Supramolecular solid-state structure, potential energy surfaces and evaluation of antiproliferative effect of 2-benzothiazolylhydrazone derivatives in vitro

Article abstract of DOI:10.1007/s11224-016-0856-0

The in situ condensation reaction of 2-hydrazinobenzothiazole with salicylaldehyde, 3,4-dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, 2-hydroxy-1-naphthaldehyde, 2-methoxy-1-naphthaldehyde, 4-metho

Full text of DOI:10.1007/s11224-016-0856-0

Struct Chem
DOI 10.1007/s11224-016-0856-0
ORIGINAL RESEARCH
Supramolecular solid-state structure, potential energy
surfaces and evaluation of antiproliferative effect
of 2-benzothiazolylhydrazone derivatives in vitro
1
2
2
3
•
Robert Katava
•
Sandra Kraljevi c´ Paveli c´
1
•
Anja Harej
•
Tomica Hrenar
Gordana Pavlovic´
Received: 22 July 2016 / Accepted: 22 September 2016
Ó Springer Science+Business Media New York 2016
Abstract The in situ condensation reaction of 2-hydrazi-
nobenzothiazole with salicylaldehyde, 3,4-dihydroxyben-
zaldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxyben-
zaldehyde, 2,3-dihydroxybenzaldehyde, 2-hydroxy-1-naph-
thaldehyde, 2-methoxy-1-naphthaldehyde, 4-methoxy-1-
naphthaldehyde and 6-methoxy-2-naphthaldehyde produced
synthons into different supramolecular arrangements via
C–HÁÁÁO, C–HÁÁÁN and C–HÁÁÁS intermolecular hydrogen
bonds. The role of aryl substituents in the shaping and stabi-
lization of supramolecular architectures of L1, L7–L9 is
supported by quantum chemical calculations. Strong
antiproliferative effects on tumor cells and cytotoxic effects
on fibroblasts are shown for all ligands L1–L9 with exception
of L6 and L7 that had no effect on fibroblast cells.
9
hydrazone Schiff bases (L1–L9, respectively) which were
identified and characterized by elemental analysis, IR and
NMR spectroscopy. The crystal and molecular structures of
four Schiff bases (L1, L7–L9) have been determined by the
single-crystal X-ray diffraction method confirming the imino
form of L1 and the amino tautomeric form of L7–L9 com-
pounds. Molecular structure analysis also confirmed that
reported compounds are E-isomers relative to exo C = N
iminobond. TheNhydrazino–Hgroup ofamino tautomersforms
Keywords Schiff base ligands Á Hydrazones Á
Benzothiazoles Á Antiproliferative activity
Introduction
Nhydrazino–HÁÁÁN
intermolecular hydrogen bonds shap-
Among heterocyclic compounds, 1,3-benzothiazoles
gained special attention due to diverse biological activities
and capability to bind to DNA molecules via p–p inter-
actions. This results in antitumor, antimicrobial, antidia-
betic, anticonvulsant and anti-inflammatory activity [1–6].
Moreover, the hydrazone functional group (–C = N–NH–)
is of great importance in medicinal chemistry with a
potential in diverse applications. Combining hydrazone and
benzothiazole functionalities in a unique molecular system
thiazolyl
2
ing molecules into R (8) rings, while imino tautomer of L1
2
forms C(4) infinite helical chains via Nthiazolyl–HÁÁÁNhydrazino
type of intermolecular hydrogen bond. The methoxy group
(
L7–L9) further shaped these primary supramolecular
Electronic supplementary material The online version of this
article (doi:10.1007/s11224-016-0856-0) contains supplementary
material, which is available to authorized users.
(Bzt–NH–N = CH–Aryl; Bzt = benzothiazolyl) may thus
bring new properties and synergistic effects in both struc-
tural and electronic context.
&
Gordana Pavlovic´
gpavlovic@ttf.hr
Hydrazone Schiff bases as ligands have vast and diverse
coordination potential to metal center due to stereochemi-
cally suitable positions of donating atoms including:
endocyclic nitrogen, imine and hydrazone nitrogen atom
and other functionalities at aryl moiety. Biological effects
of 2-hydrazinobenzothiazole Schiff base metal complexes
are still a promising area of research, particularly in com-
parison with uncoordinated ligands effects since increased
1
Department of Applied Chemistry, Faculty of Textile
Technology, University of Zagreb, Prilaz baruna Filipovi c´ a
2
8a, 10000 Zagreb, Croatia
2
3
Department of Biotechnology, Centre for High-throughput
Technologies, University of Rijeka, Radmile Matej cˇ i c´ 2,
5
1000 Rijeka, Croatia
Department of Chemistry, Faculty of Science, University of
Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia
123

