Asynthesis, Crystal Structures and in Vitro Anticancer Studies of New Thiosemicarbazone Derivatives

Article abstract of DOI:10.1080/10426507.2014.995299

Thiosemicarbazones are an important class of compounds having significant biological properties. These compounds are used as stable ligands for the synthesis of coordination compounds of biological worth. The current study describes syntheses of four new

Full text of DOI:10.1080/10426507.2014.995299

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Asynthesis, Crystal Structures and in vitro Anticancer
Studies of New Thiosemicarbazone Derivatives
Mouayed A. Husseina, Muhammad Adnan Iqbala, Muhammad Asifb, Rosenani A. Haquea,
Mohammed B. Khadeer Ahamedb, Amin M. S. Abdul Majidb & Teoh Siang Guana
a School of Chemical Science, Universiti Sains Malaysia, 11800 – Mindern, Pulau Pinang,
Malaysia
b EMAN Testing and Research Laboratory, School of Pharmaceutical Sciences, Universiti Sains
Malaysia, 11800-Minden, Pulau Pinanag, Malaysia
Accepted author version posted online: 02 Feb 2015.
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To cite this article: Mouayed A. Hussein, Muhammad Adnan Iqbal, Muhammad Asif, Rosenani A. Haque, Mohammed
B. Khadeer Ahamed, Amin M. S. Abdul Majid & Teoh Siang Guan (2015): Asynthesis, Crystal Structures and in vitro
Anticancer Studies of New Thiosemicarbazone Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:
10.1080/10426507.2014.995299
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ASYNTHESIS, CRYSTAL STRUCTURES AND IN VITRO ANTICANCER STUDIES OF
NEW THIOSEMICARBAZONE DERIVATIVES
Mouayed A. Hussein,1 Muhammad Adnan Iqbal,1 Muhammad Asif,2 Rosenani A. Haque,1
Mohammed B. Khadeer Ahamed,2 Amin M. S. Abdul Majid,2 Teoh Siang Guan1 *
1School of Chemical Science, Universiti Sains Malaysia, 11800 – Mindern, Pulau Pinang,
Malaysia
2EMAN Testing and Research Laboratory, School of Pharmaceutical Sciences, Universiti Sains
Malaysia, 11800-Minden, Pulau Pinanag, Malaysia
*Corresponding author:
Dr. Teoh Siang Guan, PhD,
Professor
School of Chemical Sciences
Universiti Sains Malaysia
Penang-11800, Malaysia
E-mail: sgteoh@usm.my
teohsiang@ymail.com
H/P : (604) – 6577888
Fax : (604) – 6574854
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Abstract
Thiosemicarbazones are an important class of compounds having significant
biological properties. These compounds are used as stable ligands for the synthesis of
coordination compounds of biological worth. The current study describes syntheses of four new
N(3)-ethyl thiosemicarbazone derivatives and their in vitro anticancer testing. The purpose of the
study was to observe the effect of various subtitutions at the aromatic ring against the growth of
cancerous cell. The synthesized compounds were characterized by NMR (1H & 13C)
spectroscopy and the molecular structures of each of the compounds were determined using
single crystal X-ray crystallographic technique. Interestingly, compound 4 appeared to have two
crystallographically different units (A and B), having slightly different bond lengths and angles
compared to each other. All the compounds were tested for their in vitro anticancer properties
against Human Colon Cancer (HCT 116) and Breast Cancer (MCF-7) cell lines. The compounds
(1-2), having halide (Cl and Br) substitutions, showed significant activity compared to those
having ethoxy and allyl substitutions (3-4), indicating the possible involvement of halides against
cancerous cells.
