WO2010103273A2

WO2010103273A2 - Essential fatty acid compounds

Info

Publication number: WO2010103273A2
Application number: PCT/GB2010/000430
Authority: WO
Inventors: Mehar Manku
Original assignee: Amarin Neuroscience Limited
Priority date: 2009-03-12
Filing date: 2010-03-10
Publication date: 2010-09-16
Also published as: GB0904300D0; WO2010103273A3; JP2012520274A; CA2754902A1; AU2010222692A1

Abstract

The invention provides compounds which include L-DOPA or dopamine linked to an essential fatty acids. The general formula is set out below: R¹-Z-O-(CH₂)_n-CH(R³)-(CH₂)_mO-Y-R². R¹ is derived from a fatty acid and R² is from L-DOPA or dopamine. A preferred linker has the 1,3 propane diol structure.

Description

Essential Fatty Acid Compounds

The present invention relates to essential fatty acid compounds and their therapeutic applications.

Levodopa, or L-DOPA (3,4-dihydroxy-L-phenylalanine), is shown as Formula 1 below:

(1 )

Dopamine (DA) is formed by the decarboxylation of L-DOPA. Dopamine is shown in Formula 2 below:

(2)

L-DOPA is used in the management of Parkinson's disease (PD), as a prodrug to increase dopamine levels. L-DOPA is the only easily accessible and low-invasive therapy for PD patients. It is able to cross the blood-brain barrier whereas dopamine itself cannot. Once L-DOPA has entered the central nervous system (CNS), it is metabolized to dopamine by aromatic-L-amino-acid decarboxylase.

In the early years of the disease, L-DOPA markedly increases the quality of life of patients suffering from Parkinson's disease. However, there are major problems with L-DOPA therapy, especially after 5-10 years of continued treatment. These include fluctuations of motor responses to L-DOPA. Motor fluctuations can range from relatively simple "wearing off' of medications prior to the next dose, also called end-of-dose failure, to random, severe fluctuations in motor functioning. As such, a patient may rapidly and unpredictably alternate from having severe Parkinsonian symptoms ("off') to a near normal motor state ("on") and then to a state of marked involuntary movements (dyskinesia), the so- called "on-off' phenomenon. Motor fluctuations are related to pre-synaptic problems, including variable absorption of L-DOPA and reduced pre-synaptic storage capacity. This results in excessive pulsatile stimulation of the dopamine receptors rather than continuous delivery of the drug.

Conversion to dopamine also occurs in the peripheral tissues, causing adverse effects and decreasing the available dopamine to the CNS, so it is standard practice to co-administer a peripheral DOPA decarboxylase inhibitor, carbidopa or benserazide, and often a catechol-O-methyl transferase (COMT) inhibitor. Peripherally, L-DOPA is rapidly metabolized to DA by the enzyme aromatic amino acid decarboxylase (AADC) and to 3-0 methyl dopa by the enzyme catechol-O-methyl transferase (COMT). Its elimination half-life is approximately 90 minutes. The amount of L-DOPA that eventually reaches the brain after a single oral dose depends on the speed of gastric emptying, presence of competition for intestinal transport of the alternative amino acids, and, most of all, the degree of peripheral metabolism. After taking it, only about 5% of L-DOPA can cross the blood-brain barrier. Patients have to take a very high dose and several times a day, leading to marked plasma drug fluctuations. Increased bioavailability would have some major advantages to the PD patient, such as the decreased fluctuations, but also decreased dose-related peripheral side effects, including nausea, vomiting, and hypotension. After crossing the blood-brain barrier, L-DOPA is taken up by the surviving striatal neurons, converted by intraneuronal AADC to dopamine (DA), which is, in turn, released presynaptically. It was previously stated that high dose of L-DOPA may cause further neurodegeneration due to exacerbation of oxidative stress, as DA is a powerful oxidant. Thus, even though a very small proportion of L-DOPA reaches the brain, if it is not stored properly in a brain of a PD patient, who has reduced storage capacity due to reduced number of striatal terminal sites, unstored L-DOPA, after conversion to DA, may cause oxidative stress.

Currently marketed formulations of L-DOPA are poorly and erratically adsorbed and extensively broken down in the periphery. A low as1% of the oral dose can reach the brain.

The present invention provides compounds of L-DOPA or dopamine with an essential fatty acid. These are show in general terms in formula 3:

R¹-Z-O-(CH₂)_n-CH(R³)-(CH₂)_m-O-Y-R² (3)

wherein:

R¹ is acyl or fatty acid group derived from C₁₂-C₃₀ fatty acids, preferably C₁₆-C₃₀ fatty acids desirably with two or more cis or trans double bonds;

R³ is H or CH₃; n is O or 1 ; m is O or 1 ;

Y is a bond or a linker group having one of a -C(=0)- group or -P(=0)- group at each end (for example a linker group of formula -C(=0)-(CH2)_p-C(=0)- wherein p is O to 4);

Z is a bond or a linker group having one of a -C(=0)- group or -P(=0)- group at each end (for example a linker group of formula -C(=0)-(CH2)_p-C(=0)- wherein p is O to 4); and R² is

for example, when Y is a bond; or

for example, when Y is a linker group.

The compounds are derivatives of a fatty acid with the available carboxyl or amino group of L-DOPA, or the amino group of dopamine, such that a single, well defined chemical entity is formed. The coupling may be direct yielding bipartate compounds or spaced with an appropriate linker group, yielding tripartate compounds, in terms of the number of moieties into which the compounds split. The linker groups Y, Z may be a phosphate, succinate or moeity derived from a difunctional acid.

A preferred compound has the 1 ,3 propane diol structure: n=1 and m=1 and R³ is H:

CH₂OR¹

CH,

CH₂OR'

A phosphate, succinate or difunctional acid derived linker Z or Y may be interposed between the R¹ and/or R² group and the 1 ,3-propane diol residue, as shown in Formula 3 above. For example, a linker Y may be between the diol and the L-DOPA or dopamine moeity.

Any fatty acid, suitably C₁₂-C₃₀ or C₁₆-C₃₀ and desirably with two or more cis or trans carbon-carbon double bonds may be of use. Preferably, the fatty acid is an n-6 or n-3 series essential fatty acid or oleic acid, columbinic acid, parinaric acid or conjugated linoleic acid.

The twelve essential (n-6) and (n-3) essential fatty acids are shown in Figure 1. The essential fatty acids, which in nature are of the all - cis configuration, are systematically named as derivatives of the corresponding octadecanoic, eicosanoic or docosanoic acids, e.g. z,z-octadeca - 9,12 - dienoic acid or z,z,z,z,z,z-docosa-4,7,10,13,16,19 - hexaenoic acid, but numerical designations based on the number of carbon atoms, the number of centres of unsaturation and the number of carbon atoms from the end of the chain to where the unsaturation begins, such as, correspondingly, 18:2n-6 or 22:6n-3 are convenient. Initials, e.g., EPA and shortened forms of the name e.g. eicosapentaenoic acid are used as trivial names. The application of lipophilic essential fatty acids in the treatment of CNS disorders has been documented, for example reference is made to WO 98/16216 and WO 00/44361. The blood-brain barrier is essentially composed of lipids and the essential fatty acids are able to cross this barrier.

Gamma-linolenic acid (GLA) or dihomo-gamma-linolenic acid (DGLA) are two fatty acids which in themselves have a range of desirable effects in the treatment of CNS disorders. Other fatty acids, such as any of the essential fatty acids (EFAs) and in particular the twelve natural acids of the n-6 and n-3 series EFAs of Figure 1 , can be used. Of these twelve, arachidonic acid, adrenic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid n-3 and docosahexaenoic acid are of particular interest. Conjugated linoleic acid (cLA), oleic acid and columbinic acid (CA) are examples of further fatty acids, though not included in Figure 1 , likely to be of particular use. The present invention further provides a pharmaceutical composition comprising a compound according to the invention for the treatment of Parkinson's disease or other movement disorders including Huntington's disease and other illnesses known to be linked to excessive numbers of trinucleotide repeats in certain genes, including fragile X syndrome, Friedreich's ataxia, spinal and bulbar muscular atrophy, spinocerebellar ataxia type I, dentato-rubral-pallidoluysian atrophy, Haw River syndrome, Machado-Joseph disease, and myotonic dystrophy.

Methods of manufacture of such medicaments are provided, as are methods of treating, preventing or delaying the onset of the symptoms of these disorders.

The compounds of the invention are shown to increase significantly brain levels of dopamine. The levels are steady and there is less peripheral breakdown of L-DOPA. This leads to fewer doses per day, reducing the peaks and troughs believed to contribute to a cycle of on/off and/or dyskinesia.

The compounds of present invention provide to an improved way to incorporate L-DOPA or dopamine in the CNS.

The compounds of the present invention are stable, which gives an advantage for drug regulatory concerns. They are readily incorporated into the body as an oral, parenteral or topical formulation, which is well tolerated. The moieties are delivered simultaneously in a single molecule. This avoids the regulatory problems which can ensue when more than one molecule are administered as separate compounds, and avoids the possibility of chiral centres. Once through the blood brain barrier, the compound readily separates to release the L-DOPA or dopamine. The essential fatty acid is also released.

There is evidence that interesting specific properties in addition to ready passage of lipid barriers can be conferred on the L-DOPA or dopamine by making it more lipophilic. These properties include prolonged duration of action, reduction of side effects especially gastro-intestinal, bypassing of first- pass liver metabolism and, potentially, site specific delivery of different materials.

Various specific fatty acids are likely to be able to add to the efficacy of L-DOPA or dopamine. Of particular value in therapy is that, under most circumstances, the fatty acids are remarkably nontoxic and can be administered safely in large doses without the risk of important side effects.

The individual fatty acids may be purified from natural animal, vegetable or microbial sources or may be chemically synthesised by methods known to those skilled in the art or developed hereafter.

Derivatisation requires the formation of one or more ester bonds. Such chemistry may be achieved by any reasonable method of ester synthesis and especially:

(a) by reaction of alcohol with acid chloride, acid anhydride or suitably activated ester with or without the presence of an organic tertiary base, e.g. pyridine, in a suitable inert solvent, e.g. dichloromethane, at a temperature between O and 120 ⁰C.

(b) by reaction of alcohol with acid or acid, short or medium chain alkyl ester, in the presence of a suitable acid catalyst, e.g. 4-toluene sulfonic acid, with or without a suitable inert solvent, e.g. toluene, at a temperature between 50 and180 ⁰C such that the water formed in the reaction is removed, e.g. under vacuum.

(c) by reaction of alcohol with acid in the presence of a condensing agent, e.g. 1 ,3- dicyclohexylcarbodiimide, with or without the presence of a suitable organic tertiary base, e.g. A- (N,N-dimethylaminopyridine), in an inert solvent, e.g. dichloromethane, at a temperature between O and 50 ⁰C. (d) by reaction of alcohol with acid or acid, short or medium chain alkyl ester, or acid, activated ester, e.g. vinyl, in the presence of a hydrolase enzyme, e.g. hog liver esterase, with or without a suitable solvent, e.g. hexane, at temperatures between 20 and 80 ⁰C under conditions such that the water or alcohol or aldehyde by product is removed, e.g. under vacuum.

(e) by reaction of acid with suitable alcohol derivative, e.g. iodide, with or without the presence of a suitable base, e.g. potassium carbonate, in a suitable inert solvent, e.g. dimethylformamide, at a temperature between 0 and180 ⁰C.

(f) by reaction of alcohol with acid, short or medium chain alkyl ester, in the presence of a catalytic amount of an alkoxide of type M⁺OY^" where M is an alkali or alkaline earth metal, e.g. sodium, and Y is an alkyl group containing 1-4 carbon atoms which may be branched, unbranched, saturated or unsaturated, with or without the presence of a suitable solvent, e.g. toluene, at temperatures between 50 and 180 ⁰C such that the lower alcohol, HOY, is removed from the reaction mixture, e.g. under vacuum.

Derivatisation of some bioactives require the formation of an amide bond. Such chemistry may be achieved by any reasonable method of amide synthesis and especially:

(g) by reaction of amine with acid chloride, acid anhydride or suitably activated ester with or without the presence of an organic tertiary base, e.g. pyridine, in a suitable inert solvent, e.g. dichloromethane, at a temperature between 0 and 12O⁰C.

(h) by reaction of amine with acid in the presence of a condensing agent, e.g. 1 ,3 dicyclohexylcarbodiimide, with or without the presence of a suitable organic tertiary base, e.g. 4- (N,N-dimethylaminopyridine), in an inert solvent, e.g. dichloromethane, at a temperature between 0 and 5O⁰C.

(i) by reaction of amine with acid or acid, short or medium chain alkyl ester, or acid, activated ester, e.g. vinyl, in the presence of a hydrolase enzyme, e.g. hog liver esterase, with or without a suitable solvent, e.g. hexane, at temperatures between 20 and 8O⁰C under conditions such that the water or alcohol or aldehyde byproduct is removed, e.g. under vacuum.

In general the chemistry of course depends on the nature of the compounds to be linked and on whether links are direct or indirect. Fatty acid pairs may for example be linked directly as fatty acid- fatty alcohol esters or as anhydrides, and if diol linkers are used ether links to fatty alcohols are an alternative to the more generally convenient ester links to fatty acids as such; in all cases linking may again be by chemistry known in itself.

Examples of Pairs of Actives, joined particularly using a 1 ,3-Propane Diol link:

L-DOPA + GLA L-DOPA + DGLA L-DOPA + EPA L-DOPA + DHA Pharmaceutical Preparations

In a further aspect, the present invention provides a pharmaceutical preparation comprising the dopamine or L-DOPA compounds of the first aspect of the invention. The active ingredient of these pharmaceutical compositions may comprises essentially only the the dopamine or L-DOPA compound(s) of the invention.

The compounds may be formulated in any way appropriate and which is known to those skilled in the art of preparing pharmaceuticals, skin care products or foods. They may be administered orally, enterally, topically, parenterally (subcutaneously, intramuscularly, intravenously), rectally, vaginally or by any other appropriate route.

The 1 ,3-propane diol diesters, may be readily emulsified using phospholipid or particularly galactolipid emulsifiers. Such emulsions are particularly useful for administration via oral, enteral and intravenous routes.

Anti-microbial preservatives, for example potassium sorbate, and flavour, can also be added to an oral emulsion.

The doses of the actives to be administered largely range from I mg to 200 g per day, preferably 10 mg to 10 g and very preferably 10 mg to 3 g. The compound need only be taken once or twice a day.

Brief Description of the Drawings

Fig 1 : essential fatty acids and their metabolism;

Fig 2: open field rearing results in Experiment 1 of Example 5;

Fig 3: DA levels in the striatum in Experiment 1 of Example 5;

Fig 4: neurotransmitters in the cortex in Experiment 1 of Example 5;

Fig 5: open field behaviour results in Experiment 2 of Example 5;

Fig 6: DA levels in the striatum in Experiment 2 of Example 5;

Fig 7: neurotransmitters in the cortex in Experiment 2 of Example 5;

Fig 8: Rotarod performance Experiment 3 of Example 5;

Fig 9: open field performance results in Experiment 3 of Example 5;

Fig 10: DA levels in the striatum in Experiment 3 of Example 5;

Fig 11 : DPOA/DA levels in the striatum in Experiment 3 of Example 5;

Fig 12: L-DOPA levels in the striatum in Experiment 3 of Example 5.

Examples

Example 1 - synthesis of GLA-dopamine conjugate (Formula 5)

5-GLA-dopamine conjugate 1

Reagents

Procedure

To the solution of GLA 1 (5.56g, 0.02mol, 1.11eq) in acetonitrile (60ml) was added triethylamine (3.2ml) under nitrogen followed with isobutyl chloroformate 2 (2.32ml, 0.018mol, 1.0eq). A white precipitate formed immediately once isobutyl chloroformate was added. The solution was continued, stirred at 0⁰C for 3 hours. The mixture was evaporated on rotavap to dryness; to the resulting mixed anhydride 3 was added a solution of dopamine hydrochloride (3.2g) in anhydrous DMF (50ml) containing triethylamine (2.78ml). The resulting mixture was stirred at 0⁰C then slowly reached room temperature with continued stirring for 18 hours. The reaction mixture was diluted with water (400ml) and extracted with diethyl ether (250mlx2). The combined organic phase was washed with water (300mlx3), brine, dried over anhydrous sodium sulfate and concentrated to dryness to afford a yellowish oil (8.1g) as crude product. The crude product was purified by column eluted with 2% methanol in dichloromethane to give product 5 (5.39g, yield 72.5%)

Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.80 (d, 1H), 6.74 (s, 1H), 6.55 (d, 1H), 5.73 (t, 1H), 5.25-5.45 (m, 6H), 3.46 (m, 2H), 2.80 (m, 4H), 2.67 (t, 2H), 2.17 (m, 2H), 2.05 (m, 4H), 1.61 (m, 2H), 1.24-1.38 (m, 8H), 0.88 (t, 3H); MS (ESI): M⁺+1 : 414.1

Example 2 - Synthesis of GLA-dopamine conjugate (Formula 11)

Reagents

Procedure

To a mixture of 1 ,3-dihydroxypropane (96g, 1.26 mol) 1.S-dicyclohexylcarbodiimide (DCC) (60.35g, 0.29 mol) and 4-(N,N-dimethylamino)pyridine (39Jg₁ 0.32 mol) in DCM(1150ml), a solution of Z₁Z₁Z- octadeca-6,9,12-trienoic acid 1 (GLA, 7Og) in DCM (250ml) was added at room temperature under nitrogen. The reaction was continued with stirring for 18 hours. The crude reaction mixture was filtered, the filtrate was washed with dilute hydrochloric acid (1M), water, brine and dried over anhydrous sodium sulfate. The filtrate, after vacuum filtration, was concentrated to give crude product which was purified by column chromatograph eluted with EtOAc/Hexane (10-30%) to afford the GLA-monoester 7 as a yellow oil (68g, 80.1% yield).

Analysis

NMR (CDCI₃, 500MHz): δ_H: 5.30-5.45 (m, 6H), 4.23 (t, 2H), 3.67 (m, 2H), 2.79 (m, 4H), 2.50 (br, 1H)₁ 2.31 (t, 2H)₁ 2.08 (m, 4H)₁ 1.88 (m, 2H), 1.65 (m, 2H)₁ 1.20-1.40 (m, 8H)₁ 0.88 (t, 3H)

Reagents

Procedure

A solution of GLA-monoester 7 (40g, 0.119 mol), succinic anhydride (11.9g, 0.119mol) in THF (580ml) was cooled to 0°C(salt/ice bath), while stirring under an atmosphere of nitrogen. To this, a solution of DBU (18.1g, 0.119 mol) in THF (220 ml) was added dropwise at 0⁰C. When the addition was completed, the reaction mixture was allowed to warm-up to room temperature and stirring was continued for 6 h. The THF was removed under the reduced pressure and the residue was taken up in diethyl ether (1 L) and transferred into a separating funnel. The organic phase was washed with HCI (2M, 1L) water (1L) and brine (1L), dried over anhydrous MgSO₄, after filtered, concentrated to dryness to give crude product. The crude product was purified by passing through a silica plug, eluting with hexane initially to remove the non-polar impurity. After this impurity was removed (evidence from TLC), the product was eluted with 40% EtOAc/Hexane to afford intermediate 9 as a yellow oil (46.77g, 90% yield).

Analysis

NMR (CDCI₃, 500MHz): δ_H: 5.30-5.45 (m, 6H), 4.10-4.20 (m, 4H), 2.79 (m, 4H), 2.68 (m, 2H), 2.62 (m, 2H), 2.31 (t, 2H), 2.09 (m, 4H), 1.96 (m, 2H), 1.62 (m, 2H), 1.20-1.40 (m, 8H), 0.89 (t, 3H)

10

11-GLA-dopamine conjugate 2

Reagents

Procedure

To the solution of Fatty acid intermediate 9 (12.Og, 0.02752mol, 1.11eq) in acetonitrile (120ml) was added triethylamine (4.96ml, 0.03567mol, 1.44eq) under nitrogen followed with isobutyl chloroformate (3.21ml, 0.02477mol, 1.0eq). A white precipitate formed immediately once isobutyl chloroformate was added. The solution was continued stirred at 0⁰C for 3 h. The mixture was evaporated on rotavap to dryness (making sure any unreacted chloroformate was evaporated); to the resulting mixed anhydride 10 was added a solution of dopamine hydrochloride (4.92g, 0.026mol, 1.0eq)) in anhydrous DMF (100ml) containing triethylamine (3.616ml). The resulting mixture was stirred at 0⁰C then slowly allowed to reach room temperature. Stirring was continued at room temperature for 18 h. The reaction mixture was diluted with water (500ml) and extracted with diethyl ether (500ml). The organic phase was washed with water (500mlx3), brine, dried over anhydrous sodium sulfate and concentrated to dryness to afford a yellowish oil (14.8g) as crude product. The crude product was purified by column eluted with 1.2% methanol in dichloromethane to give product 11(7.25g, yield 50.6%)

Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.79 (d, 1H), 6.70 (s, 1H), 6.57 (d, 1H), 5.95 (t, 1H), 5.25-5.45 (m, 6H), 4.15 (m, 4H), 3.46 (m, 2H), 2.80 (m, 4H), 2.67 (m, 4H), 2.44 (t, 2H), 2.32 (t, 2H), 2.09 (m, 4H), 1.96 (m, 2H), 1.65 (m, 2H), 1.24-1.38 (m, 8H), 0.88 (t, 3H); MS (APCI): M⁺+1 : 572.3

Example 3 - Synthesis of GLA- levodopa conjugate (Formula 18)

12 14

Reagents

Procedure L-3-(3,4-dihydroxyphenyl)alanine (Levadopa) 12 (15g) was dissolved in dioxane (150ml) and 1MNaOH (120ml) was added to adjust pH«12. The resulting solution was cooled to 0⁰C and di-tert- butyl dicarbonate 13 (18.27g) was added. The reaction mixture was continued with stirring at room temperature for 4 hours. The solvent was evaporated and the resulting aqueous residue was added ethyl acetate (250ml), followed by the addition of 1MHCI solution until pH«2 with vigorous stirring. The organic layer was separated and the aqueous portion was extracted with ethyl acetate (200ml) again. The combined organic phase was washed with water (300mlx2), brine, dried over sodium sulfate and concentrated to dryness to give intermediate 14 (20.1g).

Analysis

NMR (DMSOd₆, 500MHz): δ_H: 6.92 (d, 1H), 6.61 (m, 2H), 6.47 (m, 1H), 3.99 (m, 1H), 2.82 (dd, 1H), 2.65 (dd, 1H), 1.34 (s, 9H)

TBSCI

14 15

Reagents

Procedure

A solution of Boc-Levadopa 14 (20.1g) and tert-butyldimethylchlorosilane (29.5g) in acetonitrile (250ml) was cooled down to O⁰C with an ice bath. After stirring 10 min, DBU (29.87g) was added over 10 min and the resulting mixture was stirred at 0⁰C then at room temperature 16 hours. The solvent acetonitrile was removed under vacuum, the residue was partitioned between ethyl acetate (400ml) and water (400ml), the organic phase was washed with water (400mlX3), dried over sodium sulfate and concentrated to dryness to give crude product (41.2g) which was purified by column eluted with 2% MeOH/DCM then 5%MeOH/DCM to give intermediate 15 (23.Og) Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.75 (d, 1H), 6.66 (s, 1H), 6.62 (d, 1H), 4.97 (m, 1 H), 4.57 (m, 1H), 2.99 (m, 2H), 1.42 (s, 9H), 0.98 (s, 18H), 0.18 (s, 12H)

16

Reagents

Procedure

To the solution of fatty acid alcohol 7 from Example 2 (14.7g) and TBS-boc-Levadopa 15 (23g) in DCM was added DCC (10.54g) and DMAP (7.05g), the resulting reaction mixture was stirred at room temperature for 18 hours. The solid in the mixture was filtered off, the filtrate was concentrated to dryness. The residue was partitioned between ethyl acetate (300ml) and 1MHCI (300ml). The organic phase was washed with water, brine, and dried over sodium sulfate, after concentrated to dryness to afford crude product which was purified by column eluting with 1% MeOH/DCM to give intermediate 16 (19.7g). Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.73 (d, 1H), 6.62 (s, 1H), 6.55 (d, 1H), 5.30-5.45 (m, 6H), 4.90 (m, 1H), 4.50 (m, 1H), 4.18 (m, 2H), 4.12 (m, 2H), 2.95 (m, 2H), 2.81 (m, 4H), 2.31 (t, 2H), 2.09 (m, 4H), 1.94 (m, 2H), 1.64 (m, 2H), 1.42 (s, 9H), 1.20-1.40 (m, 8H), 0.98 (s, 9H), 0.97 (s, 9H), 0.89 (t, 3H), 0.18 (s, 12H)

16

17

18-GLA-levodopa conjugate 1

Reagents

Procedure

The protected intermediate 16 (19.7g) was dissolved in THF (200ml). The solution was cooled to 0⁰C for 10 min. To this solution was added TBAF (1M solution in THF, 46.7ml). The resulting mixture was stirred at 0⁰C for 30 min. Saturated ammonium chloride water solution (400ml) was added to the reaction mixture and was extracted with ethyl acetate (500ml). The organic phase was washed with water, brine, dried over sodium sulfate and concentrated to dryness to give intermediate 17 (13.5g).

To the solution of R-NH-boc 17 (13.5g) in DCM (250ml) was added TFA (60ml) at 0⁰C. The resulting mixture was stirred at 0⁰C and then allowed to reach room temperature slowly for 16 hours. The solvent and extra TFA were evaporated on ratovap under reduced pressure. The residue was partitioned between ethyl acetate (400ml) and water (400ml). The organic phase was washed with water, brine, dried over sodium sulfate and concentrated to dryness to afford crude product. The crude product was purified by column eluting with 5% MeOH/DCM to give product 18 (9.Og).

Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.75 (s, 1 H), 6.70 (d, 1H), 6.45 (d, 1H), 5.30-5.45 (m, 6H), 4.18 (m, 3H), 4.09 (m, 2H), 3.15 (m, 1H), 3.01 (m, 1H), 2.78 (m, 4H), 2.31 (t, 2H), 2.05 (m, 4H), 1.95 (m, 2H)₁ 1.63 (m, 2H), 1.20-1.40 (m, 8H), 0.87 (t, 3H); MS (APCI): M⁺+1: 516.3

Example 4 - Synthesis of GLA-levodopa conjugate (Formula 19)

19-GLA-levodpoa conjugate 2

Reagents

Procedure

To the solution of fatty acid derivative 9 from Example 2 (15g, 0.0344mol) in acetonitrile (150ml) was added triethylamine (6.2ml, 0.0446mol) under nitrogen followed with isobutyl chloroformate 2 (4.23g, 0.031 mol). A white precipitate formed immediately once isobutyl chloroformate was added. The solution was continued stirred at 0⁰C for 3 hours. The mixture was evaporated on rotavap to dryness. Levadopa (6.41 g, 0.0325mol) was dissolved in dioxane (75ml) and NaOH (1M) was added to adjusted pH=12 (~65ml). The resulting solution was cooled to 0⁰C, then was added to the mixed anhydride 10 which was made from fatty acid and isobutyl chloroformate. The mixture was continued stirred at 0⁰C and slowly reached room temperature and stirred for 18 hours. The reaction mixture was acidified by HCI (1 M) to pH=3 and extracted with ethyl acetate (250ml) and washed with water (250mlx2), brine, dried over sodium sulfate and concentrated to dryness to afford crude product (13.8g) which was purified by column eluted with 1%~10% MeOH/DCM to give product 19 (5.Og, yield: 26%) as a yellowish oil.

Analysis

NMR (CDCI₃, 500MHz): δ_H: 6.70 (m, 2H), 6.64 (s, 1H), 6.50 (d, 1H)₁ 5.30-5.45 (m, 6H), 4.73 (m, 1H), 4.12 (m, 4H), 2.96 (m, 2H), 2.81 (m, 4H), 2.58 (m, 2H), 2.49 (m, 2H), 2.31 (t, 2H), 2.05 (m, 4H), 1.94 (m, 2H), 1.63 (m, 2H), 1.20-1.40 (m, 8H), 0.88 (t, 3H); MS (APCI): M⁺+1 : 616.6

The effects of L-DOPA have been commonly investigated in rodent models of PD, such as the reserpine or MPTP-induced model. The reserpine model is the first animal model, which was widely used to test the effects of anti-parkinsonian drugs. The mechanisms of this anti-hypertensive drug to induce parkinsonism are still not fully understood, but it involves depletion of synaptic vesicles of neurotransmitter content (in particular DA, but also other neurotransmitters) and reduced terminal reuptake of neurotransmitters. The advantage of reserpine is that it's relatively quick acting, has robust effects on behavior and is non-toxic, whereas MPTP is far more toxic to handle and has much less severe effects on animals' behavior, even though it selectively destroys nigrostriatal neurons.

Example 5 - effects of conjugates of Examples 3 and 4 5.1. Experiment 1

Animals: 56 C57BL/6 male (10-12 weeks old, 25g) from Charles River, Canada. Animals were kept in the animal holding room (21 ±1 'C) in normal 12h light-dark cycle (lights on at 6:00 am and off at 6:00 pm) for 1 week of habituation to the new environment, prior to experimental procedures. They had ad-lib access to food and water. Drugs: L-DOPA (Sigma, Canada) and DRUG 1 (conjugate of Example 3) and 2 (conjugate of Example 4) were dissolved at 1 mg/ml in vegetable oil. Prepared solutions were kept on ice and stored in 4 "C overnight.

Experimental design: Preparation of drug solutions, gavage and Open field testing were all done in the same room (the animal holding room). Drugs were administered on 2 consecutive days. At 9 pm prior to day 1 of drug administration, food was removed from the animal cages, in order to ensure an empty stomach at feeding the next day. On day 1, each animal was exposed to a gavage session, which consisted of 1) weighing the animal, 2) gavaging and 3) putting back the regular food in the cage. Animals were gavaged group by group (see below), so that time of day effects were controlled for. At 9:00 pm on day 1 , food was removed again from the cages. On day 2, animals were again exposed to gavage sessions as described above. Two hours after each gavage session, each animal was subjected to an Open Field session of 3 minutes to record locomotor and anxiety-related behavior, followed by sacrifice for biochemical analyses of neurotransmitters with HPLC. The following experimental groups were used:

Animals were split into 7 sub-groups according type and dose of drug: 1) control; 2) L-DOPA (10 mg/kg); 3) L-DOPA (100 mg/kg); 4) DRUG 1 (10 mg/kg); 5) DRUG 1 (100 mg/kg); 6) DRUG 2 (10 mg/kg) and 7) DRUG 2 (100mg/kg). Drugs were injected by gavage, using a soft feeding tube (5FR 15", MED-RX, Canada).

Behavior: The Open Field (Hall, 1934) measured spontaneous locomotor and anxiety related behavior in normal animal room lighting. In normal lighting (animal holding room lighting), it is more likely to measure locomotor activity than anxiety-related behavior, as the animals are used to the room lighting. We measured line crosses (distance moved), rearing (exploratory activity) and grooming (displacement response) manually, while the animal was in the Open Field for 3 minutes, in normal lighting.

Biochemical analyses: Immediately after an Open Field testing, the animal was brought to a surgery room and sacrificed using cervical dislocation. Striatum and other brain regions were dissected and stored in eppendorf tubes containing a buffer (250ml H2O with 2.2 mg L-ascorbic acid, 2.33 ml 70% HCIO₄ and 25 mg EDTA) for HPLC analyses. Samples were centrifuged at 11000 RPM and 4°C, for 25 minutes. DA, DOPAC and other neurotransmitters were analyzed later with HPLC equipment. This report limits to DA and DOPAC.

Statistics: On all measures (neurotransmitters and behavior), drug effects were analyzed using oneway analyses of variance (ANOVA) with factor DRUG (L-DOPA, DRUG 1 and DRUG 2). If main effects were significant (p<0.05), Bonferroni post-hoc tests were performed to compare all groups to the control condition.

5.2 Experiment 2

Animals: 40 male C57BL/6 (10-12 weeks old, 25g) from Charles River, Canada. Animals were kept in the animal holding room (21±1 °C) in normal 12h light-dark cycle (lights on at 6:00 am and off at 6:00 pm) for 1 week of habituation to the new environment, prior to experimental procedures. They had ad-lib access to food and water. Drugs: As Example 5.1 above. All experimental procedures were the same as described in Example 5.1. The following experimental groups were used: 1) control; 2) L-DOPA (100 mg/kg); 3) DRUG 1 (100 mg/kg); 4) DRUG 2(100 mg/kg).

Statistics: On all measures (neurotransmitters and behavior), drug effects were analyzed using oneway analyses of variance (ANOVA) with factor DRUG (L-DOPA₁ DRUG 1 , DRUG 2). If main effects were significant (p<0.05), Bonferroni post-hoc tests were performed to compare treatment groups with the control group.

5.3. Experiment 3

Animals: 42 male (10-12 weeks old, 25g) C57BL/6 from Charles River, Canada. Animals were kept in the animal holding room (21 ±1⁰C) in 12h light-dark cycle (lights on at 6:00 am and off at 6:00 pm) for 1 week of habituation to the new environment, prior to experimental procedures. They had ad-lib access to food and water.

Drugs: L-DOPA and DRUG 2 (see Example 5.1). Reserpine (Sigma, Canada) was dissolved in 1% acetic acid in sterile dd H2O (Visanji et al., 2006). All drugs were prepared as in Example 5.1.

Experimental procedures: Drugs were administered on 2 consecutive days. At 9:00 pm prior to day 1 of drug administration, food was removed from the animal cages, in order to ensure an empty stomach at feeding the next day. On day 1 , each animal was exposed to a gavage session, which consisted of 1) weighing the animal, 2) gavaging and 3) putting back the regular food in the cage. Animals were gavaged group by group, for which the time of day effects were controlled. Immediately following gavage on day 1 , reserpine (1 mg/kg) was administered i.p. At 9:00 pm on day 1 , food was removed again from the cages. On day 2, animals were again exposed to gavage sessions as described above. Two hours after each gavage session and 24 hours following reserpine administration, each animal was subjected to a Rotorod session, followed by an Open Field. Open Field procedures were similar as described above. The Rotorod (Rozas & Labandeira, 1997) is a test for measuring gait, locomotor co-ordination, balance and endurance, so it measures overall motor performance. Our animals were not pre-trained, because we expected drug 2 to enhance performance above baseline, but with pre-training this performance enhancement could perhaps not be measured. The animals were positioned on a rod that rotated with increasing speed until the animals fell off and automatic sensors captured the time and rotation speed. The average distance moved (m) of 2 successive trials was measured. Between each trial, animals rested for 1 minute.

All other procedures were the same as previous experiment.

Animals were split in 6 sub-groups according type of treatment: 1) control; 2) reserpine (1 mg/kg); 3) L-DOPA (100 mg/kg); 4) DRUG 2 (100 mg/kg); 5) L-DOPA + reserpine (both 100 mg/kg), 6) DRUG 2 + reserpine (both 100mg/kg).

Statistics: On all measures (neurotransmitters and behavior), drug effects were analyzed using two- way analyses of variance (ANOVA) with factors reserpine (RES) and drug (DRUG). If main effects were significant (p<0.05), Bonferroni post-hoc tests were performed. Results Experiment 1

Behavior: There was no effect of these drugs on Open Field line crosses or grooming (data not shown). As shown in Figure 2, There was an effect of drug on rearing (DRUG: F (df 6,50)= 2.332, p<0.05). Post-hoc analyses revealed that DRUG 2 100mg/kg significantly increased rearing compared to control and L-DOPA 10mg/kg (^*p<0.05).

Neurotransmitters in the striatum: For DA, there was a main effect of drug (DRUG: F(df 1,60)= 7.90, p<0.05). Post-hoc analyses revealed that DRUG 1 (100mg/kg) and DRUG 2 (100mg/kg) caused a significant increase of DA compared to control (*p<0.05). This is shown in Figure 3. No effects were found for DOPAC (data not shown).

Neurotransmitters in the cortex: For DA, there was a main effect of drug (DRUG: F(df 1 ,63)= 11.33, p<0.05). Post-hoc analyses revealed that DRUG 2 100mg/kg significantly increased DA compared to control (*p<0.05) (figure 3 upper panel). A significant main effect was also found for DOPAC (DRUG: F(df 1 ,63)= 8.52, p<0.05). Post-hoc analyses showed that the high dose of DRUG 2 significantly increased DOPAC compared to control (*p<0.05). This is shown in Figure 4, lower panel.

5.2. Experiment 2

Behavior: There was a significant effect of drug on line crosses (DRUG: F (df 3,18) = 4.510, p<0.05). Post-hoc analyses revealed that DRUG 2 significantly increased line crossings compared to control (*p<0.05). This is shown in Figure 5, upper panel. An effect of drug on rearing was also found (DRUG: F (df 3,18)= 3.261 , p<0.05). Post-hoc analyses showed that DRUG 2 significantly increased rearing compared to control (*p<0.05), shown in Figure 5, lower panel. No effects were found for grooming (data not shown).

Neurotransmitters in the striatum: A significant effect of drug was found for DA (DRUG: F (df 3,31)= 4.611 , p<0.01) . This is shown in Figure 6, upper panel. Post-hoc analyses revealed that DRUG 2 significantly increased DA compared to control (*p<0.01). For DOPAC, a significant effect of drug was found (DRUG: F (df 3,32) = 3.485, p<0.05). This is shown in Figure 6, lower panel. DRUG 2 significantly increased DOPAC compared to control (*p<0.05).

Neurotransmitters in the cortex: No significant effects were found for DA or DOPAC in cortex, but some clear trends were found for DA and NE, so the results are nonetheless shown in figure 7.

5.3. Experiment 3

Behavior- Rotarod: Results are shown in figure 8. Two-way ANOVA analyses revealed significant main effects for reserpine (RES: F(df 1 ,33)= 16.75, p<0.001) and DRUG (DRUG: F (df2,33)= 6.248, p<0.01). Post-hoc analyses revealed the following effects:

1) Reserpine significantly reduced rotarod performance when compared with controls (*p<0.05); 2) after L-DOPA treatment, reserpine-induced change were not attenuated; 3) DRUG 2 could significantly enhance rotarod performance when compared to control (^**p<0.01), and also reversed a reserpine-induce impairment (#p<0.05).

Behavior- Open Field: Results are shown in figure 9. Upper panel: On locomotion (line crosses), there was a strong main effect of reserpine (RES: F(df 1 ,34)= 329.7, p<0.0001), but not a significant main effect of DRUG. Reserpine markedly reduced line crosses in the test (***p<0.001) compared to control. L-DOPA, nor DRUG 2 increased line crosses compared to control. L-DOPA did not attenuate line crosses in the reserpine treated mice compared to reserpine treated mice, but DRUG 2 did attenuate line crossses in the reserpine treated mice compared to reserpine treated mice (#p<0.05). Similar results were found for rearing (figure 9 middle panel). There was a strong main effect of reserpine (RES: F(df 1 ,33)= 119.8, p<0.0001), but no main effect of DRUG. Reserpine reduced rearing scores (***p<0.001) and only DRUG 2, but not L-DOPA significantly reversed these changes (#p<0.05). For grooming, no main effect of reserpine was found, but an effect of drugs was found (DRUG: F(df 2,32)= 9, p<0.001), as shown in figure 9 (lower panel). Post-hoc analyses showed that reserpine did not significantly affect grooming, neither did L-DOPA or Drug 2 compared to control, although there was an increasing trend. L-DOPA did not change grooming in animals treated with reserpine compared to reserpine only treated mice, but DRUG 2 significantly increased grooming in mice treated with reserpine compared to reserpine only treated mice (##p<0.01).

Neurotransmitters in the striatum: There was a significant main effect of reserpine on striatal DA (RES: F (df 1 ,34)= 24.39, p< 0.0001), as well as DRUG (DRUG: F(df 2,33)= 3.705, p<0.05). Post- hoc analyses showed that reserpine caused a strong reduction of striatal DA compared to control (*** p<0.001), as shown in figure 10. L-DOPA did not have a significant effects on DA compared to control, but DRUG 2 significantly increased striatal DA compared to control (*p<0.05). After reserpine injection, L-DOPA did not attenuate striatal DA changes in mice treated with reserpine, whereas DRUG 2 marginally increased DA in mice treated with reserpine when compared to mice treated with reserpine alone (p=0.07 and p<0.05 with individual student t-test). When figure 8 (rotarod performance) and figure 10 (striatal DA) are compared, it shows a strong correlation between Rotarod performance and striatal DA, suggesting that the effects of DRUG 2 on rotarod performance were mediated by striatal DA.

DOPAC/DA in the striatum: There was a significant main effect of reserpine on striatal DOPAC/DA ratio (RES: F (df1 ,31)= 76,25, p<0.0001). Post-hoc analyses showed that reserpine caused a strong increase of striatal DA compared to control (*** p<0.001), which was not attenuated by L-DOPA, as shown in figure 11. DRUG 2 partially attenuated the effect of reserpine. Also, a significant decrease of DOPAC/DA was found by DRUG2 combined with reserpine, compared with reserpine only (#p<0.05).

L-DOPA concentrations in the striatum: There was a main effect of reserpine (RES: F (df 2,32)= 5.191 , p<0.05), but there was no effect of DRUG. Reserpine significantly increased L-DOPA concentration (*p<0.05), which was attenuated by both L-DOPA and DRUG 2 (##p<0.01) (figure 12).

The linking of the L-DOPA to a fatty acid enhances the access and storage of L-DOPA in striatal terminals and therefore enhanes the effect of L-DOPA on DA (and metabolite), as well as motor behaviour in the Open Field experiments 1 and 2 of Example 5. Through this mechanism, a reserpine induced DA depletion in striatum and a motor impairment in Rotarod and Open Field is more easily prevented by the L-DOPA conjugates of the invention than standard L-DOPA. Both DRUG 1 and DRUG 2 did not significantly alter line crosses and grooming, but rearing was significantly increased by DRUG 2 (100mg/kg) compared to control, while unaffected by L-DOPA or DRUG 1. Rearing has been reported to be correlated to striatal DA, thus suggesting that the effect of DRUG 2 on rearing was related to its effect on striatal DA. Indeed, both striatal and cortical DA were significantly increased by the high dose of DRUG 2 compared to control, while the other drugs did not cause any significant changes. Although cortical DOPAC was also increased by the high dose of standard L-DOPA, these results suggest that DRUG 2, rather than DRUG 1 , was more effective than standard L-DOPA in altering neurotransmitter levels.

Based on findings from experiment 1 , the high dose of DRUG 1 and DRUG 2 were again compared in a repeated experiment, Experiment 2. The results were confirmed at behavioral as well as neurotransmitter level. The pattern was very clear. Only DRUG 2 caused an increase of Open Field line crosses and rearing, as well as increases in striatal DA and DOPAC compared to control. Although not significantly, a trend of increased cortical DA and NE was found after DRUG2 treatment, compared to control, while standard L-DOPA and DRUG 1 did not produce any effects. Thus, these results again suggest that DRUG 2, rather than DRUG1 , was more effective at altering neurotransmitters and behavior than standard L-DOPA.

DRUG 2 more strongly increased Rotarod performance in Experiment 3, compared to control than L- DOPA. The difference between controls and DRUG 2 treated mice on rotarod performance was remarkable. In the Open Field, no clear effects of L-DOPA or DRUG 2 compared to control were found on distance moved and rearing, but grooming was increased (a trend) by DRUG 2, which was not found in experiment 1 and experiment 2 and this difference cannot be explained at this point of data analyses. The effects of striatal DA on grooming are complex and may vary, depending on which dopamine receptors are stimulated. Reserpine (1mg/kg) had potent effects on behavior 24 hours after administration. Rotarod performance was significantly impaired and Open Field line crosses and rearing significantly decreased. There was no effect of reserpine on grooming. L-DOPA was not able to attenuate most of the effects of reserpine, but DRUG 2 significantly reduced the effects of reserpine on rotarod performance and Open Field line crosses and rearing. It should be mentioned that reserpine treated animals that were treated with DRUG 2 were often more active in the home cage than reserpine only treated animals, but also than animals treated with reserpine combined with L-DOPA. The same animals did not always display a similar increased level of mobility in the Open Field. This finding would suggest that home cage activity measures would capture the differences between the drugs better than just the Open Field.

In line with experiment 1 and 2, DRUG 2 in Experiment 2 showed potent effects on striatal DA, whereas L-DOPA did not produce an effect on striatal DA. Reserpine, as expected, significantly decreased striatal DA and increased DOPAC/DA ratio. The effect of reserpine on DA was marginally attenuated by DRUG 2, while L-DOPA did not produce any effects. The effects of reserpine on DOPAC/DA were significantly attenuated by DRUG 2 and L-DOPA did not have any effects against reserpine. The mild effect of DRUG 2 against a reserpine-induced DA depletion is in contrast with stronger effects of DRUG 2 against reserpine on behavioral measures. As motor behavior is critically dependent on striatal DA, especially when striatal DA falls below a threshold (Dauer & Przedborski, 2003), only marginally increases of striatal DA by DRUG 2 after reserpine treatment may have relatively dramatic consequences for behavior. This may explain the discrepancy between results of striatal DA and behavior, in terms of the effects of DRUG 2 against reserpine. Figure 12 (striatal L-DOPA levels) may provide interesting clues about the effects of the drugs. Predictions about brain L-DOPA levels are hard to make, as the true mechanisms of the drugs are unknown. The hypothesis was that DRUG 2 would enhance brain access of L-DOPA and increase its neuronal absorption. Increased absorption would imply increased content of L-DOPA in the brain. The L-DOPA then becomes converted to DA. Depending on the rate of conversion to DA₁ no difference on total L-DOPA levels may be observed after DRUG 2 treatment, as all the administered L-DOPA may be converted to DA at the time of measurement. As such, you would see an increase of DA levels after DRUG 2 treatment, but not L-DOPA. This was the case. The increase of L-DOPA levels after reserpine treatment was hard to explain. The variation of this result was high and perhaps unreliable. Reserpine inhibits the vesicular monoamine transporter (VMAT) and thus less DA can be stored in vesicles and becomes metabolized by monoamine oxidase. Thus, a decrease of DA would be expected, but not an increase of L-DOPA. Reserpine does not inhibit tyrosine hydroxylase, so the increase of L-DOPA levels cannot be explained by a reduction of conversion of L-DOPA to DA. Both L-DOPA and DRUG2 attenuated this effect of reserpine.

Claims

1. Compounds of formula 3:

R¹-Z-O-(CH₂)_n-CH(R³)-(CH₂)m-O-Y-R² (3)

wherein:

R¹ is acyl or fatty acid group derived from C₁₂-C₃₀ fatty acids, preferably C₁₆-C₃₀ fatty acids, desirably with two or more cis or trans double bonds;

R³ is H or CH₃; n is 0 or 1 ; m is 0 or 1 ;

Y is a bond or a linker group having one of a -C(=O)- group or -P(=O)- group at each end;

Z is a bond or a linker group having one of a -C(=O)- group or -P(=O)- group at each end; and R² is

or R² is