Struct Chem
biological activity is noticed when organic drugs are
administered in the form of metal complex. Also, their
applications are versatile including not only medicinal, but
supramolecular chemistry and chemistry of dyes [7].
Survey of CSD [8, 9] revealed 12 structures of unco-
ordinated 2-hydrazinobenzothiazole Schiff bases that can
be found in more common amino [10, 11] or just one so far
imino tautomeric form [11] (Scheme 1), as solvates and
anhydrous compounds. The preferential supramolecular
architecture of hydrazones in the solid state is simultane-
ously dependable of the type and position of the sub-
stituents on the aryl moiety and the presence of solvate
molecules in the crystal, rather than of the tautomeric type
of hydrazone molecule.
Experimental
Materials and methods
Salicylaldehyde, 3,4-dihydroxybenzaldehyde, 2,4-dihy-
droxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 2,3-dihy-
droxybenzaldehyde, 2-hydroxy-1-naphthaldehyde, 2-methoxy-
1-naphthaldehyde, 4-methoxy-1-naphthaldehyde, 6-methoxy-
2-naphthaldehyde and 2-hydrazinobenzothiazole were pur-
chased from commercial sources Acros Organics and Sigma-
Aldrich and used as received. Infrared spectra were recorded on
a PerkinElmer FT-IR spectrometer Spectrum TWO equipped
with a PerkinElmer Universal ATR Sampler Accessory in the
-
1
1
13
4000–400 cm region. The H NMR and the C NMR
spectra were recorded on a Brucker Avance 600 MHz NMR
In the more common amino hydrazones [10, 11], inter-
1
13
Spectrometer (600 and 150 MHz, for H and C, respectively)
molecular contacts are characterized by Nhydrazino
–
HÁÁÁN
hydrogen bonds that either form symmetric
in DMSO-d solutions. Chemical shifts are reported in ppm
6
thiazolyl
2
2
R (8) dimers or infinite helical C(4) chains that are often
relative to TMS as an internal standard. The elemental analysis
was performed at the Analytical Services Laboratory of the
Ru d¯ er Bo sˇ kovi c´ Institute, Zagreb, on a Perkin Elmer CHNS/O
analyzer Series II 2400. Melting points were determined on a
Reichert Austria 7905 microscope for the determination of the
melting points. The powder X-ray diffraction data were col-
lected by the Panalytical X’Change powder diffractometer in
the Bragg–Brentano geometry using CuKa radiation. The
sample was contained on a Si sample holder. Patterns were
collected in the range of 2h = 5–50° with the step size of 0.03°
and at 1.5 s per step. The data were collected using the X’Pert
programs Suite [16] and visualized using the DiffractWD [17].
supported by pÁÁÁp interactions. In solvated compounds,
solvate molecules are involved in indirect links between
the hydrazone molecules [11].
In our effort to prepare metal complexes and investigate
their pharmacological activities, we synthesized and char-
acterized a series of nine ligands (L1–L9) functionalized
by -OH or -OCH functionalities at different phenyl or
3
naphthyl positions (Bzt–NH–N = CH–C H –R R ; Bzt–
6
4
1 2
NH–N = CH–C H –R (Scheme 2) with the aim to
3
1
0
7
(
a) enhance coordination capability of 2-benzothiazolylhy-
drazone derivatives as ligands and (b) outline
supramolecular architecture of these ligands in uncoordi-
nated state and (c) test antiproliferative effects of obtained
structures in vitro.
Preparative and crystallization procedures
The synthesis of six ligands (L1–L6) was previously
reported [12–15]. All tested compounds were identified
by elemental analysis, IR spectroscopy, NMR spec-
troscopy, and additionally, compounds L1, L7–L9 were
characterized by single-crystal X-ray diffraction method.
Structural and theoretical studies in the solid state were
performed in order to identify any common features of
intermolecular interactions that might be responsible for
biological activity of these ligand systems. In the con-
tinuation of our research, we plan to prepare metal
complexes of these ligands and investigate their biologi-
cal activity as well.
2-Benzothiazolylhydrazone derivatives were prepared by
condensation reactions of 2-hydrazinobenzothiazole
(3 mmol) with appropriate arenealdehydes (3 mmol) in
ethanol (30 mL) (Scheme 2). The mixture was refluxed
for 4 h at 70 °C. Resulting precipitates were filtered off
and washed with cold ethanol. The compounds L7–L9
were prepared according to the same synthetic procedure
as previously reported L1–L6 [12–15]. Spectral data and
elemental analysis of all prepared compounds can be
found in the Supporting Information (Table S2, Figs.
S1–S27).
Scheme 1 Tautomeric forms of
2
-benzothiazolylhydrazones
1
23

Struct Chem
Crystals of 2-hydroxybenzaldehyde-(2-benzothiazolylhy-
4-methoxy-1-naphthaldehyde-(2-benzothiazolylhydrazone)
(L8)
drazone) (L1), 4-methoxy-1-naphthaldehyde-(2-benzothia-
zolylhydrazone) (L8) and 6-methoxy-2-naphthaldehyde-(2-
benzothiazolylhydrazone) (L9) precipitated after a few days
from cooled DMSO solutions. Crystals of 2-methoxy-1-
naphthaldehyde-(2-benzothiazolylhydrazone) (L7) were pre-
pared by evaporation of pyridine solution of L7.
-
1
Yellow solid; yield: 77 %; m.p. 219–221 °C; IR (m cm
ATR): 3170 (N–H), 1609 (C = N)exo, 1560 (Carom
,
=
1
C
arom), 1268 (Carom–N), 1228 (C–O);
(600.00 MHz, DMSO-d
d, J = 8.46 Hz, 1H, H ), 8.68 (s, 1H, N = C–H), 8.30 (d,
H
NMR
, d ppm): 12.14 (s, 1H, NH), 9.02
6
(
ar
J = 8.34 Hz, 1H, Harom), 7.82–7.80 (m, 2H, Harom), 7.74
m, J = 7.56 Hz, 1H, Harom) 7.62 (t, J = 7.44 Hz, 1H,
2
-methoxy-1-naphthaldehyde-(2-benzothiazolylhydrazone)
(
(
L7)
Harom), 7.46 (d, J = 7.98 Hz, 1H, H ), 7.33 (t,
ar
J = 7.56 Hz, 1H, Harom), 7.13–7.08 (m, 2H, Harom), 4.05
13
-
1
Yellow solid; yield: 85 %; m.p. 216–218 °C; IR (m cm
,
(
s, 3H, CH3); C NMR (150.00 MHz, DMSO-d , d ppm):
6
ATR): 3168 (N–H), 1608 (C = N), 1561 (C = Carom),
1
270 (Carom–N), 1248 (C–O); H NMR (600.00 MHz,
arom
166.8, 156.3, 130.8, 129.9, 127.8, 125.9, 125.7, 125.1,
1
124.6, 122.1, 121.5, 121.4, 104.3, 55.8; Anal. calcd. for
DMSO-d , d ppm): 12.27 (s, 1H, NH), 9.31 (d,
6
C H N OS: C, 68.44; H, 4.54; N, 12.61; S, 9.62 %,
19 15 3
J = 8.70 Hz, 1H, Harom), 8.93 (s, 1H, N = C–H), 8.02 (d,
J = 9.06 Hz, 1H, Harom), 7.92 (d, J = 8.10 Hz, 1H,
Harom), 7.83 (d, J = 7.74 Hz, 1H, Harom), 7.67 (t,
J = 7.74 Hz, 1H, Harom), 7.52–7.46 (m, 3H, Harom), 7.34
Found: C, 68.33; H, 4.70; N, 12.71; S, 9.65 %.
6-methoxy-2-naphthaldehyde-(2-benzothiazolylhydrazone)
(L9)
(
t, J = 7.44 Hz, 1H, Harom), 7.14 (t, J = 7.68 Hz, 1H,
1
Harom), 4.01 (s, 3H, CH3); C NMR (150.00 MHz,
3
DMSO-d , d ppm) 166.9, 157.3, 132.2, 130.5, 128.8, 128.6,
Yellow solid; yield (based on 2-hydrazinobenzothiazole):
-
6
1
1
28.2, 125.9, 125.4, 124.0, 121.5, 114.3, 113.4, 56.7; Anal.
79 %; m.p. 235–237 °C; IR (m cm , ATR): 3183 (N–H),
1621 (C = N)exo, 1601 (C = N)endo, 1574 (Carom
calcd. for C H N OS: C, 68.44; H, 4.54; N, 12.61; S,
19 15 3
=
1
H
9
.62 %, Found: C, 68.30; H, 4.74; N, 12.81; S, 9.73 %.
Carom), 1265 (Carom–N), 1243 (C–O);
NMR
Scheme 2 Preparation and
structural formulas of
compounds L1–L9 with
torsional coordinates u
1 2
and u
(
atoms that define torsional
coordinates are presented in
Fig. S28)
123

