Process For The Preparation Of Reactive Polymers Having Chemoenzymatically Hydrolysable Functional Groups

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FORM 2
THE PATENTS ACT 1970
As amended by the Patents (Amendment) Act, 2002
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
TITLE
Process for the preparation of reactive polymers
having chemoenzymatically
hydrolysable functional groups
APPLICANTS
KOPRAN RESEARCH LABORATORIES LIMITED,
Parijat House, 1076 Dr. E. Moses Road, Worli, Mumbai 400 018, Maharashtra, India, a research organization incorporated in India under the Companies Act, 1956

The following specification particularly describes the nature of this invention and the
manner in which it is to be performed


FIELD OF THE INVENTION
This invention relates to process for the preparation of the reactive polymers having chemoenzymatically hydrolysable functional groups.
PRIOR ART
Polymers have numerous applications in various fields like textiles, paints, pigments, household articles, pharmaceuticals, agriculture or chemicals. In pharmaceutical industry, polymers are used as drug excipients, drug delivery system, therapeutic agents or prodrug. Polymers like polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, carbomer, polylactide or polymethacrylate are

known to be used as drug excipients such as binders, diluents or dispersing agents.
In drug delivery systems, biologically active compounds like ephedrine or amphetamine are ionically bonded to polymers like polystyrol resins to control the release of the active compounds and alter their bioavailability favourably or to give protection against bitterness of the active compounds or prevent their gastric destruction (Chaudhari, N.C., & Saunders, L., J. Pharm, Pharmacol, 8: 975, 1956). It is generally known that in a drug delivery system, the drug is physically entrapped in the polymeric gel, and, invivo it is released in its original chemical form without any chemical modification.
Certain polymers as such are used as therapeutic agents. For instance, US Patent No. 5162307 describes polymeric inhibitors of the enzyme elastase having the Formula P - L - R, where P is a non-biodegradable polymer, L is a covalent bond or a linker group and R is a peptide. Therapeutic peptides conjugated to polyethylene glycol chains are reported to show improved durability and reduced antigenicity (US Patent No 5183660). Anion-binding hydrophilic epichlorohydrin and 1-(3-aminopropyl) imidazole copolymeric bile acid sequestrant and its pharmaceutical compositions are reported for use in the treatment of various ailments like diarrhoea, constipation, dumping syndrome or irritable bowel syndrome (US Patent No 5900233). Polymer analogues of cis-dichlorodiamine platinum are reported for use as antineoplastic agents ['Organometallic polymers as drugs and drug delivery systems' by Gebelein C. G., Koblitz F. K., Biomedical and Dental Applications of Polymers, New York, Plenum Press 1981, p 215]. Homopolymers or copolymers formed of monomers having a pendant ammonium group and hydrophobic monomer have been reported for treatment of microbial infections (US Patent No 6034129)._PCT Publication No. WO 99/63940 discusses low molecular weight polymeric derivatives of benzimidazoles as antiulcer-


