FORM 2
THE PATENTS ACT 1970
As amended by the Patents (Amendment) Act, 2002
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
TITLE
CHEMOENZYMATICALLY HYDROLYSABLE
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
The following specification particularly describes the nature of this invention and
the manner in which it is to be performed


FIELD OF THE EVVENTION
This invention relates to chemoenzymatically hydrolysable biologically active
compounds.
This invention also relates to process for the preparation of the chemoenzymatically
hydrolysable 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 copolytneric 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 in 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 G. 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 polymers is generally
in the range of 1000 - 10,000. Such polymeric drugs get absorbed from the gastro
intestinal tract and elicit systemic activity. The above polymeric drugs are non-
biodegradable in physiological fluid and are excreted from the body.
Polymer substituted biologically active compounds have been used as prodrugs. For
instance US Patent No. 5372807 describes an intraveneus formulation comprising an
antifibrotic agent linked to a cis-4-hydroyl-L-proline polymer. US Patent No. 5622718
2

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
tnolecules 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 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 (Makromol., Chem., by Franzmann and Ringsdorf, 177, 2547, 1976).
Deacetylcolchicine or daunomycin is known to be bound to polymers of 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, Academie Press, 1980). Activated 4-alkylthioderivatives of
cyclophósphamide bound to DIVEMA (divinyl ether and maleic anhydride) copolymer
via the anhydride groups are reported (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-
3

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. at., 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 immobilisation are used for interaction with biological systems. These
conjugates on administration, under physiological pH and influence of enzymes, are
cleaved/ hydrolysed at the point of attachment of the polymer to the biologically active
agent 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 benzimidazoles of the Formula I:
4


Wherein R6= H °r CH3 X ~ "or-CONHCH2NHCO-,R7 = H,
OCOCH2COO-,
CH3, C2H5 or CONH2, Y = OH or NH2, E - -COO- and B is benzimidazole moiety of the
Formula II:

wherein each of R1, R2, R3, R4, R5 = H, CM2 alkyl, C6-i2 (un) substituted aryl, C1.8
alkoxy, C6-12 aryloxy, Ci-s 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
benzimidazoles of the Formula I are formed by condensing an antiulcer benzimidazole
and a biocompatible partially orally biodegradable synthetic crosslinked polymer of the
Formula III:
5


wherein R6, R7, X and E each is as defïned 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 hydrolysabJe
group "E" to release N-substituted benzimidazole derivatives (the benzimidazole along
with a part of the polymer), which are acid stable.
OBJECTS OF INVENTION
An object of the invention is to provide chemoenzymatically hydrolysable biologically
active compounds capable of undergoing rapid chemoenzymatic hydrolysis to release
hydroxy alkyl derivatives thereof.
Another object of the invention is to provide chemoenzymatically hydrolysable
biologically active compounds capable of undergoing rapid chemoenzymatic hydrolysis
to release hydroxy alkyl derivatives thereof showing improved lipophilicity.
Another object of the invention is to provide chemoenzymatically hydrolysable
biologically active compounds capable of undergoing rapid chemoenzymatic hydrolysis
to release hydroxy alkyl derivatives thereof showing high polarity.
6

Another object of the invention is to provide chemoenzymatically hydrolysable
biologically active compounds capable of undergoing rapid chemoenzymatic hydrolysis
to release hydroxy alkyl derivatives thereof showing high optical purity.
Another object of the invention is to provide chemoenzymatically hydrolysable
biologically active compounds capable of undergoing rapid chemoenzymatic hydrolysis
to release hydroxy alkyl derivatives thereof showing improved bioavailability and bio-
efficacy and reduced side effects.
Another object of the invention is to provide a process for the preparation of
chemoenzymatically hydrolysable biologically active compounds capable of undergoing
rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof.
Another object of the invention is to provide a process for the preparation of
chemoenzymatically hydrolysable biologically active compounds capable of undergoing
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
chemoenzymatically hydrolysable biologically active compounds capable of undergoing
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
chemoenzymatically hydrolysable biologically active compounds capable of undergoing
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
chemoenzymatically hydrolysable biologically active compounds capable of undergoing
7

rapid chemoenzymatic hydrolysis to release hydroxy alkyl derivatives thereof showing
improved bioavailability and bioefficacy and reduced side effects.
DESCRIPTION OF INVENTION
According to the invention there is provided chemoenzymatically hydrolysable
biologically active compounds of the Formula VI:

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

8


L = spacer comprising (un)substituted alkyl, hydroxya.lkyl Or alkoxy alkyl with the
condition that the carbon chain length contains 2 - 6 Carbon atoms when

and pharmaceutically acceptable acid addition salts and ehantiomers thereof.
According to the invention there is also provided a process for the preparation of chemo
enzymatically hydrolysable biologically active compoxmcis of the Formula VI:

wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6-12aryl,
9

R4 = CONH2,-COOR6 (R6 = H or CC1-6 alkyl) or CN
D = Biologically active agent having functional groups such as

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

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

with a reactive polymer of the Formula IV
10


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

R3 = Cl, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OSO2CF3, OH, NH2, NHR5
(R =alkyl), SH, epoxide or aziridine,
R4 = CONH2,-COOR6 (R6 = H or Ci-e 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, in a polar solvent at 20-90°C and pH 2-10, cooling the reaction
mixture to ambient temperature, isolating the biologically active compound followed by
drying and if desired, converting the resulting biologically active compound into
pharmaceutically acceptable acid addition salts and enantiomers thereof by known
methods.
11

Preferably in the formula IV R1 is CH3, R2 is H and R3 is I or OT5 and R4 is
COOH and in the formula VI R1 is CH3 and R2 is Hand R4 is COOH.
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 compounds of the Formula VI may be isolated by solvent extraction and identified
by LCMS (Liquid Chromatography Mass Spectra) analysis.
The condensation may be preferably carried out at 40 - 80 C and pH 4 to 9.
The biologically active compound may be dried at 25 - 50°C.
The polar solvent may be water, alcohol such as methanol, isopropyl alcohol or
mixtures thereof preferably water or water: methanol or water : isopropyl alcohol.
12

The reactive polymers of the Formula IV have been describe in our patent Application
No. 962/MUM/2002.
The biologically active compounds i.e. the conjugates of the invention are unreported and
novel. They comprise a side chain having a hydrolysabje ester group viz., -COO-
attached to the polymeric backbone thereof through a spacer or linkage L. Because of the
spacer the compounds of the invention have reduced steric hinderance and are capable of
undergoing rapid enzymatic hydrolysis. On being chemoenzymatically hydrolysed/
cleaved at the hydrolysable group viz. -COO- group, the compounds of the invention
release hydroxyalkyl derivatives thereof i.e. chemically modified biologically active
compounds represented by the Formula VII.

wherein R2 = H, CC1-12 alkyl, C6-12 aryl, or -OH and D = Biologically active agent having
functional groups such as

L = spacer comprising (un)substituted alkyl, hydroxyallkyl or alkoxy alkyk with the
condition that the carbon chain length comprises 2 t3 6 carbon atoms when

13

Since the biologically active agents are attached to the hydrolysable group through a
spacer or linkage group viz. L, the hydroxy alkyl derivatives of the biologically active
compounds represented by the Formula VII released therefrom also comprise the spacer
or linkage which renders them highly lipophilic. Synthetic biologically active
compounds of the Formula VII have been described in our Patent Application No.
964/MUM/02. 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 - l
Step l : 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 VIII:

Step 2 : Preparation of substituted pseudoephedrine
14

10 gm of the above reactive polymer of Formula VIII was mixed with 5 gm of
pseudoephedrine hydrochloride dissolved in a sol vent system of methanol + water (1:1)
4
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 vacuüm at
50°C for 12 hours to obtain 16.0 gm of polymer-substituted pseudoephedrine of the
Formula IX.
Step l : The copolymer was prepared in the same way as in example l (Step 1), except
that 45 gm of 2-chloro ethyl acrylate was used in place of 2-(paratoluenesulphonyl) ethyl
methacrylate and l g of ethylene glycol dimethacrylate was also added. 35 g of polymer
of Formula X was obtained.
15


Step 2 : Preparation of substituted 2-mercaptobenzothiazole
5 g of the polymer of step l (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 vacuüm at 50°C for 12 hours to obtain 6.0 g of the polymer substituted 2-
mercaptobenzothiazole of the Formula XI.
16


Formula XI
EXAMPLE - 3
Step l : The copolymer was prepared in the same way as in example l (Step 1), except
that 45 gm of 3-iodopropyl Methacrylate was used in place of 2-(paratoluenesulphonyl)
ethyl methacrylate and l g of ethylene glycol dimethacrylate was also added. 37 g of
polymer of Formula XII was obtained.
17


Step 2 : Preparation of substituted fluoxetine
3.5 g of this reactive polymer of Formula XII was mixed witn 1.75 g of fluoxetine
dissolved in aqueous medium at pH 9.5. The reaction misture 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 vacuüm at 45°C for 12 hours to obtain 49 g of polymer-substituted
fluoxetine of the Formula XIII.
18


Formula XIII
EXAMPLE - 4
Step l (a) : The copolymer was prepared in the same way as Example l (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 place of 2-
(paratoluenesulphonyl) ethyl methacrylate. 46 g polymer of Formula XIV was
obtained.
19


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 ( l : 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 rrom Amlodipine and then dried under vacuüm at 45°C for
12 hours to obtain 13.8 g of polymer-substituted Amlodipine of the Formula XV.

EXAMPLE - 5
Step l : The copolymer was prepared in the same way as in Example l (Step 1), except
that 45 gm of 2-(methanesulphonyl) ethyl methacrylate (prepared as per methods
20

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 a 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 vacuüm at 50°C for 12 hours to obtain 6.8g of the polymer substituted
Alendronate of the formula XVII.

BIOLOGICAL ACTIVITY
ANTI-DEPRESSANT ACTIVITY
Principle
It is known that mice or rats forced to swim in a restricteq 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.
21

Weight of animals : 30 - 40 g
Drugs : Dose (mg/kg)
1. Compound of formula XIII : 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 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.
Resul t and discussion
Table - l : Effect of test and standard drug 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*
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 drug Compound of formula XIII showed less
immobility time (sec) when compared to control group.
22

Conclusion
Standard drug fluoxetine and test compound Compound of formula XIII 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 method s :
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 T]
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

23

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

No. Group Dose (mg /
kg) Preconvulsion time
(secs.) 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 1 16.66 ± 11.81
After treatment 256.88 ±11.58* 54.59
N=6,
* PO.005 as compared to control
* Pseudoephedrine manufactured by Avon Organics Ltd., India
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-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.
24

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 l, 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 III 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 't' test. The value of P less than
5% (P < 0.05) was considered to be statistically significant.
Table l : 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
P < 0.05 Significant'
N = 4
* Amlodipine Brin'sh Pharmacopoeial grade manufacturen by Kopran Limited, India
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.
25

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 préparé a stock solution, 200 mg of the Standard (2-mercaptobenzothiazole), was
dissolved in l ml of Dimethylformamide (DMF), which was used as solvent. This stock
was then diluted serially to get the concentrations of 5 mg/ml and l mg/ml. These
concentrations were selected so as to determine the ratige 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 minirnum 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 platecl 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 m^de for application of standard
solution of 2-mercaptobenzothiazole, compound of Fornmla 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 prediffusioft of compound, for l hr. Pïates
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.
26

Minimum Inhibitory Concentration values of compounds tested against Aspergillus
niger