Struct Chem
(
600.00 MHz, DMSO-d , d ppm) 12.25 (s, 1H, NH), 8.30
s, 1H, N = C–H), 8.02 (s, 1H, Harom) 7.95 (d,
L8. Programs CrysAlis CCD [18] and CrysAlis RED [18]
(Version 1.171.37.35) were employed for data collection,
cell refinement and data reduction. Structures were solved by
direct methods, and all non-hydrogen atoms were refined
6
(
J = 8.46 Hz, 1H, Harom), 7.89 (t, J = 8.58 Hz, 2H, Harom),
7
.80 (d, J = 7.56 Hz, 1H, Harom), 7.49 (d, J = 7.62 Hz,
2
1
H, Harom), 7.36–7.33 (m, 2H, Harom), 7.23 (d,
anisotropically based on F by weighted full-matrix least
J = 8.04 Hz, 1H, H
), 7.14 (t, J = 6.96 Hz, 1H, Harom),
squares. Programs SHELXST-2014 [19] and SHELXL-2014
[19] integrated in the WinGX v. 2014.1.v [20] software
system were used to solve and refine structures. Hydrogen
arom
1
3
3
.91 (s, 3H, CH3); C NMR (150.00 MHz, DMSO-d , d
6
ppm) 167.0, 158.1, 135.0, 129.8, 129.8, 128.2, 127.8,
27.4, 125.9, 122.8, 121.5, 119.0, 106.4, 55.3; Anal. calcd.
for C H N OS: C, 68.44; H, 4.54; N, 12.61; S, 9.62 %,
2
3
1
atoms belonging to Csp and Csp carbon atoms were placed
in geometrically idealized positions, and they were con-
strained to ride on their parent atoms using the appropriate
SHELXL-2014 [19] HFIX instructions. The positions of
hydrogen atoms belonging to the oxygen O1 and nitrogen N1
in compound L1, nitrogen N2 in compound L8 and nitrogen
atoms N21 and N22 in compounds L7 and L9 were deter-
mined from difference Fourier syntheses, and their coordi-
nates were refined freely, while isotropic displacement
parameters were refined with U (H) = 1.2 U (N). The
1
9 15 3
Found: C, 68.51; H, 4.71; N, 12.72; S, 9.70 %.
Single-crystal X-ray diffraction experiment
The general and crystallographic data for compounds L1,
L7–L9 are listed in Table 1. Crystallographic data were
recorded on Oxford Xcalibur diffractometer, equipped with
a CCD area detector, using Mo Ka radiation (k = 0.71,073
iso
eq
˚
A) at T = 296 K for L1, L7 and L9 and on Oxford Diffrac-
molecular geometry calculations were performed by PLA-
TON [21], and the molecular graphics were generated using
Mercury program [22].
tion Xcalibur Nova R, equipped with a CCD area detector,
˚
using Cu Ka radiation (k = 1.54,184 A) at T = 296 K for
Table 1 General and crystal data and summary of intensity data collection and structure refinement for compounds L1, L7–L9
Compound
L1
L7
L8
L9
Brutto chemical formula
C
14 11 3
H N OS
C
H N
19 15 3
OS
C
19 15
H N
3
OS
19 15 3
C H N OS
M
r
269.3
Monoclinic, yellow, Monoclinic, yellow, Monoclinic, yellow, Monoclinic, yellow,
prism prism prism needle
P 2 /n I 2/a P 2 /n P 2 /c
333.4
333.4
333.4
Crystal system, color, habit
Space group
3
Crystal dimensions (mm )
1
1
1
0.49 9 0.16 9 0.10
0.35 9 0.23 9 0.20
0.37 9 0.20 9 0.12
0.62 9 0.17 9 0.08
Unit cell parameters
˚
a/A
15.6365 (10)
4.5903 (3)
17.6379 (13)
91.813 (6)
1265.35 (3)
4
21.0027 (12)
12.2101 (6)
24.9320 (14)
99.759 (5)
6301.2 (6)
16
12.0230 (2)
6.3698 (1)
20.7480 (2)
93.318 (1)
1586.30 (1)
4
17.5548 (10)
7.3052 (3)
26.3033 (15)
107.263 (6)
3221.2 (3)
8
˚
b/A
˚
c/A
b/°
V/A
3
˚
3
Z
-3
q(calculated)/g cm
1.41
1.41
1.40
1.37
-
1
l/mm
F(000)
0.250
0.216
1.894
0.211
560
2784
696
1392
o
h–range/
4.6–27.5
5702, 2875, 1718
4.2–27.5
14,250, 7203, 3690
4.1–70.0
13,595, 3009, 2866
4.3–27.5
15,369, 7376, 4334
Reflections collected, unique (Rint.),
observed [I [ 2r(I)]
Number of parameters refined
178
441
221
441
R(F
0
)
0.060
0.068
0.035
0.073
2
0
R
w
(F
)
0.112
0.092
0.100
0.117
2
Goodness of fit on F , S
0.986
1.004
1.071
1.038
Max. and min. electron density, Dqmax
Dqmin/e A
,
-0.222, 0.245
-0.241, 0.215
-0.259, 0.197
-0.281, 0.222
-
3
˚
1
23

