Novel carbohydrazide and hydrazone biomarkers based on 9-substituted acridine and anthracene fluorogens

Article abstract of DOI:10.3987/COM-09-S(S)83

Three series of carbohydrazides or hydrazones bearing either acridine or anthracene pharmacophores were synthesized as potential noncovalent DNA-binding antitumor agents. Carbohydrazides with an acridine or anthracene moiety were prepared from appropriate acridine or anthracene carbaldehydes via cyclocondensation with selected hydrazides whilst hydrazones with a 10H-acridin-9-ylidene moiety were obtained by condensation of (acridin-9-yl)hydrazine with various aldehydes or ketones. The spectroscopic properties of the first two series revealed efficient fluorescence implying that the compounds could be amenable for use as biomarkers. The structures of the compounds were characterized by spectral methods (UV-vis, fluorescence, IR, and ¹H, ¹³C, and 2D NMR) and quantum-chemical calculations (DFT, ZINDO, and AM1). The first carbohydrazide series was also tested against human leukemia cell line HL-60 wherein the phenyl-substituted derivative was found to possess the highest activity.

Full text of DOI:10.3987/COM-09-S(S)83

HETEROCYCLES, Vol. 80, No. 2, 2010
1047
HETEROCYCLES, Vol. 80, No. 2, 2010, pp. 1047 - 1066. © The Japan Institute of Heterocyclic Chemistry
Received, 28th July, 2009, Accepted, 4th September, 2009, Published online, 8th September, 2009
DOI: 10.3987/COM-09-S(S)83
NOVEL CARBOHYDRAZIDE AND HYDRAZONE BIOMARKERS
BASED ON 9-SUBSTITUTED ACRIDINE AND ANTHRACENE
FLUOROGENS†
Zdenka Bedlovičová,1 Ján Imrich,1* Pavol Kristian,1 Ivan Danihel,1 Stanislav
Böhm,2 Danica Sabolová,1 Mária Kožurková,1 Helena Paulíková,3 and Karel
D. Klika4
1
Institute of Chemistry, Faculty of Science, P. J. Šafárik University, SK-04167
Košice, Slovak Republic. Email: jan.imrich@upjs.sk
2
Department of Organic Chemistry, Institute of Chemical Technology, CZ-16628
Prague, Czech Republic
3
Department of Biochemistry and Microbiology, Faculty of Chemical and Food
Technology, Slovak Technical University, SK-81237 Bratislava, Slovak Republic
4 Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Abstract – Three series of carbohydrazides or hydrazones bearing either acridine
or anthracene pharmacophores were synthesized as potential noncovalent
DNA-binding antitumor agents. Carbohydrazides with an acridine or anthracene
moiety were prepared from appropriate acridine or anthracene carbaldehydes via
cyclocondensation with selected hydrazides whilst hydrazones with a
10H-acridin-9-ylidene
moiety
were
obtained
by
condensation
of
(acridin-9-yl)hydrazine with various aldehydes or ketones. The spectroscopic
properties of the first two series revealed efficient fluorescence implying that the
compounds could be amenable for use as biomarkers. The structures of the
compounds were characterized by spectral methods (UV-vis, fluorescence, IR, and
13
1H, C, and 2D NMR) and quantum-chemical calculations (DFT, ZINDO, and
AM1). The first carbohydrazide series was also tested against human leukemia cell
line HL-60 wherein the phenyl-substituted derivative was found to possess the
highest activity.
† Dedicated to Prof. Emeritus Akira Suzuki on the occasion of his 80th birthday.

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HETEROCYCLES, Vol. 80, No. 2, 2010
INTRODUCTION
The chemistry and biological properties of acridines13, especially their proclivity towards
spiroopen-chain tautomerism1gj as well as intercalating1ag, fluorescent4, and antitumor activities5, have
been the focal point of our research interest over two decades. Similarly too, because of their intercalating
and fluorescent properties, we have paid attention to analogous anthracene substrates.6 Analogously,
azomethines represent a class of compounds which are interesting not only from chemical1b and
spectroscopic2 points of view, but also because they are the subject of biological and pharmaceutical
research endeavors.7,8 Structural studies on the imine double bond of azomethines and ketimines have
been conducted9 where the azomethines were found to occur as only one geometrical isomer whilst the
ketimines were found to be present in the reaction mixture as two geometrical isomers. Recently, we have
reported the physico-chemical properties of potentially intercalating 4-acridonecarboxamide imines.2
The aim of this contribution is to explore the effect of acridine vs. anthracene and the effect of
substitution in the side-chain on the properties of analogous 9-substituted acridine/anthracene
carbohydrazide and hydrazone compounds with respect to conjugation, prototropic tautomerism, intra-
and interunit geometry, and spectral properties, in particular fluorescence, as well as their biological
activities. The three series of novel carbohydrazides and hydrazones prepared here, together with a
previous set,2 afforded a comparison between the various permutations regarding the positional placement
of the tricyclic aromatic system, i.e. the acridine or anthracene moiety, with respect to the substituent unit.
RESULTS AND DISCUSSION
Synthesis
The synthesis of the first series of carbohydrazides 5ae (Scheme 1) started from commercially available
diphenylamine (1) giving, after modification of Bernthsen’s reaction,10 9-methylacridine (2). Following
radical bromination of 2, 9-bromomethylacridine (3) was obtained11 and this provided (acridin-9-yl)
carbaldehyde (4) after oxidation with sodium and 2-nitropropane.12 Subsequent condensation with
various hydrazides (methyl, a; phenyl, b; 3-pyridyl, c; 4-pyridyl, d; and (9-oxoacridin-4-yl), e) then
yielded substituted N'-(acridin-9-ylmethylene)hydrazides 5ae in yields up to 74%. Anthracenyl
derivatives of the second series were prepared by the condensation of commercially available
9-anthraldehyde
(6)
with
the
same
set
of
hydrazides
to
provide
substituted
N'-(anthracen-9-ylmethylene)hydrazides 7ae in excellent yields up to 90%. The third series of
disubstituted hydrazones of 9,10-dihydroacridine, together with two trisubstituted ones, were prepared as
indicated in Scheme 2 starting from the UllmannJourdan condensation of o-chlorobenzoic acid (8) and
aniline (9) to provide N-phenylanthranilic acid (10). Compound 10 upon treatment with POCl3 yielded

HETEROCYCLES, Vol. 80, No. 2, 2010
1049
Br
H2C
Me
i
ii
N
N
N
H
1
2
3
7
O
8
6
HC
H
C
11
H
N
13
8a
14
5
R
iii
iv
N
10a
9
N 10
4a
12
9a
O
1
N
4
2
4
5a-e
3
H
H
H
N
C
R
iv
O
N
9
10
O
1
4
6
7a-e
R = methyl (a), phenyl (b), 3-pyridyl (c), 4-pyridyl (d), 9-oxoacridin-4-yl (e)
Scheme 1. Reaction sequence leading to carbohydrazide series 5ae and 7ae. Reagents and conditions:
(i) ZnCl2, acetic acid, 190 C; (ii) NBS, AIBN, CCl4, reflux; (iii) DMF, MeOH, Na, 2-nitropropane, rt;
(iv) MeOH, H+, corresponding hydrazides, rt.
Cl
NH2
CO2H
CO2H
ii
i
+
N
N
H
Cl
11
9
8
10
R1
R2
13
NH2
12
N
N
11
N
1
iv
iii
9
N
H
N
H
13a-g
12
13
R1
a
b
c
d
e
f
g
H
H
H
H
H
methyl methyl
R2 phenyl 2,4,6-trimethoxyphenyl 4-bromophenyl 4-methoxyphenyl 2,4,6-trimethylphenyl methyl phenyl
Scheme 2. Reaction sequence leading to hydrazone series 13ag. Reagents and conditions: (i) Cu, K2CO3,
2-methoxyethanol, reflux; (ii) POCl3, reflux; (iii) NH2NH2·H2O, MeOH, reflux; (iv) EtOH, H+,
appropriate aldehyde or ketone, reflux.

