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Synthesis and Molecular Field Similarity Study for P53 Inhibitory Activity of Thiazol-2-yl Dithiocarbamate esters

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Various thiazol-2-yl dithiocarbamate esters were synthesized by thiolation of amino group at position-2 of 2-aminothiazole. Treatment of 2-aminothiazole with carbon disulphide and alkyl or acyl halide in the presence of strong base in dimethylformamide afforded the corresponding dithiocarbamate esters. Furthermore, all synthesized derivatives were evaluated for molecular field similarity required for p53 inhibitory activity through molecular modelling study.
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______________________________________________________________________Research Paper
Synthesis and Molecular Field Similarity Study for P53 Inhibitory
Activity of Thiazol-2-yl Dithiocarbamate esters.
S. K. Nayaka, H. K. Chopra*a and P. S. Panesarb
aDepartment of Chemistry, Sant Longowal Institute of Engineering & Technology (Deemed
University), Longowal, Sangrur, India.
bDepartment of Food Technology, Sant Longowal Institute of Engineering & Technology
(Deemed University), Longowal, India.
ABSTRACT
Various thiazol-2-yl dithiocarbamate esters were synthesized by thiolation of amino group at position-2 of 2-
aminothiazole. Treatment of 2-aminothiazole with carbon disulphide and alkyl or acyl halide in the presence of
strong base in dimethylformamide afforded the corresponding dithiocarbamate esters. Furthermore, all
synthesized derivatives were evaluated for molecular field similarity required for p53 inhibitory activity through
molecular modelling study.
Key Words: 2-Aminothiazole, dithiocarbamate, molecular field and molecular similarity.
INTRODUCTION
Numerous studies regarding dithiocarbamates, have
demonstrated that these compounds have potential
anticholinergic1-3, tuberculostatic4, antimicrobial5-7,
antiviral activities8,9 and anti-p53 activity10. The p53
protein acts as labile transcription factor and
regulates the expression of a wide variety of genes
involved in cell cycle arrest, apoptosis, DNA repair
and differentiation. The induction of apoptosis by
p53 activation results in a variety of diseases such as
arthrosclerosis11, diabetes12, osteoarthritis and
neuronal disorders (such as Alzheimer’s disease,
Parkinson’s disease, Huntington’s disease,
amyotrophic lateral sclerosis)13,14. Apoptosis has also
been reported in other pathological dysfunctions, for
example, T-cell depletion in HIV infection and
mononuclear cell loss in P. falciparum and S.
typhimurium infection15. In a study, Wu and Momand
reported that pyrrolidine dithiocarbamate inhibits p53
nuclear translocation and transactivation through the
cysteine residue oxidation10. In search of novel p53
inhibitors, the synthesis of various novel
dithiocarbamate esters of 2-aminothiazole was
carried out.
________________________________________
*Address for correspondence:
E-mail: hk67@rediffmail.com
All synthesized agents were evaluated for molecular
similarity pattern required for p53-inhibition through
molecular modelling study.
MATERIALS AND METHODS
The molecular modelling study was carried out using
software packages FieldAlign and FieldTemplater
(both trial versions) from Cresset Biomolecular
Discovery Ltd. 1H and 13C NMR spectra were
recorded on Bruker Avance 400 FT spectrometer in
deuteriochloroform and deuteriodimethyl sulfoxide
with tetramethysilane as internal standard. Chemical
shifts were reported in parts per million. Mass spectra
(MS) were measured by the EI method. Melting
points are uncorrected. Silica gel (60-120 mesh) was
used for column chromatography. All the reactions
were monitored by TLC using 0.25 mm silica gel
plates (Merck 60F-254) with or without UV
indicator. N, N-Dimethylformamide was distilled
from anhydrous magnesium sulfate prior to use. All
other reagents were commercially available (Merck,
Fluka) and were used without further purification.
Thiazol-2-yl dithiocarbamic acid (2a)
To a ice cold mixture of carbon disulfide (456 mg, 6
mmol) and KOH (336 mg, 6 mmol) in dry DMF, a
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solution of compound 1 (600 mg, 6 mmol) in dry
DMF was added and stirred at 0 °C for 3 hrs, then
warmed to room temperature and stirring was
continued until reaction was complete (monitored by
TLC). The solvent was removed under vacuo and
mixture was treated with aqueous PTSA. The
aqueous mixture was extracted with a mixture of
acetone and ethyl acetate (10:90). The removal of
solvent afforded solid which was recrystalized by
ethanol. Yield: 89%. 1H-NMR (CDCl3) 7.25 (d, 1H,
CH), 7.43 (d, 1H, CH), 8.67 (bs, 1H, NH), 14.35 (s,
SH); TOF-MS (ES+) m/z= 177 (M+1).
