Hammett plot (dH(NH) versus sþp (X)) for
substituent X variations in the series of N-hexyl-1,8-naphthalimide
hydrazones (3ae3g) is in fact biphasic concave upwards (Fig. 2)
with a break at sþp ¼0.00 (X¼H). The point of the unsubstituted
compound and the points of the hydrazones with ED substituents
are on a line with rED¼0.157ꢀ0.005 (n¼4, r¼0.9991). The second
segment of the plot, which includes EW substituents is steeper
The amine nitrogen lone pair conjugates with the naphthalimide
moiety leading to the formation of resonance structure 10 and 11
(Scheme 3). It conjugates also with the other part of the molecule
(Scheme 2) thus connecting the benzylideneamino and naph-
thalimide moieties. The presence of such conjugation is demon-
strated by using the LFER model (Fig. 3), where the excellent
correlation between dH(H-5) and R substituent constants with
(
rEW¼0.36ꢀ0.04; n¼3, r¼0.9872). We established that the plot of
Hirshfeld charges of the amine nitrogen atom of 3ae3g versus spþ
r
¼0.115ꢀ0.007 (n¼6, r¼0.9923) is observed. This effect could be
substituent constants was also biphasic concave upwards with
explained by the resonance form 12 (Scheme 3), in which the

I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
715
Electrospray ionization (ESI) in positive and negative mode was
used for the structure elucidation of the investigated compounds
3ae3h, as the hydrazones form stable protonated [MþH]þ or
deprotonated [MꢁH]ꢁ ions. High resolution MS/MS experiments in
the positive mode proved to be more informative to study these
compounds due to the formation of more ions during the frag-
mentation of [MþH]þ. The mass spectrum of unsubstituted
hydrazone 3d is presented in Fig. 4 and the characteristic ions for
this series of hydrazones are given in Table 2. The main fragmen-
tation pathway of compounds 3ae3h is presented in Scheme 4. The
proposed fragmentation patterns are supported by high resolution
mass spectrometry (HRMS). The proposed structure of the ions is
given in Fig. S4 in the Supplementary data.
Scheme 3. Resonance structures for the series of p-substituted hydrazones 3ae3g
involving benzylidene hydrazine and naphthalimide moieties.
LCeESI-MS/MS experiments showed that the fragmentation of
þ
[MþH]þ ions involves loss of 1-hexene leading to [MHꢁC6H12
]
ions with a high relative intensity (Scheme 4; route a). The for-
mation of these ions takes place throughout McLafferty rear-
rangement. There is a dependence between the electronic effect of
substituents and the relative intensity of these ions. Hydrazones
with strong ED substituents (3a, 3b and 3h) have a relative in-
tensity of 100%, whereas for those with EW suþbstituents the in-
tensity is below 50% (Table 2). The [MHꢁC6H12
] ions give rise to
ions of moderate intensity with m/z 212 (Table 2). Accurate mass
measurement of the ion m/z 212 in the mass spectrum of 3d is
212.0587. The measured mass of this ion in the other hydrazones is
practically the same. However, m/z 212.0587 does not give the
correct composition of the ion with relative error less than 10 ppm.
The unsubstituted hydrazone 3d contains naphthalene and cyclic
imide moieties. The fragmentation of naphthalene and its de-
rivatives is characterized by a loss of acetylene.43,44 On the other
hand, the aromatic cyclic imides,45 including naphthalimides46 lose
CO to give fragment ions. Ions with m/z 212 can be formed in two
ways (Scheme 4; route a1 and a2). Through the route a1, acetylene
and the related benzene are lost corresponding to 212(I) ion with
the theoretical mass of m/z 212.0454. According to the route a2,
after the loss of CO and the corresponding cyclohexa-1,3-dien-5-
yne, an ion 212(II) with theoretical mass of m/z 212.0818 should
be formed. In our experimental conditions, even at high resolu-
tions, these two ions could not be resolved. These ions are formed
also from the m/z 296 ion, which is the most intense in the mass
spectra of the hydrazones (Table 2). In compounds 3ce3g m/z 296
shows a relative intensity of 100%, whereas in the hydrazones with
substituents with strong ED properties this intensity is between
58% and 86%. However, the analysis of the results showed that it is
composed of two unresolved ions: 296(I) (theoretical mass
296.1393; route b) and 296(II) (theoretical mass 296.1757; route c).
The m/z 296(I) is formed by a cleavage of the bond between the
imine carbon atom and the benzene ring47 accompanied by a loss of
acetylene. The m/z 296(II) ion is formed by cleavage of the same
bond but CO is lost instead of acetylene in this case. The loss of 1-
hexene from these two ions (296(I) and 296(II)) leads to the for-
mation of ions 212(I) and 212(II) correspondingly. An ion with m/z
279 of low intensity (Scheme 4, route d) is observed in the mass
spectra of hydrazones. It is formed throughout a loss of neutral
fragments in the route c accompanied by elimination of NH3. After
the loss of the respective phenylmethanimine from the molecular
ion (route f) an ion with m/z 295 is formed. Its relative intensity
varied from moderate (3b) to very low (3f and 3g). Except for 3a,
the fragmentation of [MþH]þ ions by the loss of corresponding
benzonitrile (route e) after the cleavage of NeN bond,47 leads to the
formation of the m/z 297 ion of low or very low intensity (Table 2).