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HETEROCYCLES, Vol. 80, No. 2, 2010
9-chloroacridine (11), which then readily yielded (acridin-9-yl)hydrazine (12) by the application of
hydrazine monohydrate. Disubstituted 13ae and trisubstituted 9-(alkyl/benzylidenehydrazono)-9,10-
dihydroacridines 13f,g were then prepared by condensation of 12 with a set of aldehydes and ketones
(benzaldehyde, a; 2,4,6-trimethoxybenzaldehyde, b; 4-bromobenzaldehyde, c; 4-methoxybenzaldehyde,
d; 2,4,6-trimethylbenzaldehyde, e; acetone, f; acetophenone, g) under acidic catalysis in yields of
4193% for the 13ae and 38% and 42% for 13f,g, respectively.
NMR spectroscopy
In general, the N-10 nitrogen located in the structural framework of the acridine moiety has an extremely
strong tendency to retain a proton,1b,f,g,in,3,13 i.e. if tautomeric interchanges are possible, then the H-10
tautomer usually dominates and the predominance of the equilibrium can easily be ascertained by
standard methods of appraisal (correlations and diagnostic NMR chemical shifts compared to previous
reports1b,fn,3). Thus determination that the H-13 tautomer as depicted in Scheme 1 dominates over the
H-10 tautomer for the compounds of the acridine series 5ae provides a rare exception1h against this
general rule, and despite the potential gain from the additional conjugation with the carbonyl double bond
that would arise. The H-13 tautomeric structure permits free rotation about the C9C11 bond and as a
consequence the outer rings of the acridine moiety are rendered equivalent on the NMR timescale.
Interestingly, the only two compounds of the three series to displayed two sets of signals in solution for a
major and minor species, the first one, 5a, in the integral intensity ratio 67 : 33 (Figure 1). Upon heating
1
to 80° C the corresponding signals in the H NMR spectrum started to coalescence but resumed their
normal appearance upon lowering the temperature back to 25° C. It appears that this equilibrium is due to
E/Z configurational interchange about the amide bond, i.e. geometrical isomerism, based on the chemical
shift differences which exist only in the side-chain for these two species. The other derivatives
500
10000
400
300
5000
200
100
0
0
10.0
5.0
10.0
5.0
ppm (f1)
ppm (f1)
Figure 1. 1H NMR spectrum of the compound 5a at 25 ºC (left) and 80 ºC (right).

HETEROCYCLES, Vol. 80, No. 2, 2010
1051
5be in the series and compounds 7be in the second series are likely to be simply strongly biased in this
respect and thus they exhibit only the one set of signals. The corresponding methyl-substituted derivative
7a in the anthracene series also showed two species in the ratio 69 : 31, the minor one characterized by
some typical chemical shifts with other signals overlapped by the signals of the major species.
For the predominant tautomeric structures of 5ae, there are two competing mechanisms for conjugation
of the lone pair of the N-13 nitrogen atom (structure A, Figure 2). The NMR chemical shifts for C-9,
C-11, and the carbonyl carbon C-14 as well as the chemical shifts of the protons in the side-chain are all
strongly affected by these mechanisms, in particular if there is a dominant one in effect.
O
R1
H
N
N
R
N
H
N
N
A
N
H
B
Figure 2. Structures showing the conjugation pathways in the first series for compounds 5ae (structure
A) and the third series for compounds 13ag (structure B).
Conjugation of the considered lone pair with the carbonyl oxygen is affected by the substituents R
attached to the carbonyl carbon whilst conjugation extending down into the tricyclic aromatic system is
obviously determined to a large extent by whether an acridine or an anthracene moiety is present since
replacement of the nitrogen atom N-10 in the acridinyl series by a carbon atom in the anthracenyl series
will clearly mitigate any conjugation arising from the tricyclic aromatic system. Hence, comparison of the
observed nuclear shifts in series 5ae with the analogous series 7ae should thus allude to any
dominating mechanism. The pertinent chemical shifts presented in Table 1 are in accordance with the
expected electronic character of the substituents. For example, the effect of the electron donating
character of the methyl substituent in 5a on carbon C-11 is unequivocal (138.9 ppm) whilst the electron
accepting character of the 4-pyridinyl-substituent of 5d (146.1 ppm) is also evident. Conversely, a reverse
substituent effect is obvious for the carbonyl carbon C-14, cf. 5a, 172.4 ppm and 5d, 161.9 ppm, and this
same effect is also observed for the acridinyl carbon C-9. While the chemical shifts of carbons C-4a and
C-10a are affected only very slightly by the substituents, those of carbons C-8a and C-9a are not affected
at all. The effects of the substituents on the chemical shifts of the amidic hydrogen H-13 are likewise in
concert with the electronic character of the substituents, e.g. cf. 5a, 11.79 ppm and 5d, 12.56 ppm.

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HETEROCYCLES, Vol. 80, No. 2, 2010
Table 1. Selected 1H and 13C NMR chemical shifts of compounds 5ae, 7ae, and 13ag
Cmpd
5a, major
5a, minor
5b
H-10

H-11
9.25
9.42
9.69
9.85
9.68
9.75
9.21
9.34
9.71
9.69
9.70
9.76

H-13
C-9
C-10
C-11
138.9
142.0
144.8
144.9
146.1
145.9
C-13
C-14
172.4
166.2
163.4
161.8
161.9
162.7
171.9
165.6
163.0
161.7
161.5
163.9

11.79 136.8













-
a
12.16
-

12.35 134.6
12.96 137.9
12.56 134.3

5c

5d


a
5e
12.70
7a, major
7a, minor
7b
8.70
8.64
8.74
8.73
8.75
8.75
10.83
10.60
10.89
10.73
10.78
10.43
10.68
11.47 125.1 129.1 141.4
11.69 144.5
a
a
12.14 125.0 129.6 146.9
12.34 124.5 129.8 147.7
12.34 124.7 129.9 148.3
12.47 124.5 130.1 148.3