Keywords
Thiosemicarbazones, crystal structures, cytotoxicity, MCF-7, HCT 116, SAR
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INTRODUCTION
Thiosemicarbazones are an important class of compounds having general structure R1R2-
C=N-NH-CS-NH2 and can be synthesized by condensation reaction of semicarbazide with a
1
suitable aldehyde or ketone . Moreover, suitable substitutions at either thioamide (a), sulfur (b)
or hydrazine (c) nitrogen provide versatile derivatives (Figure 1). For example, according to the
general structure (a), the substitutions R3 and R4 may either be alkyl, aryl or hydrogen. In the
general structure (b), the reported R3 substitutions are propyl and iso-propyl 2. Such substitutions
in (b) are adopted to inhibit the coordination of sulfur with the metal ion. In structure (c), the
substitution R3 is commonly a hydrogen.
Thiosemicarbazones are popular because of their metal chelating properties in order to generate
metal-complexes having interesting geometries. Metal-thiosemicarbazone complexes have been
3-6
found to have interesting catalytic
as well as biological applications 5,7-24. However, it has
been also reported that by suitable structural variations thiosemicarbazone ligands could also
become potential anticarcinogenic agents 25-30
.
Thiosemicarbazones belong to a large group of thiourea derivatives, whose biological activities
are a function of parent aldehyde or ketone moiety 31-33. Conjugated N-N-S tridentate ligand
system of thiosemicarbazone seems necessary for anticancer activity, probably due to the
observation that structural changes that hamper a thiosemicarbazone’s capability to function as a
34
chelating agent to demolish or reduce its medicinal activity . Recent studies showed that the
substitutions at the terminal positions (R1R2 and R3R4 in Figure 1a) of thiosemicarbazones have
much improved their anticancer activity 35-37. For example, the presence of aromatic group at
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N(4) of isatin-β thiosemicarbazones derivatives have shown increased cytotoxicity against the
parental KB-3-1 cell lines and the P-glycoprotein-expressing cell line KB-V1 38. Herein, we
report the synthesis and characterization of some new thiosemicarbazone derivatives having
variation of substitutions at the benzene ring at R1R2-terminal position (scheme 1) to further
observe this phenomenon.
RESULTS AND DISCUSSION
Synthesis
New thiosemicarbazones (1-4) were prepared by condensation of 4-ethyl-3-
thiosemicarbazide with corresponding aldehyde derivatives. Compounds were collected either as
white or yellow powderous material. Each of the synthesized compounds was recrystalized using
diethyl ether/ethanol solvent system before characterization. The compounds were characterized
by 1H & 13C NMR and elemental analysis which confirmed their assigned structures (scheme 1).
Single crystals of each of the synthesized compound were obtained by slow evaporation of
respective solutions either in ethanol or DMF, see the experimental part for detail.
Characterization by NMR
In general, for the all the synthesized compounds, the aromatic C-H chemical shifts
appeared in the range 6.75-8.07 δ ppm. Importantly, for compounds 1-2 these aromatic C-H
chemical shifts appeared in relatively downfield region (7.73-8.07 δ ppm) which might be due to
the presence of electron withdrawing effect of halide groups (R1R2 = Br & Cl) attached to the
benzene ring. In rest of the compounds (3-4) this range remained relatively upfield (6.75-7.49 δ
ppm). Furthermore, the chemical shifts for –CH=N- group appeared in the range 8.39-9.09 δ
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25,26
1
ppm which is in accordance with the previous reports
on thiosemicarbazones. H NMR
signals for -N-NH- proton peak appeared at comparable region (11.44±0.04 δ ppm). The
chemical shifts for rest of the substitutions (methoxy and allyl) appeared in the range below 6.00
δ ppm.
Similarly, 13C NMR chemical shifts for the aromatic carbons appeared in the range 113.9-147.8 δ
ppm. Noticeably, for the compounds 1-2, having halide substitutions, the Ar-C range remained
relatively upfield (122.5-137.5) which might be due to resonantly electron donating ability of
39
halides . Furthermore, the –CH=N- and -C=S groups chemical shifts appeared at 150±5 and
176.5±0.2 δ ppm, respectively 4,25,40. 13C NMR signals for terminal N(4)-ethyl group appeared in
the range 14.4-30.6 δ ppm and the ethoxy group in 3 at 64.1 and 50.0 for O-CH2CH3 in order.
