P1519PC00

TAMSULOSIN DERIVATIVES

BACKGROUND OF THE INVENTION

The present invention relates to certain derivatives of the known pharmaceutically active compound tamsulosin, to methods of making such derivatives and to

pharmaceutical compositions comprising them. The tamsulosin derivatives of the present invention may be used in medicine, particularly as prodrugs for the delivery of tamsulosin to patients in need of tamsulosin therapy.

Tamsulosin is the generic name for (R)-5-[ 2-[[2-(2-ethoxyphenoxy)ethyl]-amino] propyl]-2-methoxy-benzenesulfonamide of the formula (1).

It has been disclosed in EP 34432 and US 4731478 as a pharmaceutically active substance having alpha-adrenergic blocking activity and being useful for the treatment of cardiac insufficiencies and benign prostatic hyperplasia.

Tamsulosin medicaments are marketed under various tradenames, including FLOMAX ^® (Boehringer Ingelheim) in the US, HARNAL ^® (Yamanouchi) in Japan and OMNIC ^® (Astellas) in Europe, for the treatment of symptoms of benign prostatic hyperplasia (also known as BPH) such as urinary volume and frequency problems. The approved drug products include a capsule dosage form for oral administration that comprises 0.4 mg of the tamsulosin hydrochloride within a plurality of coated pellets. The capsule provides controlled release of the tamsulosin from the pellets and is a once daily dosage form. US 4772475 is listed in the US Food and Drug Administration's Approved Drug Products with Therapeutic Equivalence Evaluations (the "Orange Book") as corresponding to FLOMAX ^® .

US 4772475 (EP 194838, EP 533297) discloses controlled-release pharmaceutical dosage forms comprising multiple granulate units containing tamsulosin, microcrystalline cellulose and a release control agent. The granulate gradually releases tamsulosin from the granulate matrix.

WO 2004/043449 of Synthon discloses a pharmaceutical pellet composition comprising tamsulosin as an active ingredient and having an advantageous coating layer with respect to obtaining an extended release profile. Each pellet comprises a pellet core, which has a diameter within the range of 0.1-1.5 mm, comprising a tamsulosin salt, an inert pellet-forming carrier, a release control agent, and optionally water. Each pellet core is surrounded by an outer layer coat, which comprises a pharmaceutically acceptable acid-resistant polymer, in an amount, calculated on a dry pellet core basis, that is within the range of 1 to 25 weight %.

In medical treatment, it is often considered as advantageous to administer tamsulosin together with another active substance. Many such suggestions were disclosed in the prior art. In an example, WO 03/090753 suggests the possibility of a combination medicament of tamsulosin and finasteride or dutasteride. WO 2006/055659 suggests a fixed dose composition (FDC) of dutasteride and tamsulosin, wherein dutasteride is formulated in a soft gel and tamsulosin is formulated in the form of beads, said beads comprising a multilayer composition with tamsulosin incorporated in one of these layers.

According to the present medical experience, the desired therapeutic effect of tamsulosin is obtained when tamsulosin is administered in a polymeric matrix that modifies the release rate of tamsulosin in body fluids according to the therapeutic demands, whereby the release rate in the stomach is limited. Thus, the formulation of tamsulosin in the form of coated monolithic pellets as suggested by WO 2004/043449 of Synthon, whereby the coating material prevents the release in the stomach and the matrix material modifies the release in the intestines, is advantageous from a therapeutic point of view.

While various possibilities of how to formulate tamsulosin into pharmaceutical compositions with the desired release rate exist, there still exists a need for an improvement. In particular, a technical solution leading to further simplification of the dosage form while maintaining the desired release rate of tamsulosin would be advantageous. BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of certain derivatives of tamsulosin having advantageous stability characteristics in the environment of the human body. In particular, the compounds are stable at the pH of stomach fluid, while being unstable at the pH of intestinal tract fluid, whereby they selectively decompose to tamsulosin. Thereby, the compounds of the present invention may primarily serve as prodrugs of tamsulosin, whereby the desired sustained release of tamsulosin from a pharmaceutical composition may be obtained without the need of special excipients.