7c

7d

7e

13a
8.55
8.80
8.53
8.48
8.87

149.8
148.8
149.9
149.2
149.6
144.2
145.5
154.4
150.9
153.1
154.3
155.6
158.0
155.8














13b


13c


13d


13e


13f


13g



a Not detected.
Similar substituent effects are also manifested in the anthracene series 7ae. For example, the chemical
shift of C-14, cf. 7a, 171.9 ppm and 7d, 161.5 ppm, are comparable to the comparison of 5a. The evident
slight difference between the two series is due to the electron accepting character of the N-10 nitrogen
atom in acridine compared to the C-10 carbon atom in the anthracene unit.
Thus, from the chemical shift values, it can be concluded that there is an efficient transfer mechanism of
electronic effects from the substituents throughout the molecules in these two series of compounds 5ae
and 7ae but it appears there is considerable participation from both mechanisms with neither
dominating.
In the case of derivatives 13ag, the values of various diagnostic 13C NMR chemical shifts (e.g. C-4a and
C-10a, 138142; C-8a and C-9a, 114120; and, in particular,1i,3 C-4 and C-5, 115116 ppm) in
comparison to previous reports1b,fn,3 clearly indicate that the dihydroacridine structure of the acridine
moiety is predominant, i.e. the H-10 tautomer dominates over the H-11 tautomer. For the H-10 tautomer,

HETEROCYCLES, Vol. 80, No. 2, 2010
1053
interchange between the conformers is slow and as a consequence the outer rings of the acridine moiety
are rendered inequivalent on the NMR timescale.
The conjugation between the acridine unit, emanating from the nitrogen heteroatom, and the side-chain is
mitigated by the substituents R1 and R2 with the phenyl substituents aiding conjugation whilst the methyl
substituents in ketimines 13f,g reduce this conjugation by their inductive effects. These inductive effects
manifest themselves on the chemical shift of the carbon of the azomethine group (C=N) with some
deshielding evident for C-13 in 13g (phenyl and methyl substituents present) and with greater deshielding
for C-13 in 13f (two methyl substituents present). Conversely, there is some shielding observed for C-9 in
13g in comparison to the very consistent values for aldimines 13ae (149150 ppm) with greater
deshielding evident for the carbon atom C-9 in 13g. The aiding of conjugation by the phenyl substituents
was also evident from the UV spectra (vide infra).
UV-vis absorption spectroscopy
Electronic absorption spectra of compounds 5ad, 7ae, and 13ag measured in methanol are presented
in Table 2. Compounds 5ad exhibited broad absorption bands with their principal maxima lying in the
range 363367 nm together with a shoulder in the range 402407 nm. Compounds 7ad each displayed
only a clear single band in the range 386413 nm whilst compounds 13ag each displayed a long
wavelength maximum in the range 406441 nm together with one or more two bands at ca. 320 and 280
nm. These absorbances are characteristic for nπ* transitions and the long wavelength maxima of
compounds 13 are shifted to longer wavelengths compared to series 5ad and 7ae as a consequence of
extended conjugation (cf. Figure 2).
In compounds 5ad, the positions of the absorption maxima are only slightly influenced by the nature of
the substituents (Figure 3) whereas by comparison the changes were more notable for 7ae and 13ag
(Table 2). The largest bathochromic shift was observed for 13c due to the presence of the 4-bromophenyl
substituent which not only extends the conjugation by way of the phenyl unit, but the large effect of
which could be attributed also to the electron accepting character of the bromine atom. On the other hand,
the shorter conjugation system due to the absence of the phenyl ring in 13f was confirmed by a distinct
hypsochromic shift of the long wavelength maximum to 406 nm.
Conjugation and transitions in the UV-vis spectra for the phenyl-substituted compounds 5b, 7b, and 13a
were interpreted by orbital analysis using the semiempirical ZINDO method on structures optimized by
DFT at the B3LYP/6-311+G(d,p) level of theory. From the orbitals depicted in Figure 4 it follows that the
HOMO and LUMO represent net π and π* orbitals and these contribute strongly to the conjugation of the
acridine moiety and the side-chain. Intriguingly, the phenyl substituent in 5b and 7b does not participate
in the extended conjugation in contrast to 13a where the phenyl substituent does appear to participate.

1054
HETEROCYCLES, Vol. 80, No. 2, 2010
Table 2. UV-vis absorption maximaa of compounds 5ad, 7ae, and 13ag
Cmpd
λmax (nm)
Absorb.
log ε
Cmpd
λmax (nm)
Absorb.
0.216
log ε
402
363
0.246
0.337
4.71
4.85
5a
7e
386
4.65
434
323
275
0.479
0.258
0.438
5.00
4.73
4.96
407
367
0.224
0.273
4.67
4.75
5b
13a
407
360
284
0.192
0.229
0.241
4.60
4.68
4.70
436
325
290
0.630
0.369
0.433
5.12
4.89
4.96
5c
5d
7a
13b
13c
13d
402
367
0.173
0.205
4.56
4.63
441
275
0.526
0.487
5.04
5.01
434
320
275
0.580
0.372
0.505
5.08
4.89
5.02
395
411
0.408
0.200
4.93
4.62
434
326
289
0.419
0.246
0.297
4.94
4.71
4.79
7b
13e
406
288
0.441
0.419
4.96
4.94
7c
413
390
0.372
0.444
4.89
4.97
13f
421
275
0.417
0.459
4.94
4.98
7d
13g
a In methanol at 24 C; conc., 4.8  106 M.
In the case of 5b, the contribution to the absorbance band of the lone electron pair of N-10 in the acridine
moiety is via the HOMO-4 and HOMO-5 orbitals. A similar contribution by the analogous lone pair in
13a was not found and it participates via the π (and π* accordingly) HOMO, HOMO-1, HOMO-2, and
LUMO orbitals.
0.4
13a
13b
13c
13d
13e
13f
5a
5b
5c
5d
0.6
0.4
0.2
0
0.3
0.2
0.1
0
13g
300
350
400
450
500
550
300
400
500
600
Wavelength (nm)
Wavelenght (nm)
Figure 3. UV-vis spectra of compounds 5ad (left) and 13a-g (right) (in methanol at 24 C; conc., 4.8 
106 M).

HETEROCYCLES, Vol. 80, No. 2, 2010
1055
a
b
c
Figure 4. HOMO of derivatives 5b (a), 7b (b), and 13a (c).
Fluorescence emission spectroscopy
Due to the planar aromatic units they contain, acridine and anthracene derivatives express unique
chemical and physical properties with substitution at the C-9 position able to improve their luminescent
properties.14 Fluorescence emission spectra taken in methanol of compounds 5ad, 7ad, and 13b,c were
recorded at excitation wavelengths corresponding to their absorbance maxima. It can be seen from Figure
5 that compounds 5ad each had one emission maximum in the range 479482 nm but that of the
reference compound 9-isothiocyanatoacridine (acridine-ITC) exhibited three emission peaks at 479, 457,
and 429 nm (Table 3). The highest fluorescence observed, that for derivative 5c, is almost 14-fold higher
by comparison to acridine-ITC, a compound known as a fluorescence marker for amino and thiol groups
present in biomacromolecules.15 The fluorescence emission spectra of compounds 4, 7ad, 13b,c, and the
reference compound acridine are summarized in Table 4 wherein it can be seen that prepared compounds
700
5a
600
500
400
5b
5c
5d
Acridine-ITC
300
200
100
0
400
450
500
550
600
650
700
Wavelength (nm)
Figure 5. The emission fluorescence spectra of compounds 5ad and 9-isothiocyanatoacridine
(acridine-ITC) in methanol at 24 C (conc., 6.0  106 M).