The most downfield chemical shifts for the aromatic substitutions appeared for the allyl group
(113.1-117.4 δ ppm).
Crystallographic study
The single crystals of each of the synthesized compounds were obtained by the slow
evaporation method. According to this method, a saturated solution of the compound in a
suitable solvent (~0.5 mL) was sealed in a small glass bottle (2.0 mL) using parafilm. Small
holes, using needles, were then made to let the solvent evaporate slowly at room temperature.
The crystals appeared and suitable of them were then picked up under microscope and were
analyzed by Bruker SMART APEXII CCD area-detector diffractometer. Crystallographic data
for the compounds have been listed in Table 1 whereas the selected bond lengths and angles are
listed in Table S2 (supplementary file).
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The molecular structures of compounds 1-4 have been shown in Figures 2-3. The molecular
structures of all the compounds contain anti orientation of N1 in relation to S1 and syn in relation
to N3. These geometrical features are in accordance with the previous reports containing
compounds having similar structures 27,33,41-43. However, in some cases this orientation inverses
27, influenced by the nearby substitutions and H-bondings. Similarly, the terminal ethyl C9 atom
is anti in relation to N2, and syn in relation to S1 atom. Figure 2 shows that in compound 1, the
chain C8-C10 has positional disorder. Such disorders have been frequently observed in
crystallography at the terminal chains 44,45. Furthermore, the salicylaldimine plane of 1 which
includes O1, N1 and C1-C7 found to be rotated approximately 180o around C6-C7 bond
compared to that in the other compounds (2-4). This led to change the anti geometry between the
C1 or C5 atom and N1 atom to syn geometry.
The asymmetric unit of 4 contains two C14H19N3O2S molecules (A and B). Figure 3 shows an
arrangement for the molecular structure of 4, which reveals that A and B are crystallographically
different units having slightly different bond lengths and angles compared to each other. In
Figure 3A, the syn conformation of C8A atom with respect to C10A atom [torsion angle C8A–
N3A–C9A–C10A = 101.5(4)o] and the anti conformation of the C6A with respect to O1A atom
[torsion angle C6A–C1A–C2A–O1A = -179.0(3)o], C3A atom with respect to C12A atom
[torsion angle C3A–C4A–C11A–C12A = - 90.5(4)o] and C4A atom with respect to C13A atom
[torsion angle C4A–C11A–C12A–C13A = -119.4(4)o] corresponded to, the anti conformation
[torsion angle = -77.9(4)o] and the syn conformations [torsion angle = 179.2(3)o], [torsion angle
= 174.9(3)o] and [torsion angle = 116.8(4)o] for ligand 4B, respectively.
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The S1-C8 bond distance is longer than compared to that of N2-C8 which indicates that the
compounds are present in thione form. The N3-C8 [1.268(17) Å] bond distance of 1 is shorter
that compared to that of N3-C9 [1.51(2) Å] that is different compared to the other compounds (2-
4). This phenomenon leads to decrease the S1-C8-N3 [117.5(8)o] bond angle and increases the
C8-N3-C9 [130.0(14)o] bond angle compared to other compounds (2-4). Simultaneously, the N2-
C8-N3 [121.4(8)o] bond angle increases as long as the S1-C8-N3 bond angle decreases.
In vitro anticancer study
All the synthesized compounds were tested for their cytotoxicity against two cancerous cell
lines, namely Human Colon Cancer (HCT 116) and Breast Cancer (MCF-7), using MTT assay.
The compounds showed dose dependent cytotoxic behavior as the population of viable cells
reduced abruptly with increasing concentration of the compounds. Please see the supplementary
material file for related experimental and discussion parts.
EXPERIMENTAL
Materials and Instrumentation
The melting points were determined using a Stuart Scientific SMP1 melting point
13
apparatus. NMR spectra (1H and C) were recorded on a Bruker spectrophotometer at 400 and
500 MHz NMR machines. Tetramethylsilane was used as an internal standard and the samples
were prepared in deuterated dimethyl sulfoxide (DMSO-d6). Elemental analysis was carried out
using a Perkin Elmer 2400 series-11 CHN analyzer. X-ray crystallographic data were recorded
on a Bruker SMART APEXII CCD area-detector diffractometer using graphite monochromated
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Mo Kα radiation (λ = 0.71073 Å) at 100 K. The data were collected and reduced using APEX2
and SAINT programs. The molecular structures of each of the compounds were solved using the
SHELXS-97 program package, and refined using the SHELXL-97 program 46. All non-hydrogen
atoms were anisotropically refined. The molecular graphics were created using SHELXTL. All
chemicals, including thiosemicarbazide, aldehydes, and solvents, were purchased from Sigma-
Aldrich.
Synthesis
Compounds 1-4 were prepared by the following general procedure according to which
equimolar ethanolic solutions of aldehyde derivative (4.19 mmol in 20 mL ethanol) and the
corresponding thiosemicarbazide (4.19 mmol in 20 mL ethanol) were refluxed with stirring for 2
h. The product was filtered, washed with ethanol and air-dried. The crystals for 1 and 2 were
obtained by slow evaporation of ethanol solution at room temperature. The crystals for 3 and 4
were obtained by slow evaporation of DMF solution at room temperature.
N-ethyl-2-(3,5-dibromo-2-hydroxybenzylidene)-hydrazine-carbothioamide (1)
A solution of 3,5-dibromo-2-hydroxybenzaldehyde (1.17 g, 4.19 mmol) in ethanol (20 mL)
was added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 mL).
The resulting yellow solution was refluxed with stirring for 2 h and then filtered. The filtrate was
left to stand at room temperature for two days, and needle colorless crystals were
obtained. Yield: 77% (1.22 g); mp. 194 oC; Anal. Calcd. for C10H10Br2N3OS: C, 31.5; H, 2.6; N,
11.0%; Found: C, 31.5; H, 3.0; N, 10.9% ; 1H NMR ( 500MHz, DMSO-d6, δ/ppm): 1.15 (t, J =
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7.5 Hz, 3H, CH3), 3.62 (q, 2H, CH2), 7.74, 8.07 (s, 2H, H-aromatic), 8.27 (s, 1H, CS-NH), 8.74
(s, 1H, CH=N), 11.46 (N-NH); 13C NMR (125.1 MHz, DMSO-d6, δ/ppm): 14.4 (CH3), 30.6
(CH2), 122.5, 124.5, 125.0, 129.5, 137.5 (C-aromatic), 150.5 (C=N), 176.6 (C=S).
N-ethyl-2-(3,5-dichloro-2-hydroxybenzylidene)-hydrazine-carbothioamide (2)
A solution of 3,5-dichloro-2-hydroxybenzaldehyde (0.80 g, 4.19 mmol) in ethanol (20 mL)
was added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 mL).
The resulting yellow solution was refluxed with stirring for 2 h and then filtered. The filtrate was
left to stand at room temperature for two days, and block colorless crystals were obtained. Yield:
o
79% (0.96 g); m.p. 186 C; Anal. Calcd. for C10H11Cl2N3OS: C, 41.0; H, 3.7; N, 14.3%. Found:
1
C, 40.9; H, 3.4; N, 13.9% ; H NMR (500 MHz, DMSO-d6, δ/ppm): 1.13 (t, J = 7.5 Hz, 3H,
CH3), 3.50-3.62 (CH2), 7.73, 8.05 (s, 2H, H-aromatic), 8.28 (s, 1H, CS-NH), 8.73 (s, 1H,
CH=N), 11.47 (s, 1H, N-NH); 13C NMR (125.1 MHz, DMSO-d6, δ/ppm): 14.4 (CH3), 30.6
(CH2), 122.5, 123.2, 123.7, 129.5, 137.5 (C-aromatic), 150.5 (C=N), 176.6 (C=S).
N-ethyl-2-(3-ethoxy-2-hydroxybenzylidene)-hydrazine-carbothioamide (3)
A solution of 3-ethoxy-2-hydroxybenzaldehyde (0.69 g, 4.19 mmol) in ethanol (20 mL) was
added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20 mL). The
resulting yellow solution was refluxed with stirring for 2 h. A white fluffy product was formed
when the solution cooled down to room temperature, then filtered, washed with ethanol and air
dried. Needle colorless crystals were obtained by slow evaporation of a DMF solution at room
o
temperature. Yield: 86% (0.96 g); m.p. 222 C; Anal. Calcd. for C12H17N3O2S: C, 53.8; H, 6.3;
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N, 15.7%. Found: C, 54.1; H, 6.5; N, 15.2% ; 1H NMR (500 MHz, DMSO-d6, δ/ppm): 1.13 (t, J
= 7.5 Hz, 3H, CH3), 1.36 (t, J = 7.0 Hz, 3H, ethoxy-CH3), 3.01 (q, 2H, CH2), 4.04 (q, 2H,
ethoxy-CH2), 6.75 (d, J = 6.5 Hz, 1H, H-aromatic), 6.97 (d, J = 6.5 Hz, 1H, H-aromatic), 7.49
(m, 1H, H-aromatic), 8.39 (s, 1H, CH=N), 11.41 (s, 1H, N-NH); 13C NMR (125.1 MHz, DMSO-
d6, δ/ppm): 14.5 (CH3), 30.8 (CH2), 50.0 (ethoxy-CH3), 64.1 (ethoxy-CH2), 113.9, 118.2, 119.1,
121.2, 139.1, 146.0 (C-aromatic), 146.9 (C=N), 177.4 (C=S).
N-ethyl-2-(5-allyl-3-methoxy-2-hydroxybenzylidene-hydrazine-carbothioamide (4)
A solution of 5-allyl-3-methoxy-2-hydroxybenzaldehyde (0.80 g, 4.19 mmol) in ethanol (20
mL) was added to a solution of 4-ethyl-3-thiosemicarbazide (0.5 g, 4.19 mmol) in ethanol (20
mL). The resulting colorless solution was refluxed with stirring for 2 h. A white fluffy product
was formed when the solution cooled down to room temperature, which was then filtered,
washed with ethanol, and air dried. Plate colorless crystals were obtained by slow evaporation of
o
a DMF solution at room temperature. Yield: 80% (0.98 g); m.p. 194 C. Anal. Calcd. for
1
C14H19N3O2S: C, 57.2; H, 6.4; N, 14.3%. Found: C, 57.1; H, 6.5; N, 14.2% ; H NMR (400
MHz, DMSO-d6, δ/ppm): 1.15 (t, J = 7.5 Hz, 3H, CH3), 3.30 (d, J = 6.0 Hz, CH2-Ph), 3.60 (q,
2H, CH2), 3.79 (s, 3H, O-CH3), 5.02 (q, 2H, CH2=), 6.02 (m, 1H, =CH), 6.78 (s, 1H, H-
romatic), 7.34 (s, 1H, H-aromatic), 8.37 (s, 1H, CS-NH), 9.09 (s, 1H, CH=N) , 11.40 (s, 1H, N-
13
NH); C NMR (100.1 MHz, DMSO-d6, δ/ppm): 14.6 (CH3), 38.2 (CH2), 55.8 (O-CH3), 113.1
(CH2-Ph), 115.4 (CH2=), 117.4 (=CH), 120.8, 130.3, 138.1, 139.2, 147.8 (C-aromatic), 155.8
(C=N), 177.5 (C=S).
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CONCLUSIONS
Four new thiosemicarbazone derivatives were synthesized characterized by NMR and
structurally elucidated using X-ray crystallographic technique. The crystal structures of
compounds revealed syn and anti geometries compared to each other. Specifically, compound 4
revealed rarely observed crystallographically different units of the same molecule which have
slightly different bond lengths and angles. Such crystallographically different units have been
rarely isolated 47. Furthermore, the compounds were tested for their cytotoxic properties and
found to have considerable antiproliferative abilities. However, among the tested compounds, 1-
2 (having Br and Cl substitution on benzene ring) showed more pronounced anti-proliferative
effects than compared to that of 3-4. The current research concludes that as the electronegativity
of substituted halide group increases the biological significance of the compounds further
enhances.
