NZ521857A - Watersoluble prodrugs of propofol - Google Patents

Abstract

Stable and water-soluble propofol derivatives of formula (I), wherein R1 is a cyclic or linear amino acid; said compounds being readily metabolized to release propofol in vivo. Also disclosed is the use of such compounds to anesthetize.

Description

52 1 8 5 7 intellectual property office of n.z. - 8 OCT 2002 RiCIIViD Patents Form No. 5 Our Ref: JC218059 \ NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION WATERSOLUBLE PRODRUGS OF PROPOFOL We, BIOTECHNOLOGIE - GESELLSCHAFT MITTELHESSEN MBH, a body corporate organised under the laws of Germany of Kerkraderstr. 7, D-35394 Giessen, Federal Republic of Germany hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: -1 - followed by Page 1 a PT0541615 100031728_1 PA342 64 - 1 a - WATERSOLUBLE PRODRUGS OF PROPOFOL Field of the invention The present invention relates to esters of propofol (2,6-diisopropylphenol), a method for anesthetizing a mammal as well as a method for treating convulsions, migraine or related diseases, or for inhibition of free radicals in a mammal using said compounds. Furthermore, the present invention relates to said compounds for use as a medicament and the use of said compounds for the preparation of a medicament for anesthetizing a mammal or for treating convulsions, migraine or related diseases, or for inhibition of free radicals in a mammal.

Background of the invention Propofol (2,6-diisopropylphenol, see compound 1 of Fig. 1) is an important intravenous agent in the practice of anesthesia. Due to its very low solubility in water, propofol was initially formulated as a 1 % w/v solution in the presence of Cremophor EL (a solubilizing surfactant), but the anaphylactic reactions associated with its administration have led to a search for alternative formulations (Trapani G, Altomare C, Sanna E, Biggio G, Liso G. 2000. Propofol in anesthesia. Mechanism of action, structure-activity relationships, and drug delivery. Curr. Med. Chem. 7: 249-271. Franks NP, Lieb WR. 1994. Molecular and cellular mechanisms of general anaesthesia. Nature (Lond). 367: 607-614.). Presently, propofol is formulated in an oil-in-water emulsion (1 % w/v) of soya bean oil, glycerol and purified egg phosphatide (Diprivan®, Zeneca UK). Intravenous (i.v.) injection of Diprivan® pro-Wices hypnosis rapidly (usually within 40 sec) and smoothly with minimal excitation, but pain at the site of injection is a major adverse effect (Prankerd RD, Stella VJ. 1990. Use of oil-in-water emulsions as a vehicle for parenteral drug administration. J. Parent. Sci. Technol. 44: 139-149.). As a lipid-based emulsion, it suffers from a number of limitations, such as poor physical stability, potential for embolism, and need for strictly aseptic handling (Bennett SN, Mc Neil MM, Bland LA, Arduino MJ, Villarino ME, Perrotta DM. 1995. Postoperative infections traced to contamination of an intravenous anesthetic, propofol. New England Journal of Medicine 333: 147-154.). Moreover, particular care is required in patients with disorders of fat metabolism (Dollery C. (ed.). 1991. Propofol. In Therapeutics Drugs, followed by Page 2 PA34264 2 Churchill Livingstone, London, Vol 2 pp. 269-271), and the material of the tubes used for infusing the emulsion must be carefully selected.

To avoid these drawbacks, safe alternative dosage forms, in particular aqueous formulations are needed. Approaches in this direction include the complexation of propofol with hydroxypropyl-p-cyclodextrin (Brewster M. 1991. Parenteral safety and applications of 2-hydroxypropyi-p-cyclodextrin. In Duchene D, editor. New Trends in Cyclodextrins and Derivatives, Paris: Editions de Sante, pp. 313-350. Trapani G, Lopedota A, Franco M, Latrofa A, Liso G. 1996. Effect of 2-hydroxypropyl-p-cyclodextrin on the aqueous solubility of the anaesthetic agent propofol (2,6-diisopropylphenol). Int. J. Pharm. 139: 215-218. Trapani G, Latrofa A, Franco M, Lopedota A, Sanna E, Liso G. 1998. Inclusion complexation of propofol with 2-hydroxypropyl-p-cyclodextrin. Physicochemical, nuclear magnetic resonance spectroscopic studies, and anesthetic properties in rat. J. Pharm. Sci. 87: 514-518.) and Chemical delivery systems. The main objectives of these approaches are to increase the hydrosolubility of propofol, improve patient acceptance, e.g. reduced pain at the site of injection, and a decrease in side-effects as well as prolonged action (Pop E, Anderson W, Prokai-Tatrai K, Vlasak J, Brewster ME, Bodor N. 1992. Syntheses and preliminary pharmacological evaluation of some chemical delivery systems of 2,6-diisopropylphenol (propofol). Med Chem. Res. 2: 16-21.). Water-soluble prodrugs of propofol have also been prepared as suitable formulations for parenteral administration (Morimoto BH, Barker PL. Preparation of phosphochoiine iinkea prodrug-aerivatives. PCT int. Appl., 2000, WO 0048572. Stella VJ, Zygmunt J J, Geog IG, Safadi MS. Water-soluble prodrugs of hindered alcohols or phenols. PCT Int. Appl., 2000, WO 0008033. Sagara Y, HendierS, Khon-Reiter S, Gillenwater G, Carlo D, Schubert D, Chang J. 1999. Propofol hemisuccinate pro-0pcts neuronal cells from oxidative injury. J. Neurochem. 73: 2524-2530. Hendler SS, Sanchez RA, Zielinski J. Water-soluble prodrugs of propofol. PCT Int. Appl., 1999, WO 9958555.) a-Aminoacid ester derivatives of propofol (see compounds 2a-c of Fig. 1) (Trapani G, Latrofa A, Franco M. Lopedota A, Maciocco E, Liso G. 1998. Water-soluble salts of amino acid esters of the anesthetic agent propofol. Int. J. Pharm. 175: 195-204.) were investigated as prodrugs, which demonstrated good aqueous solubility and stability. But the resistance of these compounds against hydrolytic activation in plasma and brain homogenate is much too high for them to actually be considered true prodrugs. Interestingly, some of them were found to interact with the subtype A of the y-aminobutyric acid (GABAa) receptor, a major fAJ^<4 0y 3 target mediating the pharmacological actions of propofol and other general anesthetics (Trapani G, Altomare C, Sanna E, Blgglo G, Liso G. 20D0. Propofol In anesthesia, Mechanism of action, structure-activity relationships, and drug delivery. Curr. Med. Chem. 7: 249-271. Franks NP, Lleb WR. 1994. Molecular and cellular mechanisms of general anaesthesia. Nature (Lond). 367: 607-614.). Nevertheless, due to their binding affinity to the GABA* receptors, similar to that of parent propofol, some of them were suggested as promising candidates for In vivo pharmacological evaluation.