The elimination of 1-hexene from this ion leads to the protonated
form of 4-amino-1,8-naphthalimide with m/z 213 (Scheme 4). The
intensity of m/z 213 depends on the electronic effect of substituent:
it varies from moderate for ED substituents to very low for EW
substituents.
Fig. 3. Hammett type plot of the H-5 hydrogen atom NMR chemical shifts for the
series of p-substituted hydrazones 3ae3g in CD3SOCD3 versus R substituent constants.
Compound 3g (X¼NO2) is outside of the established trend for the series studied.
substituents withþR-effectstabilizethestructure, while substituents
with ꢁR-effect destabilize it. The hydrazone 3g (X¼NO2) is outside of
the established trend for the series studied, probably due to the large
contribution of the resonance structure 8 for this compound.
In a previous work we performed a full geometry optimization
for N-methyl analogues of hydrazones 3ae3g in acetonitrile by
using PCM computations.14 According to these results ED sub-
stituents shorten the C10eC(H) bond length thus making the NeN
bond longer as a result of the stabilization by resonance structure 9
and make the resonance stabilization 64749 favourable (Scheme
2). The considerable contribution of the resonance form 9 is sup-
ported by the bathochromic effect caused by ED substituents in the
absorption spectra of hydrazones in acetonitrile.14 EW substituents
make the C]N longer and the bond NeN shorter in compliance
with the increased contribution of the resonance stabilization
44548. The ED and EW kinds of substituents have opposite ef-
fects on the NeC6 bond length. ED substituents make this bond
shorter by the stabilization effect 11412 (Scheme 3), while EW
substituents make it longer by destabilizing these resonance forms.
2.3. Electrospray ionization mass spectrometry
Hydrazones are important derivatives for structural and quan-
titative characterization of carbonyl compounds, including carbo-
hydrates, in environmental analysis, food chemistry and industrial
analysis by chromatography, fluorescence spectroscopy, and mass
spectrometry.39,40 The detailed cognition of the fragmentation be-
haviour of hydrazones is an additional demonstration for their
structure and is significant to the analytical practice.41,42

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I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
Fig. 4. High resolution ESI(þ) mass spectrum of the unsubstituted hydrazone 3d.
Table 2
Selected general fragments in the ESI(þ) mass spectra of hydrazones 3ae3h and their relative intensity. Elemental composition could be assigned to experimental m/z values
with relative errors below 10 ppm
Ion
Compound (X/Y)
Calcd m/z
Elemental
Composition
3a
N(CH3)2
H
3b
OCH3
H
3c
CH3
H
3d
H
H
3e
Cl
H
3f
CN
H
3g
NO2
H
3h
OCH3
OCH3
Accurate m/z determination (relative error [ppm]; relative intensity [%])
M-83a
359.1515
(3.6; 100)
Calcd 359.1502
297.1601
(1.1; 82)
296.1519
(80; 58)
346.1182
(1.1; 100)
Calcd 346.1186
297.1590
(2.6; 8)
296.1516
(81; 86)
296.1516
(41; 86)
295.1439
(0.6; 26)
279.1491
(0.3; 13)
213.0656
(1.2; 27)
212.0579
(113; 18)
212.0579
(58; 18)
330.1239
(0.7; 80)
Calcd 330.1237
297.1571
(8.9; 11)
296.1520
(80; 100)
296.1520
(43; 100)
295.1445
(1.3; 16)
279.1494
(1.0; 12)
213.0660
(0.7; 17)
212.0583
(111; 16)
212.0583
(60; 16)
316.1090
(2.9; 50)
Calcd 316.1080
297.1606
(3.0; 4)
296.1529
(77; 100)
296.1529
(46; 100)
295.1452
(3.6; 8)
279.1501
(3.2; 17)
213.0665
(3.0; 13)
212.0587
(109; 26)
212.0587
(63; 26)
350.0689
(0.5; 44)
Calcd 350.0691
297.1587
(3.4; 2)
296.1516
(81; 100)
296.1516
(41; 100)
295.1441
(0.1; 10)
279.1481
(3.9; 13)
213.0663
(2.2; 9)
341.1039
(1.7; 27)
Calcd 341.1033
297.1608
(3.7; 1)
296.1524
(79: 100)
296.1524
(44: 100)
295.1444
(1.0; 1)
279.1497
(1.9; 8)
213.0662
(1.7; 1)
212.0584
(110; 10)
212.0584
(61; 10)
361.0937
(1.6; 31)
Calcd 361.0931
297.1585
(4.2; 11)
296.1524
(79; 100)
296.1524
(44; 100)
295.1447
(2.2; 1)
279.1496
(1.6; 11)
213.0645
(6.2; 1)
212.0582
(111; 17)
212.0582
(60; 17)
376.1290
(0.4; 100)
Calcd 376.1292
297.1597
(0.1; 8)
296.1519
(80; 70)
296.1519
(42; 70)
295.1444
(0.9; 17)
279.1493
(0.4; 12)
213.0659
(0.1; 20)
212.0580
(112; 19)
212.0580
(59; 19)
297.1597
C18H21N2O2
296.1757
(II) C18H22N3O
296.1393
(I) C17H18N3O2
295.1441
C18H19N2O2
279.1492
C18H19N2O
213.0658
C12H9N2O2
212.0818
(II) C12H10N3O
212.0454
296.1519
(42; 58)
295.1450
(3.2; 11)
279.1500
(2.9; 13)
213.0664
(2.7; 27)
212.0586
(109; 11)
212.0586
(62; 11)
212.0574
(115; 18)
212.0574
(57; 18)
(I) C11H6N3O2
a
m/z of ions [MHꢁC6H12]þ.
