WO2000024358A2 - Synthesis and uses of thiopentone enantiomers - Google Patents

Abstract

The present invention relates to the synthesis of R- and S- thiopentone and to an improved anaesthetic agent including R-thiopentone.

Description

Synthesis and uses of thiopentone enantiomers

Field of Invention

The present invention relates to the synthesis of R- and S- thiopentone and to an improved anaesthetic agent including R-thiopentone.

Background to the Invention

Thiopentone has been the principal injectable anaesthetic agent since the 1930's. Like many barbiturates, thiopentone contains a chiral centre and is synthesised and used clinically as a racemate consisting of equal quantities of the R- and S-enantiomers. These enantiomeric forms result from asymmetry of the alpha-carbon of the 5-(l-methylbutyl) side chain. Use of the racemic thiopentone in clinical medicine has overlooked differences in the pharmacology of the individual enantiomers, Enantioselective pharmacology can occur at any site in the body where a drug interacts with an endogenous chiral centre. Most often, this can be a binding protein in a receptor (thus affecting drug action), metabolising enzymes (thus affecting drug elimination) and/or other macromolecules (thus affecting drug distribution). With current methodology, it is not usually possible to predict from first principles to what extent different drug enantiomers will interact differently with proteins or enzymes: the differences in the kinetics and dynamics must be determined experimentally.

As well as being used as an anaesthetic agent, thiopentone has been an important agent for the treatment of severe head injury since the 1980's. It is used to manage the complications of traumatic head injuries and following neurosurgery, and to treat uncontrollable fitting.

The use of thiopentone, however, is subject to limitations due to direct effects on the heart muscle and prolonged recovery from anaesthesia or prolonged time to recovery of clinical responsiveness after infusion for cerebral protection.

The present inventors have found a method for the efficient large-scale synthesis of both S- and R-thiopentone. Further, via pharmacological and pharmacokinetic studies of enantiopure thiopentone, the present inventors have surprisingly found that R-thiopentone has a pharmacological advantage over the traditional clinically used racemic thiopentone. More specifically, the present inventors have found that R-thiopentone when used as an intravenously injectable anaesthetic agent has faster clearance from the body and is associated with relatively less depressant effects on the brain and heart muscle than either the racemate or S-thiopentone.

Summary of the Invention

In a first aspect, the present invention is directed to a process of preparing an enantiomer of thiopentone which includes the following steps: (a) providing an R- or S-citronellol according to the formula (I)

(I)

where Z is H or a protecting group

(b) oxidative-cleavage of the double bond of compound (I) to provide the aldehyde (II)

(II)

where Z is H or a protecting group

(c) reduction of the aldehyde and where Z is a protecting group, deprotection of the hydroxy group of compound (II) to provide the alcohol

(III)

(III)

(d) oxidisation of the alcohol (III) to the acid (IV) followed by esterification to give the ester (V)

(IV) (V)

where R=an alkyl group

(e) condensation of the ester (V) with dialkyl oxalate under basic conditions to give compound (VI)

(VI)

where R and R'= alkyl groups

(f) decarbonylation of compound (VI) to give malonate (VII) followed by ethylation to give compound (VIII)

(VII) (VIII)

where R and R' = alkyl groups

(g) condensation of compound (VIII) with thiourea to give R- or S- enantiomer of thiopentone (IX)

(IX)

The steps for preparing R- or S-thiopentone according to the first aspect of the invention are outlined in Figure 1.

Protected R- or S-citronellol (I) may be formed by converting R- or S- citronellol to the acetate or a similar carboxylic acid ester derivative by reaction with acetic acid or an acetic acid derivative such as acetic anhydride or with an alkylcarboxylic acid or alkylcarboxylic acid derivative in the presence of an acidic or basic reagent. For example, one of the following methods may be used:

(i) reaction of R- or S-citronellol with acetic acid or alkylcarboxylic acid in the presence of strong acid catalyst such as hydrochloric acid, sulfuric acid, /7-toluenesulfonic acid, boron trifluoride or zinc chloride

(ii) reaction of R- or S-citronellol with acetic anhydride or alkylcarboxylic acid anhydride in the presence of reagents such as pyridine or zinc chloride or (iii) reaction of R- or S-citronellol with acetyl chloride or alkylcarboxylic acid chloride alone or in the presence of bases such as pyridine, triethylamine, or finely powdered sodium bicarbonate.

Protected R- or S-citronellol (I) may also be prepared by reaction of the R- or S-citronellyl bromide with acetic acid or an alkali metal or alkaline earth metal salt of acetic acid.

Preferably, the oxidative-cleavage step (step b) is carried out using one of the following methods:

(i) reaction of compound (I) in methanol at -78°C with ozone followed by treatment with dimethyl sulfide or zinc/acetic acid (ozonolysis);

(ii) reaction of compound (I) in aqueous f-butanol, pyridine or dioxane with sodium periodate and a catalytic amount of potassium permanganate

(Lemieux-Rudloff oxidation);

(iii) reaction of compound (I) in aqueous dioxane or 80% aqueous acetic acid with sodium periodate and a catalytic amount of osmium tetroxide

(Lemieux-Johnson oxidation); or

(iv) reaction of compound (I) in aqueous acetone with sodium periodate and a catalytic amount of ruthenium tetroxide (Pappo-Becker oxidation).

Preferabfy, the reduction/deprotection step (step c) is carried out using one of the following methods:

(i) reaction of compound (II) in dioxane with hydrazine hydrate followed by heating, cooling and addition of potassium hydroxide and further heating

( Wolff -Kishner reduction);

(ii) reaction of compound (II) in dioxane with p-toluenesulfonyl hydrazine to give the tosylhydrazone followed by sodium borohydride reduction and base hydrolysis;

(iii) reaction of compound (II) in aqueous dioxane with hydrazine dihydrochloride followed by addition of hydrazine hydrate, heating, cooling and addition of potassium hydroxide and further heating. If step (iii) is used for the reduction then it is not necessary to first protect the hydroxy group of the citronellol starting material.

Preferably, the oxidation step (step d) is carried out by treating the alcohol (III) with aqueous potassium permanganate or chromic acid in aqueous sulfuric acid. Preferably, the esterification step (step d) is carried out by treating the acid (IV) with an alcohol such as methanol or ethanol, and an acid catalyst. The acid catalyst may be selected from the group consisting of sulfuric acid, -toluenesulfonic acid, boron trifluoride and zinc chloride.

Preferably, the condensation reaction step (step e) is carried out by treating the ester (V) with a base such as sodium ethoxide or sodium hydride in ethanol and dialkyl oxalate followed by treatment with aqueous acid.

Preferably, diethyl oxalate is used.

Preferably, the decarbonylation step (step f) is carried out by heating compound (VI).

Preferably, the ethylation reaction in step (f) is carried out with a base such as sodium ethoxide or sodium hydride/ethanol, in ethanol, DMF or

DMSO followed by the addition of an alkyl halide such as ethyl bromide or ethyl iodide.

Preferably the condensation reaction of compound (VIII) with thiourea in step (g) is carried out in sodium ethoxide or sodium hydride in either ethanol or DMSO with heating.

More preferably, the process of preparing R- and S-thiopentone includes the following steps:

(a) reacting R- or S-citronellol with acetic anhydride in pyridine to provide compound (I); (b) treating compound (I) in methanol with ozone followed by treatment with dimethyl sulfide (DMS) to provide aldehyde (II).

(c) treating aldehyde (II) with hydrazine hydrate followed by treatment with potassium hydroxide to provide the alcohol (III);

(d) treating the alcohol (III) with potassium permanganate to form the acid (IV) followed by treatment of the acid (IV) with ethanol and sulfuric acid to give ester (V);

(e) treatment of ester (V) with sodium hydride/ethanol in diethyl ether and diethyl oxalate followed by treatment with aqueous acid to give compound (VI); (f) heating compound (VI) to provide the malonate (VII). Treatment of the malonate (VII) with sodium hydride in DMF followed by treatment with ethyl iodide to give compound (VIII) ;

(g) condensation of compound (VIII) with thiourea in sodium hydride/ethanol to give R- or S-thiopentone (IX). In a second aspect, the present invention is directed to a process of preparing the R- and S- enantiomers of thiopentone, wherein the alcohol (III) according to the first aspect of the invention is alternatively formed by catalytic asymmetric hydrogenation of the allylic alcohol (X). Preferably, the asymmetric hydrogenation is carried out using a catalytic amount of a chiral transition metal complex, such as Ru-(S)-BINAP or Ru-(R)-BINAP, to lead to the formation of R- or S-thiopentone. The allylic alcohol (X) may be formed by:

(i) reaction of 2-pentanone with Wittig reagent prepared from ethyl bromoacetate and triethyl phosphite to give ethyl 3-methylhex-2E-enoate; followed by (ii) treatment of ethyl-3-methylhex-2E-enoate with a reducing agent such as lithium aluminium hydride to give E-3-methylhex-2-en-l-ol (X).

A preferred embodiment of the invention according to the second aspect of the invention is outlined in Figure 2.

In a third aspect, the present invention is directed to a process of preparing the R- and S- enantiomers of thiopentone, wherein the acid (IV) according to the first aspect of the invention is alternatively formed by asymmetric hydrogenation of the acrylic acid (XI). Preferably, the asymmetric hydrogenation is carried out using a catalytic amount of a chiral transition metal complex, such as Ru-(S)-BINAP or Ru-(R)-BINAP, to lead to the formation of R- or S-thiopentone. The acrylic acid (XI) may be formed by:

(i) reaction of 2-pentanone with Wittig reagent prepared from ethyl bromoacetate and triethyl phosphite to give ethyl 3-methylhex-2E-enoate; followed by (ii) hydrolysis of ethyl 3-methylhex-2E-enoate to give E-3-methylhex-2- enoic acid (XI).

A preferred embodiment of the invention according to the third aspect of the invention is outlined in Figure 3.

In a fourth aspect, the present invention is directed to a process of preparing the R- and S- enantiomers of thiopentone, wherein the diethylmalonate (VII) according to the first aspect of the invention is alternatively formed by asymmetric hydrogenation of the vinylidene dicarboxylate (XII). The enantioselectivity of the reduction may be modified by full or partial hydrolysis of the alkyl carboxylate group. Preferably, the asymmetric hydrogenation is carried out using a catalytic amount of a chiral transition metal complex, such as Ru-(S)-BINAP or Ru-(R)-BINAP, to lead to the formation of R-thiopentone. The vinylidene dicarboxylate (XII) may be prepared by:

(i) condensation of 2-pentanone with dialkyl malonate, for example diethyl malonate, using piperidine and acetic acid as a catalyst with the azeotropic removal of water; or

(ii) bromination of racemic dialkyl 2-(2-pentyl)malonate, for example diethyl 2-(2-pentyl)malonate, to give diethyl 2-bromo-2-(2-pentyl)malonate followed by dehydrobromination with, for example, sodium ethoxide. A preferred embodiment of the invention according to the fourth aspect of the invention is outlined in Figure 4.