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HETEROCYCLES, Vol. 80, No. 2, 2010
Table 3. Fluorescence spectral data for 5ad and 9-isothiocyanatoacridine (acridine-ITC)
a
Compound
λem (nm)
479
F (a.u.)
299.14
F/Fo
5a
6.72
5b
482
487.49
10.95
13.82
12.92
1.00
5c
5d
482
615.02
480
575.51
acridine-ITC
479, 457, 429
44.51, 84.5, 97.66
a Relative fluorescence related to fluorescence of a long wavelength maximum of 6.0  106 M solution of acridine-ITC in
methanol, slit width 10 nm.
Table 4. Fluorescence spectral data for 4, 7ad, 13b,c, and acridine
a
Compound
λem (nm)
427
F (a.u.)
305.33
341.43
97.28
F/Fo
3.24
3.63
1.03
2.57
2.20
0.18
0.61
1.00
4
7a
459
7b
477
7c
478
242.00
206.70
16.96
7d
485
13b
13c
acridine
510
501
57.78
418
94.15
a Relative fluorescence related to fluorescence maximum of 6.0  106 M solution of acridine in methanol, slit width 20 nm.
5d
600
5d*
7d
400
7d*
200
0
400
500
600
700
800
Wavelength (nm)
Figure 6. The emission fluorescence spectra of compounds 5d and 7d (in methanol at 24 C; conc., 6.0 
106 M) and their excimer species 5d* and 7d* (in methanol at 24 C; conc., 3.8  103 M).
excepting 13b,c exhibited stronger fluorescence than the reference. For samples of varying concentration,
the observation of a non-linear concentration dependence of fluorescence is an indication of a change in
the oligomeric state of a fluorogen with the most common oligomeric forms of fluorogens, J-aggregates

HETEROCYCLES, Vol. 80, No. 2, 2010
1057
and excimers, evident by the sharpening of their absorbance spectra and broad red-shifted fluorescence,
respectively.16 At high concentration, 3.8 x 10-3 M, a red-shifted fluorescence ca. 40–70 nm was observed
for the acridine derivative 5d as well as for the analogous anthracene derivative 7d (Figure 6) but, notably,
without any significant perturbations in their absorbance spectra. These changes in the fluorescence
spectra are thus ascribed15,16 to the formation of excimer species for these two compounds (5d* and 7d*,
respectively).
The observed reduced fluorescence in 13b,c and its underlying cause is of interest and the explanation
may lie in the structural differences between the ground and the first excited states. For the
DFT-optimized structures at the B3LYP/6-311+G(d,p) level of theory of the methyl-substituted
derivatives 5a and 7a, amongst the probable structures, the most preferable were found to be the
E,E-configurations (Figure 7a,b) where the HC=NNC=O segment is also planar. By contrast, whilst
the acridine and anthracene moieties in 5a and 7a, respectively, are perfectly planar, the dihydroacridine
moiety in the dimethyl-substituted derivative 13f is considerably deformed (Figure 7c) and no longer
forms an ideal plane and instead it forms a shallow and a slightly twisted butterfly conformation folded
about the C-9···N-10 axis. This has been confirmed by X-ray analysis in the case of spiroacridine and
acridinium derivatives containing a dihydroacridine ring.17 The calculated first excited state (Figure 7d),
however, is essentially planar and thus the supposition that the structural differences between the ground
and the first excited states may limit the ability for fluorescence to occur appears to be valid.
a
b
c
d
Figure 7. Optimized structures of 5a (a) and 7a (b) calculated by DFT at the B3LYP/6-311+G(d,p) level
of theory and 13f in the ground state (c) and in the first excited state (d) as by provided by AM1
calculations.

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Biological activities
To evaluate the biological activity potential of the compounds, preliminary investigations were conducted
by testing compounds 5ad for their cytotoxic effect against human leukemia cell line HL-60. From the
concentrations that produced 25% inhibition (IC25) of cell viability (Table 5), it is evident that the tested
Table 5. Cytotoxic effect of compounds 5ad on the human leukemia cell line HL-60 cells
Compound
IC25 (µM)
IC50 (µM)
5a
5b
5c
5d
10.5 ± 1.2
> 1000
1.0 ± 1.3
100 ±2.5
2.0 ± 0.8
> 1000
5.6 ± 0.5
> 1000
derivatives do clearly possess mild cytotoxic properties wherein the most significant activity was
observed for the phenyl-substituted derivative 5b. A more than 100-fold increase of IC50 compared to
IC25 in all studied compounds evidences undoubtely a good HL-60 cell reparation at higher
concentrations of 5. This can be ascribed either to a hampered incorporation of 5 into tumor cells nuclei
due to a binding into other cell organelles or an induction of a repair cell mechanisms. For example,
enzymatic pathways enhancing the synthesis of low-molecular intracelular thiols as glutathione (GSH),
which can interact by its thiol group with studied derivatives to protect DNA, were proven in a similar
case.18 The protection extent may be high when glutathione is bound to strongly intercalating compound.
EXPERIMENTAL
General
Melting points were determined on a Boetius block and are uncorrected. Elemental analyses were
performed using a PerkinElmer CHN 2400 analyzer. IR spectra were recorded on a Nicolet 6700 FT IR
instrument.
NMR spectra were obtained at room temperature in hexadeuteriodimethyl sulfoxide (DMSO-d6) using a
1
Varian Mercury Plus NMR spectrometer operating at 400.13 MHz for H and 100.61 MHz for 13C.
Tetramethylsilane was used as an internal standard for both 1H and 13C nuclei (TMS = 0.00 ppm for both).
NMR chemical shift assignments were rendered by standard application of 1-D and 2-D correlation
spectra wherein heteronuclear HSQC and HMBC experiments were optimized on 145 Hz for one-bond
couplings and on 8 Hz for long-range couplings. Full descriptions of the NMR methodology have been
described elsewhere.2,3,19,20
UV-vis spectra were measured on a Varian Cary 100 UV-vis spectrophotometer in 0.01 M Tris buffer (pH
7.4). The working concentration of all compounds was 4.8  106 M. All measurements were performed at
24 C.

