Title of Invention


Abstract a synthetic diaminoketothiazole, its process of preparation and its use as a microtubule inhibitor, a probe ibr tubulin-microtubule system and a cytotoxic agent Diaminoketothiazole of the formula:
This invention relates to a New solid phase method for the preparation of Diaminoketothiazoles.
Further this invention also relates to the use of Diaminoketothiazole (DATI) as a microtubule inhibitor, a probe for tubulin-microtubule system and a cytotoxic agent.
Microtubules are a topic of intense research because of their important and multiple functions in the cell. Many of the potential anticancer agents act on microtubules and arrest mitosis as during mitotic cell division, microtubules play a crucial role by maintaining proper spindle function. Microtubule effectors work in two ways, they can interfere with microtubule dynamics and they can shift the tubulin-microtubule equilibrium in the cell by either inducing of inhibiting microtubule polymerization. There £ire three major classes of microtubule effectors. Taxanes stabilize microtubules by blocking disassembly. Vinca alkaloids and colchicine site binders destabilize microtubules by the inhibition of assembly of tubulin molecules, the major component of microtubules. Taxanes like Paclitaxel, docetaxel and vinca alkaloids like vincristine and vinblastine are well characterized and widely used clinically in different types of malignancies.
The main drawback of Taxanes and vinca alkaloids is that their use is limited by the development of drug resistance, neurotoxicity and limited availability leading to very high expenses involved.
The derivatives of diaminoketothiazoles have received much attention lately as inhibitors of cyclin-dependant kinases and glycogen synthase kinase-3. These are thus claimed to be useful for the treatment of malignancies and Alzhemer's disease, impaired glucose tolerance. Type 1 and 2 diabates.
For the synthesis of diaminoketothiazoles, ther exists only few methods. The
first method makes use of a cyanothiaourea derivative to provide the (C-N-C-S) atoms required for the thiazole construction and tlie remaining C atom is sourced from an alpha-haloketone. The second medod utilizes thiocarbamoylamine derivatives as the source of the (C-N-C-S) four-atom complement.
In the third approach, an S-alkyldithiobiuret serves as synthon for providing the (C-N-C-S) four-atom complement. These methods are usually suited for the solution phase synthesis of the title compounds. However, in the light of combinatorial library synthesis, solid phase methods are much more desirable. Such approaches allow rapid synthesis of a large number of analogue molecules that can be later subjected to bioactivity screening.
An object of this invention is to propose a Dianunoketothia2X)le (DATl) and a
novel process for the preparation thereof.
Another object of this invention is to propose a new solid phase synthesis of diaminoketothiazoles.
Further object of this invention is to propose a useful methd for the synthesis of diaminoketothiazoles on a solid support
Still further object of this invention is to propose a process of synthesis of diaminoketothiazoles (DATl) which is cheap and cost effective.
Another object of this invention is to propose DATl as a microtubule inhibitor and cytotoxic agent.
still another object of this invention is to propose diaminoketothiazoles for the treatment of cancer and other disease using its microtubule inhibition activity.
Yet another object of this invention is to propose diaminoketothiazoles as a probe for structure-function studies of tubulin-microtubule system.
According to this invention there is provided a Diaminoketothiazole (DATI).
Further, according to this invention there is also provided a new solid phase method for the preparation of diaminoketothiazole (DATI) comprising reacting aminomethylpolystrene beads and 1-[N-(aiylthiocarbamoyl) amidino]-3,5-dimethylpyrazole to produce N-(N-arylthiocarbamoyl)-N-guanidinomethyll polystyrene (2);
reacting and N-(N-arylthiocarbamoyl)-N-guanidinomethyll polystyrene with alphahaloketones in the presence of a base which produces the acyclic S-alkyl intermediate derivative (3);
subjecting the said intermediate to the step of cyclisation to obtain the intermediate cyclic thiazoline (4);
subjecting to intermediate to a step of eliminative aromatization step to produce diaminoketothiazole in the solution;
filtering the said solution to remove the impurities and then precipitately isolate 5-aroyl-4-amino-2-arylaminothiazoles from the solution.