Several characteristic ions are observed in the mass spectra of
hydrazones with ED substituents 3a, 3b and 3h (Scheme S1 in
Supplementary data). In these three compounds an ion with m/z
322 is detected. Its intensity is high for 3a and low for 3b and 3h.
This ion is obtained from [MþH]þ ions through the loss of the
corresponding substituted benzene. The following elimination of 1-
hexene leads to an m/z 238 ion. For 3a and 3h, ions of low intensity
are registered. They are formed after a loss of NH3 or HCN from
[MþH]þ ions. In the mass spectrum of 3a (X¼N(CH3)2) a specific ion
with m/z 398 and middle intensity is observed. It is formed by
elimination of dimethylamine. A specific ion (m/z 333) for 3h
(X¼Y¼OCH3) results after the loss of CO, HCN and pentane.
properties of the crystals were studied for all hydrazones. Using
violet light, the fluorescence is yellow-green and red, whereas
green light leads to a red fluorescence (Fig. S5). According to Gan
et al.12 the fluorescence of the hydrazone of 4-hydrazino-N-butyl-
1,8-naphthalimide with p-dimethylaminobenzaldehyde is rosy in
colour and of low intensity. We observed a highly intensive red
fluorescence of the dimethylamino substituted hydrazone (3a). On
the other hand, the p-nitro substituted compound 3g does not
fluoresce.
Next, we studied the effect of the substituents on the solid-state
(crystals) fluorescence properties at lex¼390 nm. The normalized
fluorescence spectra for the entire series of compounds are shown
in Fig. 5. The spectral shapes of substituted aryl hydrazones of N-
hexyl-1,8-naphthalimide are similar to each other. In the spectral
band a long wavelength shoulder is observed, which is only hinted
at in the cases of 3d and 3e. The emission band of 3a (X¼N(CH3)2) is
situated in the red light area (Fig. 5). That is why the fluorescence
colour is red only. The emission maxima (Fmax) and full width at the
2.4. Solid-state fluorescence properties
Previously, we synthesized the hydrazone of 4-hydrazino-N-
hexyl-1,8-naphthalimide with piperonal (3i).29 Its solid-state fluo-
rescence was excited by violet and green light. The fluorescent

I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
717
Scheme 4. The main fragmentation pathway of hydrazones 3ae3h under ESI(þ) MS conditions.
Table 3
The emission maxima (Fmax) and full width at the half maxima of hydrazones 3ae3f,
3h and 3i in solid-state (lex¼390 nm)
Compound
X
Fmax (nm)
D
F
a (nm)
FWHM (nm)
Y
3a
3b
3c
3d
3e
3f
N(CH3)2
OCH3
CH3
H
Cl
CN
OCH3
OCH2O
H
H
H
H
H
H
OCH3
643
622
582
567
562
560
608
580
73
76
49
41
39
48
59
33
90
82
80
76
74
73
103
61
3h
3ib
a
D
F¼Fmax(solid)ꢁFmax(acetonitrile solution).
b
The hydrazone 3i was synthesized as described in Ref. 29.
charge transfer takes place from the aryl moiety towards the
naphthalimide part of the molecule in the hydrazones excited
state.14 In the solid state, this transition is facilitated by the sub-
stituents with þR-effect, whereas their inductive effect does not
Fig. 5. Normalized emission spectra (lex¼390 nm) of hydrazones 3ae3f, 3h and 3i in
solid-state (crystals).
influence Fmax
.
