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 synthesis of hydroxyalkyl derivatives of biologically active compounds
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
Original GRANTED
964/MUM/02
5/11/02
The following specification particularly describes the nature of this invention and the manner in which it is to be performed

-9 MAR 2004
9-3-2004

FIELD OF THE INVENTION
This invention relates to hydroxyalkyl derivatives of biologically active compounds
This invention also relates to a process for the synthesis of the hydroxyalkyl derivatives of the biologically active compounds.
PRIOR ART
Biologically active agents substituted with polymers by covalent conjugation are reported to show therapeutic activity. 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 l-(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 also 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]. PCT Publication No WO 99/63940 discusses low molecular weight polymeric derivatives of benzimidazoles as antiulcer agents. The molecular weight of such polymer is generally in the range of 1000 -10000. Such polymeric drugs get absorbed from the gastro intestinal tract and elicit systemic activity.
Biologically active agents substituted with polymers are also administered as prodrugs. For instance US Patent No 5372807 describes an intravenous Formulation comprising an antifibrotic agent linked to a cis-4-hydroxyl-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 polyscchride and an unoxidised, oxidation-sensitive substance, conjugated via amine or imine bonds. US Patent No 4587046 describes biologically active drug such a catecholamine hormones coupled to carrier molecules like monodisperse peptides. US Patent Publication No. 20010031262 describes 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 @ -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 (Makromol, Chem., by Franzmann and Ringsdorf, 177, 2547, 1976). Deacetylcolchicine / daunomycin are known to be bound to N-(2-hydroxypropyl) methacrylamide (Synthese und Untersuchung von potentiell spaltbaren spacergruppen zur Polymer-fixierung von NOR-stickstoff-LOST und den Anthracyclinen Daunomycin und Adriamycin, Ph.D Thesis, Johannes Guttenberg University Mainz, FRG 1982). Daunomycin has also been attached to polymeric carriers to form amino sugar daunosamine (Shih etal., 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-alkylthioderivaties of cyclophosphamide bound to DIVEMA (divinyl ether and maleic anhydride) copolymer via the anhydride groups are reported (Hirano et al, 1980, Cancer Res., 40 : 2263). 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, erythroprotein, or polynucleotides with block copolymer of ethyleneoxy monomer or a mixture of ethyleneoxy and the -OCH(CH3)CH2-monomers through bifunctional 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 conjugate of 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 immobilization are used for interaction with biological systems. These compounds on administration , under physiological pH and influence of enzymes, are hydrolysed/cleaved at the point of attachment of the polymer to the drug to release the drug in its original chemical form.
There is described in our PCT Publication No WO 01/62248, orally administrable acid stable polymer substituted antiulcer benzimidazoles of the Formula I:

R6—CH CH—R6
I I
Y—CH CH—Y
I I
CH2 CH2
I I
B B
Formula I
wherein R6 = H or CH3, X = -OCOCH2COO-, or - CONHCH2NHCO-, R7 = H,
CH3, C2H5 or CONH2, Y = OH or NH2, E = -COO- and B is benzimidazole moiety of the Formula II:

Formula II
wherein each of R1, R2, R3, R4, R5 = H, C1-12 alkyl, C6-i2 (un)substituted aryl, Ci-g alkoxy, C6-n 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 III

Formula III
wherein R6, Y' and B each is as defined above, is released invivo. Such benzimidazole derivatives are reported to be acid stable.
BACKGROUND OF INVENTION
We have described chemoenzymatically hydrolysable biologically active compounds of the Formula VI in our Patent Application No. 963/MUM/2002.

wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6-12 aryl,
R4 = CONH2,-COOR6 (R6 = H or C1-6 alkyl) or CN
D = Biologically active agent having functional groups such as
—NH,—NH2, SH,—OH, CI, Br, 1, F, OTs, OMs, epoxy or aziridine

and Z' - —N—NH, —0—> —S— > —CH—CH2— or —CH—CH2-

1 OH NH2
X represents a cross linking group such as —C-O-CH2CH2-O-C— , —C6H4— or —CONHCH2NHCO— which is optional
o o
L = spacer comprising (un) substituted alkyl, hydroxyalkyl or alkoxy alkyl (having carbon chain length with more than one carbon atom when R3 = epoxy or aziridine) and pharmaceutically acceptable acid addition salts and enantiomers thereof.
As described in our Patent Application No. 963/MUM/2002, the biologically active compounds when chemoenzymatically hydrolysed/ cleaved at the hydrolysable ester group viz. -COO-thereof, release hydroxyalkyl derivatives thereof i.e. chemically modified biologically active compounds represented by the Formula VII.
R2 I
HO—L—Z'—D
Formula - VII
wherein R2 = H, C1-12 alkyl, C6-12 aryl, or -OH and D = Biologically active agent having functional groups such as
I
—NH,—NH2, —SH,—OH, —CI,—Br, 1,—F, OTs, OMs, epoxy or aziridine

L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl (having carbon chain length with more than one carbon atom when R3 = epoxy or aziridine)
OBJECTS OF INVENTION
An object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula VII which have improved lipophilicity.

Another object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula VII, which are polar.
Another object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula VII, which have improved bioavailability and bioefficacy and reduced side effects.
Another object of the invention is to provide a process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the formula VII, which have improved lipophiliciry.
Another object of the invention is to provide a process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the formula VII, which are polar.
Another object of the invention is to provide a process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the formula VII, which have improved bioavailability and bioefficacy and reduced side effects.
DESCRIPTION OF INVENTION
According to the invention there is provided hydroxyalkyl derivatives of biologically active compounds represented by the formula VII
R2
I HO— L— Z'— D
Formula - VII
wherein R2 = H, C1-12 alkyl, C6-12 aryl, or -OH and D = Biologically active agent having functional groups such as
—NH,—NH2, SH,—OH, CI, Br, 1, F, OTs, OMs, epoxy or aaridine
and Z' = —N>—NH>—o—, —S—, —CH—CH.,— or—CH—CH2—
I OH NH2

L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl (having carbon chain length with more than one carbon atom when R3 = epoxy or aziridine) and pharmaceutically acceptable acid addition salts and enantiomers thereof.
According to the invention there is also provided a process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the Formula VII
R2
I HO—L—Z'—D
Formula VII
wherein R2 = H, C1-12 alkyl, C6-12 aryl, or -OH and D = Biologically active agent having functional groups such as
I
—NH,—NH2, SH,—OH, CI, Br, 1, F, OTs, OMs, epoxy or aziridine
' S ' CH—CH2— or CH—CH2—
I I
OH NH2
L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl (having carbon chain length with more than one carbon atom when R3 = epoxy or aziridine) and pharmaceutically acceptable acid addition salts and enantiomers thereof, the process comprising ;
a) condensing biologically active agents having functional groups such as
I
—NH,—NH2, SH,—OH, —CI, Br, 1, F, OTs, OMs, epoxy or aziridine
with substituted alkyl derivatives under alkaline conditions, at 20 - 80°C, in an organic solvent,
b) diluting the reaction mass with aqueous medium,
c) extracting the biologically active compounds from the reaction mixture with an organic solvent; and
d) evaporating the organic solvent.
The biologically active agents may be antibacterial such as, Ciprofloxacin; antiamoebic such as secnidazole; antifungal such as fluconazole or 2-mercaptobenzothiazole; antithelmintic such as albendazole; antitubercular such as ethambutol; anti-inflammatory such as mefenamic acid; anti-

ulcer such as omeprazole; antiosteoporotic such as alendronate; respiratory drugs such as albuterol, astemizole, ephedrine, Montelukast, pseudoephedrine, terbutaline, fenoterol, salmeterol; antidiabetic such as metformin, Pioglitazone, rosiglitazone, troglitazone, glipizide, glimepiride, tolbutamide, gliclazide; anticoagulant such as warfarin, antimigraine such as sumatriptane, CNS drugs such as amphetamine, paroxetine, fluoxetine, sertraline, Zolpidem, citalopram, risperidone, talyetant, vilazodone, lamictal, seroxat; diuretic such as Furosemide; anabolic steroids such as Trenbolone; cardiovascular such as atorvastatin, rosuvastatin, losartan, valsatran, amlodipine, atenolol, captopril, lisinopril, carvedilol, crestor, exnta, accupril; anorexic such as Fenfluramine; peristaltic stimulative agent such as Cisapride; anticancer drugs such as cycloserine, tamoxifen, gemcitabine, capecitabine, chlorambucil, methotrexate, fluorouracil, faslodex, iressa, repifermin, ethynylcytidine, epothizone; vaccines such as typhoid vaccine, polio vaccine; peptides such as Insulin; anti - HIV such as acyclovir, valacyclovir, lamivudine, stavudine, zidovudine, efavirenz, nevirapine, ziagen, EPIVIR, atazanavir or reversible proton pump inhibitors.
The substituted alkyl derivatives may be chloroethanol, iodoethanol, iodopropanol or 2-mercaptoethanol, preferably chloroethanol or iodoethanol or iodopropanol.
The alkali used may be sodium hydride, triethylamine or ammonia solution or potassium carbonate, preferably potassium carbonate or triethylamine.
The condensation is preferably carried out at 30 - 50°C.
The organic solvent may be tetrahydrofuran, dichloro methane, methanol or methylene chloride, preferably methanol methylene chloride or tetrahydrofuran.
Evaporation of the solvent may be carried out under vacuum.
The hydroxyalkyl derivatives of the biologically active compounds of the invention are novel. They are highly lipophilic due to the presence of the spacer or linkage group viz. L. Because of the high lipophilicity they show improved absorption and hence high bioefficacy. Because of the high absorption and bio-efficacy, the compounds of the invention are effective at low doses and have correspondingly reduced side effects. The compounds of the invention are also polar due to the presence of hydroxyl group(s). Because of the high polarity, they show better ionization and absorption.

The following experimental examples are illustrative of the invention but not limitative of the scope thereof.
EXAMPLE -1
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. It has the structure of the following Formula XVIII.

Formula - XVIII
The chemical identity of the compound of Formula XVIII synthesized as above was the same as that of compound of Formula XVIII as released from compound of the following Formula IX as described in our Patent Application No. 963/MUM/2002.
CH3
( CH2-CH— CH2-C— CH2— )n
COOH CO—O—CH2

Formula IX
Biological activities of compound of Formula XVIII synthetically obtained and compound of Formula XVIII as obtained from compound of Formula IX 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 drugs.
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 T1 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
T1
% Protection =1 x 100
T2
Results:
Table 1: Effect on histamine aerosol - induced bronchospasm in guinea pigs.

No. Group Dose (mg /
kg) Preconvulsion time
(sees.) Protection (%)
1 Pseudoephedrine (std.)+ 10
Before treatment 111.20 ±10.28
After treatment 296.36 ±11.56* 63.0
2 Compound of formula IX 65
Before treatment 116.66± 11.81

After treatment 256.88 ±11.58* 54.59
3 Synthetic compound of formula XVIII 10
Before treatment 119.58 ± 11.21
After treatment 287.67 + 11.58* 58.43
N=6,
* PO.005 as compared to control
+ Pseudoephedrine manufactured by Avon organics Ltd India
Conclusion
Test and standard drugs significantly prolonged the latent period of convulsion as compared to control following exposure to histamine aerosol.
EXAMPLE - 2
25 ml Methanol, 5 g 2-mercaptobenzothiazole and 7.67 g of potassium carbonate were stirred mechanically for 15 min at 30°C. 5.66 g of 2-iodoethanol was added dropwise to the reaction mass at 30°C over a period of 15 min. The reaction mass was refluxed for further 20 hours and then poured slowly in 25ml of water. The product was extracted twice with 25 ml methylene chloride, the solvent was removed under vacuum at 40°C. The solid product obtained was purified by column chromatography and identified by LCMS (Liquid Chromatography Mass Spectra) as 2-mercaptobenzothiazole with an ethanol moiety showing molecular ion peak at 211.25 nm. It has the structure of the following Formula XIX.

S SCH2CH2OH Formula - XIX
The chemical identity of the compound of Formula XIX synthesized as above was the same as that of compound of Formula XIX as released from compound of the following Formula XI as described in our Patent Application No 963/MUM/2002.


n

Formula XT
Biological activities of compound of Formula XIX synthetically obtained and compound of Formula XIX as obtained from compound of Formula XI were compared with 2-mercaptobenzothiazole as below.
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 compounds 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 4 cups were made for application of standard solution of 2-mercaptobenzothiazole, compound of Formula XI, synthetic compound of formula XIX 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

Concentration (mg/ml) Compound of
formula XI
(Zone diameter) Synthetic compound of formula XIX (Zone diameter) 2- Mercaptobenzo-
thiazole (Zone
diameter) Dimethyl formamide (DMF) Control (Zone diameter)
1.0 27 mm 25 mm 26 mm 15 mm
5.0 40 mm 41 mm 38 mm 15 mm
+ 2-Mercaptobe nzothiazole manu: actured by Loba Chemie Ltd, India
Results
Significant anti-fungal activity was observed with the test compounds and the activity was comparable to 2-mercaptobenzothiazole.
EXAMPLE - 3
25 ml Methanol, 5 g fluoxetine hydrochloride and 3.7 g of potassium carbonate were stirred mechanically for 15 min at 30°C. 2.73 g of 3-iodopropanol was added dropwise to the reaction mass at 30°C over a period of 15 min. The reaction mass was refluxed for further 20 hours and then poured slowly in 25ml of water. The product was extracted twice with 25 ml methylene chloride, the solvent was removed under vacuum at 40°C. The solid product obtained was purified by column chromatography and identified by LCMS (Liquid Chromatography Mass Spectra) as fluoxetine hydrochloride with an ethanol moiety showing molecular ion peak at 367.33 nm. It has the structure of the following Formula XX.

HO—CH2—CH2—CH2

CH3
Formula - XX
The chemical identity of the compound of Formula XX synthesized as above was the same as that of compound of Formula XX as released from compound of the following Formula XIII as described in our Patent Application No. 963/MUM/2002.


n

Formula XIII
Biological activities of compound of Formula XX synthetically obtained and compound of Formula XX as obtained from compound of Formula XIII were compared with fluoxetine hydrochloride as below.

ANTI-DEPRESSANT ACTIVITY
Principle
It is known 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 XIII: 42mg/kg
2. Fluoxetine (Standard) : 20mg/kg
3. Synthetic compound of formula XX : 20 mg/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 hr 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
Table -1 : Effect of test and standard drugs on swimming model

Group Dose (mg/kg) Immobility Time (Sec)
Vehicle control - 179.83 ± 22.44
Fluoxetine+ 20 26.17 ±5.93*
Compound of formula XIII 42 57.67 ±10.03*
Synthetic compound of formula XX 20 33.33 + 9.15

N=6
*P<0.05 significant as compared to control
Results are in Mean ± SEM
+ Fluoxetine manufactured by Zydus Cadila, India
Standard drug fluoxetine and test drugs showed less immobility time (sec) when compared to control group.
Conclusion
Standard drug fluoxetine and test compounds showed significant antidepressant activity when compared to control group.
EXAMPLE - 4
25 ml Methylene chloride, 5 g Amlodipine base and 5 ml of triethylamine were stirred mechanically for 15 min at 30°C. 10 g of 1, 4-butanediol diglycidylether was added dropwise to the reaction mass at 30°C over a period of 15 min. The reaction mass was refluxed for further 20 hours and then poured slowly in 25 ml of water. The product was extracted twice with 25 ml methylene chloride, the solvent was removed under vacuum at 40°C. The solid product obtained was purified by column chromatography and identified by LCMS (Liquid Chromatography Mass Spectra) as Amlodipine base with an ethanol moiety showing molecular ion peak at 672.88 nm. It has the structure of the following Formula XXI.
HO—(CH2)2—0--CH2—CH—CH2
OH O—(CH2)4—O—CH2
CH— OH

Formula - XXI
The chemical identity of the compound of Formula XXI synthesized as above was the same as that of compound of Formula XXI as released from compound of the following Formula XV as described in our Patent Application No. 963/MUM/2002.

CH3

( CH2—CH—CH2-C—CH2—) n
COOH CO—O—(CH2)2—O—CH2—CH—CH2
OH 0—(CH2)4—O—CH2

CH—OH

Formula XV
Biological activities of compound of Formula XVIII synthetically obtained and compound of Formula XVIII as obtained from compound of Formula IX were compared with Amlodipine base as below.
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-II 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 anaesthetic 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 forcep. 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 4 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 III were the hypertensive controls
Group IV received 0.9 mg/kg of synthetic compound of formula XXI
All the compounds were administered personally between 3 pm - 4 pm
Expression of results and statistics
The results were analysed statistically using Student's 'f test. The value of P less than 5% (P < 0.05) was considered to be statistically significant.
Table-1 : Effect of compound of Formula XV on hypertensive rats at different time intervals

Compounds Mean blood pressure (mmHg) at hours

1 2 3 4
Compound of Formula XV 77.0 ±0.86* 75.0 ±5.31* 67.5 ±0.91* 63.0 ±2.68*
Amlodipine BP+ 79.1 ±2.15* 78.67 ±5.1* 69.65 ±1.13* 66.66 ±2.71*
Control 122.0 ±3.63 122.25 ±3.97 119.0 ±4.73 117.25 ±4.92
Synthetic compound of formula XXI 78.0 + 2.35 77.2 ±1.26 74.0 + 0.85 69.60 ±1.11
P < 0.05 Signaficant
N = 4
+ Amlodipine British Pharmacopoeial grade manufactured by Kopran Limited, India
Results
In the present investigation, potent antihypertensive effect was observed with the test compounds of the invention. This anti-hypertensive effect was comparable to amlodipine. The test compounds were statistically significant anti-hypertensive compounds.
EXAMPLE - 5
25 ml Tetrahydrofuran, 5 g Alendronate and 2.4 g of potassium carbonate were stirred mechanically for 15 min at 30°C. 5.3 g of lodoethanol was added dropwise to the reaction mass at 30°C over a period of 15 min. The reaction mass was refluxed for further 20 hours and then poured slowly in 25ml of water. The product was extracted twice with 25 ml methylene chloride, the solvent was removed under vacuum at 40°C. The solid product obtained was purified by column chromatography and identified by LCMS (Liquid Chromatography Mass Spectra) as Alendronate with an ethanol moiety showing molecular ion peak at 325.12 run. It has the structure of the following Formula XXII.


Formula - XXII
The chemical identity of the compound of Formula XXII synthesized as above was the same as that of compound of Formula XXII as released from compound of the following Formula XVII as described in our Patent Application No. 963/MUM/2002.
6H

Formula XVII
Biological activities of compound of Formula XXII synthetically obtained and compound of Formula XXII as obtained from compound of Formula XVII were compared with Alendronate as below.
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 |J.g/kg) orally to 7 weeks old male rats. At 5th day, first dose of standard and test drugs 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.

Result
Table 1 : Effect of alendronate and test compounds on plasma calcium concentration in rats
treated with bPTH

Treatment Plasma Calcium concentration (mg/dl)

1Day 2 Day 3 Day 4 Day 6 Day 9 Day
Control (+PTH) 11.07± 11.34 + 11.53 + 10.59 ± 10.90 ± 10.93 ±
0.14 0.13 0.31 0.57 0.33 0.12
Alendronate (+PTH)+ 10.39 ± 9.92 + 9.72 ± 10.28 ± 10.56 ± 10.68 +
(1.25mg/kg) 0.26* 0.46* 0.24* 0.23 0.14 0.15*
Compound of formula 10.67 ± 10.20 ± 10.14 + 10.76 ± 10.60 ± 10.59 +
XVII (+PTH) (5.2 0.08* 0.34* 0.19* 0.46 0.10 0.30
mg/kg)
Synthetic compound 10.43 ± 10.39 ± .10.20 ± 10.35 ± 10.36 ± 10.53 ±
of formula XXII (1.25 0.28* 0.43* 0.19* 0.20 0.25 0.27*
mg/kg)
* P < 0.05 Significant
N = 5
+ Alendronate manufactured by Nivedita Chemicals Ltd, India
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 the test drugs reduce the increment of plasma calcium level induced by bPTH.