Caffeine-hydrazones as anticancer agents with pronounced selectivity toward T-lymphoblastic leukaemia cells

Article abstract of DOI:10.1016/j.bioorg.2015.03.003

We report design and synthesis of set of novel anticancer agents based on caffeine-hydrazones bearing 2-hydroxyaryl- or 2-N-heteroaryl moiety. Anticancer activity evaluation using seven cancer cell lines and two non-malignant cell lines demonstrated that several derivatives display significant anticancer activity and great selectivity index toward T-lymphoblastic leukaemia cells. In general, hydrazones bearing 2-N-heteroaryl moiety are more active and selective than those with 2-hydroxyaryl moiety. Tested compounds exhibit dose-dependent inhibition of both RNA and DNA synthesis, with some exceptions. Antimicrobial activities were tested on set of twelve bacterial and yeast strains, however prepared compounds were not active, suggesting for a molecular target specific for eukaryotic cells.

Full text of DOI:10.1016/j.bioorg.2015.03.003

Accepted Manuscript
Caffeine-hydrazones as anticancer agents with pronounced selectivity toward
T-lymphoblastic leukaemia cells
Robert Kaplánek, Milan Jakubek, Jakub Rak, Zdeněk Kejík, Martin Havlík,
Bohumil Dolenský, Ivo Frydrych, Marián Hajdúch, Milan Kolá ř, Kateřina
Bogdanová, Jarmila Králová, Petr Džubák, Vladimír Král
PII:
S0045-2068(15)00021-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.03.003
YBIOO 1798
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
30 October 2014
Please cite this article as: R. Kaplánek, M. Jakubek, J. Rak, Z. Kejík, M. Havlík, B. Dolenský, I. Frydrych, M.
Hajdúch, M. Kolá ř, K. Bogdanová, J. Králová, P. Džubák, V. Král, Caffeine-hydrazones as anticancer agents with
pronounced selectivity toward T-lymphoblastic leukaemia cells, Bioorganic Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bioorg.2015.03.003
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Caffeine-hydrazones as anticancer agents with pronounced selectivity toward
T-lymphoblastic leukaemia cells
Robert Kapláneka, Milan Jakubeka, Jakub Raka, Zdeněk Kejíka, Martin Havlíka, Bohumil Dolenskýa, Ivo
Frydrychb, Marián Hajdúchb, Milan Kolářb,c, Kateřina Bogdanováb, Jarmila Královád, Petr Džubákb* and
Vladimír Krála*
a
Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemical
Technology, Technická 5, 166 28 Prague 6, Czech Republic
b
Institute of Molecular and Translational Medicine, Palacky University and University Hospital in
Olomouc, Faculty of Medicine and Dentistry, Hněvotínská 5, 775 15 Olomouc, Czech Republic
c
Department of Microbiology, Palacky University and University Hospital in Olomouc, Faculty of
Medicine and Dentistry, Hněvotínská 5, 775 15 Olomouc, Czech Republic
d
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20
Prague 4, Czech Republic
Corresponding authors: medicinal chemistry and biochemistry: Vladimir.Kral@vscht.cz Tel.:
+420220445027; biology: Petr.Dzubak@upol.cz Tel.: +420585632150
Keywords: Anticancer agents; Caffeine-hydrazones; Cancer treatment; Leukaemia; Selectivity
Abstract:
We report design and synthesis of set of novel anticancer agents based on caffeine-
hydrazones bearing 2-hydroxyaryl- or 2-N-heteroaryl moiety. Anticancer activity evaluation using
seven cancer cell lines and two non-malignant cell lines demonstrated that several derivatives display
significant anticancer activity and great selectivity index toward T-lymphoblastic leukaemia cells. In
general, hydrazones bearing 2-N-heteroaryl moiety are more active and selective than those with 2-
hydroxyaryl moiety. Tested compounds exhibit dose-dependent inhibition of both RNA and DNA
synthesis, with some exceptions. Antimicrobial activities were tested on set of twelve bacterial and
yeast strains, however prepared compounds were not active, suggesting for a molecular target
specific for eukaryotic cells.
Graphical Abstract:
1

1. Introduction
Cancer is a leading cause of death worldwide and the disease treatment remains serious
medical and societal problem. There are numerous compounds with anticancer activity, but many of
them display serious side effects, including tissuel toxicity and low selectivity resulting in ultimate
damage of healthy tissues and organs. Moreover, the primary or secondary cancer cells drug
resistance limits efficient therapy and thus, identification of new therapies and agents is urgently
needed.
Heterocylic hydrazones are important class of biologically active compounds with a wide
range of biological activity. Hydrazones and related transition metal chelators were already
successfully used for regulation of iron balance in the body, thus they can serve as a therapeutics in
the treatment of iron overload diseases, namely hereditary or transfusional hemochromatosis [1-5].
However, some hydrazones display antimicrobial (antibacterial, antimycobacterial, antiviral,
antimalaric), anti-inflammatory and cytostatic activity [1-4, 6-10].
The mechanism of action of hydrazones as anticancer agents is based on chelation of metal
ions, mainly iron that is necessary to ensure needs of rapidly proliferating tumor tissues. . Another
possible mechanisms are inhibition of enzymes (ribonucleotide reductase, histon deacetylase or acyl
transferases), toxicity of formed metal complexes (redox systems producing toxic reactive oxygen
species) as well as intercalation to DNA [3, 9, 11-13]. Their mechanism of action is still not fully
understood and warrants further investigation.
It is known that compounds contained in coffee, mainly caffeine, has chemopreventive effect
in several types of cancer [14]. Thus, caffeine skeleton remains in the focus of interest for further
modification and preparation of compounds with anticancer activity.
Our approach for synthesis of novel anticancer agents is based on combination of two active
moieties, eg. hydrazone and caffeine. The design using molecular hybridization technique, synthesis
and evaluation of anticancer and antimicrobial activity and metal-binding properties of novel
caffeine-hydrazones bearing substituted 2-hydroxyaryl- or 2-N-heteroaryl moiety are reported
herein.
2. Results and discussion
2.1. Synthesis of target hydrazones 4-35
The target hydrazones 4-35 were prepared in three synthetic steps (Scheme 1). The synthesis
starts by reaction of caffeine (1) with chlorine in chloroform solution [15]. 8-Chlorocaffeine (2) was
then converted into 8-hydrazinocaffeine (3) by heating with large excess of hydrazine hydrate in
ethanol [16] with the yield of 88%. In the last step, the 8-hydrazinocaffeine (3) was reacted with the
excess of the substituted 2-hydroxyaryl- or 2-N-heteroaryl- aldehydes and ketones, respectively
(molar ratio 1:2) in refluxing ethanol containing catalytic amount of acetic acid providing the
hydrazones 4-35 with high yields, 77-96%. Structures of hydrazones 4-35 and yields are shown in
Table 1. It is known that hydrazones due to geometric isomerism can exist as E/Z isomers at the C=N
1
bond of the imino group [17, 18]. As a result, H NMR shows splitting for the CH=N or C(CH3)=N and
NH protons. In our case, 1H NMR of hydrazones showed only one isomer (CH=N proton is
represented by only one singlet peak, the same for NH proton).
2

Scheme 1. General scheme of preparation of the caffeine-hydrazones 4-35. R, Z and Y are defined in
Table 1.
Table 1. Structures and yields of of prepared hydrazones
4 (88%)
11 (86%)
18 (80%)
25 (90%)
5 (79%)
12 (83%)
19 (94%)
26 (81%)
33 (81%)
6 (86%)
13 (79%)
20 (91%)
27 (86%)
34 (77%)
7 (83%)
14 (86%)
21 (91%)
28 (96%)
35 (79%)
8 (77%)
15 (89%)
22 (88%)
29 (83%)
9 (80%)
16 (92%)
23 (77%)
30 (79%)
10 (92%)
17 (93%)
24 (93%)
31 (87%)
32 (85%)
3

2.2. Biological activity
Some caffeine-hydrazones and structurally related xanthine-8-hydrazones were prepared
and tested for their antimicrobial activity [19-21], as antioxidants [22], EGFR dimerization inhibitors
[23] or HIV-1 integrase inhibitors [24].
2.2.1. Cytostatic activity
The in vitro cytotoxic activity of hydrazones 4-35 was tested on the seven cancer cell lines:
CCRF-CEM (T-lymphoblastic leukaemia), CEM-DNR (T-lymphoblastic leukaemia, daunorubicin
resistant), K562 (acute myeloid leukaemia) and K562-TAX (acute myeloid leukaemia, paclitaxel
resistant), A549 (human lung adenocarcinoma), HCT116 (human colorectal cancer), HCT116p53-/-
(human colorectal cancer, p53-deficient) and on the two representatives of non-malignant cell lines:
BJ (human fibroblast) and MRC-5 (human lung fibroblasts). Cytotoxic activities are presented as the
IC50 values (Table 2). Data were expressed as the average values of IC50 from six independent
experiments with SD ranging from 5-50% of the average values.
The tested compounds (4-35) showed cytotoxic activity at concentrations of 0.34 to > 25 µM.
Majority of the compounds were more active against highly chemosensitive the T-lymphoblastic
leukaemia cell line CCRF-CEM compared to corresponding multidrug resistant subline CEM-DNR
overexpressing the multidrug resistance protein 1 (MRP-1) [25]. However, in case of K562 myeloid
leukemia cells and their drug resistant counterparts K562 overexpressing membrane transporter P-
glycoprotein 1 (Pgp-1) [25], the cytotoxic activities were similar against both cell lines (except for
hydrazones 9, 21 and 28), suggesting involvement of MRP-1 but not Pgp-1 in drug efflux and
resistance mechanisms. The IC50 values for the human lung adenocarcinoma cell line A549 were in
the range from 2 to >25 µM. The inhibitory activities for the human colorectal cancer cell line HTC116
and its p53-deficient counterpart (HTC116p53-/-) were similar (except for hydrazone 11). Caffeine (1)
and 8-hydrazinocaffeine (3) were used as controls and showed no activity. In general, the highest
cytotoxic effect was observed for CCRF-CEM cell line, while non-malignan fibroblasts BJ and MRC-5
were not affected, suggesting good therapeutic index.
Comparison of biological activities of prepared derivatives with those of other heteroaryl
hydrazone-based compounds against the same cell lines described in the literature is difficult due to
various descriptors of activity (IC50, EC50, GI50, etc.) and experimental conditions. For example, (2-
hydroxyaryl)ydene hydrazones derived from quinoxalin-2-hydrazine were active in the range from
1.8 to 14.2 µM (HCT116) [26]; from benzoisoindoline-based hydrazides in the range from 5.8 to > 50
µM (HCT116) and >50 µM (A549) [27]; benzoisothiazol-based hydrazides from 0.5 to > 100 µM
(CCRF-CEM); (2-N-heteroaryl)ydene hydrazones derived from bezoxazole-2-hydrazine showed IC50
values in the range from 0.01 to > 50 µM (CCRF-CEM)[28, 29]. These examples indicate that
anticancer effect is strongly dependent on the structure of both aldehyde/ketone and hydrazine part
of hydrazone molecule.
The selectivity indexes toward cancer cells (Table 3) were calculated as a ratio of the average
IC50 values for BJ and MRC-5 fibroblast cells and the IC50 values for the corresponding cancer cell line,
with the respect to measurement limit (25 µM). Overall selectivity index were calculated as a ratio of
the average IC50 values for BJ and MRC-5 fibroblast cells and the average IC50 values for all cancer cell
lines. Thus, the selectivity index represents the selectivity for a specific cancer cell line. In Table 3, the
highest selectivity index values are highlighted in blue (SI > 5) and green (5 > SI > 2). Compounds 29
(SIoverall = 5.08), 32 (SIoverall = 12.85) and 34 (SIoverall = 8.00) display the best selectivity toward all cancer
cells (Figure 1). In general, prepared hydrazones have the best selectivity toward CCRF-CEM cell line
4

(T-lymphoblastic leukaemia cell line); the best selectivity for this cell line display compounds 34
(SICCRF-CEM = 71.1), 18 (SICCRF-CEM = 50.8), 32 (SICCRF-CEM = 36.5), 29 (SICCRF-CEM = 22.2) and 25 (SICCRF-CEM
=
18.3) (Figure 2).
Figure 1. Overall selectivity index for compounds 4-35
Figure 2. Selectivity index toward CCRF-CEM for compounds 4-35
5

From a structural perspective, the best results (anticancer activity and anticancer selectivity)
were obtained for compounds possessed 2-N-heterocyclic moiety: compound 29 (derivative of
pyridin-2-carbaldehyde), 32 (derivative of di(2-pyridyl)ketone)) and 34 (derivative of acetylpyrazine).
In the series of 2-hydroxyaryl-substituted hydrazones (compounds 4-28) no correlation was observed
between electronic effect of substituents and anticancer activity.
It is well known that lipophilicity of the compounds plays an important role in their
penetration into cells. The lipophilicities of the all synthesized compounds were expressed as
partition coefficient logP values (determined using ChemBioDraw Ultra v14; data are not shown).
Unfortunately, logP values did not correlate with the anticancer activity, although in the series of 2-
hydroxyaryl-substituted hydrazones (compounds 4-28), the most active and selective were
compounds bearing bulky lipophilic substituents (compounds 11, 16, 18, 21, 23-26). On the other
hand, higher lipophilicity does not seem to be only criterion for higher activity; for example
anticancer activity and selectivity of compounds 21 and 22, both having hydroxynaphthalene moiety
(1-hydroxynaphthalen-2-yl in the case of 21, 2-hydroxynaphthalen-1-yl in the case of 22,
respectively) are very different.
In an effort to understand mechanism of action, cell cycle and nucleic acids synthesis analysis
of the most active compounds (with IC50/CCRF-CEM < 5 µM) was performed on T-lymphoblastic
leukaemia cells (CCRF-CEM) at the concentration corresponding to 1 × IC50 and 5 × IC50 after 24 h
incubation (Table 4). All tested compounds (7-9, 11, 16, 18, 21, 23-26, 29, 32, 34 and 35) induced
apoptosis at the 5 × IC50 concentration, while at the 1 × IC50 concentration, only compounds 23, 32
and 35 induced higher programmed cell death. In addition to cell death induction, compounds 8, 11,
21, 23, 24, 29, 32, 34 and 35 also inhibited at 5 × IC50 DNA and RNA synthesis and decreased
percentage of mitotic pH3ser10+ cells. In general, most of the compounds exhibited dose-dependent
inhibition of RNA and DNA synthesis (Figure 3), with only few exceptions.
Above mentioned results indicate, that structure-activity relationship and mechanism of
action of caffeine-hydrazones are complex and involve inhibition of nucleic acid synthesis and
mitosis, ultimately resulting in cell death.
6

Figure 3. Example of cell cycle, apoptosis (sub G1), mitosis (pH3Ser10+), and DNA (BrdU+) and RNA
(BrU+) synthesis analyses in CCRF-CEM cells in compound 32. The compound showed the highest
overall in vitro therapeutic index and inhibit the DNA and RNA synthesis at both 1 × and 5 x IC50, and
induced massive apoptosis following 24 hour treatment. Histograms show the cell cycle
mathematical modelling analysis using the ModFit algorithm.
2.2.2. Antimicrobial activity
The prepared compounds (4-35) were also tested for their antimicrobial activity against the
twelve standard reference Gram-positive and Gram-negative bacterial and yeast strains and strains
obtained from biological material from patients: Enterococcus faecalis CCM 4224, Staphylococcus
aureus CCM 3953, Escherichia coli CCM 3954, Pseudomonas aeruginosa CCM 395, methicillin-
resistant Staphylococcus aureus MRSA 4591, fluoroquinolone resistant Staphylococcus haemolyticus
A/16568, Escherichia coli C/16702 and Pseudomonas aeruginosa A/16575, Candida albicans, Candida
krusei, Candida tropicalis, and Candida parapsilosis). Hydrazones 4-35 did not show any antimicrobial
activity in the concentration ranges tested (up to 200 µM), thus providing additional evidence for
specificity of the anticancer effect.
2.3. Binding ability toward metal ions
The chelation of iron and other biologically important metal ions that are necessary to ensure
the growth and division of tumour cells might be the primary mechanism of action for some
hydrazone-based anticancer agents. For this reason, we have tested the ability of hydrazones 4-35 to
bind the Fe2+, Fe3+, Co2+, Cu2+, Ni2+, Zn2+, Mn2+ and Cr3+ ions by UV-Vis titration (Figure 4). In parallel
the chelation of Ca2+ and Mg2+ that could induce serious adverse events after in vivo administration
was also investigated. Indeed, tested hydrazones are strong binders of Fe2+, Fe3+, Co2+ and Cu2+ while
the binding capability for Ni2+ and Zn2+ is much weaker. Importantly, interactions with other metal
ions (Ca2+, Mg2+, Mn2+ and Cr3+) were not observed, thus indicating therapeutic potential of caffeine-
7

hydrazones. Although the metal chelating activity of the compounds was proved under in vitro
conditions, its contribution to the cytotoxic effect in cancer cells is questionable, since metal
chelation is also effective strategy to inhibit bacterial growth and antimicrobial activity of those
molecules was not demonstrated.
Figure 4. UV-Vis spectra of hydrazone 11 and its complex with biologically important metal ions.
8

Table 2. IC50 values [µM] for hydrazones 4-35.
Cell line
HCT116
Compound
CCRF-
CEM
> 25
> 25
20.25
5.02
24.40
3.79
4.25
4.40
22.73
3.59
15.80
24.63
15.15
15.37
3.37
24.45
0.49
> 25
> 25
3.16
> 25
4.73
3.64
1.37
3.04
> 25
> 25
1.04
> 25
> 25
0.51
> 25
0.34
2.84
CEM-
DNR
> 25
K562-
TAX
HCT116
p53-/-
> 25
K562
A549
BJ
MRC-5
1
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
24.92
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
22.51
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
23.25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
24.23
> 25
> 25
> 25
24.23
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
18.03
> 25
> 25
> 25
> 25
> 25
> 25
20.97
> 25
> 25
6.09
23.57
23.18
22.74
3
> 25
> 25
> 25
4
> 25
> 25
21.36
24.10
> 25
> 25
23.80
20.12
> 25
> 25
5
> 25
19.77
> 25
17.12
> 25
> 25
6
> 25
> 25
7
22.99
19.24
11.96
16.59
15.07
19.81
> 25
> 25
24.95
16.84
4.15
24.12
20.64
19.21
> 25
24.82
17.30
15.24
> 25
> 25
8
13.78
> 25
23.69
> 25
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
> 25
> 25
> 25
14.15
16.04
> 25
13.11
16.79
21.47
6.48
4.93
16.21
13.63
> 25
15.01
23.14
> 25
14.42
> 25
> 25
4.31
> 25
23.65
20.96
15.87
23.30
19.41
> 25
> 25
24.76
> 25
> 25
> 25
> 25
23.69
> 25
15.49
> 25
18.42
> 25
16.13
> 25
23.10
> 25
> 25
14.64
> 25
18.38
19.90
> 25
19.97
> 25
24.00
> 25
> 25
> 25
> 25
20.45
5.70
20.93
5.86
20.89
> 25
16.27
> 25
13.15
> 25
4.30
> 25
> 25
> 25
> 25
15.34
20.35
16.24
15.32
14.91
4.40
12.56
15.51
13.16
14.16
> 25
15.57
16.32
16.92
15.11
20.58
7.35
22.71
15.38
15.59
14.65
> 25
16.74
16.73
15.07
13.78
> 25
16.56
22.22
23.47
20.43
> 25
21.48
3.33
> 25
> 25
24.41
3.70
24.50
8.04
> 25
4.97
6.76
3.83
> 25
> 25
> 25
> 25
24.34
2.89
> 25
> 25
> 25
24.34
1.40
> 25
0.88
> 25
1.05
1.40
2.00
> 25
> 25
> 25
> 25
> 25
4.19
2.79
8.93
3.74
2.97
2.56
4.50
8.99
14.73
14.77
4.75
3.33
Average values of IC50 from 6 independent experiments with SD ranging from 5 to 25% of the average
values. Dose response curves for CCRF-CEM cancer cell line compared to MRC-5 fibroblast cell line
are in Supplement Figure S1-S8.
9

Table 3. Selectivity indexes (SI) toward cancer cells for hydrazones 4-35.
Selectivity indexes (SI)
Compound
CCRF-
CEM
CEM-
DNR
K562-
TAX
HCT116
p53-/-
K562
HCT116
A549
Overall
4
1.23
4.98
1.02
6.49
5.88
5.68
1.10
6.86
1.58
1.02
1.65
1.63
7.42
1.02
50.78
0.95
1.00
7.92
0.86
5.29
6.87
18.27
8.23
1.00
0.97
22.15
1.00
1.00
36.45
0.97
71.13
8.41
1.00
1.00
1.00
1.07
1.30
2.09
1.51
1.63
1.26
1.00
1.00
1.00
1.06
1.00
1.00
0.95
1.00
1.54
0.86
1.63
1.23
1.54
1.63
1.68
5.49
4.62
1.00
1.03
6.43
0.97
5.75
1.62
1.00
1.26
1.00
0.98
1.81
1.00
1.00
1.74
1.56
1.00
5.80
1.00
1.61
1.00
1.71
0.95
1.00
1.90
0.86
1.99
1.61
1.90
1.77
1.00
1.12
6.90
1.00
1.00
21.13
0.97
8.63
2.67
1.17
1.04
1.00
0.98
1.48
6.02
1.00
1.88
1.49
1.16
3.86
1.00
1.36
1.00
1.36
1.19
1.00
5.82
0.86
1.61
1.53
1.48
1.65
1.21
3.28
3.40
1.00
1.00
17.7
0.97
6.44
1.62
1.00
1.46
1.00
1.02
1.21
1.30
1.00
5.00
1.73
1.00
1.06
1.19
1.58
1.07
1.29
0.95
1.22
4.38
0.86
1.1
1.05
1.24
1.00
0.99
1.45
1.64
1.00
1.52
1.83
1.00
1.00
1.00
1.55
1.00
1.25
0.95
1.19
4.27
0.86
1.49
1.49
1.66
1.81
1.00
0.99
6.21
1.00
1.03
13.28
0.97
9.41
7.16
1.00
1.00
1.00
0.98
1.06
1.00
1.00
1.64
1.08
1.00
1.01
1.00
1.08
1.00
1.04
0.95
1.20
1.00
0.86
1.51
1.12
1.07
1.22
1.00
0.98
2.86
1.00
1.00
9.30
0.97
5.36
2.65
1.06
1.29
1.00
1.14
1.51
1.67
1.07
2.10
1.46
1.02
1.41
1.08
1.51
1.01
1.44
0.98
1.08
2.38
0.86
1.68
1.59
1.72
1.81
1.09
1.28
5.08
1.00
1.01
12.85
0.97
8.00
2.86
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
1.63
1.60
1.71
1.00
0.97
5.99
1.00
1.00
13.28
0.97
8.12
5.03
SI = [average IC50 values for BJ and MRC-5 cells] / [IC50 values for corresponding cancer cell line];
Overall SI = [average IC50 values for BJ and MRC-5 cells] / [average IC50 values for all cancer cell lines],
with the respect to measurement limit (25 µM). The highest SI values are highlighted in blue (SI > 5)
and green (5 > SI > 2).
10

Table 4. Summary of cell cycle, apoptosis (sub G1), mitosis (pH3Ser10+), and DNA (BrdU+) and RNA
(BrU+) synthesis analyses in CCRF-CEM cells (for compounds with IC50 cell line < 5 µM). Data are
expressed as a percentage of the total cellular population.
% of total cell populations
BrdU+
(DNA
synthesis) synthesis)
BrU+
(RNA
Compound
Sub-G1
(apoptosis)
pH3Ser10
+
(mitosis)
G0/G1
S
G2/M
1.51
34.13
47.53
18.85
1.49
64.32
47.58
Control
7 1 × IC50
7 5 × IC50
8 1 × IC50
8 5 × IC50
9 1 × IC50
9 5 × IC50
11 1 × IC50
11 5 × IC50
16 1 × IC50
16 5 × IC50
18 1 × IC50
18 5 × IC50
21 1 × IC50
21 5 × IC50
23 1 × IC50
23 5 × IC50
24 1 × IC50
24 5 × IC50
25 1 × IC50
25 5 × IC50
26 1 × IC50
26 5 × IC50
29 1 × IC50
29 5 × IC50
32 1 × IC50
32 5 × IC50
34 1 × IC50
34 5 × IC50
35 1 × IC50
35 5 × IC50
4.94
7.55
5.04
24.99
5.51
10.51
6.31
20.28
4.79
7.62
2.81
3.85
5.76
9.26
8.09
39.75
7.47
24.05
5.85
8.72
7.20
16.20
6.93
23.81
9.30
35.21
5.13
15.38
8.32
27.57
29.30
28.25
31.38
32.03
30.87
34.56
30.53
46.08
24.54
41.09
32.90
34.82
30.36
43.54
38.76
51.65
20.69
51.25
29.87
21.48
28.46
43.18
25.44
52.17
45.02
58.84
31.41
45.67
28.72
52.77
46.97
53.73
43.92
48.09
41.74
38.90
45.19
25.72
53.80
37.07
42.83
41.20
42.72
36.07
39.14
27.81
69.22
22.44
40.63
65.99
40.51
34.88
46.44
19.67
33.29
25.05
40.52
31.97
37.96
20.92
23.73
18.02
24.70
19.87
27.39
26.54
24.28
28.19
21.66
21.83
24.27
23.98
26.92
20.39
22.10
20.54
10.09
26.31
29.50
12.53
31.03
21.94
28.12
28.16
21.69
16.11
28.07
22.36
33.31
26.31
0.95
0.59
1.12
0.43
0.81
0.47
0.49
0.04
0.83
0.11
1.98
1.19
0.52
0.04
0.18
0.13
0.33
0.02
1.16
0.32
0.79
0.11
0.86
0.05
0.04
0.05
1.35
0.06
0.61
0.02
60.61
46.02
54.02
24.79
59.37
48.93
61.19
3.82
3.62
0.78
48.67
5.87
36.49
6.80
29.12
3.27
69.15
27.04
52.30
53.83
69.98
15.34
44.85
6.51
95.83
55.12
48.66
5.24
66.04
33.56
44.48
1.63
69.53
2.45
50.21
2.23
57.41
66.51
61.33
44.60
62.66
2.46
39.00
24.88
36.18
32.67
49.44
16.20
0.51
7.13
0.36
0.20
56.50
33.76
64.42
1.65
44.85
55.91
54.47
1.49
11

3. Conclusions
In conclusion, we prepared series of caffeine-hydrazones possessing 2-hydroxyaryl- (4-28) or
2-N-heteroaryl group (29-35), respectively. Target compounds were prepared in three synthetic steps
with high overall yields. The anticancer activity of the prepared hydrazones was tested on nine cell
lines. Six compounds (11, 21, 29, 32, 34 and 35) show high cytotoxic activity. The best results were
obtained for compounds possessed 2-N-heterocyclic moiety (29, 32 and 34) which show superior
selectivity against malignant versus non-malignant cells. Majority of tested compounds exhibits dose-
dependent inhibition of both RNA and DNA synthesis, mitosis and induced apoptosis, however, they
failed to show any significant antimicrobial activity. Since the metal chelating capacity of these
compounds was not parallelised with antimicrobial activity, we hypothesize that anticancer activity is
independent of metal chelation.
Our results demonstrate that caffeine-hydrazones are a promising class of potent anticancer
agents with promising selectivity against cancer cells and warrant further attention and preclinical
development.
4. Experimental
4.1. Chemistry
4.1.1. Measurements and Materials
All chemicals were purchased from commercial suppliers and used without further
purification. NMR spectra were recorded on Varian Gemini 300 HC (300.077 MHz) at room
temperature in DMSO-d6 or pyridine-d5. The chemical shifts (δ) are presented in ppm, and coupling
constants (J) are presented in Hz. In some cases, 13C NMR spectra were not recorded due to
insufficient solubility. The program MestReNova ver. 8 was used for processing the NMR spectra.
Mass spectra were obtained using electrospray ionisation (ESI) with triple quadrupole mass
spectrometer TSQ Quantum Access. Elemental analysis was carried out using Elementar Vario El III
analyser.
4.1.2. Preparation of 8-chlorocaffeine (2)
Caffeine (1) (4.86 g; 25 mmol) was dissolved in chloroform (500 mL) at 50°C and chlorine gas
was then passed through the solution for 2 h while stirred at 50°C. Then solution was evaporated to
dryness yielded yellowish solid. 1H NMR (CDCl3) showed no starting material (signal at 7.53
disappeared). Crude product was used without further purification in the following step. Yield: 5.71 g
(100 %). 1H NMR (CDCl3): δ 3.38 (s, 3H); 3.53 (s, 3H); 3.94 (s, 3H)
4.1.3. Preparation of 8-hydrazinocaffeine (3)
8-Chlorocaffeine (2) (4.57 g; 20 mmol) was dissolved in ethanol (50 mL) and hydrazine
hydrate (10 mL; 200 mmol) was added and reaction mixture was heated at 80°C for 1 h. It is
necessary vigorous stirring, because voluminous precipitate of 3 was formed. Then reaction mixture
was poured on crushed ice (100 mL) and precipitate was filtered off, washed with water (3x 20 mL),
cold ethanol (10 mL) and diethyl ether (20 mL) and dried in vacuo at 50°C. The yield of 3 was 3.95 g
(88 %). 1H NMR (DMSO-d6): δ 3.16 (s, 3H); 3.36 (s, 3H); 3.54 (s, 3H); 4.32 (bs, 2H); 8.09 (bs, 1H).
12

4.1.4. Preparation of hydrazones 4-35
General procedure: 8-Hydrazinocaffeine (3) (112 mg; 0.5 mmol) and carbonyl compound (1 mmol; 2
eq.) were mixed in ethanol (20 mL, containing 0.2 mL of acetic acid) and heated at 75°C for 3 days.
After cooling down, reaction mixture was evaporated to dryness and solid residue was suspended in
diethyl ether (50 mL), filtered off, washed with additional portion of diethyl ether (3 25 mL) and
dried in vacuo at 50°C to obtain the hydrazone. NMR spectra of selected active compounds are in
Supplement Figure S9-S18.
1
Hydrazone 4: Carbonyl compound: 2-hydroxybenzaldehyde (122 mg). Yield: 145 mg (88%). H NMR
(DMSO-d6): δ 3.18 (s, 3H); 3.37 (s, 3H); 3.80 (s, 3H); 6.86 (m, 2H); 7.21 (m, 1H); 7.54 (dd, J = 7.6, 1.5
Hz, 1H); 8.37 (s, 1H); 10.71 (s, 1H); 11.40 (s, 1H). 13C NMR (DMSO-d6): δ 27.3 29.3 31.5 103.0 116.2
119.3 119.6 127.8 130.5 142.7 147.9 150.2 150.9 153.4 156.5. ESI-MS (m/z): 329 [M+H]+. Anal. Calcd
for C15H16N6O3: C, 54.87; H, 4.91; N, 25.60. Found: C, 54.86; H, 4.93; N, 25.58.
Hydrazone 5: Carbonyl compound: 2-hydroxy-3-methylbenzaldehyde (136 mg). Yield: 135 mg (79%).
1H NMR (DMSO-d6): δ 2.48 (s, 3H); 3.19 (s, 3H); 3.38 (s, 3H); 3.81 (s, 3H); 6.86 (d, J = 8.7 Hz, 1H); 7.35
(dd, J = 8.7, 2.6 Hz, 1H); 7.73 (d, J = 2.6 Hz, 1H); 8.34 (s, 1H); 10.86 (s, 1H); 11.52 (s, 1H). 13C NMR
(DMSO-d6): δ 18.0; 27.3; 29.4; 31.5; 103.1; 110.6; 118.5; 122.0; 129.1; 132.7; 140.4; 147.8; 149.9;
150.9; 153.5; 155.5. ESI-MS (m/z): 343 [M+H]+. Anal. Calcd for C16H18N6O3: C, 56.13; H, 5.30; N, 24.55.
Found: C, 56.14; H, 5.30; N, 24.56.
Hydrazone 6: Carbonyl compound: 2-hydroxy-3-methoxybenzaldehyde (152 mg). Yield: 166 mg
1
(86%). H NMR (DMSO-d6): δ 3.17 (s, 3H); 3.36 (s, 3H); 3.79 (s, 3H); 3.80 (s, 3H); 6.80 (dd, J = 7.9 Hz,
1H); 6.96 (d, J = 7.9 Hz, 1H); 7.14 (d, J = 7.9 Hz, 1H); 8.37 (s, 1H); 10.24 (s, 1H); 11.41 (s, 1H). 13C NMR
(DMSO-d6): δ 27.3; 29.3; 31.6; 55.8; 103.9; 109.6; 113.0; 119.0; 119.1; 119.9; 141.9; 142.5; 146.0;
147.9; 150.2; 153.5. ESI-MS (m/z): 359 [M+H]+. Anal. Calcd for C16H18N6O4: C, 53.63; H, 5.06; N, 23.45.
Found: C, 53.65; H, 5.07; N, 23.44.
Hydrazone 7: Carbonyl compound: 2,3-dihydroxy-3-methoxybenzaldehyde (138 mg). Yield: 143 mg
(83%). 1H NMR (DMSO-d6): δ 3.19 (s, 3H); 3.38 (s, 3H); 3.80 (s, 3H); 6.70 (m, 1H); 6.80 (dd, J = 7.8, 1.7
Hz, 1H); 6.98 (dd, J = 7.8, 1.7 Hz, 1H); 8.36 (s, 1H); 9.27 (s, 1H); 10.40 (s, 1H); 11.40 (s, 1H). 13C NMR
(DMSO-d6): δ 27.3; 29.3; 31.3; 102.9; 116.5; 118.7; 119.1; 119.6; 143.9; 145.2; 145.6; 147.8; 150.0;
150.9; 153.4. ESI-MS (m/z): 345 [M+H]+. Anal. Calcd for C15H16N6O4: C, 52.32; H, 4.68; N, 24.41.
Found: C, 52.31; H, 4.70; N, 24.38.
1
Hydrazone 8: Carbonyl compound: 3-ally-2-hydroxybenzaldehyde (162 mg). Yield: 142 mg (77%). H
NMR (DMSO-d6): δ 3.17 (s, 3H); 3.35 (m, 2H); 3.38 (s, 3H); 3.72 (s, 3H); 5.03 (m, 2H); 5.98 (m, 1H);
6.86 (t, J = 7.6 Hz, 1H); 7.12 (d, J = 6.9 Hz, 1H); 7.27 (d, J = 7.6 Hz, 1H); 8.34 (s, 1H); 11.43 (s, 1H);
11.46 (s, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.4; 30.7; 33.3; 102.9; 115.7; 117.9; 119.1; 127.0; 128.4;
130.8; 136.7; 146.0; 147.8; 149.7; 150.8; 153.4; 154.7. ESI-MS (m/z): 369 [M+H]+. Anal. Calcd for
C18H20N6O3: C, 58.69; H, 5.47; N, 22.81. Found: C, 58.67; H, 5.49; N, 22.78.
Hydrazone 9: Carbonyl compound: 2-hydroxy-4-methoxybenzaldehyde (152 mg). Yield: 143 mg
(80%). 1H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.37 (s, 3H); 3.75 (s, 3H); 3.78 (s, 3H); 6.47 (m, 2H); 7.44 (d,
J = 8.2 Hz, 1H); 8.31 (s, 1H); 11.02 (s, 1H); 11.25 (s, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.2; 31.2; 55.2;
13

101.1; 102.8; 106.1; 112.5; 129.5; 143.6; 147.8; 150.2; 150.9; 153.3; 158.3; 161.4. ESI-MS (m/z): 359
[M+H]+. Anal. Calcd for C16H18N6O4: C, 53.63; H, 5.06; N, 23.45. Found: C, 53.60; H, 5.07; N, 23.43.
Hydrazone 10: Carbonyl compound: 2,4-dihydroxy-3-methoxybenzaldehyde (138 mg). Yield: 158 mg
(92%). 1H NMR (DMSO-d6): δ 3.17 (s, 3H); 3.36 (s, 3H); 3.77 (s, 3H); 6.32 (m, 2H); 7.30 (m, 1H); 8.28 (s,
1H); 10.45 (bs, 1H); 11.12 (bs, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.2; 31.1; 102.6; 102.7; 107.5; 111.2;
129.8; 144.3; 147.8; 150.3; 150.8; 153.2; 158.5; 160.0. ESI-MS (m/z): 345 [M+H]+. Anal. Calcd for
C15H16N6O4: C, 52.32; H, 4.68; N, 24.41. Found: C, 52.29; H, 4.70; N, 24.40.
Hydrazone 11: Carbonyl compound: 4-(diethylamino)-2-hydroxybenzaldehyde (193 mg). Yield: 172
mg (86%). 1H NMR (DMSO-d6): δ 1.10 (t, J = 7.1 Hz, 6H); 3.18 (s, 3H); 3.34 (q, J = 7.1 Hz, 4H); 3.37 (s,
3H); 3.75 (s, 3H); 6.11 (s, 1H); 6.24 (d, J = 8.9 Hz, 1H); 7.22 (d, J = 8.9 Hz, 1H); 8.21 (s, 1H); 10.87 (s,
1H); 11.01 (s, 1H). 13C NMR (pyridine-d5): δ 13.1; 28.0; 29.9; 31.9; 44.8; 99.3; 104.4; 104.6; 108.5;
131.8; 147.7; 148.9; 150.9; 151.8; 152.1; 154.7; 160.8. ESI-MS (m/z): 400 [M+H]+. Anal. Calcd for
C19H25N7O3: C, 57.13; H, 6.31; N, 24.55. Found: C, 57.11; H, 6.34; N, 24.51.
Hydrazone 12: Carbonyl compound: 8-hydroxyjulolidine-9-carbaldehyde (217 mg). Yield: 176 mg
(83%). 1H NMR (DMSO-d6): 1.85 (m, 4H); 2.60 (m, 4H); 3.14 (m, 4H); 3.18 (s, 3H); 3.38 (s, 3H); 3.70 (s,
3H); 6.70 (s, 1H); 8.11 (s, 1H); 10.96 (s, 1H); 11.29 (s, 1H). 13C NMR (DMSO-d6): δ 20.4; 20.8; 21.6;
26.6; 27.3; 29.4; 30.6; 48.8; 49.3; 102.8; 106.2; 106.5; 112.3; 127.7; 144.7; 147.7; 148.1; 150.4; 150.9;
153.4; 153.9. ESI-MS (m/z): 424 [M+H]+. Anal. Calcd for C21H25N7O3: C, 59.56; H, 5.95; N, 23.15.
Found: C, 59.56; H, 5.99; N, 23.11.
Hydrazone 13: Carbonyl compound: 2-hydroxy-5-methylbenzaldehyde (136 mg). Yield: 135 mg (79%).
1H NMR (DMSO-d6): δ 2.23 (s, 3H); 3.19 (s, 3H); 3.38 (s, 3H); 3.81 (s, 3H); 6.79 (d, J = 8.2 Hz, 1H); 7.04
(d, J = 8.2 Hz, 1H); 7.34 (s, 1H); 8.35 (s, 1H); 10.52 (s, 1H); 11.38 (s, 1H). 13C NMR (DMSO-d6): δ 20.1;
27.3; 29.3; 31.4; 40.4; 103.1; 116.1; 119.1; 127.8; 127.8; 131.2; 143.0; 150.2; 151.0; 153.5; 154.4;
156.3. ESI-MS (m/z): 343 [M+H]+. Anal. Calcd for C16H18N6O3: C, 56.13; H, 5.30; N, 24.55. Found: C,
56.13; H, 5.32; N, 24.49.
Hydrazone 14: Carbonyl compound: 2-hydroxy-5-methoxybenzaldehyde (152 mg). Yield: 154 mg
(86%). 1H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.37 (s, 3H); 3.71 (s, 3H); 3.83 (s, 3H); 6.82 (m, 2H); 7.10 (m,
1H); 8.34 (s, 1H); 10.18 (s, 1H); 11.44 (bs, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.3; 31.6; 55.4; 103.0;
110.9; 117.1; 119.8; 142.0; 147.8; 150.1; 150.5; 150.9; 152.1; 153.4. ESI-MS (m/z): 359 [M+H]+. Anal.
Calcd for C16H18N6O4: C, 53.63; H, 5.06; N, 23.45. Found: C, 53.60; H, 5.07; N, 23.46.
Hydrazone 15: Carbonyl compound: 2,5-dihydroxy-3-methoxybenzaldehyde (138 mg). Yield: 153 mg
(89%). 1H NMR (DMSO-d6): δ 3.19 (s, 3H); 3.38 (s, 3H); 3.83 (s, 3H); 6.68 (m, 2H); 7.00 (dd, J = 2.9, 1.4
Hz, 1H); 8.32 (s, 1H); 8.91 (s, 1H); 9.85 (s, 1H); 11.34 (s, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.3; 31.6;
103.0; 112.3; 116.9; 118.0; 119.9; 142.2; 147.9; 149.3; 149.9; 150.2; 150.9; 153.4. ESI-MS (m/z): 345
[M+H]+. Anal. Calcd for C15H16N6O4: C, 52.32; H, 4.68; N, 24.41. Found: C, 52.29; H, 4.70; N, 24.38.
Hydrazone 16: Carbonyl compound: 5-chloro-2-hydroxybenzaldehyde (157 mg). Yield: 167 mg (92%).
1H NMR (DMSO-d6): δ 3.19 (s, 3H); 3.38 (s, 3H); 3.82 (s, 3H); 6.91 (m, 1H); 7.25 (m, 1H); 7.60 (s, 1H);
8.34 (s, 1H); 10.85 (s, 1H); 11.54 (s, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.3; 31.5; 103.0; 118.0; 121.4;
14

123.0; 126.2; 129.8; 140.4; 147.7; 149.8; 150.9; 153.4; 155.1. ESI-MS (m/z): 363 [M+H]+. Anal. Calcd
for C15H15ClN6O3: C, 49.66; H, 4.17; N, 23.17. Found: C, 49.65; H, 4.20; N, 23.15.
Hydrazone 17: Carbonyl compound: 5-bromo-2-hydroxybenzaldehyde (201 mg). Yield: 189 mg (93%).
1H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.39 (m, 3H); 3.74 (s, 3H); 6.82 (t, J = 7.5 Hz, 1H); 7.15 (d, J = 7.5
Hz, 1H); 7.24 (d, J = 7.5 Hz, 1H); 8.35 (s, 1H); 11.37 (s, 1H); 11.42 (s, 1H). 13C NMR (DMSO-d6): δ 27.3;
29.4; 30.8; 103.1; 117.7; 118.9; 124.9; 127.9; 131.7; 146.1; 147.9; 149.9; 151.0; 153.6; 155.2. ESI-MS
(m/z): 407, 409 [M+H]+. Anal. Calcd for C15H15BrN6O3: C, 44.24; H, 3.71; N, 20.64. Found: C, 44.21; H,
3.70; N, 20.62.
Hydrazone 18: Carbonyl compound: 5-tert-butyl-2-hydroxybenzaldehyde (178 mg). Yield: 154 mg
1
(80%). H NMR (DMSO-d6): δ 1.25 (s, 9H); 3.19 (s, 3H); 3.37 (s, 3H); 3.82 (s, 3H); 6.82 (d, J = 8.6 Hz,
1H); 7.26 (dd, J = 8.6, 2.5 Hz, 1H); 7.51 (d, J = 2.5 Hz, 1H); 8.37 (s, 1H); 10.55 (s, 1H); 11.41 (s, 1H). 13
C
NMR (DMSO-d6): δ 27.5; 29.5; 31.4; 31.5; 33.9; 103.8; 116.0; 118.7; 124.3; 124.7; 127.9; 128.0; 141.6;
143.7; 151.2; 153.7; 154.5. 13C NMR (pyridine-d5): δ 28.0; 29.9; 31.8; 32.3; 34.4; 104.6; 117.2; 119.9;
126.1; 128.7; 142.5; 145.4; 148.8; 151.3; 152.1; 154.8; 156.4. ESI-MS (m/z): 385 [M+H]+. Anal. Calcd
for C19H24N6O3: C, 59.36; H, 6.29; N, 21.86. Found: C, 59.35; H, 6.33; N, 21.84.
Hydrazone 19: Carbonyl compound: 2-hydroxy-5-nitrobenzaldehyde (167 mg). Yield: 175 mg (94%).
1H NMR (DMSO-d6): δ 3.19 (s, 3H); 3.39 (m, 3H); 3.83 (s, 3H); 7.07 (d, J = 9.0 Hz, 1H); 8.11 (d, J = 9.01
Hz, 1H); 8.44 (s, 1H); 8.50 (d, J = 3.0 Hz, 1H); 11.65 (s, 1H); 12.00 (s, 1H). ESI-MS (m/z): 374 [M+H]+.
Anal. Calcd for C15H15N7O5: C, 48.26; H, 4.05; N, 26.26. Found: C, 48.26; H, 4.07; N, 26.24.
Hydrazone 20: Carbonyl compound: methyl 3-formyl-4-hydroxybenzoate (180 mg). Yield: 176 mg
1
(91%). H NMR (DMSO-d6): δ 3.21 (s, 3H); 3.41 (s, 3H); 3.37 (s, 3H); 3.83 (s, 3H); 7.01 (d, J = 8.6 Hz,
1H); 7.83 (dd, J = 8.6, 2.2 Hz, 1H); 8.25 (d, J = 2.2 Hz, 1H); 8.45 (s, 1H); 11.52 (bs, 1H). ESI-MS (m/z):
387 [M+H]+. Anal. Calcd for C17H18N6O5: C, 52.85; H, 4.70; N, 21.75. Found: C, 52.86; H, 4.73; N, 21.72.
1
Hydrazone 21: Carbonyl compound: 1-hydroxy-2-naphthaldehyde (172 mg). Yield: 172 mg (91%). H
NMR (DMSO-d6): δ 3.19 (s, 3H); 3.43 (s, 3H); 3.78 (s, 3H); 7.51 (m, 4H); 7.85 (m, 1H); 8.27 (m, 1H);
8.51 (s, 1H); 11.52 (s, 1H); 12.20 (s, 1H). 13C NMR (DMSO-d6): δ 27.2; 29.3; 30.8; 102.9; 112.1; 118.7;
122.4; 124.4; 125.5; 126.1; 127.4; 127.5; 134.1; 145.5; 147.7; 149.7; 150.8; 153.3; 153.6. ESI-MS
(m/z): 379 [M+H]+. Anal. Calcd for C19H18N6O3: C, 60.31; H, 4.79; N, 22.21. Found: C, 60.29; H, 4.79; N,
22.19.
1
Hydrazone 22: Carbonyl compound: 2-hydroxy-1-naphthaldehyde (172 mg). Yield: 167 mg (88%). H
NMR (DMSO-d6): 3.20 (s, 3H); 3.41 (s, 3H); 3.79 (s, 3H); 7.38 (m, 1H); 7.57 (m, 1H); 7.87 (m, 2H); 8.29
(d, J = 8.3 Hz, 1H); 9.18 (s, 1H); 11.45 (s, 1H); 12.20 (s, 1H). 13C NMR (DMSO-d6): δ 27.4; 29.3; 30.9;
108.5; 118.7; 121.8; 127.6; 127.9; 128.9; 131.3; 131.8; 132.4; 134.8; 143.0; 147.3; 150.0; 151.0;
156.8; 160.1. ESI-MS (m/z): 379 [M+H]+. Anal. Calcd for C19H18N6O3: C, 60.31; H, 4.79; N, 22.21.
Found: C, 60.32; H, 4.80; N, 22.18.
Hydrazone 23: Carbonyl compound: 3,5-di-tert-butyl-2-hydroxybenzaldehyde (234 mg). Yield: 169 mg
(77%). 1H NMR (DMSO-d6): δ 1.28 (s, 9H); 1.41 (s, 9H); 3.20 (s, 3H); 3.41 (s, 3H); 3.75 (s, 3H); 7.21 (s,
1H); 7.27 (s, 1H); 8.38 (s, 1H); 11.39 (s, 1H); 11.74 (s, 1H). 13C NMR (pyridine-d5): δ 28.0; 30.0; 30.1;
15