According to another embodiment of this invention, there is provided the use of diaminoketothiazole as a microtubule inhibitor, a tubulin binding agent and a cytotoxic agent.
Fig 1. Time-course of inhibition of in vitro microtubule assembly by DATl. 1.2 rag/ml of 3X MTP was incubated with different concentrations of DATl for 2 min at 240C in PEM buffer. Subsecpuently 1 mM GTP was added and polymerization was followed by the turbidity at 345 nm for 20 min at 3TC. Control MTP (1), MTP with 10 µM DATl (2), 20 µM DATl (3) and 40 µM DATl (4).
Figure 2: Effect of DATl on the microtubule networic. HeLa cells were exposed to DMSO (A & B), 1 µM (C ) or 0.2 µM (D) DATl and 0.1 µM (E) or 0.02 µM (F) vinblastine. After 24 h, microtubules were visualized by indirect immunofluorescence microscopy using an antibody against p-tubuiia
The present invention relates to the solid phase synthesis of diaminoketothiazoles on polymer beads. The details of the new invention is described below. Polymer beads comprising DVB-cross linked chloromethyylpolystyrene such as 2% by wt, was converted to aminometthylpolystyrene (AMPS) by a reported method The amioomethylpolystyrene beads so obtained were then reacted by a new method with l-[(N-arylthiocarbamoyl)amidino]-3,5-dimethylpyrazole 1 which acts as a thiocarbamoyl group transfer agent This converts the amino group on the polymer bead into a N-(N-arylthiocarbamoyl)guanidine group giving novel N-(N-arylthiocarbamoyl)-N'-guanidinomethyl polystyrene 2 (AGMPS). his on reaction with alpha haloketones in the presence of a base gives the acyclic S-alkyl intermediate derivative 3, which then directly and in citu undergoes a cyclisation to the next intennediate cyclic thiazoline 4, followed by an eliminative aromatisation step in which the aminomethyl polystyrene acts as a leaving group, thus leading to the release of diamiuoketothiazole 5 in solution, A ftlbatiou
noves the polymer, dilution of the solvent with with water cleanly precipitates the product 5-aroyl-4-amino-2-3arylamincthiazoles 5.
Reaction scheme
DATl distorts microtubules in HeLa cells as well as inhibits the in vitro assembly of raicrotubular proteins. It exhibits cytotoxicity in different types of cancer ceil lines and is much more active than paclitaxel and somewhat more active than vinblastine in drug resistant cancer cells. It is much less toxic to normal cells than cancer cells. Moreover, its synthesis is quite cheap compared to the cost involved in the synthesis of taxanes and vinca alkaloids.
The compound DATl was tested for its cytotoxicity on human cervical, uterus and colon cancer cell lines and mouse fibrocercoma cells. Cell lines were obtained from ATCC, USA and NCCS, Pune, India MTT assay, which correlates a formzan dye formation with the number of viable cells, was used for this purpose. The widely used anticancer drugs paclitaxel, vinblastine or the antimitotic drug colchicine were used for comparison . In 5 out of the 9 cell lines tested, DATl showed activity with IC50 values in a range of 0.05-0.3 µM, and in 2 cell lines, the values were in the range of 1-5 µM (Table 1). These values were either comparable or 5-20 times lower than paclitaxel and vinblastine. Subsequently, DATl was tested on the multidrug resistant cell line MES-SA/DX5, which is resistant to a number of important antimitotic and anticancer agents, viz, colchicine, paclitaxel, vinca alkaloids, doxorubicin etc. It was found to be 15 and 2 times more active than paclitaxel and vinblastine respectively.
The cell survival in the normal immortalized cell line IMR 90 (lung epidielial) was good after the treatment of DATl in a concentration which was much more than an tlie IC50 values in all the cancer cell lines tested. In comparison, (he cell survival was less upon vinblastine treatment and similar upon paclitaxel heatnnent in similar - concentrations (data not shown).