The solid-state fluorescence spectra of the crystalline forms are
red-shifted by 33e76 nm compared to those in solution (Table 3)
indicating the presence of intermolecular interactions in the solid
half maxima (FWHM) are presented in Table 3. The electron
accepting cyano group causes a small (7 nm) hypsochromic shift in
Fmax. The ED substituents generally lead to large bathochromic
shifts of the emission maxima, especially in the cases of 3b
(Fmax¼622 nm) and 3a (Fmax¼643 nm) compared to Fmax¼567 nm
for the unsubstituted compound (Table 3). The experimental
emission wavelengths for p-substituted hydrazones correlate ex-
state. The red-shift (
DF, Table 3) for the substituted hydrazones is
larger than F of the unsubstituted compound (3d), except for 3e
D
(X¼Cl) and 3i (X¼Y¼OCH2O). This shift increases with the en-
hancement of the þR-effect of the substituent. The lower red shift
(DF) of p-chloro derivative leading to a decline from the Hammett
type correlation is most probably due to the particular type of its
crystal structure. Most probably the stacking interactions in the
solid state prevent the intramolecular charge transfer.
cellently with Rþ constants with
pound 3e (X¼Cl) being out of the trend (Fig. 6). An intramolecular
r¼ꢁ43ꢀ3 (n¼5, r¼0.9915), com-

718
I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
be achieved by varying the aldehyde used as a visualization agent.
Usually, fluorochromes with emission maxima in the red area of the
visible spectrum are preferred due to the lack of background noise
caused by tissues autofluorescence.48
We tested different aldehydes for the localization of dipeptidyl
peptidase IV (DPPIV) and gamma-glutamyl transpeptidase (GGT)
activities in cryosections of rat organs according to the above
principle. Examples of the enzymes activities imaging are given in
Fig. 8. Best results were obtained with 1a, 1b,1e, 1f and 1h. Using all
these aldehydes, precise enzyme activity localization was observed.
The specificity of the enzyme reactions was confirmed by inhibition
studies using irreversible inhibitors. The fluorescent signal in the
preparations was strong and stable upon excitation (Fig. 8b, d). At
the same time, the red colour of the corresponding hydrazones
permits observations by light microscopy as well (Fig. 8a, c). The
histochemical samples are stable and can be studied several weeks
after the preparation.
Fig. 6. Dependence between experimental emission maxima and Rþ Hammett con-
stants for the series of p-substituted hydrazones 3ae3f. Compound 3e (X¼Cl) is out-
side of the trend established for this series.
3. Conclusions
Eight substituted aryl hydrazones of 4-hydrazino-N-hexyl-1,8-
naphthalimide were synthesized. A linear correlation between
chemical shift of the hydrogen, bonded to the amine type nitrogen
atom (NH) and sp constants of the substituents was observed for
the series of p-substituted hydrazones (3ae3g). The influence of
both field and resonance electronic effects and the contribution of
different resonance forms were evaluated using a model based on
the dual substituent parameters. The Hammett type correlation
between dH(NH) and sþp constants of the substituents was biphasic
concave upwards with a break at sþp ¼0.00 (X¼H). The excellent
correlation between dH(H-5) from naphthalimide moiety and R
substituent constants was observed to show that the amine nitro-
gen puts together the two parts (benzylideneamino and naph-
thalimide) in the hydrazone molecule in the ground state. The main
fragmentation pathway of hydrazones 3ae3h was determined us-
ing electrospray ionization mass spectrometry and high resolution
MS/MS. The highest relative intensity in the mass spectra of the
The full width at the half maximum depends on the electronic
effect of substituents. It varies from 61 nm for 3i to 103 nm for 3h
(X¼Y¼OCH3). The spectral band of 3h covers the area from 420 to
800 nm (Fig. 5). For the series of p-substituted hydrazones a Hammett
type correlation (n¼6, r¼0.9823) is determined between FWHM and
sþp constants of substituent X (Fig. 7). The slope is negative
(
r
¼ꢁ7.4ꢀ0.7) to show that EW substituents shorten the full width at
the half maximum, whereas ED substituents lengthen it (Table 3).
compounds was observed for [MHꢁC6H12
]
þ ions and the ions with
m/z 296. The solid-state fluorescence of all the hydrazones was
yellow-green and red, except for the p-nitro substituted compound,
which did not fluoresce. It was shown that a correlation exists
between experimental emission maxima and Rþ Hammett con-
stants for the series of p-substituted hydrazones 3ae3f (compound
3e (X¼Cl) is outside of the trend). A linear correlation of full width
at the half maxima of these hydrazones against sþp Hammett pa-
rameters was observed. It was shown that the newly developed
histochemical method based on the formation of fluorescent
hydrazones as final reaction products is suitable for the in situ
imaging of the enzyme activity. The proposed histochemical
method allows a precise localization of the enzymes since the red
emission eliminates the fluorescent background noise. The fluo-
rescence in the preparations is stable upon excitation and histo-
chemical samples can be used at least for several weeks.
Fig. 7. Linear correlation of the full width at the half maxima for the series of p-
substituted hydrazones 3ae3f against sþp Hammett parameters.