Thiazol-2-yl dithiocarbamic acid esters (3-11)
To a ice cold mixture of carbon disulfide (456 mg, 6
mmol) and KOH (336 mg, 6 mmol) in dry DMF, a
solution of compound 1 (600 mg, 6 mmol) in dry
DMF was added and stirred at 0 °C and at end of
reaction stirring was continued at room temperature
(monitored by TLC). Then appropriate alkyl or acyl
halide (6 mmol) was added dropwise at 0 °C and
reaction mixture was stirred at room temperature
until reaction was complete (monitored by TLC). The
solvent was removed under vacuo and mixture was
quenched by addition of cold water and was extracted
with EtOAc, dried over anhyd. Na2SO4. The product
was purified by silica gel (60-120 mesh) column
chromatography eluted with petroleum ether and
ethyl acetate (20:80). Evaporation of the solvent
afforded NMR pure product.
Thiazol-2-yl dithiocarbamic acid ethyl ester (3).
Yield: 88%. 1H-NMR (CDCl3): 1.57 (t, 3H, CH3),
3.27 (q, 2H, CH2), 7.17 (d, 1H, CH), 7.34 (d, 1H,
CH), 8.83 (bs, 1H, NH); 13C-NMR (CDCl3): 20.5,
31.7, 109.8, 138.2, 172.1, 197.2; TOF-MS (ES+)
m/z= 204.5 (M+1).
Thiazol-2-yl dithiocarbamic acid isopropyl ester
(4). Yield: 79%. 1H-NMR (CDCl3): 1.46 (d, 6H,
2CH3), 2.94 (m, 1H, CH), 7.25 (d, 1H, CH), 7.31 (d,
1H, CH), 8.12 (bs, 1H, NH); 13C-NMR (CDCl3):
26.2, 38.3, 128.1, 132.8, 170.9, 198.2; TOF-MS
(ES+) m/z= 219.3 (M+1).
(Thiazol-2-ylthiocarbamoylsulfanyl)-acetic acid
(5). Yield: 66%. 1H-NMR (CDCl3): 3.87 (s, 2H,
CH2), 7.25 (d, 1H, CH), 7.36 (d, 1H, CH), 8.41 (bs,
1H, NH), 10.37 (s, 1H, COOH); 13C-NMR (CDCl3):
42.1, 127.3, 133.9, 171.7, 178.6, 199.3; TOF-MS
(ES+) m/z= 235.6 (M+1).
Thiazol-2-yl-dithiocarbamic acid 2-oxo-propyl
ester (6). Yield: 82%. 1H-NMR (CDCl3): 2.12 (s,
3H, CH3), 3.79 (s, 2H, CH2), 6.92 (d, 1H, CH), 7.20
(d, 1H, CH), 8.36 (bs, 1H, NH); 13C-NMR (CDCl3):
26.4, 46.9, 128.7, 136.8, 172.2, 198.6, 212.8; TOF-
MS (ES+) m/z= 233.4 (M+1).
Thiazol-2-yl dithiocarbamic acid (2-chloro) acetyl
ester (7). Yield: 84%. 1H-NMR (CDCl3): 4.16 (d,
2H, CH2), 6.99 (d, 1H, CH), 7.28 (d, 1H, CH), 8.28
(bs, 1H, NH); 13C-NMR (CDCl3): 62.4, 126.2,
136.5, 172.2, 188.4, 196.8; TOF-MS (ES+) m/z=
253.6 (M+1).
Thiazol-2-yl dithiocarbamic acid benzoyl ester (8).
Yield: 69%. 1H-NMR (CDCl3): 6.88 (d, 1H, CH),
7.33-7.62 (m, 6H, CH and C6H5), 8.89 (bs, 1H, NH);
13C-NMR (CDCl3): 122.4, 127.8, 128.3, 132.6,
134.2, 142.4, 171.6, 188.5, 198.2; TOF-MS (ES+)
m/z= 281.4 (M+1).
Thiazol-2-yl dithiocarbamic acid 5-Bromo-pentyl
ester (9). Yield: 72%. 1H-NMR (CDCl3): 1.29-1.38
(m, 6H, 3CH2), 3.36 (t, 2H, Br-CH2), 3.98 (d, 2H,
SCH2), 7.14 (d, 1H, CH), 7.38 (d, 1H, CH), 9.22 (bs,
1H, NH); 13C-NMR (CDCl3): 28.0, 32.5, 34.6, 42.8,
128.1, 137.4, 174.5, 196.9; TOF-MS (ES+) m/z=
326.2 (M+1).
Thiazol-2-yl dithiocarbamic acid benzyl ester (10).
Yield: 83%. 1H-NMR (CDCl3): 4.32 (s, 2H, SCH2),
7.14 (d, 1H, CH), 7.21-7.53 (m, 6H, CH & C6H5),
8.74 (bs, 1H, NH); 13C-NMR (CDCl3): 44.2, 124.6,
128.6, 130.1, 140.3, 142.3, 172.6, 196.3; TOF-MS
(ES+) m/z= 267.7 (M+1).
Thiazol-2-yl dithiocarbamic acid 4-methylbenzene
sulfonyl ester (11). This compound did not form and
mixture of unidentified product was obtained. It may
be expected possibly due to unstable nature of the
expected product.
RESULTS AND DISCUSSION
The main synthetic route to dithiocarbamates is based
on the interaction between the corresponding amine,
CS2 and alkyl or acyl halide in the presence of KOH
in dry DMF. The 2-aminothiazole (1) was used as
starting material that afforded dithiocarbamate salt
(2). Furthermore, a part of this salt was converted
into thiazol-2-yl dithiocarbamic acid (2a) and into
various dithiocarbamate esters (3-11) through the
treatment with aqueous p-toluene sulphonic acid (p-
TSA) and alkyl or acyl halides, respectively
(Scheme-1 and Table-1).
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S
NN
HS
S
R
S
NNH2
CS2, KOH
(1) (3-11)
DMF, 0 C-rt
S
NN
HS
S
DMF, 0 C-rt
K
(2)
RX
S
NN
HSH
S
(2a)
aq. p-TSA
Scheme 1: Synthesis of thiazol-2-yl dithiocarbamate esters (3-11).
In earlier study the use of aprotic solvent has been
reported for better results16. However, the solubility
of KOH in aprotic solvents is very low. In aprotic
systems, the rate of the process increases with the
increase of the dielectric permittivity of the solvent.
Table 1: Synthesis of thiazol-2-yl dithiocarbamate
esters
Product
entry RX Yield (%)
CH
2
CH
3
88
CH(CH
3
)
2
79
CH
2
COOH
66
CH
2
COCH
3
82
COCH
2
Cl
84
COC
6
H
5
69
CH
2
(CH
2
)
3
CH
2
Br
72
(10)
CH
2
C
6
H
5
83
(11)
*
SO
2
C
6
H
4
(p
-
CH
3
)
---
*Unidentified product was formed.
Molecular Modelling
Previously, Cheeseright et al. have been described
molecular fields in a form that enables similarity
comparisons across molecules in three dimensions
and demonstrated how molecular fields can be used
as non-structural templates for defining similar
biological behaviour17. It has so far shown that field
patterns can be used to align molecules that act at the
same target by their common field pattern and derive
the biologically active conformation of a ligand
without access to any protein structural data (Field
Templating). The virtual screening of all synthesized
compounds was carried out using field patterns for
potential hits (Field Aligning). Field Templating and
Field Aligning rely on the assumption that those
molecules whose field patterns are most similar to
those of an active search molecule will be the ones
most likely to show the same patterns of biological
activity and should be chosen for further
investigation.
Template generation
It has been reported that PFT- (12), amifostine (13)
and PDTC (14) are structurally diverse inhibitors of
p53 protein10,18. Among these PFT-, and amifostine
metabolised into active metabolites PFT- (15) and
WR1065 (16), respectively, under physiological
conditions 19,20. The PDTC is an ammonium salt
which may also expected to convert into pyrrolidine
dithiocarbamic acid (17), under physiological
conditions (Scheme-2). Thus, in the study compound
(15), (16) and (17) were selected as potent inhibitors
of p53 protein and used to generate templates with
their own field pattern using “FieldTemplater”
software21.
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N
S
O
NH
CH3
N
S
N
CH3
N S
S
(12)(15)
(13)
(14)
H2N N
H
SH
N SH
S
(16)
(17)
Phy siolo gical cond ition s
H2N N
H
SPOH
O
HO
NH 4
Scheme 2: Conversion of p53-inhibitors (12-14) into their active metabolites (15-17).