Struct Chem
Cell culturing
correspond to the minimum on the potential energy surface.
Standard Gibbs energies of formation were calculated at
T = 298.15 K and p = 101,325 Pa.
Cell lines HeLa (cervical carcinoma), SW620 (colorectal
adenocarcinoma, metastatic), HepG2 (hepatocellular car-
cinoma), A549 (lung adenocarcinoma) and 3T3 (mouse
embryonic fibroblasts) were cultured as monolayers and
maintained in Dulbecco’s modified Eagle medium
Potential energy surfaces (PES) scans for monomers
were conducted by varying the torsional coordinates u and
1
u (Scheme 2) using the automatic conformational gener-
2
ator implemented in program qcc [26, 27]. Data from PES
scans were arranged in one- or two-way arrays. Parallelized
combinatorial optimization algorithm for the arbitrary
number of ways (dimensions) implemented in program
moonee [28] was used to determine all local minimums on
the investigated PES. All local minimums were reopti-
mized at the B3LYP/6-311 ??G(d,p) level of the theory.
All quantum chemical calculations were performed using
the Gaussian 09 program [29].
(
(
DMEM) supplemented with 10 % fetal bovine serum
FBS), 2 mM L-glutamine, 100 U/ml penicillin and
1
00 lg/ml streptomycin in a humidified atmosphere with
5
% CO at 37 °C.
2
Proliferation assays
The panel cell lines were inoculated onto a series of stan-
dard 96-well microtiter plates on day 0, at 5000 cells per
well according to the doubling times of specific cell line.
Test agents were then added in five, 10-fold dilutions
Results and discussion
Chemistry
(
0.01–100 lM) and incubated for further 72 h. Working
dilutions were freshly prepared on the day of testing in the
growth medium. The solvent (DMSO) was also tested for
eventual inhibitory activity by adjusting its concentration
to be the same as in the working concentrations (DMSO
concentration never exceeded 0.1 %). After 72 h of incu-
bation, the cell growth rate was evaluated by performing
the MTT assay: experimentally determined absorbance
values were transformed into a cell percentage growth (PG)
using the formulas proposed by NIH and described previ-
ously (1). This method compares the growth of treated cells
with the growth of untreated cells in control wells on the
same plate in comparison with the starting cell number
values. Results are therefore a percentile difference from
the calculated expected value.
Target compounds were prepared in good yields by con-
densation reactions of 2-hydrazinobenzothiazole with
appropriate arenealdehydes following a general procedure
(Scheme 2). The proposed structural formulas of com-
pounds L1, L7–L9 were verified by single-crystal X-ray
1
13
diffraction, spectroscopic methods (FT-IR, H and
C
NMR) and elemental analysis. Compared to starting
materials, the most notable changes in the IR spectra of
prepared compounds are: disappearance of the absorption
-
1
band at 3319 cm assigned to m(NH ) of the 2-hydrazi-
2
nobenzothiazole, disappearance of the absorption band in
-
1
the 1631-1679 cm
region assigned to m(C = O) of
arenealdehydes and appearance of an intense band in the
The IC50 values for each compound were calculated
from dose–response curves using linear regression analysis
by fitting the mean test concentrations that give PG values
above and below the reference value. If, however, all of the
tested concentrations produce PGs exceeding the respec-
tive reference level of effect (e.g., PG value of 50) for a
given cell line, the highest tested concentration is assigned
as the default value (in the screening data report that
default value is preceded by a ‘‘ [ ’’ sign). Each test point
was performed in quadruplicate in three individual
experiments.
-
1
1608–1621 cm region assigned to the m(C = N) of both
imino functional group and benzothiazole moiety [30]. In
compound L9 two medium intensity bands appear at 1621
-
1
and 1600 cm and can be assigned to the m(C = N)exo of
the imino functional group and to the m(C = N)endo of the
benzothiazole moiety, respectively. IR spectra of com-
pounds L7–L9 show weak intensity band in
-
1
3168-3183 cm region assigned to m(NH) and strong
intensity band in the 1228–1248 cm region assigned to
-
1
m(C–O) of the methoxy group. Condensation reactions of
2
-hydrazinobenzothiazole with appropriate arenealdehydes
1
1
Quantum chemical calculation
are also confirmed with H NMR spectral data. H NMR
spectra of compounds L7–L9 exhibit singlets in
12.14–12.27, 8.30-8.93 and 3.93-4.05 ppm regions
Geometry optimization for ground states of monomers,
dimers or tetramers was performed using hybrid functional
B3LYP [23, 24] with D3 version of Grimme’s dispersion
assigned to the N–H, N = C–H and CH protons, respec-
3
tively. Compared to theoretically calculated values for
monomeric compounds of benzothiazolyl hydrazones
reported by Lindgren et al. [10], chemical shifts assigned to
N–H protons, in the 12.14–12.27 ppm region, show
[
25] in combination with the 6-311 ??G(d,p) basis set.
For all optimized structures, harmonic vibrational fre-
quencies were calculated to insure that obtained geometries
123