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1059
Fluorescence measurements were scanned on a Varian Cary Eclipse spectrofluorimeter. The excitation
and emission slits were 10 nm for compounds 5ad and 9-isothiocyanatoacridine and 20 nm for
compounds 4, 7ad, 13b,c and acridine. Emission spectra were recorded in the range 350600 nm at
concentrations of 6.0  106 and 3.8  103 M. The excitation wavelengths, λex, were: 4, 400; 5ad, 384;
7a, 388; 7b, 434; 7c, 394; 7d, 397; 13b, 436; 13c, 440; acridine, 355; and 9-isothiocyanatoacridine, 400
nm. Fluorescence intensities are expressed in arbitrary units. All measurements were performed at 24 C.
Semiempirical AM1 calculations21 were first performed to obtain the input geometries for DFT
calculations at the B3LYP/6-311+G(d,p) level of theory22 using Gaussian03 software.23 UV spectra were
analyzed using the semiempirical ZINDO method.24
(Acridin-9-yl)carbaldehyde (4)
Compound 4 was prepared according to literature methodology starting from commercially available
diphenylamine 1 giving, after modification of Bernthsen’s reaction,10 9-methylacridine (2). Following
radical bromination of 2, 9-bromomethylacridine (3) was obtained11 which then provided (acridin-9-yl)
carbaldehyde (4) after oxidation using sodium and 2-nitropropane.12
General procedure for the preparation of carbohydrazides 5ae
To a solution of the appropriate hydrazide (1.46 mmol) in anhydrous methanol, one drop of conc. aq. HCl
and (acridin-9-yl)carbaldehyde (4, 1.47 mmol) was added. The reaction mixture was left to stir ca. 3½ h
at rt until a precipitate had formed. The precipitate was filtered off, washed with methanol and then dried
followed by recrystallization from methanol.
N'-(Acridin-9-ylmethylene)acetohydrazide (5a). Yield 59%; mp 244–247 ºC; IR (KBr) ν: 3211 (NH),
3076 (CHarom), 1668 (C=O), 1633 (C=N) cm1. Major species: 1H NMR δ: 11.79 (s, 1H, NH), 9.25 (s, 1H,
H-11), 8.67 (m, 2H, H-1,8), 8.25 (d, 2H, H-4,5, J = 8.4 Hz), 7.96 (m, 2H, H-3,6), 7.76 (m, 2H, H-2,7),
2.32 (s, 3H, CH3); 13C NMR δ: 172.4 (C-14), 146.7 (C-4a,10a), 138.9 (C-11), 136.8 (C-9), 131.4 (C-3,6),
1
128.0 (C-4,5), 127.3 (C-2,7), 125.3 (C-1,8), 123.3 (C-8a,9a), 20.4 (CH3). Minor species: H NMR δ:
12.16 (s, 1H, NH), 9.42 (s, 1H, H-11), 8.67 (m, 2H, H-1,8), 8.25 (d, 2H, H-4,5, J = 8.4 Hz), 7.96 (m, 2H,
H-3,6), 7.76 (m, 2H, H-2,7), 2.13 (s, 3H, CH3); 13C NMR δ: 166.2 (C-14), 142.0 (C-11), 21.7 (CH3); Anal.
Calcd For C16H13N3O (263.30): C, 72.99; H, 4.98; N, 15.96. Found: C, 72.61; H, 5.07; N, 15.75%.
N'-(Acridin-9-ylmethylene)benzohydrazide (5b). Yield 43%; mp 262–264 ºC; IR (KBr) ν: 3213 (NH),
3057 (CHarom), 1647 (C=O), 1601 (C=N) cm1; 1H NMR δ: 12.35 (s, 1H, NH), 9.69 (s, 1H, H-11), 8.78 (d,
2H, H-1,8, J = 7.2 Hz), 8.23 (d, 2H, H-4,5, J = 8.4 Hz), 8.06 (m, 2H, H-2',6'), 7.91 (m, 2H, H-3,6), 7.75

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HETEROCYCLES, Vol. 80, No. 2, 2010
13
(m, 2H, H-2,7), 7.68 (m, 1H, H-4'), 7.64 (m, 2H, H-3',5'); C NMR δ: 163.4 (C-14), 148.4 (C-4a,10a),
144.8 (C-11), 134.6 (C-9), 133.0 (C-1'), 132.2 (C-4'), 130.3 (C-3,6), 129.8 (C-4,5), 128.7 (C-3',5'), 127.7
(C-2',6'), 127.2 (C-2,7), 125.3 (C-1,8), 123.4 (C-8a,9a); Anal. Calcd For C21H15N3O (325.37): C, 77.52; H,
4.65; N, 12.91. Found: C, 77.31; H, 4.87; N, 12.65%.
N'-(Acridin-9-ylmethylene)nicotinohydrazide (5c). Yield 64%; mp 263–266 ºC; IR (KBr) ν: 3209 (NH),
3063 (CHarom), 1651 (C=O), 1620 (C=N) cm1; 1H NMR δ: 12.96 (s, 1H, NH), 9.85 (s, 1H, H-11), 9.30 (s,
1H, H-2'), 8.87 (m, 1H, H-6'), 8.82 (d, 2H, H-1,8, J = 8.8 Hz), 8.52 (d, 1H, H-4', J = 6.4 Hz), 8.29 (d, 2H,
13
H-4,5, J = 8.8 Hz), 8.02 (m, 2H, H-3,6), 7.81 (m, 2H, H-2,7), 7.70 (m, 1H, H-5'); C NMR δ: 161.8
(C-14), 152.3 (C-6'), 148.5 (C-2'), 145.6 (C-4a,10a), 144.9 (C-11), 137.9 (C-9), 136.2 (C-4'), 132.2
(C-3,6), 128.7 (C-3'), 127.6 (C-2,7), 126.9 (C-4,5), 125.7 (C-1,8), 123.9 (C-5'), 123.4 (C-8a,9a); Anal.
Calcd For C20H14N4O (326.36): C, 73.61; H, 4.32; N, 17.17. Found: C, 74.01; H, 4.09; N, 16.75%.
N'-(Acridin-9-ylmethylene)isonicotinohydrazide (5d). Yield 74%; mp 249–252 ºC; IR (KBr) ν: 3257 (NH),
3072 (CHarom), 1678 (C=O), 1632 (C=N) cm1; 1H NMR δ: 12.56 (s, 1H, NH), 9.68 (s, 1H, H-11), 8.87 (d,
2H, H-2',6', J = 4.8 Hz), 8.73 (d, 2H, H-1,8, J = 8.4 Hz), 8.22 (d, 2H, H-4,5, J = 8.8 Hz), 7.95 (d, 2H,
H-3',5', J = 4.8 Hz), 7.91 (m, 2H, H-3,6), 7.74 (m, 2H, H-2,7); 13C NMR δ: 161.9 (C-14), 150.5 (C-2',6'),
148.3 (C-4a,10a), 146.1 (C-11), 140.1 (C-4'), 134.3 (C-9), 130.3 (C-3,6), 129.8 (C-4,5), 127.3 (C-2,7),
125.2 (C-1,8), 123.4 (C-8a,9a), 121.6 (C-3',5'); Anal. Calcd For C20H14N4O (326.36): C, 73.61; H, 4.32;
N, 17.17. Found: C, 73.97; H, 4.02; N, 16.57%.
N'-(Acridin-9-ylmethylene)-9-oxo-9,10-dihydroacridine-4-carbohydrazide (5e). Yield 28%; mp 331334
1
C; IR (KBr) ν: 3275 (NH), 3066 (CHarom), 1647 (C=Oacridone), 1616 (C=Oamide), 1587 (C=N) cm1; H
NMR δ: 12.70 (s, 1H, NH), 11.92 (s, 1H, H-10'), 9.75 (s, 1H, H-11), 8.87 (d, 2H, H-1,8, J = 8.8 Hz), 8.57
(d, 1H, H-1', J = 7.6 Hz), 8.47 (d, 1H, H-3', J = 7.6 Hz), 8.27 (m, 3H, H-4,5,8'), 7.94 (m, 2H, H-3,6), 7.92
13
(m, 1H, H-5'), 7.79 (m, 3H, H-2,7,6'), 7.50 (m, 1H, H-2'), 7.35 (m, 1H, H-7'); C NMR δ: 176,4 (C-9'),
162.7 (C-14), 145.9 (C-11), 134.0 (C-6'), 133.6 (C-3'), 130.8 (C-1'), 130.3 (C-3,6), 129.8 (C-4,5), 127.3
(C-2,7), 125.7 (C-8'), 125.1 (C-1,8), 121.8 (C-7'), 119.9 (C-2'), 118.6 (C-5'); Anal. Calcd For C28H18N4O2
(442.48): C, 76.01; H, 4.10; N, 12.66. Found: C, 76.28; H, 4.27; N, 12.25%.
General procedure for the preparation of carbohydrazides 7ae
To a solution of the appropriate hydrazide (1.46 mmol) in anhydrous methanol, one drop of conc. aq. HCl
and 9-anthraldehyde (6, 1.47 mmol) was added. The reaction mixture was stirred ca. 3½ h at rt. The
precipitate that had formed was filtered off, washed with methanol and dried followed by recrystallization