2.5. Enzyme activity imaging
The synthesized hydrazones have the useful quality of stable red
colour fluorescence in solid state. The study presented here allows
predicting their fluorescent properties depending on the electronic
effect of substituents in order to choose the most appropriate
compound for any particular application.
The synthesized 1,8-naphthalimide hydrazones can be used for
the fluorescent in situ imaging of enzyme activities according to the
previously described principle.29 Briefly, the enzyme substrates
represent amino acid or peptide hydrazides of 4-hydrazino-N-
hexyl-1,8-naphthalimide. The enzyme cleaves the hydrazide bond
and the released hydrazine reacts simultaneously with an aromatic
aldehyde to give a water-insoluble hydrazone precipitating on the
enzyme sites and marking them by an intensive fluorescence. As it
was described above, the emission maxima and full width at the
half maxima of hydrazones in the solid state depend on the elec-
tronic effect of the benzene ring substituents. Thus, a fluorescent
enzyme imaging of different fluorescence colour and intensity can
4. Experimental section
4.1. General
1H NMR spectra were obtained using a Bruker Avance AV
IIþ600 MHz NMR spectrometer and tetramethyl silane as an in-
ternal standard in CD3SOCD3 as a solvent. Chemical shifts (d) are

I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
719
Fig. 8. Fluorescent imaging of enzymes in the cryosections of rat organs using substrates based on 4-hydrazino-N-hexyl-1,8-naphthalimide and different aldehydes as visualization
agents. DPPIV activity localized by the substrate 4-Gly-Pro-hydrazido-N-hexyl-1,8-naphthalimide and 4-dimethylaminobenzaldehyde (a, b)dthe enzyme is active in the brush
border area of small intestinal enterocytes. GGT activity visualized by
g-Glu-hydrazido-N-hexyl-1,8-naphthalimide and 4-chlorobenzaldehyde (c, d)dthe enzyme activity is re-
stricted to the stereocilia of epithelial cells of the epididymis channels. Light microscopy (a, c); fluorescent microscopy (b, d). Original magnifications 200ꢂ.
expressed in parts per million and coupling constants (J) in hertz.
Multiplicity and qualifier abbreviations are as follows: s¼singlet,
d¼doublet, dd¼doublet of doublet, t¼triplet, br s¼broad singlet
and m¼multiplet.
MS/MS experiments were carried out in the positive mode of
operation using normalized collision dissociation (CID) energy
values of 30% AU for all of the compounds. The fragment ions were
detected in OrbitrapÒ analyzer at 15,000 preselected resolution.
Data acquisition and processing were carried out with XCaliburÒ
software package (ThetmoScientific Co, USA).
IR spectra were scanned on FTIR (Shimadzu IRPrestige-21) in
Nujol.
The chromatographic separation of the compounds was carried
out on an SurveyorÒ Plus HPLC system, which consisted of a binary
gradient pump, autosampler and PDA detector (ThermoScientific
Co, USA) by X-Bridge C18 column (I.D. 2.1 mmꢂ150 mm, particle
Measurements of fluorescence spectra were performed on
Varian Cary Eclipse (lex¼390 nm).
Elemental analyses were performed by VarioEL V5, CHNS Mode,
Elementar Analysensysteme GmbH. Microanalyses (C, H, N) of new
compounds were in agreement with the theoretical values within
ꢀ0.3%. Melting points were determined on a Botius micro melting
point apparatus and were uncorrected.
size 3.5 mm, Waters Corporation, USA) using as eluents: Ad0.1%
formic acid in water; Bd0.1% formic acid in acetonitrile at flow rate
of 250 L/min. The following binary gradient was used: 60e100% B
m
for 10 min; 100% B for 5 min and 100e60% B for 3 min.
Mass spectrometric investigations were performed on LTQ
OrbitrapÒ Discovery mass spectrometer (ThetmoScientific Co, USA)
equipped with electrospray ionization module IonMaxÒ (Thetmo-
Scientific Co, USA).
Full-scan spectra over the m/z range 200e1000 were acquired in
the positive and negative ion modes. The OrbitrapÒ was operated at
resolution settings of 30,000. All the OrbitrapÒ MS parameters were
optimized using the instrument control software program. The
following parameters were used: spray voltage 3.8 kV, spray cur-
Tissue sections were cut on cryotome Reichert-Jung, model
2800 (Germany). Enzyme reactions were observed under micro-
scope OPTON IM 35 (Germany).