As many of the potencial . anticancer drugs gre antimitotic and microtubule effectors, DATl was tested for its effect on microtubule assembly. A spectrophotoraetric assay was used for this purpose where turbidity at 350 nm was used to quantitate the amount of microtubule polymers formed from microtubule proteins. Figure 1 shows that it inhibited microtubule ibrmation in a concentration dependent manner.
The in vivo effect of DATl on microtubules was tested on HeLa (cervical cancer cell line) cells after an incubation of 48 hours and staining the microtubule network by an antitubulin antibody followed by a Rhodamine labeled secondary antibody. Figure 2 shows that microtubule network was destroyed by DATl in a similar manner to the anticancer drug, vinblastine.
As tubulin is the major component of microtubules, the effect of DATl on purified tubulin was checked. DATl absorbs light with absorption maxima at 211 nm, 283 nm and 374 nm in methanol. Although DATl doesn't exhibit any fluorescence by itself in aqueous solution, when incubated with tubulin, it showed fluorescence with an emission maximum of 457 nm upon excitation at 374 nm The fluorescence intensity increased with the increase in concentration of tubulin showing that it bound to tubulia
To measure the binding affinity and stoichiometry of DATl binding to tubulin, a titration of tubulin with DATl at 24 °C was performed and the fluorescence values at 450 nm were noted upon excitation at 374 nm. A Kd (Dissociation constant) value of 2.9 ± 1 JJM and a stoichiometry of 1 were calculated (mean of three expeiimeuts) from a scatchard plot
All these observations place DATl in a suitable position for consideration as a good microtubule inliibitor, a suitable probe for (he structure-fimction studies of tubulin-microtubule system and a potential anticancer agent. The physical (IR spectra], NMPv and MS spectral) data are shown in Table 2.

1. Conversion of aminomethylpolystyrene (AMPS) to N-(N-arylthiocarbamoyl)-N'-guanidinomethyl polystyrene 2 (AGMPS)-General Procedure: Aminomethylpolystyrene resin beads (2 g, 2.13 meq.NH2/g resin) was swelled in acetonitrile (5 ml). To the swelled resin, a solution of 1-[(N-arylthiocarbamoyl)amidino]-3,5-dimethylpyrazole 1 (2 molar equivalents) in acetonitrile (10 mL) was added The mixture was then refluxed for 12-15 h. The resin beads were then removed by filtration, washed repeatedly with warm and then cold acetonitrile (3 X 10ml), then with petroleum ether (60-800 b.p) (2 X10 ml) and then dried in vacuum The S capacity of the resin was then estimated by digestion and gravimetry by standard procedures. This was found to be in the range 0.98-1.32 meg/g resia
2. Synthesis of 5-acyl-4-amino-2 arylaminothiazoles 5: General Procedure:
The above arylthiocarbamoyi resin (AGMPS) was swelled in N,N-dimetfayl formamide (DMF) (5 ml). To this, die respective a-bromoketone (molar equivalent as per S-capacity) in DMF (2 ml) was added followed by two molar equivalents of triethylamine.
The mixture was wanned to 50-6(fC for 2-5 h. The resin beads were removed by filteration washed with DMF and the pooled filtrate and washings were carefully diluted by ice-cold water (100ml). The precipitated 5-aroyl-4-araino-2-arylaminothiazoles 5 were collected by filtration and purified by crystallization or column chromatography on silica gel. A few typical results in the preparation of thiazole 5 is given below.

(Table Removed)
3. Cell Viability Assay: MTT assay was used to determine the number of viable cells upon drug addition. Cells were seeded in microtitre plates (generally 5 X 103 cells per well) and were incubated with different concentrations of the cytotoxic agents for 48 h. Subsequently, 100 µl of MTT solution (0.6 mg/ml) was added per well and incubated at 37°C for additional 2 h. The amount of formazan salt was quantified in quadruplicates by recording the absorbance at 570 nm using a Biorad Plate reader. The growth inhibition constants (IC50) were calculated from the semi logariflanic dose response plots using the nonlmear regression program Origin. All the experiments were done for at least three times.