agents. The molecular weight of such polymers is generally in the range of 1000 - 10000. Such polymeric drugs get absorbed from the gastrointestinal tract and elicit systemic activity.
Prodrugs comprise biologically active agents substi.tuted with reactive polymers through covalent conjugation. For instance, US Patent No. 5372807 describes an intravenous formulation comprising an antifibrotic agent linked to cis-4-hydroxy-L-proline polymer. US Patent No. 5622718 describes an alginate conjugated with antineoplastic agent such as daunomycin or doxorubicin via an acid labile biodegradable spacer linkage. US Patent No. 6011008 describes water-soluble conjugates of a polysaccharide and an unoxidised, oxidation-sensitive substance, conjugated via amine or imine bonds. US Patent No. 4587046 describes biologically active drug such as catecholamine hormones coupled to carrier molecules like monodisperse peptides. US Patent Publication No. 20010031262 describe polylactide-CO-glycolide copolymers in the form of particles or a gel, lipid vesicles or liposomes, which are stabilized or targeted to enhance the delivery of antigens. US Patent No. 6254854 describes biodegradable porous particles incorporating a therapeutic agent which may be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of the therapeutic agent. These biodegradable particles are formed of a functionalized polyester graft copolymer consisting of a linear a-hydroxy-acid polyester backbone having an amino acid group incorporated therein and polyamino acid side chain extending from an amino acid group in the polyester backbone. Chlorambucil i.e. 4[4-bis (2-chloroethyl) amino phenyl) butyric acid] has been bound to vinylpyrrolidone and vinylamine copolymers via an amide bond (Franzmann and Ringsdorf, Makromol., Chem., 177, 2547, 1976). Deacetylcolchicine or daunomycin is known to be bound to polymers of N- (2-hydroxypropyl) methacrylamide (Synthese and Untersuchung von potentiell spaltbaren spacergruppen zur Polymer-fixierung von NOR-stickstoff-LOST und den Anthracyclinen Daunomycin und Adriamycin, Ph.D. Thesis, Johannes Gutenberg University Mainz, FRG 1982). Daunomycin has also been attached to polymeric carriers to form amino sugar daunosamine (Shih et al., 1991, Cancer Res, 51 : 4192). Polymers like poly [N-2-hydroxypropyl) methacrylamide] containing hydroxyl groups activated by BrCN have been used to bind insulin (Sung Wan Kim et. al. in Polymeric Drug Delivery Systems, Drug Design, Volume X, Academic Press 1980). Activated 4-alkylthioderivatives of cyclophosphamide bound to DIVEMA (divinyl ether and maleic anhydride) copolymer via the anhydride groups are reported by Hirano et. al. (Cancer Res., 40:2263, 1980). Oligopeptide sequences can be incorporated into N-(2-hydroxypropyl) methacrylamide copolymers, which have been reported to serve as potential drug attachment/release sites. Progesterone has been conjugated with aliphatic polyesters such as poly- (s-Caprolactone), poly- [e- (+, -)-Calactone], polypivalolactone and poly -

(+, -) - dilactide through an ester linkage [Biomed. Mater. Res, Pitt et al, 1979, 13, 491; "Polymer conjugates with Anticancer Activity", Advances in Polymer Science, D Putnam et al, 1995, Vol. 122, page 55 - 123, Springer Verlag Berlin]. US Patent No. 4587046 describes covalent conjugation of naturally occurring catecholamines and autocoid moieties with monodisperse amino acid polymers or peptides having an alkyl group through ester/amide linkages. US Patent No. 5783178 describes conjugation of vinca alkaloids, mitomycins, bleomycins, fluconazole, amphotericin B, paclitaxel derivatives, cytokines, erythropiotein or polynucleotides with block copolymer of ethyleneoxy monomer or a mixture of ethyleneoxy and the OCH(CH3)CH2- monomers through Afunctional linking group. US Patent No. 5510418 describes covalent conjugation of glycosaminoglycan with polyethylene glycol through an ether linkage and is useful for hard/soft tissue augmentation. Biphenylamine derivatives have been conjugated with polymethacrylic acid (Baker et. al. J. Pharm. Sci, 68, 20, 1979). US Patent No. 5889078 describes conjugates of cytostatic fluoro uracil with homopolymer of acrylic acids through ester or amide linkages. US Patent No. 5037883 describes conjugates of active such as anticancer daunomycin with copolymer of N-(2-hydroxypropyl) acrylamide, N-methacrylamide, N-methacrylic acid and/or N-methacryloylated amino acid through peptide group. US Patent No. 5976527 describes conjugates of proteins such as albumin, immunoglobulins, blood clotting factors and peptide hormones with polymethylmethacrylate or polymethacrylamide comprising reactive oxirane groups, which after immobilisation are used for interaction with biological systems. These conjugates on administration, under physiological pH and the influence of enzymes are hydrolysed/cleaved at the point of attachment of the polymer to the drug to release the drug in the original chemical form.
There is described in our Indian Patent Application No IN/PCT/2002/01098/MUM (PCT Publication No WO 01/62248), orally administrable acid stable polymer substituted antiulcer benzimidazole derivatives of the Formula I:

Formula II
wherein each of R1, R2, R3, R4, R5 = H, C1-12 alkyl, C6-12 (un)substituted aryl, C|.g alkoxy, C6-12 aryloxy, C1-5 alkoxy carbonyl, C6-12 aryloxy carbonyl, C1-5 alkoxy alkyl, C6-12 alkoxyaryl, C1-5 haloalkyl, C1-5 alkyl or C6-12 aryl thioethers, (un)substituted amines or diamines, (un)substituted amides, halo, cyano, nitro, carboxylic acid or carbocyclic or O, N, S containing heterocyclic ring systems or enantiomers thereof. The polymeric benzimidazole of the Formula I are formed by condensing an antiulcer benzimidazole and a biocompatible partially orally biodegradable synthetic polymer of the Formula HI


wherein R.6, R7, X and E each is as defined above and Y' = O or N. On oral administration, cleavage of the polymeric benzimidazole takes place in the gastrointestinal fluid under the influence of enzymes/chemicals at the hydrolysable group "E" to release a modified drug viz. n-substituted benzimidazole derivative (the benzimidazole alongwith a part of the polymer) which is acid stable.
OBJECTS OF INVENTION
An object of the invention is to provide reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof.
Another object of the invention is to provide reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing improved lipophilicity.
Another object of the invention is to provide reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing high polarity.

Another object of the invention is to provide reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing high optical purity.
Another object of the invention is to provide reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing improved bioavailability and bioefficacy and reduced side effects.
An object of the invention is to provide a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, TSH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof.
Another object of the invention is to provide a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reactive with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing improved lipophilicity.
Another object of the invention is to provide a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing high polarity.
Another object of the invention is to provide a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine to form chemoenzymatically hydrolysable biologically active compounds

which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing high optical purity.
Another object of the invention is to provide a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and capable of reacting with biologically active agents having reactive groups like -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, chemoenzymatically hydrolysable biologically active compounds which undergo rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing improved bioavailability and bidefficacy and reduced side effects.
DESCRIPTION OF INVENTION
According to the invention there is provided reactive polymers having chemoenzymatically hydrolysable functional groups and of the Formula IV:

wherein R1 = H or CH3, R2 = H, C,.8 alkyl or C6-i2 aryl, X represents a cross linking group such as

R3 = CI, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OS02CF3, OH, NH2, NHR5 (R5=alkyl),
SH, epoxide or aziridine,
R4 = CONH2-COOR6 (R6 = H or C1.6 alkyl) or CN
L' = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that
the carbon chain length comprises 2 to 6 carbon atoms when R3 = epoxy or aziridine.

According to the invention there is also provided a process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and of the Formula IV:

wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6-12 aryl, X represents a cross linking group such as

R3 = CI, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OS02CF3, OH, NH2, NHR5 (R5=alkyl),
SH, epoxide or aziridine,
R4 = CONH2-COOR6 (R6 = H or C1.6 alkyl) or CN
L' = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that
the carbon chain length comprises 2 to 6 carbon atoms when R3 = epoxy or aziridine, the process
comprising refluxing an acrylic monomer with a monomer of the Formula V having an ester
linkage

wherein R , R , R and L' are as defined above, in an organic solvent in the presence of an initiator and optionally in the presence of a crosslinking agent under an inert atmosphere.