31.7; 31.9; 34.6; 35.8; 104.6; 118.9; 126.1; 126.2; 137.1; 141.7; 148.8; 148.9; 151.1; 152.1; 154.9;
155.6. ESI-MS (m/z): 441 [M+H]+. Anal. Calcd for C23H32N6O3: C, 62.71; H, 7.32; N, 19.08. Found: C,
62.69; H, 7.25; N, 19.06.
Hydrazone 24: Carbonyl compound: 2-hydroxy-3,5-dichlorobenzaldehyde (191 mg). Yield: 185 mg
(93%). 1H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.39 (s, 3H); 3.75 (s, 3H); 7.52 (d, J = 2.5 Hz, 1H); 7.58 (d, J =
2.5 Hz, 1H); 8.31 (s, 1H); 11.87 (bs, 2H). 13C NMR (DMSO-d6): δ 27.4; 29.4; 30.7; 110.3; 121.5; 123.0;
127.3; 129.3; 142.2; 149.5; 149.7; 150.9; 150.9; 151.4; 153.6. ESI-MS (m/z): 397 [M+H]+. Anal. Calcd
for C15H14Cl2N6O3: C, 45.36; H, 3.55; N, 21.16. Found: C, 45.33; H, 3.56; N, 21.13.
Hydrazone 25: Carbonyl compound: 3,5-dibromo-2-hydroxybenzaldehyde (280 mg). Yield: 219 mg
1
(90%). H NMR (DMSO-d6): δ 3.13 (s, 3H); 3.35 (s, 3H); 3.69 (s, 3H); 7.63 (m, 2H); 8.18 (s, 1H); 11.70
(bs, 1H); 12.03 (bs, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.4; 30.9; 103.3; 110.6; 121.7; 124.5; 131.1;
134.7; 142.7; 147.7; 149.5; 150.9; 152.8; 153.6. ESI-MS (m/z): 485, 487, 489 [M+H]+. Anal. Calcd for
C15H14Br2N6O3: C, 37.06; H, 2.90; N, 17.29. Found: C, 37.03; H, 2.92; N, 17.28.
Hydrazone 26: Carbonyl compound: 5-allyl-2-hydroxy-3-methoxybenzaldehyde (192 mg). Yield: 161
mg (81%). 1H NMR (DMSO-d6): δ 3.20 (s, 3H); 3.31 (m, 2H); 3.39 (s, 3H); 3.79 (s, 3H); 3.82 (s, 3H); 5.07
(m, 2H), 5.96 (m, 1H); 6.80 (s, 1H); 6.98 (s, 1H); 8.38 (s, 1H); 10.06 (s, 1H); 11.39 (s, 1H). 13C NMR
(DMSO-d6): δ 27.3; 29.3; 31.5; 55.7; 66.4; 103.0; 113.1; 115.7; 118.5; 119.5; 130.3; 137.9; 142.5;
144.4; 147.8; 147.8; 150.1; 150.9; 153.4. ESI-MS (m/z): 399 [M+H]+. Anal. Calcd for C19H22N6O4: C,
57.28; H, 5.57; N, 21.09. Found: C, 57.28; H, 5.59; N, 21.10.
Hydrazone 27: Carbonyl compound: 5-bromo-2-hydroxy-3-methoxybenzaldehyde (231 mg). Yield:
1
188 mg (86%). H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.38 (s, 3H); 3.81 (s, 3H); 3.83 (s, 3H); 7.10 (d, J =
2.2 Hz, 1H); 7.33 (d, J = 2.2 Hz, 1H); 8.34 (s, 1H); 10.39 (s, 1H); 11.52 (s, 1H). ESI-MS (m/z): 437, 439
[M+H]+. Anal. Calcd for C16H17BrN6O4: C, 43.95; H, 3.92; N, 19.22. Found: C, 43.92; H, 3.94; N, 19.20.
Hydrazone 28: Carbonyl compound: 2-hydroxy-3-methoxy-5-nitrobenzaldehyde (197 mg). Yield: 194
mg (96%). 1H NMR (DMSO-d6): δ 3.18 (s, 3H); 3.39 (s, 3H); 3.83 (s, 3H); 3.94 (s, 3H); 7.70 (s, 1H); 8.13
(s, 1H); 8.44 (s, 1H); 11.57 (s, 2H). ESI-MS (m/z): 404 [M+H]+. Anal. Calcd for C16H17N7O6: C, 47.64; H,
4.25; N, 24.31. Found: C, 47.64; H, 4.27; N, 24.30.
Hydrazone 29: Carbonyl compound: pyridine-2-carbaldehyde (107 mg). Yield: 130 mg (83%). 1H NMR
(DMSO-d6): δ 3.19 (s, 3H); 3.38 (s, 3H); 3.94 (s, 3H); 7.35 (m, 1H); 7.86 (m, 2H); 8.13 (s, 1H); 8.57 (m,
1H); 11.71 (s, 1H). 13C NMR (DMSO-d6): δ 27.3; 29.4; 32.4; 103.3; 119.4; 123.7; 136.8; 143.1; 147.7;
149.4; 149.7; 150.9; 153.3; 153.5. ESI-MS (m/z): 314 [M+H]+. Anal. Calcd for C14H15N7O2: C, 53.67; H,
4.83; N, 31.29. Found: C, 53.65; H, 4.87; N, 31.26.
Hydrazone 30: Carbonyl compound: 6-methylpyridine-2-carbaldehyde (138 mg). Yield: 129 mg (79%).
1H NMR (DMSO-d6): δ 2.47 (s, 3H); 3.20 (s, 3H); 3.38 (s, 3H); 3.95 (s, 3H); 7.21 (dd, J = 6.8, 1.7 Hz, 1H);
7.69 (m, 2H); 8.07 (s, 1H); 11.70 (s, 1H). 13C NMR (DMSO-d6): δ 23.9; 26.6; 29.2; 31.9; 103.2; 116.3;
123.0; 137.0; 143.3; 147.0; 150.0; 151.0; 152.7; 154.5; 157.7. ESI-MS (m/z): 328 [M+H]+. Anal. Calcd
for C15H17N7O2: C, 55.04; H, 5.23; N, 29.95. Found: C, 55.00; H, 5.25; N, 29.93.
16

Hydrazone 31: Carbonyl compound: 6-methoxypyridine-2-carbaldehyde (122 mg). Yield: 149 mg
1
(87%). H NMR (DMSO-d6): δ 3.21 (s, 3H); 3.39 (s, 3H); 3.88 (s, 3H); 3.97 (s, 3H); 6.79 (d, J = 8.1 Hz,
1H); 7.46 (d, J = 7.3 Hz, 1H); 7.75 (m, 1H); 8.03 (s, 1H); 11.68 (s, 1H). ESI-MS (m/z): 344 [M+H]+. Anal.
Calcd for C15H17N7O3: C, 52.47; H, 4.99; N, 28.56. Found: C, 52.44; H, 5.03; N, 28.60.
1
Hydrazone 32: Carbonyl compound: di(2-pyridyl)ketone (184 mg). Yield: 166 mg (85%). H NMR
(DMSO-d6): δ 3.22 (s, 3H); 3.41 (s, 3H); 3.97 (s, 3H); 7.48 (m, 1H); 7.59 (m, 2H); 7.89 (m, 1H); 7.99 (m,
2H); 8.61 (m, 1H); 8.89 (m, 1H). 13C NMR (DMSO-d6): δ 27.4; 29.5; 31.8; 103.6; 123.6; 124.4; 126.4;
137.3; 137.7; 141.7; 147.6; 147.7; 148.4; 149.1; 150.9; 151.6; 153.7; 155.6. ESI-MS (m/z): 391 [M+H]+.
Anal. Calcd for C19H18N8O2: C, 58.45; H, 4.65; N, 28.70. Found: C, 58.46; H, 4.70; N, 28.72.
1
Hydrazone 33: Carbonyl compound: quinoline-2-carbaldehyde (157 mg). Yield: 147 mg (81%). H
NMR (DMSO-d6): δ 3.21 (s, 3H); 3.40 (s, 3H); 4.00 (s, 3H); 7.59 (m, 1H); 7.75 (m, 1H); 7.99 (m, 3H);
8.27 (s, 1H); 8.34 (d, J = 8.6 Hz, 1H); 11.84 (bs, 1H). ESI-MS (m/z): 364 [M+H]+. Anal. Calcd for
C18H17N7O2: C, 59.50; H, 4.72; N, 26.98. Found: C, 59.46; H, 4.76; N, 26.95.
1
Hydrazone 34: Carbonyl compound: acetylpyrazine (122 mg). Yield: 126 mg (77%). H NMR (DMSO-
d6): δ 2.39 (s, 3H); 3.22 (s, 3H); 3.41 (s, 3H); 4.01 (s, 3H); 8.58 (d, J = 2.5 Hz, 1H); 8.62 (m, 1H); 9.19 (s,
1H); 10.77 (bs, 1H). 13C NMR (pyridine-d5): δ 12.2; 28.1; 29.8; 34.2; 111.8; 124.1; 143.2; 143.8; 144.0;
146.8; 148.1; 151.0; 151.7; 154.9. ESI-MS (m/z): 329 [M+H]+. Anal. Calcd for C14H16N8O2: C, 51.21; H,
4.91; N, 34.13. Found: C, 51.18; H, 4.94; N, 34.10.
1
Hydrazone 35: Carbonyl compound: 3-methyl-2-acetylpyrazine (136 mg). Yield: 135 mg (79%). H
NMR (DMSO-d6): δ 2.40 (s, 3H); 2.73 (s, 3H); 3.20 (s, 3H); 3.39 (s, 3H); 3.84 (s, 3H); 8.48 (d, J = 2.5 Hz,
1H); 8.50 (d, J = 2.5 Hz, 1H); 10.31 (s, 1H). 13C NMR (DMSO-d6): δ 14.7; 24.2; 27.4; 29.3; 32.5; 103.7;
124.8; 140.8; 142.3; 147.4; 149.1; 150.8; 150.9; 151.9; 153.7. ESI-MS (m/z): 343 [M+H]+. Anal. Calcd
for C15H18N8O2: C, 52.62; H, 5.30; N, 32.73. Found: C, 52.61; H, 5.33; N, 32.70.
4.2. Biological activity
4.2.1. Cytotoxic MTT assay
All cells were purchased from the American Tissue Culture Collection (ATCC) except
HCT116p53-/-cells (Horizon Discovery, UK); K562-TAX were selected in our laboratory by the
cultivation of maternal cell lines in increasing concentrations of paclitaxel. The cells were maintained
in Nunc/Corning 80 cm2 plastic tissue culture flasks and cultured in cell culture medium
(DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, 10% foetal calf serum and NaHCO3). Cell suspensions were prepared and diluted
according to the particular cell type and the expected target cell density (25,000–30,000 cells/well
based on cell growth characteristics). Cells were added by pipette (80 µL) to 96-well microtiter plates.
Inoculates were pre-incubated for 24 h at 37°C and 5% CO2 for stabilisation. Four-fold dilutions, in 20
µL aliquots, of the intended test concentration were added to the microtiter plate walls at time zero.
All test compound concentrations were examined in duplicate. Incubation of the cells with the test
compounds was performed for 72 h at 37°C in a 5% CO2 atmosphere at 100% humidity. At the end of
the incubation period, the cells were assayed using MTT. Aliquots (10 µL) of the MTT stock solution
were pipetted into each well and incubated for a further 1–4 h. After this incubation period, the
produced formazan was dissolved by the addition of 100 µL/well 10% aq SDS (pH 5.5), followed by
17