In summary, there is still a need for stable and water soluble propofol derivatives, that are capable of hydrolytic activation under physiological conditions. Specifically, there is a need for stable and water soluble prodrugs of propofol that are readily metabolized to release propofol in vivo. wherein R1 is a cyclic or linear amino acid and their diastereomers or enantiomers In the form of their bases or salts, and wherein the amino acid Is optionally further substituted.

According to the present invention the term Mamino acid" means any artificial or naturally occurring amino acid characterized by the presence of an amino or Imlno group and a car-boxy group. The term encompasses cyclic and non-cyclic compounds, wherein the cyclic compound may be aromatic or alicyclic. Preferably, the amino acid is a naturally occurlng amino acid or a derivative thereof. Preferably, the amino acid .is an alpha-, beta-, gamma-, delta- or epsllon-amino acid.

Disclosure of the invention The present Invention provides In one aspect compounds having the formula: Preferably, the amino acid is C-termlnally linked to propofol or the amino acid. 1b N-termina'lly linked to propofol.

INTELLECTUAL property office of n.z. - 8 JAN 2004 received It is preferred that the salts include chloride, sulphate, (hemi)tartrate, (hemOsuccinate, (bemi)malate, acetate, lactate and similar anions.

According to a preferred embodiment of the present invention the compounds have the formula: /•\ wherein the heterocyclic group comprises 4 to 5 methylene groups and wherein the heterocyclic group is optionally further substituted, with one or more aromatic and/or heteroaromatic rings.

Preferably, does not comprise a tertiary nitrogen.

It is further preferred that the compounds are cleaved rapidly by esterases. Exemplarily, reference is made to compound 6a which is cleaved more rapidly than compound 6b.

More preferably, R, is selected from proline and the three positional isomers of piperidine i.e., pipecolinic, nipecotic, and isonipecotic acid.

It is also more preferred that R1 is selected from tyrosine, tryptophan, phenylalanine or histidine. The aromatic ring may also be further substituted to create condensed or fused aromatic compounds of the naphthalin, anthracen or phenanthren- type. It is however required that said compounds are essentially water-soluble.

More preferably, said compounds are selected from a-proiine, a-pipecofinic acid, ft-nipecotic acid and y-isonipecotic acid.

For migraine application the preferred compounds are those which act as depot form i.e. are cleaved more slowly like 6c>6d and new examples 4 and 5 PA34264 In a further preferred embodiment of the present invention, said compounds are selected from a-proline, a-pipecolinic acid, or B-nipecotic acid, preferably from a-proline or a-pipecolinic acid, and most preferably said compound is a-proline.

The amino acid compound may also be a linear amino acid. Preferably, the amino acid is selected from glycine, alanine, valine, leucine, isoleucine, glutamine, glutamic acid, aspara-gine, aspartic acid, cysteine, methionine, serine, or threonine.

^The skilled person is aware that the amino acid component of propofol derivatives according to the invention is of a basic nature due to the secondary nitrogen atom within the cyclic structure. Therefore, the compounds of the present invention tend to form salts. Preferred salts of the propofol derivatives of the present invention comprise hydrogen and any suitable pharmaceutically acceptable counterion, preferably selected from the group of chloride, sulphate, (hemi)tartrate, (hemi)succinate, (hemi)malate, acetate, lactate and similar anions.

According to another aspect the compounds of the present invention have the formula wherein X and Y denote linker groups and wherein the carbohydrate is optionally further substituted.

The linker group may be any linker group known in the art provided that the produced compound is still sufficiently water-soluble. Linker of the hydrazine or glutaric acid type and ho-mologs thereof are preferred.

Preferably, X has the formula: 6 intellectual property office of n.z. - 8 JAN 2004 received X =—HN—(CH2)—NH— wherein n is an integer from 0 to 10, most preferably n = 0.

Preferably, Y has the formula: 0 9 II II Y = —C—(CH2)—C— wherein m is an integer from 0 to 5, most preferably m = 0 or 2.

According to a preferred embodiment R2 is a monosaccharide, disaccharide oligosaccharide or polysaccharide comprising at least one moiety selected from allose, altrose, glucose, mannose, gulose, idose, galactose, talose, sucrose, lactose, maltose, isomaltose, cellobio-se, maltobionic acid, and lactobionic acid.

Patricluarly preferred, R2 is maltotriose, lactobionic acid or hydroxyethyl starch.

Also more preferred, R2 comprises up to 40 lactobionic acid moieties, preferably 2 to 7.

Preferably, R2 comprises up to 40 maltotriose moieties, preferably 2 to 7. ^ ) Particularly preferred, R2 comprises at least 2 hydroxyethyl glucose moieties wherein the hydroxy ethyl glucose moieties may be substituted. Reference is made to German patent application DE 10209822.0. The disclosure of which, in particular with respect to the glyco-syiation pattern of hydroxyethyl starch is incorporated herewith.

In one specific embodiment of the present invention, water-soluble derivatives of propofol are preferably prepared by esterifying the drug with cyclic amino acids, preferably with four specific cyclic aminoacids (compounds 6a-d, see Fig. 1), namely proline and the three positional isomers of piperidine carboxylic acids (i.e., pipecolinic, nipecotic, and isonipecotic acids).

PA34264 7 In another specific embodiment, water-soluble derivatives of profolol are obtained by esterifying a saccharide either directly or indirectly via linker groups with propofol. Examples for synthesis are given in the detailed description below.

Their properties such as e.g. solubility, lipophilicity, stability in aqueous solutions, susceptibility to enzymatic hydrolysis in animal plasma and liver, and their ability to interact with GABAa receptors make them excellent candidate substances for promoting anesthesia and for treating convulsions, migraine or related diseases and for inhibiting free radicals.

Three of the preferred amino acids that were esterified with propofol (with the exception of pipecolinic acid (3b)) are pharmacologically active. (S)-Proline is an inibitory amino acid, (R)-nipecotic acid is an inhibitor of GABA uptake, and isonipecotic acid is a specific GABAa ^agonist (Referenz 16).16 Thus, with the exclusion of propofol pipecolinate (6b), the preferred ester derivatives 6a, 6c and 6d may be considered rather "dual prodrugs", that are converted in vivo into two active molecules. Except proline, taken in its natural enantiomeric form (S), the other chiral amino acids (i.e., pipecolinic and nipecotic acids) were used in synthesis as racemates. The influence of their steric conformation was not further investigated at this point.

Prolinate derivative 6a is particularly well suited as a water-soluble prodrug. Said compound protects animals against pentylenetetrazole-induced convulsions, and induces an anesthetic action in a short time of a duration that is comparable with that of the marketed propofol emulsion Diprivan®. Its high solubility and stability in water at physiological pH allow to prepare freeze-dried formulations for parenteral administration. The prolinate derivative 6a is a most preferred embodiment of the present invention. in a preferred embodiment, the present invention relates to a freeze-dried pharmaceutical composition comprising at least one of the compounds of the present invention, more preferably comprising an a-proline propofol ester.

The susceptibility of the preferred proline ester 6a to enzymatic cleavage by ester hydrolases in plasma and liver affords conversion in vivo to the parent drug. Consequently, a 17 mg/mL aqueous solution of proline ester 6a, is equivalent to the commercial oil-in-water emulsion Diprivan® containing 10 mg/mL of propofol.

PA342 64 8 Prolinate 6a as well as piperidine-2-carboxylate 6b bind as such, i.e. as intact non-hydrolyzed molecules, to the propofol binding site of GABAa receptors, with IC5o values of 30-40 nM (one log unit lower than propofol).

The compounds of the present invention have demonstrated their pharmacological potential in an in vitro [35SJTBPS binding assay using rat brain and electrophysiological studies using Xenopus oocytes. Moreover, said compounds have demonstrated a pharmacologically effective anticonvulsant and anesthetic activity in vivo.

In general, the compounds of the present invention demonstrate high solubility and stability in aquous solutions and also in physiological media in vitro. They are readily hydrolyzed in ^plasma and liver esterase solutions, many of them even quantitatively within a few minutes.

The compounds of the present invention are also efficacious in vivo. Because said compounds readily hydrolize under physiological conditions and release propofol, they are excellent prodrugs for propofol action.

Therefore, the present invention is also directed at a method for anesthetizing a mammal or a method of treating convulsions, migraine or related diseases or for inhibiting in a mammal, wherein a therapeutically effective amount of a compound according to the invention is administered to said mammal.

The term "treating" is intended to refer to all processes, wherein there may be a slow-^>g, interrupting, arresting, or stopping of the progression of a convulsion or convulsions, but does not necessarily indicate a total elimination of all symptoms.

The term "anesthetizing" as used herein is to be understood in the context of the pharmaceutical action of the parent compound propofol.

As used herein, the term "mammal" refers to a warm blooded animal. It is understood that guinea pigs, dogs, cats, rats, mice, horses, cattle, sheep, monkeys, chimpanzees and humans are examples of mammals and within the scope of the meaning of the term. Humans are preferred.

In effecting treatment of a mammal in need of anesthetic treatment or suffering from convulsion, the compounds disclosed by the present invention for said purpose can be administered in any form or mode which makes the therapeutic compound bioavailable in an effective amount, including oral or parenteral routes. For example, products of the present invention can be administered intraperitoneally, intranasally, buccally, topically, orally, sub-cutaneously, intramuscularly, intravenously, transdermal^, rectally, and the like.

Parenteral administration of the compounds of the present invention is preferred.

One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition, and other relevant circumstances. (Remington's Pharmaceutical Sciences, Mack Publishing Co. (1990)). The products of the present invention can be administered alone or in the form of a pharmaceutical preparation in combination with pharmaceutical^ acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the product selected, the chosen route of administration, and standard pharmaceutical practice. For oral application suitable preparations are in the form of tablets, pills, capsules, powders, lozenges, sachets, cachets, suspensions, emulsions, solutions, drops, juices, syrups, while for parenteral, topical and inhalative application suitable forms are solutions, suspensions, easily reconstitutable dry preparations as well as sprays. Compounds according to the invention in a sustained-release substance, in dissolved form or in a plaster, optionally with the addition of agents promoting penetration of the skin, are suitable percutaneous application preparations. The products of the present invention, while effective themselves, may be formulated and administered in the form of their pharmaceutical^ acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, modulation of hydrophobicity, increased solubility, and the like.

The amount of active agent to be administered to the patient depends on the patient's weight, on the type of application, symptoms and the severity of the illness. Normally, 0.1 mg/kg to 25 mg/kg of at least one substance of the general formula I is administered, but when applied locally, e.g. intracoronary administration, much lower total doses are also possible.

PA34264 For practicing the methods of the present invention, said compound of the present invention is preferably administered by all possible routes (intraperitoneal, transdermal, intravenous, intravascular, intramuscular, inhalation, preferred route being as sterile solution for intravenous injection.

Thus, the esters of propofol according to the invention are useful as a medicament. Preferably, said compounds are used for the preparation of a medicament for anesthetizing a mammal or for treating convulsions, for treating migraine or related diseases or for inhibiting free radicals in a mammal.

' Figures Fig. 1 shows the structure of propofol (1) and propofol amino acid esters of the prior art (2a-c). A schematic diagram of the preferred method for preparing the compounds of the present invention is depicted in the middle of Fig. 1. The abbreviations used for reagents and substituents are well known to those in the art and explained in example 1. For further details, see example 1. Compounds 6a-d in combination with the substituents a-d at the end of Fig. 1 relate to preferred embodiments of the present invention.

Ei£L_2 The plot in Figure 2 shows an S (slope) versus log k"w plot of the data obtained in the lipophilicity studies in example 3 and demonstrates that, equal to the poiycratic capacity factors, slope values of the H-acceptors 2a-c are smaller than those of the amphiprotics 6a-d, proving the ability of the S parameter for encoding the total HB capacity of the compounds. m. 3 shows the effects of propofol 1 and compounds 6a-d on [35S]TBPS binding to unwashed rat cortical membranes. Rat cortical membranes were incubated with 2 nM [35S]TBPS for 90 min in the presence of different concentrations of propofol 1, or compounds 6a, 6b, 6c. and 6d. The data is expressed as a percentage of binding measured in the presence of solvent and are means of two experiments.

EiSL _4 shows the modulatory action of compound 6a (a) and 6d {b) at human a1[32y2 GABAa receptors expressed in Xenopus laevis oocytes. Values are expressed as mean PA342 64 11 (6-13 different oocytes) ± s.e.m. percentage of the potentiation of the control response to GABA (EC2Qi 2-10 nM).

Fi&jj shows the synthesis of activated precursors for use in subsequent synthesis of sac-charide-conjugates with propofol. shows the synthesis of saccharide-conjugates with propofol either by direct conjugation (Fig. 6A) or via linker groups (Fig. 6B-D).

The following examples further illustrate the best mode contemplated by the inventors for carrying out their invention. The examples relate to preferred embodiments and are not to be construed to be limiting on the scope of the invention.

Examples Chemicals -Propofol (1, see Fig. 1), dicyclohexylcarbodiimide (DCC), (S)-proline (3a, see Fig. 1), pipecolinic acid (3b, see Fig. 1), nipecotic acid (3c, see Fig. 1), isonipecotic "acid (see Fig. 1 3d), and all other reagents were purchased from Sigma-Aldrich (taufkirchen, Germany). Rat serum (lyophilized powder) and porcine liver esterase (suspension in 3.2 M (NH4)2S04 solution, pH 8) were also purchased from Sigma-Aldrich. Reagents used for the preparation of the buffers were of analytical grade. Fresh deionized water was used in the preparation of alt the solutions.

Apparatus Melting points were determined by the capillary method on a BQchi apparatus and are uncorrected. IR spectra were recorded as Nujol films for liquids and KBr pellets for solids on a Perkin-Elmer 283 spectrophotometer. 1H-NMR spectra were recorded on a Varian EM 390 spectrometer operating at 90 MHz (Varian, Milan Italy). Chemical shifts are expressed in 6 PA34264 12 values downfield from tetramethylsilane (TMS) used as internal standard. Mass spectra were recorded on a Hewlett-Packard 5995c GC-MS low resolution spectrometer (Hewlett-Packard, Milan, Italy) operating in electron impact mode. Elemental analyses were performed on a Hewlett-Packard 185 C, H, N analyzer and agreed with theoretical values to within ± 0.40%. High-performance liquid chromatography (HPLC) analyses were carried out on a Waters Associates Model 600 pump equipped with a Waters 990 variable wavelength UV detector and a 20 jjL loop injection valve (Waters, Milan, Italy). HPLC mobile phase was prepared using HPLC-grade methanol. For analysis, a phenomenex Ci$ column (25 cm x 3.9 mm; 5 pm particles) was used as the stationary phase. A flow rate of 1 mUmin was maintained and the column effluent was monitored continuously at 210 or 270 nm. Quantification of the compounds was carried out by measuring the peak areas in relation to those ^^Df external standards. Stability studies were carried out at controlled temperature of 37 ± 0.2 °C in a water bath.

Animals Male Sprague-Dawley CD® rats (Charles River, Como, Italy) weighing 180-200 g were used. The animals were kept on a controlled light-dark cycle (light period between 8:00 a.m. and 8:00 p.m.) in a room with constant temperature <22 ± 2°C) and humidity (65%). Upon arrival at the animal facilities there was a minimum of 7 days of acclimation during which the ^^animals had free access to food and water.

Animal care and handling throughout the experimental procedure were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The experimental protocol were approved by the Animal Ethical Committee of the University of Cagliari.

Example 1 Synthesis of cyclic aminoacid esters of propofol The propofol esters 6a-d were prepared according to the procedure illustrated in Fig. 1, by reacting the BOC-protected cyclic amino acids 4a-d with propofol 1 in the presence of DCC to give the corresponding esters 5a-d, which when deprotected with HCI gas yielded de rivatives 6a-d as hydrochlorides (physical and spectral data of newly synthesized compounds 4a, 5a-d, and 6a-d are shown below in Table I).

BOC-protected amino acids: preparation of 1-(tert-butoxvcarbonvnproline (4a) To a stirred mixture of proline (4.60 g, 40 mmol) in H20 (25 ml.) containing triethylamine (8.3 mL, 60 mmol), a solution of 2-(ferf-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON, 10.58 g, 43 mmol) in acetone (25 mL) was added. Stirring was prolonged for 12 h, and then 125 mL of a mixture of ethyl acetate:water (1:1, v/v) was added. The aqueous phase, combined with water (55 mL), used for washing the organic phase, was further washed with 50 mL of ethyl acetate, and then acidified with cold 0.1 N HCI (pH 2) to give compound 4a as a white precipitate.

N-BOC-piperidin carboxylic acids 4b-d were prepared in 87-89% yields, according to the above procedure (analytical data in agreement with those reported in literature (Ho B, Venkatarangan PM, Cruse SF, Hinko C.N, Andersen PH, Crider AM, Adloo AA, Roane DS, Stables JP. 1998. Synthesis of 2-piperidinecarboxylic acid derivatives as potential anticonvulsants. Eur. J. Med. Chem. 33: 23-31. Bonina FP, Arenare L, Palagiano F, Saija A, Nava F, Trombetta D, De Caprariis P. 1998. Synthesis, stability/and pharmacological evaluation of nipecotic acid prodrugs. J. Pharm. Sci. 8: 561-567. Freund R. Mederski WKR. 2000. A convenient synthetic route to spiro[indole-3,4'-piperidin]-2-ones. Helv. Chim. Acta. 83: 1247-1255.).

Esterification of BOC-protected cyclic amino acids: preparation of 2.6-diisopropvlphenyl 1-ftert-butoxvcarbonvnpirroDdin-2-carboxvlate (5a) as a typical procedure To a stirred solution of 1 (0.40 g, 2.25 mmol), compound 4a (0.57 g, 2.50 mmol), and di-methylaminopyridine (0.1 g, 0.82 mmol) in dry dichloromethane (15 mL), a solution of DCC (1.4 g, 6.8 mmol) in dry dichloromethane (10 mL) was added dropwise during 10 min. Stirring was continued at room temperature for 24 h, and then the dicyclohexylurea (DCU) precipitate was filtered off. The solution was evaporated under reduced pressure to give a residue which was purified by column chromatography on silica gel (petroleum ether-ethyl acetate 98:2 v/v as eluent) to give compound 5a.

PA34264 14 Removal of the tert-butoxvcarbonvl group: preparation of 2.6-diisopropvlphenvl pirrolidin-2-carboxvlate hydrochloride (6a) as a typical procedure To a stirred and ice-cooled solution of ester 5a (0.50 g, 1.33 mmol) in chloroform (20 mL) HCl gas was bubbled for 5 min. Evaporation of the solvent under reduced pressure gave compound 6a as a white solid.

Table I. Physical and spectral characteristics of newly synthesized compounds.

Comp. Yield Mp IR (KBr) ~ (%) (°C) (Y max), 5a 72 69-71 1770,170 0 5b 68 100-104 1760,170 0 5c 70 80-84 1755,170 0 5d 67 94-97 1750,170 0 6a 90 189-192 1760 6b 87 225-228 1770 6c 95 154-156 1760 6d 90 234-236 1740 Example 2 Solubility of cyclic aminoacid esters of propofol The solubility of the propofol derivatives 6a-d (6b-d as hydrochloride salts) in deionized water at 25 °C was determined by adding excess amount of compound to 1-2 mL of water in screw-capped test tube. The resulting mixture was vortexed for 10 min and then mechanically shaken in a thermostatic bath shaker (100 rpm) for 72 h to attain equilibrium. Next, the mixture was filtered through a 0.45 ^im membrane filter (Millipore®, cellulose acetate) and an aliquot was diluted with an appropriate amount of water and analyzed for the aminoacid ester prodrug content spectrophotometrically at 210 nm. All of the manipulations PA34264 were made without removal of the test tubes from the water bath, using thermostated pipettes, syringes, and buffer solutions. In Table I!, as shown below, solubility data is compared with the data previously determined for propofol derivatives 2a-c (Trapani G, Latrofa A, Franco M, Lopedota A, Maciocco E, Liso G. 1998. Water-soluble salts of amino acid esters of the anesthetic agent propofol. Int. J. Pharm. 175:195-204.).

Table II. Aqueous solubility, , stability in physiological media, and GABAa receptor binding of amino acid esters (as hydrochloride salts) of propofol Solubility Half-lives at 37 °Ca Cmp (mg/mL) [3SSJTBPS in deion- pH 7.4 50% rat Porcine binding » ized water a buffer serum liver esterase (13 U/mL) lCso^ pM0, g 6a 350.0 6 h 17 min 17 min 31.7 6b 13.4 7 h 2.5 min 13 min. 39.5 6c 525.0 f 6 h 3 h 152 6d 29.7 f M 45 h h 2a < 0.058b,c b, f b, i 2b 0.735b b.f b, f 6.52a 2c 0.213b b.f b,f b, i la>Data are means of two determination (less than 10% of difference). ^Previously deter- ^lined in phosphate buffer at pH 7.4.14 (c)Determined as free base.. ^Stable after 48 h. fe^sjTBPS binding in unwashed rat brain; for propofol ICSo = 4.17 pM. (h)No displacement, but a % increase in [35S]TBPS binding was observed. ®No displacement.

Compared to propofol, whose solubility under the same conditions was about 0.15 mg/mL, two derivatives, prolinate 6a and nipecotate 6c, their solubilities being 350 and 525 mg/mL, respectively, afforded a strong increase of the aqueous solubility of the anesthetic drug. None of the equations proposed for computing intrinsic solubilities gave accurate predictions (Peterson DL, Yalkowsky SH. 2001. Comparison of two methods for predicting aqueous solubility. J. Chem. Inf. Comput. Sci. 41:1531-1534. Teitko IV, Yu V, Kasheva TN, Villa AEP. 2001. Estimation of aqueous solubility of chemical compounds using E-state indices.

PA34264 16 J. Chem. Inf. Comput. Sci. 41: 1488-1493.), though it was apparent that the most relevant differences in solubility of derivatives 6 could be, at least in part, accounted for by large differences in crystal lattice energies. In fact, the most soluble 6a and 6c have a melting point lower than 200°C whereas pipecolinate 6b and isonipecotate 6d melt at 225 and 234DC, respectively.

Example 4 Chemical hydrolysis of cyclic aminoacid esters of propofol The hydrolysis of the propofol esters 6a-d was studied in aqueous buffer solutions (0.05 M ^phosphate buffers; ionic strength of 0.5 maintained by adding a calculated amount of KCI) at pH values of 4, 6, and 7.4 and temperature of 37 ± 0.2 eC. The reactions were initiated by adding 100 pi of a stock solution of the ester (13 mg/mL methanol) to 20 mL of the buffer solution preheated at 37 °C, in screw-capped test tubes (final concentration about 2.0 x 10"4 M). The solutions were kept in a water bath at a constant temperature, and at appropriate intervals aliquots of 20 pL were withdrawn and analyzed by HPLC. Pseudo-first-order rate constants for the hydrolysis were determined from the slopes of linear plots of the logarithm of residual propofol ester against time.

Example 5 Hydrolysis of cyclic aminoacid esters of propofol in physiological solution The susceptibility of the derivatives 6a-d to undergo conversion to the parent propofol was ^tudied in 0.05 M phosphate buffer (pH 7.4) containing 50% of rat serum at 37 °C. Each reaction were initiated by adding 100 pL of the methanolic stock solution of compound under examination to 1.6 mL of preheated serum solution (final concentration about 1 x 10"3. M) and the mixture was maintained in water bath at 37 °C. At appropriate times, 100 pL samples were withdrawn and added to 500 pL of cold acetonitrile in order to deproteinize the serum. After mixing and centrifugation (10 min at 4000 rpm), 20 pL of the clear supernatant were filtered through 0.2 pm membrane filter (Waters, PTFE 0.2 pm) and analyzed by HPLC.

Hydrolysis of compounds 6a-d in the presence of porcine liver esterase was followed using a reported procedure (Bonina FP, Arenare L, Palagiano F, Saija A, Nava F, Trombetta D, PA342 64 17 De Caprariis P. 1998. Synthesis, stability, and pharmacological evaluation of nipecotic acid prodrugs. J. Pharm. Sci. 8:561-567.)- Results: The kinetics of hydrolysis of the derivatives 6a-d were determined in 0.05 M phosphate buffers at pHs 4.0, 6.0 and 7.4, as well as in rat serum solution and in the presence of porcine liver esterase, at 37°C.

All the examined derivatives were stable at pH values of 4.0 and 6.0 for 48 h, whereas at physiological pH the hydrolysis of prolinate 6a and pipecolinate 6b followed first-order kinetics with half-lives of 6 and 7 h, respectively. The derivatives 6a and 6b, but not 6c and 6d, were found to be cleaved quantitatively to the parent drug in rat serum and porcine liver ^ppsterase solutions at 37°C, and the observed half-lives are reported in Table II.

Kinetic data showed that 6a and 6b are stable enough in solution buffered at pH 7.4, their half-lives exceeding 6 h, but undergo a fast cleavage at conditions similar to those prevailing in vivo, providing propofol within few minutes. Conversely, compounds 6c and 6d were found to be stable enough both in buffer solution and less susceptible than the a-amino acid esters to esterases' catalysis. The observed high stability toward the chemical hydrolysis can be ascribed to the steric protection of the C(0)0- bond by bulky flanking diisopropyl groups on the phenyl ring. The fact that the proline (6a) and pipecolinic acid (6b) esters, similarly to a-amino acid esters or related short-chained aliphatic amino acid esters (Bund-gaard H, Larsen C, Thorbek P. 1984. Prodrugs as drug delivery systems. XXVI. Preparation and enzymic hydrolysis of various water-soluble amino acid esters of metronidazole. Int. J. Pharm. 18: 67-77.), are less resistant than compounds 6c and 6d to chemical and enzyme-catalyzed hydrolysis could result from either the electron withdrawing effect of the protonated amino group, which activates the ester linkage toward OH' attack, and (predominantly) the intramolecular catalysis (i.e., intramolecular N CO 1, 2 proton shift) by the neighboring amino group (protonated or not protonated) that promotes ester cleavage. The above finding demonstrates that prolinate 6a is highly soluble, stable in water at physiological pH and rapidly hydrolyzed in plasma. Therefore, compound 6a is an excellent prodrug of propofol for parenteral administration.

Example 6 In vitro [35S]TBPS Binding Assay Experimental set up: PA34264 18 Rats were killed by decapitation and their brains rapidly removed on ice. The cerebral cortex was dissected out and homogenized in 50 volumes of ice-cold 50 mM Tris-citrate buffer (pH 7.4 at 25DC) containing 100 mM CaCfe using a Polytron PT 10 (setting 5, for 20 sec) and centrifuged at 20.000 * g for 20 min. The resulting pellet was resuspended in 50 volumes of 50 mM Tris-citrate buffer (pH 7.4 at 258C) and used for the assay. [35S]TBPS binding was determined in a final volume of 500 mL consisting of. 200 \il of tissue homogenate (0.20-0.25 mg protein), 50 pL of [35S]TBPS (final assay concentration of 1 nM), 50 pi 2 M NaCI, 50 pL of drugs or solvent and buffer to volume. Incubations (25°C) were initiated by addition of tissue and terminated 90 min later by a rapid filtration through glass-fiber filter strips (Whatman GF/B, Clifton, NJ), which were rinsed twice with a 4 mL portion of ice-cold Tris-citrate buffer using a Cell Harvester filtration manifold (model M-24m Brandel, ^Gaithersburg, MD). Filter bound radioactivity was quantitated by liquid scintillation spectrometry. Nonspecific binding was defined as binding in the presence of I00 pM picrotoxin and represented about 10% of total binding. Protein content was determined by the method of Lowry20 using bovine serum albumin as a standard.

Results: Receptor binding.

GABAa receptors are sensitive targets for the action of propofol and other general anesthetics (Trapani G, Aitomare C, Sanna E, Biggio G, Liso G. 2000. Propofol in anesthesia. Mechanism of action, structure-activity relationships, and drug delivery. Curr. Med. Chem. 7:249-271. Franks NP, Lieb WR. 1994. Molecular and cellular mechanisms of general anaesthesia. Nature (Lond). 367: 607-614.). Binding of [35S]TBPS, a cage convulsant which ^|nds in close proximity to the chloride channel portion of the GABAa receptor at level of the picrotoxin binding site, constitutes a tool for studying the function of the GABAa receptor complex (Squires RF, Casida JE, Richardson M, Saederup E. 1983. [35S]t-Butylbicyclophosphorothionate binds with high affinity to brain-specific sites coupled to y-aminobutyric acid-A and ion recognition sites. Mol. Pharmacol. 23: 326-336.). Propofol, mimicking the action of other general anesthetics, such as alphaxalone and pentobarbital (Concas A, Santoro G, Serra M, Sanna E, Biggio G. 1991. Neurochemical action of the general anaethetic propofol on the chloride ion channel coupled with GABAa receptor. Brain Res. 542: 225-232.), reduces [35S]TBPS binding in a concentration-dependent manner. The ability of the compounds 6a-d to interact with [35S]TBPS binding sites was measured and compared with that of propofol. Affinity data, expressed as IC5o values (see Table II PA34264 19 above), demonstrates that compounds 6a and 6b are able to reduce the [^SjTBPS binding, with IC50 values one magnitude order higher than ICso value of propofol (4.17 pM). A similar effect, at doses higher than 100 \iM, was shown by nipecotate 6c, whereas compound 6d displayed an increase of [^SjTBPS binding, an effect similar to that of the antagonist bicu-culfine (Concas A, Sanna E, Mascia MP, Serra M, Biggio G. 1990. Diazepam enhances bicuculline-induced increase of t-[35S]butylbicyclophosphorothionate binding in unwashed membrane preparations from rat cerebral cortex. Neurosci. Lett. 112: 87-91.)- Fig. 3 shows the competitive inhibition curves of the examined cyclic amino acid ester derivatives. ^:xample 7 Electrophysiological measurements using Xenopus Oocytes Experimental set up: Complementary DNAs encoding the human a1, (32, and y2 GABAa receptor subunits were subcloned into the pCDM8 expression vector (Invitrogen, San Diego, CA). The cDNAs were purified with the Promega Wizard Plus Miniprep DNA Purification System (Madison, Wl) and then resuspended in sterile distilled water, divided into portions, and stored at - 20 "C until used for injection. Stage V and VI oocytes were manually isolated from sections of Xenopus laevis ovary, placed in modified Barth's saline (MBS) containing 98 mM NaCI, 1 mM KCI, 10 mM Hepes-NaOH buffer (pH 7.5), 0.82 mM MgS04, 2.4 mM NaHC03, 0.91 mM CaCl2, and 0.33 mM Ca(NOs)2 and treated with 0.5 mg/mL of collagenase Type IA ^Sigma) in collagenase buffer (83 mM NaCI, 2 mM KCI, 1 mM MgCh, 5 mM Hepes-NaOH ^^uffer, pH 7.5) for 10 min at room temperature, to remove the follicular layer. A mixture of GABAa receptor a1, [32, and y2 subunit cDNAs (1.5 ng/30 nL) was injected into the oocyte nucleus using a 10 pL glass micropipette (10-15 pm tip diameter). The injected oocytes were cultured at 19 DC in sterile MBS supplemented with streptomycin (10 ^ig/mL), penicillin (10 U/mL), gentamicin <50 pg/mL), 0.5 mM theophylline, and 2 mM sodium pyruvate. Electrophysiological recordings began approximately 24 h following cDNA injection. Oocytes were placed in a 100-pL rectangular chamber and continuously perfused with MBS solution at a flow rate of 2 mL/min at room temperature. The animal pole of oocytes was impaled with two glass electrodes (0.5 to 3 MQ) filled with filtered 3 M KCI and the voltage was clamped at -70 mV with an Axoclamp 2-B amplifier (Axon Instruments, Burlingame, CA). Currents were continuously recorded on a strip-chart recorder. Resting membrane potential PA342 64 usually ranged from -30 to -50 mV. Drugs were perfused for 20 s (7-10 s were required to reach equilibrium in the recording chamber). Intervals of 5 to 10 min were allowed between drug applications.

Results: Expression of human a1, (32i and y2 GABAa receptor subunit constructs in Xenopus-laevis oocytes was utilized in a voltage-clamp electrophysiological assay. Figure 4 shows the profiles of prolinate 6a and isonipecotate 6d. Consistent with binding data, GABA-evoked chloride currents elicited at cloned GABAa receptors were enhanced by 6a and diminished by 6d, both in a concentration-dependent manner with their maximal effects apparent at the concentration of 50 and 100 pM, respectively. ^pfaken together, the in vitro results demonstrated that all the ester derivatives 6a-d modulate GABAa receptors and possess intrinsic activity, though lower than that of the parent compound 1. Three amino acid esters, 6a-c, behaved like propofol, whereas isonipecotic acid ester revealed a bicuculline-like profile.

Example 8 In vivo screening of anticonvulsant and anesthetic activities Experimental set up: Rats received an intraperitoneal administration of propofol 1 (40 mg/kg, suspended in saiine with a drop of Tween 80 per 5 mL) and equimolar doses of compounds 6a and 6d. The anticonvulsant activity against pentylenetetrazole-induced seizures (55 mg/kg) was measured. Rats treated with pentylenetetrazole were considered "protected" when clonic or tonic sei-^lures and death did not occur.

The loss and re-establishment of righting responses, time of anesthesia induction, and sleeping time were also assessed. Rats (five per group) were treated with propofol and Diprivan®, both at a dose of 60 mg/kg, and an equimolar dose of compound 6a (105 mg/kg) and were continuously monitored for the loss of righting reflex (onset and duration). Propofol and its derivative 6a were dissolved in saline with a drop of Tween 80 per 5 mL and administered intraperitoneally in a volume of 0.3 mL per 100 g of body mass. Anesthesia induction (sleep onset) was defined as the time from drug administration to loss of righting reflex, whereas the sleeping time was the time from the loss of the righting reflex until the animals were plantigrade on all four legs. The significance of differences in behavioral data were analyzed by the AN OVA test.

PA34264 21 Results: Compounds 6a and 6d, displaying in vitro agonist and antagonist behavior, respectively, were tested in vivo for their anticonvulsant activity, whereas 6a, the derivative showing the best prodrug properties, was compared to propofol, administered either as an oil/water emulsion or as the commercial formulation Diprivan®, for the in vivo anesthetic activity. As shown in Table III, compound 6a, like propofol, protected completely the animals from the pentylenetetrazole-induced convulsions. In contrast with its in vitro GABAa antagonist behavior (see Figs 3 and 4), compound 6d appeared to protect animals, though only 60%, against convulsions. Among the hypotheses that may be formulated to explain this result, it may not be excluded that propofol isonipecotate 6d, stable in vitro in rat serum solution, can instead be hydrolyzed in vivo, releasing propofol and isonipecotic acid (Anderson A, Belleli D, Bennett DJ, Buchanan KJ, Casula A, Cooke A, Feilden H, Gemmel DK, Hamilton NM, Hutchinson EJ, Lambert JJ, Maldment MS, McGuire R, McPhall P, Miller S, Muntoni A, Peters JA, Sansbury FH, Stevenson D, Sundaram H. 2001. a-Amino acid phenolic esters derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABAa receptors. J. Med Chem. 41: 3582-3591 .),15 a known GABAa agonist, at anticonvulsant concentrations.

The anesthetic activity of compound 6a was investigated by measuring onset and duration of loss of righting reflex, in comparison with that elicited by the clinical propofol formulation (Diprivan®), and an oil/water emulsion of 1 in the presence of Tween 80 (Table III). Induction time of loss of righting reflex subsequent to intraperitoneal administration of compound 1 was notably shorter than that observed for Diprivan®. Compound 6a showed an induction time intermediate between the emulsion formulation and Diprivan®, whereas the duration of anesthesia followed the order propofol emulsion < 6a ~ Diprivan®. Therefore, compound 6a could be considered an efficacious anesthetic with the same duration of action of Diprivan® but a considerably shorter induction time than the marketed formulation.

Table III. In vivo anticonvulsant and anesthetic activities of propofol ester derivatives Anticonvulsant activity* Loss of righting reflex, LRR (sec) Compounds No. of rats protected/ tested Onset Duration PA34264 22 1 /10 114.4 ±9.5 2245 ±252 6a /10 162.7 ±4.3c'd 2403 ±592 6/10 Diprivan® 289 + 14.8C 3895±1113 ^Protection against clonic and tonic seizures induced by pentylenetetrazole (55 mg/kg, i.p.). Compounds 6a and 6d were tested at doses equimolar to 40 mg/kg propofol. <b'Anesthetic activity measured as onset and duration of LRR (mean ± s.e.m.). Compound 6a was administered i.p. at a dose of 105 mg/kg equimolar to 60 mg/kg dose of propofol; <e)p < 0.01 vs. propofol-treated animals, (d)p < 0.01 vs. Diprivan-treated animals.

Example 9: Synthesis of Saccharide-conjugates of Propofol In a 50ml round bottom flasklml of propofol has been mixed with 2,5ml of TEA at room temperature. When the mixture looked homogeneous 5,5 mmol of succinnic anhydride have been added. The reaction was allowed to proceed under moderate stirring conditions for 22h. The reaction run could be followed by TLC monitoring or simply looking at the disappearance of succinnic anhydride whose solubility in the mixture isn't high, so most of it remains in the reaction vessel as a white solid.. After 22h the reaction was stopped, the solution looked brownish. After elimination of most of the TEA under vacuum, 10ml of 0,2N HCI were added to the solution which was vigorously stirred and kept in an ice bath for 30min. Thereafter a white swaying precipitate was removed from the reaction by filtration on a proper funnel filter. The precipitate dissolved once more in EtOH was precipitated a second timeby adding cold water, filtrated and kept at-20°C. —■ ^^Three grams of lactobionic acid were dissolved in 5ml of warm DMSO (~70°C). After the complete dissolution 7,5 mmol of mono chloride salt of hydrazine were added to the reaction vessel. The solution was stirred at 45°C for 20h. The proceeding of the hydrazide formation was monitored using TLC coupled with a ninhydrin test to reveal the presence of amino groups. The protonated amine looked yellow in the ninhydrine test. When the reaction looked complete, an excess of water was added and NaOH 0,1 dropwise until pl-MO. The mixture was frozen and lyophilised. The dry product can be dissolved in water and lyophilised once more to eliminate the last traces of DMSO.

In alternative, the reaction mixture can be diluted once with water, lyophilised and then incubated overnight on AgC03 to eliminate the chloride ions. Before making the last PA34264 23 lyophilisation a short passage through cation exchanger resins is needed to get rid of possible Ag+ ions.

One mmol of the succinnic acid mono-propofol ester and 370mg of the lactobionic acid hydrazide were dissolved in 3ml of DMF and stirred at room temperature. A 1:1 molar amount of DCC was added to the solution and the temperature was decreased to 0°C. The reaction was allowed to run one hour under these conditions before switching gradually the temperature to 25°C. The reaction was monitored by TLC coupled with a ninhydrin test. The disappearing of the amino functions indicated the end of the reaction (normally after 2h). The reaction was then stopped by adding dilute HCI. The precipitate was washed three ^iimes with cold water and then eliminated. The aqueous fractions were frozen and lyophilised. The purity of the product has been checked by TLC.

Propofol - Maltotriose prodrug In 10ml of a 3:1 DMSO:MeOH mixture were dissolved 200mg of Propofol, a three times molar excess of maltotrionic acid, and a catalytic amount of DMAP (dimethylamino pyridine). The solution was left stirring at room temperature for 10min. In a separate vessel 350mg of DCC were dissolved in 5ml of the same solution and added to the previous mixture dropwise in a time period of 10min. The reaction was allowed to run under the same conditions for 20h and then stopped and filtrated. The coupling product was ^recovered by precipitation in acetone (50ml) and washed several times with EtOH (100ml), ^^cOEt (100ml) and finally acetone (100ml). The reaction has been monitored by TLC and the purity of the product has been confirmed also by RP-HPLC on a C-18 column.

Propofol - oxHESlOkD prodrug In 10ml of a 5:1 DMSO:MeOH mixture were dissolved 200mg of Propofol, a three times molar excess of oxHESlOkD, and a catalytic amount of dimethylamino pyridine. The solution was left stirring at the temperature of 40°C. In a separate vessel 350mg of DCC were dissolved in 5ml of the same solution and added to the previous mixture dropwise in a time period of lOmin. The reaction was allowed to run under the same conditions for 30h and then stopped and filtrated. The coupling product was recovered by precipitation in PA3 42-54 24 acetone (50ml) and washed several times with MeOH (100ml), AcOEt (100mi) and finally acetone (100ml). The reaction has been monitored by TLC and the purity of the product has been confirmed also by RP-HPLC on a C-18 column.

Claims (29)

1. Claims: .1, Compound having the formula: wherein R1 Is a. cyclic or linegr amino acid and their, diastereomers or enantiomers in the form of their bases or salts, and wherein the amino acid is optionally further substituted.
2. 2, Compound according to claim. 1 wherein the amino acid is C-terminally linked to propofol or wherein the amino acid is N-terminally linked to propofol.
3. 3. Compound according to claim 1 or 2 having the formula vyherein the heterocyclic group comprises 4 to 5 methylene groups and wherein the heterocyclic group Is optionally further substituted.
4. 4.■ Compound according to claim 3, wherein the^amlno acid component is selected from proline, pipecolinic acid, nipecotic acid and isonipecotlc acid.
5. 5. Compound .according to. claim 4, wherein the amino acid component is selected from a-proline, a-plpecolinic acid, li-nlpecotlc add and Y-iso.nipecotic acid. .. 26 intellectual property office of n.z. - 8 JAN 2004 received
6. 6. Compound according to claim 5, wherein the amino acid component is selected from a-proline, a-pipecolinic acid, or (^-nipecotic acid.
7. 7. Compound according to claim 1 wherein the amino acid is selected from glycine, alanine, valine, leucine, isoleucine, glutamine, glutamic acid, asparagine, aspartic acid, cysteine, methionine, serine, threonine
8. 8. Compound having the formula wherein R2 denotes a saccharide with a reducing end group, preferably an aldose, and wherein X and Y denote linker groups and wherein the cartohydrate is optionally further substituted.
9. 9. Compound according to claim 8 r2— x-y— c wherein X = —HN—(CH2)—NH n=0 to 10 and wherein II • II Y - —C—(CH2)—C— 27 m= 0 to 5.
10. 10. A compound according to claim 9 wherein n=0.
11. 11. A compound according to claim 9 or 10 wherein m= 0 or 2.
12. 12. Compound according to any one of claims 8 to 11 wherein R2 is a monosaccharide, disaccharide oligosaccharide or polysaccharide comprising at least one moiety selected from allose, altrose, glucose, mannose, gulose, idose, galactose, talose, sucrose, lactose, maltose, isomaltose, cellobiose, maltobionic acid, and lactobionic acid.
13. 13. Compound according to claim 12 wherein R2 is maltriose, lactobionic acid or hydroxyethyl starch.
14. 14. Compound according to claim 13, wherein R2 comprises up to 40 lactobionic acid moieties.
15. 15. Compound according to claim 14 wherein R2 comprises 2 to 7 lactobionic acid moieties.
16. 16. Compound according to claim 13, wherein R2 comprises up to 40 maltotriose moieties.
17. 17. Compound according to claim 16 wherein R2 comprises 2 to 7 maltotriose moieties.
18. 18. Compound according to claim 11, wherein R2 comprises at least 2 hydroxyethyl glucose moieties wherein the hydroxyl ethyl glucose moieties may be substituted.
19. 19. A method for anesthetizing a non-human mammal, wherein a therapeutically effective amount of a compound according to any one of claims 1 to 18 is administered to said mammal.
20. 20. A method of treating convulsions in a non-human mammal, migraine or for inhibiting free radicals, wherein a therapeutically effective amount of a compound according to any one of claims 1 to 18 is administered to said mammal.
21. 21. A compound according to any of claims 1 to 18 for use as a medicament. intellectual property office of n.z. - 8 JAN 2004 received
22. 22. Use of a compound according to any of claims 1 to 18 for the preparation of a medicament for anesthetizing a mammal. 28
23. 23. Use of a compound according to any of claims 1 to 18 for the preparation of a medicament for treating convulsions, migraine or for inhibiting free radicals in a mammal.
24. 24. A pharmaceutical composition comprising at least one of the compounds according to any one of claims 1 to 18.
25. 25. A pharmaceutical composition according to claim 24 comprising an a-proline propofol ester.
26. 26. Freeze-dried pharmaceutical composition comprising at least one of the compounds according to any one of claims 1 to 18.
27. 27. A freeze-dried pharmaceutical composition according to claim 26 comprising an a-proline propofol.
28. 28. A compound according to any one of claims 1 to 7 or claims 9 to 18 substantially as herein described with reference to the examples and figures thereof.
29. 29. A pharmaceutical composition or freeze-dried pharmaceutical composition according to claims 24, 25 or 26 and 27 substantially as herein described with reference to the figures and examples thereof. intellectual property office of n.z. - 8 JAN 2004 RECEIVED PA34264 Abstract The present invention relates to esters of propofol (2,6-diisopropylphenol), a method for anesthetizing a mammal as well as a method for treating convulsions, migraine or related diseases or for inhibiting free radicals in a mammal using said compounds. Furthermore, the present invention relates to said compounds for use as a medicament and the use of said compounds for the preparation of a medicament for anesthetizing a mammal or for treating convulsions, migraine or related diseases or for inhibiting free radicals in a mammal.