In a fifth aspect, the present invention is directed to a process of preparing the R- and S- enantiomers of thiopentone, wherein the dialkyl 2- ethyl-2-(2-R-pentyl)malonate (VIII) according to the first aspect of the invention is alternatively formed by asymmetric hydrogenation of dialkyl 2- ethyl-2-(pent-2-en-2-yl)malonate (XIII). Preferably, the asymmetric hydrogenation is carried out using a catalytic amount of a chiral transition metal complex such as Ru-(S)-BINAP or Ru-(R)-BINAP to give R- or S- thiopentone. The enantioselectivity of the reduction may be modified by full or partial hydrolysis of the alkyl carboxylated group. Dialkyl 2-ethyl-2-(pent- 2-en-2-yl)malonate (XIII) may be prepared as follows:

(i) reduction of pent-3-en-2-one with a reagent such as sodium borohydride to give ρent-3-en-2-ol followed by reaction with phosphorous tribromide to give 2-bromopent-3-ene; and (ii) reaction of 2 bromopent-3-ene with the sodium salt of dialkyl 2-ethyl malonate gives dialkyl 2-ethyl-2-(pent-3-en-2-yl)malonate; followed by

(iii) rearrangement of the double bond under strong basic conditions such as potassium butoxide/t-butanol to give the more thermodynamically stable product, dialkyl 2-ethyl-2-(pent-2-en-2-yl)malonate (XIII).

A preferred embodiment of the invention according to the fifth aspect of the invention is outlined in Figure 5.

In a sixth aspect, the present invention is directed to a process of preparing the R- and S- enantiomers of thiopentone, wherein the dialkyl 2- ethyl-2-(2-R-pentyl)malonate (VIII) according to the first aspect of the invention is alternatively formed by asymmetric hydrogenation of diethyl 2- ethyl-2-(pent-l-en-2-yl)malonate (XIV). Preferably, the asymmetric hydrogenation is carried out with catalytic amounts of a chiral transition metal complex, such as Ru-(S)-BINAP or Ru-(R)-BINAP, to lead to the formation of R-thiopentone. Dialkyl 2-ethyl-2-(pent-l-en-2-yl)malonate (XIV) may be prepared as follows:

(i) epoxidation of 1 pentene to give 1,2-epoxypentane followed by reaction with hydrogen bromide to give 2-bromopentan-l-ol and treatment with acetic anhydride to give 2-bromopentyl acetate; and (ii) reaction of 2-bromopentyl acetate with the sodium salt of dialkyl 2- ethylmalonate to give dialkyl 2-ethyl-2-(l-acetoxypentan-2-yl)malonate followed by treatment with a strong organic base such as DBU to give dialkyl 2-ethyl-2-(pent-l-en-2-yl)malonate (XIV).

A preferred embodiment of the invention according to the sixth aspect of the invention is outlined in Figure 6.

In a seventh aspect, the present invention is directed to an improved intravenous injectable anaesthetic agent including R-thiopentone and/or its alkali metal or alkaline earth metal salt together with a pharmaceutically acceptable carrier. Preferably, the salt is a sodium salt.

Preferably, the R-thiopentone is obtained according to the first aspect of the invention. The thiopentone is preferably in a concentration range of 1.0 mg/mL to 100 mg/mL. More preferably, the concentration of R- thiopentone is 25 mg/mL.

The pharmaceutically acceptable carrier may be selected from one or more of the group consisting of water and basic solubilizing agent. Preferably, the basic solubilizing agent is sodium hydroxide, sodium bicarbonate and/or sodium carbonate. More preferably, the basic solubilizing agent is sodium carbonate.

In an eighth aspect, the present invention is directed to a method of anaesthetising a patient including the steps of administering to said patient an effective amount of R-thiopentone.

In a ninth aspect, the present invention is directed to a method of treating a patient with intracranial hypertension, abnormally increased or excessive cerebral blood flow and/or oxidative metabolism including the steps of administering to said patient an effective amount of R-thiopentone.

Preferably, the R- thiopentone is contained in an anaesthetic agent described in the seventh aspect of the invention. R-thiopentone is more rapidly eliminated from the body than racemic- and S-thiopentone. The present inventors have unexpectedly found that R- thiopentone has relatively less depressant effects on the brain and heart muscle when compared with racemic- and S-thiopentone. Due to these characteristics, R-thiopentone has been found to be a more suitable anaesthetic agent than racemic or S-thiopentone and is particularly useful when used in the treatment of acute neurological and neurosurgical emergencies including closed head injury, refractory status epilepticus due to its ability to reduce intracranial pressure, cerebral blood flow and oxidative metabolism.

A higher clearance from the body is demonstrated by the relatively lower concentrations of R-thiopentone in plasma over that of S-thiopentone and racemically administered thiopentone.

A relatively more favourable distribution into the central nervous system of R-thiopentone is demonstrated by its higher distribution coefficients (between tissue and plasma) compared to racemic or S- thiopentone.

The lower effect of R-thiopentone on the heart is exhibited by a lower relative concentration of R-thiopentone compared to that for both the S- and racemic-thiopentone in the heart compared to the brain.

By examining the enantioselectivity of thiopentone clearance and distribution into the CNS of rats, the present inventors found that plasma and

CNS tissue concentrations for S-thiopentone were approximately 10 -20% higher than those of R-thiopentone, corresponding to a higher clearance of R- thiopentone from the body. Further, it was found that halothane reduced the uptake of R-thiopentone into brain tissue enantioselectively. Thiopentone is known to cause depression of the force of contractility of the heart (negative inotropy). The effects are due to both local effects on the heart as well as effects on the central nervous system control of the heart and other reflex mechanisms affecting heart performance. By measuring myocardial contractility, the present inventors have surprisingly found that R-thiopentone has less effect on the heart than S-thiopentone.

The present inventors have established that the qualitative nature of electroencepholagram (EEG) changes are the same for RS-thiopentone, R- and S-thiopentone. Surprisingly, the present inventors have found that although qualitatively similar in effects, significant quantitative differences exist between RS-thiopentone and its enantiomers in the heart and CNS tissue. It has been found that the relative distribution into the heart compared to the brain was twice as high for S-thiopentone than for R-thiopentone. Further, the therapeutic index (ratio of lethal to anaesthetic dose) of R-thiopentone was considerably more favourable than either RS-thiopentone or S- thiopentone. This would seem to derive from a relatively greater distribution of R-thiopentone into CNS tissues and relatively less into the heart but wherein much larger doses of R- compared to S-thiopentone can be tolerated.

Studies investigating the effect of racemic thiopentone on the CNS activity using electroencephalogram (EEG) as a continuous measure of drug effect is known to have a biphasic activation-depression concentration effect relationship in both rat and humans. In order to determine whether the biphasic effect of RS-thiopentone on CNS activity resulted from a differential rate uptake of the enantiomers into the CNS, the mass balance of influx and efflux of thiopentone enantiomers from a sheep brain was studied. The present inventors surprisingly found that there was no significant difference in brain influx or efflux between the enantiomers suggesting that the biphasic effect was not due to differences between enantiomers in the rate of blood-brain equilibration. Both thiopentone enantiomers produced biphasic CNS effects consisting of initial EEG activation followed by depression and was found to be essentially the same as that for RS- thiopentone. Quantitative differences were, however, observed with RS- producing a greater EEG depression than the depression produced by R- but less than that produced by S-thiopentone.

Further, by carrying out microdialysis studies, the present inventors have found that although the rate of equilibration of thiopentone between plasma and brain extracellular fluid was slow, the differences in the potency between the thiopentone enantiomers cannot be ascribed to differences in their rates of equilibration across the blood-brain barrier.

In order that the present invention be more clearly understood, preferred forms will be described with reference to the following examples and figures.

Brief Description of the Drawings

Figure 1 outlines the process for the synthesis of R- and S-thiopentone. Figure 2 outlines a synthesis of R-3-methylhexanol. Figure 3 outlines a synthesis of R-3-methylhexanoic acid. Figure 4 outlines a synthesis of diethyl 2-(2R-pentyl)malonate.

Figure 5 outlines a synthesis of diethyl 2-ethyl-2-(2R-pentyl)malonate. Figure 6 outlines an alternate synthesis of diethyl 2-ethyl-2-(2R- pentyl)malonate.

Figure 7 shows the effect on the left ventricular pressure (dP/dt-_na- versus time for a range of doses of thiopentone. This shows the effects of thiopentone and its enantiomers on myocardial contractility.

Modes for Carrying out the Invention

The following examples illustrate the preparation of S-thiopentone in accordance with the first aspect of the invention and experimental details for studies of thiopentone and its enantiomers to support the second to fourth aspects of the invention.

The starting material S-citronellol is commercially available as are all other materials used in the process according to the present invention. Preparation of S-thiopentone: Preparation of S-citronellol acetate S-Citronellol (5 g, Merck Art. 818378) was mixed with acetic anhydride

(lOmL) and pyridine (5 mL) and the homogeneous mixture left at room temperature for 18 hours. The reaction products were washed with ice-cold water and extracted with petroleum spirit (bp 60-80°C). The petroleum extract was washed with 1 M HCl in 20% aqueous NaCl, washed 3 times with 20% aqueous NaCl, dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (5.9 g) identified as S-citronellol acetate. 1H-NMR spectrum (solvent CDC1₃, 300 MHz, δ (ppm)) Abbreviations are: s, singlet; d, doublet; t, triplet; m, multiplet; b, broad, δ 0.91 (3H, d, J = 6.5 Hz, 3-Me), 1.1-1.7 (5H, m. H-2, H-3, H-4), 1.60 (3H, s, 7-Me),L68 (3H, s, 7- Me), 1.98 (2H, m, H-5), 2.04 (3H, s, MeCO), 4.09 (2H, m, H-l), 5.10 (1H, m, H-

6); ¹³C-NMR spectrum (solvent CDC1₃, 75.4 MHz, δ(ppm)) δ 17.68 (MeCO), 19.44 {7 ,Me), 21.07 (3-Me), 22.21 (C-5), 25.41 (C-8), 25.74 (C-3), 29.50 (C-2), 35.45 (C-4), 63.06 (C-l), 124.59 (C-6), 131.35 (C-7), 171.24 (CO). Preparation of S-6-acetoxy-4-methylhexanal

S-Citronellol acetate (5.9 g) was dissolved in methanol (40 mL) and cooled to approximately -78°C using a dry-ice/acetone cooling bath then ozone in a stream oxygen gas from an ozone generator was bubbled through the stirred solution for 1 hour 15 minutes. After a further 1 hour dimethyl sulfide (4.5 mL) was added to the solution which kept at approximately -78PC for another 2 hours then at 0°C with an ice-bath for 2 hours and at room temperature for 18 hours. The methanol was removed by evaporation under reduced pressure and the residue partitioned between ethyl acetate and 20% aqueous NaCl. The ethyl acetate extract was washed 3 times with 20% aqueous NaCl, dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (5 g) identified as S-6-acetoxy-4- methylhexanal. 1H-NMR spectrum (solvent CDC1₃, 300 MHz, δ(ppm)) δ 0.91 (3H, d, J = 6.6 Hz, 4-Me), 1.5-1.7 (5H, m, H-3, H-4, H-5), 2.02 (3H, s, MeCO), 2.42 (2H, m, H-2), 4,09 (2H, m, H-6), 9.76 (1H, s, H-l). Preparation of S-3-methylhexanol S-6-Acetoxy-4-methylhexanal (5 g) was mixed with ethylene glycol (16 mL) and hydrazine hydrate (12 mL) and the solution stirred and heated at 110°C for 1 hour, cooled with an ice-bath and potassium hydroxide pellets (5 g) added. The solution was heated to reflux at 150-200°C and distillate collected with a stillhead temperature of 110-150°C. The distillate was partitioned between water and petroleum spirit (bp 60-80°C). The petroleum extract WHS washed with 1M HCl, washed 10 times with water, dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (2.4 g) identified as S-3-methylhexanol. 1H-NMR spectrum (solvent CDC1₃, 300 MHz, δ (ppm)) δ 0.89 (3H, d, J = 6.6 Hz 3-Me), 0.89 (3H, t, J = 7.1 Hz, 1.13 (1H, m, H-3), 1.25-1.45 (4H, m, H-4, H-5), 1.60 (1H, bs,

OH), 1.60 (2H, M, H-2), 3.68 (2H, m, H-l); ¹³C-NMR spectrum (solvent CDC1₃₎ 75.4 MHz, δ (ppm)) δ 14.36 (C-6), 19.62 (3-Me), 20.07 (C-5), 29.27 (C-3), 39.47 (C-4), 40.00 (C-2), 61.28 (C-l). Preparation of S-3-methylhexanoic acid S-3-Methylhexanol (10 g) was mixed with water (100 mL), potassium permanganate (29 g) and acetic acid (1 mL) and the mixture was stirred at room temperature for 48 hours. Methanol (10 mL) was added and the mixture stirred until there was no purple colour in the solution due to excess potassium permanganate. The black solid was removed by filtration through No. 1 filter paper and the filtrate was extracted with petroleum spirit

(bp 60-80°C). The aqueous phase was acidified with sulfuric acid (10 mL, 60%) and extracted twice with ethyl acetate. The ethyl acetate extract was dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (9.67 g) identified as S-3-methylhexanoic acid. 1H-NMR spectrum (solvent CDC1₃), 300 MHz, δ(ppm)) δ 0.89 (3H, t, J = 7.0 Hz, H-6),

0.96 (3H, d, J = 6.6 Hz, 3-Me), 1.15-1.40 (4H, m, H-4, H-5), 1.97 (1H, m, H-3), 2J5 (IH, dd, J = 14.9, 8.0 Hz, H-2), 2.35 (IH, dd, J = 14.9, 6.0 Hz, H-2'), 10.3 (IH, bs, COOH); ¹³C-NMR spectrum (solvent CDC1₃, 75.4 MHz, δ (ppm)) δ 14.16 (C-6), 19.67 (3-Me), 20.03 (C-5), 29.93 (C-3), 38.93 (C-4), 41.66 (C-2), 180.0 (C-l). Preparation of (-) ethyl 3S-methyIhexanoate

S-3-Methylhexanoic acid (22.87 g) was dissolved in ethanol (180 mL), sulfuric acid (9.6 mL) added and the mixture stirred at room temperature for 36 hours. Petroleum spirit (bp 60 - 80°C) (400 mL) and water (600 mL) was mixed with the reaction mixture. The petroleum spirit soluble fraction was separated, washed with water (200 mL), dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (24.9 g) identified as (-) ethyl 3S-methylhexanoate. 1H-NMR spectrum (solvent CDC1₃, 300 MHz, δ (ppm)) δ 0.89 (3H, t, J = 7.0 Hz, H-6), 0.93 (3H, d , J = 6.6 Hz, 3-Me), 1.15-1.40 (4H, m, H-4, H-5), 1.26 (3H, t, J = 7.1 Hz, Et), 1.97 (IH, m, H-3), 2.10 (IH, dd, J = 14.4, 8.1 Hz, H-2), 2.35 (IH, dd, J = 14.4 5.9 Hz,

H-2'), 4.13 (2H, q, J = 7.1 Hz, Et); ¹³C-NMR spectrum (solvent CDC1₃, 75.4 MHz, δ (ppm)), δ 14.16 (Et), 14.27 (C-6), 19.66 (3-Me), 20.01 (C-5), 30.11 (C- 3), 39.00 (C-4), 41.93 (C-2), 60.04 (Et), 173.34 (C-l). Preparation of ethyl 2-ethoxalyl-3S-methylhexanoate Sodium hydride (7.2 g, 60% in oil) was mixed with anhydrous diethyl ether (150 mL) and absolute ethanol (10 mL) added under an atmosphere of nitrogen. After reaction of sodium hydride with ethanol was complete, as indicated by cessation of hydrogen bubble formation, (-) ethyl 3S- methylhexanoate (10.39 g) and diethyl oxalate (15 mL) was added. The mixture was stirred under nitrogen for 2 hours and then the apparatus was set up for vacuum distillation. At a pressure of approximately 30 mm of Hg, diethyl ether was removed by distillation at approximately 25°C. The pot temperature was raised from 25°C to 80°C at 20°C/hour. Acetic acid (25 mL) was added with stirring and after 10 minutes, ice-cold water (100 mL) was added and the mixture extracted 3 times with petroleum spirit (bp 60-8CPC)

(3 x 200 mL). The petroleum spirit soluble fraction was separated, dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (10.24 g after subtracting the calculated oil content of 2.88 g) identified as a mixture of diastereoisomers of ethyl 2-ethoxalyl-3S- methylhexanoate. 1H-NMR spectrum (solvent CDC1₃, 300MHz, δ (ppm)) δ 0.86 (3H, t, J = 7.0 Hz, H-6), 0.90 (3H, t, J = 7.0 Hz, H-6), 0.96 (3H, d, J = 6.8 Hz, 3-Me), 1.01 (3H, d, J = 6.8 Hz, 3-Me), 1.15-1.45 (8H, m, H-4, H-5), 1.25 (6H, t, J = 7.2 Hz, Et), 1.37 (6H, t, J = 7.2 Hz, Et), 2.35 (2H, m, H-3), 3.95 (IH, d, J = 5.1 Hz, H-2), 3.97 (IH, d, J = 6.0 Hz, H-2), 4.19 (4H, q, J = 7.2 Hz, Et), 4.32 (4H, q, J = 7.2 Hz, Et).

Preparation of diethyl 2-(2S-pentyl)malonate

Ethyl 2-ethoxalyl-3S-methylhexanoate (55 g) was placed in a vacuum distillation apparatus with a reduced pressure of approximately 25 mm of Hg and the pot temperature was increased to 160°C. With the pot temperature slow rising to 165 °C a distillation fraction was collected and identified as (-) ethyl 3S-methylhexanoate. With the pot temperature rising from 165°C to 200°C the main distillation fraction was collected as a colourless liquid (39.7 g) identified as diethyl 2-(2S-pentyl)malonate. ¹H-NMR spectrum (solvent CDC1₃), 300 MHz, δ (ppm)) δ 0.89 (3H, t, J = 6.8 Hz, H-5'), 0.98 (3H, d, J = 6.8 Hz, H-l'), 1.15-1.45 (4H, m, H-3', H-4'), 1.27 (6H, t, J = 7.1 Hz, Et),

2.25 (IH, m, H-2'), 3.22 (IH, d, J = 8.1 Hz, H-2, 4.19 (4H, q, J = 7.1 Hz, Et); ¹³C-NMR spectrum (solvent CDC1₃, 75.4 MHz, δ (ppm)) δ 14.11 (Et), 14.12 (Et), 14.13 (C-5'), 20.00 (C-l'), 20.04 (C-4¹), 33.18 (C-2'), 36.60 (C-3'), 57.89 (C-2), 61.10 (Et), 61.15 (Et), 168.92 (CO), 169.08 (CO). Preparation of diethyl 2-ethyl-2-(2S-pentyl) malonate

Diethyl 2-(2S-pentyl)malonate (39.7 g) was added slowly to a stirred mixture of N,N-dimethylformamide (100 mL) and sodium hydride (60% in oil, 12 g) under an atmosphere of nitrogen at room temperature. Once hour later, iodoethane (30 mL) was added and after 5 minutes solid formed. The mixture was left standing under nitrogen for 18 hours then stirred while being cooled with an ice-bath. Acetic acid (50 mL) was stirred in then water (250 mL) added and the mixture extracted twice with light petroleum (bp 60- 80°C) (2 x 300 mL). The light petroleum extract was washed with water, dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a colourless liquid (47 g) identified as ethyl 2-ethyl-2-(2S-pentyl)malonate. 1H-NMR spectrum (solvent CDC1₃), 300 MHz, δ (ppm)) δ 0.87 (3H, t, J = 6.8 Hz, H-5'), 0.90 (3H, t, J = 6.8 Hz, H-2"), 0.94 (3H, d, J = 6.9 Hz, H-l'), 1.20 (2H, m, H-4'), 1.27 (6H, t, J = 7.1 Hz, Et), 1.49 (2H, m, H-3¹), 1.94 (2H, q, J = 6.8 Hz, H-l"), 2.06 (IH, m, H-2'), 4.19 (4H, q, J = 7.1 Hz, Et); ¹³C-NMR spectrum (solvent CDC1₃), 75.4 MHz, δ (ppm)) δ 9.3 (C-2"), 14.16 (2 x Et), 14.20 (C-5¹), 15.01 (C-l'), 21.37 (C-4¹), 26.89 (C-l"), 35.08 (C-2'), 36.60 (C-3'), 60.2 (2 x Et), 62.52 (C-2), 171.28 (CO), 171.47 (CO). Preparation of S-thiopentone

To anhydrous ethanol (250 mL), kept at 0°C with an ice-bath and maintained under a nitrogen atmosphere, was added sodium hydride (60% in oil, 22 g). After reaction of sodium hydride with ethanol was complete, as indicated by cessation of hydrogen bubble formation, ethyl 2-ethyl-2-(2S- pentyl)malonate (47.5 g ) and thiourea (27 g) was added and the mixture was heated to reflux under a nitrogen atmosphere for 40 hours. To the cooled reaction mixture was added light petroleum (bp 60-80°C) (200 mL) and water

(300 mL). The aqueous phase was separated and washed twice with light petroleum. After the addition of concentrated HCl (75 mL), the aqueous phase (pH 2) was extracted with light petroleum ethyl acetate (40:60) (500 mL). The light petroleum/ethyl acetate extract was dried with Na₂S0₄, filtered and concentrated under reduced pressure to give a yellow powder

(32.6 g). The solid was dissolved in dichloromethane, filtered through a bed of silica gel 60H (TLC grade, Merck Art.7736, bed 70 mm diameter x 30 mm) and recrystallised from a mixture of dichloromethane and light petroleum to give a pale yellow crystalline solid (23.7 g) identified as S-thiopentone. Melting point 152-152.5°C; [α]_D ²⁰ = -12.0° (2.2 g/100 mL, ethanol); 1H-NMR spectrum (solvent CDC1₃, 300 MHz, δ (ppm)) δ 0.87 (3H, t, J = 7.4 Hz, H-2"), 0.88 (3H, t, J = 7.0 Hz, H-5'), 1.05 (3H, d, J = 6.9 Hz, H-l'), 1.18 (2H, m, H-4'), 1.45(2H, m, H-3'), 2.13 (2H, q, T = 7.4 Hz, H-l"), 2.15 (IH, m, H-2'), 9.60 (2H, bs, NH); "C-NMR spectrum (solvent CDC1₃, 75.4 MHz, δ (ppm)) δ 9.86 (C-2"), 13.96 (C-l'), 14.37 (C-5¹), 20.74 (C-4¹), 28.66 (C-l"), 33.92 (C-2'), 42.98 (C-3'),

61.28 (C-5), 170.32 (CO), 170.68 (CO), 176.22 (CS); Chemical ionisation mass spectrometry (CIMS, methane reagent gas) m/z [M + 41]⁺, 283(7), [M + 29]⁺, 271(19), [M + 1]⁺, 243 (100), 213 (5), 173 (9).

All of the reactions carried out in the syntheses of the S-thiopentone enantiomer involved high yield preparation of intermediate liquid products which were readily isolated and purified by simple distillation. Relatively low volumes of solvents were required in the course of the syntheses and no purification by chromatography was necessary. The final product S- thiopentone was formed in high yield as a crystalline solid with no major by- products. The method is such that that the substance may be prepared on a large to very large scale from the commercially available chiral starting material.

Studies of thiopentone and its enantiomers:

1) Enantioselectivity of thiopentone distribution into CNS tissue:

(From paper entitled: "Enantioselectivity of thiopentone distribution into central neural tissue of rats- an interaction with halothane")

Background: Thiopentone is a racemate consisting of R-thiopentone and S-thiopentone enantiomers in equimolar amounts; these are known from previous work to possess pharmacologic and pharmacokinetic differences. This study examined the enantioselectivity of thiopentone clearance and distribution into central neural tissue of rats. Methods: Two groups of rats, initially conscious and restrained, and initially anesthetized with halothane were studied. Sequential steady state targets of

5, 10 and 20 μg/ml rαc-thiopentone were attained with a computer controlled infusion pump technique. Sequential arterial plasma and steady state samples of brain and spinal cord were assayed enantiospecifically for thiopentone. Animals: Young adult male Wistar rats (330 to 415g) were housed in groups of four, maintained on a constant 12/12 hour light dark cycle at 23°C, and allowed free access to food and water. After surgery, the animals were housed individually. Experimental procedures were performed within 4 to 8 days of surgery, when body weight had returned to within 5% of baseline values.

Preparative Surgery: Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous i.v. drug infusion and arterial blood sampling. Rats were anesthetized with pentobarbitone and ketamine: an anesthetic induction dose of pentobarbitone (30 mg/kg in 1 ml 0.9% saline), was given i.p. 5 minutes prior to ketamine (45 mg/kg, i.p. in 1 ml 0.9% saline). Body temperature was maintained by a heating pad and monitored with a rectal probe. A 1 cm thoracic incision was made just lateral to the midline, and the jugular vein and carotid artery were exposed. Silastic laboratory tubing, respectively 0.025" ID x 0.047" OD and 0.020" ID x 0.037" OD, was then inserted 2.5 cm into the jugular vein and 2.0 cm into the common carotid artery. Both cannulae were tunnelled under the skin and exteriorized above the neck, anterior to the shoulder blades. In order to maintain patency each cannula was filled with a solution of 6 g polyvinylpyrrolidone (molecular weight 40,000) in 5 ml 1000 i.u./ml sodium heparin. Post-operatively, rats were administered 0.9% saline (10 mL, s.c.) for hydra tion, amoxycillin trihydrate (85 mg/kg, i.m.) for antimicrobial cover, and buprenorphine (0J5 mg/kg, s.c.) for post-operative analgesia. A subsequent dose of amoxycillin (85 mg/kg, i.m.) was administered the following morning. Progressive bodyweights and fluid intake were recorded. Restrained conscious rats: For one week prior to the experimental procedure the animals were acclimatised to being immobilised for progressively longer periods in acrylic rat restrainers. On the day of the procedure sampling and infusion lines were attached, the rat was placed in the restrainer, and the thiopentone infusion procedure was subsequently commenced. Halothane anesthetized rats: Rats were placed in an induction chamber and anesthesia was induced with 5% halothane in oxygen at a flow rate of 1 1/min. Once induced, the head of the rat was rapidly placed in an anesthetic mask consisting of a 3 cm length of a 50 ml plastic syringe with an inlet for the gaseous anesthetic mixture and an outlet for the scavenger line. One end was sealed with masking tape, while a piece of latex rubber tubing was secured to the other end to form an airtight seal around the head of the rat.

A flow rate of 0.5 1/min containing 2.5% halothane was used while the rat was secured in the mask; the concentration of halothane was subsequently reduced to 1%. Infusion and sampling lines were then attached, the thiopentone infusion commenced at a target level of 5 μg/mL, with the rat maintained on 1% halothane. The oxygen flow was maintained at 0.5 1/min; halothane was reduced to 0.5% when the infusion was increased to a target level of 10 μg/mL, then ceased when increased to 20 μg/ml. Drug infusion procedure and blood sampling regimens: Studies were performed between 10.00 and 14.00 hours. Body temperature throughout the procedure was monitored with a rectal probe and maintained as close as possible to 37-38°C by a heating pad. At the commencement of each study, the previously implanted chronic indwelling venous and arterial cannulae were attached to 25 cm infusion and sampling lines made from relevant silastic laboratory tubing. Each animal was infused into the jugular vein cannula with (racemic) thiopentone (sodium salt, 10 mg/ml in 0.9% saline containing 2 i.u./ml heparin). The infusions were performed with a Harvard Apparatus model 22 pump, controlled by Stanpump software on an IBM PS/2-50 computer, to maintain constant target plasma concentrations of thiopentone of 5, 10, and 20 μg/ml sequentially. Target concentrations of 5 and 10 μg/ml were each maintained for 1 hour, while that of 20 μg/ml was maintained for 2 hours. The total dose delivered by the conclusion of the infusion was 84 mg/kg. The target drug concentration was reached rapidly using a "BET algorithm" and consists of the simultaneous sum of three components: a bolus to load the initial dilution volume to the required target concentration, a constant rate infusion to match the target concentration to clearance and an exponentially decreasing infusion matched to tissue saturation as described by Schwilden et al. The algorithm was implemented using previously described body weight normalised pharmacokinetic data for thiopentone in the intended population. The mamillary model parameters for thiopentone in the rat were: N, = 0.122 L.Kg^"1, k₁₀ = 0J425 min^"\ k₁₂ = 2.084 min^" \ k₁₃ = 0.310 min^"1, k₂₁ = 0.620 min^"1, k₃₁ = 0.048 min ¹ as reported by Gustafsson et al.

Arterial blood samples, each 0.1 mL, were taken at 0 (pre-drug), then at 2, 5, 10, 20, 40, 60, 62, 65, 70, 80, 100, 120, 122, 125, 130, 140, 160, 180, 210, and 240 minutes after commencing the drug infusion. Each sample was replaced with 3 volumes 0.9% saline given as an initial flush of 0.2 ml 0.9% saline followed by 0.1 ml 10 i.u./ml heparin in 0 9% saline. At the conclusion of the infusion, rats were sacrificed by cervical dislocation; the brain and spinal cord were rapidly removed. Tissue samples of frontal cortex, striatum, hippocampus, brachial intumescence, and sacral intumescence were taken and these, along with 50 μl plasma aliquots, were stored frozen at -20°C, for not longer than 4 weeks, until analysis of thiopentone enantiomer concentrations. Significant thiopentone decomposition does not occur under these conditions.

Plasma binding analysis: Plasma binding of the thiopentone enantiomers was determined by equilibrium dialysis against phosphate buffer (0.067M, pH 7.4) of rat plasma adjusted to pH 7.4 and spiked with rac- thiopentone to concentrations of 20, 40 and 100 μg/ml. Dialysis was performed at 37°C with gentle shaking for 8 hours in Plexiglas cells (2.5 cm diameter, 2.5 ml volume) separated by cellulose dialysis membrane that had been previously prepared by heating to boiling twice in deionized water.

Thiopentone enantiomer assays: Liquid chromatographic separation of the enantiomers was performed on a chiral stationary phase (AGP 100 mm x 4 mm column, ChromTech, Sweden) with a Waters 600 MS system employing spectrophotometric detection with a Waters 991 Photodiode Array Detector. Concentrations of R- and S-thiopentone were determined by the following modifications of our previously reported procedure (Huang JL, Mather LE, Duke CC: High Performance liquid chromatographic determination of thiopentone enantiomers in sheep plasma. / Chromatogr

1995; 673:245-250). The non-therapeutic 5-ethyl-5-hexylbarbituric acid was used as an internal standard in place of the previously described phenylbutazone and the extraction was scaled to small samples. To a 50 μl plasma aliquot in a 1.5 ml Eppendorf tube was added internal standard (50 μL, 50 μg/mL) and phosphoric acid (10 μL, 2 M). The sample was briefly mixed, ethyl acetate in hexane (1.1 mL, 5% v/v) was added, and the sample was vortex mixed (1 min). After centrifugation to separate the layers, the sample was frozen on dry ice (15 min), and the organic layer was decanted and evaporated to dryness in a rotary vacuum bench evaporator at 40°C. The residue was reconstituted in sodium phosphate (200 μL, 10 n M) containing isopropanol (30% v/v); 10 μl was injected onto the column. The mobile phase consisted of phosphate buffer (100 mM, pH: 6.3) in isopropanol (4.5%v/v). Detection was performed at 287 nm. Tissue samples were initially homogenised into 1 ml 100 mM Na₂HP0₄. (to give 50-100 mg/mL) prior to the addition of the internal standard (50 μL, 50 μg/mL) and an initial extraction with hexane (1.0 mL) which was discarded. The sample was subsequently acidified by the addition of H₃P0₄ (50 μL, 2 M) and as described above.

Statistical analysis: Plasma and tissue drug concentrations, areas under the plasma drug curves (AUCs, linear trapezoid calculation) and fractional plasma binding were compared between enantiomers using

Student's t-test for paired data. Enantiomeric bias was deemed if the ratio of the relevant parameter for S-thiopentone to that of R-thiopentone was significantly different from unity by Student's one-sample t-test. Multiple comparisons across drug concentrations in different regions were performed by two-factor analysis of variance controlling for factors of site and animal group. Comparisons between mean multiple values was made with post-hoc Scheffe F-tests. This approach is particularly robust when enantiomers were concurrently measured in samples subject to the same physiological conditions. Comparison of relevant parameters between the halothane anesthetized and restrained groups was performed using Student's t-test. The tests were performed using Statistix® version 4 (Analytical Software,

Tallahassee, FL. USA) on a personal computer.

Results: Clear demonstration of enantioselectivity was found in both groups. Concurrent total and unbound plasma concentrations of S-thiopentone were always approximately 10-20% higher than those of R-thiopentone, corresponding to a higher clearance of R-thiopentone. Similarly, concentrations of S-thiopentone in frontal cortex, striatum, and hippocampus and brachial and lumbar spinal cord were always approximately 20% higher than those of R-thiopentone. The distribution coefficients for brachial and lumbar spinal cord were 2-fold greater than those for brain. However, the relative tissue: plasma distribution coefficients were approximately 7-10% greater for R-thiopentone than S-thiopentone in both tissue types. Comparison of the two groups indicated that plasma concentrations were lower and tissue : plasma distribution coefficients of both enantiomers were approximately 20% lower in the halothane-anesthetized group with a slightly greater effect on R-thiopentone distribution than on S-thiopentone.

Conclusions: The present inventors concluded that the total body clearance of R-thiopentone was significantly greater than that of S-thiopentone, that halothane enantioselectively reduced the relative uptake of R-thiopentone into brain tissue, that tissue composition is important, in determining CNS tissue concentrations of thiopentone during prolonged infusions, and that halothane by way of cardiovascular effects, along with a "solvent" effect, may "trap" thiopentone in the tissues.

2) Effects of thiopentone and its enantiomers on the heart: (Paper entitled: "Pilot study of the effects of thiopentone and its enantiomers on myocardial contractility")

Background: Thiopentone is known to cause depression of the force of contractility of the heart (negative inotropy). The effects are due to both local effects on the heart as well as local effects on the central nervous system control of the heart and other reflex mechanisms affecting heart performance. At the time of this study, it was not known whether the effects of the thiopentone enantiomers and the racemate were equal as enantiomeric differences at the various sites of action could all contribute to the observed effect.

Methods: The effects of RS-thiopentone, R-thiopentone and S-thiopentone on myocardial contractility were determined in an adult female sheep that had previously been prepared with a left ventricular pressure transducer catheter and a catheter for drug injection with its tip in the left main coronary artery. The left ventricular pressure was measured and the maximum value of the first differential of the left ventricular pressure (dP/dt„_ιax) was calculated numerically and was used as an index of myocardial contractility. In this way, the local effects of the drugs on the myocardium could be observed without complications due to effects on the brain or other control mechanisms. Doses of (0, 7.5, 15, 30, 45, 60 and 75 milligrams) were scaled from fractions of those that might be injected intravenously doses by the coronary fraction of cardiac output (approximately 8%). The time course and the time integral (AUC) of the effect on dP/dt.,,-,.. were both determined. Results: It was found that the values of dP/d^-^. decreased as doses were increased from 0 to 75 milligrams and to 0 again. In every case the effects of R-thiopentone were no greater than either RS-thiopentone or S-thiopentone at the same dose (See Figure 7) and were normally less.

3) Equilibration of thiopentone enantiomers across the blood-brain barrier (Paper entitled: Microdialysis study of the equilibrium of thiopentone enantiomers across the blood-brain barrier) Background: Previous work has found that S-thiopentone is more potent than

R-thiopentone. To ascertain whether an enantiomeric difference in the rate of equilibrium across the blood/brain barrier could contribute to this difference, the plasma and brain apparent extra-cellular fluid (aECF) thiopentone concentrations of rats having infusion of RS-thiopentone were measured. Methods:

Overview: Two groups of animals were studied. In this first group the emphasis was placed upon determining enantioselectivity in the rate of attaining equilibrium across the blood-brain barrier by comparing serial concentrations of thiopentone enantiomers in plasma and brain microdialysate. In the second group the emphasis was placed upon measuring total and unbound plasma thiopentone enantiomer concentrations as well as muscle and fat concentrations as these are generally believed to be important sites of redistribution of thiopentone related to its duration of action. The studies were approved by the institutional Animal Care and Ethics Committee.

Two groups of rats underwent computer-controlled IV infusion with RS-thiopentone to a total enantiomer plasma concentration of 40 mg/L for 20 min (total dose 54.5 mg/kg). One group had serial sampling for 60 minutes of arterial blood and brain aECF from microdialysis probes placed in either striatum or hippocampus. The other group were sacrificed at 20, 40 and 60 minutes to determine the thiopentone enantiomer concentrations in plasma, plasma dialysate, CNS tissues, muscle and fat. Thiopentone enantiomer concentrations were determined by CSP - HPLC.

Animals and techniques: Young adult male Wistar rats (350-400 g) were housed in groups of four, maintained on a constant 12/12 hour light dark cycle at 23°C, and allowed free access to food and water. After surgery the animals were housed individually. Experimental procedures were performed within 3 to 5 days of surgery, when body weight had returned to within 5% of baseline values. Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous venous infusion and arterial sampling. For this surgery, the animals were anaesthetized with pentobarbitone (30 mg/kg, ip, in 1 mL 0.9% saline) given 5 minutes prior to ketamine (45 mg/kg, ip, in 1 mL 0.9% saline) and body temperature was maintained with a heating pad monitored with a rectal probe. The jugular vein and carotid artery were exposed through a 1 cm incision just lateral to the midline. Silastic laboratory tubing (respectively, 0.025 in ID x 0.047 in OD, and 0.020 in ID x 0.037 in OD, Dow Corning) was inserted then fixed 2.5 cm into the vein and 2.0 cm into the artery. The cannulae were tunnelled under the skin, externalized above the neck anterior to the scapulae and filled with a solution of 6 g polyvinylpyrrolidone (MW 40,000, Sigma Chemical Co) dissolved in sodium heparin (5 mL 1000 U/mL) to maintain patency. At the completion of surgery the animals were administered amoxycil in (85 mg/kg, im), buprenorphine (0J5 mg/kg, sc) and given 0.9% saline (10 mL, sc) as fluid replacement. A subsequent dose of amoxycillin (85 mg/kg, im) was administered the following morning. Post-operative body weights and fluid intake were monitored.

Microdialysis study: Microdialysis probes (CMA12 with a 3 mm dialysis membrane) were perfused with Ringer's solution (140 mM NaCl, 4 mM KC1, & 2.5 mM CaCl₂) delivered from a microsyringe (2.5 mL CMA) by a microinjection pump (CMA/100). The microdialysis probe was connected to the microsyringe and to the fraction collector used to collect the microdialysate samples by FEP tubing. The fraction collector (CMA/170) was controlled by a personal computer. Prior to each study the performance of the probe was assessed by examining volume delivery and recovery in vitro from a solution of sodium thiopentone (100 μg/mL in Ringer's solution) at a flow rate of 10 μl/min. Probes performing satisfactorily were reused. After the estimation of recovery, the probe was allowed a minimum of 2 h washout time before the commencement of a study. Animal preparation: Animals, at the commencement of each microdialysis study, were placed in an induction chamber and anesthesia was induced with 5%. halothane in oxygen delivered at 1 1/min. Once induced, the flow rate was reduced to 0.5 1/min and the rat was mounted in a stereotaxic frame (Kopf model 900). An anesthetic mask, consisting of a 3 cm length of a 50 mL syringe with an inlet for the gaseous anesthetic mixture and an outlet for the scavenger line, was fitted around the rat's head and the nose bar of the stereotaxic frame: latex rubber tubing at each end of the mask ensured an air-tight seal. The halothane concentration was reduced to 2.5% while the mask was fitted, and to 2.0% until implantation of the microdialysis probe was completed. Body temperature was maintained with a heating pad monitored with a rectal probe. A 30 cm infusion line and a 25 cm sampling line were attached to the chronic indwelling venous and arterial lines, respectively.

A midline incision was made to expose the skull, and co-ordinates for the corpus striatum (A/P: +0.4 mm, L: 3.0 mm, D/V: -6.6 mm) and hippocampus (A/P:-5.2 mm, L: 5.0 mm, D/V: -6.0 mm) were measured from bregma. A circular region of the skull was removed with a dental drill to the expose the brain surface, and the dura was gently removed using the tip of 23G needle. The dorsal/ventral coordinate was taken from the surface of the brain, the probe was gradually lowered into position, and the halothane was reduced to 1.0%. The probe was allowed to settle for 30 minutes, during which time the blood brain barrier was considered to have restabilized. The halothane was subsequently turned off until the rat showed signs of arousal prior to the commencement of the thiopentone infusion. Oxygen flow was maintained at 0.5 1/min for the duration of the study procedure. Drug infusion and sampling regimens: RS-thiopentone sodium (10 mg/mL, Pentothal, Abbott Australasia Pty. Ltd, in deionized water containing 2 U/mL heparin) was infused into the indwelling jugular vein cannula. The infusion (by Harvard Apparatus 22 Pump controlled by Stanpump software, run on a personal computer) was delivered to provide a constant target thiopentone plasma concentration in of 40 μg/mL for 20 minutes (total dose

54.5 mg/kg). Sampling was performed during and for 40 minutes after the infusion. Microdialysate sampling was commenced immediately after the infusion began at a perfusion rate of 5 μl/min. Blood samples (0.1 mL) were collected into tubes (1.5 mL Eppendorf, containing 5 μL 1000 U/mL sodium heparin) at 0, 1, 2, 5, 10, 20, 21, 22, 25, 30, 40, and 60 minutes: each sample was replaced with 3 volumes of 0.9% saline. Plasma was separated by centrifugation (7000 rpm, 2 minutes), and aliquots (50 μL) were stored frozen (-20°C, for not longer than 4 weeks) until analysis. Microdialysate samples were collected over 5 minute intervals for 60 minutes into sample vials (250 μL, polypropylene) containing internal standard (50 μL, 2 μg/mL in Ringer's solution) and assayed for thiopentone enantiomers immediately after completion of the study.

Microdialysis probe calibration: The microdialysate concentration of thiopentone was corrected for recovery using a technique where the loss of drug across the probe was used as an estimate of recover. However, although the recovery of an ideal solute across a probe membrane into the perfusate is equivalent to the loss of the solute across the probe membrane from the perfusate, directional dependence of diffusion may occur with real solutes due to an interaction with the membrane. In this instance recovery will no longer be equivalent to loss. The ratio of recovery to loss in vitro for a solution of RS-thiopentone sodium (100 μg/mL in Ringer's solution) at a flow rate of 5 μl/min was found to be 0.64. The loss of thiopentone across the probe was estimated at the conclusion of microdialysis sampling. A 100 μg/mL solution of RS-thiopentone sodium was infused through the probe at a flow rate of 5 μl/min. The probe was allowed to equilibrate with the surrounding tissue for 20 minutes prior to collection of a microdialysate sample over a 10 minute interval. Microdialysate concentrations were corrected for recovery by equation (i) and referred to as apparent extracellular fluid (aECF) concentrations.

aECF concentration = microdialysate concentration / loss*0.64 (i)

The lag time for the microdialysis system was determined separately. A microdialysis probe was perfused from a reservoir of Ringer's solution at a flow rate of 5 μl/min. After 30 seconds the probe was placed in a solution of RS-thiopentone sodium (500 μg/mL in Ringer's solution) for 3 minutes then returned to the reservoir of Ringer's solution. Microdialysate fractions were collected into sampling vials (250 μL, polypropylene, containing 50 μL Ringer's solution) over 30 second intervals for 10 minutes. At the conclusion of each study the brain was removed and placed in a formalin solution (10% in 0.9% saline) for a period of 2-3 days. The brain was subsequently sliced with a scalpel blade, through the coronal plain, to expose the cannula tract then examined with a hand lens. Comparison with photographic plates in the atlas of Paxinos and Watson (1986) was used to determine whether the cannula was correctly placed in the striatum or hippocampus. Tissue uptake and plasma binding studies: The jugular vein of rats was cannulated as described above; after a recovery period of 2-3 days an infusion of RS-thiopentone sodium was performed as before to maintain a constant plasma concentration of 40 μg/mL for 20 minutes. In successive groups of (anaesthetized) animals, 20, 30, and 60 minutes after the commencement of the infusion blood was withdrawn from the heart by cardiac puncture (10 mL syringe, 21G needle filled with 1000 U/mL sodium heparin). The plasma was separated by centrifugation (3000 rpm, Beckman bench centrifuge). CNS tissue samples were collected from the cortex, striatum, hippocampus, cerebellum, brachial and sacral spinal intumescences; adipose tissue and skeletal muscle were respectively sampled from the epididymal fat pad and gluteus muscle. Plasma and tissue samples were stored frozen at -70°C until the time of analysis.

The concentrations of unbound thiopentone enantiomers were determined in duplicate by equilibrium dialysis of the harvested plasma (adjusted to pH 7.4 with 0.5 M NaH P0₄ immediately prior to dialysis) against phosphate buffer

(0.067 M, pH:7 4) in cells (2.5 mL, Plexiglas, 2.5 cm diameter, 0.5 cm depth) separated by cellulose membranes (prepared by heating to boiling in deionized water x3). The cells were incubated at 37°C for 8 hours with gentle shaking.

Thiopentone enantiomer assays: The concentrations of R-thiopentone and S-thiopentone in the samples were determined by a liquid chromatographic procedure. The non-therapeutic 5-ethyl-5-hexylbarbituric acid was used as an internal standard. Plasma aliquots (50 μL, in 1.5 mL Eppendorf tubes) were extracted with ethyl acetate in hexane (1.1 mL, 5% v/v) after the addition of internal standard (50 μL, 50 μg/mL) and H₃P0₄ (10 μL, 2 M). The samples were shaken vigorously (1 minute), centrifuged (7000 rpm, 2 minutes in an Eppendorf Microfuge) and frozen on dry ice (15 minutes) before the organic layer was decanted and evaporated to dryness in a rotary vacuum bench evaporator (40°C). The residue was reconstituted in Na₂HP0₄ (200 μL, 10 mM containing 30% v/v isopropanol; an aliquot (10 μL) was injected onto the column. Tissue samples were homogenized (50-100 mg/mL) into Na₂HP0₄ (0.2 M). An aliquot (200 μL) was taken, internal standard (100 μL, 50 μg/mL) was added, and the sample extracted with hexane (1.0 mL) by shaking vigorously (1 minute). After centrifugation (2 min, 7000 rpm) the organic layer was decanted and discarded, the sample was resuspended by sonication and vortexing, acidified by the addition of

H₃Pθ4 (20 μL, 2 M), and extracted with ethyl acetate in hexane (5% v/v) as before. Microdialysate samples were injected (60 μL) without prior extraction. The mobile phase consisted of phosphate buffer (100 mM, pH: 6.3) in isopropanol (4.5% v/v) . Data analysis: The area under the respective curves (AUCs) for RS- thiopentone concentrations in plasma and aECF were compared between the two sites of microdialysis by Student's --test. Within each group, pairwise comparisons of AUCs of R- and S-thiopentone concentrations were performed by Student's t-test for paired data; enantiomeric bias was tested for by comparing the ratio of the AUCs for S- to R-thiopentone to unity with Student's one sample t-tes In tissue uptake studies the CNS tissue concentrations of thiopentone enantiomers, as well as the S- to R- enantiomeric ratios for both and the tissue :plasma distribution coefficients, were compared across region and time by two factor analysis of variance. Peripheral tissue samples were compared by one factor analysis of variance. Pairwise comparisons were performed by the method of Least Significant Differences (LSD). Distribution coefficients for peripheral tissues and plasma binding (30 and 60 minute intervals) were compared by Student's f-test. Pairwise comparisons of data for thiopentone enantiomers in tissue uptake and plasma binding studies were performed with Student's f-test for paired data. Again, enantiomeric bias was tested for by comparing the ratios of data for S- to R-thiopentone to unity with Student's one sample f-test. Results: In the animals undergoing microdialysis, target plasma thiopentone concentrations were maintained during the infusion, then decayed biphasically. aECF concentrations increased slowly to maxima at 25 to 30 min from approximately 3% of the corresponding plasma concentration at 1 min, to 9% at the cessation of infusion to 12% at 60 min. The concurrent plasma unbound fraction of R-thiopentone was slightly but significantly greater than that of S-thiopentone. Enantioselectivity in the rate of plasma- aECF equilibration was not found. In the animals undergoing tissue distribution analysis, CNS tissues were highest at 20min, muscle concentrations at 30 min and fat concentrations at 60 min. Distribution coefficients into all tissues sampled favour R-thiopentone with a calculated from total or unbound plasma concentrations. Conclusions: The inventors surprisingly found that the rate of equilibration of thiopentone between plasma and brain extra cellular fluid was remarkably slow. There was no evidence to support differences in potency between the thiopentone enantiomers being due to differences in their rates of equilibration across the blood-brain barrier. 4) Effects of thiopentone and its enantiomers on CNS tissue: (Paper entitled: "Electroencephalographic effects of thiopentone and its enantiomers in the rat")

Background: Previous electrophysiological studies with some chiral barbituates have shown that one enantiomer can be excitant while the other is depressant. Other behavioural studies with some chiral barbituates have shown there to be quantitative differences in potency between enantiomers.

Thiopentone is known to have both differences in potency between enantiomers as well as biphasic effects on the electroencephalogram. At the time of this study, it was unknown whether this was due to differential electrophysiological effects between its enantiomers. Methods: A study was performed in rats with RS-thiopentone, R-thiopentone and S-thiopentone to determine the nature and time course of the electroencephalographic effects. Two paradigms of computer-controlled infusions of the drugs were performed in groups of animals previously prepared with EEG electrodes and/or blood sampling cannulae. The first used sequentially increasing stepwise increments for 10 minutes each followed by washout. The second used a brief (4 minute) infusion followed by washout. Plasma thiopentone enantiomer concentrations were determined by CSP-HPLC.

Animals and their preparation: Young adult male Wistar rats (350- 400 g) were housed in groups of four, maintained on a constant 12/12 hour light dark cycle at 23 °C, and allowed free access to food and water. After surgery rats were housed individually, and post-operative body weights and fluid intake were monitored.

EEG recordings: EEG recordings were taken from a single pair of electrodes positioned contralaterally across the frontal and occipital lobes. The signal was collected with a Biopac EEG100 amplifier module (gain 5000,

1-30 Hz band pass filter) connected to a MP100 analogue to digital converter, and acquired by a Pentium 120 computer using Acqknowledge III software (Biopac Systems, Inc). Recording electrodes were made from 0-08 x 3/32 stainless steel screws soldered to 1.5 cm lengths of IDC computer cable and connected to the EEG100 amplifier module by a recording cable (2 m length,

7 core shielded electrical cable). The EEG electrodes were previously implanted under halothane in oxygen anaesthesia induced with the animals mounted in a stereotaxic frame (Kopf model 900). A midline incision was made to expose the skull, and 4 holes were made with a 2 mm dental drill, approximately 2-3 mm on each side of bregma and lambda. A fifth screw was inserted to act as an anchor, 34 mm lateral to the midline, midway between bregma and lambda. Heat shrink tubing, approximately 4-5 mm in height, was placed around the perimeter of the screws and filled with acrylic dental cement. The electrode ends were subsequently soldered to an 8-pin IC socket (Newark Electronics), the exposed electrical wire and IC socket were then embedded in acrylic dental cement. The wound was closed by sutures placed on either side of the electrode block. During surgery, body temperature was maintained with a heating pad and monitored with a rectal probe. Rats received amoxycillin (85 mg/kg, im) and buprenorphine (0.2 mg/kg, sc) post- operatively, and a subsequent dose of amoxycillin (85 mg/kg, im) was administered the following morning. Body weight was allowed to return to baseline before vascular cannulation was performed.

Vasculai' cannulation: Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous venous infusion and arterial sampling. For this surgery, the animals were anesthetized by pentobarbitone (30 mg/kg, ip in 1 mL 0.9% saline) followed 5 minutes later by ketamine (45 mg/kg, ip in 1 mL 0.9% saline). Body temperature was maintained with a heating pad and monitored with a rectal probe. A 1 cm thoracic incision was made just lateral to the midline, and the jugular vein and carotid artery were exposed. The jugular vein was cannulated with Silastic laboratory tubing (Dow Corning, 0.025 in ID x 0.047 in OD) inserted 2.5 cm; the carotid artery was cannulated with Silastic laboratory tubing (Dow Corning, 0.020 in ID x 0.037 in OD) inserted 2.0 cm. The cannulae were tunnelled under the skin and exteriorized above the neck anterior to the scapulae. Each line was filled with a solution of 6 g polyvinylpyrrolidone (MW 40,000; Sigma Chemical Co) in sodium heparin (5 mL, 1000 U/mL) to maintain line patency. At the completion of surgery rats were administered amoxycillin (85 mg/kg, im), buprenorphine (0.15 mg/kg, se), and given 0.9% saline (10 mL, sc) for fluid replacement. A subsequent dose of amoxycillin (85 mg/kg, im) was administered the following morning. Experimental procedures were performed 2 days later.

Drugs: RS-thiopentone sodium (Pentothal, Abbott Australasia Pty Ltd) was dissolved in deionized water to a final concentration of 10.0 mg/mL (= 9.2 mg/mL as thiopentone); R- and S- thiopentone were each dissolved in a minimum volume of 0JM NaOH, then diluted to a final concentration of

10.0 mg/mL containing 0.06% Na₂C0₃ (w/v). All solutions contained 2 U/mL heparin.

Experimental: On the day of the study rats were placed in the recording chamber and allowed to acclimatize for 1 hour. After a 75 cm infusion line and a 45 cm sampling line were attached to the chronic indwelling venous and arterial lines, and the recording cable was attached, rats were allowed a further 30 minutes to settle before commencing the study. An infusion of RS-thiopentone, R-thiopentone, or S-thiopentone was delivered by a Harvard Apparatus 22 Pump controlled by Stanpump software run on a personal computer. A rectal probe to monitor body temperature was inserted as soon as possible, and body temperature was maintained with a heating lamp.

Study 1: After a baseline recording for 20 minutes, sequential stepwise target-controlled infusions were used to produce target plasma concentrations of 10, 20, and 40 mg/L for 10 minutes then 60 mg/L for 5 minutes (total dose: 72 mg/kg) for RS-thiopentone and R-thiopentone; due to the greater potency, animals treated with S-thiopentone were maintained at 40 mg/L for 15 minutes instead of being increased to 60 mg/L (total dose: 57 mg/kg). Study 2: After a baseline recording for 20 minutes, a step target plasma concentration of 60 mg/L was maintained over a 4 minute interval (total dose: 42 mg/kg). Arterial blood (0J mL) was sampled at the conclusion of the infusion, then 1, 2, 5, 10, 20, 40, 60, 90, 120, 150, 180, and 210 minutes later. Each sample was replaced with 3 volumes 0.9% saline (initial flush 0.2 mL 0.9% saline, followed by 0J mL 10 U/mL heparin in 0.9% saline, to ensure the dead volume of the sampling line was filled with heparinized saline between sampling intervals).

EEG signal analysis: The product of the rectified signal amplitude (μV) and the rate of signal crossing through 0 μV (Hz) derived from the filtered EEG signal was used as a surrogate measure of CNS activity. This is effectively a null variable derived from the inverse relationship existing between the dominant frequency and amplitude within a given EEG sample. A data acquisition integral function was used to determine the area under the curve (AUC) of the product of amplitude and frequency for 10 second epochs over the duration of the recording; from this value the μV*Hz per second for each epoch was determined. Individual maximum and minimum values of μV*Hz per second as well as the times at which these values occurred were determined. Maximum and minimum values were expressed as a percentage of the mean value of μV*Hz per second for the 20 minute baseline period before drug infusion.

Thiopentone enantiomer assays: The plasma concentrations of R- and S-thiopentone were determined by HPLC-CSP. A chiral-AGP column (Chrom Tech, Sweden) was used with a Waters 600 MS system and spectrophotometric detection at 287 nm with a Waters 991 Photodiode Array Detector. The non-therapeutic 5-ethyl-5-hexylbarbituric acid was used as an internal standard. Plasma aliquots (50 μL) in Eppendorf tubes (1.5 mL) were extracted with ethyl acetate in hexane (1.1 mL, 5% v/v) after the addition of internal standard (50 μL, 50 mg/L) and H₂P0₄ (10 μL, 2 M). The samples were shaken vigorously (1 minute), centrifuged (7000 rpm, 2 minutes) in an Eppendorf Microfuge, and frozen on dry ice (15 minutes) before the organic layer was decanted and evaporated to dryness in a rotary vacuum bench evaporator at 40°C. The residue was reconstituted in Na₂HP0₄ (200 μL, 10 mM) containing isopropanol (30% v/v);10 μL was injected onto the column.

Data analysis: The areas under the relevant plasma drug concentration-time curves (AUCs) for each of thiopentone enantiomers were determined by the linear trapezoid method. Student's Mest for paired data was used for comparisons between the AUCs for R- and S-thiopentone in animals infused with RS-thiopentone. Enantiomeric bias was tested for by comparing the ratio of the AUCs to unity with Student's one sample f-test. Student's t-test was used for between group comparisons of the AUCs for R- and S-thiopentone in rats infused with the separate enantiomers of thiopentone.

Results: Each of the drugs tested caused biphasic changes to the EEG: an initial activation was followed by deactivation. However, clear evidence for quantitative enantioselectivity was found in that the maximum value of depression was substantially less for R-thiopentone than for either S- thiopentone or RS-thiopentone; moreover, S-thiopentone caused a greater incidence of fatality than did R-thiopentone or RS-thiopentone for the same doses. Plasma concentrations of R-thiopentone were approximately 10% less than those for S-thiopentone for the same doses. Conclusions: Although both enantiomers exhibited qualitatively similar effects on the EEG, the quantitative effects of R-thiopentone were less and recovery from maximal effects was faster than with either S-thiopentone or RS-thiopentone.

5) Effects of thiopentone and its enantiomers on CNS tissue and the brain: (Paper entitled: "Electro-encephalographic effects of thiopentone enantiomers in the rat:-Correlation with drug tissue distribution") Background: Previous qualitative electrophysical studies with several chiral barbiturate enantiomers have shown that one enantiomer can be excitant while the other is depressant. Other studies have shown there to be quantitative differences in potency between barbiturate enantiomers including thiopentone for the same pharmacological end point. The relationships between the various pharmacological indices and distribution of the enantiomers into vital tissue is in need of clarification. Methods: Rats were infused with RS-thiopentone, R-thiopentone or S-thiopentone at a constant rate of 4 mg/kg/min until fatal. EEG signal and arterial plasma thiopentone concentrations were sampled constantly to determine the relationships between them. At the end of the infusion, the animals were dissected to determine whether there was enantioselectivity in thiopentone uptake into tissues. Animals and their preparation: Young adult male Wistar rats (350-400 g) were obtained from the Gore Hill Animal Research Facility. The animals were housed in groups of four, maintained on a constant 12/12 hour light dark cycle at 23°C, and allowed free access to food and water. After surgery rats were housed individually, and post-operative body weights and fluid intake were monitored.

EEG recordings: EEG recordings were taken from a single pair of electrodes positioned contralaterally across the frontal and occipital lobes. The signal was collected with a Biopac EEG100 amplifier module connected to a MPlOO analogue to digital converter, and acquired by a Pentium 120 computer using Acqknowledge III software (Biopac Systems, Inc). Recording electrodes were made from 0-08 x 3/32 stainless steel screws soldered to 1.5 cm lengths of IDC computer cable and connected to the EEG100 amplifier module by a recording cable (2 m length, 7 core shielded electrical cable). The EEG electrodes were previously implanted under halothane in oxygen anaesthesia induced with the animals mounted in a stereotaxic frame (Kopf model 900). A midline incision was made to expose the skull, and 4 holes were made with a 2 mm dental drill, approximately 2-3 mm on each side of bregma and lambda. A fifth screw was inserted to act as an anchor, 34 mm lateral to the midline, midway between bregma and lambda. Heat shrink tubing, approximately 4-5 mm in height, was placed around the perimeter of the screws and filled with acrylic dental cement. The electrode ends were subsequently soldered to an 8-pin IC socket (Newark Electronics), the exposed electrical wire and IC socket were then embedded in acrylic dental cement. The wound was closed by sutures placed on either side of the electrode block. During surgery, body temperature was maintained with a heating pad and monitored with a rectal probe. Rats received amoxycillin (85 mg/kg, im) and buprenorphine (0.2 mg/kg, sc) post-operatively, and a subsequent dose of amoxycillin (85 mg/kg, im) was administered the following morning. Body weight was allowed to return to baseline before vascular cannulation was performed. Vascular cannulation: Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous venous infusion and arterial sampling. For this surgery, the animals were anesthetized by pentobarbitone (30 mg/kg, ip in 1 mL 0.9% saline) followed 5 minutes later by ketamine (45 mg/kg, ip in 1 mL 0.9% saline). Body temperature was maintained with a heating pad and monitored with a rectal probe. A 1 cm thoracic incision was made just lateral to the midline, and the jugular vein and carotid artery were exposed. The jugular vein was cannulated with Silastic; laboratory tubing (Dow Corning, 0.025 in ID x 0.047 in OD) inserted 2.5 cm; the carotid artery was cannulated with Silastic laboratory tubing (Dow Corning, 0.020 in ID x 0.037 in OD) inserted 2.0 cm.

The cannulae were tunnelled under the skin and exteriorized above the neck anterior to the scapulae. Each line was filled with a solution of 6 g polyvinylpyrrolidone (MW 40,000; Sigma Chemical Co) in sodium heparin (5 mL, 1000 U/mL) to maintain line patency. At the completion of surgery rats were administered amoxycillin (85 m /kg, im), buprenorphine (0.15 mg/kg, se), and given 0.9% saline (10 mL, sc) for fluid replacement. A subsequent dose of amoxycillin (85 mg/lcg, im) was administered the following morning. Experimental procedures were performed 2 days later.

Drugs: RS-thiopentone sodium (Pentothal, Abbott Australasia Pty Ltd) was dissolved in deionized water to a final concentration of 9.2 mg /ml; R- and S- thiopentone (Huang et al., 1996) were each dissolved in a minimum volume of 0.1M NaOH, then diluted to a final concentration of 10.0 mg/mL containing 0.06% Na₂C0₃ (w/v). All solutions contained 2 U/mL heparin. Experimental: On the day of the study rats were placed in the recording chamber and allowed to acclimatise for 1 hour. After a 75 cm infusion line and a 45 cm sampling line were attached to the chronic indwelling venous and arterial lines, and the recording cable was attached, rats were allowed a further 30 minutes to settle before commencing the study. An infusion of RS-thiopentone, R-thiopentone, or S-thiopentone was delivered at 4 mg/min until fatal. A rectal probe to monitor body temperature was inserted as soon as possible, and body temperature was maintained with a heating lamp.

EEG data analysis: The product of the rectified amplitude (μV) and frequency (Hz) derived from the 1-30 Hz band-pass filtered EEG signal was used as a surrogate measure of CNS activity. This is effectively a null variable derived from the inverse relationship existing between the dominant frequency and amplitude within a given EEG sample. A data acquisition integral function was used to determine the area under the curve (AUC) of the product of amplitude and frequency for 10 second epochs over the duration of the recording; from this value the μV*LIz per second for each epoch was determined. Individual maximum and minimum values of μV*Hz per second as well as the times at which these values occurred were determined. Maximum and minimum values were expressed as a percentage of the mean value of μV*Hz per second for the 20 minute baseline period. Thiopentone enantiomer assays: The plasma concentrations of R- and

S-thiopentone were determined by HPLC-CSP. A chiral-AGP column (Chrom Tech, Sweden) was used with a Waters 600 MS system and spectrophotometric detection at 287 nm with a Waters 991 Photodiode Array Detector. The non-therapeutic 5-ethyl-5-hexylbarbituric acid was used as an internal standard. Plasma aliquots (50 μL) in Eppendorf tubes (1.5 mL) were extracted with ethyl acetate in hexane (1.1 mL, 5% v/v) after the addition of internal standard (50 μL, 50 mg/L) and H₃P0₄ (lOμL, 2 M). The samples were shaken vigorously (1 minute), centrifuged (7000 rpm, 2 minutes) in an Eppendorf Microfuge, and frozen on dry ice (15 minutes) before the organic layer was decanted and evaporated to dryness in a rotary vacuum bench evaporator at 40°C. The residue was reconstituted in Na₂HP0₄ (200 μL, 10 mM) containing isopropanol (30% v/v); 10 μL was injected onto the column.

Data analysis: One way ANOVA was used to compare between groups having infusions of R-, S- or RS-thiopentone. Student's two sample t- test was used for between group comparisons of the parameters of the EEG effects for R- and S-thiopentone in rats infused with the separate enantiomers of thiopentone. Student's t-test for paired data was used to compare between enantiomers when RS-thiopentone was infused. Results: Anaesthetic and fatal doses (mg/kg) as well as plasma drug concentrations (μg/mL) decreased in parallel in the order: R-thiopentone > RS-thiopentone > S-thiopentone.

Initial activation of the EEG was similar for all infusions but the respective plasma drug concentrations for the same extent of deactivation was parallel to the anaesthetic doses. Tissue:plasma distribution coefficients after infusion of RS-thiopentone were higher for R- than for S-thiopentone in brain and visceral regions but not in fat or muscle. After administering the separate enantiomers, it became clear that the relative distribution into the heart compared to the brain was twice as high for S-thiopentone than for R- thiopentone.

Conclusion: The present inventors have surprisingly found that although qualitatively similar in effects, significant quantitative differences exist between RS-thiopentone and its enantiomers. The therapeutic index (ratio of lethal to anaesthetic dose) of R-thiopentone was considerably more favourable than either RS-thiopentone or S-thiopentone. This would seem to derive from a relatively greater distribution into CNS tissues and relatively less into the heart.

Summary The inventors have conducted studies that point to an enantioselective advantage of R-thiopentone over S-thiopentone and RS-thiopentone on the following grounds:

(a) the therapeutic index (ratio of lethal to anaesthetic doses) clearly favours R-thiopentone over RS- and S-thiopentone. This was known from prior art and was confirmed by the present inventors.

(b) pharmacokinetic studies indicate a relatively higher clearance of R- than S-thiopentone. This difference, although small, is favourable towards faster termination of effects of R-thiopentone than either RS- or S- thiopentone. A higher clearance of R- than of S-thiopentone after administration of RS-thiopentone in humans and sheep was known from prior art. It was not previously known also to occur in the rat.

(c) the negative inotropic effect (of the sheep heart after coronary arterial injection in vivo) is greater for S-thiopentone than for RS- or R- thiopentone; that for R-thiopentone is generally less than that from RS- thiopentone. (d) higher relative distribution coefficients into CNS of R- than of S- thiopentone indicate a larger fraction of the dose partitions into desired tissues. In contrast, lower heart:brain distribution ratio for R-thiopentone suggests that this is a significant reason for its greater tolerability than either RS- or S-thiopentone.

The evidence is as follows:

(i) Enantioselectivity of CNS distribution coefficients: Sequential increasing steady state plasma concentration targets of RS-thiopentone were attained in rats and serial arterial plasma and terminal steady state brain and spinal cord samples were assayed enantiospecifically for thiopentone.

Concurrent total and unbound plasma concentrations of S-thiopentone were —5-20% higher than those of R-thiopentone and, although concentrations of S-thiopentone in frontal cortex, striatum, hippocampus and brachial and lumbar spinal cord were —20% higher than those of R-thiopentone, the respective tissue :plasma distribution coefficients were —10% greater for R- thiopentone.

(ii) Kinetics in plasma and brain after RS-thiopentone. A study of the time courses of plasma and brain microdialysate (essentially ECF) during and after administration of RS-thiopentone found that plasma and ECF concentrations of R-thiopentone decreased more rapidly than those of S- thiopentone after infusion. The unbound plasma fraction was slightly higher for S-thiopentone.

(iii) Enantioselectivity of the EEG effect. Two studies, performed with ascending and descending thiopentone plasma concentrations of RS-, R- and S-thiopentone found qualitatively similar EEG patterns with RS-, R- and

S-thiopentone but there were quantitative differences in the extent and duration of EEG deactivation. The relative extent of EEG deactivation increased in the order S-> RS-> R-thiopentone. The rate of EEG recovery was the fastest from R-thiopentone. The washout of R-thiopentone plasma concentrations was faster than that of S-thiopentone.

(iv) Systemic kinetics, EEG effects and therapeutic index of infused rac-, R- and S-thiopentone. Constant rate infusions of RS-, R- and S-thiopentone were used to determine the doses producing EEG changes, anaesthesia, and death. Although S-thiopentone was more potent than RS- thiopentone or R-thiopentone, the therapeutic index (ratio of lethal to anaesthetic doses) of R-thiopentone was significantly greater than for RS- thiopentone or S-thiopentone. Higher relative distribution coefficients of R- thiopentone pertained for every tissue except heart where S-thiopentone was higher. Heart:brain concentration ratios increased in the order R- < RS- < S-thiopentone. Both the EEG pattern and relatively favourable heart:brain distribution of R-thiopentone was consistent with its greatest tolerability.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A process of preparing an enantiomer of thiopentone which includes the following steps:

(a) providing a R- or S-citronellol according to the formula (I)

(I)

where Z is H or a protecting group

(b) oxidative-cleavage of the double bond of compound (I) to provide the aldehyde (II)

(II)

where Z is H or a protecting group

(c) reduction of the aldehyde and where Z is a protecting group, deprotection of the hydroxy group of compound (II) to provide the alcohol (III)

(HI) (d) oxidisation of the alcohol (III) to the acid (IV) followed by esterification to give the ester (V)

(IV) (V)

where R=an alkyl group

(e) condensation of the ester (V) with dialkyl oxalate under basic conditions to give compound (VI)

'

(VI)

where R and R'= alkyl groups

(f) decarbonylation of compound (VI) to give malonate (VII) followed by ethylation to give compound (VIII)

(VII) (VIII) where R and R' = alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

2. A process according to claim 1 wherein compound (IX) formed in step (g) is the R- enantiomer.

3. A process according to claim 1 wherein compound (IX) formed in step (g) is the S- enantiomer.

4. A process according to any one of the preceding claims wherein the protected R- or S-citronellol according to the formula (I) is formed by converting R- or S- citronellol to a carboxylic acid derivative thereof by reaction with a compound selected from the group consisting of alkylcarboxylic acids, derivatives thereof, and anhydrides of alkyl carboxylic acids.

5. A process according to any one of the preceding claims wherein oxidative-cleavage step (b) is carried out by reacting compound (I) in a method selected from the group consisting of ozonolysis, a Lemieux-Rudloff oxidation, a Lemieux-Johnson oxidation and a Pappo-Becker oxidation.

6. A process according to any one of the preceding claims wherein reduction/deprotection step (c) is carried out by a method selected from the group consisting of a (i) a Wolff-Kishner reduction of compound (II), (ii) reaction of compound (II) in dioxane with p-toluenesulfonyl hydrazine to give the tosylhydrazone followed by sodium borohydride reduction and base hydrolysis, (iii) reaction of compound (II) in aqueous dioxane with hydrazine dihydrochloride followed by addition of hydrazine hydrate, heating, cooling and addition of potassium hydroxide and further heating.

7. A process according to claim 6 wherein the reduction/deprotection step is method (iii) and Z=H.

8. A process according to any one of the preceding claims wherein the oxidation in step (d) is carried out by treating the alcohol (III) with aqueous potassium permanganate or chromic acid in aqueous sulfuric acid.

9. A process according to any one of claim 1 to 7 wherein the esterification in step (d) is carried out by treating the acid (IV) with an alcohol and an acid catalyst.

10. A process according to any one of the preceding claims wherein the condensation reaction in step (e) is carried out by treating ester (V) with a base followed by treatment with aqueous acid.

11. A process according to any one of the preceding claims wherein the decarbonylation in step (f) is carried out by heating compound (VI).

12. A process according to any one of the preceding claims wherein the ethylation in step (f) is carried out with a base followed by the addition of an alkyl halide.

13. A process according to any one of the preceding claims wherein the condensation reaction of compound (VIII) with thiourea in step (g) is carried out in the presence of an alkali metal ethoxide or hydride in a suitable solvent with heating.

14. A process of preparing an enantiomer of thiopentone including the following steps: (a) reacting R- or S-citronellol with acetic anhydride in pyridine to provide compound (I); (b) treating compound (I) in methanol with ozone followed by treatment with dimethyl sulfide (DMS) to provide aldehyde (II).

(c) treating aldehyde (II) with hydrazine hydrate followed by treatment with potassium hydroxide to provide the alcohol (III); (d) treating the alcohol (III) with potassium permanganate to form the acid

(IV) followed by treatment of the acid (IV) with ethanol and sulfuric acid to give ester (V);

(e) treatment of ester (V) with sodium hydride/ethanol in diethyl ether and diethyl oxalate followed by treatment with aqueous acid to give compound (VI);

(f) heating compound (VI) to provide the malonate (VII). Treatment of the malonate (VII) with sodium hydride in DMF followed by treatment with ethyl iodide to give compound (VIII);

(g) condensation of compound (VIII) with thiourea in sodium hydride/ethanol to give R- or S-thiopentone (IX).

15. A process of preparing an enantiomer of thiopentone which includes the following steps:

(c) carrying out a catalytic asymmetric hydrogenation of compound (X) to provide the alcohol (III)

(X)

(III)

(d) oxidisation of the alcohol (III) to the acid (IV) followed by esterification to give the ester (V)

(IV) (V)

where R=an alkyl group

(e) condensation of the ester (V) with dialkyl oxalate under basic conditions to give compound (VI)

(VI)

where R and R' = alkyl groups

(f) decarbonylation of compound (VI) to give malonate (VII) followed by ethylation to give compound (VIII)

(VII) (VIII)

where R and R¹ = alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

16. A process of preparing an enantiomer of thiopentone which includes the following steps:

(d) carrying out asymmetric hydrogenation of a compound of formula (XI) to form the acid (IV) followed by esterification to give the ester (V)

(XI)

(IV) (V)

where R=an alkyl group

(e) condensation of the ester (V) with dialkyl oxalate under basic conditions to give compound (VI) '

(VI)

where R and R¹ = alkyl groups

(f) decarbonylation of compound (VI) to give malonate (VII) followed by ethylation to give compound (VIII)

(VII) (VIII)

where R and R' = alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

17. A process of preparing an enantiomer of thiopentone which includes the following steps:

(f) asymmetric hydrogenation of a compound of formula (XII) to give malonate (VII) followed by ethylation to give compound (VIII)

(XII)

(VII) (VIII)

where R, R' and R"=H or alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

18. A process of preparing an enantiomer of thiopentone which includes the following steps:

(f) carrying out asymmetric hydrogenation of a compound of formula (XIII) to form a compound of formula (VIII)

(VIII)

where R' and R"=H or alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

19. A process of preparing an enantiomer of thiopentone which includes the following steps:

(f) carrying out asymmetric hydrogenation of a compound of formula

(XIV) to form a compound of formula (VIII)

(XIV)

(VIII)

where R. R' and R"=H or alkyl groups

(g) condensation of compound (VIII) with thiourea to give an R- or S- enantiomer of thiopentone (IX)

(IX)

20. A process according to any one of claims 15 to 19 wherein the asymmetric hydrogenation is carried out using a catalytic amount of a chiral transition metal complex.

21. A process of preparing an enantiomer of thiopentone which includes:

condensation of an R- or S- enantiomer of compound (VIII) with thiourea to give an R- or S-enantiomer of thiopentone (IX)

(IX)

where R and R'=H or alkyl groups

22. An intravenous injectable anaesthetic agent including R-thiopentone and/or its alkali metal or alkaline earth metal salt together with a pharmaceutically acceptable carrier.

23. An intravenous injectable anaesthetic agent according to claim 22 wherein the R-thiopentone is present in a concentration in the range of 1.0 mg/ml to 100 mg/ml.

24. An injectable anaesthetic agent according to claim 22 or 23 wherein the R-thiopentone is obtained by a process in accordance with any one of claims 1 to 21.

25. A method of anaesthetising a patient including the steps of administering to said patient an effective amount of R-thiopentone.

26. A method of treating a patient with intracranial hypertension, abnormally increased or excessive cerebral blood flow and/or oxidative metabolism including the steps of administering to said patient an effective amount of R-thiopentone.

27. A method according to claim 25 or 26 wherein the R-thiopentone is obtained by a process in accordance with any one of claim 1 to 21.