HETEROCYCLES, Vol. 80, No. 2, 2010
1061
from methanol.
N'-(Anthracen-9-ylmethylene)acetohydrazide (7a). Yield 85%; mp 231–235 C; IR (KBr) ν: 3199 (NH),
3066 (CHarom), 1666 (C=O), 1624 (C=N) cm1; Major species: 1H NMR δ: 11.47 (s, 1H, NH), 9.21 (s, 1H,
H-11), 8.70 (s, 1H, H-10), 8.59 (d, 2H, H-1,8, J = 8.4 Hz), 8.15 (d, 2H, H-4,5, J = 8.4 Hz), 7.65 (m, 2H,
H-2,7), 7.58 (m, 2H, H-3,6), 2.28 (s, 3H, CH3); 13C NMR δ: 171.9 (C-14), 141.4 (C-11), 130.8 (C-4a,10a),
129.4 (C-8a,9a), 129.1 (C-10), 128.9 (C-4,5), 127.0 (C-2,7), 125.4 (C-3,6), 125.1 (C-9), 124.5 (C-1,8),
20.4 (CH3); Minor species: 1H NMR δ: 11.69 (s, 1H, NH), 9.34 (s, 1H, H-11), 8.64 (s, 1H, H-10), 2.08 (s,
3H, CH3); 13C NMR δ: 165.6 (C-14), 144.5 (C-11), 21.7 (CH3). Anal. Calcd For C17H14N2O (262.31): C,
77.84; H, 5.38; N, 10.68. Found: C, 77.42; H, 5.07; N, 10.45%.
N'-(Anthracen-9-ylmethylene)benzohydrazide (7b). Yield 90%; mp 279–282 ºC; IR (KBr) ν: 3219 (NH),
3061 (CHarom), 1647 (C=O), 1622 (C=N) cm1; 1H NMR δ: 12.14 (s, 1H, NH), 9.71 (s, 1H, H-11), 8.79 (d,
2H, H-1,8, J = 8.4 Hz), 8.74 (s, 1H, H-10), 8.17 (d, 2H, H-4,5, J = 8.0 Hz), 8.06 (d, 2H, H-2',6', J = 7.2
Hz), 7.69–7.58 (m, 7H, H-2,3,6,7,3'5'); 13C NMR δ: 163.0 (C-14), 146.9 (C-11), 133.3 (C-1'), 131.8
(C-4'), 130.9 (C-4a,10a), 129.6 (C-10), 129.6 (C-8a,9a), 128.9 (C-4,5), 128.5 (C-2,7), 127.6 (C-2',6'),
127.1 (C-3',5'), 125.5 (C-3,6), 125.0 (C-9), 124.8 (C-1,8); Anal. Calcd For C22H16N2O (324.38): C, 81.46;
H, 4.97; N, 8.64. Found: C, 81.08; H, 4.67; N, 9.02%.
N'-(Anthracen-9-ylmethylene)nicotinohydrazide (7c). Yield 83%; mp 265–268 C; IR (KBr) ν: 3202
(NH), 3084 (CHarom), 1643 (C=O), 1624 (C=N) cm1; 1H NMR δ: 12.34 (s, 1H, NH), 9.69 (s, 1H, H-11),
9.28 (s, 1H, H-2'), 8.88 (m, 1H, H-6'), 8.73 (m, 3H, H-1,8,10), 8.39 (m, 1H, H-4'), 8.13 (m, 2H, H-4,5),
13
7.65 (m, 1H, H-5'), 7.64 (m, 2H, H-2,7), 7.57 (m, 2H, H-3,6); C NMR δ: 161.7 (C-14), 152.2 (C-6'),
148.5 (C-2'), 147.7 (C-11), 135.5 (C-4'), 130.8 (C-4a,10a), 129.8 (C-10), 129.6 (C-8a,9a), 129.0 (C-4,5),
127.2 (C-2,7), 125.5 (C-3,6), 124.8 (C-1,8), 124.7 (C-3',5'), 124.5 (C-9); Anal. Calcd For C21H15N3O
(325.37): C, 77.52; H, 4.65; N, 12.91. Found: C, 77.23; H, 4.21; N, 12.42%.
N'-(Anthracen-9-ylmethylene)isonicotinohydrazide (7d). Yield 85%; mp 263–265 C; IR (KBr) ν: 3205
(NH), 3086 (CHarom), 1693 (C=O), 1635 (C=N) cm1; 1H NMR δ: 12.34 (s, 1H, NH), 9.70 (s, 1H, H-11),
8.88 (m, 2H, H-2',6'), 8.77 (d, 2H, H-1,8, J = 8.4 Hz), 8.75 (s, 1H, H-10), 8.18 (d, 2H, H-4,5, J = 8.8 Hz),
13
7.96 (d, 2H, H-3',5', J = 4.8 Hz), 7.68 (m, 2H, H-2,7), 7.61 (m, 2H, H-3,6); C NMR δ: 161.5 (C-14),
150.4 (C-2',6'), 148.3 (C-11), 140.4 (C-4'), 130.8 (C-4a,10a), 129.9 (C-10), 129.6 (C-8a,9a), 129.0 (C-4,5),
127.3 (C-2,7), 125.5 (C-3,6), 124.7 (C-1,8,9), 121.5 (C-3',5'); Anal. Calcd For C21H15N3O (325.37): C,
77.52; H, 4.65; N, 12.91. Found: C, 77.27; H, 4.79; N, 13.05%.