In a first aspect, the invention relates to a compound of general formula (2)

wherein R is hydrogen or a Ci-Cio alkyl/aryl/aralkyl group, and Ri is a Ci-Cio alkyl/aryl/aralkyl group. In particular, R is hydrogen or a methyl group and Ri is a 2-propyl group. The general formula (2) also embraces single enantiomers and/or diastereomers as well as mixtures thereof. Furthermore the compounds of general formula (2) may form various hydrates and solvates and may react with acids and bases under formation of salts. The compounds of general formula (2) are a a specific class of compounds of general formula (2-1)

wherein Z is -0-, -S- or -NH- linkage and R, Ri have the above meaning.

In a second aspect, the invention relates to a process for making compounds of general formula (2) as defined above comprising reacting tamsulosin of formula (1)

with a haloalkylhaloformate of general formula (3)

in which R is hydrogen or a C ₁ -C ₁₀ alkyl/aryl/aralkyl group , X and Y

independently represent a halogen atom,

ielding an intermediate of general formula (4),

which further reacts with a carboxylic acid of general formula (5)

R _r COOH or an activated derivative thereof, wherein Ri is a C ₁ -C ₁₀ alkyl/aryl/aralkyl group, yielding the compound of formula (2).

In an alternate aspect, the compound of formula (2) may be produced by reacting tamsulosin of formula (1) with a compound of formula (6)

wherein R is hydrogen or a C ₁ -C ₁₀ alkyl/aryl/aralkyl group, Ri is a C ₁ -C ₁₀ alkyl/aryl/aralkyl group and L is a leaving group suitable for reaction with an amine, instance an aromatic leaving group, preferably a p-nitrophenyloxy group. In a third aspect, the invention relates to pharmaceutical compositions comprising the compound of formula (2) and at least one pharmaceutically acceptable excipient. In a particular aspect, the pharmaceutical compositions are designed for oral administration.

The invention also relates to the compounds of formula (2) and compositions comprising them for use in therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new compounds of general formula (2),

wherein R is hydrogen or a Ci-Cio alkyl/aryl/aralkyl group, and Ri is a Ci-Cio alkyl/aryl/aralkyl group.

The "alkyl", as used herein, refers to a saturated or unsaturated, straight-chain, branched or cyclic monovalent hydrocarbon radical. The "aryl" refers to a monovalent hydrocarbon radical derived from any aromatic ring system; more particularly, it includes a phenyl radical, or phenyl substituted by one or more alkyl groups. The "aralkyl" refers to an acyclic alkyl group in which one hydrogen atom, typically on a terminal carbon atom, is replaced by an aryl group.

Preferably, the R is a Ci-Cio alkyl group and most preferably R is a methyl group. Preferably, the Ri is a Ci-Cio alkyl group and most preferably Ri is a 2-propyl group.

Those skilled in the art will appreciate that the compounds of general formula (2) are derivatives of the known pharmaceutically active compound tamsulosin of formula

(1),

(1) to which a certain side chain has been attached. As tamsulosin is a chiral compound characterised by having (R)-orientation of substituents on the chiral carbon in the position 2 of the propyl-group attached to the methoxybenzenesulfonamide moiety, compounds of the present invention have the same orientation of substituents in the corresponding position, unless specifically disclosed to the contrary.

In addition to one chiral carbon in the tamsulosin core, the compounds of the present invention may comprise also (a) further chiral carbon(s) in the attached side chain. Thus, the compounds of formula (2) may exist as single enantiomers or diastereomers and/or as mixtures of the same including racemic compounds. Any such possibility is covered by the formula (2).

The formula (2) also embraces various hydrates and/or solvates, whenever formed and/or obtained by making any of the compounds of general formula (2) in an isolated form. The compounds of general formula (2) may be isolated in crystalline state, in amorphous state or as oils.