(a) (b)
Fig 1: (a) Field alignment of template molecules, silver (15), dark brown (16), and golden (17) in lowest energy conformations; the positive (red),
negative (Blue), Van der Waals (yellow), and hydrophobic (orange) field point are represented as balls or cubes or polygons, while black arrows
indicate the essential field points; (b) Field alignment of synthesized database molecules (silver) over reference template (17) (black); the
essential field points are represented by black arrows.
The energy minimization and subsequent molecular
field alignment indicates that only four field points-
one positive, one negative, one Van der Waal and one
hydrophobic ( in the form of polygons) of template
(15) overlie. Similarly, four field points- one positive,
two negative and one hydrophobic (in the form of
cubes) of template (16) overlie after alignment.
While, five field pints- one positive, two negative,
one Van der Waal and one hydrophobic (in the form
of balls) of template (17) overlie after alignment
(Figure-1a).
The overlaid five field points of template (17) are
those in which field points of both templates (15) and
(16) are also included. Thus, these five field points
of template (17) may be expected as essential
molecular field points for anti-p53 activity. Each
template set in the series was ranked according to
how well its constituents overlaid in field and volume
space using the Gsim score. Of three highest ranked
template sets from FieldTemplater, only top-ranking
template set was chosen as the master template set on
which to base the calculation of field similarities
across the whole data set. The scores for field
alignment of the top-rank template set are given in
table-2.
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Table 2: Scores for top-rank template after alignment of compound (15), (16) and (17).
S. No. Template scores Values of scores
a
1 Molecular Similarity 0.611
2 Field Similarity 0.615
3 Raw Field Score -32.505
4 Shape Similarity 0.607
5 Raw Shape Score 71.939
a All scores are for top rank set of aligned templates from total of five sets.
Finally, it was easy to make selection of constituent
molecule 17 from master template set due to presence
of all five essential field points in a single molecule
and was used as a reference template.
Molecular field alignment of database molecules
Once a reference template has been generated, it can
be used to align multiple database molecules as part
of lead optimization process. The template molecule
(17), black coloured, was entered in its 3D bioactive
conformation in FieldAlign software package21
(Figure-1b). While, ten database molecules (2a-11)
were entered in 2D structures. FieldAlign generates
3D conformers from a 2D molecule and then
calculates a field similarity score for each conformer.
FieldAlign works with just one bioactive conformer
as a field template or with a template made up from
the fields of several individual structures. In this way
it identifies, scores, and displays the conformers of a
database molecule that best match the template. In
present study, three overlay scores were computed
from which a single overall score for molecular
similarity (Gsim) was derived; raw overlay energy
(E0), field similarity (Fsim), and a volume overall
similarity (Vsim). The FieldAlign protocol was used
to score the molecular similarity (Gsim) of 50
representative conformations of ten database
molecules (2-11) against the reference template (17).
The most probable conformer of each database
molecules was selected on the basis of pair wise
matching and close observation of molecular field
point alignment. The alignment scores of selected
conformers are represented in quantitative terms of
molecular similarity (Table-3).
Table-3: Scores values for database molecules (2a-11) after alignment over reference template (17).
Database
Molecules
Conformer
Number
Molecular
Similarities Field Similarities Field Scores Shape
Similarities Shape Scores
(2) 3 0.775 0.695 - 29.694 0.856 82.436
(3) 20 0.706 0.671 - 30.61 0.741 80.120
(4) 12 0.711 0.682 - 31.830 0.739 82.897
(5) 8 0.703 0.651 - 34.521 0.754 83.218
(6) 4 0.702 0.664 - 35.538 0.739 83.696
(7) 6 0.679 0.698 - 34.812 0.661 76.678
(8) 2 0.638 0.655 - 36.449 0.622 79.911
(9) 14 0.621 0.614 - 34.274 0.628 83.917
(10) 5 0.625 0.608 - 32.623 0.643 81.743
(11) 2 0.598 0.613 - 35.246 0.582 81.398
854
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ACKNOWLEDGMENT
We are grateful to Head of Department of Chemistry,
Sant Longowal Institute of Engineering and
Technology (SLIET), for providing work facilities.
The authors are also thankful to Sophisticated
Analytical Instrumentation Facilities (SAIF), Punjab
University, for providing spectral analysis facilities.
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