Preferably, in the formula IV R1 is CH3, R2 is H, R3 is I or OTs and R4 is COOH and in
the formula V R1 is CH3, R2 is H and R3 is I orOTs
The acrylic monomers are acrylic acid, methacrylic acid, acrylamide, methacrylamide, acrylonitrile,
ethylacrylate, methylacrylate, butylacrylate or 2-hexylethyl methacrylate,, preferably acrylic acid
or acrylamide.
The preferred monomer of the formula V is 2-hydroxyethyl methacrylate, 2-iodoethyl
methacrylate, 2-(paratoluene sulphonyl) ethyl methacrylate, 2-chloroethyl methacrylate, 2-
(methane sulphonyl)ethyl methacrylate or 3-[epoxy-(2-methacrylate)]-l-[n-butoxy-(4-
glycidylether)]-2-propanol.
The crosslinking agent may be methylene bis acrylamide, ethylene glycol dimethacrylate, glycol dimethacrylate, or divinyl benzene preferably ethylene glycol dimethacrylate.
The organic solvent may be a mixture of ethyl acetate with methanol, ethanol, isopropyl alcohol or acetone, preferably ethylacetate: acetone.
The initiator may be benzoyl peroxide, lauryl peroxide, azobis isobutyronitrile, or ammonium persulphate, preferably benzoyl peroxide.
The inert atmosphere may be provided by argon or nitrogen preferably nitrogen.
The solid reactive polymer obtained on refluxing is cooled, filtered and washed with a solvent like methanol or acetone to free it from residual monomers and dried at 25-75°C.
The reactive polymers of the invention comprise a side chain having a hydrolysable ester group viz. - COO - attached to the polymeric backbone and a spacer viz. L. The side chain further has a terminal reactive group capable of reacting with biologically active agents having functional groups such as
I
—NH,—NH2> —SH,—OH, —CI,—Br, 1,—F, OTs, OMs, epoxy or aziiidine

The reactive polymers of the Formula IV on conjugation with biologically active agents having reactive groups -NH-, -NH2, -OH, -SH, -CI, -Br, -I, -F, OTs, OMs, epoxy or aziridine form chemoenzymatically hydrolysable biologically active compounds of the Formula VI.

wherein
L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that
the carbon chain length comprises 2 to 6 carbon atoms when
and R1, R2, R4, X are as defined above, D = Biologically active agent having functional groups as defined above

The biologically active compounds of the formula VI have been described in our Patent Application No. 963/MUM/02.

The reactive polymers of the invention are unreported and novel. Because of the spacer the polymers of the invention have reduced steric hindrance and are capable of undergoing rapid chemoenzymatic hydrolysis of the ester linkage -COO-. Therefore, the biologically active compounds formed with the polymers also undergo rapid chemoenzymatic hydrolysis thereby rapidly releasing hydroxy alkyl derivatives thereof i.e. chemically modified biologically active compounds represented by the Formula VII.

wherein R2, Z', L and D are as defined above.
Synthetic hydroxyalkyl derivatives of the biologically active compounds represented by the Formula VII have been described in our Patent Application No. 964/MUM/02. Because of the spacer the hydroxy alkyl derivatives of the biologically active compounds are highly lipophilic. Due to the hydroxyl groups they have increased polarity and better ionisation and absorption. The hydroxyalkyl derivatives of the biologically active compounds are capable of stereo selective hydrolysis to form optically pure isomers thereof. Because the hydroxyalkyl derivatives of the biologically active compounds are optically pure and highly lipophilic, their bioavailability and bioefficacy are improved. Due to the improved bioavailability and bioefficacy, the hydroxyalkyl derivatives of the biologically active compounds are effective at low doses thereby correspondingly reducing the side effects.
The following experimental examples are illustrative of the invention but not limitative of the scope thereof.
EXAMPLE - 1
Step 1: 45 gm of 2-(paratoluenesulphonyl) ethyl Methacrylate (prepared as per methods known in the art), 5 gm of acrylic acid and 0.6 gm of benzoyl peroxide were mixed together and added to 200 ml of ethyl acetate + Acetone (1:1) mixture. The reaction mass was agitated at 80 rpm and refluxed for 4 hours under inert conditions. The material obtained was cooled and stirred in 200 ml of ethyl acetate + acetone (1:1) mixture for one hour. The product was filtered and dried at 50°C for 12 hours (Yield: 51 gm) to obtain polymer of the Formula VIH:


Formula VIII
Step 2: Preparation of substituted pseudoephedrine
10 gm of the above reactive polymer of Formula VIII was mixed with 5 gm of pseudoephedrine hydrochloride dissolved in a solvent system of methanol + water (1: 1) at pH 9.5. The reaction mixture was refluxed for 36 hours. The product was washed with water (100 ml x 5) till free from pseudoephedrine and then dried under vacuum at 50°C for 12 hours to obtain 16.0 gm of polymer-substituted pseudoephedrine of the Formula IX.