The 4-hydrazino-N-butyl-1,8-naphthalimide was synthesized as
described before.29 The DPPIV inhibitor (N-(Phe-Pro)-O-(4-
nitrobenzoyl)hydroxylamine hydrochloride) was synthesized after
Demuth et al.49 The GGT inhibitor 6-diazo-5-oxo-norleucine and all
the organic solvents and aldehydes were purchased from Sigma-
eAldrich (Germany). Acetonitrile LCeMS ChromasolvÒ, formic acid
eluent additive for LCeMS and silica gel 60 (0.063e0.200 mm) for
column chromatography were from Fluka (Germany). Aluminium
oxide 90 neutral (0.063e0.250 mm) for column chromatography
was from Merck (Germany).
rent 100 mA, sheath gas flow rate 30 AU, auxiliary gas flow rate 0,
sweep gas flow rate 5 AU, capillary voltage 10 V, capillary tem-
perature 300 ꢃC and tube lens voltage 70 V.

720
I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
4.2. Synthesis of hydrazones
J¼7.4 Hz, 2H, H-30, H-50), 7.44 (t, J¼7.3 Hz, 1H, H-40), 4.05e4.00 (m,
2H, NCH2), 1.65e1.58 (m, 2H, NCH2CH2), 1.37e1.27 (m, 6H,
A mixture of 2 (0.062 g, 0.2 mmol) and the corresponding
benzaldehyde (0.2 mmol) in ethanol (2.8 mL) was refluxed for
5e15 min in the presence of acetic acid (10 mL). The solution was
allowed to crystallize at room temperature. The obtained pre-
cipitate was filtered, washed with cold ethanol and purified by
recrystallisation (3a) or column chromatography on silica gel
(3be3f, 3h) or aluminium oxide (3g).
CH2CH2(CH2)3), 0.87 (t, J¼7.0 Hz, 3H, CH3). Selected IR data
n
(cmꢁ1): 3283 (NH), 1685 (C]O), 1632 (C]O), 1616 (C]N); HRMS
calcd for C25H26N3O2 [MþH]þ m/z 400.2019, found HRMS-ESI(þ) m/
z 400.2021. Anal. Calcd for C25H25N3O2: C, 75.16; H, 6.31; N, 10.52%,
found: C, 74.96; H, 6.59; N, 10.37%.
4.2.5. 6-(2-(4-Chlorobenzylidene)hydrazinyl)-2-hexyl-1H-benzo[de]
isoquinoline-1,3(2H)-dione (3e). The crude product was chromato-
graphed (dichloromethane/hexane/isopropanol, 1:5:0.1) to yield
compound 3e (0.050 g, 58%) as yellow crystals; mp 188e190 ꢃC; 1H
4.2.1. 6-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-2-hexyl-1H-
benzo[de]isoquinoline-1,3(2H)-dione (3a). To the cold solution,
water (250
recrystallized from ethanol/water to yield compound 3a (0.048 g,
54%) as dark purple crystals; mp 215e217 ꢃC; 1H NMR
(ppm):
m
L) was added. The obtained precipitate was filtered and
NMR
d
(ppm): 11.54 (s, 1H, NHN]), 8.78 (d, J¼8.4 Hz, 1H, H-9), 8.47
(d, J¼7.2 Hz, 1H, H-7), 8.42 (s, 1H, CH]N), 8.36 (d, J¼8.5 Hz, 1H, H-
4), 7.83 (d, J¼8.5 Hz, 2H, H-20, H-60), 7.81e7.77 (m, 1H, H-8), 7.73 (d,
J¼8.5 Hz, 1H, H-5), 7.53 (d, J¼8.4 Hz, 2H, H-30, H-50), 4.02e3.98 (m,
2H, NCH2), 1.64e1.56 (m, 2H, NCH2CH2), 1.31 (dt, J¼14.0, 7.3 Hz, 6H,
d
11.25 (s, 1H, NHN]), 8.78 (d, J¼8.4 Hz, 1H, H-9), 8.47 (d, J¼7.2 Hz,
1H, H-7), 8.35 (s, 1H, CH]N), 8.35 (d, J¼8.9 Hz, 1H, H-4), 7.76 (dd,
J¼8.0, 7.6 Hz, 1H, H-8), 7.65 (d, J¼8.5 Hz, 1H, H-5), 7.63 (d, J¼8.8 Hz,
2H, H-20, H-60), 6.79 (d, J¼8.8 Hz, 2H, H-30, H-50), 4.04e3.99 (m, 2H,
NCH2), 3.00 (s, 6H, eN(CH3)2), 1.65e1.57 (m, 2H, NCH2CH2),
1.37e1.25 (m, 6H, CH2CH2(CH2)3), 0.87 (t, J¼6.9 Hz, 3H, CH3). Se-
CH2CH2(CH2)3), 0.86 (t, J¼6.9 Hz, 3H, CH3). Selected IR data
n
(cmꢁ1): 3309 (NH), 1695 (C]O), 1638 (C]O), 1616 (C]N), 1093 (C-
Cl); HRMS calcd for C25H25ClN3O2 [MþH]þ m/z 434.1630 and
436.1600, found HRMS-ESI(þ) m/z 434.1631 and 436.1595 (30%).
Anal. Calcd for C25H24ClN3O2: C, 69.20; H, 5.57; N, 9.68%, found: C,
69.23; H, 5.77; N, 9.56%.
lected IR data
n
(cmꢁ1): 3308 (NH), 1685 (C]O), 1635 (C]O), 1616
(C]N); HRMS calcd for C27H31N4O2 [MþH]þ m/z 443.2441, found
HRMS-ESI(þ) m/z 443.2445. Anal. Calcd for C27H30N4O2: C, 73.28;
H, 6.83; N, 12.66%, found: C, 73.01; H, 6.65; N, 12.77%.
4.2.6. 4-((2-(2-Hexyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]iso-
quinolin-6-yl)hydrazono)methyl)benzonitrile (3f). The crude prod-
uct was chromatographed (dichloromethane/hexane/isopropanol,
1:3:0.1) to yield compound 3f (0.036 g, 42%) as yellow crystals; mp
4.2.2. 2-Hexyl-6-(2-(4-methoxybenzylidene)hydrazinyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (3b). The crude product was chro-
matographed (dichloromethane/hexane/isopropanol, 1:3:0.1) to
yield compound 3b (0.038 g, 44%) as red crystals; mp 195e197 ꢃC;
175e177 ꢃC; 1H NMR
d (ppm): 11.72 (s, 1H, NHN]), 8.79 (d,
J¼8.5 Hz, 1H, H-9), 8.48 (d, J¼7.2 Hz, 1H, H-7), 8.45 (s, 1H, CH]N),
8.38 (d, J¼8.4 Hz, 1H, H-4), 7.97 (d, J¼8.1 Hz, 2H, H-20, H-60), 7.91 (d,
J¼8.1 Hz, 2H, H-30, H-50), 7.81 (t, J¼7.8 Hz,1H, H-8), 7.78 (d, J¼8.4 Hz,
1H, H-5), 4.00 (t, J¼7.5 Hz, 2H, NCH2), 1.64e1.57 (m, 2H, NCH2CH2),
1.36e1.25 (m, 6H, CH2CH2(CH2)3), 0.87 (t, J¼6.5 Hz, 3H, CH3). Se-
1H NMR
d
(ppm): 11.39 (s, 1H, NHN]), 8.79 (d, J¼8.4 Hz, 1H, H-9),
8.48 (d, J¼7.3 Hz, 1H, H-7), 8.42 (s, 1H, CH]N), 8.42 (d, J¼8.5 Hz, 1H,
H-4), 7.79 (dd, J¼8.2, 7.4 Hz, 1H, H-8), 7.76 (d, J¼8.7 Hz, 2H, H-20, H-
60), 7.70 (d, J¼8.5 Hz, 1H, H-5), 7.05 (d, J¼8.8 Hz, 2H, H-30, H-50),
4.04e3.99 (m, 2H, NCH2), 3.83 (s, 3H, CH3OC6H4), 1.65e1.57 (m, 2H,
NCH2CH2), 1.37e1.25 (m, 6H, CH2CH2(CH2)3), 0.87 (t, J¼7.0 Hz, 3H,
lected IR data n
(Nujol, cmꢁ1): 3307 (NH), 2228 (C^N), 1691 (C]O),
1638 (C]O), 1616 (C]N); HRMS calcd for C26H25N4O2 [MþH]þ m/z
425.1972, found HRMS-ESI(þ) m/z 425.1973. Anal. Calcd for
C26H24N4O2: C, 73.56; H, 5.70; N, 13.20%, found: C, 73.37; H, 5.64; N,
12.98%.
CH3). Selected IR data
n
(cmꢁ1): 3315 (NH), 1683 (C]O), 1641 (C]
O), 1616 (C]N), 1251 (CeOeC), 1029 (CeOeC); HRMS calcd for
C26H28N3O3 [MþH]þ m/z 430.2125, found HRMS-ESI(þ) m/z
430.2126. Anal. Calcd for C26H27N3O3: C, 72.71; H, 6.34; N, 9.78%,
found: C, 72.94; H, 6.25; N, 9.62%.
