Title of Invention

PROCESS FOR THE PREPARATION OF THIAZOLO PYRIMIDINES OF FORMULA I

Abstract The present invention provides novel thiazolo pyrimidine derivatives of general formula/structure (I) and a method for preparing general structure/compounds (I.) The compounds, derivatives and analogs thereof, are useful for treatment, management, or prevention of, or amelioration of one or more more symptoms of cancer, HIV, tuberculosis and other microbial and viral infection.
Full Text FORM 2
The Patent Act 1970,
(39 of 1970)
&
The Patent rule 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
1. TITLE OF THE INVENTION
Thiazolo pyrimidine derivatives and their medicinal applications.

2.APPLICANT (S)
(a) NAME:
(b) NATIONALITY:
(c) ADDRESS:

Shelar Ashok Ranganath Indian

Dr.Ashok Shelar (Ph.D)
Plot No.8,Unit No.1
Ambai Defence Colony, Kolhapur-416008 Maharashtra State(lndia)
2. COMPLETE SPECIFICATION
DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.


TITLE
Thiazolo pyrimidine derivatives and their
Medicinal applications
Field of Invention:
Heterocyclic compounds occur widely throughout the nature; indeed, probably the majority of known organic compounds are heterocyclic in structure. The pyrimidine ring system occurs in thymine,cytosine,uracil, adenine and guanine which are component structures of nucleic acids and certain coenzymes.





Pyrimidine nucleosides have an important role in medicinal chemistry. Many antibiotics are nucleosides of pyrimidines which are effective in halting growth of cancer and can inhibit growth or destroy microorganisms ex.Puromycins.
Many pyrimidines of synthetic origin are widely used as therapeutic drugs, of these is a sulfonamide drug, sulfadiazine.Other class of pyrimidine medicinals are barbituric acid and phenobarbitals.



Barbutric acid Phenobarbutric acid
Literature is replete with medicinal uses of pyrimidines and purines.For instance 2, 6-dimercapto purines are used as anticancer agents.
References:
D.J.Brown, "The Pyrimidines" Wiley (Interscience), New York, 1962
T.L.V.Ulbricht, "Purines,Pyrimidines and Nucleotides" Pergamon, Oxford, 1964

PRIOR ART OF INVENTION:
THAZOLO PYRIMIDINE DERIVATIVES
Among different derivatives of pyiamidines, the thiozolo pyrimidines play very important role in the biological processes. The pyrimidine derivatives show profound physiological activities.They show wide spectrum of activity including antiviral,antibacterial,antitumor activity. The fused bicyclic compounds from Biginelli products are well documented for their useful biological activities.These products are synthesized by classical Biginelli reactions.
SYNTHETIC REFERENCES:
1. A.Mobinkhaledi et al.,Phosphorous,Sulphur and Silicon, 178,495,
2003
2. M.Amrollahi, Asian J.Chem., 17,2,902,2005
BIOLOGICAL ACTIVITY REFERENCES:
1. E.Maquoi et al.,Clin.Cancer Res., 10,4038,2004
2. M.Huang,Y.Wang et al.,Mol.Pharmacol,62,463,2002
3. N.V.Bubno et al.,J.Cancer Res.,63,6395,2003

4. T.Kato,JP,59,190,974,1984 (Chem.Abs. 102:132067,1985)
5. A.Dondoni et al.,J.Org.Chem.67,6979,2002

STATEMENT OF INVENTION (OBJECT OF INVENTION)
There is ever present need for efficacious,nontoxic,with least side effects, drugs against a variety of human diseases. Alleviation of human sufferings has to be the primary aim of every scientific endevour, particularly in the fields of drug discovery.Towards this end an attempt is being made to provide novel drug molecules that have immense potential of treatment and cure against infectious diseases and deadly forms of cancer as also viral infections like HIV.

DESCRIPTION OF INVENTION IN DETAIL:
The compounds of folio wing general formula/structure ( I ) have been prepared.







PROCEDURE FOR PREPARATION OF COMPOUNDS OF
GENERAL FORMULA ( I )
Equimolar quantity of the desired 3,4-dihydro pyrimidine-one and chloro
acetylchloride were taken in a round bottom flask to which a pinch of
silveracetate and catalytic amount of 1:1 HCL were added.Ethanol was
used as a solvent and the contents were refluxed till the completion of
reaction indicated on TLC runs( 20% of ethylacetate used for TLC
elutions).

After the completion of reaction the contents were poured on ice to get the desired product.
The structures of the compounds prepared were checked by spectroscopic
and physical analytical methods.
Ethyl 5- (phenyl)-7-methyl-3-oxo-2, 3-dihydro-5 H-[l, 3] thiazolo[3,2-a]
pyrimidine -6-carboxylate:
IR band at 1711 cm-1
NMR triplet at d 1.34 and quartet at d 4.30 (ethoxy group)
3 singlets at d 2.23,2.98 and 5.43 (vinyl methyl protons -S-CH2 protons
Doublet of doublet at 7.43-7.50(J=7.5 Hz) and 8.15-8.98(J=7.5 Hz) for
the 4 aromatic protons
Elemental Analysis:
Mol.Formula:C 15H13N304S
Elements Found C 54.41%, H 3.93, N 12.70%


Structures and IUPAC Names of Compounds






















IUPAC Chemical Names Structure nos
ethyl 5-(4-methoxy phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H-[1,3] thiazolo[3,2-a] 1 pyrimidine-6-carboxylate
ethyl 5-(4-nitro phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H-[1,3] thiazolo[3,2-a] 2
pyrimidine-6-carboxylate
ethyl 5-(3-nitro phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H[1,3]thiazolo[3,2-a] 3
pyrimidine-6-carboxylate
ethyl 5-(4-hydroxy phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H[1,3]thiazolo[3,2-a] 4
pyrimidine-6-carboxylate
ethyl 5-(4-chloro phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H[1,3]thiazolo[3,2-a] 5
pyrimidine-6-carboxylate
ethyl 5-( phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H [1,3]thiazolo [3,2-a] 6
pyrimidine-6-carboxylate
ethyl 5-(4-fluoro phenyl)-7-methyl-3-oxo-2,3-dihydro-5 H[1,3]thiazolo[3,2-a] 7
pyrimidine-6-carboxylate



BIOLOGICAL ACTIVITY PROFILE FOR THE COMPOUNDS:
Following Methods have been employed for screening for biological activities.
Antimicrobial activities were determined by the Microbroth Dilution Method ( NCCLS )
Anti Tubercular activities were determined against the H 37 Rv strain of M.tuberculosis
Cyto toxic effects was evaluated using the human HeLa cell line srocured from the National Center For Cell Science,Pune
Anti retro viral tests for evaluation of anti HIV activity was performed by the Method of Viral Load by RT-PCR
The details of procedure are given in the following pages

Determination of Minimum Bactericidal Concentration:
Microbroth Dilution Method(NCCLS)
Materials and Reagents
Mueller-Hinton broth for performance of MIC test
1.0 and 0.5 McFarland turbidity standards
Scrupulously clean,acid washed borosilicate glass tubes
Vortex mixer
Eppendrof or similar micropipette
Nutrient agar plates,such as blood agar,for subculture fro broth
PROCEDURE
Preparation of Inoculation
Subcultured the test organism and quality contro organisms into
Appropriate medium(a blood agar plate)and incubated overnight at 35C Inoculate a tube that contains 2 ml of saline or Mueller-hinton broth With five or more colonies from the agar plate and adjust turbidity to Match a McFarland standard(approximately 10 CFU/ml) Transfer 0.1 ml of the turbid broth into 10 ml of Mueller-Hinton Broth,inoculate in shaking water bath or equivalent at 35C until Turbid(5 to 6 hours for rapid growth).The relevant control organism Should be inoculated into 3 ml of broth and incubated without shaking Until turbid.This represents a midexponential phase of growth. Prepare twofold serial dilutions of the antibiotic in 2 ml of Mueller-Hinton broth.Use acid washed borosilicated glass tubes. Standardise the inoculum of the patients organism and the control

Strain by adjusting to a McFarland 0.5 standard in 3 ml of saline broth.
Dilute the adjusted inocula l:10(approx 5X10 CFU/ml)
Using an Eppendorf or equivalent pipette,dispence 0.1 ml of the diluted
Inoculum intothe tubes that contain serial dilutions of antibiotic.Insert the
Tip well under the surface of the broth;avoid any contact between the tip and the walls of the tube.Rinse the tip five times.The same tip may be used
If the tubes are inoculated from the lower to higher concentration of the
antibiotic.The final inoculum size is approximately 2.5X10 CFU/ml.
incubate the tubes for 20 hours at 35C.
QUANTITATION OF THE INOCULUM
From the final dilution of the inoculum(approximately 5X10 CFU/ml)
perform four serial tenfold dilutions in Mueller-Hinton broths (final
Concentration of 5X10 CFU/ml).
In duplicate dispense 0.1ml onto the surface of a blood agar plate.Spread
the inoculum evenly with sterile bent glass rod.
incubate the plates overnight at 35C.
DETERMINATION OF MIC
After overnight incubation determine the MIC of quantity control strain by
virtual inspection of the tubes.
For the patient's sample only,vortex the tubes without growth vigorously
for 15 seconds to resuspend any bacteria that might have adhered to the
walls of the tube(avoid aerosols).
reincubate the tubes for additional four hours.

Determine the MIC of patient's sample by visual inspection of the tubes.
CONCLUSIONS:
The compounds of General Formula (I) have shown excellent activity against Staph.Aureus,E-Coli.Klebsiella,Enterococcus fecalis.Antifungal activity against Fluconazole Aspergillus and Candida albicas.

Anti T.B. tests
Anti tubercular activity was tested against H37 Rv strain of M.tuberculosis maintained in the laboratory. Middle brook 7H9 medium was prepared in bottles and sterilized by autoclaving.ADC growth supplement containing bovine albumin fraction V,dextrose and catalase was added to each bottle with sterile precautions. A sterile suspension(10 mg/ml) of each compound to be tested was prepared using appropriate solvents under sterile conditions.Each compound was tested at a concentration say of 5 micro grams/ml and the required amount was added to each bottle of middle brook
medium.M.tuberculosis culture suspension was added at a concentration of 10 raise to 5 organisms per ml and the media were incubated at 37 degree centigrade in a humidified chamber for at least 4 weeks. Controls of antimycobacterial agents i.e. ciprofloxacin 5micro grams/ml,streptomycin 7.5 micro grams/ml and pyrazinamide 7.5 micro grams /ml were set up with each batch.A medium control and growth control tubes also were included.

Each bottle was examined periodically to observe for growth or contamination if any. At the end of four weeks a smear was prepared from each bottle showing turbidity stained with Z-N stain to confirm the presence of M.tuberculosis.Those media with the presence of organisms were considered as resistant to the compound and those without any bacteria were deemed as sensitive. CONCLUSIONS:
The compounds of General Formula (I) have shown excellent activity against the H37 Rv strain of M.Tuberculosis.The lab results were further confirmed by field tests on Actual samples from patients.

Anti Cancer Tests
In vitro evaluation of cytotoxic effects on human tumour cell lines:
Cell culture :The HeLa cell line(procured from National Centre For Cell Science Pune
University,India)was maintained in the labs by serial sub cultures. The cell line was
maintained as mono layers in 75 cm square culture flasks in Eagles minimum essential
medium supplemented with 10% fetal calf serum 2mg of gentamicin, 100 Unit/ml of
penicillin and 100 micro gram/ml of streptomycin. The cells were grown in humidified
37 degree incubator with a regulated supply of 5% carbon dioxide. Medium was changed
every 5-6 days.
On the day of the experiment cells were washed with phosphate buffered saline and were detached from the surface of the flask by addition of 0.25% trypsin in EDTA buffer.The cells were then plated at a density of 5X10 to the power of 3 cells per well in 96 well micro plates containing 250 micro litre of cell culture medium per well and allowed to adhere over night. Stock solutions(10 micro gram per ml) of the compounds to be tested were freshly prepared just before use under sterile conditions.Each compound was tested in two concentration 10 and 100 micro grams per ml.Requisite amount of the compound was added to each well and were allowed to act on tumour cells for a period of 72 hours.

A cell control was also set up with each compound for comparison. At the end of the incubation period, each well was studied under 40X power inverted microscope and analyzed for cytotoxic effects of each concentration of the compound on tumour cell lines.
CONCLUSIONS:
The compounds of General Formula (I) have shown excellent anti cancer activity.The
Photographs are records of the cell growth inhibition.

WE CLAIM:
1. A process for the preparation of compounds of the general formula (I)










General structure / formula (I)
2. A process of the claim 1,wherein desired 3,4-dihydro pyridine-one and chloroacetyl chloride in equimolar quantity are mixed well;
3. A process of the claim 2, wherein a pinch of silveracetate, catalytic amount of 1:1 hydrochloric acid are added and refluxed with ethanol as a solvent.
4. A process of the claim 1, wherein the substituents are as Rl=H,OH,OMe,Cl,F,N02 etc. R2=H,OH,OMe,Cl,N02 etc.

R3=H,0H,Cl,0Me etc.
5. A method for inhibiting the growth of pathogenic microorganisms (ex: bacteria,fungi,M.tuberculum etc.) wherein the chemotherapeutic agent is a compound of general formula ( I ),its salt or a pharmaceutically acceptable form.
6. A method for inhibiting the growth/replication of any form of cancer (ex: tumor etc.) wherein the chemotherapeutic agent is a compound of general formula ( I ), its salt or a pharmaceutically acceptable form.
7. A method for inhibiting the replication of viruses (ex: HIV etc.) wherein the chemotherapeutic agent is a compound of general formula ( I ), its salt or a pharmaceutically acceptable form.
8. A method for use as CNS stimulant( ex: Nootropic or anti Alzheimer agent etc.) wherein the chemotherapeutic agent is a compound of general formula ( I ), its salt or a pharmaceutically acceptable form.

ABSTRACT
The present invention provides novel Thiazolo pyrimidine derivatives of general formula/structure ( I ) and a method for preparing general structure/compounds ( I.) The compounds,derivatives and analogs
thereof, are useful for treatment,management,or prevention of, or amelioration of one or more more symptoms of cancer, HIV , tuberculosis and other microbial and viral infections.





Documents:

533-MUM-2008-ABSTRACT(GRANTED)-(12-10-2011).pdf

533-mum-2008-abstract.doc

533-mum-2008-abstract.pdf

533-MUM-2008-CANCELLED PAGES(9-5-2011).pdf

533-mum-2008-claims(16-2-2011).pdf

533-MUM-2008-CLAIMS(AMENDED)-(17-2-2011).pdf

533-MUM-2008-CLAIMS(AMENDED)-(2-3-2011).pdf

533-MUM-2008-CLAIMS(AMENDED)-(9-5-2011).pdf

533-MUM-2008-CLAIMS(GRANTED)-(12-10-2011).pdf

533-mum-2008-claims.doc

533-mum-2008-claims.pdf

533-mum-2008-correspondence(16-2-2011).pdf

533-MUM-2008-CORRESPONDENCE(9-5-2011).pdf

533-mum-2008-description (complete).pdf

533-MUM-2008-DESCRIPTION(GRANTED)-(12-10-2011).pdf

533-mum-2008-diagram.doc

533-MUM-2008-DRAWING(GRANTED)-(12-10-2011).pdf

533-mum-2008-drawings.pdf

533-mum-2008-form 13(9-5-2011).pdf

533-mum-2008-form 18(18-3-2008).pdf

533-MUM-2008-FORM 2(GRANTED)-(12-10-2011).pdf

533-MUM-2008-FORM 2(TITLE PAGE)-(18-3-2008).pdf

533-MUM-2008-FORM 2(TITLE PAGE)-(9-5-2011).pdf

533-MUM-2008-FORM 2(TITLE PAGE)-(GRANTED)-(12-10-2011).pdf

533-mum-2008-form 9(27-5-2008).pdf

533-mum-2008-form-1.pdf

533-mum-2008-form-18.pdf

533-mum-2008-form-2.doc

533-mum-2008-form-2.pdf

533-mum-2008-form-3.pdf

533-mum-2008-form-5.pdf

533-MUM-2008-REPLY TO EXAMINATION REPORT(17-2-2011).pdf

533-MUM-2008-REPLY TO EXAMINATION REPORT(2-3-2011).pdf

533-MUM-2008-SPECIFICATION(AMENDED)-(9-5-2011).pdf

abstract1.jpg


Patent Number 249237
Indian Patent Application Number 533/MUM/2008
PG Journal Number 42/2011
Publication Date 21-Oct-2011
Grant Date 12-Oct-2011
Date of Filing 18-Mar-2008
Name of Patentee SHELAR ASHOK RANGANATH
Applicant Address PLOT NO.8, UNIT NO.1, AMBAI DEFENCE COLONY, KOLHAPUR-416008,
Inventors:
# Inventor's Name Inventor's Address
1 SHELAR ASHOK RANGANATH ASHOK SHELAR(PH.D) PLOT NO.8, UNIT NO.1, AMBAI DEFENCE COLONY, KOLHAPUR 416008.
2 SHELAR MILIND ASHOK MILIND SHELAR(PH.D) IPER PLOT NO.8, UNIT NO.1, AMBAI DEFENCE COLONY, KOLHAPUR 416008.
3 SHELAR BAIKRISHNA ASHOK B.A.SHELAR(PH.D) IPER PLOT NO.8, UNIT NO.1, AMBAI DEFENCE COLONY, KOLHAPUR 416008.
PCT International Classification Number A61K31/519C07D513/04
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA