AU3624193A - New cyclodextrins and new formulated drugs - Google Patents

Description

NEW CYCLODEXTRINS AND NEW FORMULATED DRUGS

CROSS REFERENCE TO RELATED APPLICATIONS

The subject matter of this application relates to that of PCT Application AU89/0025S, published as WO 90/02141 on 3 March 1990 (hereinafter "the '359 Application"), and PCT Application AU91/00071 filed March 1, 1991 (hereinafter "the '071 application"), the disclosures of which are expressly incorporated herein by reference. BACKGROUND OF THE INVENTION

Many of today's commonly prescribed drugs have undesirable properties or delivery profiles which detract from their efficacy and/or beneficial nature. For example, some drugs may exhibit a poor or unpredictable bioavailability profile, which may be due, in part, to the drug's disadvantageous solubility characteristics. Two examples of such drugs which have a disadvantageous solubility profile are Propofol, i.e., 2,6-bis(1-methylethyl) phenol, and Alfaxalone, i.e., 3-hydroxypregnane-11,20-dione, which are well-known anesthetics. Indeed, those drugs might find increased usage if their solubility profiles were improved.

Accordingly, there is a continuing need for new drug delivery systems for Propofol and Alfaxalone and pharmacologically active derivatives or metabolites thereof. SUMMARY OF THE INVENTION

It is an object of this invention to provide inclusion complexes and pharmaceutical compositions comprising Propofol or Alfaxalone with substituted or unsubstituted cyclodextrins.

It is another object of this invention to provide methods for increasing the solubility, stability and/or bioavailability of Propofol and Alfaxalone.

It is yet another object of this invention to provide methods for treating mammals in need of such treatment with therapeutically effective amounts of the foregoing pharmaceutical compositions.

Accordingly, embodiments of this invention provide inclusion complexes and pharmaceutical compositions comprising Propofol or Alfaxalone or a pharmacologically active derivative or metabolite thereof included in a substituted or unsubstituted cyclodextrin or salt thereof.

Another embodiment provides methods for increasing the solubility of Propofol or Alfaxalone or a derivative or metabolite thereof in a neutral or acidic aqueous solution, comprising the step of forming one of the above-described inclusion complexes.

Another embodiment of this invention provides a method for improving the bioavailability. of Propofol or Alfaxalone or a derivative or metabolite thereof in a host mammal comprising the step of forming one of the above-described inclusion complexes.

Another embodiment, provides a method for treating a host mammal in need of such treatment, comprising orally or parenterally administering to said mammal a therapeutically effective amount of the aforementioned pharmaceutical compositions.

Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and the advantages of this invention may be realized and obtained by means of the compositions of matter and methods particularly pointed out in the appended claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. DEFINITIONS

The following definitions are provided for some basic terms that are used throughout this specification.

Cyclodextrin - refers to α- , β- , γ-, or δ- cyclodextrins, which are those that are generally available. It will be appreciated, however, that if other cyclodextrins are discovered or become available in sufficient commercial quantities, such cyclodextrins shall also be encompassed by this invention.

Cyclodextrin Derivative - refers to a cyclodextrin-containing compound in which one or more atoms or groups of atoms are substituted for a C2, C3 or C6 hydroxyl or hydroxyl hydrogen, i.e., "modified cyclodextrins." The term cyclodextrin derivative also encompasses "linked cyclodextrins" where two or more cyclodextrins are linked together, and compounds where a useful agent such as a pharmaceutical is covalently bonded to a cyclodextrin, such that the covalent bond, when broken will yield the agent in active form. This term also includes any salt or hydrate which can be formed from the cyclodextrin derivative.

Modified cyclodextrin - refers to a species of cyclodextrin derivatives that contains one or more atoms or groups of atoms substituted for a C2, C3 or C6 hydroxyl or hydroxyl hydrogen. The term modified cyclodextrin will not be meant to include compounds where two or more cyclodextrins are linked together, or compounds where a useful agent such as a pharmaceutical is covalently bound to a cyclodextrin. This term also includes any salt or hydrate which can be formed from the modified cyclodextrin.

Linked cyclodextrins - refers to two or more cyclodextrins linked together by one or more bridging groups. The bridging groups can link a C2, C3 or C6 position of one cyclodextrin to any one of the C2, C3, or C6 positions of the other cyclodextrin. This term includes any salt or hydrate which can be formed from the linked cyclodextrins.

Cyclodextrin inclusion-Association Complex - refers to an inclusion complex in which there are one or more associable groups or portions of a group located on a substituent that is substituted at a C2, C3 or C6 position of a cyclodextrin, which groups or portions form an association with one or more associable groups or portions of a guest atom or molecule. The associable portions can include polar or charged groups or portions, or groups or portions capable of hydrogen bonding. This term also includes any salt or hydrate which can be formed from the inclusion-association complex.

Cyclodextrin Inclusion Salt - refers to an inclusion-association complex in which the associable group or portion of the cyclodextrin substituent carries a net positive or negative charge which causes it to associate with an oppositely charged group or portion of a guest atom or molecule. This term also includes any other salt or a hydrate which can be formed from the cyclodextrin inclusion salt.

Solubility - refers to solution in water or other aqueous-based media. II. CYCLODEXTRIN DERIVATIVES

The cyclodextrin derivatives which can be used to prepare the inclusion complexes and pharmaceutical compositions in accordance with this invention are discussed below.

A. Modified Cyclodextrins

Modified cyclodextrins in accordance with this invention can comprise an otherwise substituted or unsubstituted cyclodextrin in which at least one C2, C3 or C6 hydroxyl is substituted with a group selected from -XR¹, YR³, siR⁴R⁵R⁶, and -R⁷,

wherein X can represent

- S - , , , ,

, , , , , ,

SUBSTITUTE SHEET

Y can represent , , ,

and wherein R¹ to R¹¹ can each represent the same or different groups selected from: the groups -XR¹, YR³, siR⁴R⁵R⁶, and -R⁷ are as defined above, hydrcgen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, and wherein any two or three groups bonded to the same substituent can be taken together to represent a single group multiply bonded to said same substituent, and wherein R^l to R¹¹ may be further substituted by at least one -XR¹, -YR²R³, -siR⁴R⁵R⁶, -R⁷, halogen, and OR¹², wherein R¹² is as defined for R^l to R¹¹.

Cyclodextrins in which one or more C2, C3 or C6 hydroxyls are selectively substituted by ether substituents are also encompassed. For example, cyclodextrin derivatives thus substituted only on one or more secondary carbons, or uniformly substituted only on one, two or three, etc. primary carbons are encompassed. The ether substituents may be further substituted with any of the foregoing groups. Of the above recited inclusion complexes, preferred groups include the substituted amino cyclodextrins, i.e., cyclodextrins wherein at least one substitution for said C2, C3 or C6 hydroxyl is of the formula -YR²R³ , wherein Y is N, and R² and R³ are as previously defined. Also of particular interest are the inclusion complexes wherein R² is hydrogen and R³ represents amino, hydroxyl, carboxyl, sulfonate (SO₃), phosphate (PO₄ ^-3), substituted alkyl, cylcoalkyl, or aryl, or wherein R² and R³ are taken together to represent a hereto substituted multiply bonded alkyl group .

Many modified cyclodextrins in accordance with this invention will possess one or more pendant arms as described in the '359 and '071 Applications. One general formula for preferred pendant arm cyclodextrin derivative are of the formula CD - w - R¹³- L, wherein

CD represents an otherwise substituted or unsubstituted cyclodextrin,

W represents a functional linking group,

R¹³ represents a group defined the same as R¹-R¹² above, and

L represents a group selected from reactive, charged, polar or associating groups. Advantageously,

W represents an optional, functional linking group such as amino, amide, ester, thioether, thioamide, thioester, etc., R¹³ represents an optional arm such as substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl, and

L represents an optional group selected from reactive, charged, polar or associating groups, e.g., amino, carboxyl, hydroxyl, sulfonate, phosphate, acyloxy, alkyloxy and thiyl.

While cyclodextrin derivatives having at least one of each substituent constitute a preferred group, each of the foregoing groups is optionally present. For example, the reactive, charged, polar or associating species can be bonded directly to the cyclodextrin or to the functional linking group. Likewise, a reactive, charged, polar or associating species may not always be desired. Moreover, such species can be anywhere on the arm, and there can be more than one such species on the arm, e.g, the arm could possess multiple groups that could associate with multiple groups on an included or associated molecule, such as biological molecules which may contain many .repeating groups such as amino, carboxyl, and hydroxyl. The arm can also contain other functional or reactive groups which, in turn, may be used to link yet other arms and charged, polar or associating species. Preferred modified cyclodextrins of CD-W-R-L group also include those in which a carboxyl-substituted alkyl group is linked to a C2, C3 or C6 position of the cyclodextrin through an amino, ester, amide, thioether, thioester, thioamide or other functional linking group. Alkyl groups of from 1-3, 1-6, and 1-10 carbons comprise preferred groups. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprise yet another preferred group.

It has been found that, in some instances, advantageous results can be obtained using cyclodextrin derivatives in which at least one C2, C3 or C6 hydroxyl is substituted with a group having a net negative charge, or with a substituent that contains a group having a net negative charge. Examples of groups that can carry a net negative charge include hydroxyl, carboxyl, phosphate (PO₄ ^-3) or sulfonate (SO₃ ^-1). Other groups having net negative charges will be readily apparent to those skilled in the art.

Preferred modified cyclodextrins of this group include those in which a carboxyl-substituted alkyl group is linked to a C2, C3 or C6 position of the cyclodextrin through an amino, ester, amide, thioether, thioester, thioamide or other functional linking group. Alkyl groups of from 1-3, 1-6, and 1-10 carbons comprise preferred groups. Those of from 10-20 comprise another preferred group, and those of greater than 20 carbons comprise yet another preferred group. One example of such compounds is 6^A-amino-6^A-deoxy-6^A-N-(3-carboxypropanoyl)-β-cyclodextrin (hereinafter "β-CDNSc").

The modified cyclodextrin, β-CDNSc, is also an example of another preferred group of cyclodextrin derivatives having the formula CD - X - R¹⁴- Q, wherein:

X represents or

R¹⁴ and R¹⁵ represent groups as defined, by R¹-R¹³ above, and Q is a carboxylic acid group. Of these derivatives, those where R¹⁴ is alkyl comprise a preferred group of the alkyl groups, those of from 1-3, 1-6, and 1-10 carbons comprise preferred groups. Those of from 10-20 comprise another preferred group, and those of grearer than 20 carbons comprises yεt another preferred group. Preparation of such compounds is described in the '359 and '071 Applications.

For Propofol, it will generally be desirable to employ derivatives of β-cyclodextrin such as β-amino cyclodextrin and β-CDNSc. For Alfaxalone, in addition to the β-cyclodextrin derivatives, in some instances it may be desirable to use γ-cyclodextrin or derivatives thereof such as γ-amino cyclodextrin and γ-CDNSc.

B Linked Cyclodextrins

Another preferred embodiment of this invention comprises inclusion complexes and pharmaceutical compositions comprising Propofol and Alfaxalone included in at least two otherwise substituted or unsubstituted cyclodextrins covalently bonded to each other by at least one linking group. The at least one linking group links a first cyclodextrin at a C2, C3 or C6 position to a second cyclodextrin at a C2, C3 or C6 position. The cylodextrins can be substituted as described above. When there are only two cyclodextrins which are not otherwise substituted and are linked by only one linking group, that linking group is other than a disulfide that links the two cyclodextrins at the C6 position. A preferred group of the linked cyclodextrins are those in which only two cyclodextrins are linked together. As discussed in the '359 and '071 applications, a first otherwise substituted or unsubstituted cyclodextrin can be linked through one of its primary (i.e., C6) carbons or one of its secondary (i.e., C2 or C3) carbons to a primary (i.e., C6) carbon or a secondary (i.e., C2 or C3) carbon of a second otherwise substituted or unsubstituted cyclodextrin. Thus, otherwise substituted or unsubstituted cyclodextrins which are linked by C6-C6, C2-C2, C3- C3, C2-C3, C6-C3 or C6-C2 linkages comprise preferred embodiments of this invention.

Also, as discussed in the '359 Application, the linked cyclodextrins are preferably linked by at least one linking group of the formula - X - R¹⁶ - Y - or - R¹⁷ - , wherein

X and Y can be the same or different, and represent functional linking groups, and

of the formula - X - R¹ - Y - or - R² - , wherein

X and Y can be the same or different, and represent functional linking groups such as ether, thioether, ester, thioester, amide, thioamide, and amine, and

R¹⁶ and R¹⁷ represent groups as defined by R¹-R¹⁵ above, and are advantageously selected from substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and heterocyclyl. The otherwise substituted or unsubstituted cyclodextrins which are linked may also be the same or different. For example, an α-cyclodextrin can be linked to a β- , γ- or δ-cyclodextrin.

IV. COMPOSITIONS AND METHODS OF USE

It will be readily apparent that this invention encompasses many different inclusion complexes comprising Propofol and Alfaxalone. When employed as pharmaceutical compositions, the compositions may be in any pharmaceutically acceptable form for any type of administration, including topical, oral, rectal or parenteral administration. Methods of treating a patient, including a human, will comprise administering therapeutically effective amounts of such compositions. Preparation and administration of such compositions will be within the skill of persons in such arts.

Other advantages can also accrue from the aforementioned pharmaceutical compositions, including protecting the pharmaceutical agents from enzymes and acids of the gastrointestinal tract, lytic agents in the body such as in the saliva, enzymes in the lungs and in the body, particularly at the surface and within cells such as polymorphic nuclear leukocytes, maσrophages and other cells, and possible also from the destructive mechanisms found within the cytoplasm of most cells.

Preparation of the cyclodextrin derivatives and linked

cyclodextrins discussed above is set forth in detail in the "359 and '071 applications.

Also included in the scope of the inventions therein is new cyclodextrin methods of synethsis and uses thereof including but no limited to uses with pharmaceuticals cosmetics

agricultural diagnostic and diagnosis of illness and the conducting of electricl impulses and used in biomedical and bioeleσtrical and the uses of such cyclodextrins to encapsulate conductors.

Also included in the scope is inclusion complexes formed with mambutone and cyclodextrins and methods for measuring inclusion complexes. The invention described include the formation of Metalo-Cyclodextrins by 6^A-deoxy-B-Cyclodextrins by

6^A-(3-Aminopropyl-Amino)-6^A-deoxy-B-cyclodextrins and their chiral discrimination in complexing R- and S- Tryptophan Anions in Aqueous Solution. Solubility of Propofol - water at 25° is 0.28 mg/ml

Solubility of Propofol in 25% w/v

6^A-amino-6^A-deoxy-cyclodextrin hydrochloride salt is 10.7 mg/ml a 1:1 mixture of β-amine hydrochloride propofol gave a white powder. When 73.6 mg of this complex (64 mg CND . CHI : 9.6 mg propofol) was added to 1 ml of water and sonicated for 2 hours the solution contained propofol at approx 0.2 mg/ml.

6^A-Amino-6^A-N-(6-aminohexyl) B-CD (B-CDN6N)

B-CDOTs (5g, 3.75 m mole) was heated in 1,6 - diaminohexane (5g, 43 m mole) at 80 - 90°C for 5 hours. The hot mixture was poured into and stirred ethanol (100 ml) and allowed to stand overnight. The solid was filtered and the filter cake was washed with acetone (20 ml) and diethyl ether (20 ml) and then air dried. The solid was dissolved in 5 ml of hot water and then poured slowly into stirred ethanol (25 ml). The mixture was allowed to stand overnight and the solid was filtered off. The solid was crystalised from hot water (10 ml). On cooling, a white solid was produced which was filtered and dried in vacuo to yield 2 g of B-CDN6N. 6^A-Cyano-6^A-deoxy-β-cyclodextrin (β-CDCN)

Impure β-CDOTs (5 g) and sodium cyanide (1 g) were dissolved in water (150 ml) with heating on a boiling water bath. Heating was continued and the reaction followed by t.l.c. (solvent A). After 3 hours no β-CDOTs remained. The reaction mixture was concentrated in vacuo to 50 ml, filtered (0.22 urn) and loaded, via the pump, onto a C18 ^u-Bondapack column (19 × 150 mm). The column was eluted with 5% aqueous methanol in water at 15ml minute^-1 and two peaks were observed both eluting very close to the void. Both contained the desired product with the first eluted peak also containing cyanide salts (3.2 g total). Peak 2 gave pure β-CDCN

(2.2g) as a glass on drying. This material was dissolved in water

(15 ml) and filtered into acetone (200 ml). The resultant precipitate was collected by filtration and rinsed with acetone (10 ml) and ether (2 × 10ml) to give the product as a white powder

(1.84 g). T.l.c. (solvent A) of the product showed: R_c (relative to β-CD), β-CDCN, 1.0 HPLC of the product using a 65% acetonitrile

- water eluent showed: t_R (relative to β-CD), β-CDCN, 0.85. The yield is believed to be low due to overloading of the column and should be -90% when chromatography is repeated.

6^A-Amino-6^A-N-(2-aminoethyl) 6^A-deoxy-α-cyclodextrin (α-CDN2N) FABMS (DMSO) 1015 (M), 1055 (M+40), 1113 (M+98)

6^A-Amino-6^A-N-(3-aminopropyl) 6^A-deoxy α-cyclodextrin (α-CDN3N) Purification of the crude product was attempted by Soxhlet extraction, followed by precipitation from water with ethanol. The compound was not satisfactorily pure.

N4Nlb

Diaminobutne (10g) was dissolved in dichloromethane (50 ml). The solution was stirred vigorously whilst IbCl (1g) was added drop wise, and then allowed to stand for 4h. The mixture was added to dilute NaHCO₃ solution with ether (100ml). The mixture was shaken, then the organic layer was washed twice with water (40ml), dried

(Na₂SO₄) and evaporated. The residue was recrystallized twice from CHCl₃/petrol to give product (800 mg) as off-white crystals.

N2Nlb

Diaminoethane (10 g) was dissolved in dichloromethane (50 ml). The solution was stirred vigorously whilst IbCl (1 g) was added dropwise, and then allowed to stand for 4 h. The mixture was added to dilute NaHCO₃ solution with ether (100 ml). The mixture was shaken, then the organic layer was washed twice with water (40 ml), dried (Na₂SO₄) and evaporated. The residue was recrystallised twice from CHCl₃/petrol to give product (800 mg) as off-white crystals. N6NIb

vmax 3350 (NH) 1640 (CO) 1540 1500 840 770cm^-1

N2NIb

Prepared similarly from dry piperazine and IbCl.

3-Nitrophenyl-((+)-6-methoxy-α-methyl-2-naphthaleneacetate) (NpNP) A mixture of (+)-6-Methoxy-α-methyl-2-naphthaleneacetate (5g), m-nitrophenol (3.7 g) and N,N'-dicyclohexylcarbodiimide (6.8 g) in dry ether (100 ml) was left stirring at room temperature overnight. The solid was filtered off, and the filtrate evaporated. The residue- was dissolved in chloroform (5 ml) and ether (50 ml) was added. On standing overnight at 5°C crystals of dicyclohexylurea formed, which were filtered off. The filtrate was evaporated and yellow crystals (10 g) of product formed. This was recrystallised from chloroform - hexane to give colourless product. Yield 8 g.

6^A-Deoxy-6^A-[4-N-(6'-methoxy-α-methyl-2-naphthaleneacetyl) piperizin-1-yl] β-cyclodextrin (β-CDN2,2NNp)

Impure β-CDN2,2N (5g) and NpNP (1.6 g) were heated together in dimethyl-formamide (10 ml) at 70°C overnight. The mixture was then poured into acetone (100 ml). The solid was collected by filtration, then dissolved in hot 50% aqueous ethanol (30 ml). The ethanol was evaporated in vacuo and the precipitate (6 g) was filtered off. TLC showed this to be a new compound, with minor amounts of β-CD and β-CDN2,2N. Recrystallisation from aqueous ethanol did not remove all the β-CD, but did remove β-CDN2,2N. T.l.c. (solvent B) of the product showed: R_c (relative to β-CD), β-CDN2,2N, 0.2; β-CD,1; β-CDN2, 2NNp, 1.1. HPLC of the product using a 65% acetonitrile - water eluent showed: t_R (relative to β-CD), β-CDN2,2NNp, 0.65; β-CD, 1; unknown, 1.6; β- CDN2,2N, 4.7.

The compound was isolated using preparative HPLC (65% acetonitrile-water) gave a fraction (10 mg).

6^A-Amino-6^A-deoxy-N-[6'-N'-(6"-methoxy-α-methyl-2-naphthaleneacetyl) 6-aminohexyl] β-cyclodextrin (β-CDN6NNp)

Prepared in exactly the same way as β-CDN2,2NNp, but because the diamine is free of β-CD, purification is easier. Yield from 5g of β-CDN6N, 5g (80%), after rextn from aq ethanol. T.l.c. (solvent B) of the product showed: R_c (relative to 0-CD), β-CDN6N, 0.1; β- CDN6NNp, 1.5

2-(4-Nitrophenyl) dioxolane

4-netrobenzaldeyhde (15.1 g, 100 mmol) was heated in toluene under a dean-stark trap with dihydroxyethane (20 ml) and TFA (0.5 ml) overnight. The mixture was cooled and stirred with sodium carbonate (1 g) for 5 minutes, then sodium carbonate solution (50 ml) was added. After an hour, the layers were separated and the toluene was washed with brine (50 ml) then dried and evaporated, giving 18.1 g of pale yellow powder (90%).

2-(4-Aminophenyl) dioxolane

sulphur (11 g, 0.3 mol) was dissolved in hot cone sodium hydroxide solution (10 g in 80 ml). Nitrophenyldioxolane (17.8g, 91 mmol) was dissolved in hot ethanol (50ml) and added dropwise to the polysulfide solution over 3 h. After a further 2h, the mixture was cooled and extracted twice with ether (50ml). The extracts were dried (Na2SO₄) and evaporated to an orange gum that crystallised on standing. This was recrystallised from toluene/petrol to give 998

(x). β-CD (OTs) ₂ + N4N*

Complete reaction of a di-tosylate mixture gave a FAB spectrum containing; 1187 (cap), 1205 (from mono OTs), 1275 (β-(N4N)₂). α-CDN3N+α-CDOTs*

The FAB showed no signals attributable to dimers. With DMF, DMSO or pyridine as solvent, only starting materials and αCD were detected by FAB. HPLC shows new components, but in small quantities, and these were not isolated. The ratio of reactants was varied from 1:2 to 2:1. β-CDN2N + β-CDOTs*

β-CDN4N + β-CDOTs*

α-CDNH₂ + α-CDOTs*

FAB 972 (α-CDNH₂) 1034 (?)*

β-CDN4N + β-CD(OTs)₇*

β-CD(OTs)₇ (1 g) and dry β-CDN4N were heated together in pyridine

(1 ml) at 80°C overnight.

FABMS 2276 2124 (152=Ts) and many others.

6^A-Deoxy-α-Cyclodextrin (α-CDH)

α-CDOTs (100 mg) was dissolved in water (1 ml) with sodium sulfide (100 mg). after 3 h at 70°C, the mixture was poured into acetone (10 ml). The solid was centrifuged down, redissolved in hot water and precipitated with ethanol. This solid was removed and dried in vacuo.

FABMS 976,978 clean [small 959, M-18]

FABMS results suggests that the product is 6-deoxy-a-CD (aH-H2,

955g/mol) and thus 978 is 955+Na, [tosylates can easily be reduced to alkanes by hydride donors].

collected by vacuum filtration, rinsed with cold ethyl acetate (20 ml) and dried in vacuo to give bis-(m-nitrophenyl)-succinate (21.34 g). ii) Succinic acid (5.9g, 0.05 mol) and 3-nitrophenol (13.9g, 0.1 mol) were dissolved in dry dimethylformamide (300 ml) and the solution allowed to stir at room temperature for 15 minutes. N,N'-dicyclohexylcarbodiimide (20 g, 0.1 mol) in dry dimethylformamide (50 ml) was added and the solution allowed to stir at room temperature for 48 hours during which time the solution became a bright opaque yellow. The resultant dicyclohexylurea was removed by vacuum filtration through a sintered glass funnel, leaving the filtrate as a bright clear yellow solution. The dimethylformamide was removed in vacuo to leave a pale yellow solid which was recrystallised from dry ethyl acetate (200 ml), during which dicyclohexylurea was removed by hot filtration. After standing for 90 minutes the remaining dicyclohexylurea crystallised out as fine off-white needles and collected by gravity filtration (Whatman N° 1 filter paper). The filtrate was placed in a fridge at 4°C overnight and the resulting product collected by vacuum filtration as a fine white powder (6.5g, 36%, m.p. 153-154°C).

Bis (2^A-deoxy-2^A-β-cyclodextrin) succinate (β-CD₂OSc)

β-CD (1.1 g) was dissolved in water (50 ml) and ScNP (0.15 g) in acetonitrile (10 ml) added in one portion. The reaction mixture turned yellow. After stirring for 5 min, dilute HCl was added to the reaction mixture until it reached pH-3. The solution was cooled and filtered to remove any unreacted β-CD. The filtrate was extracted twice with ether and the solvent removed in vacuo. T.l.c. analysis of the residue showed two components to be present, however chromatography isolated only β-CD. We were unable to prove that the dimer had been formed.

Reaction of β-CDSH with β-CDNHCOCHCH₂

A mixture of β-CDSH (250 mg) and sodium hydroxide (5 mg) was dissolved in water (1 ml) while being flushed with nitrogen. The reaction mixture was evaporated to dryness under reduced pressure (water pump with heating for ca. 2 hours) and the residue was dissolved in dimethylformamide (2 ml) while again being flushed with nitrogen. To this solution was added β-CDNHCOCHCH₂ (260 mg) and the resulting mixture stirred at 60°C under an atmosphere of nitrogen for 18 hours. The solvent was removed in vacuo and the residue dissolved in water (2 ml) followed by the addition of excess acetone to precipitate the crude product mixture (200 mg) . HPLC of the crude product using a 70% acetonitrile - water eluent showed the following: t_R (relative to β-CD), 0.54, 1.00, 2.30. The product at t_R 2.30 was isolated by preparative HPLC and identified as (β-CDS) ₂. ie the competing dimerisation was favoured.

6^A-Amino-6^A-deoxy-N-(3-phenylamino carbonyl propanoyl) β-cyclodextrin (β-CDNScNPh)

A solution of β-CDNScNP (140 mg) and benzylamine (17 mg) in dimethyl-formamide (1 ml) was stirred at room temperature for 5 hours. Evaporation in vacuo to an oil followed by the addition of acetone (20 ml) with stirring gave a white solid which was collected by vacuum filtration and washed with acetone (2 × 10 ml) and ether (2 × 5 ml). Drying to constant weight over P₂O₅ in vacuo gave β-CDNScNPh (125 mg). T.l.c. (solvent B) of - the product showed: R_c (relative to β-CD) , β-CDNScNPh, 1.1. HPLC of the product using a 75% acetonitrile - water eluent showed:

(relative to β-CD) , β-CDNScNPh, 0.86.

2^A(3^A)-O-(2-ethanal)-α-cyclodextrin (α-2CDOCH₂CHO)

A 10% sodium hydroxide solution (13.2 ml) was added to α-CD (30 g) . The slurry was heated to 50°C and 2,3-epoxy-l-propanol (3.6 ml) was added with stirring. After stirring at 50°C for 16 h, the mixture was cooled and diluted with water (100 ml). The intermediate glycol was obtained by precipitation. This material was dissolved in water (188 ml) and reacted with a solution of NaIO₄ (8.8 g) in water (188 ml) with vigorous shaking for 5 min only. Work up included quenching the reaction mixture with potassium iodide (6.2 g) in water (37.5 ml) and Na₂SO₃ (1.9 g). Excess iodine was removed by continuous extraction with ether (CCI₄ would be much better). 2^A(3^A)-O-(carboxy methyl)-β-cyclodextrin (β-2CDOCH₂CO₂H)

An 80% NaH dispersion (282 mg) was stirred in dry dimethylsulfoxide (20 ml) at 80°C for 2 h. To this green solution, under nitrogen, was added a solution of ICH₂CO₂-Na+ (368 mg) and dry β-CD (9.82 g) in dry dimethylsulfoxide (30 ml). The reaction mixture was left stirring at 50°C for 48 h, acidified with 1N HCl and poured into acetone (100 ml). The crude product was collected and subjected to, firstly Sephadex G15 chromatography and secondly Sephadex A-25-DEAE chromatography.

6^A-Deoxy-6^A-thio-α-cyclodextrin (α-CDSH)

A mixture of α-CDOTs (2g) and thiourea (2g) in 80% aqueous methanol (150 ml) was heated at reflux for 48 hours. The solvent was removed in vacuo and the residue extracted with methanol (20 ml). The resulting solid was separated by filtration, transferred to a flask and heated with 10% aqueous sodium hydroxide (80 ml) at 50°C for 18 hours under an atmosphere of nitrogen. The reaction mixture was acidified to pH2 with dilute hydrochloric acid and stirred at room temperature with tricloroethylene (5 ml). The white solid which separated was collected by vacuum filtration, resuspended in water (15 ml) and heated on a boiling water bath until the solution became clear. The solution was evaporated to dryness under reduced pressure and the resulting residue dissolved in water (15 ml). Concentration of the solution volume and repeated crystallizations removed sodium chloride from the mixture. The addition of acetone to the solution resulted in the separation of α-CDSH as a white solid in a crude yield of ca 10%. HPLC of the product using a 70% acetonitrile - water eluant showed: t_R (relative to α-CD), 0.81. 6^A-Deoxy-6^A-thio-β-cyclodextrin (β-CDSH)

A solution of β-CDOTs (5g) and thiourea (5g) in DMF (50 ml) was heated at 80°C for 18 hours. The reaction mixture was concentrated and the solid dissolved in 10% sodium hydroxide (173 ml) and kept without stirrng²⁴ at 50°C under an atmosphere of nitrogen for 5 hours²⁵. The solution pH was adjusted to 2 by the addition of 10% HCl and tetrachloroethane (10ml) added. After stirring vigorously overnight, the precipitate was collected, washed with water and the TCE removed by boiling the compound in water until it dissolved. Upon cooling needle like crystals formed (3.85 g). T.l.c. (ethylacetate - propanol - water, 7» 7: 5) of the product showed:

R_c (relative to β-CD) , βCDSH, 12.5. HPLC using a 70% acetonitrile - water eluant showed the product was essentially pure, with only minor impurities: t_R (relative to β-CD), β-CDOTs, 0.6; β-CDSH, 0.77; β-CD, 1.0. decomp 245-247 °C ^vmax no diagnostic peaks. FABMS M+H⁺ requires 1151 found 1151

6^A-Amino-6^A-deoxy-6^A-N-propenoyl-β-cyclodextrin (β-CDNHCOCH=CH₂) (i) To a solution of βCDNH₂ (1.0 mg, 0.8 mmol) and sodium hydroxide (1.7 mg, 4.2 × 10^-5 mol) in water (2 ml) was added acryloyl chloride (4.5 mg, 4.4 × 10^-5 mol) with vigorous stirring at 0°C for 10 minutes. The reaction mixture was poured into acetone (20 ml) and the precipitated product collected by centrifugation (1700 r.p.m., 5 min) as a fine white powder (0.73 g). T.l.c. (solvent B) of the product showed: R_c (relative to β-CD), β-CDNHCOCH=CH₂, 1.2. HPLC of the product using a 65% acetonitrile - water eluant showed: t_R (relative to β-CD), β-CDNHCOCH=CH₂, 0.89; β-CDNH₂, 1.16. Separation of the product from β-CDNH₂ was achieved by adding a solution of the crude product mixture (100 mg) in water (0.5 ml) to a slurry of Bio-Rad 70^R in water (5 ml). After stirring for 1 hour at room temperature the solid was filtered and the solvent distilled in vacuo to give a white solid. HPLC of the product using a 70% acetonitrile - water eluant showed: t_R (relative to β-CD), β-CDNHCOCH=CH₂, 0.89.

( β-CDNHCOCH₂CH₂S-β-CD)

A solution of β-CDNHCOCH=CH₂ (117 mg), β-CDSH (124 mg) and sodium hydroxide (4.3 mg) in water (5 ml) was stirred at room temperature for 4 days. The reaction mixture was poured into acetone (20 ml) and the precipitated product collected by centrifugation (1700 r.p.m., 5 minutes) as a fine white powder (200 mg). T.l.c.

(solvent B) of the product showed: R_c (relative to β-CD) , β-CDNHCOCH=CH₂, 1.2; β-CDNHCOCH₂CH₂-S-β-CD, baseline. HPLC of the product using a 70% acetonitrile - water eluant showed: tR

(relative to β-CD), β-CD, 1.0; β-CDNHCOCH₂CH₂-S-β-CD, 5.1

FABMS M+H⁺ requires 2339 found 2339. β-CDNHCOCH₂CH₂-β-CD

(i) A mixture of β-CDNHCOCH=CH₂ (100 mg), NaBH₄ (4 mg), β-CDI (104.5 mg) and tributyltin chloride (5.5 mg) in oxygen free ethanol (5 mis) was stirred vigorously whilst under ultra-violet irradiation at room temperature for 18 hours. T.l.c. (solvent B) showed no reaction had occurred. (ii) A solution of β-CDNHCOCH=CH₂ (50 mg, 4.2 × 10^-5 mol) and β-CDI (52.3 mg, 4.2 × 10^-5 mol) with 2,2'-azobis(isobutyronitrile) (AIBN) ( 1 mg) in 50% aqueous methanol (2 ml) was degassed and heated at 100°C in a sealed ampoule for 24 hours. T.l.c. (solvent B) of the product showed: R_c (relative to β-CD), β-CDNHCOCH=CH₂?; β-CDNHCOCH₂CH₂-β-CD,?. HPLC of the product using a 65% acetonitrile- water eluant showed: t_R (relative to β-CD), β-CDNHCOCH=CH₂, 0.88; β-CDNHCOCH₂CH₂-β-CD, 3.77. (β-CDNHCOCH₂CH₂SCH₂) ₂*

To a solution of ethanediol (2.6 mg) and NaH (2 mg) in dry dimethylformamide (1 ml) at room temperature was added β-CDNHCOCH=CH₂ (100 mg) and the mixture stirred at 80°C for 18 hours under an atmosphere of nitrogen.

T.l.c. analysis revealed a streak from the baseline to an R_f corresponding to that of the acrylamide. The reaction mixture was diluted with water and upon addition of acetone a flocculant precipitate appeared. This material was collected by centrifuge to give a light brown powder. N.m.r. analysis showed vinyl signals still to be present.

This reaction needs further investigation since the starting material has been found to be contaminated. It should also be kept in mind that under the reaction conditions described, the formation of (β-CDS)₂ is also a possibility.

6^A-deoxy-6^A-Thio-6^A-S-((+)-6-methoxy-α-methyl-2-naphthaleneacetyl)-β-cyclodextrin (β-CDSNp)

A solution of β-CDSH (150 mg), m-NO₂PhNp (50 mg) and triethylamine (1-drop) in dry dimethylformamide (0.5 ml) was stirred at room temperature for 48 hours. T.l.c. analysis (acetonitrile - methanol - acetic acid, 3:2:1.5) indicated that there could be a faint spot of higher R_f than the starting material. The reaction mixture was warmed to 50°c and left stirring overnight (18 hours). T.l.c. analysis (acetonitrile - methanol - acetic acid, 3:2:1.5) indicated no β-CDSH remaining. Precipitation with acetone gave a white powder. HPLC of the product using a 70% acetonitrile - water eluant showed: T_R (relative to β-CD), (β-CDSNp), 4.6 6^A-O-(4-bromobutyl)-6^A-deoxy-β-cyclodextrin (β-CDO(CH₂)₄Br*

To a mixture of β-CD (1.0 g), KOH (49 mg) and methyl-tridecyl-ammonium-chloride (433 mg, 0.62 ml of a 70% solution in water) was added 1,4-dibromobutane (190 mg). The mixture was left stirring at room temperature for 66 hours. It was observed that not all of the KOH had dissolved in this time period. T.l.c. of the concentrated reaction mixture showed only a streak with an R_f corresponding to that of β-CD.

6^A-amino-6^A-deoxy-6^A-N-benzoly-α-cyclodextrin (α-CDNHCOPh)*

To a solution of α-CDNH₂ (10 mg) in aqueous 10% sodium hydroxide (2.5 ml) was added benzoyl chloride (0.2 ml) and the mixture shaken vigorously for 20 minutes. T.l.c. showed lots of benzoic acid and no CD material.

1,4-Bis (2^A,2^B,2^C,2^D,2^E,2^F, 3^A,3^B,3^C,3^D,3^E,3^F,3^G, 6^A,6^B,6^C,6^D,6^E,6^F-eicosabenzoate-β-cyclodextrin-6^G-oxy) butane ((β-CD(OBz)₂₀O)₂(CH₂)₄)*

To a solution of β-CDOTs(OBz)₂₀ (500 mg) in dry dimethylformamide (1.5 ml) at room temperature was added a mixture of 1,4-butanediol (12.7 mg) and NaH (6.8 mg) in dry dimethylformamide (0.5 ml) at 70°C for 16 hours. T.l.c. indicated starting material and a new spot with a slightly lower R_f. Chromatography with 20% ethyl acetate - hexane gave starting material but no new compound; even after shipping column with ethyl acetate.

Later analysis revealed β-CDOTs(OBz)₂₀ to in fact β-CDCl(OBz)₂₀

1,4-Bis (2^A,2^B,2^C,2^D,2^E,2^F, 3^A,3^B,3^C,3^D,3^E,3^F,3^G, 6^A,6^B,6^C,6^D,6^E,6^F-eicosabenzoate-β-cyclodextrin-6^G-oxy) benzene ((β-CD(OBz)₂₀O)₂Ph)*

A solution of quinol (15.5 mg) and 80% NaH (8.5 mg) in dry dimethylformamide (0.5 ml) was added to a solution of β-CDOTs(OBz)₂₀ (500 mg, 0.15 mmol) in dry dimethylformamide (1.5 ml) at 70ºC for 70 hours. The black reaction mixture was poured into water and the resultant precipitate found to contain neither product nor starting material. 4-Bis ( β-cyclodextrin-6^A-oxy) benzene ( β-CDO) ₂Ph*

(i) A mixture of β-CDOTs (500 mg), K₂CO₃ (118 mg), KI (64 mg) and quinol (42.7 mg) in dry dimethylformamide (2.5 ml) was stirred at 70°C under an atmosphere of nitrogen, for 24 hours. The reaction mixture was diluted with acetone and the resultant precipitate collected. N.m.r. showed that none of the desired product was present.

(ii) The above reaction was repeated with the exception that ratio of reactants were changed to 2.2 eq of β-CDOTs, leq of quinol, 2 eq of K₂CO₃ and I eq of KI. Appears that we have formed β-CDI.

6^A-O-(4-hydroxyphenyl)-β-cyclodextrin (β-CDOPhOH)*

(i) A mixture of β-CDOTs (500 mg) K₂CO₃ (540 mg) KI (130 mg) and quinol (215 mg) in dry dimethylformamide (10 ml) was stirred at 70°C under an atmosphere of nitrogen for 24 hours. T.l.c. revealed a streaky mess. Precipitation with acetone gave a water soluble black gum.

(ii) A mixture of β-CDOTs (500 mg) K₂CO₃ (1.1 g) and quinol (430 mg) in dry dimethylformamide (25 ml) was stirred at 70°C under an atmosphere of nitrogen, for 72 hours. T.l.c. (acetonitrile -methanol - acetic acid, 3:2:1.5) revealved a spot with the same R_f as β-CD as well as another with a slightly higher R_f. Water (1 ml) was added and the reaction mixture diluted with 20 volumes of acetone yielding a brown precipitate which according to t.l.c. was a decomposition product.

6^A-O-Butyl-β-cyclodextrin (β-CDOBu)*

A mixture of β-CD (2 g), KOH (100 mg) and butylbromide (320 mg) in water (20 ml) was heated at reflux for 24 hours. T.l.c. indicated no reaction had taken place, only starting material was detected. 6^A-O-(4-hydroxybutyl)-β-cyclodextrin (β-CDOBuOH)*

(i) A solution of β-CDOTs (500 mg) and 1,4 butanediol (1 ml) in dry dimethylformamide (10 ml) was stirred with NaH (500 mg) at 100°C for 19 hours. T.l.c. revealed a new spot with the same R_f as β-CD. The reaction mixture was quenched with water (1 ml) and diluted with acetone to give a gluggy precipitate. Attempted recystallization from water to water - acetone failed.

(ii) A solution of β-CDOTs (500 mg) in dry 1,4-butadiol (6 ml) was added NaH (500 mg) carefully and the reaction mixture stirred vigorously at 70°C for 18 hours. T.l.c. revealed a new spot with the same R_f as β-CD. Radial chromatography (acetonitrile - methanol - acetic acid, 3:2:1.5) was performed on the reaction mixture and precipitation of the collected fractions with ehtyl acetate gave a fine white powder (240 mg). N.m.r. gave signals at δ 1.8 and 2.8 indicating possible product. HPLC showed major peak was not β-CD.

6^A-Amino-6^A-N-(2L-amino-3-(4-bis(2-chloroethylamino)-phenyl)propanoyl-α-cyclodextrin (α-CDNHCOMel)*

To a solution of Melphalan (78.1 mg) and ethyl chloroformate (27.9 mg) in dry dimethylformamide (0.5 ml) containing triethylamine (26 mg) at room temperature was added α-CDNH₂ (50 mg). The reaction mixture was heated at 80°C for 18 hours.

2A(3A)-O-(ethoxycarbonylmethyl)-β-cyclodextrin (β-2CDOCH₂CO₂Et)* Oven dried β-CD (908 mg) was stirred with dibutyltin oxide (600 mg) in dimethylformamide (20 ml) under an atmosphere of nitrogen at 95-100°C. After 2 hours Ethyl 2-iodo-acetate (334 mg) was added and stirring continued at 95-100°C for a further 18 hours. T.l.c. analysis (dimethylformamide - water - butan-2-one, 1:1:5) revealed new spots as well as β-CD. HPLC revealed 3 new peaks and chromatography of the crude mixture gave fraction(s) which on evaporation gave a crystalline powder. 6^A-Carboxy-6^A-deoxy-β-cyclodextrin (β-CDO₂H)*

A slurry of platinum oxide (100 mg) in a solution of β-CDI (500 mg) in water (15 ml) at 55°C was stirred vigorously under a head of oxygen for 18 hours. Analysis by t.l.c. showed no change. m-Nitrophenyl-acryloate

Acryloyl chloride (10 g) was added dropwise to a solution of sodium hydroxide (3.6 g, 89.3 mmol) and m-nitrophenol (7 g) in water (500 ml) at 0°C.

Alphaxalone The solubility of alphaxalone is 0.0036 mg/ml. In a 1:1 complex with BCDNH₂ formulation it can easily be solubilised up to 12.44 mg/ml which is an increase of solubility of up to 3,455 times.

The following GLP toxicity studies have been carried out in respect of BCDNH₂ used to solubilise alphaxalone: 1. Acute intravenous toxicity study in rats.

2. Primary dermal irritation study in rabbits.

3. Acute oral toxicity study in rats.

4. Test for mutagenic activity with salmonella typhimuium TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

5. Mouse lymphoma ratation assay.

6. Micronucleus test in bone marrow of CD-1.

7. Chromosomal aberrations assay with Chinese hamster ovary cells in vitro.

The solubility of alphaxalone in BCDNH₂ is as follows:

Solubility of Alphaxalone in BCDNH

% β-CDNH₂ Solubility of Alphaxalone

mg/ml

0 0.0036

1.43 1.770

3.6 4.160

7.1 8.320

10.7 12.44 The potency of the commercial product of Alphaxalone, Althesin 9.0 mg/ml.

The following graphs illustrate the alphaxalone BCDNH₂. The ascending linear part of the solubility diagram is generally ascribed to the formation of a 1:1 complex when the slope is less than 1. Assuming such a complex to be formed, the apparent formation constant can be determined from the initial straight line portion according to the equation described by Higuchi and Connors as follows:

Therefore, the solubility constant of alphaxalone with ACRD's new CD is 59723M^-1. The work carried out in respect of the BCDNH₂ formulations provides for a viable commercial product. BCDNH is not toxic and causes no problems up to 2 grams/kilogram IV and up to 5 grams/kilogram oral and that it is non-mutagenic.

The solubiliser used by commercial companies who have sold Alphaxalone in the past has been highly toxic causing Alphaxalone to be withdrawn from the market. The BCDNH₂ complex with Alphaxalone provides a new and improved formulation for patients. IMPROVED SOLUBILITY OF PROPOFOL

Propofol (2,6-diisopropylphenol) is an intravenous anaesthetic that can be administered either by repeated bolus dose or by continuous infusion. It is formulated in a white, aqueous and isotonic emulsion of pH 6 to 8.5 for intravenous injection containing propofol 10 mg/ml. The vehicle contains glycerol, soybean oil and purified egg phosphatide. According to the available information, it should not be diluted below 2 mg/ml (5:1 dilution) with dextrose 5% in water for injection as this is the point at which the emulsion cracks. It is therefore, desirable to formulate the drug as an aqueous injection. In this report, we use two chemically modified cyclodextrins (β CDN4N, β CDN6N) to solubilise the compound.

MATERIALS AND METHODS

Propofol was supplied by Sigma Chemical Company. ACRD CDs were synthesized in our laboratory. Methanol used in the experiment was supplied by Waters (Division of Millipore). De-ionized water from Milli-Q water system (Millipore, Mass, USA) was used throughout the experiment. All chemicals used were of analytical grade. SOLUBILITY STUDIES

Saturated solutions of propofol were made by adding about 30 mg of the drug to 1 ml of water with respective 0, 1, 2, 5, 10, 15, 20, 25% of cyclodextrin. The solutions were mixed on a Coulter Mixer for over 24 hours to ensure saturation. The suspensions were then centrifuged (Dupont, Sorval, GLC-2B) at 3000 rpm for 15 min. The thin upper layer of oil was excavated off under vacuum and the clear solution was then collected with a 1 ml syringe and filtered through 0.45 filter, 4mm (Alltech, cat. no. 2091). The concentration of the drug in the filtrates were determined by HPLC analysis. HPLC ANALYSIS

Concentrations of propofol in the filtrates were determined by HPLC. The HPLC system consisted of a Waters model 501 pump, a Waters Lambda-Max Model 481 LC spectrophotometer with detection set at 270 nm, and a Waters 745B data module recorder. Propofol was separated on a 10um C-18 ODS uBondapak column (3.9 × 300 mm, Millipore). The mobile phase consisted of methanol:water (70:30), the flow rate was 1 ml/min and propofol eluted at 3.4 min. Concentrations were quantitated by measuring the peak heights. Standard curve for propofol was found to be linear (r exceeding 0.999) over the examined concentration range (Figs 5 & 6).

RESULTS AND DISCUSSIONS

The phase solubility diagram in Fig 7 & 8 indicated that the solubility of propofol increased linearly in increasing concentrations of β CDN4N and β CDN6N. In a solution of 25% of β-CDN6N gave a solubility of 10.24 mg/ml was achieved and a solution of 25% of β CDN6N gave a solubility of 12.88 mg/ml. This solubilities are exceeding the strength of 10 mg/ml used in Diprivan, the therpeutic formulation. A 5:1 dilution of the 25% solution (i.e. the 5% solution of either of the cyclodextrins) gave a solubility of propofol over 2 mg/ml. This is the concentration, at which the emulsion of the current therapeutic formulation cracks during the clinical application.

Conc. of β-CDN4N (%) Conc. of Propofol (mg/ml)

0 0.1

1 0.5

2 0.6

5 2.44

10 4.28

15 7.3

20 7.3

25 10.24 Cone, of β CDN6N (%) Cone, of Propofol (mg/ml)

0 0.08

1 0.49

2 0.87

5 2.5

10 3.72

15 10.4

20 10.24

25 12.88

IMPROVE SOLUBILITY OF ALPHAXALONE

Alphaxalone, a steroidal anaesthetic, is extremely isoluble in water (3.5 ug/ml). It is formulated in a non-ionic detergent, Cremaphor (polyoxyethylenated castor oil), for clinical administration. However, the excipient, Cremaphor is associated with numerous allergic reactions. It would be desirable to solubilize alphaxalone without this detergent in the aqueous solution. In this report, we use two chemically modified cyclodextrins (β CDN4N, β CDN6N) to solubilise alphaxalone.

MATERIALS AND METHODS

Alphaxalone was supplied by Pharmatec, Inc. (Alachua, Florida, USA, Fl 32615). The chemically modified cyclodextrins were synthesized in our laboratory. Methanol used in the experiment was supplied by Waters (Division of Millipore). De-ionized water from Milli-Q water system (Millipore, Mass, USA) was used throughout the experiment. All chemicals were weighed on Mettler AJ 100 analytical balance. SOLUBILITY STUDIES

Saturated solutions of alphaxalone were made by adding 30 mg of the drug to 1 ml of water with respective 0, 1, 2, 5, 10, 15, 20, 25% of cyclodextrins. The solutions were mixed on a Coulter Mixer for over 24 hours to ensure saturation. The suspensions were then centrifuged (Dupont, Sorval, GLC-2B) at 3000 rpm for 15 min. The upper layers of the solutions were collected with a 1 ml syringe and filtered through 0.45 urn filter, 4mm (Alltech, cat. no. 2091). The concentrations of the drug in the filtrates were determined by HPLC analysis.

HPLC ANALYSIS

Concentrations of alphaxalone in the filtrates were determined by HPLC. The HPLC system consisted of a Waters model 501 pump, a Waters Lambda-Max Model 481 LC spectrophotometer with detection set at 208 nm, and a Waters 745B data module recorder. Alphaxalone was separated on a 10um C-18 ODS uBondapak column (3.9 × 300 mm, Millipore). The mobile phase consisted of methanol:water (70:30), the flow rate was 1 ml/min and alphaxalone eluted at 6.1 min. Concentrations were quantitated by measuring the peak heights. Standard curves for alphaxalone were found to be linear (r exceeding 0.999) over the examined concentration range (Fig 9).

RESULTS AND DISCUSSIONS

The phase solubility diagrams in Fig 10 & 11 indicated that the solubility of alphaxalone increased linearly in increasing concentrations of β CDN4N and β CDN6N. In a solution of 25% of cyclodextrin, a solubility of 26.3 mg/ml was achieved in β CDN4N and 28.1 mg/ml in β CDN6N. This solubilities are over 7300 and 7809 times the aqueous solubility of alphaxalone in water (3.6 ug/ml), exceeding the strength of 9 mg/ml used in Althesin, the therapeutic formulation. This result showed that an unsaturated solution of the therapeutic strength can be prepared with over 10% of the cyclodextrins. Previous preliminary studies also indicated that the β CDN4N and β CDN6N alphaxalone solutions did not give precipitation upon storage in refrigeration. This property is essential when these cyclodextrin are considered for intravenous applications.

Conc. of β CDN4N (%) Conc. of Alphaxalone (mg/ml)

0 under detection limit

1 1.42

2 2.97

5 3.13

10 11.9

15 16.9

20 22.7

25 26.3

Conc. of β CDN6N (%) Conc. of Alphaxalone (mg/ml)

0 Under detection limit

1 1.3

2 2.7

5 3.3

10 11.9

15 18.1

20 24.2

25 28.1

6^A-Amino-6^A-N-(4-aminohexyl)-6^A-deoxy-β-cyclodextrin dihydrochloride (β-CDN6N/2HCl)

Concentrated hydrochloric acid was added dropwise to a stirred suspension of β-cCDN6N (6g) in water (30 ml) at 0°C until the solution reached pH2 (ca. 25 drops). The cold solution was filtered through a plug of cotton wool into acetone (300 ml) and the precipitate collected by vacuum filtration and air dried to give 5.6g of a white powder of β-CDN6N/2HCl. SOLUBILIZATION OF ALPHAXALONE WITH β-CDN4N AND β-CDN6N

Alphaxalone, a steroidal anaesthetic, is extremely insoluble in water (3.6 microg/ml). It is formulated in a non-tonic detergent, cremaphor(polyoxyethylenated caster oil) for clinical administration. However, the excipient, cremaphor is associated with numerous allergic reactors. it would be desirable to solubilize alphaxalone without this detergent in the aqueous solution. In this report, we use two new ACRD cyclodextrins β- CDN4N and β-CDN6N. MATERIALS AND METHODS

Alphaxalone was supplied by Pharmatec, Inc. (Alachua, Florida USA, FL 32615). The two ACRD cyclodextrins β-CDN4N and β-CDN6N were synthesized in our laboratory. Methanol used in the experiment was supplied by Waters (Division of Millipore). De-ironized water from Milli-Q water system (Millipore, Mass, USA) was used throughout the experiment. All chemicals were weighed on Mettler AJ 100 analytical balance.

PRELIMINARY SOLUBILITY TEST

Saturated solutions of alphaxalone were made by adding 25 mg of the drug to half a.ml of solutions with respective 10.04 and 10.75% of β-CDN4N and β-CDN6N cyclodextrin. The solutions were mixed on a Coulter Mixer for over 24 hours to ensure saturation. The suspensions were then centrifuged (Dupont, Sorval, CLC-2B) at 3000 rpm for 15 minutes. The upper layers of the solutions were collected with a 1 ml syringe and filtered through 0.45 micron filter, 4 mm (Alltech, Cat. No. 2091). The concentrations of the drug in filtrates were determined by spectrosphotometer (Phillips) at 208 mm. RESULTS AND DISCUSSIONS

Calibration, curve of absorbance of alphaxalone vs concentration is shown in Figure 13. The solubilities of alphaxalone at 10.04 and 10.76% were found to be 13.4 and 16.1 mg/ml respectively. No precipitation was observed when the solutions were stored overnight in refrigeration. This finding appeared to be an advantage over other cyclodextrins as precipition on storage in the cold is often observed with other cyclodextrins.

When β-CDN4N and β-CDN6N alphaxalone solutions were injected i.p. in rats anaesthesic effect was observed in the rat receiving β-CDN4N alphaxalone solution, but not in the rat receiving CDN6 alphaxalone solution. In conclusion, β-CDN4N cyclodextrin appears to be a suitable carrier for alphaxalone I.V. injection. This warrants further studies on its phase solubility and stability. 6^A-Amino-6^A-N-(6-aminohexyl)β-CD (β-CDN6N) β-CDOTs (5g, 3.75 m mole) was heated in 1,6 - diaminohexane (5 g, 43 m mole) at 80 - 90°C for 5 hours. The hot mixture was poured into stirred ethanol (100 ml) and allowed to stand overnight. The solid was filtered and the filter cake was washed with acetone (20 ml) and diethyl ether (20 ml) and then air dried. The solid was dissolved in 5 ml of hot water and then poured slowly into stirred ethanol (25 ml). The mixture was allowed to stand overnight and the solid was filtered off. The solid was crystallised from hot water (10 ml). On cooling, a white solid was produced which was filtered and dried in vacuo to yield 2 g of β-CDN6N.

6^A-Amino-6^A-N-(6-aminohexyl)β-CD (β-CDN6N)

β-CDOTs (20g, 15.0 m mole) was heated in 1,6 - diaminohexane (20 g, 172 m mole) at 80 - 90°C for 5 hours. The hot mixture was poured into stirred ethanol (400 ml) and allowed to stand overnight. The solid was filtered and the filter cake was washed with acetone (80 ml) and diethyl ether (80 ml) and then air dried. The solid was dissolved in 20 ml of hot water and then poured slowly into stirred ethanol (100 ml). The mixture was allowed to stand overnight and the solid was filtered off. The solid was crystallised from hot water (40 ml). On cooling, a white solid was produced which was filtered and dried in vacuo to yield 12.5 g of β-CDN6N.

6^A-Amino-6^A-N-(4-aminobutyl)6^A-deoxy β-cyclodextrin (β-CDN4N) β-CDOTs (5g, 3.75 m mol) was heated in tetramethylenediamine (Putrascine, note STENCH) (5g, 5.67 m mol) at 80 - 90°C for 5 hours. The mixture was poured into stirred ethanol (100ml) and allowed to stand overnight. The solid was filtered, washed with acetone (25 ml) and diethyl ether (25 ml) and then air dried. The solid was dissolved in hot water (5 ml), filtered and poured into stirred ethanol (25 ml). The filtered solid was dissolved in 15 ml of hot water and cooled in fridge overnight. The crystalline solid was filtered and dried in vacuo to yield 1.3 g β-CDN4N.

6^A-Amino-6^A-N-(4-aminobutyl)6^A-deoxy β-cyclodextrin (β-CDN4N) β-CDOTs (20g, 15.0 m mol) was heated in tetramethylenediamine (Putrascine, note STENCH) (20g, 22.68 m mol) at 80 - 90°C for 5 hours. The mixture was poured into stirred ethanol (400ml) and allowed to stand overnight. The solid was filtered, washed with acetone (100 ml) and diethyl ether (100 ml) and then air dried. The solid was dissolved in hot water (20 ml), filtered and poured into stirred ethanol (100 ml). The filtered solid was dissolved in 60 ml of hot water and cooled in fridge overnight. The crystalline solid was filtered and dried in vacuo to yield 7.7 g β-CDN4N. Purification of Toluenesulphonyl Chloride

10 g of toluenesulphonyl chloride was dissolved in 25ml of chloroform and filtered then diluted with 125 ml of petroleum ether (bpt 60 - 80°C) to precipitate impurities. The solution was filtered, clarified with charcoal and concentrated to 40 ml by evaporation. Further evaporation to a very small volume gave 7g of white crystals on cooling, m.p. 67 - 68 °C.

6^A-O-p-Toluenesulfonyl-y-Cyclodextrin (y-CDOTs)

Dry y-CD (5 g, 3.85 m mol) was dissolved in dry pyridine by gentle warming and shaking. Freshly purified 4-toluenesulfonyl chloride (0.8 g, 3.67 m mol) was added in one portion and the solution stirred at room temperature for 16 hours. The pyridine was removed in vacuo and the resulting glassy residue was triturated with acetone (50 ml). The solidified residue was collected by filtration after standing 1 hour. The solid was dissolved in 50 ml boiling water and the milky solution was allowed to cool overnight. The fine white precipitate was filtered and dried under vacuum. T.l.c. using actonitile/water (2:1) showed that solid is unchanged starting material together with impurities. 6^A-O-p-Toluenesulfonyl-y-Cyclodextrin (y-CDOTs)

Dry y-CD (10 g, 7.6 m mol) was dissolved in dry pyridine by gentle warming and shaking. Freshly purified 4-toluenesulfonyl chloride (1.6 g, .7.34 m mol) was added in one portion and the solution stirred at room temperature for 16 hours. The pyridine was removed in vacuo and the resulting glassy residue was triturated with acetone (100 ml). The solidified residue was collected by filtration after standing 1 hour. The solid was dissolved in 100 ml boiling water and the milky solution was allowed to cool overnight. The fine white precipitate was filtered and dried under vacuum. T.l.c. using actonitile/water (2:1) showed that solid is unchanged starting material together with impurities. STUDY TITLE: A non-Surfactant Formulation of Alphaxalone based on a Chemically Modified Cyclodextrin: Activity studies in rats.

I. OBJECTIVE

The purpose of this study was to compare the activity of a formulation, consisting of an anaesthetic, alphaxalone solubilised by an ACRD chemically modified cyclodextrin (ACRD CD), with the standard formulation, Saffan.

II. ANIMALS AND ANIMAL HUSBANDRY

A. Animals

Young adult Sprague Dawley rats, 6 weeks of age at study initiation were obtained from the breeding laboratory in the University of Queensland. This was a species and strain historically used for this type of study. Body weight was appropriate to age and sex.

B. Quarantine

Animals were held in quarantine for a minimum of one day prior to initiation of the study. Animals were examined for general health before release from quarantine.

C. Animal Husbandry

The animal care and husbandry met or exceeded the standards of the Australian code of practice and National Health and Medical Research Council guidelines. The animals were housed in a facility with temperature control (18 - 26º). They received drinking water via water bottles and standard rodent chow ad libitum, apart from an overnight fast prior to dosing. III. TEST ARTICLE

The chemically modified cyclodextrin was synthesised in the chemistry laboratory in Lowood. The drugs alphaxalone was supplied by Pharmatec Co. in the USA. The formulation will be prepared as: alfaxalone 10 mg/ml in 12% ACRD CD. IV. EXPERIMENTATION

The rats were divided into two groups. Each group either received alphaxalone solubilised by the ACRD CD or the standard clinical formulation. The formulations were given intraperitoneally. The doses and dosing schedules are as follows:

Group A B

6 rats 5 rats

Saffan Alphaxalone

24 mg/kg 24 mg/kg During each session after administering the doses, several parameters were measured. Duration of loss of righting response after drug administration was recorded as an index of sleeping time. The degree of anaesthesia during sleeping was estimated every 5 minutes by the presence or absence of a visible response to auditory stimulation (hand clap 10 cm over the supine animmal) and response to abdominal pinch stimulation (tissue forceps pinch). All animals surviving at termination of the study were killed by CO₂ asphyxiation or high doses of anaesthetics.

V. RESULTS AND DISCUSSION

The duration of anaesthesia after i.p. injection of saffan of alfaxalone - CD is shown in Table 1. The mean duration of anaesthesia after injection of Saffan was 43.6 min and after injection of alphaxalone - CD, was 59 min. In one animal, a dose of alphaxalone - CD 48 mg/kg was given, the duration of anaesthesia in that animal was found to be 100 min.

The ACRD CD increased the solubility of alphaxalone by over 7000 times (refer to previous internal report). The CD encapsulated alphaxalone formulation maintains the pharmacological effect of anaesthesia. Therefore, it is able to achieve an aqueous formulation of alphaxalone with the ACRD chemically modified cyclodextrin. All ACRD cyclodextrins being chemically modified cyclodextrins used in this study were hydrochloride salts of such cyclodextrins and are prepared in standard manner as previously disclosed in earlier applications by Australian Commercial Research & Development Ltd.

Table 1. Duration of anaesthesia after i.p. injection of Saffen or Alphaxalone - CD

Saffan Alphaxalone - CD

Dose 24 mg/kg Dose 24 mg/kg

Duration of anaesthesia Duration of anaesthesia

40.3 min 57 min

18.0 40

44.0 70

52.0 69

35.5

72.0

- - - - - - - - - - - - - - - - - -

43.6 min 59 min

Dose 48 mg/kg

100 min The monotosylates, obtained by the treatment of α- and β-Cyclodextrin with p-methylbenzenesulphonylchloride in pyridine, reacted with ammonia in N,N-dimethylformamide at 10⁶ Nm^-2 and room temperature to give the title compounds, pure and in good yield. These amines are of usually low basicity, with pK_bs of 5.30 and 5.28 respectively. The solubility in water at 25° of the amine derived from β-Cyclodextrin is 3.75 gm/100ml, while that of the corresponding hydrochloride salt is 70.5 gm/100 ml. An aqueous solution saturated with that hydrochloride salt dissolves Nabumetone to 674 mg/100 ml at 25° whereas, under similar conditions, the solubility of Nabumetone in water saturated with β-Cyclodextrin is only 36 mg/100 ml and the solubility of Nabumetone alone in water is even lower at 0.8 mg/100ml. The ability of the cyclodextrins (1a-c) and their derivatives to act as host molecules in the formation of inclusion complexes is well established. ^1-9 As part of a program ¹⁰'¹¹ aimed to exploit this behaviour in the administration of pharmaceuticals, we have studied inclusion complexes of a range of modified cyclodextrins. The low solubility of β-Cyclodextrin (1b) in water, which is restricted to 1.85 g/100 ml [0.019 M] at 25° ,² is a principal limitation to the study and application of inclusion complexes involing this compound. Derivatives of β-Cyclodextrin with enhanced solubility in aqueous solution are therefore of particular interest. To be suitable for pharmaceutical applications, the modified cyclodextrins must also be discrete chemical entities, easily obtained pure on a large scale.

At the outset of our work, there had been one report¹² of the synthesis of 6^A-amino-6^A-deoxy-α-cyclodextrin (4a) via the azide (3a) Figure 17 & 18. A number of groups ^13-16 had used a similar approach to obtain the corresponding β-Cyclodextrin derivative (4b), although full details were only reported ¹⁶ during the course of the present investigation. We set out to obtain the amines (4a) and (4b), in order to study their properties and those of their inclusion complexes, and as starting materials for the synthesis of other modified cyclodextrins. We found that samples obtained in the reported manner were contaminated with residual inorganic azide used to form the azides (3a) and (3b), and with the palladium catalyst used in the reduction of the azides (3a) and (3b) to the corresponding amines (4a) and (4b). In addition, these synthetic procedures are undesirable for large scale application, due to the use of sodium azide and hydrogen under pressure. Consequently, we have developed an alternative method for the synthesis of the amines (4a) and (4b), in pure form, directly from the tosylates (2a) and (2b), respectively. In this report we describe that synthetic procedure and we give details of the unusual basicity of the amines (4a) and (4b). We report the high solubility of the amino-β-cyclodextrin hydrochloride salt (5b), relative to that of the parent (1b) and we discuss the use of that salt to improve the solubility of pharmaceuticals. RESULTS AND DISCUSSION

The α-cyclodextrin tosylate (2a) was synthesized by the method of Melton and Slessor,¹² except that the crude product was purified by reverse-phase chromatography rather than by passage through a column of activated charcoal. The corresponding derivative of β-cyclodextrin (2b) was prepared using a similar procedure.

Initially aqueous ammonia was used in an attempt to convert the tosylates (2a) and (2b) directly to the corresponding amines (4a) and (4b) however the tosylates (2a) and (2b) were susceptible to hydroylsis particularly in the case of the α-cyclodextrin derivative (2a). Treatment of the tosylates (2a) and (2b) with anhydrous saturated solutions of ammonia in pyridine or N,N-dimethylformamide gave the corresponding amines (4a) and (4b) without hydrolysis but, at atmospheric pressure and room temperature, more than 50% of each of the respective starting materials (2a) and (2b) remained after two weeks. Finally, solutions of the tosylates (2a) and (2b) in N,N-dimethylformamide were placed in a pressure reactor. Condensed ammonia was added and the reactor was sealed and allowed to warm to room temperature while the pressure increased to 10 Nm^-2. Work-up of the reactions after 18 h gave the amines (4a) and (4b), the latter of which was purified by ion-exchange chromatography.

The amines (4a) and (4b) were completely characterized. In particular, the regiospecificity of incorporation of the amino group was confirmed by proton-coupled ¹³C n.m.r. spectroscopy. The α-cyclodextrin derivative (4a) displayed a triplet at δ 42.8, characteristic for the C6^A carbon, while the corresponding signal for the β-cyclodextrin derivative (4b) was observed at δ 42.1. As part of the characterization process, the amine (4a) was found to have a pK_b = 5.30 ± 0.02 (1 = 0.500, 298.2 K), while the β-Cyclodextrin derivative (4b) was determined to have a pK_b = 5.28 ± 0.02 under the same conditions. This value for the β-cyclodextrin derivative (4b) is well outside the range of 5.8-6.5 reported¹⁶ during the current work and there is no obvious explanation for the discrepancy. Nevertheless it is clear that both the compounds (4a) and (4b) have unusually high pK_b values for primary amines. By way of comparison, the pK_b of ethylamine is 3.34, while that of 1-aminobutane is 3.30¹⁷. It may be that the low basicity of the amines (4a) and (4b) results, in each case, from the effect of the hydrophobic cyclodextrin annulus adjacent to the amino group, to limit the extent of stabiliztion of the protonated form through solvation. Alternatively, hydroxyl residues and the ether linkage in the vicinity of the amino group may affect the pK_b. By analogy, 2-aminoethanol, with a pK_b of 4.38, and 2-methoxyethylamine, with a pK_b of 4.70¹⁷ are each considerably less basic than ethylamine.

The amine hydrochloride salt (5b) was prepared by titration of an aqueous solution of the amine (4b) with hydrogen chloride. The solubility of the salt (5b) in water at 25° was found to be 70.5 gm/100 ml [0.60 M], which is substantially higher than that of either the free base (4b) (3.75 gm/100ml [0.033 M]) or the parent cyclodextrin (1b) (1.85 gm/100 ml [0.016 M]). On this basis, the salt (5b) is particularly suitable for use in studies and applications of cyclodextrin inclusion complexes where a high concentration of the cyclodextrin derivative is required.

One area of application of cyclodextrin inclusion complexes is in the preparation of water-based soluble formulations of pharmaceuticals.^10,11 As an example of the utility of the hydrochloride (5b) in this area, an aqueous solution saturated with the salt (5b) dissolves the nonsteroidal antiinflammatory drug Nabumetone. (4-[6-methoxynaphth-2-yl]-2-butanone) (6) to 674 mg/100 ml at 25° whereas, under similar conditions, the solubility of Nabumetone (6) in water saturated with β-cyclodextrin is only 36 mg/100 ml and the solubility of Nabumetone (6) alone in water is even lower at 0.8 mg/100 ml. The relatively greater effect of the amine salt (5b) compared to that of β-cyclodextrin (1b) is not due to a relatively greater tendency of the cyclodextrin salt (5b) to bind Nabumetone (6) in an inclusion complex. In fact, the stability constant for the inclusion complex formed between the amine salt (5b) and Nabumetone (6) is only 1830 + 185 mol^-1 dm³, whereas that of the complex with β-cyclodextrin is 4400 + 480 mol^-1 dm³. Instead the fact that the cyclodextrin derivative (5b) increases the solubility of the drug (6) by a factor of more than 800, while the solubility enhancement by β-cyclodextrin (1b) is only a factor of 45, must reflect the relatively greater solubility of the amine salt (5b)

EXPERIMENTAL

Ultraviolet spectra were recorded on a Pye Unican SP8-100 spectrophotometer. ¹H and ¹³C n.m.r. spectra were recorded on either a Bruker CXP-300 or a Bruker ACP-300 spectrometer. Fast atom bombardment mass spectra were recorded on a Vacuum Generators ZAB 2HF spectrometer. Thin layer chromatography (t.l.c.) was performed using Kieselgel 60 F₂₅₄ (Merck) on aluminium back plates, eluting with 14:3:3 butanone-methanol-water (Solvent A) or 8:1:1 acetic acid-chloroform-water (Solvent B), and visualized by wetting with a 1.5% solution of sulfuric acid and heating (the R,. value of a cyclodextrin derivative indicates the R_f value relative to that of the parent cyclodextrin). High performance liquid chromatography (h.p.l.c.) was carried out using an ICI LC1500 solvent delivery system coupled to a Knauer differential refractometer. Analytical h.p.l.c. was performed on a Waters Carbohydrate Analysis column (3.9 × 300 mm), eluting at 1.5 ml min^-1 with acetonitrile-water (70%, v/v) (the t_r value of a cyclodextrin derivative indicates the retention time relative to that of the parent cyclodextrin). Preparative h.p.l.c. was performed using a Waters C₁₈ μ-Bondapak column (19 × 150 mm). Solvents were analytical reagent grade and were used as supplied, except that pyridine and N,N- dimethylformamide were dried by strorage over 4Aº molecular sieves. Ether refers to diethyl ether. The cyclodextrins (1a) and (1b) were supplied by Nihon Shokuhin Kako Co. and contained up to 10% water. They were dried under reduced pressure over phosphorus pentoxide, to constant weight, before use.

6^A-O-(4-methylbenzenesulphonyl)α-cyclodextrin (2a)

The tosylate (2a) was prepared using a modification of the procedure of Melton and Slessor.¹²

α-Cyclodextrin (1a) (8.0 g, 8.22 mmol) was dissolved in pyridine (800 ml) by gentle warming and shaking. 4-Methylbenzenesulfonyl chloride (8.0 g, 42.1 mmol) was then added in one portion and the solution was stirred at room temperature for 2 h. The resultant mixture was poured onto ice-cold acetone/ether (6:1, v/v, 6L). The fine white precipitate that formed was allowed to settle over 1h, then most of the supernatant was decanted and the solid was subsequently collected by gravity filtration (Whatman No. 1 filter paper). The solid was then washed with cold acetone (100 ml) and allowed to dry overnight, then it was dissolved in aqueous methanol (30%, v/v 100 ml), and the solution was filtered (0.22 μm membrane) and loaded in one portion, through the pump, onto the C₁₈ h.p.l.c. column. Elution with aqueous methanol (30%, v/v, 100 ml) at 15 ml min^-1 gave fractions containing α-cyclodextrin (1a) (0-35 min). Continued elution (45-120 min) gave fractions that were concentrated under reduced pressure to give the monotosylate (2a) (1.83 g, 19.7%) as a colourless powder. T.l.c. (solvent A) R_c =1.5. Analytical h.p.l.c. t_R = 0.5. Mass spectrum: m/z 1149 (M + Na), 1127 (M + H), ¹H n.m.r. (d₆-DMSO/CDCL₃) 52.45 (S, 3H), 3.1-5.6 (m, 59H), 7.41 (d, J8 Hz, 2H), 7.78 (d, J8 Hz, 2H). ¹³C n.m.r. (d⁶-DMSO) 525.2, 64.0, 73.0, 73.7, 75.7, 76.1, 77.1, 77.3, 85.6, 86.1, 105.6, 106.0, 131.7, 134.0, 136.5 148.9. The preparative h.p.l.c. column was washed with several volumes of methanol to remove polytosylated cyclodextrins.

6^A-O-(4-methylbenzenesulphonyl)-β-Cyclodextrin (2b)

β-Cyclodextrin (13.0 g, 11.45 mmol) was dissolved in pyridine (100 ml). 4-Methylbenzenesulfonyl chloride (1.7 g, 8.94 mmol) was added over a period of 0.75 h, with stirring, and the resultant clear solution was allowed to stand at room temperature for 18 h. The mixture was then concentrated under reduced pressure and the residual oil was triturated with acetone (100 ml). The solid that formed was separated by filtration and twice recrystallized from water to give the monotosylate (2b) (4.5 g, 30.5%) as a colourless powder. T.l.c. (Solvent B) R_c = 1.6. Analytical h.p.l.c. t_r =

0.55. Mass spectrum: m/z 1311 (M + Na), 1289 (M + H) . % n.m.r.

(d₆-DMSO) 52.47 (s, 3H), 3.2-5.1 (m, 69H), 7.46, (d, J8 Hz, 2H), 7.76 (d, J8 Hz, 2H). ¹³C n.m.r. (d₆-DMSO/D₂O) 525.1, 63.9, 75.8, 76.3, 76.7, 81.9, 82.4, 82.8, 85.6, 105.9, 131.5, 133.5, 136.5, 148.6.

6^A-Amino-6^A-deoxy-α-cyclodextrin (4a)

The monotosylate (2a) (1.0g, 0.89 mmol) was dissolved in N,N-dimethylformamide (50 ml) in a 400 ml Parr pressure reaction vessel. Condensed ammonia (100 ml) was added carefully and the vessel was sealed. The mixture was then allowed to warm to room temperature, while the pressure inside the vessel increased to 10⁶ Nm^-2. After stirring the mixture for 18 h at room temperature, the pressure was released, allowing the excess ammonia to evaporate, and the residual solution was concentrated under reduced pressure.

The residual solid was dissolved in water (10 ml) and the solution was concentrated under reduced pressure, then the process was repeated, in order to remove residual N,N-dimethylformamide. The remaining solid was dissolved in aqueous ammonia (20%, v/v, 10 ml) and the solution was added dropwise to acetone (200 ml). The precipitate that formed was collected by filtration under reduced pressure and washed with acetone (5 ml) and then with ether (5 ml), to give the amine (4a) as a colourless powder (670 mg, 70%), with spectral and physical properties consistent with those of a sample obtained using the literature procedure.¹² T.l.c. (solvent A) R_c = 0.3. Analytical h.p.l.c. t_r = 1.2. Mass spectrum: m/z 994 (M + Na), 972 (M + H). ¹³C n.m.r. (D₂O) 542.8, 61.7, 73.2,

73.7, 74.7, 82.5, 84.3, 102.8.

6^A-Amino-6^A-deoxy-β-cyclodextrin (4b)

A crude sample of the amine (4b) was prepared from the monotosylate (2b), using the procedure described above for the synthesis of the amine (4a). The material obtained in that manner contained small quantities of cyclodextrin-based impurities. To remove those impurities, the material was dissolved in water (5 ml) and the solution was added to a stirred suspension of BioRex 70 ion-exchange resin (3g, acid form) in water (20 ml). The mixture was stirred for 4 h, then the resin was separated by filtration and washed with water. Subsequent elution of the resin with aqueous ammonia (20%, v/v) and concentration of the eluant under reduced pressure gave the pure amine (4b) (450 mg, 54%) as a colourless powder. T.l.c. (solvent B) R_c = 0.6. Analytical h.p.l.c. t_r = 1.3. Mass spectrum: m/z 1134 (M +H). ¹³C n.m.r. (D₂O) 542.1, 61.2, 72.5,

72.8, 73.0, 74.0, 81.9, 82.1, 83.1, 102.6, 102.8.

6^A-Amino-6^A-deoxy-β-cyclodextrin hydrochloride (5b)

A solution of the amine (4b) (5gm, 4.4 mmol) in water (20 ml) was adjusted to pH 6 with hydrochloric acid (0.5 M, c. 9ml) then it was filtered and added dropwise to acetone (200 ml). The precipitate that formed was collected by filtration and washed with acetone and ether, then it was allowed to dry. The residual solid was dissolved in water (20 ml) and the solution was concentrated under reduced pressure, then the process was repeated twice. The residue was then dried under reduced pressure over phosphorus pentoxide, to give the amine hydrochloride (5b) (5gm, 97%) as a colourless powder, with spectral and physical properties consistent with those reported previously. Determination of the pK_bs of the amines (4a) and (4b)

The pK_bs of the amines (4a) and (4b) were determined from pH titrations carried out by hand using a Ross combination pH electrode (Orion Research Inc. Model No. 81-03) with a Radiometer PHM64a pH meter and a conventional semi-micro burette Alternatively, titrations were carried out automatically using a Ross combination liquid junction pH electrode (Orion Research Inc. Model No 81-72) with an Orion Research Inc. pH meter Model SA 720 and a 665 Dosimat autoburette (Metrohm) interfaced to a Laser XT/3 8085 personal computer. Titrations were carried out in three-necked water-jacketted glass vessels which alowed room for a pH electrode, a titrant delivery tube and a high purity nitrogen delivery tube for blanketting the solution with CO₂-free nitrogen. A magnetic stirring bar was used to ensure complete mixing. The pH meter was standardized with a phosphate buffer mixture (pH 6.865) and potassium hydrogen phthalate (pH 4.005) at 298.2 K. prior to the titration, a solution of the amine (4a) or (4b), of known volume and concentration was placed in the titration vessel and acidified with a known volume of standardized acid. Nitrogen was then bubbled through the solution for two hours to remove any contaminating CO₂.

Titration of the protonated amine (4a) or (4b) was then carried out by the addition of standarized 0.100 mol dm^-3 sodium hydroxide or potassium hydroxide. The pK_bs were computed from the titration data using the program SUPERQUAD,¹⁸ optimizing both the value of pKw and the value of the purity of the amine (4a) or (4b).

Determination of the Solubility of Nabumetone (6) in Water and in Saturated Aqueous Solutions of β-Cyclodextrin (1b) and the Amine Hydrochloride Salt (5b) Suspensions of Nabumetone (6) in water and in saturated aqueous solutions of β-Cyclodextrin (1b) and the salt (5b) were stirred at 25º for 3 days, then they were filtered (0.22 urn membrane). The filtrates were analysed using ultraviolet spectroscopy, from which the concentration of Nabumetone (6) in each solution was calculated, based on the predetermined molar extinction coefficient for Nabumetone (6) of _max 331 nm, ∈1550. Control experiments established that no more Nabumetone (6) dissolved when the suspensions were stirred for longer than three days and the molar extinction coefficient of Nabumetone (6) at 331 mn in water was unaffected by the presence of either β-cyclodextrin (1b) or the salt (5b).

Determination of the Stability Constants of the Inclusion Complexes Formed Between Nabumetone (6) and β-Cyclodextrin (1b) and the Amine Hydrochloride Salt (5b)

Suspensions of Nabumetone (6) in solutions of β-cyclodextrin (1b) or the salt (5b) in phosphate buffer (0.1 M, pH 7.4) ranging in cyclodextrin concentration from 1 × 10^-4 M to 3 × 10^-3 M, were stirred at 25' for 3 days. The mixtures were then filtered (0.22um membrane) and the filtrates were analysed using ultraviolet spectroscopy, from which the concentration of dissolved Nabumetone (6) in each solution was calculated, as described above. The solubility of Nabumetone (6) in the phosphate buffer was determined in a similar manner.

The solubility of Nabumetone (6) in the buffer, in the presence and absence of the cyclodextrins (1b) and (5b), as a function of the concentration of the cyclodextrins (1b) and (5b), was used to calculate the stability constants of the inclusion complexes.⁹ A potentiometric titration study in aqueous solution (I = 0.10, KCl) of the complexation of benzoic, 4-methylbenzoic, RS-2-phenylpropanoic acid (HA) and their conjugate bases (A-) with β-cyclodextrin, β-CD, and its substituted analogue, 6^A-Amino-6^A-deoxy-β-cyclodextrin, βCDNH₂, in which a primary hydroxyl group is replaced by an amino group which may be protonated to produce a singly charged species, βCDNH₃+, is reported. At 298.2 K the stability constants for the complexes have the values (in dm³ mol^-1) shown in parentheses: benzoic acid. βCD (K_1HA = 590 + 60); benzoate. βCD (K_1A = 60 + 10); benzoic acid. βCDNH₃ + (K_2HA = 340

± 30); benzoate, βCDNH₃ + (K_2A = 120 + 20); benzoate, βCDNH₂ + (K_3A

= 50 ± 20); 4-methylbenzoic acid, βCD (K_1HA = 1680 + 90); 4-methylbenzoate βCD (K_1HA = 110 ± 1); 4-methylbenzoic acid, βCDNH₃+

(K_2HA = 910 ± 20); 4-methylbenzoate βCDNH₃ (K_2A = 330 ± 20); and 4-methylbenzoate βCDNH₂ (K_3A = 100 + 20). These data indicate that for a given cyclodextrin the guest carboxylic acids form complexes of higher stability than do their conjugate base analogues, and that βCDNH₃+ forms more stable complexes with the conjugate bases than do βCD and βCDNH₂. These trends are also observed for the complexation of RS-2-phenylpropanoic acid and RS-2-phenylpropanoate where the complexes indicated are characterised by the stability constants (in dm³ mol^-1) shown in parentheses: RS-2-phenylpropanoic acid. βCD (K_1RHA = 1090 ± 30, K_1SHA = 1010 ± 40); RS-2-phenylpropanoate. βCD (K_1RA = 63 ± 8,K_1SA = 52 ± 5); RS-2-phenylpropanoic acid. βCDNH₃+ (K_2RHA = 580 ± 20, K_2SHA = 480 ± 10); RS-2-phenylpropanoate. βCDNH₃+ (K_2RA = 150 ± 8, K_2SA = 110 ± 10); and RS-2-phenylpropanoate. βCDNH₂ (K_3RA = 36 ± 6, K_3SA = 13 ± 7). These data also show that while K_1RHA and K_1SHA, and K_1RA and K_1SA are indistinguishable for RS-2-phenylpropanoic acid. β-CD and RS-2-phenylpropanoate. βCD, chiral discrimination is indicated by K_2RHA > K_2SHA for RS-2-phenylpropanoic acid. βCDNH₃+, K_2RA > K_2SA for RS-2-phenylpropanoate. βCDNH₃+, and K_3RA > K_3SA for RS-2-phenylpropanoate. βCDNH₂. In contrast, the ¹H NMR spectra of the methyl groups of the enantiomers of RS-2-phenylpropanoic acid appear as two separate doublets indicating chiral discrimination when, complexed by βCD or βCDNH₃+, but such chiral discrimination is not observed for RS- 2-phenylpropanoate when complexed by β-CDNH₃+. The implications of these observations are discussed.

The ability of the chiral α-1, 4-linked cyclic oligomers of D- glucopyranose, or cyclodextrins (CDs), to act as host species in the formation of inclusion complexes with a wide range of guest species is well established.^1-9 Because CDs only exist as D enantiomers, two diastereomeric complexes are formed with racemic guest species, which may result in distinct NMR spectra being observed for each of the R and S guest enantiomers in

solution,^10-12 partial resolution of racemic guests through preferential precipitation of one diastereomeric complex,^13-15 and the chromotographic separation of enantiomers on columns where the stationary phase consists of CDs bonded to silica:^16-18 More recently the effect of substitution of CD hydroxyl groups by other moieties on the complexation characteristics of CDs has become an area of active study as a consequence of the modification of complexation characteristics and solubilities of CDs which can result from such substitution.^19-24 We are particularly interested in the influence of such substition, and the effect of substituent charge, on the complexation process and chiral discrimination between enantiomeric guests; an area which has not previously been subjected to systematic study. Accordingly, we have selected β-cyclodextrin, βCD, and 6^A-amino-6^A-deoxy-β-cyclodextrin, βCDNH₂, in which a primary hydroxyl group of βCD is replaced by an amino group which may be protonated to produce a positively charged species, βCDNH₃+,^20,21 to examine the effect of substitution and charge on the complexation and chiral discrimination characteristics of βCD. The guest species, benzoic acid, 4-methylbenzoic acid, RS-2-phenylpropanoic acid, and their conjugate bases provide convenient conjugate acid-base pairs to test the effect of changing the guest charge from neutral to negative on complexation by these three CD hosts.

EXPERIMENTAL

βCD (Sigma) and βCDNH₂, prepared as in the literature,^20'21 were dried to constant weight and stored over P₂O₅ in a vacuum dessicator prior to use. The carboxylic acids (Sigma) were used as received. The enantiomeric purities of R- and S-2-phenylpropanoic acid were determined to be >95% and >97%, respectively, after HPLC analysis of their diastereomers formed with S-1-phenylethylamine. These purity limits were used in calculations of error limits of the stability constants characterising the complexation of these enantiomers by CDs. Deionised water was purified with a MilliQ-Reagent system to produce water with a specific resistance of >15 Mohm cm, which was then boiled to remove CO₂. All solutions were prepared from this water, and were 0.10 mol dm^-3 in KCl which acted as the supporting electrolyte. Titrations were performed using a Metrohm - Dosimat E665 titrimator, an Orion SA 720 potentiometer, and an Orion 8103 Ross combination pH electrode which was filled with 0.10 mol dm^-3 KCl and calibrated before use with appropriate buffer solutions. Throughout a titration a stream of fine nitrogen bubbles (previously passed through aqueous 0.10 mol dm^-3 KCl) was passed. through the titration solution which was magnetically stirred and thermostatted at 298.2 ± 0.1K in a water-jacketted titration vessel which was closed to the atmosphere with the exception of a vent which permitted egress of the nitrogen stream. A pKw value was determined by titration of 1.0 × .10^-2 mol dm^-3 HCl (8.0 or 2.0 cm³) with standardised 5.0 × 10^-2 mol dm^-3 NaOH. The pKa values of the carboxylic acids and βCDNH₃+ were determined by titration of 2 × 10^-3 mol dm^-3 aqueous solutions (8.0 or 2.0 cm³) with standardised 5.0 × 10^-2 mol dm^-3 NaOH. To determine the stability constants for the complexation of a guest carboxylic acid and its conjugate base by βCD, the burette contained a solution of 1.5 × 10^-2 mol dm^-3 βCD at pH 7. The pH of each 2 × 10^-3 mol dm^-3 carboxylic acid/carboxylate solution (2.0 cm³) in the titration vessel was adjusted to a value near the pK_a of the carboxylic acid. Up to 3 cm³ of βCD solution were titrated into the carboxylic acid/carboxylate solutions in increments not greated than 0.05 cm³, and the observed pH increased by 0.4-0.9 pH units in total depending on the carboxylic acid being studied. At least three similar titrations, with starting pHs in the range 3.9-4.8, were performed for each carboxylic acid system studied.

To determine the stability constants for the complexation of 4-methylbenzoic and R- and S-2-phenylpropanoic acids and their conjugate based by βCDNH₃+, the burette contained a solution of 1.6 × 10^-2mol dm^-3 βCDNH₃+ at pH 6. The pH of each 2 × 10^-3mol dm^-3 carboxylic acid/carboxylate solution (2.0 cm³) in the titration vessel was adjusted to a value near the pK_a of the carboxylic acid. Up to 3 cm³ of βCDNH₃+ solution were titrated into the carboxylic acid/carboxylate solutions in increments not greater than 0.05cm and the pH increased by approximately 0.4 pH units in total. At least three similar titrations, with starting pHs in the range 3.9-4.5, were performed for each carboxylic acid system studied. To determine the stability constants for the complexation of benzoic acid and benzoate by βCDNH₃+, the burette contained a solution of 6.0 x 10^-3mol dm^-3 benzoic acid/benzoate at pH 4. The pH of each 5 x 10^-3mol dm^-3 βCDNH₃+ solution (2.0 cm³) in the titration vessel was adjusted to a value near pH 4. Up to 1 cm³ of benzoic acid/benzoate solution was titrated into the βCDNH₃+ solution in increments of 0.01cm³, and the observed pH increased by approximately 0.3 pH units in total. At least three similar titrations, with starting pHs in the range 3.5-4.5, were performed. To determine the stability constants for the complexation of a guest carboxylate with βCDNH₃+ and its conjugate base βCDNH₂, the burette contained a solution of 0.01 - 0.02 mol dm^-3 carboxylate at pH 7. The pH of each 2 × 10^-3 mol dm^-3 βCDNH₂ solution (2.0 cm³) in the titration vessel was adjusted to a value near the pK_a of βCDNH₃+. Up to 3cm³ of carboxylate solution were added to the βCDNH₃+/βCDNH₂ solutions in increments not greater than 0.05 cm³, and the pH increased by 0.1 - 0.3 pH units, depending on the carboxylate being studied. At least three similar titrations, with starting pHs in the range 8.2-8.8 were performed for each carboxylate system studied.

¹H NMR spectra were run in D₂O solution in 5mm diameter NMR tubes on a Bruker ACP 300 spectrometer. The sweep width was 4000 Hz and typically 100 transients were collected prior to Fourier transformation. Chemical shifts were measured from external sodium 3-(trimethylsilyl)-1-propanesulfonate (TPS) in D₂O. Solutions of βCD or βCDNH₃+ and RS-2-phenylpropanoic acid were adjusted to pH1 with DCl/D₂O, and solutions of βCDNH₃+ and RS-2-phenylpropanoate were buffered at pH 6.4 with phosphate buffer (0.20 mol dm^-3) made up in D₂O. The concentration of βCD or βCDNE₃+ was in the range 1.0 × 10^-2 - 9.0 × 1^0-2 mol dm^-3, and that of RS-2-phenylpropanoic acid or RS-2-phenylpropanoate was in the range 1.0 × 10^-3 - 1.0 × 10^-2 mol dm^-3 so that≥ 90% of the guest species as complexed. When separate resonances were observed in the RS-2-phenylpropanoic acid systems for the guest enantiomers in the two diastereomers, the resonances were assigned by adding resolved R-2-phenylpropanoic acid to the solution and observing which resonance increased in intensity. The βCDNH₃+ and RS-2-phenylpropanoate solutions were prepared at the higher end of the concentration scales indicated above, which together with the high phosphate buffer concentration, may result in an increased solution viscosity which may explain the broader ¹H resonances observed in these solutions. RESULTS AND DISCUSSION

The complexation of a carboxylic acid, HA, and its conjugate base, A-, by βCD may be expressed as in Figure 17 where the acidity constant, K_a, characterises the carboxylic acid, K_1HA and K_1A are the stability constants for the complexation of HA and A-, respectively, by βCD, and K_á characterises HA in the HA.βCD complex.

The variation of the pH of a benzoic acid/benzoate solution in the vicinity of the pK_a (=-log₁₀K_a) of benzoic acid as it was titrated with βCD solution Figure 17A arises because pK_a = pK_á or K_1HA = K (and analogous inequalities hold for the other titrations discussed herein). The best fit of the data to the expressions for K_1HA and K_1A, employing the independently determined value of K_a using program SUPERQUAD²⁵ yields the curve through the data points in Figure 17A (the value of K_á was subsequently calculated from these three values). Similar curves were obtained for the titration of 4-methylbenzoic acid/4-methylbenzoate and R- and S-2-phenylpropanoic acid/phenylpropanoate by βCD, and the data were fitted in a similar manner. The pK_a and pK_á values for benzoic, 4-methylbenzoic, and R- and S-2-phenylpropanoic acid appear in Table 1, as do the K_1HA and K_1A values for the complexation of these carboxylic acids and their conjugate bases by βCD. The complexation of a guest carboxylate (A-) by βCDNH₃+ and its conjugate base, βCDNH₂, may be expressed as in Scheme (2), where K_a is the acidity constant of βCDNH₃+, K_2A and K_3A are the stability constants for the complexation of A- by βCDNH₃+ and βCDNH₂ respectively, and pK_á characterises βCDNH₃+ in the A-.βCDNH₃+ complex.

The variation of the pH of solutions of βCDNH₃+/βCDNH₂ in the vicinity of the pK_a of βCDNH₃+ as they were titrated with a solution of either R- or S-2-phenylpropanoate are shown in Figure 17B. The best fit of the data to the expressions for K_2A and K_3A, employing the independently determined value of K_a, using program SUPERQUAD²⁵ yields the curve through the data points in Figure 17B (the value of Ká was subsequently calculated from these three values). Similar curves were obtained for the titration of βCDNH₃+/βCDNH₂ by benzoate and 4-methylbenzoate, and the data were similarly fitted. The pK_a and pK_á values for βCDH₃+ appear in Figure 16 and the K_2A and K_3A values for the complexation of benzoate, 4-methylbenzoate, and R- and S-2-phenylpropanoate by βCDNH₃+ and βCDNH₂ also appear in Figure 16.

The complexation of HA and A- by βCDNH₃+ may be expressed as in Scheme (3), where K_a, K_á, K_2HA and K_2A are constants characterising the equilibria, and whose values appear in Figure 16. The variations of the pH of R- and S-2-phenylpropanoic acid/phenylpropanoate solutions in the vicinity of the pK_a of RS-2-phenylpropanoic acid as they were titrated with βCDNH₃+ solutions are shown in Figure 17C. The best fit of the data to the expressions for K_2HA and K_2A' employing the independently determined value of K_a, using program SUPERQUAD²⁵ yields the curve through the data points in Figure 17C (The value of K_á was subsequently calculated from these three values). Similar curves were obtained for the titration of 4-methylbenzoic acid/4-methylbenzoate by βCDNH3+, and for the titration of βCDNH₃+ by benzoic acid/benzoate, and the data were similarly fitted. The PK_a and PK_á values for benzoic, 4- methylbenzoic, and RS-2-phenylpropanoic acid appear m Figure 16, and the K_2HA and K_2A values for the complexation of these carboxylic acids and their conjugate bases by βCDNH₃+ also appear in Figure 16 A typical plot of the variation of species concentration with pH, calculated from data in Figure 16, is shown in Figure 17D

(3)

Figures 17A-B and Figure 16

The formation of a CD inclusion complex involves dipolar, hydrogen bonding and van der Waals interactions to varying degrees depending on the nature of the CD and the guest species, and also solvent interactions with the CD and the guest species.^1-4,26 The general structure of the inclusion complex usually shows the aromatic moiety of the guest species to be in the annulus in the vicinity of the hydrophobic ring delineated by the ether oxygens,^27-30 with the dipole moment of the guest species aligned antiparallel to that of the CD.^31-33 The CD dipole moment is in the range 10 - 20D* with the positive and negative poles adjacent to the primary and secondary hydroxyl groups delineating the narrow and wide ends of the CD annulus, respectively.^31-33 It has been observed that the carboxylic acid group of benzoic acid is in the vicinity of the primary hydroxyl groups of αCD in the benzoic acid αCD complex consistents with an antiparallel alignment of the αCD and benzoic acid dipole moments³⁰, and similar antiparallel dipolar orientations are assumed in the complexes appearing in Figure 16. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * 1D = 3.33564 × 10^-30 C m

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

The magnitude of the CD inclusion complex stability constant reflects the competative abilities of the CD to complex the guest species and water to solvate it, and accordingly the data in Figure 16 indicate the changes in these abilities as both the natures of the host CD and the guest species are varied. Six distinct trends emerge from the data in Figure 16 and are now discussed. i) For all systems the carboxylic acid complex is of high stability than the analogues carboxylate complex. This suggests that the negative charge of the carboxylates causes a stronger solvation than in the case of the uncharged conjugate carboxylic acids with the consequence that, although van der Waals interations between the aromatic moieties of the guests and the hydrophobic interior of the CD annulus still result in complexation, the stronger solvation of the carboxylates results in a lower stability for their inclusion complexes. i) The stabilities of the carboxylate complexes increase in the sequence A-.βCDNH₂≤ A-.βCD<A-.βCDNH₃+. This trend may be rationalised on the basis that the positive charge on βCDNH₃+ increases its dipole moment, and provides an increased electrostatic interaction with the carboxylate with a consequent increase in stability of the inclusion complex. iii) For a given CD complex, stability increases with the nature of the carboxylic acid in the sequence benzoic acid <RS-2- phenylpropanoic acid ≤R-S-phenylpropanoic acid <4- methylbenzoic acid. The areas of the hydrophobic surfaces of RS-s-phenylpropanoic acid and 4-methylbenzoic acid are greater than that of benzoic acid and they probably have more extensive van der Waals interactions with the CD as a consequence. In addition the carboxylic acid group, at which significant solvation may occur, represents a proportionately smaller part of their total surface area than is the case for benzoic acid.

It is also possible that the fit of the guest species to the CD cavity improves in the sequence benzoic acid < RS-2- phenylpropanoic acid < 4-methylbenzoic acid. The combination of these effects produces a lower stability constant characterising the benzoic acid complex. iv) For a given CD complex stability increases with the nature of the carboxylate in the sequence S-2-phenylpropanoate ≤ benzoate≤ R-2-phenypropanoate <4-methylbenzoate. Clearly the factors discussed under iii) apply here also, but it is evident that the increase in solvation arising from the negative carboxylate charge causes this factor to become more important, with the consequence that the stabilities of the benzoate and RS-2-phenylpropanoate complexes become comparable. v) Significant chiral discrimination in favour of complexation of R-2-phenylpropanoic acid and R-2-phenylpropanoate over the S-enantiomers is observed for βCDNH₃+ and βCDNH₂, but not for βCD. The replacement of a primary hydroxyl group by -NH₃+ or -NH₂ increases the asymmetry of the CD annulus and accordingly βCDNH₃+ and βCDNH₂ are more likely to discriminate between guest enantiomers through the formation of diastereomeric complexes than is βCD. vi) For the guest carboxylic acids and for βCDNH₃+, pKa < pK_á, which indicates that in the inclusion complex the conjugate base is destabilised relative to the conjugate acid, by comparision with the situation in the uncomplexed state. Because of its charge the carboxylate will be more strongly solvated that the carboxylic acid, and evidently the decreased solvation resulting from inclusion in the CD annulus has a greater effect on the carboxylate with the consequence that it is destabilised by comparison to the included carboxylic acid. This has the overall effect of descreasing the acidity of the carboxylic acid. In the case of βCDNH₃+ the decrease in acidity occuring on formation of the inclusion complex may be a consequence of disruption of the interactions between the -NH₃+ substituent and adjacent hydroxyl residues and the ether linkages which are thought to confer the rather low pK_a value on βCDNH₃+.²¹ In addition, the cancellation of the βCDNH₃+ charge by that of the included carboxylate may decrease the acidity of βCDNH₃+

In the presence of βCD in D₂O/CDl at pH 1, the methyl group of RS-2-phenylpropanoic acid is characterised by two ¹H NMR doublet resonances arising from the R-enantiomer (5 = 1.409 ppm, J_H._H = 6.6 Hz) and the S-enantiomer (5 = 1.421 ppm, J_H-H = 6.9 Hz), which compares with the doublet observed for RS-2-phenylpropanoic acid (δ = 1.289 ppm, J_H-H = 7.2 Hz) in the absence of βCD as seen in Figure 17E. The identification of the enantiomer resonances was made by adding R-2-phenylpropanoic acid to RS-2-phenylpropanoic acid in the presence of βCD and noting the relative increase in amplitude of the upfield doublet. In the presence of βCDNH₃+ in D₂O/DCl at pH 1, the methyl group of RS-2-phenylpropanoic acid is characterised by two doublet resonances arising from the R-enantiomer (δ = 1.428 ppm, J_H-H = 6.9 Hz) and the S-enantiomer ( δ = 1.440 ppm, J_H._H = 6.9 Hz) and the enantiomer resonances were identified as for the previous system. In contrast, in the presence of βCDNH₃+ in D₂O/phosphate buffer at pH 6.4 the resonance of the methyl group of RS-2-phenylpropanoate appears as a doublet ( δ = 1.315 ppm, J_H-H = 6.9Hz), which compares with the doublet observed for RS-2-phenylpropanoate ( δ - 1.357ppm, J_H-H = 7.2 Hz) in the absence of βCDNH₃+. (It was not possible to carry out similar experiments with RS-2-phenylpropanoate in the presence of βCD or βCDNH₂ as K_1RA, K_1SA, K_3RA and K_3SA are too small to give sufficient concentrations of the inclusion complexes within the solubility limits of the systems). The observation of separate resonances for RS-2-phenylpropanoic acid in the presence of βCD and βCDNH₃+ indicates that the magnetic environment of the methyl protons in the diastereomeric complexes are different, while the absence of separate resonances for RS-2-phenylpropanoate in the presence of βCDNH₃+ indicates that the magnetic environment of the methyl protons in the diastereomeric complexes are not signficantly different. Thus, a spectroscopic chiral discrimination occurs when RS-2-phenylpropanoic acid is complexed by either βCD or βCDNH₃+, but not when RS-2-phenylpropanoate is completed by βCDNH₃+. In contrast, a significant thermodynamic chiral discrimination occurs in the complexation of both RS-2-phenylpropanoic acid and RS-2-phenypropanoate by βCDNH₃+, but no significant thermodynamic chiral discrimination occurs in the complexation of these guests by βCD. This demonstrates that although the titrimetric method may detect a significant thermodynamic chiral discrimination. such discrimination does not necessarily induce sufficient magnetic inequivalence in the diastereomeric complexes to be detectable by 1H NMR spectroscopy. Conversely, although different diastereomeric complexes may be identified by ¹H NMR spectroscopy, this does not necessarily imply the existence of a significant thermodynamic chiral discrimination as the energy differences involved in the spectroscopic distinction are small.

THE ACID DISSOCATION CONSTANTS OF PROTONATED 6^A-AMINO-6^A-N-(2-N, 2- N-(DI-2(AMINOETHYL))-2-AMINOETHYL)6^A-DE0XY-0-CYCLODEXTRIN (β- CDTren) AND 6^A-AMINO-6^A-N-(3-(N-(2-N-,2-N-(DI-2-AMINOETHYL)-2- AMINOETHYL))-CARBAMOYLPROPANOYL-6^A-DEOXY-β-CYCLODEXTRIN (β- CDNScTren); AND THE FORMATION OF METALLOCYCLODEXTRINS BY THE LATTER CYCLODEXTRIN IN AQUEOUS SOLUTION

In aqueous solutions of protonated 6^a-amino-6^A-N-(2-N,2-N-(di)-(2- aminoethyl)-)2-aminoethyl) 6^A-deoxy-β-cyclodextrin (βCDNTren) the following acid dissociations occur at 298.2 K and at I = 0.1 (NaNO₃):

The pK_a magnitudes characterising these acid dissociations were derived from data in the pH range 2.0 - 12.0. However, there is some possibility of that a minor contaminant may be present and accordingly these pK_a magnitudes require further checking. In acqueous solutions of protonated 6^A-amino-6^A-N-(3-N-(2-N,2-N- (di-2-aminoethyl)-2-aminoethyl))-carbomylpropanoyl)6^A-deoxy-β- cyclodextrin (β-CDNScTren) the following acid dissociations occur at 298.2 K and at I = 0.1 (NaNO₃):

The pK_a magnitudes characterising these acid dissociations were derived from data in the pH range 2.0 - 12.0. It should be noted that 6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di-2-aminoethyl)-2-aminoethyl))- carbomylpropanoyl)-6A-deoxy-β-cyclodextrin decomposes in aqueous solution in the matter of several hours.

In acqueous solutions of 6^A-amino-6^A-N-(3-N-(2-N,2-N-(di-2- aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β- cyclodextrin (β-CDNScTren) and divalent metal ions (M²⁺ = Co²⁺, Ni²⁺, Cu²⁺ or Zn²⁺), metallo-6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di-2- aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β- cyclodextrins, [M.β-CDNScTren]²⁺, are formed:

and are characterised by the apparent constant (K_mcd) shown above the equilibrium sign. The magnitutde of log (Kmcd/dm³ mol^-1) = 6.65 ± 0.15, 9.08 ± 0.15, 13.0 ± 0.2 and 7.62 ± 0.15 when M = Co, Ni, Cu and Zn, respectively, at 298.2 K and I = 0.1 (NaNO₃). This variation in log (Kmcd/dm³ mol^-1) is as anticipated from the Irving- Williams series.¹'² Due to the decomposition of 6^A-amino-6^A-N-(3- (N-(2-N,2-N-(di-2-aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)- 6^A-deoxy-β-cyclodextrin mentioned above, the log (Kmcd/dm³ mol^-1) requires further experimental confirmation. (None of the metallo-6^A-amino-6^A-N- (3- (N- (2-N,2-N- (di-2-aminoethyl) -2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β-cyclodextrins were isolated from solution and no studies of metallo-6^A-amino-6^A-N-(2-N,2-N-(di-(2-aminoethyl)-2-aminoethyl)-6A-deoxy-β-cyclodextrins have been made.)

Studies of the formation of metallocyclodextrins similar to this study have been reported in the literature.^3-10

Schematic representations of metallo-6^A-amino-6^A-N-(2-N,2-N-(di-(2-aminoethyl)-2-aminoethyl)-6^A-deoxy-β-cyclodextrin,(β-CDNTren.M]²⁺, and metallo-6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di-2-aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6A-deoxy-β-cyclodextrin, [β-CDNScTren.M]²⁺, are shown in Figure 1. These presentations are indicative of the probably structures, but are not claimed to be definitive.

- - - - - - - - - - - - - - - - - - -

Figure 24 here

- - - - - - - - - - - - - - - - - - -

Experimental

6^A-amino-6^A-N-(2-N,2-N-(di-(2-aminoethyl))-2-aminoethyl)-6^A-deoxy-β-cyclodextrins¹¹ (β-CDTren) and 6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di- 2-aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β-cyclodextrins¹² (β-CDNScTren) were prepared as previously described, and were dried to constant weight and stored in the dark over P₂O₅ in a vacuum dessicator prior to use. Metal perchlorates (Fluka) were twice recrystallised from water, and were dried and stored over P₂O₅ under vacuum. (CAUTION. Anhydrous perchlorate salts are potentially powerful oxidants and should be handled with care.)

All solutions were prepared using dionised water purified with a

MilliQ-Reagent system to produce water with a specific resistance of >15 M cm, which was then boiled to remove CO₂. Titrations were performed using a Metrohm Dosimat E665 titrimator, an Orion SA 720 potentiometer, and an Orion 8103 Ross combination pH electrode which as filled with 0.10 mol dm^-3 NaNO₃. Throughout a titration a stream of fine nitrogen bubbles (previously passed through aqueous 0.10 mol dm^-3 NaNO₃) was passed through the titration solution which was magnetically stirred and thermostatted at 298.2 ± 0.10K in a water-jacketted 20cm³ titration vessel which was closed to the atmosphere with the exception of a vent which permitted egress of the nitrogen stream. Standarised 0.100 mol dm^-3 NaOH was used as titrant in all titrations. All 0.001 mol dm^-3 cyclodextrin stock solutions were also 0.005 mol dm^-3 in HNO₃ and 0.095 mol dm^-3 in NaNO₃. The Ni(ClO₄)₂, Cu(ClO₄)₂ and Zn(ClO₄)₂ stock solutions (0.1004, 0.1007 and 0.1003 mol dm^-3, respectively) were prepared in water and standardised by edta titration in the presence of Murexide indicator in the first two cases and Eriochrome Black T in the case of Zn(ClO₄)₂.¹³ Ion exchange of Co²⁺ on an Amberlite HRC-120 cation exchange column in the acid form followed by back titration of the liberated H⁺ was used as the standardisation method for the 0.1005 mol dm^-3 Co(ClO₄)₂ stock solution. E₀ and ply. values were determined by titration of 0.005 mol dm^-3 HNO₃ (0.095 mol dm^-3 in NaNO₃) against 0.100 mol dm_-3 NaOH.

The pK_a values of 6^A-amino-6^A-N-(2-N,2-N-(di-(2-aminoethyl))-2 -aminoethyl)-6^A-deoxy-β-cyclodextrin and 6^A-amino-6^A-N-(3-(N-(2-N,2 -N-(di-2-aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β -cyclodextrin were determined by titration of 10cm³ aliquots of 0.001 mol dm^-3 solutions. The apparent stability constants for the formation of the metallo-6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di-2 -aminoethyl)-2-aminoethyl))-carbamoylpropanoyl)-6^A-deoxy-β -cyclodextrins were determined by titration of 10cm³ aliquots of 0.001 mol dm^-3 6^A-amino-6^A-N-(3-(N-(2-N,2-N-(di-2-aminoethyl)-2- aminoethyl) ) -carbamoylpropanoyl) -6^A-deoxy-β-cyclodextrin with 95mm³ or 45mm³ of divalent metal perchlorate solution added. Derivation of the stability constants was performed using the program SUPERQUAD.¹⁴ Errors quoted for K_MCD represent the standard deviation for the best fit of the variation of pH with added volume of NaOH titrant obtained through SUPERQUAD. Errors quoted pK_a are similarly calculated.

THE FORMATION OF METALLO-CYCLODEXTRINS BY 6^A-(3-AMINOPROPYLAMINO)- 6^A-DEOXY-β-CYCLODEXTRIN AND THEIR CHIRAL DISCRIMINATION IN COMPLEXING R- AND S-TRYPTOPHAN ANIONS IN AQUEOUS SOLUTION

In aqueous solutions of 6^A-(3-aminopropylamino)-6^A-deoxy-β- cyclodextrin (sometimes abbreviated as β-CDN3N, but herein abbreviated to CD), divalent metal ions (M²⁺ = Co²⁺, Ni²⁺, Cu²⁺ or Zn²⁺) and tryptophan anion (T-) at I = 0.1 (NaCIO₄) following equilibria shown below are established, and are characterised by the apparent stability constants (K_subscript) shown above the equilibrium sign. The magnitudes of these apparent stability constants are given in Table 2. It should be noted that not every divalent metal formed all of the complexes shown below as is indicated by the absence of a K_subscript in Table 1 (some of the magnitudes quoted in Table 25 differ from those in the ARC report, and are the finally refined magnitudes). None of the metallo-6^A- (3-aminopropylamino)-6A-deoxy-β-cyclodextrin or other complexes formed were isolated from solution.

1) The acid dissocations of diprotonated 6^A-(3-aminopropylamino)- 6^A-deoxy-β-cyclodextrin are represented by:

The K_a1 and K_a2 magnitudes (Table 1) were derived from data in the pH range 6.0 - 11.5.

2) The formation of 6^A-(3-aminopropylamino)-6^A-deoxy-β- Gyclodextrin tryptophan anion complexes:

shows no chiral discrimination between _R- and S-tryptophan anion. (K_CDRT and K_CDST were derived from data in the pH range 8 - 10).

3) The stability of the metallocyclodextrin formed by 6^A-(3- aminopropylamino)-6^A-deoxy-β-cyclodextrin:

varies with the nature of M as shown by the variation of the magnitude of K_MCD in the sequence Co < Ni < Cu > Zn (Table 2) and as anticipated from the Irving-Williams series.^1,2 (K_MCDRT characterising the complexation of M by CD, shown above, were derived from data in the pH ranges 6.0 - 8.5, 5.5 - 8.5, 5.5 - 9.0 and 5.5 - 7.5 when M = Co, Ni, Cu and Zn, respectively.)

4) The stability of the metallocyclodextrin formed by metallo- 6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin with the tryptophan anion:

also varies with the nature of M as shown by the variation of the magnitude of K_MCDRT and K_MCDST in the sequence Co < Ni < Cu > Zn as shown in Table 1 and as anticipated from the Irving-Williams series. ^1'2 In addition there is a substantial chiral discrimination for S- tryptophan when M = Ni as a comparison of K_MCDRT and K_MCDST (Table 2) shows. When M = Co and Cu there is a moderate chiral discrimination for S-tryptophan, but when M = Zn, no chrial discrimination is observed.

The protonated and deprotonated metallo-6^A-(3-aminopropylamino)- 6^A-deoxy-β-cyclodextrin tryptophan anion complexes are also formed when M = Cu:

But no chiral discimination between R- and S-tryptophan anion is observed within experimental error (Table 2) . (The apparent stability constants characterising the complexations shown above, were derived from data in the pH ranges 7.5 - 8.7, 7.0 - 9.2, 4.5 - 9.5 and 6.5 - 8.0 when M = Co, Ni, Cu and Zn, respectively.)

Studies of the formation of metallocyclodextrins and their chiral discrimination between ligand species such as the trypotphan anion similar to this study have been reported in the literature.^3-10 5) The formation of metal tryptophan anion complexes:

also occurs. They also exhibit stability variations consistent with the Irving-Williams series.^1,2 The apparent stability constants characterising the complexations of M by Tr shown above, were derived from data in the pH ranges 6.5 - 8.5, 5.0 - 9.0, 3.0 -6.5 and 5.5 - 7.0 when M = Co, Ni, Cu and Zn, respectively.

Under the conditions of this work monoprotonated tryptophan is characterised by pK_a1 = 2.40 ± 0.02 and pK_a2 = 9.28 ± 0.01 derived from data in the pH ranges 2.0 - 3.0 and 8.0 - 10.5, respectively. Schematic representations of metallo-6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin and the metallo-6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin-tryptophan anion complex are shown in Figure 25. These representations are indicative of the probable structures, but are not claimed to be definitive. - - - - - - - - - - - - - - - - - - - - - - - - - - - Figure 25 and Figure 26 here

- - - - - - - - - - - - - - - - - - - - - - - - - - -

Experimental

6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin, prepared as in the literature,¹¹ βCD (Sigma), R-, S- and R,S-tryptophan (Sigma), were dried to constant weight and stored in the dark over P₂O₅ in a vacuum dessicator prior to use. The enantiomeric purities of R- and S-tryptophan were determined to be > 99% after HPLC analysis (Pirkle covalent L-phenylglycine column) of the respective esters formed from thionyl chloride pretreated methanol at 348K. These purity limits were used in calculations of error limits of the stability constants characterising the complexation of these enantiomers. Metal perchlorates (Fluka) were twice recrystallised from water, and were dried and stored over P₂O₅ under vacuum. (CAUTION. Anhydrousperchlorate salts are potentially powerful oxidants and should be handled with care.) All solutions were prepared using deionised water purified with a MilliQ-Reagent system to produce water with a specific resistance of >15M cm, which was then boiled to remove CO₂. Titrations were performed using a Metrohm Dosimat E665 titrimator, an Orion SA 720 potentiometer, and an Orion 8103 Ross combination pH electrode which was filled with 0.10 mol dm^-3 NaClO₄. Throughout a titration a stream of fine nitrogen bubbles (previously passed through aqueous 0.10 mol dm^-3 NaClO₄) was passed through the titration solution which was magnetically stirred and thermostatred at 298.2 + 0.1K in a water-jacketted 20cm³ titration vessel which was closed to the atmosphere with the exception of a vent which permitted egress of the nitrogen stream. Standarised 0.100 mol dm^-3 NaOH was used as the titrant in all titrations. All 0.001 mol dm^-3, tryptophan and cyclodextrin stock solutions were also 0.01 mol dm^*3 in HClO₄ and 0.09 mol dm^-3 in a Ni(ClO₄)₂, Cu(ClO₄)₂ and Zn(Cl₄)₂ stock solutions (0.1002, 0.1018 and 0.1003 mol dm^-3, respectively) were prepared in water and standardised by edta titration in the presence of Murexide indicator in the first two cases and Eriochrome Black T in the case of Zn(ClO₄)₂ ¹² Ion exchange of Co²⁺ on an Amberiite HRC-120 cation exchange column in the acid form followed by back titration of the liberated H⁺ was used as the standarisation method for the 0.1003 mol dm^-3 Co(ClO₄)₂ stock solution. E₀ and pK_w values were determined by titration of 0.010 mol dm^-3 HClO₄(0.09 mol dm^-3 in NaClO₄) against 0.100 mol dm^-3 NaOH.

The pK_a values of 6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin and tryptophan were determined by titration of 10cm³ aliquots of 0.001 mol dm^-3 solutions. The apparent stability constants for the formation of the β-cyclodextrin-tryptophan and 6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin-tryptophancomplexeswere determined by titration of 5cm³ each of 0.001 mol dm^-3 solutions of either R- or S-tryptophan and the appropriate cyclodextrin. The apparent stability constants for the formulation of the metal-tryptophan complexes and metallo-6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrins were determined by titration of 10cm³ aliquots of 0.001 mol dm^-3 either tryptophan or cyclodextrin with either 95mm³ or 45mm³ of divalent metal perchlorate solution added. The apparent stability constants for the formation of the metallo-6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin-tryptophan anion complexes were determined by titration of 5cm³ each of 1.0 × 10^-3 mol dm^-3 solutions of R- or S-tryptophan and 6^A-(3-aminopropylamino)-6^A-deoxy-β-cyclodextrin with 45mm³ of divalent metal perchlorate solution added. Derivation of the stability constants was performed using the program SUPERQUAD.¹³ At least three runs were performed for each system, and at least two of these runs were average; the criterion for selection for this averaging being that X for each run was < 12.6 at the 95% confidence level.¹³

FIGURE CAPTIONS FIGURE 19 - Variation of the pH of a solution (2.0 cm₃) of benzoic acid/benzoate (1.04 × 10^-3 mol dm^-3) with volume of added βCD (1.51 × 10^-2 mol dm^-3) at 298.2K and I = 0.10 (KCl). The curve through the data points represents the best fit of the data to the equilibria shown in (1) using SUPERQUAD. FIGURE 20 - Variation of the pH of a solution (2.0 cm³) of βCDNH₃+/βCDNH₂ (2.21 × 10^-3 mol dm^-3) with volume of added R- r S-2-phenylpropanoate (1.40 × 10^-2 and 1.50 × 10^-2 mol dm^-3, respectively) at 298.2K and I = 0.10 (KCl). The upper and lowed data sets refer to s- and R-2-phenylpropanoate, respectively. The curves through the data points represent the best fits of the data to the equilibria shown in (2) using SUPERQUAD.

FIGURE 21 - Variation of the pH of a solution (2.0 cm³) of R- or S-2-phenylpropanoic acid/phenylpropanoate (2.28 × 10^-3 and 2.10 × 10^-3 mol dm^-3, respectively) with volume of added βCDNH₃+ (1.58 × 10^-2 mol dm^-3) at 298.2K and I = 0.10 (KCl). The upper and lower data sets refer to R- and S-2-phenylpropanoic acid/phenylpropanoate, respectively. The curves through the data points represent the best fits of the data to the equilibria shown in (3) using SUPERQUAD. FIGURE 22 - Speciation plot for the βCDNH₂/βCDNH₃+/S-2-phenylpropanoic acid/S-2-phenylpropanoate system calculated from pK_a, K_2SHA, K_2SA and K_3SA (Table 1). The total concentration of S-2-phenylpropanoic acid/S-2-phenylpropanoate is 10^-3 mol dm^-3 and the total concentration of βCDNH₂/βCDNH₃+ is 1.5 × 10^-2 mol dm^-3. The total concentration of S-2-phenylpropanoic acid/S-2-phenylpropanoate is defined as 100% and the free βCDNH₂/βCDNH₃+ concentration is not shown. The curves represent: a) S-2-phenylpropanoic acid. βCDNH₃+, b) S-2-phenylpropanoic acid, c)S-2-phenylpropanoate. βCDNH₃+, d)S-2-phenylpropanoate, and e)S-2-phenyl+propanoate. βCDNH₂.

FIGURE 23 - ¹H NMR (300 M Hz) spectra of themethyl groups of: a)RS-2-phenylpropanoic acid (0.005 mol dm^*3) at pH 1. b) RS-2-phenylpropanoic acid (0.003 mol dm^-3 in the presence of βCD (0.011 mol dm^-3) at pH 1. c) RS-2-phenylpropanoic acid (0.001 mol dm^-3) with added R-2-phenylpropanoic acid (0.0003 mol dm^-3) in the presence of βCD (0.011 mol dm^-3) at pH 1. d) RS-2-phenylpropanoic acid (0.005 mol dm^-3) in the presence of βCDNH₃+ (0.03mol dm^-3) at pH 1. e) RS-2-phenylpropanoic acid (0.002 mol dm^-3) with added RS-2-phenylpropanoic acid (0.002 mol dm^-3) in the presence of βCDNH₃+ (0.03mol dm^-3) at pH 1. f) RS-2-phenylpropanoate (0.01 mol dm^-3 ) at pH 6.4. g) RS-2-phenylpropanoate (0.01 mol dm^-3) in the presence of βCDNH₃+ (0.09 mol dm^-3) at pH 6.4. The chemical shifts are downfield from TPS. The origin of the broader resonances observed in f) and g) is thought to be the higher viscosity of these solutions. REFERENCES

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Example 53 in Cyclodextrin compositions and methods for pharmaceutical and industrial applications. International Patent Application No. PCT/AU91/00071; 1991 J.H. Coates, CJ. Easton, S.F. Lincoln, SJ van Eyk, BL May, ML Williams, SE Brown, A Lepore, ML Liao, V Macolino, DS Schiesser, P Singh, CB Whalland and I McKenzie.

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Claims (29)

WHAT IS CLAIMED IS :

1. An inclusion complex comprising (1) the pharmaceutical agent Propofol (2,6-bis(1-methylethyl)phenol) or Alfaxalone, i.e., 3- hydroxypregnane-11,20-dione, and (2) a cyclodextrin derivative comprising an otherwise substituted or unsubstituted cyclodextrin in which at least one C2, C3 or C6 hydroxyl is substituted with a group selected from -XR¹, -YR²R³, -SiR⁴R⁵R⁶, and -R⁷ wherein X can represent

Y can represent

and wherein R¹ to R¹¹ can each represent the same or different groups selected from the groups -XR¹, -YR²R³, -SiR⁴R⁵R⁶, and -R⁷ are as defined above, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, and wherein any two or three groups bonded to the same substituent can be taken together to represent a single group multiply bonded to said same substituent, and wherein R¹ to R¹¹ may be further substituted by at least one -XR¹, -YR²R³, -SiR⁴R⁵R⁶, -R⁷, halogen, and OR¹², wherein R¹² is as defined for R¹ to R¹¹, and wherein when said cyclodextrin comprises at least one substitution as described above, or is substituted soley for one or more hydroxyls selected from the group of C2 and C3 hydroxyls, said cyclodextrin may also be an substituted by an ether group R¹²-O-, wherein R¹² is as defined above for R¹ to R¹¹.

2. An inclusion complex according to claim 1, wherein said at least one substitution is of the formula -YR²R³, wherein Y is N, and R² and R³ are as previously defined.

3. An inclusion complex according to claim 2, wherein R² is hydrogen and R³ represents amino, hydroxyl, carboxyl, sulfonate (SO_3-), phosphate (PO₄ ^-3), substituted alkyl, cycloalkyl, or aryl, or R² and R³ are taken together to represent a hereto substituted multiply bonded alkyl group.

4. An inclusion complex according to claim 1, having the formula CD - W - R¹³ - L, wherein CD represents an otherwise substituted or unsubstituted cyclodextrin, W represents a functional linking group,

R¹³ represents a group defined the same as R¹-R² above, and L represents a group selected from reactive, charged, polar, or associating groups selected from amino, carboxyl, hydroxyl, sulfonate, phosphate, acyloxy, alkyloxy and thiyl.

5. An inclusion complex according to claim 4, wherein W represents amino, amide, ester, thioether, thioamide, thioester or thiol,

R¹³ represents an otherwise substituted or unsubstituted: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl heteroaryl and heterocyclyl, and

L represents a carboxyl group.

6. An inclusion complex according to claim 5, wherein said otherwise substituted or unsubstituted alkyl group is selected from the group of alkyls having from 1-3, 1-6, 1-10 10-20 and greater than carbons.

7. An inclusion complex according to claim 6, wherein said otherwise substituted or unsubstituted alkyl group is selected from the group of alkyls having from 1-3 carbons.

8. An inclusion complex according to claim 6, wherein said otherwise substituted or unsubstituted alkyl group is selected from the group of alkyls having from 1-6 carbons.

9. An inclusion complex according to claim 6, wherein said otherwise substituted or unsubstituted alkyl group is selected from the group of alkyls having from 1-10 carbons.

10. An inclusion complex according to claim 7, comprising a 6^A-amino-6^A-deoxy-6^A-N-(3-carboxypropanoyl) cyclodextrin.

11. An inclusion complex according to claim 10, which is a β-cyclodextrin.

12. An inclusion complex according to claim 4, wherein L represents a group having a net negative charge.

13. An inclusion complex according to claim 12, wherein L represents a group selected from hydroxyl, carboxyl, phosphate (PO₄ ^-3) or sulfonate (SO₃ ^-1).

14. An inclusion complex according to claim 13, wherein R represents an otherwise substituted or unsubstituted alkyl group selected from the group of alkyls having from 1-3, 1-6, 1-10, 10-20 and greater than 20 carbons.

15. An inclusion complex according to claim 14, wherein R represents an otherwise substituted or unsubstituted alkyl group selected from the group of alkyls having from 1-3, 1-6 and 1-10 carbons.

16. An inclusion complex according to any of claims 4-15, wherein L is amino, ester or amide.

17. An inclusion complex according to claim 1, wherein the cyclodextrin derivative is β-amino cyclodextrin.

18. An inclusion complex according to claim 1, wherein the cyclodextrin derivative is -amino cyclodextrin.

19. An inclusion complex according to claim 10, which is a - cyclodextrin.

20. An inclusion complex according to any of claims 1-10 and 12-16 wherein said cyclodextrin is a β-cyclodextrin.

21. An inclusion complex according to any of claims 1-10 and 12-16 wherein said cyclodextrin is a -cyclodextrin.

22. An inclusion complex comprising Alfaxalone and unsubstituted -cyclodextrin.

23. A pharmaceutical composition comprising an inclusion complex according to any of claims 1-22, which is in form suitable for oral delivery.

24. A pharmaceutical composition according to claim 23 which is in form of an aqueous solution.

25. A pharmaceutical composition comprising a cyclodextrin derivative according to any of claims 1-22, which is in form suitable for parenternal delivery.

26. A process for increasing the solubility of Propofol or Alfaxalone comprising the step of forming an inclusion complex to any of claims 1-22.

27. A process for improving the in vitro or in vivo stability of Propofol or Alfaxalone comprising the step of forming an inclusion complex to any of claims 1-22.

28. A process for improving the bioavailability of Propofol or Alfaxalone comprising the step of forming an inclusion complex to any of claims 1-22.

29. A process for the treatment of a patient in need of an aesthetic comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition according to any of claims 23-25.