EXAMPLE - 2
Step 1 : The copolymer was prepared in the same way as in example 1 (Step 1), except that 45 gm of 2-chloro ethyl acrylate was used in place of 2-(paratoluenesulphonyl) ethyl methacrylate and 1 g of ethylene glycol dimethacrylate was also added. 35 g of polymer of Formula X was obtained.


Step 2: Preparation of substituted 2-mercaptobenzothiazole
5 g of the polymer of step 1 (Formula X) was mixed with 2.5 g of 2-mercaptobenzothiazole dissolved in a solvent system of methanol + water (9:1) at pH 9.3. The reaction mixture was refluxed at 65°C for 36 hours. The resultant product was washed with the methanol (100 ml x 4) will free from 2-mercaptobenzothiazole and dried under vacuum at 50°C for 12 hours to obtain 6.0g of the polymer substituted 2-mercaptobenzothiazole of the Formula XL


EXAMPLE-3
Step 1: The copolymer was prepared in the same way as in example 1 (Step 1), except that 45 gm of 3-iodopropyl Methacrylate was used in place of 2-(paratoluenesulphonyl) ethyl methacrylate and 1 g of ethylene glycol dimethacrylate was also added. 37 g of polymer of Formula XII was obtained.


Step 2: Preparation of substituted fluoxetine
3.5 g of this reactive polymer of Formula XII was mixed with 1.75 g of fluoxetine dissolved in aqueous medium at pH 9.5. The reaction mixture was stirred at 70°C for 24 hours. The resultant product was washed with water (100 ml x 5) till free from fluoxetine and dried under vacuum at 45°C for 12 hours to obtain 4.9 g of polymer-substituted fluoxetine of the Formula XITf.


EXAMPLE - 4
Step 1 (a): The copolymer was prepared in the same way as Example 1 (step 1), except that 45 g of 3-[Epoxy-(2-methacrylate)]-l-[n-butoxy-(4-glycidylether)]-2-propanol (prepared as per methods known in the art) was used in pace of 2-(paratoluenesulphonyl) ethyl methacrylate. 46 g polymer of Formula XIV was obtained.


Step 2: Preparation of substituted amlodipine
10.0 g of this reactive polymer of formula XIV was mixed with 5.0 g of Amlodipine base dissolved in a solvent of isopropyl alcohol + water (1: 1) at pH 9.5. The reaction mixture was stirred at 30°C for 72 hours. The product was washed with the same solvent system (100 ml x 5) till free from Amlodipine and then dried under vacuum at 45°C for 12 hours to obtain 13.8 g of polymer-substituted Amlodipine of the Formula XV.

Formula XV EXAMPLE - 5
Step 1 : The copolymer was prepared in the same way as in Example 1 (Step 1), except that 45 gm of 2-(methanesulphonyl) ethyl methacrylate (prepared as per methods known in the art) was used in place of 2-(paratoluenesulphonyl) ethyl methacrylate. 43 gm of the polymer of the Formula XVI was obtained.

Step 2: Preparation of substituted Alendronate
5.0 g of the above reactive polymer of formula XVI was mixed with 2.5 g of Alendronate sodium dissolved in an aqueous medium at pH 9.5. The reaction mixture was stirred at 30°C for 24 hours.


The resultant product was washed with water (100 ml x 7) and dried under vacuum at 50°C for 12 hours to obtain 6.8 g of the polymer-substituted Alendronate of the formula XVII.
Formula XVII BIOLOGICAL ACTIVITY
ANTI-DEPRESSANT ACTIVITY
Principle
It is known suggested that mice or rats forced to swim in a restricted space from which they cannot
escape are induced to characteristic behaviour of immobility. This behaviour reflects a state of
despair, which can be reduced by several agents, which are therapeutically effective in human
depression.
Materials & Methods
Animals
Swiss albino mice of either sex.
Weight of animals: 30 - 40 g
Drugs: Dose (mg/kg)
1. Compound of formula XHI: 42mg/kg
2. Fluoxetine (Standard): 20mg/kg
Method
Swiss albino mice of either sex weighing about 30 - 40 g were used. They were brought to the laboratory and acclimatized for 7 days. Mice were individually forced to swim inside a vertical Plexiglas cylinder; mice placed in cylinders for the first time were initially highly active, after 2-3 . min activity began to subside and phases of immobility or floating increased. Mice were immobilized approximately for 80% of the time, They were again placed in the. cylinder 24 hi later and total duration of immobility was measured during a 5 min test. Floating behaviour during this 5 min period has been found to be reproducible in different groups of mice. An animal was judged to be immobile whenever it remains floating passively in water. The drugs were administered one hour prior to testing.

Evaluation
Duration of immobility was measured in controls and drug treated animals. Significance was calculated using 't' test.
Result and discussion

Standard drug fluoxetine and test drug Compound of formula XHI showed less immobility time (sec) when compared to control group.
Conclusion
Standard drug fluoxetine and test compound Compound of formula Xm showed significant antidepressant activity when compared to control group.
ANTI-ASTHMATIC ACTIVITY
Principle : Exposure of spasmogen like Acetylcholine chloride or Histamine causes contraction of
bronchial smooth muscle. This method permits the evaluation of bronchodilator drugs by
measuring time required to produce convulsion after exposure to spasmogens.
Materials and methods :
Animals : Adult guinea pigs of either sex.
Weight of animals : 300 to 350gms.
Materials : Aerosol chamber with 2 compartments and with a central spout for introduction of atomized histamine.
Drugs : Histamine, Test and standard drug.

Experimental procedure : Protection against histamine Aerosol induced Bronchospasm. Experimental bronchial asthma was induced in guinea pigs by exposing them to 10% histamine under constant pressure in an aerosol chamber. The animals exposed to histamine aerosol showed progressive dyspnoea. The end point preconvulsive dyspnoea (PCD) was determined from the time of aerosol exposure to the onset of dyspnoea leading to the appearance of convulsion. As soon as PCD was commenced the animals were removed from the chamber and placed in fresh air. This PCD was taken as Ti Guinea pigs were administered with test and standard drugs, two hours after the dose administration the time for the onset of PCD was recorded as T2 and protection offered by the treatment was calculated by following formula

Conclusion
Test and standard drug significantly prolonged the latent period of convulsion as compared to control following exposure to histamine aerosol.
ANTI-HYPERTENSIVE ACTIVITY
Principle
Ischemia of kidneys causes elevation of blood pressure by activation of renin-angiotensin system. This principle can be used for inducing acute renal hypertension by clamping the left renal artery. The protease renin catalyses the first and rate limiting step in the formation of angiotensin-!! leading to acute hypertension. This test was used to evaluate antihypertensive activities of drugs.

Procedure
Male Sprague-Dawley rats weighing about 200 - 250 g were anesthetized by anesthetic ether. The fur was shaved and the skin was disinfected. In left lumbar area a flank incision was made parallel to long axis of the rat. The renal pedicel was exposed with the kidney retracted to abdomen. The artery was dissected clean and a U-shaped silver clip was clipped around it near the aorta, using special forceps. The size of the clip was adjusted so that internal gap ranges form 0.25 to 0.38 mm. The right kidney was removed through a flank incision after tying off renal pedicle.
The skin incisions were closed by wound clips. Four to five weeks after clipping, the blood pressure was measured and rats with higher than 150 mm Hg selected for the experiments. Blood pressure reading was taken at 1, 2, 3 and 4 hrs after drug treatment.
Drug treatment schedule
The animals were divided into 3 groups.
Group I received 25mg/kg of compound of Formula XV
Group II received 0.9 mg/kg of Amlodipine (Manufactured by Kopran Ltd.)
Group m were the hypertensive controls
All the compounds were administered personally between 3 pm - 4 pm
Expression of results and statistics
The results were analysed statistically using Student's Y test. The value of P less than 5% (P < 0.05) was considered to be statistically significant.

Results
In the present investigation, potent antihypertensive effect was observed with the test compound of the invention. This anti-hypertensive effect was comparable to amlodipine. The test compound was statistically significant anti-hypertensive compound.

ANTI FUNGAL ACTIVITY Principle
Inhibition of microbial growth under standardized conditions may be utilized for demonstrating the therapeutic efficacy of antibiotics. The microbiological assay is based upon the comparison of inhibition of growth of microorganisms by measured concentration of antibiotics to be examined with that produced by known concentration of the antibiotic having known activity. For such screening cylinder plate (or cup-plate) method and turbidimetric (or tube assay) methods are used.
Preparation of antibiotic solution
To prepare a stock solution, 200 mg of the standard (2-mercaptobenzothiazole), was dissolved in 1 ml of Dimethylformamide (DMF), which was used as solvent. This stock was then diluted serially to get the concentrations of 5 mg/ml and 1 mg/ml. These concentrations were selected so as to determine the range at which the compound is effective against the selected organism. Once the range is determined, further dilutions within the range are tested to determine the minimum inhibitory concentration. Preparation of the test compound is same as the standard.
Determination of antifungal activity using agar cup method
0.1 ml of standardized inoculum of Asp. niger was plated on to muller hinter agar plate, using surface spread method. Cups upto 8 mm in diameter were bored in the inoculated agar with a sterile borer. In one plate 3 cups were made for application of standard solution of 2-mercaptobenzothiazole, compound of Formula XI and DMF as a control respectively, of the same concentration. After application of above mentioned solution to the plate, plates were kept in a refrigerator for prediffusion of compound, for 1 hr. Plates were removed from the refrigerator after an hour and incubated for 3 days at 30°C. Results were noted after 24 hrs, 48 hrs and 72 hrs.
Minimum Inhibitory Concentration values of compounds tested against Aspergillus niger

Results
Significant anti-fungal activity was observed with the test compound and the activity was comparable to 2-mercaptobenzothiazole.

ANTI-OSTEOPOROTIC ACTIVITY
Principle
Parathyroid Hormone (PTH) increases plasma calcium by stimulating bone resorption mediated through osteoclastic activity and reabsorption of calcium by the kidney. Hypercalcemia induced by PTH were reduced by drug like alendronate, so this model is used to test the antiosteoporotic activity of the test compound.
Procedure
PTH induced hypercalcemia
To establish experimental hypercalcemia, PTH was administered (30 (ig/kg) orally to 7 weeks old male rats. At 5th day, first dose of standard and test drug was administered. Blood was collected from fundus oculi at 1, 2, 3, 4, 6 and 9 days after the single dose of drugs. The results were analysed statistically using student's't' test.

Conclusion
Plasma calcium was significantly increased above normal by intravenous injection of bPTH. One hour after injection of bPTH, the plasma calcium level of bPTH injected animals was increased above the normal range at each time point. Standard drug alendronate and test drug reduces the increment of plasma calcium level induced by bPTH. IN-VITRO RELEASE STUDIES
a) 1.0 g of compound of Formula IX was accurately weighed and suspended in 100 ml of 0.1N
sodium hydroxide in a round bottom flask at room temperature. Temperature of reaction mass

was increased and maintained at 37°C ± 1°C. The contents were stirred mechanically at 37°C + 1°C for 5 hrs. At the end of reaction the reaction mass was extracted twice with 50 ml of methylene chloride. The two extracts of methylene chloride were mixed and methylene chloride evaporated under vacuum at room temperature.
The residue was analysed by LCMS and found to have molecular ion peak of 209.23 nm. It has a chemical structure of the following Formula XVHL

This suggests cleavage of compound of Formula IX at hydrolysable ester link and formation of a chemically modified pseudoephedrine i.e. hydroxyalkyl derivative of pseudoephedrine with an ethanol moiety.
b) Synthesis of the compound of Formula XVIII
25 ml Methanol, 7 g pseudoephedrine hydrochloride and 8.89 g of potassium carbonate were stirred mechanically for 30 min at 30°C. 7.04 g of chloroethanol was added dropwise to the reaction mass at 30°C over a period of 20 min. The reaction mass was refluxed for further 24 hours and then poured slowly in 25ml of water. The product was extracted twice with 20 ml methylene chloride, the solvent was removed under vacuum at 40°C. The solid product obtained was purified by LCMS (Liquid Chromatography Mass Spectra) as Pseudoephedrine with an ethanol moiety showing molecular ion peak at 209.23 nm.
This shows that the compound of Formula XVITI as released from the compound of Formula IX as well as that synthesized from pseudoephedrine have the same structure and properties, thereby establishing their chemical identity.
Biological activities of compound of Formula XVEH as obtained from compound of Formula IX and compound of Formula XVHI as synthetically obtained from pseudoephedrine were compared with pseudoephedrine as below.