Concentration Compound of 2- Mercaptöbenzo- Dimethyl
(mg/ml) formula XI (Zone thiazole (Zone formamide (DMF)
diameter) diameter) Control (Zone
diameter)
1.0 27 mm 26 mm 15 mm
5.0 40 mm 38 mm 15 mm
' 2-Mercaptobenzothiazole manufactured by Loba Chemie Ltd, India
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
medïated 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 5th day, fïrst dose of Standard and test drug was administered.
Blood was collected from fundus oculi at l, 2, 3, 4, 6 and 9 days after the single dose of
drugs. The results were analysed statistically using student's 't' test.
27

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

Treatment Plasma Calcium concentration (mg/dl)
1Day 2Day 3 Day 4Day 6Day 9 Day
Control (+PTH) 11.07 ±0.14 11.34 + 11.53 ± 10.59 ± 10.90 + 10.93 +
0.13 0.31 0.57 0.33 0.12
Alendronate* (+PTH) 10.39 ± 9.92 + 9.72 + 10.28 ± 10.56 + 10.68 +
(1.25 mg/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)
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 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 roünd 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 vacuüm 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 XVIII.
28


Formula- XVIII
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 vacuüm 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 XVIII 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 XVIII as obtained from compound of
Formula IX and compound of Formula XVIII 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.
29

Materials and methods :
Animals : Adult guinea pigs of either sex.
Weight of anima Is : 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

30

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

No. Group Dose (mg /
kg) Preconvulsion time
(secs.) Protection
(%)
1 Pseudoephedrine (std.)+ 10
Before treatment 11 1.20 ±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,
* P<0.005 as comparcd to control
+ Pseudoephedrine manufactured by Avon Organics Ltd., India
Conclusion
Test and Standard drug significantly prolonged the latent period of convulsion as
compared to control following exposure to histamine aerosol.
31

We Claim:
l Chemoenzymatically hydrolysable biologically active compounds of the
Formula VI:

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 suth as


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

and pharmaceutically acceptable acid addition salts and enantiomers thereof.
2. Biologically active compounds of the formula VI as claimed in
claim l, wherein R1 is CH3 and R2 is H and R4 is COOH.
3. Process for the preparation of chemoenzymatically hydrolysable
biologically active compounds of the Formula VI:

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
33

D = Biologically active agent having functional groups such as

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

and pharmaceutically acceptable acid addition salts and enantiomers thereof, the process
comprising, condensing a biologically active agent having ftmctional groups such as
l
—NH, NH2, SH,—OH,—Cl,—Br, 1, F, OTs,OMs,epoxy oraziridine
with a reactive polymer of the Formula IV
34


wherein R1 = H or CH3, R2 = H, C1-8 alkyl or C6_12 aryl,
X represents a cross linking group such as

R3 - Cl, Br, I, F, OTs, OMs, p-nitrobenzene sulphonate, OS02CF3, OH, NH2, NHR5
(R5=alkyl), SH, epoxy or aziridine,.
R4 = CONH2-GOOR6 (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 contains 2 - 6 carbon atoms when R3 =
epoxy or aziridine, in polar solvent at 20-90°C and pH 2-10, cooling the reaction
mixture to ambient temperature, isolating the biologically active compound followed by
drying and if desired, converting the resulting biologically active compound into
pharmaceutically acceptable acid addition salts and enantiomers thereof by known
methods.
4. Process as claimed in claim 3, wherein in the formula IV R1 is CH3, R2 is
H and R3 is I or OTS and R4 is COOH and in the formula VI R' is CH3, R2 is
H and R4 is COOH.
35

5. Process as claimed in claim 3, wherein the condensation is carried out at
40 - 80°C and pH 4 to 9.
6. Process as claimed in claim 3, wherein the polar solvent is water or water:
methanol or water : isopropyl alcohol.
7. Process for the preparation of chemoenzymatically hydrolysable biologically
active compounds of the Formula VI:

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

X represents a cross linking group such as
36