Struct Chem
significant deshielding due to involvement of these protons
in the formation of intermolecular hydrogen bonds.
According to the literature data, chemical shifts assigned to
N–H protons bond in the 9-12 ppm region are character-
istic for E-isomeric form relative to exo C = N imino
bond, contrary to chemical shift assigned to N–H protons in
and L9, molecule 1), and S12, C12–C62, N12 and C72 (L7
and L9, molecule 2)] and arene planes [defined by atoms:
C9–C14 (L1), C9–C18 (L8), C91, C101–C181 (L7 and L9,
molecule 1) and C92, C102–C182 (L7 and L9, molecule 2)]
of: 5.41(15)° (L1), 9.96(5)° (L8), 4.47(11)° (L7, molecule
1), 8.82(10)° (L7, molecule 2), 3.37(8)° (L9, molecule 1),
6.03(9)° (L9, molecule 2). Molecular structure analysis also
confirmed that reported compounds are E-isomers relative
to exo C = N imino bond. Imino tautomeric form of L1 and
amino form of L7–L9 has been confirmed by geometric
parameters listed in Tables 2 and 3. The N1–C7 bond
1
4–15 ppm region found in Z-isomeric form [31, 32].
Accordingly, all prepared compounds (L7–L9) as well as
previously reported (L1–L6) are E-stereoisomers. Calcu-
lated PXRD patterns of 2-benzothiazolylhydrazones L1
and L7 from the single-crystal X-ray crystallography
studies, whose single crystals were obtained from DMSO
˚
(1.335(3) A) in L1 that exists in imino tautomeric form is
(
L1) and pyridine (L7) solutions, are in agreement with
predominantly r in character and significantly longer in
comparison with N1–C7 (L8), N11–C71 (L7, L9) and N12–
˚
C72 (L7, L9) bonds (1.294(3)–1.303(3) A) in compounds
PXRD patterns of L1 and L7 prepared by condensation
reactions in ethanol solutions (Supporting Information
Figs. S29, S30).
L7–L9 that exist in amino form. The N2–C7 bond in L1
˚
(1.299(3) A) is predominantly p in character and signifi-
Molecular structures of L1, L7–L9
cantly shorter in comparison with N2–C7 (L8), N21–C71
˚
L7, L9) and N22–C72 (L7, L9) bonds (1.346(4) A–
(
˚
1.360(3) A). The N3–C8 (L1, L8), N31–C81 (L7, L9) and
Mercury–rendered ORTEP drawings of asymmetric units of
compounds L1, L7-L9 are depicted in Fig. 1. Asymmetric
units of compounds L1 and L8 consist of a single molecule,
while asymmetric units of compounds L7 and L9 consist of
two independent and conformationally slightly different
molecules marked 1 and 2. The dihedral angle is calculated
between the benzothiazolyl and arene plane. All compounds
are moderately to strongly planar with dihedral angles
between the benzothiazolyl [defined by atoms: S1, C1–C6,
N1 and C7 (L1 and L8), S11, C11–C61, N11 and C71 (L7
N32–C82 (L7, L9) bonds are predominantly p in character
˚
(1.269(3)–1.290(3) A) in all compounds. Values of selected
bonds and angles in Tables 2 and 3 are in good agreement
with values found in amino forms and one imino form of
2-benzothiazolylhydrazones reported by Nogueira et al.
[11] (REFCODES: SAJQAX, SAJQEB, SAJQIF, SAJQOL,
SAJQUR, SAJRAY, SAJREC, SAJRIG).
Intramolecular hydrogen bonds are found in structures
L1, L7 and L8 (Table 4). Imino nitrogen atoms N3 (L1,
Fig. 1 Mercury–rendered
ORTEP view of the molecular
structure of compounds L1, L7–
L9 showing the crystallographic
atom-numbering scheme (the
displacement ellipsoids are
drawn at the 50 % probability
level at 296(2) K, and the
hydrogen atoms are drawn as
spheres of arbitrary radius)
1
23