Salts of the compounds of general formula (2) with acids and bases, whenever formed, are also within the scope of the present invention and are covered by the formula (2)·

The compounds of general formula (2) may be prepared by various procedures. Any of these procedures are within the scope of the present invention. In a preferred aspect, the processes leading to compounds of formula (2), and, per analogiam, to compounds of general formula (2-1), use tamsulosin and/or a salt thereof as the starting material. In other words, the preferred synthetic route leading to the compounds of the invention comprises at least one synthetic step resulting in attaching the desired

(alkyl)oxycarbonyl-side chain to the tamsulosin skeleton.

Tamsulosin is a well known compound and is commercially available or can be prepared by well-known synthetic methods.

In particular, the compounds of the present invention may be prepared according to a first process, which is illustrated on the scheme 1 below.

(2)

Scheme 1

In the first step, tamsulosin (or a salt thereof) reacts with a compound of the formula (3),

in which, typically, X and Y independently represent a halogen atom and R is hydrogen or a Ci-Cio alkyl/aryl/aralkyl group. Most preferably, both X and Y are chlorines. The halogen atoms, particularly in the position X, may be replaced by other suitable groups, e.g. a hydroxy-group, a sulfonyloxy-group (e.g. a methanesulfonyloxy group) or an aryloxy-group (e.g. a p-nitrophenyloxy group). In some embodiments, such replacement of halogen in the position X is performed after the coupling of tamsulosin with compound (3), i.e. on the intermediate of general formula (4).

The reaction between tamsulosin of formula (1) and the compound of formula (3) typically proceeds in an inert solvent, preferably in the presence of a base. The inert solvent may be, without limitation, a C5-C1 ₀ hydrocarbon, for instance n-hexane, cyclohexane, benzene, toluene etc., a halogenated hydrocarbon, for instance

dichloromethane or chloroform. The base may be an inorganic or organic base. The inorganic base may advantageously comprise an alkali metal or an alkali earth metal hydroxide or carbonate. The organic base is typically an amine, which may be a primary, secondary or tertiary amine. The course of the reaction may be monitored by a suitable analytical technique, e.g. by HPLC or TLC. The reaction temperature is typically ambient or close to ambient (e.g., 0-50°C, preferably 10 to 45°C). After the reaction, the intermediate of formula (4) may be isolated from the reaction mixture by conventional means, and purified, if necessary. In addition, the compound of formula (4) may be resolved into enantiomers, if this is desirable or advantageous. In an alternate procedure, the intermediate of formula (4) is not isolated from the reaction mixture and is subjected to the next reaction step within that mixture, optionally with some necessary pre-treatment, e.g. filtration or pH-adjustment.

In the second step, the compound of formula (4) in an inert, preferably anhydrous, organic solvent reacts with a carboxylic acid Ri-COOH, wherein Ri is typically a Ci-Cio alkyl/aryl/aralkyl group and most preferably Ri is a 2-propyl group, optionally in the presence of a base, which may be an organic or inorganic base as discussed above. Alternately, an activated derivative of the carboxylic acid, such as an acylchloride, anhydride or ester may be used. After elaboration of the reaction mixture, which typically comprises an extraction with water, the product is isolated from the reaction mixture and purified, if necessary, for instance by chromatography or by crystallization.

Based on the conformation of the starting compound (4), the product may be isolated as a racemate or as a single enantiomer. If a racemate is produced, it may be resolved into enantiomers by conventional means.

In a second process leading to the compounds of the present invention, tamsulosin reacts, in an inert, typically organic, solvent, with a compound of general formula (6)

in which R and Ri are as defined above and L is a leaving group suitable for reaction with an amine, for instance an aromatic leaving group, preferably a

p-nitrophenyloxy group. After elaboration of the reaction mixture, which typically comprises an extraction with water, the product is isolated from the reaction mixture and purified, if necessary, for instance by chromatography or by crystallization.