further incubation at 37°C overnight. The optical density (OD) was measured at 540 nm with a
Labsystem iEMS Reader MF. Tumour cell survival (IC50) was calculated using the following equation:
IC = (ODdrug-exposed well/mean ODcontrol wells) x 100%. The IC50 value, the drug concentration lethal to 50%
of the tumour cells, was calculated from the appropriate dose-response curves by the Dotmatics
software platform.
4.2.2. Flow cytometric analysis
4.2.2.1. Cell Cycle and Apoptosis Analysis
A suspension of CCRF-CEM cells (ATCC), seeded at a density of 1.106 cells/ mL in 6-well
panels, was cultivated with the 1 × IC50 or 5 × IC50 of the tested compound in a humidified CO2
incubator at 37°C in RPMI 1640 cell culture medium containing 10% fetal calf serum, 10 mM
glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The treated cells and control sample
containing vehicle were harvested at the same time point (24 h). After a further 24 hours, cells were
washed with cold 1x PBS and fixed in 70% ethanol added dropwise and then stored overnight at -
20°C. Cells were then washed in hypotonic citrate buffer, treated with RNase (50 μg/mL) and stained
with propidium iodide. Flow cytometry using a 488 nm single beam laser (Becton Dickinson) was
used for measurement. The cell cycle profile was analysed by the ModFitLT (Verity) software
algorithm where G0/G1, S and G2/M phases of the cell cycle are calculated as a parts of 100% cellular
population. Apoptosis was measured independently using a logarithmic model expressed as the
percentage of the particles with propidium iodide content lower than cells in the G0/G1 phase (<G1)
of the cell cycle. Half of the sample was used for pH3Ser10 antibody (Sigma) staining and subsequent
flow cytometry analysis of mitotic cells), positively labeled cells were calculated as a percentage from
total population in the program CellQuest (Becton-Dickinson) [30].
4.2.2.2. BrdU Incorporation (DNA Synthesis) Analysis
The same cultivation procedure used in the previous method was applied for BrdU analysis.
Before harvesting, 10 μM 5-bromo-2-deoxyuridine (BrdU) was added to the cells for pulse labelling
for 30 min. The cells were fixed with ice-cold 70% ethanol and stored overnight. Before analysis, they
were incubated on ice for 30 min, washed with PBS, and resuspended in 2 M HCl for 30 min at room
temperature to denature DNA. Following neutralisation with 0.1 M Na2B4O7 (Borax), the cells were
washed with PBS containing 0.5% Tween-20 and 1% BSA. The cells were then stained with primary
anti-BrdU antibody (Exbio) for 30 min at room temperature in the dark. Cells were washed with PBS
and stained with secondary anti-mouse-FITC antibody (Sigma). The cells were then washed with PBS
and incubated with propidium iodide (0.1 mg/mL) and RNase A (0.5 mg/mL) for 1 h at room
temperature in the dark. Afterwards, stained cells were analysed by flow cytometry using a 488 nm
single beam laser (FACSCalibur, Becton); positively labelled cells were calculated as a percentage
from total population in the program CellQuest (Becton-Dickinson) [30].
4.2.2.3. BrU Incorporation (RNA Synthesis) Analysis
Cells were cultured and treated as above. Before harvesting, cells were pulse labelled with 1
mM 5-bromouridine (BrU) for 30 min. The cells were then fixed in 1% buffered paraformaldehyde
with 0.05% NP-40 at room temperature for 15 min and then stored at 4°C overnight. Before
measurement, they were washed in 1% glycine in PBS, washed in PBS, and stained with a primary
anti-BrDU antibody that crossreacts with BrU (Exbio) for 30 min at room temperature in the dark.
After another washing step in PBS, cells were stained with secondary anti-mouse-FITC antibody
18

(Sigma). Following the staining, cells were washed with PBS and fixed with 1% PBS-buffered
paraformaldehyde with 0.05% NP-40 for 1 hour. The cells were then washed with PBS, incubated
with propidium iodide (0.1 mg/mL) and RNase A (0.5 mg/mL) for 1 h at room temperature in the dark
and finally analysed by flow cytometry using a 488 nm single beam laser (FACSCalibur, Becton) [30].
4.2.3. Antimicrobial activity
The tested bacterial strains were selected to include both Gram-positive and Gram-negative
standard reference bacteria with good susceptibility to antibiotics. In addition, multidrug-resistant
bacteria were tested simultaneously because their frequency in both the human population and the
environment continues to increase and must be considered. The following reference strains (labelling
according to the Czech Collection of Microorganisms, Czech Republic) were used: Enterococcus
faecalis CCM 4224, Staphylococcus aureus CCM 3953, Escherichia coli CCM 3954, Pseudomonas
aeruginosa CCM 3955. We also used multi-resistant strains isolated from the clinical specimens of
patients hospitalised at the University Hospital Olomouc (Czech Republic): methicillin-resistant
Staphylococcus aureus MRSA 4591 and fluroquinolone resistant Staphylococcus haemolyticus
A/16568), Escherichia coli C/16702 and Pseudomonas aeruginosa A/16575. Similarly, the
effectiveness against Candida albicans, Candida krusei, Candida tropicalis and Candida parapsilosis
was also determined.
Bacterial and yeast suspensions were prepared as follows. The tested strains were inoculated
on blood agar and Sabouraud dextrose agar and incubated for 24 hours/35°C. Four to five isolated
colonies were dissolved in 2 mL of Mueller Hinton broth (Himedia) and incubated for 1 hour/35°C.
The broth was then diluted in a Petri dish in 10 mL of distilled water and inoculated in microtiter
plates.
MIC assessment: antimicrobial efficacy was tested by the standard dilution micromethod,
and the minimal inhibition concentrations (MIC) required for inhibition of bacterial growth were
determined. Samples were diluted exponentially and tested in microtiter plates in Brain Heart
Infusion broth (Himedia). Plates were inoculated into a standard quantity of microbes (106 CFU/mL).
The MIC was read after 24 and 48 hours of incubation at 35°C for bacteria and yeast, respectively, as
the minimum inhibitory concentration of the tested substance that inhibited the growth of the
bacterial or Candida strain.
MBC/MBS assessment (minimal bactericidal/bacteriostatic concentration): to assess
bactericidal (ability of the compound to kill bacteria) or bacteriostatic (ability of the compound to
inhibit bacterial growth) properties of the compound, 1 µl of the bacterial suspension from microtiter
plates with the determined MIC was inoculated on blood agar (bacteria) or on Sabouraud dextrose
agar (yeasts) and incubated for 24 or 48 hours at 35°C for bacteria and yeast, respectively. The
bactericidal/bacteriostatic properties of the compounds were determined by the negative or positive
growth of bacterial colonies.
4.3. Metal-binding studies
Binding studies of hydrazones 4-35 with the chlorides of metal ions (Fe2+, Fe3+, Co2+, Cu2+,
Cr3+, Ni2+, Zn2+, Mn2+, Ca2+ and Mg2+) were performed using UV/Vis spectroscopy in DMSO-water (1:1,
v/v). The corresponding hydrazone (0.03 mM) solution in DMSO-water (1:1, v/v, 100 mL) was titrated
using a semi-automatic titration system (peristaltic pump, flow-cuvette, syringe pump) with the
continuous addition of a stock solution of metal ions (6 mM, 10 mL/h). The spectra were collected
every 30 sec.
19

Acknowledgements
The authors are grateful to the Ministry of Education, Youth and Sport of the Czech Republic,
for the project CZ.1.07/2.3.00/30.0060 and CZ.1.07/2.3.00/30.0041 supported by the European
Social Fund and program NPU I LO1304. This work was supported by the Technology Agency of the
Czech Republic, project TE01020028.
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21

Highlights
Caffeine-hydrazones were prepared in three-step synthesis
Anticancer activity tested on the 7 cancer and 2 healthy cell lines
High activity and great selectivity toward T-lymphoblastic leukaemia cells
Antimicrobial activity tested on 13 bacterial stains, compounds were not active
Binding properties toward biologically important metal ions
22