Water soluble prodrugs of hindered alcohols or phenols

Description of the invention

The present invention relates to novel water-soluble aliphatic prodrugs of a sterically injected drug containing a hydroxyl group. In particular, the present invention relates to novel water-soluble propofol drug phosphonooxymethyl ethers, their use, their preparation process, and pharmaceutical compositions containing the new compounds. The present invention also relates to novel intermediates used to prepare the final prodrugs as well as to the method of their preparation.

Successful delivery of a pharmaceutical to a patient is of great importance in the treatment of disease. However, the clinical use of many drugs with known properties is limited by their very low water solubility. Due to their low solubility in water, these drugs have to be formulated with co-solvents as pharmaceutical carriers containing surfactants. These surfactants have been shown to lead to severe side effects in humans, limiting the clinical safety of these drugs and thus the treatment options for serious diseases.

An example of sterically injected, poorly water-soluble phenol is the anesthetic propofol.

<img file="PL198141B1_D0001.tif" />

propofol (2,6-diisopropylphenol)

Propofol is formulated for clinical use intravenously as an o / w emulsion. Not only is propofol poorly soluble in water, it also causes pain at the injection site. This pain must be relieved by the use of lidocaine. Due to the fact that it is formulated as an emulsion, adding other drugs is difficult and questionable, and changes to the physical form, such as increasing the size of the oil droplets, can lead to pulmonary embolism. A water-soluble and stable prodrug of propofol would have many advantages. This form could be a plain aqueous solution which could be mixed with other drugs. If the prodrug as such did not cause pain, the prodrug could be more patient friendly and, as a result, there would be no carrier toxicity.

The present invention provides a water-soluble drug form of propofol. The compounds of the invention are the free acid phosphonooxymethyl ethers of propofol and their pharmaceutically acceptable salts. The solubility of the acid and salt in water facilitates the preparation of pharmaceutical forms. All prodrugs of the invention show excellent water solubility compared to their respective parent drugs.

The invention relates to novel water-soluble phosphonoxymethyl derivatives of propofol represented by the general formula I:

<img file="PL198141B1_D0002.tif" />

Formula I above represents a derivative of ROH where ROH is the drug propofol. In the above formula, lm is an integer of at least 1, n is an integer of 1 or 2. R¹ is a hydrogen or alkali metal ion, including sodium, potassium or lithium, or a protonated amine or a protonated amino acid or any other pharmaceutically acceptable cation. Following intravenous or oral administration, the derivatives of formula I are converted back to the parent drug by hydrolysis and / or phosphatase.

It is therefore an object of the present invention to develop water-insoluble drug derivatives which exhibit good activity and water solubility.

It is a further object of the invention to provide pharmaceutical compositions of these water-soluble compounds containing an amount of a compound of formula I and a pharmaceutically acceptable carrier.

A further object of the invention is to develop propofol derivatives which act as potential prodrugs, which have good stability at pharmaceutical formulation pH values, but which disintegrate rapidly in vivo under physiological conditions.

Brief description of the figures of the drawing

Figure 1 illustrates the in vivo enzymatic conversion of the propofol prodrug to propofol.

Figure 2 illustrates the change in blood concentration of propofol over time after administration of the propofol or Diprivan® prodrug in a dog study.

In this specification, unless otherwise indicated or otherwise apparent from the context, the following definitions apply.

"Phosphono is the group -P (O) (OH) 2, and" phosphonooxymethoxy or "phosphonooxymethyl ether is generally the group -OCH2OP (O) (OH) 2. "Methylthiomethyl refers to the group -CH2SCH3. The present invention also includes compounds in which n = 2 such that "phosphonodi (oxymethyl) ether is -OCH2OCH2OP (O) (OH) 2.

"Phosphono protecting groups are moieties that can be used to block or protect phosphono functional groups. Preferably, such protecting groups are groups that can be removed by methods not substantially affecting the rest of the molecule. Suitable phosphonooxy protecting groups include, for example, benzyl (designated with "Bn), t-butyl, and allyl.

"Pharmaceutically acceptable salt means a metal or amine salt of an acidic phosphonic group in which the cation does not contribute significantly to the toxicity or biological activity of the active compound." Suitable metal salts include lithium, potassium, sodium, calcium, barium, magnesium, zinc and aluminum salts. The preferred salts are the sodium and potassium salts. Suitable amine salts are, for example, the salts of ammonia, tromethamine, triethanolamine, ethylenediamine, glucamine, N-methylglucamine, glycine, lysine, ornithine, arginine, ethanolamine to name but a few. Preferred amine salts are the salts of lysine, arginine, N-methylglucamine and tromethamine.

In the description and in the claims, the term -OCH2OP (O) (OH) 2 includes both the free acid and its pharmaceutically acceptable salts, unless the context expressly indicates that it is a free acid.

A first aspect of the invention relates to propofol prodrugs represented by formula I:

<img file="PL198141B1_D0003.tif" />

wherein:

RO- stands for the rest of propofol,

R¹ is a hydrogen or alkali metal ion or a protonated amine or a protonated amino acid,

R² is a hydrogen or alkali metal ion or a protonated amine or a protonated amino acid, m is an integer of at least 1, n is an integer from 1 or 2; and pharmaceutically acceptable salts thereof.

The alkali metal ion of R1 and R2 may each be independently selected from the group consisting of sodium, potassium and lithium.

PL 198 141 B1

An embodiment of a compound of formula I is a compound of formula

<img file="PL198141B1_D0004.tif" />

wherein Z is selected from the group consisting of hydrogen, alkali metal ion and amine, and pharmaceutically acceptable salts thereof.

One embodiment of the invention is a compound of the above formula wherein n is 1 and each Z is independently selected from the group consisting of sodium, tromethamine, triethanolamine, triethylamine, arginine, lysine, ethanolamine, and N-methylglucamine.

The invention also relates to a pharmaceutical composition comprising an effective amount of a compound of formula I as defined above and a pharmaceutically acceptable carrier, especially a composition as defined for use as a medicament.

The invention also relates to the use of a compound of formula I as defined above for the manufacture of a medicament, in particular a medicament intended for oral administration or a medicament intended for parenteral administration.

In a preferred embodiment, the above use relates to a medicament having an anesthetic effect.

Derivatives of formula I can be prepared directly from propofol (represented by © -OH according to the reaction sequence outlined in Scheme 1:

Scheme 1

OH -► --► (r) —O^{/ X}OP (OXOR)₂

ΠΙ IV ο ^ ί ^ οζχοζ) · * II where ROH stands for propofol. It is understood that the above route is only one of several alternative possibilities. These alternative routes will become apparent upon reviewing the following disclosure and examples.

The invention also relates to a process for the preparation of a compound of formula I as defined above, which comprises removing the phosphono protecting group from a compound of formula IV:

Wherein Y is a protecting group, a phosphono group, and n is an integer equal to 1 or 2, and the product is isolated.

The invention also relates to an intermediate of formula IV:

<img file="PL198141B1_D0005.tif" />

wherein Y is a phosphono protecting group and n is an integer equal to 1 or 2.

The phosphono protecting group may be selected from the group consisting of benzyl, t-butyl, allyl, and other acceptable phosphate protecting groups.

The invention also relates to a process for the preparation of a compound of formula I as defined above, which comprises reacting a compound of formula III

<img file="PL198141B1_D0006.tif" />

with N-iodosuccinimide and a protected phosphoric acid of formula HOP (O) (OY), where Y is a protecting group, a phosphono group and the product is isolated.

In a preferred embodiment, the phosphono protecting group is selected from the group consisting of benzyl, t-butyl, and allyl.

The invention also relates to an intermediate of formula III

<img file="PL198141B1_D0007.tif" />

The invention also relates to a process for the preparation of the compound of formula III above, which comprises reacting propofol with chloromethyl methyl sulfide in the presence of sodium hydride, and isolating the product.

The preparation of the compound of formula I is illustrated in detail in the examples below.

In a first step, the free hydroxyl group of propofol is converted to the corresponding methylthiomethyl ether -OCH2SCH3. This conversion can be accomplished by reaction with chloromethyl methyl sulfide.

In the second step of the reaction sequence, the methylthiomethyl ether is converted to the corresponding protected phosphoxymethyl ether.

In the third step of the reaction sequence, the phosphono group protection is removed. Deprotection is performed by conventional methods well known in the art, such as acid or base catalyzed hydrolysis, hydrogenolysis, reduction and the like. For example, hydrogenolysis can be used to remove the benzyl protecting group from the phosphono group. The deprotection methodology is

Described in standard texts such as TW Green and PGM Wutz, Protective groups in organic synthesis, J. Wiley publishers, New York, 1991, pp. 47-67.

Base salts of a compound of Formula II can be prepared by conventional techniques, including contacting the free acid of compound II with a metal base or amine. Suitable metal bases include sodium, potassium, lithium, calcium, calcium, barium, magnesium, zinc and aluminum hydroxides, carbonates and bicarbonates, and suitable amines include triethylamine, ammonia, lysine, arginine, N-methylglucamine, ethanolamine, procaine, benzatin, dibenzylamine, tromethamine (TRIS), chloroprocaine, choline, diethanolamine, triethanolamine and the like. The base salts can be further purified by chromatography followed by lyophilization or crystallization.

The compounds of the invention are propofol phosphonooxymethyl ethers. The pharmaceutically acceptable salt form exhibits improved water solubility over the parent compounds, which allows for more convenient preparation of drug forms. Without being bound by theory, it is believed that the phosphonooxymethyl ethers of the invention are prodrugs of the parent compound; the phosphonooxymethyl moiety in contact with the phosphatase in vivo cleaves to give the parent compound. As indicated above, the compounds of the invention are effective pharmaceuticals or therapeutic agents.

An anesthetist skilled in the art of anesthesia will be able to judge, without undue experimentation, an appropriate treatment protocol for administering a compound of the invention. The dose, regimen and schedule of administration of the compounds of the invention are not particularly limited, and will depend on the particular compound employed. Thus, the compound of formula I may be administered by any suitable route of administration, preferably parenterally; the dose may, for example, be in the range of about 0.5 to 10 mg / kg body weight, administered according to procedures for inducing or maintaining general anesthesia. Alternatively, a compound of formula I may be administered by parenteral infusion, at a dose, for example, in the range of 2 pg / kg / min to 800 pg / kg / mm, administered according to the procedures of general anesthesia maintenance, initiation and maintenance of MAC sedation or initiation and maintaining the sedation of the ICU.

The invention also provides pharmaceutical compositions comprising a pharmaceutically effective amount of a compound of Formula I in combination with one or more pharmaceutically acceptable carriers, vehicles, diluents or adjuvants. For example, the compounds of the invention may be formulated into tablets, pills, powder mixes, capsules, injections, solutions, suppositories, emulsions, dispersions, food premixes, and other suitable forms. They can also be manufactured in the form of sterile solid compositions, for example, lyophilized, and combined with other pharmaceutically acceptable carriers if necessary. Such solid compositions may be reconstituted with sterile water, saline, or a mixture of water and an organic solvent such as propylene glycol, ethanol, and the like, or other sterile injection medium immediately prior to use of the parenteral preparation.

Typical pharmaceutically acceptable carriers are, for example, mannitol, urea, lactose, non-reducing sugars, potato and corn starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly (vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl benzoate. , calcium carbonate, sodium carbonate, gelatin, potassium carbonate, silicic acid. The pharmaceutical preparation may also contain non-toxic excipients such as emulsifying, preserving, wetting agents and the like, such as, for example, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene monostearate, glyceryl tripalmitate, sodium dioctyl sulfosuccinate and the like.

In the following experimental procedures, all temperatures are given in degrees Celsius (C) unless otherwise indicated. The nuclear magnetic resonance characteristics report chemical shift values ​​(δ) in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. The relative surface area reported for the various shifts in the proton NMR spectral data corresponds to the number of hydrogen atoms of a given functional type in the molecule. The type of shifts in terms of the multiplicity are given as wide singlet (bs), wide doublet (bd), wide triplet (bt), wide quartet (bq), singlet (s), multiplet (m), doublet (d), quartet (q ), triplet (t), doublet of doublets (dd), doublet of triplets (dt) and doublet of quartets (dq). As solvents for making NMR spectra, acetone-d6 (deuterated acetone), DMSO-d6 (perdeuterodimethylsulfoxide), D2O (deuterated water), CDCL (deuterochloroform) and other common deuterated solvents were used.

The abbreviations used are common abbreviations used in the art. Some of them are: MS (mass spectrometry); HRMS (high resolution mass spectrometry); Ac (acetyl); Ph (phenyl);

PL 198 141 B1

FAB (fast atom bombardment); min (minute); h (hour (s)); NIS (N-iodosuccinimide); DMSO (dimethylsulfoxide); THF (tetrahydrofuran).

The following examples are intended to illustrate the synthesis of representative compounds of the invention and are not intended to limit the scope of the invention in any way. One skilled in the art will be able to adapt these methods, without undue experimentation, to the synthesis of compounds falling within the scope of the invention but not specifically mentioned. For example, specific salts have been used in the following examples, however, these salts are not to be construed as limiting. An example of such a situation is the repeated use of silver dibenzyl phosphate. Tetraalkylammonium salts such as tetramethylammonium salts can be used in place of the silver salt.

Examples

I. Synthesis of O-phosphonooxymethylpropofol

<img file="PL198141B1_D0008.tif" />

<img file="PL198141B1_D0009.tif" />

To a stirred suspension of sodium hydride (150 mg, 6.2 mmol) in dry HMPA (10 ml) under argon, propofol (1.1 ml, 97%, 5.7 mmol) was added dropwise over 15 minutes. The reaction mixture was then stirred at room temperature for an additional 30 minutes. To this mixture, chloromethyl methyl sulfide (550 µΙ, 95%, 6.2 mmol) was added dropwise and then stirred at room temperature. After 20 h, the reaction mixture was partitioned with stirring between water (10 ml) and benzene (20 ml). The aqueous layer was separated and extracted with benzene (10 ml). The benzene fractions were combined, washed with water (2 x 3 ml), dried over sodium sulfate and evaporated under reduced pressure. The resulting oily residue was subjected to column chromatography (silica gel, hexane then 4: 1 hexane / chloroform) to give 1.15 g (85% yield) of the title compound as a colorless oil.

EIMS^: [M⁺L m /^with 238.

¹H NMR (300 MHz, CDCl 3, δ): 1.24 (d, J = 6.9 Hz, 12H), 2.37 (s, 3H), 3.37 (hept, J = 6.9 Hz, 2H ), 4.86 (s, 2H), 7.12 (s, 3H). ⁿC NMR (75 MHz, CDCl 3, δ): 15.40, 23.98, 26.68, 78.12, 124.04, 125.05, 141.74, 152.20.

Ib. Synthesis of O-chloromethylpropofol

<img file="PL198141B1_D0010.tif" />

To a stirred solution of O-methylthiomethylpropofol (3.00 g, 12.5 mmol) in dry methylene chloride (30 mL) under argon, was added a 1M solution of SO2Cl in dry methylene chloride (12.2 mL, 12.2 mmol) at 5 ° C for five minutes. The reaction mixture was stirred for 10 minutes at the same temperature and then three hours at room temperature. The solvent was evaporated under reduced pressure and the brown oily residue was purified by flash column chromatography (silica gel, hexane / ethyl acetate 1:20) to give 2.36 g (83% yield) of the title compound as yellow oil.

CIMS ⁽NH^3): [M]⁴; m / z: 226^, [MH + NH³]^+, m /^with 244.

¹H NMR (300 MHz, CDCl3, δ): 1.22 (d, J = 6.9 Hz, 12H), 3.35 (hept, J = 6.9 Hz, 2H), 5.76 (s, 2H ). 7.15 (m, 3H). 1³C NMR (75 MHz, CDCl3, δ) 23.93, 26.84, 83.34, 124.34, 125.95, 141.34, 150.93.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (route 1)

<img file="PL198141B1_D0011.tif" />

A mixture of O-chloromethylpropofol (2.20 g, 9.7 mmol), silver dibenzyl phosphate (3.85 g, 10.0 mmol) and dry toluene (50 ml) was refluxed under argon for 45 minutes. The mixture was cooled to room temperature and filtered. After evaporation of the solvent, the oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 9: 1 then hexane / ethyl acetate 1: 1) to obtain 4.43 g (98% yield) of the title compound.

CIMS ⁽NH₃^): m / z 469^, ^ H ^ H ^ m /^with 486.

¹H NMR (300 MHz, CDCl₃, δ): 1.17 (d, J = 6.8 Hz, 12H), 3.33 (hept, J = 6.9 Hz, 2H), 5.00 (d, J = 7.8 Hz, 2H ), 5.01 (d, J = 7.8 Hz, 2H), 5.42 (d, J = 9.9 Hz, 2H), 7.12 (m, 3H), 7.32 (m, 10H ). 13 C NMR (75 MHz, CDCl₃, δ): 23.79, 26.57, 69.15, 69.23, 94.14, 94.20, 124.07, 125.62, 127.70, 128.44, 135.42, 135, 51, 141.50, 151.07.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (alternative route 1)

<img file="PL198141B1_D0012.tif" />

To a stirred solution of O-methylthiomethylpropofol (1.45 g, 6.08 mmol) in dry methylene chloride (15 ml) under argon at 0-5 ° C was added 1M SO2O2 in dry methylene chloride (6.5 ml, 6 , 5 mmoles) within five minutes. The reaction mixture was stirred for 10 minutes at 5 ° C and for 3 hours at room temperature. Then the solvent was evaporated under reduced pressure. The residual oil was dissolved in toluene (ACS grade, 20 mL), silver dibenzyl phosphate (3.50 g, 9.1 mmol) was added and the resulting mixture was refluxed for 45 minutes. The brown reaction mixture was cooled to room temperature and filtered. After evaporation of the solvent in vacuo, the oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 9: 1 then hexane / ethyl acetate 1: 1) to give 2.41 g (85% yield) of the title compound as a yellow oil . The product had the same Rf (TLC) and spectrum¹H NMR as authentic sample.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (alternative route 2)

<img file="PL198141B1_D0013.tif" />

PL 198 141 B1

Propofol (200 μl, 97%, 1.04 mmol) was added dropwise over 5 minutes to a stirred suspension of sodium hydride (41 mg of 60% dispersion in mineral oil, 1.02 mmol) in dry dimethoxyethane (1.5 ml) under argon. ) and the reaction mixture was stirred for an additional 15 minutes. The resulting homogeneous solution was added dropwise over 15 minutes to a stirred solution of chloroiodomethane (4.0 mL, 53 mmol) in dry dimethoxyethane (4 mL). This reaction mixture was stirred for two hours, filtered and then the solvent and excess chloroiodomethane were evaporated. The residual oil was dissolved in toluene (HPLC grade, 10 ml). To this solution was added silver dibenzyl phosphate (400 mg, 1.04 mmol) and the resulting mixture was refluxed for 10 minutes. After the reaction mixture was cooled to room temperature and filtered, the solvent was evaporated in vacuo. The oily residue was purified by silica gel flash column chromatography (9: 1 hexane / ethyl acetate, then 1: 1 hexane / ethyl acetate) to give 205 mg (42% yield) of the title compound as a yellow oil. This product had the same Rf (TLC) and spectrum¹H NMR (CDCl3, 300 MHz) as authentic sample.

In addition to the above reaction Ic (alternative route 2), it is understood that other reagents may be used depending on the target compound. For example, when a compound of Formula I is needed where n = 2, chloroiodomethane can be replaced with a compound such as X-CH₂-O-CH₂-Cl where X is a good leaving group.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (alternative route 3)

<img file="PL198141B1_D0014.tif" />

Powdered, activated 4A molecular sieves (100 mg) were added to a stirred solution of O-methylthiomethylpropofol (91 mg, 0.38 mmol) in dry methylene chloride (2 ml) under argon, followed by dibenzyl phosphate solution (127 mg, 0.45 mmol). ) and N-iodosuccinimide (95%, 102 mg, 0.43 mmol) in tetrahydrofuran (2 ml). The reaction mixture was stirred at room temperature for one hour, filtered and diluted with methylene chloride (30 ml). The resulting solution was washed with sodium thiosulfate solution (2 mL of a 1M solution), saturated sodium bicarbonate solution (3 mL), brine (5 mL), dried over sodium sulfate and magnesium sulfate, filtered and concentrated in vacuo. The oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 1: 1) to give 120 mg (67% yield) of the title compound as^yellowthese^gooh ^P.ro^dat^kt mta^Ł dad alone ^Rf (^TLC) ^and wtomo ¹IH ^NMR (^{CDCl3, 300 MH}z) i^kpr^óbkand authentic.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (alternative route 4)

<img file="PL198141B1_D0015.tif" />

To a solution of propofol (97%, 38 mg, 0.21 mmol) in methylene chloride (1 ml) was added tetrabutylammonium bromide (10 mg, 0.03 mmol) and a solution of sodium hydroxide (40 mg, 1 mmol) in water (0.1 2 ml). The heterogeneous mixture was stirred for 15 minutes. A solution of chloromethyl dibenzylphosphate (104 mg, 0.32 mmol) in methylene chloride (1 mL) was then added and the reaction mixture was stirred vigorously for eight hours. The mixture was then diluted with methylene chloride (10 ml), washed with water (2 ml), dried over sodium sulfate, filtered and evaporated in vacuo. Oily leave10

The solid was purified by silica gel flash column chromatography (hexane / ethyl acetate 20: 1 then hexane / ethyl acetate 10: 1) to give 44 mg (yield 45%) of the title compound as yellow^gooh Produfó irna^Ł the same ^Rf (TLC) ^and second ¹H NMR (CDC ^ 3.00 MHz) i^kpdo^kand authentic.

With respect to the above reaction Ic (alternative route 4) it should be understood that the reagent

<img file="PL198141B1_D0016.tif" />

ο

<img file="PL198141B1_D0017.tif" />

can generally be represented by the following formula:

<img file="PL198141B1_D0018.tif" />

<img file="PL198141B1_D0019.tif" />

wherein X is a leaving group, each R³ and r⁴ is a hydrogen atom, an organic group or an inorganic group and Y is a phosphate protecting group. Examples of leaving groups include chlorine, bromine, iodine, tosylate or any other suitable leaving group. Examples of phosphate protecting groups include protecting groups that temporarily block the reactivity of the phosphate group and allow for selective nucleophilic substitution. Examples of such blocking groups include, but are not limited to, benzyl, allyl, tert-butyl and isopropyl, ethyl and β-cyanoethyl.

Ic. Synthesis of O-phosphonooxymethylpropofol dibenzyl ester (alternative route 5)

<img file="PL198141B1_D0020.tif" />

Propofol (97%, 172 µL, 0.90 mmol) was added dropwise over 5 minutes to a stirred suspension of sodium hydride (36 mg 60% dispersion in mineral oil, 0.91 mmol) in dry dimethoxyethane (2 ml) under argon. The resulting mixture was stirred at room temperature for an additional 20 minutes. A solution of formaldehyde bis (dibenzylphosphono) acetal (500 mg, 0.88 mmol) in dry dimethoxyethane (3 mL) was then added to the mixture. This reaction mixture was stirred at room temperature for 20 hours and then at 70 ° C for 2.5 hours. The mixture was then filtered and the solvent evaporated in vacuo. The oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 10: 1 then hexane / ethyl acetate 1: 1) to give 29 mg (7% yield) of the title compound as a yellow oil. The product had the same Rf (TLC)^and in^idmo ^{1h nmr} (^{cdc | 3}, ^{300 MH}z) i^kpr^óbkand authentic.

Id. Synthesis of O-phosphonooxymethylpropofol

Palladium on carbon (10%, 20 mg) was added to a solution of O-phosphonooxymethylpropofol dibenzyl ester (115 mg, 0.245 mmol) in methanol (10 ml). This mixture was stirred under an atmosphere of hydrogen (1 atm) for 1.5 h. The catalyst was removed by filtration through celite and the filtrate was evaporated under reduced pressure to give 70.5 mg (100% yield) of the title compound as a colorless oil, unstable on standing at temperature. room.

FABMS^{- (}GLY^): [M^-H] 'm /^with 287.

¹H NMR (300 MHz, acetone-de, δ): 1.19 (d, J = 6.8 Hz, 12H), 3.46 (sext, J = 6.8 Hz, 2H), 5.45 (d , J = 9.7 Hz, 2H), 7.15 (m, 3H). 1³C NMR (75 MHz, acetone-de, δ): 24.2178, 27.1496, 94.63, 94.65, 124.08, 126.30, 142.46, 152.32.

le. Synthesis of O-phosphonooxymethylpropofol disodium salt

<img file="PL198141B1_D0021.tif" />

Water (5 ml) and palladium on carbon (10%, 300 mg) were added to a solution of O-phosphonooxymethylpropofol dibenzyl ester (1.05 g, 2.24 mmol) in tetrahydrofuran (100 ml). This mixture was stirred under hydrogen (1 atm) for one hour. The catalyst was removed by filtration through celite and the filtrate was treated with a solution of sodium carbonate hydrate (263 mg in 3 mL of water, 2.12 mmol). THF was evaporated under reduced pressure and the remaining aqueous solution was extracted with ether (3 x 3 ml). The aqueous layer was evaporated to dryness (argon stream or rotary evaporator) and the resulting solid was dried overnight in vacuo, washed with ether (4x4 ml), hexane (2x4 ml) and dried again in vacuo to yield 655 mg (93% yield) of the title compound as a white powder. FABMS^-(GLY^{): [}M.^-2Na + H.^]; m /^with 287.

¹H NMR (300 MHz, D2O, δ): 1.22 (d, J = 7.0 Hz, 12H), 3.46 (hept, J = 6.9 Hz, 2H), 5.27 (d, J = 7.5 Hz, 2H), 7.28 (m, 3H).

II. Synthesis of phosphonooxymethylating agents

IIa. Synthesis of chloromethylbenzyl phosphate θ m τ^Ο _ Η Li-do-l! and

Ag (- OP (() Bn&gt;₂ -—— ► Cl-CĄ-O-POBBn)²

To a boiling solution of chloroiodomethane (97%, 25 g, 0.14 mol) in toluene (H-LC grade, 30 ml) was added silver dibenzyl phosphate (7.0 g, 0.018 mol) in several portions over 20 minutes. Heating to reflux was continued for one hour. The reaction mixture was cooled to room temperature, filtered and the solvent was evaporated under reduced pressure. The oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 7: 3) to give 3.63 g (62% yield) of the title compound as yellow oil.

FABMS + ⁽NBA^{): [}MH^]+ m /^with 327 ¹H NMR (300 MHz, CDCl, δ): 5.10 (d, J = 8.0 Hz, 4H), 5.63 (d, J = 15.7 Hz, 2H), 7.36 (s, 10H ).

13 C NMR (75 MHz, CDCl 3, δ): 69.68, 69.75, 73.33, 73.42, 127.93, 128.51, 128.63, 135.07.

Ilb. Synthesis of dibenzyl (p-toluenesulfonomethyl) phosphate

<img file="PL198141B1_D0022.tif" />

Chloromethyl dibenzylphosphate (150 mg, 0.46 mmol) was added to a stirred solution of silver p-toluenesulfonate (600 mg, 2.15 mmol) in dry acetonitrile (3 mL) under an argon atmosphere.

PL 198 141 B1

The reaction mixture was stirred for 21 hours at room temperature, then the solvent was removed and the residue was extracted with diethyl ether (3 x 3 ml). The combined extracts were filtered, evaporated and dried in vacuo to afford 210 mg (99% yield) of the title compound as a white solid.

EIMS: [MH] + m /^with 463.

¹H NMR (300 MHz, CDCl 3, δ): 2.37 (s, 3H), 4.91 (2 xd, J = 7.9 Hz, 4H), 5.61 (d, J = 14.2 Hz, 2H), 7.29 (m, 12H), 7.78 (d, J = 8.4 Hz, 2H).

Regarding the above reaction IIb, as also explained above for Ic, the reagent of formula:

ff

Cl — CH2^_O ~ P (OBn) 2 can generally be represented by the following formula:

R3 O ^ P — O — Y ^XR4 ^ABOUT OY with all symbols having the meanings defined above.

lc. Synthesis of formaldehyde bis (dibenzyloxyphosphono) acetal^about about . -zs Ł ™ s I-CHb-I || at^{Ag +} O ^ OBn ^ - _9θ% ► BiO ^ PO-CiłrPOBBnh

Silver dibenzyl phosphate (3.0 g, 7.8 mmol) was added to a solution of diiodomethane (4 mL, 50 mmol) in dry toluene (15 mL). The resulting mixture was refluxed for 15 minutes under argon. The mixture was then cooled to room temperature and filtered. The solvent was then evaporated in vacuo. The oily residue was purified by silica gel flash column chromatography (hexane / ethyl acetate 1: 1 and then ethyl acetate) to give a yellowish oil which then crystallized to give 1.97 g (90% yield) of the title compound as a white solid, m.p. 39-42 ° C.

CIMS ⁽NH³): [MH] + m / z 569.

¹H NMR (300 MHz, CDCl 3, δ): 5.03 (d, J = 7.9 Hz, 8H), 5.49 (t, J = 14.3 Hz, 2H), 7.30 (m, 20H ).

1³C NMR (75 MHz, CDCl 3, δ): 69.54, 69.61.86.48, 127.88, 128.48, 128.55, 135.10, 135.20.

Biological research

The compounds of the invention are new pharmaceutical agents.

Representative compounds of formula I were tested in in vitro and in vivo conversion studies. In all of these studies, the prodrugs were converted to the pharmaceutically active parent drug.

(1) Evaluation of the water solubility of the propofol prodrug

The solubility of the propofol prodrug in water is approximately 500 mg / ml based on HPLC analysis of a saturated aqueous solution.

(2) In vitro conversion of propofol prodrug to propofol

In vitro conversion studies of the propofol prodrug to propofol were performed using alkaline phosphatase in glycine buffer at pH 10.4. 25 ml of a propofol prodrug solution in glycine buffer with a propofol concentration of 100 µg / ml was prepared. One milliliter was allowed for a time zero evaluation and the remaining 24 mL was placed in a 37 ° C bath. To 24 ml of propofol prodrug solution, 960 µl of an alkaline phosphatase solution in glycine buffer with a phosphatase concentration of 0.1 mg / ml was added and returned to the water bath. 1.5 ml samples were taken at 5, 10, 20, 30, 40, 60, 90, 120, 180, 240, 300 and 360 minutes. 10 µl of glacial acetic acid was added immediately to each sample to stop the enzymatic reaction. The samples were analyzed by HPLC to quantify the concentration of propofol prodrug and propofol. The in vitro conversion results are shown in Figure 1. These results indicate that the propofol prodrug is a substrate for alkaline phosphatase.

(3) General toxicity study in rats

An iv injection propofol prodrug formulation was prepared at a concentration of 68 mg / ml in 0.9% Sodium Chloride Injection, USP. This concentration corresponded to a concentration of 36 mg / ml propofol. The propofol prodrug solution was filtered through a 0.22 µm nylon membrane prior to administration.

A study of the propofol prodrug in rats was carried out on two male Harlen Sprague-Dawley strains weighing 820 g and 650 g. A rat weighing 820 g received 200 µl of an iv formulation of propofol prodrug (corresponding to 9 mg / kg propofol) via the tail vein. The blood sample was drawn from the tail vein (into a heparinized syringe) after approximately 12 minutes. A 650 g rat received a dose of Metaphane® mild sedative prior to administration of the propofol prodrug formulation. A 650 g rat was administered 125 µl of a propofol prodrug formulation via the tail vein and a blood sample was drawn from the same vein (with a heparinized syringe) after approximately 6 minutes. Blood samples of both rats were tested for propofol by HPLC.

The results of injection of the propofol prodrug in both rats were similar. Both rats became restless after injection but did not lose their righting reflex. Based on observation, rats fully recovered after injection of propofol. The presence of propofol was confirmed in blood collected from both rats by HPLC analysis. The rats showed no signs of discomfort from the propofol prodrug.

(4) Canine pharmacokinetic study

A pharmacokinetic study using either Diprivan® or a propofol prodrug was performed in a dog with an appropriate washout period between studies. Blood concentrations were determined using HPLC with fluorescence detection, and brain activity was monitored by two-lead electroencephalography (EEG). Prior to administration, the dog was blindfolded, cotton wool was placed in the ears, and limbs were tied to minimize movement and limit external stimuli so that the effect of propofol on the dog's brain waves could be monitored as effectively as possible.

A blood propofol concentration over time study was conducted on a Beagle dog weighing approximately 13 kg. Approximately 8 ml of blood was drawn prior to injection for standard curve and time zero determination of blood level. The dog received a volume of the Diprovan® or propofol formulation corresponding to 7 mg / kg of propofol by injection into the head vein.

2 ml blood samples were drawn from the head vein (other than that through which the formulation was administered), jugular or tibial (with heparinized syringe) at 1, 3, 5, 10, 15, 20, and 30 minutes after injection. Blood samples were also taken at 60, 90, 120, 180, 240, 300, 360, 480, and 1440 minutes. Blood samples were extracted to obtain propofol immediately after being collected from the dog. The dog was fasted for approximately 20 hours prior to receiving either the Diprivan® formulation or the propofol prodrug. After sampling at 120 minutes, the dog was allowed to drink water. Food was administered after sampling at 480 minutes. The dog was fed Hill's Science Diet Maintenance. The light / dark cycle consisted of 12 hours of light throughout the day.

The concentration of propofol in the blood samples was determined by HPLC with fluorescence detection. The results are shown in Fig. 2. The blood extraction and HPLC methods used were as described by Plummer (1987) with slight modifications. The sample preparation and test run procedures were as follows:

To a 1 ml blood sample were added 10 µl of thymol as an internal standard (20 µg / ml) and 1 ml of phosphate buffer (0.1 M, pH 7.3), shaking after each addition. Then 5 ml of cyclohexane was added and the samples were mixed at 75 rpm for 20-30 minutes. The organic layer was separated by centrifugation for 1 minute at approximately 2000 rpm. Approximately 4.5 ml of the organic layer was transferred to a tube containing 50 µl of diluted tetramethylammonium hydroxide (TMAH) solution at a concentration of approximately 1.8% (w / v). The solvent was evaporated to dryness under a stream of nitrogen and taken up in 200 µl of mobile phase A. The samples were centrifuged at 15,000 rpm for 30 seconds to separate the solids and the supernatant was injected on the HPLC. Samples for the standard curve were prepared by adding propofol to 1 ml of blood at a concentration of 5, 1.0.5, 0.1 and 0.01 µg / ml. These standards were treated analogously to the samples.

The HPLC system included the following Shimadzu components: LC-10AT pumps, SCL-10A controller, RF 353 fluorescence detector and SIL-10A autosampler. The following HPLC parameters were used: excitation at 275 nm and emission at 320 nm; flow 1 ml / min; injection volume 3-30 µl depending on propofol concentration. A Zorbax RX-C18 HPLC column, 15 cm x 4.6 mm diam, was used. ext., particle size 5 gm. Mobile phase A was a mixture of acetonitrile and 25 mM phosphate buffer, 15 mM TBAP, pH 7.1, 60:40 by volume. Phase

Mobile B was a mixture of acetonitrile, water and THF in a volume ratio of 80:10:10. Mobile phase B was used to clean the column after elution of thymol and propofol with mobile phase A (4.2 min. And 7.4 min. Respectively).

After injection of both formulations, the dog was found to be showing signs of anesthesia based on observation and EEG picture. The dog awoke from anesthesia with both formulations after 20-30 minutes. Blood levels of propofol following injection of the propofol prodrug were similar to those caused by Dipivan® injection .