Microtubule and Tnbulin Preparation: Microtubular protein was prepared from goat brains by two cycles of temperature dependent assembly-disassembly process in PEM buffer (100 mM PIPES, pH 6.9, 1 mM MgCl2 & 1 mM EGTA ) with 1 mM GTP at 37 °C .For the polymeriztion experiments, it was followed by one more cycle in PEM. Tubulin was purified from 2X MTP using glutamate buffer for assembly.
5. Polymerization assay: MTP polymerization, in the presence or absence of DATl, was measured by the time course of the turbidity at 37°C at 345 nnt A ShimazuUV-1601 double beam spectrophotometer fitted with a temperature-
ccnlrolled circulating water bath was used for this purpose.
6. Immunoiluoreseence assay; HeLa cells were incubated with the drug for 24 h at 37 "C, washed with PBS and fixed with 4% paraformaldehyde at 4 C. Subsequently, they were washed and penmeabilized with 0.2 % Triton X-100 in PBS for 20 min at 37 °C. Microtubules were stained by a mouse monoclonal antibody against p-tubulin in 1:100 dilution followed by a Rhodamine conjugated goat antimouse antibody in 1:50 dilution and were observed by a Nikon Eclipse TE300 microscope.
7. Tubulin binding: Emission spectra of DATl in presence of tubulin were recorded from 400 nm to 600 am using an excitation wavelength of 374 nm Excitation and emission bandpasses are 2.5 nm each and the fluorescence values recorded are uncorrected. All fluorescence measurements were performed in a Perkin-Elmer model LS50B Luminescence spectrometer.
The binding parameters of DATl binding to tubulin were measured from fluorescence data by the standard Scatchard analysis. The binding constants and stoichiometries were determined from Scatchard plot using 2 µM tubulin and varying DATl over 0.2-20 µM Fluorescence values were recorded at 450 nm using an excitation wavelength of 350 ran to reduce the absorbance of DATl. Inner filter effect coirection were perfonned to minimize the effect of high absorbance of the fluorophore.
Table 1: Cytotoxic activity of DATl against different tumour cell lines
Different concentrations of DATl, paclitaxel and vinblastine or colchicine were incubated at 37°C with the different cell lines. After 48 hours, drug containing media were removed and MTT assay were done as described in the examples. IC50 values (giowtfa iniiibition constants) were calculated using the nonlinear regression program Origin. The average of three experiment.s is shown.
(Table Removed)

Diaminoketothiazole of the formula:
(Formula Removed)
Wherein Ar is 4-Ome-C6H5
Ar' is C6H5
2. A new solid phase method for the preparation of
diaminoketothiazole (DATl) comprising reacting
aminomethylpolystrene beads and l-[N-(arylthiocarbamoyl)
amidino]-3,5-dimethylpyrazole to produce N-(N-
arylthiocarbamoyl)-N-guanidinomethyll polystyrene (2);
reacting and N-(N-arylthiocarbamioyl)-N-guanidinomethyll
polystyrene with alphahaloketones in the presence of a base which produces the acyclic S-alkyl intermediate derivative (3);
subjecting the said intermediate to the step of cyclisation to obtain the intermediate cyclic thiazoline (4);
subjecting to intermediate to a step of eliminative aromatization step to produce diaminoketothiazole in the solution;
filtering the said solution to remove the impurities and then precipitately isolate 5-aroyl-4-amino-2-arylaminothiazoles from the solution.
3. A method as claimed in claim 2, wherein said L-haloketones is L-bromoketone.






789-del-2004-description (complete).pdf








Patent Number 239492
Indian Patent Application Number 789/DEL/2004
PG Journal Number 14/2010
Publication Date 02-Apr-2010
Grant Date 23-Mar-2010
Date of Filing 27-Apr-2004
# Inventor's Name Inventor's Address
PCT International Classification Number C07D 297/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA