EP1744785A1

EP1744785A1 - Hydroxyalkyl derivatives of biologically active compounds

Info

Publication number: EP1744785A1
Application number: EP04821934A
Authority: EP
Inventors: Jayant Venkatesh Kopran Res. Lab. Ltd DESHPANDE; Vaishali Madhukar Kopran Res. Lab. Ltd KADAM; Vandana Sandeep Kopran Res. Lab. Ltd GUPTE; Kamlesh Jayantilal Kopran Res. Lab. Ltd RANBHAN
Original assignee: Kopran Research Laboratories Ltd
Current assignee: Kopran Research Laboratories Ltd
Priority date: 2004-04-30
Filing date: 2004-04-30
Publication date: 2007-01-24
Also published as: WO2005105153A1

Abstract

Hydroxyalkyl derivatives of biologically active compounds represented by the formula VII wherein R2 = H, CI-12 alkyl, C6-12 aryl, or -OH and D = Biologically active agent having functional groups such as Formula (a) epoxy or aziridine and Z' = Formula (b) L= spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that the carbon chain length contains 2 to 6 carbon atoms when Z' = Formula (c) and pharmaceutically acceptable acid addition salts and enantiomers thereof Also process for the synthesis of compounds of the Formula VII and pharmaceutically acceptable acid addition salts and enantiomers thereof comprising condensation of the biologically active agents having functional groups such as Formula (d) epoxy or azifidine with substituted alkyl derivatives under alkaline conditions, at 20 - 80°C, in an organic solvent.

Description

TITLE OF INVENTION

Hydroxyalkyl derivatives of biologically active compounds

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 polyscchri.de 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 α-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 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-alkylthioderivaties of cyclophosphamide bound to DINEMA (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-(ε-Caprolactone), poly-[ε-(+, -)-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, mitornycins, bleomycins, fluconazole, amphotericm 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-methacryiic 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 Indian Patent Application No /PCT/2002/01098/MUM (PCT Publication No WO 01/62248), orally administrable acid stable polymer substituted antiulcer benzimidazoles of the Formula I:

Formula I

wherein R⁶ = H or CH₃, X = -OCOCH₂COO-, — >~ or - CONHCH-NHCO-, 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, C].] alkyl, C₆-ι₂ (un)substituted aryl, Ci-g alkoxy, C₆_ι aryloxy, Cι-₅ alkoxy carbonyl, C₆-ι aryloxy carbonyl, Cι_₅ alkoxy alkyl, C₆_ι₂ alkoxyaryl, C₁.₅ haloalkyl, C₁.₅ alkyl or C₆.ι₂ aryl thioethers, (un)substituted amines or diamines, (un)substituted amides, halo, cyano, nitro, carboxylic acid or carbocyclic or O, N, S containing heterocychc 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

Formula III

wherein R , 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.

Formula VI

wherein R¹ = H or CH₃, R² = H, Cι.₈ alkyl or C₆-ι₂ 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, — d, Br, 1, F, OTs, OMs, epoxy or aziridine

and Z' = N, — NH, — ( -> S > CH— CH₂— or CH— CH₂- I I OH NH₂

X represents a cross linking group such as which is optional — C— O— CH₂CH₂-0— C — j — c₆H₄ — or — CONHCH₂NHCO — O o

L = spacer comprising (un) substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that the carbon chain length contains 2 to 6 carbon atoms when

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 NIL

R2 I HO— L— Z'— D

Formula - VII wherein R² = H, Cι_₁₂ alkyl, C₆.ι₂ aryl, or -OH and D = Biologically active agent having functional groups such as

— Ν IH, — ΝH₂, SH, — OH, — O, — Br, 1, — F, OTs, OMs, epoxy or aziridine

and Z' = __^■ N, — NH, — ( -CH— CH₂— or- -CH— CH₂- I I OH NH₂

L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that the carbon chain length contains 2 to 6 carbon atoms when

OBJECTS OF INVENTION

An object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula Nπ which have improved lipophilicity.

Another object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula Nπ, which are polar.

Another object of the invention is to provide hydroxyalkyl derivatives of biologically active compounds represented by the formula VLI, which have improved bioavailabihty 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 Nil, which have improved lipophilicity.

Another object of the invention is to provide a process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the formula NH, 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 Nπ, which have improved bioavailabihty 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 NH R2 I HO— L— Z'— D

Formula - VII wherein R² = H, Cι_ι₂ alkyl, C₆.ι₂ aryl, or -OH and D = Biologically active agent having functional groups such as NH, NH₂, SH, — OH, — O, Br, 1, F, OTs, OMs, epoxy or aziridine

^{md Z}' ~ — N, — NH, — O ' — S > — CH— CH₂— or — CH— CH₂— ' OH NH₂

Z' = — CH— CH₂— or - -CH— CHr OH Nft

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 NH R2 I HO— L— Z'— D

Formula VII

wherein R² = H, Cι-ι₂ alkyl, C₆.₁₂ aryl, or -OH and D = Biologically active agent having functional groups such as ΝH, ΝH₂, SH, — OH, CI, Br, 1, F, OTs, OMs, epoxy or aziridine

^{and Z -}

L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that the carbon chain length contains 2 to 6 carbon atoms when Z' = — CH— Cft— or - -CH— CH2- OH NH

and pharmaceutically acceptable acid addition salts and enantiomers thereof, the process comprising ; a) condensing biologically active agents having functional groups such as

— N IH, — NH₂, SH, — OH, — O, — 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, ethynylcytidme, epothizone; vaccines such as typhoid vaccine, polio vaccine; peptides such as Insulin; anti - HIV such as acyclovir, valacyclovir, lamivudine, stavudine, zidovudine, efavirenz, nevirapine, ziagen, EPINIR, 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 XNIH.

Formula - XVIII

The chemical identity of the compound of Formula XNITJ synthesized as above was the same as that of compound of Formula XNDI as released from compound of the following Formula LX as described in our Patent Application No. 963/ UM/2002.

Formula IX

Biological activities of compound of Formula XNIH synthetically obtained and compound of Formula XNIJJ as obtained from compound of Formula LX were compared with pseudoephedrine as below.

AΝTI-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 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 Ti % Protection = 1 x 100 T₂

Results: Table 1: Effect on histamine aerosol - induced bronchospasm in guinea pigs.

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.

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.

Formula XI

Biological activities of compound of Formula XIX synthetically obtained and compound of Formula XLX 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 muUer 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 XLX 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 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.

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 XIH as described in our Patent Application No. 963/MUM/2002.

Formula XIII Biological activities of compound of Formula XX synthetically obtained and compound of Formula XX as obtained from compound of Formula XHI 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 XIH : 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

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 diglycidyl ether 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. 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 XN as described in our Patent Application No. 963/MUM/2002.

Formula XV

Biological activities of compound of Formula XNiπ synthetically obtained and compound of Formula XNHI 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-H 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 HI were the hypertensive controls

Group IN 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 't' 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

P < 0.₀₅ Significant* N = 4 ⁺ Amlodipine British Pharmacopoeial grade manufactured by Kopran Limited, India

Results h 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 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 Alendronate with an ethanol moiety showing molecular ion peak at 325.12 nm. It has the structure of the following Formula XXLI.

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

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 μg/kg) orally to 7 weeks old male rats. At 5 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

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.

Claims

We claim:

1. Hydroxyalkyl derivatives of biologically active compounds represented by the formula Nπ R2 I HO— L— Z'— D

Formula - VII wherein R² = H, Ci-₁₂ alkyl, C₆.₁₂ aryl, or -OH and D = Biologically active agent having functional groups such as

— ΝH, — ΝH₂, SH, — OH, — a, — Br, 1, F, OTs, OMs, epoxy or aziridine

and Z' -

L = spacer comprising (un)substituted alkyl, hydroxyalkyl or alkoxy alkyl with the condition that the carbon length contains 2 to 6 carbon atoms when

— CH— Cft— or — CH— ca- OH ML

and pharmaceutically acceptable acid addition salts and enantiomers thereof.

2. Hydroxyalkyl derivatives of biologically active compounds represented by the formula NH as claimed in claim 1, wherein R is H.

3. Process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the Formula Nil : R.2 I ΉO— —Z—Ό

Formula VII

wherein R² = H, Cι-ι₂ alkyl, C₆.ι₂ aryl, or -OH and D = Biologically active agent having functional groups such as

— NH, — NH₂, SH, — OH, — O, — Br, 1, F, OTs, OMs, epoxy or aziridine

and Z' = N, — NH, — O — » — S — » — CH— CH .₂,— — o uri — C ^Hπ—— CH,- i I OH NH₂

and pharmaceutically acceptable acid addition salts and enantiomers thereof, the process comprising ;

a) condensing biologically active agents having functional groups such as

— 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.

4. Process as claimed in claim 3, wherein in the compound of the formula Nπ R² is H.

5. Process as claimed in claim 3, wherein the substituted alkyl derivative is chloroethanol, iodoethanol or iodopropanol.

6. Process as claimed in claim 3, wherein the alkali is potassium carbonate or triethylamine.

7. Process as claimed in claim 3, wherein the condensation is carried out at 30 - 50°C.

8. Process as claimed in claim 3, wherein the organic solvent is methylene chloride, methanol or tetrahydrofuran.

9. Process for the synthesis of hydroxyalkyl derivatives of biologically active compounds represented by the Formula Nπ :

R2 I HO— L— Z— D

Formula VII

wherein R = H, C_M2 alkyl, C₆_ι₂ aryl, or -OH and D = Biologically active agent having functional groups such as

— Ν IH, — ΝH₂, SH, — OH, — CI, Br, 1, F, OTs, OMs, epoxy or aziridine

and Z' = __ ^■■N, — NH, — 0 — > — S . — CH— CH₂— or — CH— CH₂— I OH NH,