Struct Chem
˚
Table 2 Selected bond lengths (A)
L8), N31 (L7), N32 (L7) act as a proton acceptors and form
strong O–HÁÁÁN intramolecular hydrogen bond in L1 and
weak C–HÁÁÁN intramolecular hydrogen bond in L7 and L8.
In all cases S(6) structural motif is formed by an
intramolecular hydrogen bond.
Compound
N1–C7
N2–C7
N2–N3
N3–C8
L1
L8
1.335 (3)
1.298 (2)
N11–C71
1.294 (3)
1.303 (3)
N12–C72
1.299 (3)
1.302 (3)
1.299 (3)
1.348 (2)
N21–C71
1.360 (3)
1.353 (4)
N22–C72
1.350 (3)
1.346 (4)
1.389 (3)
1.376 (2)
N21–N31
1.372 (3)
1.372 (4)
N22–N32
1.382 (3)
1.370 (3)
1.290 (3)
1.272 (2)
N31–C81
1.269 (3)
1.281 (3)
N32–C82
1.275 (3)
1.273 (4)
L7
L9
Crystal structures of L1, L7–L9
2-hydroxybenzaldehyde-(2-benzothiazolylhydrazone) (L1)
L7
L9
Both intramolecular and intermolecular hydrogen bonds
are present in the crystal structure of L1. Hydroxy O1 atom
acts as a proton donor and forms intramolecular O1–
H11OÁÁÁN3 hydrogen bond with the imino N3 nitrogen
atom, thus shaping S(6) structural motif. Bond length
Table 3 Selected bond angles (°)
˚
(
2.656(3) A) and \D–HÁÁÁA angle (153(3) °) are in good
Compound
C6–N1–C7
C7–N2–N3
N2–N3–C8
agreement with corresponding O–HÁÁÁN intramolecular
hydrogen bond in structure of 2-benzothiazolylhydrazone
reported by Nogueira et al. [11] (REFCODE: SAJREC).
Intermolecular contacts are characterized by N–HÁÁÁN
hydrogen bonds (Fig. 2a, Table 4). Endocyclic N1 nitrogen
atom acts as proton donor and forms N1–H11 NÁÁÁN2
intermolecular hydrogen bond with hydrazone N2 nitrogen
atom of the second molecule. Resulting supramolecular
motif in the crystal structure of L1 is infinite helical C(4)
chain generated via N1–H11 NÁÁÁN2 hydrogen bond in the
direction of the b axis (Fig. 2b). That alignment of
L1
L8
115.3 (2)
111.3 (2)
114.9 (2)
109.7 (1)
115.1 (1)
116.4 (1)
C61–N11–C71
109.0 (2)
C71–N21–N31
115.6 (2)
N21–N31–C81
118.4 (2)
L7
L9
109.2 (2)
117.1 (3)
115.5 (3)
C62–N12–C72
108.9 (2)
C72–N22–N32
115.8 (2)
N22–N32–C82
117.7 (2)
L7
L9
109.4 (3)
120.4 (3)
114.9 (3)
Table 4 Geometry of hydrogen
bonds in compounds L1, L7–L9
D–HÁÁÁA
D–H
HÁÁÁA
DÁÁÁA
\D–HÁÁÁA Symmetry code
L1
O1–H11OÁÁÁN3
N1–H11 NÁÁÁN2
L7
0.84 (3) 1.88 (3) 2.656 (3)
0.83 (2) 2.05 (2) 2.877 (3)
153 (3)
174 (2)
1/2-x,1/2 ? y,1/2-z
C151–H151ÁÁÁN31
C152–H152ÁÁÁN32
C51–H51ÁÁÁO12
C52–H52ÁÁÁO11
C151–H151ÁÁÁS11
N21–H21NÁÁÁN12
0.93
0.93
0.93
0.93
0.93
2.25
2.28
2.63
2.63
2.92
2.900 (4)
2.929 (4)
3.461 (4)
3.478 (4)
3.831 (3)
126
127
150
150
168
0.87 (2) 2.19 (2) 3.040 (3)
164 (2)
173 (2)
135
1/2-x,1/-y,1/2-z
N22–H22 NÁÁÁN11 0.90 (2) 2.16 (2) 3.062 (3)
1/2-x,1/2-y,1/2-z
C22–H22ÁÁÁS11
C192–H19FÁÁÁN11 0.96
L8
0.93
2.97
3.683 (3)
x,-y ? 1/2, ? z?1/2
-x ? 1/2, -y ? 3/2, -z ? 1/2
2.70
3.597 (4)
155
C15–H15ÁÁÁN3
N2–H12 NÁÁÁN1
C19–H19BÁÁÁO1
L9
0.93
2.31
2.9578 (18)
126
0.88 (2) 2.14 (2) 3.0104 (16) 173 (2)
2-x,-1-y,-z
1-x,2-y,-z
0.96
2.54
3.440 (2)
156
N21–H21 NÁÁÁN12 0.86 (3) 2.25 (3) 3.040 (4)
N22–H22 NÁÁÁN11 0.85 (3) 2.28 (3) 3.110 (4)
155 (3)
169 (3)
140
1-x,1/2 ? y,1/2-z
1-x,-1/2 ? y,1/2-z
1 ? x,1/2-y,1/2 ? z
C42–H42ÁÁÁO12
0.93
2.55
3.317 (4)
123

Struct Chem
molecules in crystal is additionally supported by weak
aromatic stacking interaction between the thiazolyl and
phenyl rings of adjacent molecules (Table 5). Similar
supramolecular helical arrangement is found in crystal
structure of benzothiazolyl hydrazone reported by Lind-
gren et al. [10] (REFCODE: ZEZYUA).
2-methoxy-1-naphthaldehyde-(2-benzothiazolylhydrazone)
(L7)
Weak intramolecular C151–H151ÁÁÁN31 and C152–
H152ÁÁÁN32 hydrogen bonds that shape S(6) structural
motif are present in independent molecules 1 and 2 in
asymmetric unit of compound L7. Also, atom C151 (1) acts
as
a bifurcated proton donor and forms another
intramolecular C151–H151ÁÁÁS11 hydrogen bond. Inter-
molecular contacts are characterized by N–HÁÁÁN, C–HÁÁÁO,
C–HÁÁÁN and C–HÁÁÁS hydrogen bonds (Table 4). Hydra-
zino nitrogen atoms N21 and N22 act as proton donors and
form intermolecular N21–H21 NÁÁÁN12 and N22–
H22 NÁÁÁN11 hydrogen bonds that connect independent
2
molecules 1 and 2 into R (6) dimer (Fig. 3a). Independent
2
molecules 1 and 2 are further mutually connected via weak
Fig. 2 a Main supramolecular motif in compound L1 formed by N1–
H11 NÁÁÁN2 intermolecular hydrogen bond and S(6) structural motif
shaped via intramolecular O1–H11OÁÁÁN3 hydrogen bond shown as
dashed magenta lines. b Infinite helical C(4) chain in the direction of
the b axis shaped via N1–H11 NÁÁÁN2 hydrogen bond (shown as
dashed magenta lines) and supported by weak aromatic stacking
interaction between the thiazolyl and phenyl rings of adjacent
molecules (shown as dashed dark blue lines)
C51–H51ÁÁÁO12 and C52–H52ÁÁÁO11 hydrogen bonds that
2
form R (22) rings. In the crystal structure, dimers are
2
mutually connected via weak C192–H19FÁÁÁN11 and C22–
H22ÁÁÁS11 hydrogen bonds (Fig. 3b). The S11 atom acts as
a bifurcated proton acceptor. Supramolecular architecture
is additionally supported by p–p stacking (Table 5).
Table 5 Geometrical
parameters of pÁÁÁp interactions
a
b
c
d
e
Interaction
CgÁÁÁCg distance
CgÁÁÁP1
CgÁÁÁP2
a
b
Slippage
˚
(
and L8
A, °) for compounds L1, L7
L1
i
Cg1–Cg2
3.7627 (15)
3.7625 (15)
3.4252 (10)
3.4598 (11)
3.4598 (11)
3.4251 (10)
1.34 (12)
1.34 (12)
23.1
24.4
1.479
1.557
ii
Cg2–Cg1
L7
Cg1–Cg7
Cg2–Cg8
Cg8–Cg2
Cg9–Cg3
L8
3.7916 (14)
3.9374 (18)
3.9373 (18)
3.7171 (19)
3.3348 (10)
3.5156 (12)
3.6998 (12)
3.5295 (14)
3.5423 (10)
3.6999 (12)
3.5155 (12)
3.4738 (12)
11.15 (12)
10.43 (14)
10.43 (14)
3.26 (15)
20.9
20.0
26.8
20.8
1.352
1.347
1.773
1.323
i
Cg1–Cg4
3.8657 (8)
3.8657 (8)
3.6310 (5)
3.6038 (6)
3.6038 (6)
3.6310 (5)
9.98 (7)
9.98 (7)
21.2
20.1
1.399
1.326
ii
Cg4–Cg1
a
Ring Cg1 is defined by atoms S1, C1, C6, N1 and C7 (thiazolyl ring atoms) in L1 and L8 and by atoms
S11, C11, C61, N11 and C71 (thiazolyl ring atoms) in L7. Cg2 is defined by atoms C1–C6 (phenyl ring
atoms) in L1, and Cg3 is defined by atoms C91, C101–C141 (phenyl ring atoms) in L7. Cg4 is defined by
atoms C13–C18 (phenyl ring atoms) in L8. Cg7 is defined by atoms S12, C12, C62, N12 and C72 (thiazolyl
ring atoms) in L7. Cg8 is defined by atoms C12–C62 in L7 (phenyl ring atoms). Cg9 is defined by atoms
C92, C102–C142 (phenyl ring atoms) in L7
b
CgÁÁÁP1 is the perpendicular distance of corresponding centroid to a plane. Planes P1 or P2 are defined by
the atoms, which define the corresponding centroids
c
CgÁÁÁP2 is the perpendicular distance of corresponding centroid to a plane. Planes P1 or P2 are defined by
the atoms, which define the corresponding centroids
d
Dihedral angle between P1 and P2
e
Angle between CgÁÁÁCg distance and CgÁÁÁP1
i
=
x,-1 ? y, z
ii
=
x,1 ? y, z
1
23

Struct Chem
4-methoxy-1-naphthaldehyde-(2-benzothiazolylhydrazone)
(L8)
Weak intramolecular C15–H11ÁÁÁN3 hydrogen bond shapes
S(6) structural motif, similar to compound L7. Molecules
of compound L8 are connected via intermolecular N2–
H12 NÁÁÁN1 and C19–H19BÁÁÁO1 hydrogen bonds
2
2
(
Table 4), respectively, into alternating R (8) and R (6)
2 2
centrosymmetric rings which further form 2D layers
Fig. 4a). These layers are perpendicularly supported by p–
p interactions between the thiazolyl and phenyl rings
Fig. 4b, Table 5).
(
(
6-methoxy-2-naphthaldehyde-(2-benzothiazolylhydrazone)
(L9)
Intramolecular hydrogen bonds are not found in compound
L9. Similar to crystal structure of compound L7, crystal-
lographically independent molecules 1 and 2 in asymmetric
2
unit of compound L9 are mutually connected into R (8)
2
dimers via intermolecular N21–H21 NÁÁÁN12 and N22–
H22 NÁÁÁN11 hydrogen bonds (Table 4). Interestingly,
molecules 2 are mutually connected via C42–H42ÁÁÁO12
1
hydrogen bond into C (16) chains which is not the case for
1
molecules 1 (Fig. 5).
Fig. 3 a Structural motif of compound L7 generated via N–HÁÁÁN, C–
HÁÁÁO, C–HÁÁÁN and C–HÁÁÁS hydrogen bonds (shown as dashed
magenta lines). b Part of crystal packing of L7 showing mutually
connected dimers via weak C–HÁÁÁN and C–HÁÁÁS hydrogen bonds
Antiproliferative effects in vitro
We evaluated antiproliferative effects of L1–L9 on human
tumor cell lines, as well as cytotoxicity on mouse embry-
onic fibroblasts. All tested ligands exerted strong antipro-
liferative effects on tumor cells and cytotoxic effects on
Fig. 4 a Main supramolecular
motif in compound L8 shaped
via N2–H12 NÁÁÁN1 and C19–
H19BÁÁÁO1 hydrogen bonds
shown as dashed magenta lines.
2
2
2 2
b Alternating R (8) and R (6)
centrosymmetric rings shaped
via N2–H12 NÁÁÁN1 and C19–
H19BÁÁÁO1 hydrogen bonds into
2
D layers (shown as dashed
magenta lines) that are
supported by weak aromatic
stacking interaction between the
thiazolyl and phenyl rings of
adjacent chains (shown as
dashed dark blue lines)
123

Struct Chem
Fig. 5 Part of crystal structure
2
of L9 showing R (8) dimers
2
formed via intermolecular N21–
H21 NÁÁÁN12 and N22–
H22 NÁÁÁN11 hydrogen bonds
1
and C (16) chains formed via
C42–H42ÁÁÁO12 hydrogen bond
1
(shown as dashed magenta
lines)
Table 7 Calculated standard
Gibbs energies of binding at
°
-1
Table 6 IC50 values (lM)
Substance
r
D Gbinding/kJ mol
a
Substance
IC50 (lM)
Cell lines
SW620
2
98.15 K and 1 atm for dimers
or tetramers of L1–L9 (B3LYP/
-311 ??G(d,p) level of the
theory)
L1
L2
L3
L4
L5
L6
L7
L8
L9
-139.76
–
6
–
A549
HepG2
3T3
HeLa
–
L1
L2
L3
L4
L5
L6
L7
L8
L9
a
8.16
6.72
9.63
5.74
6.97*
5.20*
8.31
9.71
41.04
2.56
8.17
5.59*
4.05
5.20
5.0*
–
9.08
[100
7.57
-99.61
-110.78
-86.20
-96.24
7.19
4.55
6.50
6.44*
3.30*
49.08
89.74
32.47
4.20
[100*
3.54*
6.34
7.79*
1.98*
[100*
[100
18.18
\0.01
6.44*
4.08*
6.94
52.05
26.16
3.87
78.56
30.51
[100
tetramers. These values revealed that the stability in L6 and
L7 is bigger than in L8 (Table 7). In L7, the existence of
IC50; 50 % inhibitory concentration or compound concentration
required to inhibit tumor cell proliferation by 50 %
*
The color of the substance interferes with colorimetric measure-
weak C51–H51ÁÁÁO12 and C52–H52ÁÁÁO11 hydrogen bonds
2
2
ments at the highest tested concentration (100 lM)
that form R (22) rings was already mentioned previously.
Since L6 and L7 have the oxygen atom attached to the
same position 2, it is reasonable to predict that this position
is more favorable for binding due to the formation of two
weak hydrogen bonds. On the contrary, in L8 where the
oxygen atom of methoxy group is attached in position 4,
there is no such a possibility and consequently the structure
is less stable. Moreover, the methoxy group provides better
stabilization of dimers than the hydroxyl group (Table 7).
Since the experimental structures of L2–L5 are not avail-
able, the stability of L1 was not compared to any value.
In order to deeply understand the cause of molecular
packing in investigated systems, we calculated the poten-
tial energy surfaces of molecules spanned by torsional
coordinates (Scheme 2, Fig. S28). Changes along these
torsional coordinates could produce different conformers
that will pack diverse unit cell and therefore are important
for explanation of structures. Calculation of PES scans
fibroblasts with exception of L6 and L7 that had no effect
on fibroblast cells (Table 6). Compounds L7 and L8 are the
least potent ligands and inhibited tumor cell growth at
higher tested concentrations (10–1000 lM). Compounds
L1–L5 and L9 had non-selective strong antiproliferative
effects, on all tested cells at micromolar concentration
(\1 lM) except for L4 that had no effect on HepG2 and L9
that had no effects on HeLa cells.
Quantum chemical calculations
Standard Gibbs energies of binding were calculated by
subtraction of standard Gibbs energies of formation for
monomers from the standard Gibbs energies of dimers or
1
23

Struct Chem
1 2
Fig. 6 Potential energy surfaces spanned by two torsional coordinates u and u (defined on Scheme 2): a L1, b L2, c L3, d L4, and e L5
along the two torsional coordinates shows that the internal
rotation in L1 is very unfavorable due to the presence of
behavior, and it is reasonable to predict that the packing of
all these compounds will be similar.
intramolecular hydrogen bond (Fig. 6a, for u = 0°).
Internal rotation curves of naphthalene rings in L6, L7,
L8, (Fig. 7) and L9 (Fig. 8) show that the conformers of
minimal energy are the ones where this ring is on the same
side as lone electronic pair of imine nitrogen atom, which
is exactly the same as in obtained crystal structures.
1
Although along this PES slice there is another local mini-
mum, this structure is much higher in energy to be able to
produce packing. Potential energy surfaces calculated for
compounds L2–L5 (Fig. 6b–e) are showing the similar
123

Struct Chem
1 2
Fig. 7 Potential energy surfaces spanned by two torsional coordinates u and u (defined on Scheme 2): a L6, b L7, and c L8
potential biological activities. The compounds L1–L9 are
characterized by elemental analysis, IR and NMR spec-
troscopy, and compounds L1, L7–L9 are structurally
determined in the solid state by single-crystal X-ray
diffraction method.
In all 2-benzothiazolylhydrazones derived form 2-
methoxy-1-naphthaldehyde, 4-methoxy-1-naphthaldehyde
and 6-methoxy-2-naphthaldehyde (L7–L9, respectively)
2
2
characteristic R ð8Þ dimer is shaped via Nhydrazino–
HÁÁÁN
intermolecular hydrogen bond, but these
2
thiazolyl
dimers are differently supported (R (22) fused rings in L7,
2
2
2
2
alternating R (8) and R (6) centrosymmetric rings in L8
1
2
and C (16) infinite chains in L9) by C–HÁÁÁO intermolec-
1
Fig. 8 Potential energy surfaces spanned by two torsional coordi-
nates u and u (defined on Scheme 2) for L9
ular hydrogen bonds which are formed via -OCH group.
3
1
2
On the contrary to the amino L7–L9 dimer arrangements of
primary crystal structure, supramolecular structure of
imino L1 hydrazone is shaped into infinite helical C(4)
chains along b axis via Nthiazolyl–HÁÁÁNhydrazino inter-
molecular hydrogen bond.
Conclusions
In our aims to prepare metal complexes with 2-benzoth-
iazolylhydrazone ligands and investigate their pharmaco-
logical activities, we firstly synthesized and characterized a
series of nine 2-benzothyazolylhydrazone ligands (L1–L9)
Moreover, quantum chemical calculations confirmed
that methoxy group (L7–L9) provides better stabilization
of dimers than the hydroxyl group in the L1 hydrazone
which prefers formation of intramolecular O–HÁÁÁN
hydrogen bond over intermolecular ones. Calculated
functionalized by -OH or -OCH functionalities at dif-
3
ferent phenyl or naphthyl positions and investigated their
1
23

Struct Chem
standard Gibbs energies of formation reveal that oxygen
atom attached to the position 2 in L6 and L7 is more
favorable for binding than the position 4 (L8). Potential
energy surfaces calculated for compounds L2–L5 are
showing the similar behavior to compound L1, and it is
reasonable to predict that the packing of all these com-
pounds will be similar.
11. Nogueira AF, Vasconcelos TRA, Wardell JL, Wardell SMSV
2011) Z Kristallogr 226:846–860
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All tested ligands exerted strong antiproliferative effects
on tumor cells and cytotoxic effects on fibroblasts except
L6 and L7 that had no effect on fibroblast cells. Com-
pounds L7 and L8, characterized by methoxy-substituted
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hydroxy-substituted phenyl moiety, exerted non-selective
strong antiproliferative effects on all tested cells at
micromolar concentrations.
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Acknowledgments We acknowledge Kre sˇ imir Mol cˇ anov, PhD,
Division of Physical Chemistry, Laboratory for chemical and bio-
logical crystallography, Ru d¯ er Bo sˇ kovi c´ Institute, Zagreb, Croatia, for
X-ray single-crystal diffraction data collection for compound 8. We
greatly appreciate support of University of Rijeka research Grant No.
2
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