Based on the stereochemistry of the starting compound (6), the product may be isolated as a mixture of diastereoisomers or as a single enantiomer. If the mixture is produced, it may be resolved into enantiomers by conventional means. The starting compound of formula (6) may be typically prepared from a haloformate of formula (3)

in which R is hydrogen or a Ci-Cio alkyl/aryl/aralkyl group , Y is a halogen atom and X is a halogen atom or a OH-group, for instance according to the scheme below

(3) < ⁷ > (6)

In particular, the halo atom Y of compound (3) is replaced by leaving group L to yield the compound of formula (7), wherein R is as above defined. This intermediate reacts with the carboxylic acid Ri-COOH, (Ri is a Ci-Cio alkyl/aryl/aralkyl group), which is typically present in a suitable reactive form, e.g. in the form of a metal salt, e.g. silver or mercury salt, or as a tetraalkylammonium salt, optionally after prior replacement of the group X by a better reactive group (for instance by an iodine group).

The intermediate of formula (6) may be isolated from the reaction mixture by conventional means, and purified, if necessary. In addition, the compound of formula (6) may be resolved into enantiomers, if this is desirable or advantageous. In an alternate procedure, the intermediate of formula (6) is not isolated from the reaction mixture and is subjected to the reaction with tamsulosin within that mixture, optionally with some necessary pre-treatment, e.g. filtration or pH-adjustment.

The compounds of formula (2) of the present invention are characterised by an advantageous stability profile, particularly in an aqueous solution of different pHs. Thus, in a typical stability test, representative examples of the inventive compounds having the formula (2a) and (2b), resp.

(2a) (2b) were incubated in simulated gastric and intestinal fluids prepared according to US Pharmacopoeia and the stability of the compounds in the solution was measured at 37°C corresponding to the average human body temperature. Details of the experiment are disclosed in the Experimental Examples 1 and 2.

The results of the testing show that, surprisingly, the compounds of the present invention are stable at aqueous conditions corresponding to stomach conditions, while at conditions corresponding to the intestines they slowly and completely split the side chain moiety thus "liberating" tamsulosin within a certain, pharmaceutically advantageous, time period.

The fact that this unexpected behaviour is associated with the particular structure of the carbamate side chain has been demonstrated by the fact that a model

ethylcarbamate, a compound of formula (8)

which has been selected as a representative of the basic behaviour of a carbamate group attached to the tamsulosin skeleton, has been found stable both at simulated gastric and at simulated intestinal conditions (Experimental Example 3).

Accordingly, the compounds of formula (2) may serve as prodrugs of tamsulosin, which allow maintaining the whole bolus of tamsulosin intact during its passage through the stomach and starting to liberate tamsulosin only in the intestinal fluid. Accordingly, it was proven as essentially not necessary to prepare tamsulosin-comprising compositions containing specific means and/or excipients for obtaining a sustained release of tamsulosin (such as tamsulosin-comprising pellets, which are present in the commercial drug preparation sold under the brand name FLOMAX). Instead, the same or similar therapeutic response may be obtained by means of technologically simple

pharmaceutical compositions comprising the "tamsulosin-prodrugs" of the present invention without need of specific excipients and/or arrangements delaying or otherwise modifying the release and/or protecting the tamsulosin against contact with the stomach environment.

The present invention thus comprises the novel compounds of formula (2) for use in therapy. In particular, the compounds may be used as prodrugs for delivery of tamsulosin to a patient in need thereof, or, in other words, for treating, by an approved way, conditions that are treatable by tamsulosin.

The compounds of the present invention may be formulated into various pharmaceutical compositions comprising the therapeutic or prophylactic amount of the compound of formula (2) and at least one pharmaceutically acceptable excipient. The excipients comprise fillers, binders, diluents, pH-adjustors, release control agents, lubricants, colourants etc. In particular, the compositions are designated for oral administration and typically are solid-state compositions.

In particular, the compositions are designated as immediate release compositions, i.e. liberating the active compound (the "tamsulosin prodrug" of formula (2)) in the stomach. Nonetheless, the compositions of the present invention also comprise sustained release compositions, which refer to compositions allowing the release of the active compound from the composition over a prolonged period of time. In some embodiments, the final release of tamsulosin from the compositions of the present invention, regardless of the presence of excipients causing the sustained release, occurs over a time period of at least about 4 hours, such as at least about 8 hours, at least about 12 hours, at least about 16 hours, and in some embodiments, at least about 24 hours.

Pharmaceutical compositions comprising the compounds of the present invention may be produced by conventional methods of mixing, blending, dissolving, granulating or lyophilization, etc.

The compositions comprising the tamsulosin prodrugs of the present invention may be formulated into various final dosage forms, preferably into solid dosage forms.

Typical dosage forms are tablets, pellets or capsules. The compositions and dosage forms comprising the tamsulosin prodrug of formula (2) may be administered singly or in combination with other pharmaceutically active substances. Both substances may be formulated into the same composition or may be physically separated in two different compositions and/or dosage forms. In an example, the active substance that might be co-administered with the tamsulosin prodrug of the present invention, e.g. in the treatment of benign prostatic hyperplasia, is a testosterone- 5a-reductase inhibitor, for instance finasteride or dutasteride. The "other

pharmaceutically active substance" may also be tamsulosin iself; in this particular case, the resulting medicament may represent a medicament with a dual release of tamsulosin in the body environment.

In a non-limiting example, such combined dosage form may be advantageously represented by two concentrically placed capsules ("capsule-in-capsule"), wherein the inner, smaller one contains a pharmaceutical formulation comprising a dose of at least one active substance, which is to be co-administered with the tamsulosin prodrug, and the space between the inner and outer capsule is filled with the composition of tamsulosin prodrug of the present invention, comprising, in total, the required dose of tamsulosin.

The compounds, compositions and dosage forms according to the present invention may be used in therapy, for example, in the management of functional treatment of symptomatic benign prostatic hypertrophy or hyperplasia (BPH) or other disorders treatable by tamsulosin (the Disorders). The extended release of tamsulosin from the prodrug assures that the therapeutic concentration of tamsulosin in blood is maintained for a sufficiently long time, without initial dumping in the stomach.

Accordingly, the present invention further provides a method for treating and/or preventing any one or more Disorders which comprises orally administering an effective and/or prophylactic amount, to a person in need thereof, of the compound of formula (2) of the present invention within a pharmaceutical composition. Preferably, the compositions comprising the tamsulosin prodrugs of the invention are administered once a day, and more preferably after a meal. Administration after food intake is advantageous because of minimizing damages to tissues of the gastrointestinal tract.

The present invention also provides the use of the compound of the present invention, in a fixed-dose combination medicament, advantageously with a testosterone- 5a-reductase inhibitor, for instance finasteride or dutasteride, for treating and/or preventing any one or more of the Disorders.

The compounds of formula (2) above form a specific class of compounds of general formula (2-1)

wherein Z is -0-, -S- or -NH- linkage and R, Ri have the above meaning. While the invention has been explained and illustrated with respect to the compounds of formula (2), i.e. to compounds of formula (2-1), wherein the -Z- linkage is the -O- linkage, the other compounds of formula (2-1), i.e. compounds wherein -Z- is the -S- or - NH- linkage, are also within the scope of the present invention. In such case, the above teaching referring to the processes leading to compounds (2), to the pharmaceutical compositions and dosage forms comprising compounds (2), as well as to the uses of compounds (2) in medical treatment, applies to compounds of the general formula (2-1) mutatis mutandis.

The invention is further illustrated by the following Examples, but should not be construed as being limited thereto.

EXAMPLES

Example 1

Step 1

1 -chloroethyl 2-(2-ethoxyphenoxy)ethyl((R)- 1 -(4-methoxy-3 -sulfamoylphenyl)- propan-2-yl)carbamate

Tamsulosin-base (3.56 g) was suspended in 30 ml of dry dichloromethane (under CaCi2 tube). The suspension was cooled down to 5°C (ice-water bath). Then

1 -chloroethyl chloro formate (0.95 ml) was added. The temperature of the mixture was adjusted to 10°C-15°C and a solution of triethylamine (1.2 ml) in 5 ml dichloromethane was added. The mixture was stirred at 10°C-15°C for 1 hour.

The reaction was monitored by TLC (MeOH: dichloromethane (5 : 95 v/v)).

The mixture was processed by extraction.

Extraction: 1 x 20 ml 1 M solution of HC1 (the water layer was a thick

suspension.To the separatory funnel 45 ml of CH2CI2 was added and the water layer was separated and solids were filtered off).

1 x 30 ml sat.sol.NaCl

The organic layer was dried by MgS0 ₄ which was then filtered off. The filtrate was evaporated on a rotary vacuum evaporator (water bath 30°C).

Yield: 3.4 g

Step 2

l-((2-(2-ethoxyphenoxy)ethyl)((R)-l-(4-methoxy-3-sulfamoylph enyl)propan-2- yl)carbamoyloxy)ethyl isobutyrate (2A)

Isobutyric acid (1.9 ml) was added to the 100 ml reaction vessel and cooled down to 10°C. Then triethyl amine (2.8 ml) was added (the temperature after the addition was 15°C). The solution of the "carbamate" from Step 1 in 10 ml CH2CI2 was added. The dropping funnel was washed with 2 x 5 ml of CH2CI2 .The reaction mixture was stirred over night (room temperature). The mixture was heated for 2 hours at 40°C -42°C and again stirred over night (RT).

The reaction was monitored by TLC (EtOAc: dichloromethane (10 : 90 v/v)).

The mixture was processed by extraction with 2 x 15 ml water, 1 x 15 ml aHC0 ₃ (5 ml satsol. aHC0 ₃ + 10 ml water) and 2 x 15 ml sat.sol.NaCl. The organic layer was dried by MgS0 ₄ and evaporated on RVO (water bath 30°C).

Yield: 2.16 g

A portion of the residue (1.08 g of the product dissolved in 3 ml of eluent) was purified by column chromatography on 20 g of silica (Kieselgel 60)

Eluent: EtOAc : : CH ₂ Cl2 - 2% (fractions 1-3)

EtOAc : : CH2CI2 - 4% (fractions 3-12)

EtOAc : : CH2CI2 - 6% (fractions 12-18)

EtOAc : : CH2CI2 - 8% (fractions 18-33)

EtOAc : : CH2CI2 - 10°/ Ό (fractions 33-36)

EtOAc : : CH2CI2 - 14°/ Ό (fractions 36-43)

Combined fractions (20-42) were evaporated (water bath 30°C) on a rotary vacuum evaporator.

0.9 g of product was obtained.

Example 2

Step 1

1 -chloromethyl 2-(2-ethoxyphenoxy)ethyl((S)-l-(4-methoxy-3-sulfamoylphenyl) - propan-2-yl)carbamate

Tamsulosin-base (10 g) was suspended in 80 ml of dry dichloromethane (under CaCi ₂ tube). The suspension was cooled down to 5°C (ice-water bath). Then 1- chloromethyl chloroformate (2.2 ml) was added.The temperature of the mixture was adjusted at 10°C-15°C and a solution of triethylamine (3.4 ml) in 14 ml dichlormethane was added. The mixture was stirred at 10°C-15°C for 1 hour. The reaction was monitored by TLC (MeOH:dichloromethane (5 : 95 v/v)).

The mixture was processed by extraction with 1 x 60 ml 1 M solution of HQ (the water layer was a thick suspension.To the separatory funnel 80 ml of (¾(¾ was added and the water layer was separated) and 1 x 80 ml sat.sol.NaCl.

The organic layer was dried by MgSC^ and evaporated on a rotary vacuum evaporator (water bath 30°C).

Yield: 9.9 g

Step 2

l-((2-(2-ethoxyphenoxy)ethyl)((S)-l-(4-methoxy-3-sulfamoylph enyl)propan-2- yl)carbamoyloxy)methyl isobutyrate

Isobutyric acid (6.61 g) and triethylamine (10.3 ml) were added to a 250 ml reaction vessel under stirring. The mixture was cooled to 15°C and a solution of the "carbamate" from Step 1 in 85 ml of dry CH2CI2 was added by means of a pressure equalized funnel within about 10 minutes (inner temperature max. 25°C). The reaction mixture was stirred over night (room temperature). The mixture was then transferred into a 250 ml separatory funnel and extracted with a mixture of 25 ml of a saturated solution of sodium hydrogencarbonate with 35 ml water and then twice with 60 ml of a saturated solution of sodium chloride. After drying over magnesium sulfate, filtering and washing with dicloromethane, the solution was evaporated on a rotary vacuum evaporator. Yield: 8.09 g of light viscous mass.

The residue was dissolved in 15 ml of 10% EtOAc in dichloromethane and chromatographed on 220 g Kieselgel 60. The extraction was controlled by TLC

(Kieselgel, 10% EtOAc in dichloromethane).

Eluent: EtOAc : CH2CI2 - 10% (fractions 1-10)

EtOAc : CH2CI2 - 12.5% (fractions 11-14) EtOAc : : CH ₂ Cl2 - 16.25% (fractions 15-20)

EtOAc : : CH2CI2 - 18.75% (fractions 21-26)

EtOAc : : CH2CI2 - 20% (fractions 27-31)

EtOAc : : CH2CI2 - 22.5% (fractions 32-36)

EtOAc : : CH2CI2 - 25% (fractions 37-38)

Combined fractions (28-38) were rechromatographed under the same conditions. Desired fractions were collected and evaporated.

Experimental Example 1 - Stability of the compound (2A) (hereinunder "TSL Prodrug") in artificial gastric and intestinal fluids

a) Test media

Simulated gastric fluid (USP): Dissolve 2.0 g of sodium chloride and 3.2 g of purified pepsin that is derived from porcine stomach mucosa, with an activity of 800 to 2500 units per mg of protein, in 7 ml of hydrochloric acid and dilute with water to make 100 ml. This solution has a pH of about 1.2.

Simulated intestinal fluid (USP): Dissolve 6.8 g of monobasic potassium phosphate in 250 ml of water and add 77 ml of 0.2 N sodium hydroxide and 500 ml of water. Add 10.0 g of pancreatin, mix and adjust the solution with either 0.2 N sodium hydroxide or 0.2 N hydrochloric acid to pH 6.8 ± 0.1. Dilute with water to 100 ml.

b) Standard and sample preparation

Compound 2A Stock Preparation: Transfer approximately 20 mg of Compound 2A into a 100 ml volumetric flask, dissolve in 50 ml of ethanol and make up the volume with water. The stock solution has a concentration of approx. 0.2 mg/ml. Prepare similarly the second Stock preparation with a concentration of 0.4 mg/ml.

Tamsulosin Identification Preparation: Transfer 10 mg of tamsulosin into a 100 ml volumetric flask. Dissolve in a mixture of acetonitrile and Mobile Phase A in 1/9 volume ratio. Dilute this solution 10 times by the same mixture (10 μg/ml).

Sample Preparation 1 (GF): Transfer 5 ml of the Compound 2A Stock Preparation into a 50 ml volumetric flask. Add 25 ml of Simulated Gastric Fluid and cover by cap. Sample Preparation 2 (IF): Transfer 2 ml of the Compound 2A Stock Preparation into a 25 ml volumetric flask. Add 15 ml of Simulated Intestinal Fluid and cover by cap. c) Incubation of the Samples

Both Sample preparations are stored in a thermostated vessel at a temperature of

37°C.

d) Sampling

Gastric fluid sample : 0 0.5 1 2 3 hours

Intestinal fluid sample: 0 0.5 1 2 3 4 5 6 hours

e) Analysis

HPLC:

Analytical column: Symmetry C 18 3.5μιη, 4.6 x 75 mm

Column temperature: 40°C

Flow rate: 1 ml/min

Detection: UV 282 nm

Injection volume: 20 μΐ

Mobile Phase A: 10 ml phosphate buffer pH 4.0 (1.38% of NaH ₂ P0 ₄ .H ₂ 0 ml of water, pH adjusted with 85% H ₃ P0 ₄ p. .)

Mobile Phase B: acetonitrile

Gradient program: Time (min) cone, of B (%)

0 10

10 90

14 90

15 10

18 10 HPLC Sample after GF hydrolysis test: Add 2 ml of 1M NaOH to the incubated Sample Preparation 1 , mix, and make up the volume by Mobile Phase A. Filter the sample with a microfilter (0.45 μιη) before injecting into the HPLC.

HPLC Sample after IF hydrolysis test: Add 50 μΐ of 85% phosphoric acid, mix and make up the volume by Mobile Phase A. Centrifuge a 4 ml aliquot (5500 rpm) and use the supernatant for HPLC injection. (NB. A recovery test at t=0 should be performed). f) Quantification:

Use an external calibration curve in the range of 1-40 mg of Compound 2A and of tamsulosin.

g) Results

Gastric fluid hydrolysis tests

* 100% of "TSL-prodrug" = 19.50 μg/ml; Average measured cone. = 19.50 μg/ml (RSD = 2.17%)

19.5 μg/ml - measured cone, after incubation

Intestinal fluid hydrolysis tests

Recovery results

Recovery 100% = 34.27 μg/ml H drolysis results

calculated from the molecular weight of both monitored substances. The measured results show good correlation between the theory and the realized test.

Conclusion

It can be concluded that Compound 2A degrades in intestinal fluid very significantly. 50% decrease was observed during 1 hour of incubation. After 6 hours, only 20% of the original amount of the Compound 2A was determined. Formation of tamsulosin is in accordance with the theoretical molecular weight balance. The sum of both monitored compounds corresponds with the original amount of Compound 2A measured before incubation.

Experimental example 2 Stability of the compound (2B) (hereinunder "TSL Prodrug II") in artificial gastric and intestinal fluids

Setup of the test: See Experimental Example 1, arts, a) - f) except:

HPLC:

Analytical column: Symmetry C18 3.5μιη, 4.6 x 75 mm

Guard column: CI 8, 4 x 3 mm Phenomenex

Column temperature: 40°C

Flow rate: 0.5 ml/min Detection: UV 282 nm

Injection volume: 20 μΐ

Gradient program: Time (min) cone. of B (%)

0 10

10 100

17 100

18 10

22 10

Mobile phase A: 0.1% acetic acid (glacial, for LC-MS) in water

Mobile phase B: Acetonitrile HPLC gradient grade

Results

Gastric fluid hydrolysis test

* 100% of "TSL-prodrug Π" = 22.28 μg/ml; Average measured cone. = 22.16 μ^πιΐ (RSD = 2.19%)

** 22.28 μg/ml - measured cone, after incubation

Intestinal fluid hydrolysis test

Recovery results

Recovery 100%

Hydrolysis results

Theoretical formed amount of TSL during hydrolysis of "TSL-prodi calculated from the molecular weight of both monitored substances. The measured results show good correlation between the theory and the realized test.

NA = not analyzed

Conclusion

It can be concluded that compound (2B) degrades in intestinal fluid very fast. A

70% decrease is observed within 0.5 hour of incubation. After three hours of incubation, compound (2B) was not detected. Formation of tamsulosine is in accordance with thetheoretical molecular weight balance. The sum of both monitored compounds corresponds with the amount of compound (2B) measured before incubation. Experimental Example 3 Stability of compound (8) in artificial gastric and intestinal fluids (comparative)

The synthesis of tamsulosin-ethylcarbamate of formula (8) was performed by reaction of tamsulosin with diethyl pyrocarbonate (Scheme below). The desired product was obtained in quantitative yield.

Setup of the test: See Experimental Example

Results

Gastric fluid hydrolysis test

* 100% of TSL-ethylcarbamate = 23.44 μg/ml; Average measured cone. = 23.19 μ^πιΐ (RSD = 0.54%)

23.44 μg/ml - measured cone, after incubation

Intestinal fluid hydrolysis test

Recovery of this procedure for TSL-ethylcarbamate was more than 90% for the sample on time "0". No more recovery tests were carried out. Results

It can be concluded that TSL-ethylcarbamate is stable in intestinal fluid. After hours of incubation of TSL-ethylcarbamate no trend of significant changes of TSL- ethylcarbamate content was observed.

The invention having been described, it will be readily apparent to those skilled in the art that further changes and modifications in actual implementation of the concepts and embodiments described herein can easily be made or may be learned by practice of the invention, without departing from the spirit and scope of the invention as defined by the following claims.