ANTI-ASTHMATIC ACTIVITY
Principle : Exposure of spasmogen like Acetylcholine chloride or Histamine causes contraction of bronchial smooth muscle. This method permits the evaluation of bronchodilator drugs by measuring time required to produce convulsion after exposure to spasmogens.
Materials and methods :
Animals : Adult guinea pigs of either sex.
Weight of animals: 300 to 350gms.
Materials: Aerosol chamber with 2 compartments and with a central spout for introduction of atomized histamine.
Drugs:Histamine, Test and standard drug.
Experimental procedure: Protection against histamine Aerosol induced Bronchospasm. Experimental bronchial asthma was induced in guinea pigs by exposing them to 10% histamine under constant pressure in an aerosol chamber.The animals exposed to histamine aerosol showed progressive dyspnoea. The end point preconvulsive dyspnoea (PCD) was determined from the time of aerosol exposure to the onset of dyspnoea leading to the appearance of convulsion. As soon as PCD was commenced the animals were removed from the chamber and placed in fresh air. This PCD was taken as Ti Guinea pigs were administered with test and standard drugs, two hours after the dose administration the time for the onset of PCD was recorded as T2 and protection offered by the treatment was calculated by following formula
% Protection =1 x 100
T2


Conclusion
Test and standard drug significantly prolonged the latent period of convulsion as compared to control following exposure to histamine aerosol.

We Claim
1. Process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and of the Formula IV:

wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6-12 aryl, X represents a cross linking group such as


which is optional

R3 = CI, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OS02CF3, OH, NH2, NHR5 (R5=alkyl),
SH, epoxide or aziridine,
R4 = CONH2,-COOR6 (R6 = H or C,.6 alkyl) or CN
L' = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that
the carbon chain length comprises 2 to 6 carbon atoms when R3 = epoxy or aziridine, the process
comprising refluxing an acrylic monomer with a monomer of the Formula V having an ester
linkage


wherein R1 , R2 , R3 and L' are as defined above in an organic solvent in the presence of an initiator and optionally in the presence of a crosslinking agent under an inert atmosphere.
2. Process as claimed in claim 1, wherein in the formula IV R1 is CH3, R2 is H, R3 is I or OTs and R4 is COOH and in the formula V R1 is CH3, R2 is H, R3 is I or OTs..
3. Process as claimed in claim 1, wherein the acrylic monomer is acrylic acid or acrylamide.
4. Process as claimed in claim 1, wherein the monomer of the formula V is 2-hydroxyethyl methacrylate, 2-iodoethyl methacrylate, 2-(paratoluene sulphonyl)ethyl methacrylate, 2-chloroethyl methacrylate, 2-(methane sulphonyl)ethyl methacrylate or 3-[epoxy-(2-methacrylate)]-l-[n-butoxy-(4-glycidylether)]-2-propanol.
5. Process as claimed in claim 1, wherein the crosslinking agent is ethylene glycol dimethacrylate.
6. Process as claimed in claim 1, wherein the organic solvent is ethylacetate : acetone.
7. Process as claimed in claim 1, wherein the initiator is benzoyl peroxide.
8. Process as claimed in claim 1, wherein the inert atmosphere is nitrogen.
9. Process for the preparation of reactive polymers having chemoenzymatically hydrolysable functional groups and of the Formula IV:


wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6-12 aryl, X represents a cross linking group such as

R3 = CI, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OS02CF3) OH, NH2, NHR5 (R5=alkyl),
SH, epoxide or aziridine,
R4 = CONH2-COOR6 (R6 = H or C,.6 alkyl) or CN
L' = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that
the carbon chain length comprises 2 to 6 carbon atoms when R = epoxy or aziridine substantially
as herein described with reference to Examples 1 to 5.
Dated this 3rd day of November 2003

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