Background Co-pending patent application WO 95/05169 (Strathclyde University) relates to flavonoids and their use in methods of treating anxiety in patients. The flavonoids of WO 95/05169 are described as having anxiolytic properties without associated depression of the central nervous system (e. g. sedative and muscle relaxant effects) commonly found with benzodiazepines.

The compounds of WO 95/05169 fall under a general formula: wherein R', R2, R3 and R4, R5 and R8 are independently selected from H, OH, R, NO2, halo, OR, NH2, NHR, NR2, COOR, COOH, CN, or a sugar group; R6 and R7 are both H, or R6 and R7 together form a single bond; R is C14 alkyl or alkenyl; or dimers thereof.

Preferred compounds of WO 95/05169 are described as halo derivatives, in particular where R5 is halo at the 2'position of the above general formula.

-It has now been found that certain compounds falling within the generic formula of WO 95/05169 exhibit unexpectedly good anxiolytic activity without associated depression of the central nervous system (e. g. sedative and muscle relaxant effects) commonly found with benzodiazepines, when the flavone nucleus and/or phenyl ring is nitrated and/or halogenated. Thus, patients may be treated for anxiety without inducing sedative or myorelaxant side-effects.

It has also been found that compounds of the present invention display a substantially reduced or no anti-convulsant effect, and that memory is apparently not adversely affected, side-effects commonly found with benzodiazepines.

Statement of Invention According to the present invention there is provided a method of treating anxiety in a patient which comprises administering to the patient an effective non-toxic amount of a flavonoid of general Formula (I): wherein R1 ! R2, R3, R4 and Rs are independently selected from Cl, Br, F and H; R6 is H; R7, R9 and R10 are independently selected from H,-OH, -R,-NO2, Br, Cl, F,-OR,-NH2,-NHR,-NR2,-COOR,- COOH,-CN or a sugar group; R8 is selected from H, NO2, Br, Cl, and F; R is C1C6 alkyl or alkenyl; with the proviso that when R1 is H, R8 is No. and when R1 is selected from Cl, Br and F, R8 is selected from H, NO2 Br, Cl and F; or the administration of an effective non-toxic amount of a bi-flavonoid which is a dimer of a compound of general Formula (I) and wherein R1 to R10 and R have the meanings given for general Formula (I).

The sugar group may be any of the known sugars, including monosaccharides, disaccharides and polysaccharides; and may in particular be glycosyl, galactopyranosyl or mannopyranosyl.

Preferred compounds of Formula I for use in a method of treating anxiety in a patient include compounds wherein R1 is selected from H, Cl and Br; R2 to R7 inclusive, R9 and R10 are all H; R8 is selected from H, NO2, Br, Cl and F; with the proviso that when Ru ils H, Rs is NO2 and when Ru ils selected from Cl, Br and F, R8 is selected from H, NO2, Br, Cl, and F.

More preferred compounds of Formula (I) are those wherein R1 is selected from H and Br; R2 to R7 inclusive, R9 and R10 are all H; and R8 is selected from Br, NO2, and H; with the proviso that when Ru ils H, R8 is NO2.

Most preferred compounds of Formula (I) are those wherein R1 is Br; R2 to R7 inclusive, R9 and R10 are all H; and R8 is selected from Br and NO2.

Examples of preferred compounds of Formula (I) for use in treating anxiety in a patient include: 6, nitroflavone 3'chloroflavone 3'bromof lavone 6,3'-dibromoflavone 6, nitro, 3'bromoflavone Especially preferred are compounds (V) and (VI) above.

Compounds wherein R6 and R7 together form a single bond are flavone derivatives.

The bi-flavonoid is a dimer of two covalently bonded moieties which are each of general Formula (I) as set out above.

Bonding between the two moieties generally occurs at the 3'- position of one moiety and the 8-position of the other moiety.

The preferred bi-flavonoid has general Formula (VII) wherein to R10 and R have the same meanings as for general Formula (I): Preferred compounds of general Formula (VII) for use in a method of treating anxiety in a patient include those wherein at least R'in each of the dimer moieties of general Formula (I) is selected from H, C1, and Br; R8 is selected from Cl, Br, F, H and NO2 ; and all other R functional groups are as defined for Formula (I) with the proviso that when R'is H, R8 is NO2 and when R'is selected from Cl, Br and F, Rs is selected from H, NO2 Br, C1, and F.

The compounds of general Formula (VII) wherein the compounds are 3'halo (ie Br, Cl or F) containing compounds; Rg is selected from NO2, Cl and Br; and all other R functional groups are hydrogen are more preferred.

Pharmaceutical formulations include at least one compound of general Formula (I) and/or (VII) together with at least one pharmaceutically acceptable carrier or excipient. Naturally, the skilled addressee will appreciate that compounds of the invention employed in pharmaceutical formulations of the invention possess R groups R'to Rlo and R as defined herein. Each carrier must be "pharmaceutically acceptable"in the sense of being compatible with the other ingredients of the formulations and not injurious to the patient.

It should be understood that the flavonoid compounds of the present invention can be administered in the form of pharmaceutically acceptable salts or esters thereof. Salts are usually acid addition salts (e. g. with hydrohalogen acids) or acceptable metal salts (e. g. Na, Ca, Mg).

Formulations include those adapted for oral, rectal, nasal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention adapted for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e. g. povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e. g. sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethylcellulose) active-surface or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations are parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatis and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The dose will depend on a number of factors known to the skilled physician including the severity of the conditions, the identity of the recipient; and also the efficacy and toxicity of the particular compound of general Formula (I) which is being administered. Generally doses in the range 0.1-100 mg/kg body weight may be used, particularly 1-10 mg/kg. The frequency of administration will vary depending on the rate of metabolism or excretion of the administered compound, but may be repeated daily, optionally as two or more sub-doses. Unit doses of 20 to 500 mg, preferably 100 to 400 mg may be used.

As a further aspect of the present invention there is provided a compound of general Formula (I): wherein R1, R2, R3, R4 and R are independently selected from Cl, Br, F and H; R6 is H; R7, R9 and R10 are independently selected from H,-R, NO2, Br, Cl, F,-OR,-NH2,-NHR,-NR2,-COOR,-COOH,-CN or a sugar group; R8 is selected from H, NO2, Br, Cl, and F; R is C1C6 alkyl or alkenyl; with the proviso that when R1 is H, Ra is NO2, and when R1 is selected from Cl, Br, and F, Ra is selected from H, NO2, Br, Cl and F or a bi-flavonoid which is a dimer of a compound of general Formula (I) and wherein R1 to Rl° and R have the meanings given for general formula (I).

Preferred compounds of Formula (I) include compounds wherein R1 is selected from H, Cl and Br; R2 to R'inclusive, R9 and R10 are all H; R8 is selected from H, NO2, Br, Cl and F; with the proviso that when Ru ils H, Re is NO2 and when Ru ils selected from Cl, Br, and F, Re is selected from H, NO2, Br, Cl and F More preferred compounds of Formula (I) are those wherein R1 is selected from H and Br; R2 to R7 inclusive, R9 and R10 are all H; and R8 is selected from H, NO2 and Br; with the proviso that when Ru ils H, Re is NO2 Most preferred compounds of Formula (I) are those wherein R1 is Br; R2 to R'inclusive, R9 and R10 are all H; and Re is selected from Br and NO2.

Preferred compounds of the invention include: 6, nitroflavone 3'chlorof lavone 3'bromoflavone 6,3'-dibromoflavone 6, nitro, 3'bromoflavone Most preferred compounds of the invention are (V) and (VI).

In a further aspect of the invention there is provided use of compounds of Formula (I) in the preparation of a medicament for the treatment of anxiety in a patient. In a preferment there is provided use of a compound according to any one or more of formulae (II) to (VII) in the preparation of a medicament for the treatment of anxiety in a patient. More preferably there is provided use of a compound or a cocktail of compounds selected from compounds (V) and (VI) in the preparation of a medicament for the treatment of anxiety in a patient.

Compounds of the invention can be prepared according to the general outline provided in scheme 1 below: Scheme I The Baker-Venkataraman transformation as described by, for example, Baker, W. Molecular rearrangement of some o- acyloxyacetophenones and the mechanism of production of 3- acylchromones. J. Chem. Soc. (1933), 1381-1388; Mahal, H. S, Venkataraman, K. Synthetical experiments in the chromone group Part XIV. The action of sodamine on 1-acyloxy-2-acetonaphthones.

J. Chem. Soc. (1934), 1767-1769; Wheeler, T. S. Flavone Organic Syntheses; Wiley New York, 1963, Colleer Vol IV, pp 478-481, can be used as a method of synthesising flavone molecules of the invention.

Generally, an appropriately substituted hydroxyacetophenone (1) comprising substituents R as defined for R7, R8, R9 and Rl° of Formula (I) can be converted into a benzoyl ester of Formula (2) by reaction with an appropriately substituted benzoyl halide (B) comprising substituents R as defined for Rl, R2, R3, R4 and R5 of Formula (I). Suitable benzoyl halides include benzoyl chloride, benzoyl bromide or benzoyl fluoride having R groups as defined for R', R2, R3, R4 and R5 above. The benzoyl ester (2) may then be treated with base, such as KOH/pyridine, forming a diketone of Formula (3) comprising R and R substituents of choice as defined above for Rl to R5 inclusive and R7 to Rl° inclusive. The diketone may then be treated with acid leading to the generation of flavone of interest (4) according to the teaching of Ares J. J. et al., A. J. Med Chem. (1995) 38 pp 4937-4943.

Where R=H, nitro-substituted flavones (5) can be obtained by treating a flavone of interest (4) with HN03 in H2S04 using the nitration method as outlined by Cushman M. et al A. J. Med. Chem.

(1994) 37 pp3353-3362.

The invention will now be illustrated by reference to the following figures and examples.

Figure 1: Displacement curve of [3H] FNZ (0.5nM) binding by 6- nitro-3'-bromoflavone to washed crude synaptosomal membranes from rat cerebral cortex.

Figure 2: Displacement curve of [3H] FNZ (0.5nM) binding by 6, 3'- digromoflavone to washed crude synaptosomal membranes from rat cerebral cortex.

Figure 3: Displacement curve of [3H] FNZ (0.5nM) binding by 3' bromoflavone to washed crude synaptosomal membranes from rat cerebral cortex.

Figure 4: Displacement curve of [3H] FNZ (0. 5nM) binding by 6- nitroflavone to washed crude synaptosomal membranes from rat cerebral cortex.

Figure 5: Displacement curve of [3H] FNZ (0.5nM) binding by 3' chloroflavone to be washed crude synaptosomal membranes from rat cerebral cortex. Example 1 : A1 :- 6'nitroflavone Reference is made to scheme III: NO2 HN03/AcOH OH + NU2 OH NO t 0 ? 0 8 0 0 OH + Ct K % OH- N 02 N 02 v v 0 g 10 Br2/Pyridine . OH" n tt) I OH Br H Pyridine w NO N 0 N02 0 H Br 6-nitroflavone Nitric acid (fuming, 4mL) was added to a solution of 2- hydroxyacetophenone (1) (60mol) in glacial acetic acid (70mL) and the mixture was stirred in a round-bottomed flask equipped with a drying tube (CaCl2) at room temperature for 40 min and then at 45-50°C for 16h. Acetic acid was removed at reduced pressure, and water (100mL) was added. The yellow cloudy solution was neutralized by Na2CO3 to PH 6, and then the yellow precipitate was collected and carefully recrystallised from EtOH in several crops to give 7.

A solution of 5 g of compound 7 and 6 g of compound 9 in 50 mL of EtOH was treated with 50 g of KOH in 50 mL of water, and left a day at room temperature. Then it was acidified with HCland the intermediate 10 was used in the next step.

A 10% solution of bromine in 12mL of AcOH was added to a hot solution of 1 g of compound 10 in lOmL of AcOH, the mixture was left overnight at room temperature and the product crystallized from AcOH rendering compound 11. A solution of 1 g of 11 in pyridine (lOmL) was stirred for 30 min at room temperature. Then it was poured into a 3% aqueous HCl/ice solution with vigorous stirring.

The resulting precipitate was filtered and washed with water. The crude material was recrystallized from ethanol yielding 6-nitroflavone.

6-nitroflavone : H-NMR (200 Mhz, CDCI3) : 5 9,10 (d, J=2.1) Hz, h- 5), 8.53 (dd, J-2.0 Hz, 8.1 Hz, H-7), 7,96-7.91 (m, H-2, H-6'), 7.73 (d, J=8.1 Hz, H-8), 7.62-7.53 (m, H-3', H-4'H-5'), 6.67 (s, H-3).

Example 2: A2 :- 3'chloroflavone Reference is made to scheme II: cl 0 ruz o OHCl) Cl pyridine 3 step 1 0 00 1 2 3 step2 l pyridine/KOH Cl 0 1 H2SO4/AcOH step 3 00 0 3-chloroflavone 4 Step 1 To a solution of the acid chloride 2 (Aldrich) (15mmol) in pyridine (10ml), at 0°C, solid 2-hydroxyacetophenone 1 (9mmol) (Aldrich) was added with stirring. The reaction mixture was stirred for 15min at 0°C followed by 30min at room temperature.

Then it was poured into a 3% aqueous HCl/ice solution with vigorous stirring. The resulting precipitate was filtered and washed with water. The crude material was recrystallized from methanol yielding compound 3.

Step 2 To a solution of compound 3 (lOmmol) in pyridine (10ml), at 50°C, pulverised potassium hydroxide (15mmol) was added. The mixture was stirred for 15min and, after cooling, a 10% aqueous acetic acid solution was added. The resulting precipitate was filtered.

The crude material, containing diketone 4, was used in the next step without purification.

Step 3 A mixture of the crude material from step 2 (equivalent to lOmmol of compound 4), with concentrated sulfuric acid (0.5 ml), and glacial acetic acid (13ml) was heated at reflux for 1 h and cooled to room temperature. Then, the mixture was poured onto crushed ice (75g), and the resulting precipitate was filtered.

Recrystallization from acetone afforded product 3'- chloroflavone.

3'-chloroflavone : 1H-NMR (200Mhz, CDCI3) : 5 8.24 (dd, J=2.0.

Hz, 8.2Hz, 11-5), 7.93 (t, J=2.2 Hz, H-2g), 7.81- 7.68 (m, H-4', H-6'), 7.60-7.40 (m, H-6, H-7, H-8, H-5'), 6.81 (s, H-3).

Example 3 : A3 :- 3'-bromoflavone Reference is made to scheme II (example 2) Steps 1,2 and 3, same conditions, (Scheme II) but using compound 5 (Aldrich) and 2-hydroxyacetophenone 1 as starting materials.

Compound 5: 3'bromoflavone: 1H-NMR (200 Mhz, CDCI3) : 5 8.24 (dd, J=2.1,8.5 Hz, H-5), 8.10 (t, J=2.0 Hz, H-2'), 7.84 (dq, J=2.0,8.4 Hz, H-4'), 7.66 (m, H-6', H-6, H-7), 7.42 (m, H-5', H-8), 6.81 (s, H-3).

Example 4: A4 :-6, 3'-dibromoflavone Steps 1,2 and 3, same conditions, (scheme II example 2) but using compounds 5 and 6 (Aldrich) as starting materials.

6,3'- dibromoflavone: 1H-NMR (200 Mhz, CDCI3) : 5 8.34 (s, H-2'), 8.13 (m, H-4'H-5), 8.02 (dd J=2.0,8.2 Hz, H-7), 7.86 (d, J=8.2 Hz, H-8), 7.80 (m, H- 6'), 7.55 (t, J=8.0 Hz, H-5'), 7.20 (s, H-3).

Example 5: A5 :- 6, nitro-3'-bromoflavone Reference is made to scheme III (example 1) Same procedure as in Scheme III but using compounds 5 & 7 as starting materials.

6-nitro-3'-bromoflavone : H-NMR (200 Mhz, CDCI3) : 5 9. 11 (d, J=2.4 Hz, H=5). 8.56 (dd, J=2.4,8.5 Hz, H-7), 8.10 (t, J=2.1 Hz, H-2'), 7.85 (m, H-4'), 7.74 (d, J=8.2 Hz, H-5'), 7.73 (m, H-6'), 7.44 (t, J=8.2 Hz, H-5'), 6.87 (s, H-3).

Example 6: Binding Affinity of flavone derivatives for Benzodiazepine Receptors (BDZ-Rs) Animals Adult male Wistar rats weighing 250g were used for biochemical experiments.

Animals were housed in a controlled environment, with free access to food and water and maintained on a 12h/12h day/night cycle.

Membrane Preparation Membrane preparations were carried out according to Medina et al. (1990). Briefly, brains were rapidly dissected out on ice and the different structures were homogenized in 10 volumes of 0. 32 M sucrose and centrifuged at 900 x g for 10 min. The resulting supernatant was centrifuged at 100,000 x g for 30 min and the pellet washed twice in 25mM Tris HC1 buffer pH 7.4 at 100,000 x g for 30 min, and stored at-20°C until used.

Binding affinities to BDZ-Rs were determined by the ability of the compounds to inhibit Hoffman-La Roche 3H-flunitrazepam (3H- FNZ) binding to extensively washed rat cerebal cortical membranes following the method as described by Levi de Stein, M. et al., Mol. Brain Res (1989), 5, pp 9-15.

In brief, for each assay, triplicate samples of the membranes, containing 0.2 to 0.4 mg protein were suspended in a final volume of 1 ml of 0.25 mM Tris-Cl buffer, pH 7.3.

The incubation was carried out at 4°C for 60 min with 0.5 nM 3H-FNZ. To study the binding saturation, a range of 0.3 to 10 nM 3H-FNZ was used. Non-specific binding was determined in parallel incubations in the presence of 3pM FNZ, and represented 5-15% of total. The assays were terminated by filtration under vacuum through Whatman GF/A glass-fiber filters, and three washes with 3mL each of incubation medium. Filters were dried and counted after the addition of 5 mL of 2,5-diphenyloxazole/xylene as scintillation fluid.

Results are shown in Table 1 below.

Table 1 : Binding affinity of flavone derivatives, Al, A2, A3, A4, and AS for Benzodiazepine receptors (BDZ-Rs) of Rat Cerebral Cortex membranes.

Compound R6 R8 Rt Rl R2 R3 Kib (UM) Flavone 6 H H H H H H 1.00 Al H NO2 H H H H 0.275 A2 H H H Cl H H 0.614 A3 H H H Br H H 0.413 A4 H Br H Br H H 0.019 A5 H NO2 H Br H H 0.025 Kib SEM values are means of 3-5 independent determinations.

For values ranging from 0.001 to 4.5, the SEM varied between 6 to 13% of the values listed. The low affinity values are the result of duplicate measurements.

Biological Data Table 2 shows the mean SEM of Ksi values of different compounds, obtained from displacement curves of [3H] flunitrazepam binding to rat synaptosomal membranes. The competition curves were analyzed using Graph-Pad Prism software.

Table 2 Compound Y, (rim) n 6 nitro-3'-bromoflavone 25.172.46 7 6, 3'-dibromoflavone 18.632.18 3 3'-bromoflavone 413.646 3 6-nitroflavone 27541. 1 3 3'-chloroflavone 61432. 7 3 n represents the number of experiments performed.

Figures 1-5 show representative displacement curves of [3H] flunitrazepam (0.5 nM) binding by different flavones to washed crude synaptosomal membranes from rat cerebral cortex. The Kils obtained in each case were: Compound Ki (nM) 6-nitro-3'-bromoflavone 21.8 6,3'-dibromoflavone 22.7 3'-bromoflavone 427 6-nitroflavone 315 3'-chloroflavone 665 Results and Discussion The following structure activity relationships were obtained by analysis of the information collected in Table 1 and are expressed as flavone substitutions that produce the following changes in the binding affinity of the mother compound (flavone 6): A) Great increases f40 to 1000 times): -a bromine atom or a nitro group on carbon 6 plus a halogen group on carbon 3' (A4, A5). The effectiveness of the different substituents on carbon 6 is as follows; Br>NO2.

B) Moderate increases (3.6 to 13 times): -a nitro group on carbon 6 (A1). The effectiveness of this substituent is as indicated previously.

-a bromine or a chlorine atom on carbon 3'(A2, A3).