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HETEROCYCLES, Vol. 80, No. 2, 2010
N'-(Anthracen-9-ylmethylene)-9-oxo-9,10-dihydroacridine-4-carbohydrazide (7e). Yield 36%; mp
324327 C; IR (KBr) ν: 3209 (NH), 3046 (CHarom), 1663 (C=Oacridone), 1622 (C=Oamide), 1587 (C=N)
cm1; 1H NMR δ: 12.47 (s, 1H, NH), 12.02 (s, 1H, H-10'), 9.76 (s, 1H, H-11), 8.87 (d, 2H, H-1,8, J = 8.8
Hz), 8.75 (s, 1H, H-10), 8.54 (d, 1H, H-1', J = 8.0 Hz), 8.47 (d, 1H, H-3', J = 6.8 Hz), 8.26 (d, 1H, H-8', J
= 8.0 Hz), 8.17 (d, 2H, H-4,5, J = 8.4 Hz), 7.89 (d, 1H, H-5', J = 8.4 Hz), 7.76 (m, 1H, H-6'), 7.69 (m, 2H,
13
H-2,7), 7.62 (m, 2H, H-3,6), 7.46 (m, 1H, H-2'), 7.33 (m, 1H, H-7'); C NMR δ: 176,5 (C-9'), 163.9
(C-14), 148.3 (C-11), 140.2 (C-4a',10a'), 134.0 (C-6'), 133.5 (C-3'), 130.9 (C-4a,10a), 130.6 (C-1'), 130.1
(C-10), 129.8 (C-8a,9a), 129.1 (C-4,5), 127.4 (C-2,7), 125.8 (C-8'), 125.6 (C-3,6), 124.8 (C-1,8), 124.5
(C-9), 122.1 (C-7'), 121.7 and 118.4 (C-4´ and C-9a'), 120.5 (C-8a'), 120.0 (C-2'), 118.7 (C-5'),; Anal.
Calcd For C29H19N3O2 (441.49): C, 78.90; H, 4.34; N, 9.52. Found: C, 78.55; H, 4.13; N, 9.32%.
General procedure for the preparation of hydrazones 13ag
To a solution of the appropriate aldehyde or ketone (1.147 mmol) in ethanol (10 mL), (acridin-9-yl)
hydrazine (12, 200 mg, 0.956 mmol) and several drops of glacial acetic acid were added. The reaction
mixture was then refluxed for 3 h following which it was cooled and poured onto chilled water. The
resulting precipitate was filtered off, dried and the product recrystallized from methanol.
9-(Benzylidenehydrazono)-9,10-dihydroacridine (13a). Yield: 84%; mp 6870 C; IR (KBr) ν: 3271
(NH), 3101, 1626, 1597 (C=N) cm1; 1H NMR δ: 10.83 (s, 1H, H-10), 8.99 (dd, 1H, H-1, J = 8.8, 1.6 Hz),
8.55 (s, 1H, HC=N), 8.40 (dd, 1H, H-8, J = 8.0, 1.2 Hz), 7.89 (dd, 2H, H-2',6', J = 8.8, 1.6 Hz), 7.557.48
(m, 5H, H-3,6,3'5'), 7.30 (dd, 1H, H-5, J = 8.4, 1.2 Hz), 7.28 (dd, 1H, H-4, J = 8.4, 0.8 Hz), 7.147.05
(m, 2H, H-2,7); 13C NMR δ: 154.4 (C-13), 149.8 (C-9), 140.6 (C-4a), 138.6 (C-10a), 135.3 (C-1'), 132.3
(C-1), 131.4 (C-3), 130.9 (C-6), 130.0 (C-4'), 129.0 (C-3',5'), 128.6 (C-2',6'), 125.5 (C-8), 120.9 (C-7),
119.7 (C-2), 119.3 (C-8a), 116.2 (C-5), 115.6 (C-9a), 115.6 (C-4) ; Anal. Calcd For C20H15N3 (297.36): C,
80.78; H, 5.08; N, 14.13. Found: C, 80.54; H, 5.01; N, 13.85%.
9-(2,4,6-Trimethoxybenzylidenehydrazono)-9,10-dihydroacridine (13b). Yield 83%; mp 239240 C; IR
(KBr) ν: 3267 (NH), 3107, 1620, 1599 (C=N) cm1; 1H NMR δ: 10.60 (s, 1H, H-10), 9.87 (d, 1H, H-1, J
= 8.4 Hz), 8.80 (s, 1H, HC=N), 8.41 (d, 1H, H-8, J = 8.0 Hz), 7.47 and 7.45 (m, m, 2H, H-3,6), 7.22 (d,
1H, J = 8.4 Hz) and 7.18 (d, 1H, J = 8.4 Hz, H-4,5), 7.05 and 7.04 (m, m, 2H, H-2,7), 6.35 (s, 2H, H-3',5'),
3.94 (s, 6H, C-2',6'-OCH3), 3.87 (s, 3H, C-4'-OCH3); 13C NMR δ: 162.6 (C-4'), 160.4 (C-2',6'), 150.9
(C-13), 148.8 (C-9), 140.2 (C-4a), 138.6 (C-10a), 133.2 (C-1), 131.0 (C-3), 130.4 (C-6), 125.5 (C-8),
120.5 (C-7), 119.6 (C-2), 119.2 (C-8a), 116.4 (C-5), 115.7 (C-9a), 115.3 (C-4), 104.8 (C-1'), 91.0
(C-3',5'), 56.0 (C-2',6'-OCH3), 55.4 (C-4'-OCH3); Anal. Calcd For C23H21N3O3 (387.44): C, 71.30; H,