4.2.7. 2-Hexyl-6-(2-(4-nitrobenzylidene)hydrazinyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (3g). The crude product was chromato-
graphed on neutral aluminium oxide (dichloromethane/hexane/
isopropanol, 1:5:0.1) to yield compound 3g (0.045 g, 51%) as
4.2.3. 2-Hexyl-6-(2-(4-methylbenzylidene)hydrazinyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (3c). The crude product was chromato-
graphed (dichloromethane/hexane/isopropanol, 1:3:0.1) to yield
compound 3c (0.037 g, 45%) as orange crystals; mp 185e187 ꢃC; 1H
orange-red crystals; mp 231e233 ꢃC; 1H NMR
d (ppm): 11.82 (s, 1H,
NH), 8.88e8.77 (m, 1H, H9), 8.58e8.46 (m, 2H, H7 & CH]N), 8.41
(br s, 1H, H4), 8.35e8.26 (m, 2H, H30 & H50), 8.11e8.01 (m, 2H, H20 &
H60), 7.90e7.79 (m, 2H, H5 & H8), 4.09e3.95 (m, 2H, NCH2),
1.72e1.52 (m, 2H, NCH2CH2), 1.39e1.14 (m, 6H, NCH2(CH2)3),
NMR
d
(ppm): 11.46 (s, 1H, NHN]), 8.81 (dd, J¼8.5, 0.8 Hz, 1H, H-9),
8.49 (dd, J¼7.2, 0.9 Hz, 1H, H-7), 8.44 (s, 1H, CH]N), 8.38 (d,
J¼8.5 Hz,1H, H-4), 7.80 (dd, J¼8.4, 7.3 Hz,1H, H-8), 7.74 (d, J¼8.5 Hz,
1H, H-5), 7.72 (d, J¼8.1 Hz, 2H, H-20, H-60), 7.30 (d, J¼7.9 Hz, 2H, H-
30, H-50), 4.05e3.98 (m, 2H, NCH2), 2.37 (s, 3H, CH3C6H4), 1.66e1.58
(m, 2H, NCH2CH2), 1.37e1.26 (m, 6H, CH2CH2(CH2)3), 0.87 (t,
0.93e0.81 (m, 3H, CH3). Selected IR data n
(cmꢁ1): 3313 (NH), 1690
(C]O), 1644 (C]O), 1616 (C]N), 1512 (NO2), 1337 (NO2); HRMS
calcd for C25H25N4O4 [MþH]þ m/z 445.1870, found HR-ESI(þ) m/z
445.1864. Anal. Calcd for C25H24N4O4: C, 67.55; H, 5.44; N, 12.60%,
found: C, 67.35; H, 5.30; N, 12.78%.
J¼7.0 Hz, 3H, CH3). Selected IR data
n
(cmꢁ1): 3291 (NH), 1691 (C]
O), 1635 (C]O), 1616 (C]N); HRMS calcd for C26H28N3O2 [MþH]þ
m/z 414.2176, found HRMS-ESI(þ) m/z 414.2173. Anal. Calcd for
C26H27N3O2: C, 75.52; H, 6.58; N, 10.16%, found: C, 75.38; H, 6.44; N,
10.05%.
4.2.8. 6-(2-(3,4-Dimethoxybenzylidene)hydrazinyl)-2-hexyl-1H-
benzo[de]isoquinoline-1,3(2H)-dione (3h). The crude product was
chromatographed (dichloromethane/hexane/isopropanol, 1:6:0.1)
to yield compound 3h (0.039 g, 42%) as red crystals; mp
4.2.4. 6-(2-Benzylidenehydrazinyl)-2-hexyl-1H-benzo[de]isoquino-
line-1,3(2H)-dione (3d). The crude product was chromatographed
(dichloromethane/hexane/isopropanol, 1:6:0.1) to yield compound
3d (0.027 g, 34%) as yellow crystals. Yield 0.027 g (34%), crystals;
148e150 ꢃC; 1H NMR
d (ppm): 11.39 (s, 1H, NHN]), 8.79 (d,
J¼8.5 Hz, 1H, H-9), 8.48 (dd, J¼7.3, 1.0 Hz, 1H, H-7), 8.39 (s, 1H, CH]
N), 8.37 (d, J¼8.5 Hz, 1H, H-4), 7.79 (dd, J¼8.4, 7.3 Hz, 1H, H-8), 7.74
(d, J¼8.5 Hz, 1H, H-5), 7.45 (d, J¼1.9 Hz, 1H, H-20), 7.27 (dd, J¼8.4,
1.9 Hz, 1H, H-60), 7.05 (d, J¼8.4 Hz, 1H, H-50), 4.03e3.99 (m, 2H,
NCH2), 3.89 (s, 3H, CH3OC6H4), 3.83 (s, 3H, CH3OC6H4), 1.65e1.57
(m, 2H, NCH2), 1.36e1.28 (m, 6H) CH2CH2(CH2)3, 0.87 (t, J¼7.1 Hz,
mp 207e209 ꢃC; 1H NMR
d (ppm): 11.52 (s, 1H, NHN]), 8.82 (d,
J¼8.4 Hz, 1H, H-9), 8.50 (d, J¼7.2 Hz, 1H, H-7), 8.48 (s, 1H, CH]N),
8.40 (d, J¼8.5 Hz, 1H, H-4), 7.83 (d, J¼7.2 Hz, 2H, H-20, H-60), 7.82
(dd, J¼8.0, 7.3 Hz, 1H, H-8), 7.77 (d, J¼8.5 Hz, 1H, H-5), 7.50 (t,

I.P. Ivanov et al. / Tetrahedron 69 (2013) 712e721
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721
3H, CH3). Selected IR data
n
(cmꢁ1): 3302 (NH), 1685 (C]O), 1639
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Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2012.10.093.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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