Supplementary Material
CCDC 885657, 885660, 885704 and 958080 contain the supplementary data for 1, 2, 3 and 4,
respectively.
These
data
can
be
obtained
free
of
charge
at
http://www.ccdc.cam.ac.uk/data_request/cif. A supplementary file has been provided which
contains the additional spectral and crystallographic features.
Acknowledgements
We thank the Malaysian Government for a Research University Grant which partly supported
this work. Dr. Muhammad Adnan Iqbal is recipient of USM Post-Doctoral Fellowship in
research. The article was compiled and communicated by Dr. Muhammad Adnan Iqbal.
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Table Captions
Table 1: Crystallographic data of compounds 1-4.
Parameter
1
2
3
4
Empirical formula C10H10 Br2N3OS
C10H11 Cl2N3OS
292.19
C12H17N3O2S
267.36
100.0
C14H19N3O2S
293.39
100.0
Formula weight
Temperature (K)
380.08
100
100.0
Radiation/wavelen MoKα / 0.71073 MoKα / 0.71073 Å
MoKα / 0.71073 MoKα / 0.71073
gth
Å
Å
Å
Crystal
Needles
colorless
/ Blocks / colorless
Needles
colorless
/ Plates / colorless
morphology and
colour
Crystal system,
space group
Unit cell
triclinic, Pī
triclinic, Pī
Monoclinic,
triclinic, Pī
P2ī/c
dimensions
a = 4.288(2) Å
b = 11.893(5) Å
c = 13.181(6) Å
a = 5.3023(2) Å
b = 9.3655(3) Å
c = 12.9787(5)Å
a = 12.9464(3) Å a = 5.9197(10) Å
b = 5.9878(1) Å
c = 17.3365(3)Å
b = 13.667(2) Å
c = 18.725(3) Å
α = 97.082(9)°
β = 97.600(9)°
γ = 99.750(9)°
α = 104.444(2)°
β = 95.345(2)°
γ = 91.663(2)°
α = 90°
α = 92.552(4)°
β = 90.300(6)°
γ = 102.110(3)°
β = 99.371(1)°
γ = 90°
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Volume (Å3)
Z, Calculated
density (Mg/m3)
F(000)
649.3(5)
2, 1.944
620.51(4)
2, 1.564
1326.00(4)
4, 1.339
1479.6(4)
4, 1.317
370
300
568
624
Crystal size (mm)
Reflections
0.09 x 0.12 x 0.50 0.12 x 0.23 x 0.44
0.11 x 0.14 x 0.52 0.09 x 0.15 x 0.43
12601 / 3789
0.067
13860 / 3581
24246 / 3582
27095 / 8497
collected / unique
R(int)
0.029
0.030
0.061
Goodness of fit (S) 1.06
1.10
1.05
1.07
R[F2 > 2σ(F2)],
wR(F2)
0.0415, 0.1306
0.0335, 0.0869
0.0385, 0.1006
0.0745, 0.2575
Observed data
[I>2σ(I)]
2914
3049
3044
6499
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Figure Captions
Figure 1: The derivatives of thiosemicarbzides which can be obtained by substituting at, (a)
thioamide nitrogen, (b) sulfur, and (c) hydrazine nitrogen. The substitutions, R1 & R2
are either alkyl or aryl groups.
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Figure 2: Molecular structures of 1-3 drawn at 50% probability. The N4-ethyl unit in 1 has an
occupancy disorder.
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Figure 3: Molecular structure of 4 drawn at 50% probability. The structure was found to have
two crystallographically different units (A and B) which have slightly different bond
lengths and angles compare to each other.
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Scheme 1: Syntheses of thiosemicarbazone derivatives (1-4) by refluxing different
hydroxybenzaldehydes (A) and 4-ethyl-3- thiosemicarbazide (B) in ethanol.
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