HETEROCYCLES, Vol. 80, No. 2, 2010
1063
5.46; N, 10.85. Found: C, 71.48; H, 5.53; N, 10.77%.
9-(4-Bromobenzylidenehydrazono)-9,10-dihydroacridine (13c). Yield 93%; mp 99101 C; IR (KBr) ν:
1
1630, 1597 (C=N) cm1; H NMR δ: 10.89 (s, 1H, H-10), 8.91 (d, 1H, H-1, J = 8.0 Hz), 8.53 (s, 1H,
HC=N), 8.39 (d, 1H, H-8, J = 7.6 Hz), 7.82 (m, 2H, H-2',6'), 7.72 (m, 2H, H-3',5'), 7.51 (m, 2H, H-3,6),
7.31 (d, 1H, J = 8.4 Hz) and 7.27 (d, 1H, J = 8.4 Hz, H-4,5), 7.12 and 7.07 (m, m, 2H, H-2,7); 13C NMR
δ: 153.1 (C-13), 149.9 (C-9), 140.5 (C-4a), 138.5 (C-10a), 134.5 (C-1'), 132.2 (C-1), 131.9 (C-3',5'),
131.4 (C-6), 130.9 (C-3), 129.3 (C-2',6'), 125.4 (C-8), 123.2 (C-4'), 120.8 (C-7), 119.8 (C-2), 119.1
(C-8a), 116.1 (C-5), 115.6 (C-4), 115.4 (C-9a); Anal. Calcd For C20H14BrN3 (376.26): C, 63.85; H, 3.75;
N, 11.17. Found: C, 63.58; H, 3.53; N, 10.84%.
9-(4-Methoxybenzylidenehydrazono)-9,10-dihydroacridine (13d). Yield 78%; mp 6870 C; IR (KBr) ν:
1
3269 (NH), 3130, 1624, 1597 (C=N) cm1; H NMR δ: 10.73 (s, 1H, H-10), 9.05 (dd, 1H, H-1, J = 8.4,
1.2 Hz), 8.48 (s, 1H, HC=N), 8.37 (dd, 1H, H-8, J = 8.4, 1.2 Hz), 7.83 (m, 2H, H-2',6'), 7.517.46 (m, 2H,
H-3,6), 7.27 (dd, 1H, J = 8.4, 1.2 Hz) and 7.23 (dd, 1H, J = 8.4, 0.8 Hz, H-4,5), 7.117.03 (m, 4H,
H-2,7,3',5'), 3.84 (s, 3H, OCH3); 13C NMR δ: 160.8 (C-4'), 154.3 (C-13), 149.2 (C-9), 140.5 (C-4a), 138.5
(C-10a), 132.3 (C-1), 131.2 (C-6), 130.7 (C-3), 129.2 (C-2',6'), 127.9 (C-1'), 125.3 (C-8), 120.7 (C-7),
119.5 (C-2), 119.3 (C-8a), 116.0 (C-9a), 115.7 (C-5), 115.4 (C-4), 114.4 (C-3',5'), 55.3 (OCH3); Anal.
Calcd For C21H17N3O (327.39): C, 77.04; H, 5.23; N, 12.83. Found: C, 76.38; H, 5.53; N, 12.64%.
9-(2,4,6-Trimethylbenzylidenehydrazono)-9,10-dihydroacridine (13e). Yield 41%; mp 7981 C; IR
(KBr) ν: 3259 (NH), 3103, 1628, 1595 (C=N) cm1; 1H NMR δ: 10.78 (s, 1H, H-10), 9.06 (dd, 1H, H-1, J
= 8.0, 1.2 Hz), 8.87 (s, 1H, HC=N), 8.42 (dd, 1H, H-8, J = 8.0, 1.6 Hz), 7.49 and 7.47 (m, m, 2H, H-3,6),
7.27 (dd, 1H, J = 8.0, 0.4 Hz) and 7.25 (dd, 1H, J = 7.6, 0.8 Hz, H-4,5), 7.10 (ddd, 1H, J = 8.0, 7.2, 1.2
13
Hz H-7), 6.96 (m, 3H, H-2,3',5'), 2.51 (s, 6H, C-2',6'-CH3), 2.28 (s, 3H, C-4'-CH3); C NMR δ: 155.6
(C-13) 149.6 (C-9) 140.3 (C-4a) 138.6 (C-10a) 138.4 (C-4'), 137.5 (C-2',6'), 131.8 (C-1), 131.3 (C-3),
130.7 (C-6), 129.5 (C-3',5'), 128.9 (C-1'), 125.5 (C-8) 120.7 (C-7) 119.2 (C-8a) 119.2 (C-2) 116.0
(C-5) 115.6 (C-9a) 115.5 (C-4) 21.3 (C-2',6'-CH3), 20.7 (C-4'-CH3); Anal. Calcd For C23H21N3
(339.44): C, 81.38; H, 6.24; N, 12.38. Found: C, 80.98; H, 5.94; N, 12.06%.
9-(Propan-2-ylidenehydrazono)-9,10-dihydroacridine (13f). Yield 38%; mp 7678 C; IR (KBr) ν: 3277
(NH), 3132, 2995, 2939 (CH), 1622, 1595 (C=N) cm1; 1H NMR δ: 10.43 (s, 1H, H-10), 8.74 (d, 1H, H-1,
J = 7.6 Hz), 8.24 (d, 1H, H-8, J = 7.6 Hz), 7.40 (m, 2H, H-3,6), 7.18 and 7.15 (m, m, 2H, H-4,5), 7.02 (m,

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1H, H-7), 6.94 (m, 1H, H-2), 2.12 (s, 3H, CH3), 1.99 (s, 3H, CH3); 13C NMR δ: 158.0 (C-13), 144.2 (C-9),
140.5 (C-4a), 138.4 (C-10a), 131.7 (C-1), 130.7 (C-3), 130.1 (C-6), 125.2 (C-8), 120.5 (C-7), 119.9
(C-8a), 119.0 (C-2), 115.9 (C-9a), 115.6 (C-4), 115.2 (C-5), 24.6 (CH3), 17.9 (CH3); Anal. Calcd For
C16H15N3 (249.32): C, 77.08; H, 6.06; N, 16.85. Found: C, 76.88; H, 5.94; N, 17.06%.
9-((1-Phenylethylidene)hydrazono)-9,10-dihydroacridine (13g). Yield 42%; mp 8689 C; IR (KBr) ν:
2923, 1638, 1613 (C=N) cm1; 1H NMR δ: 10.68 (s, 1H, H-10), 8.58 (d, 1H, H-1, J = 7.6 Hz), 8.38 (d, 1H,
H-8, J = 7.6 Hz), 7.97 (m, 2H, H-2',6'), 7.527.45 (m, 5H, H-3,6,3'5'), 7.24 (m, 2H, H-4,5), 7.11 (m, 1H,
13
H-7), 6.95 (m, 1H, H-2), 2.46 (s, 3H, CH3); C NMR δ: 155.8 (C-13), 145.5 (C-9), 140.6 (C-4a), 138.4
(C-10a), 138.3 (C-1´), 131.5 (C-1), 131.1 (C-3), 130.5 (C-6), 129.1 (C-4'), 128.5 (C-3',5'), 126.0 (C-2',6'),
125.4 (C-8), 120.7 (C-7), 119.8 (C-8a), 119.4 (C-2), 115.7 (C-5), 115.4 (C-4), 115.4 (C-9a), 14.3 (CH3);
Anal. Calcd For C21H17N3 (311.39): C, 81.00; H, 5.50; N, 13.49. Found: C, 79.83; H, 5.34; N, 13.46%.
ACKNOWLEDGEMENTS
Financial support for this work by the Slovak Grant Agency VEGA (Grants No. 1/0476/08, 1/0053/08),
Slovak State NMR Program (Grant No. 2003SP200280203) and Turun Yliopistosäätiö is gratefully
acknowledged. We also thank the Center for Scientific Computing (CSC) for computational support and
services.
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