CA2347879A1 - Substituted dicinnamoylquinides and their use in augmentation of adenosine function - Google Patents

Abstract

This invention describes the novel use of alkyl, alkoxyl, halogenyl, or hydroxy substituted dicinnamoylquinides as therapeutic agents for enhancing adenosine levels in the brain and peripheral organs. These agents are particularly useful in treating human diseases or conditions that benefit from acute or chronic elevated levels of adenosine, such as reperfusion injury, coronary or cerebral ischemia, coronary vasoconstriction, paroxysmal supraventricular tachycardia, hypertension, wound healing, diabetes, inflammation, or sleep disturbances. They can also be used to protect normal cells from chemotoxicity in patients undergoing cancer therapy, and reverse the behavioral effects of caffeine intake.

Description

APPLICATION FOR LETTERS PATENT
The present patent application claims benefit of the filing date of U.S.
Provisional Patent Application entitled "Substituted Dicinnamoylquinides and their use in Augmentation of Adenosine Function", by de Paulis et al, filed May 11, 2001, which is incorporated herein by reference in its entirety. (Serial Number not yet known) Be it known that we, Tomas de Paulis, a citizen of Sweden, residing at 205 Woodland Ct., Hermitage, TN 37076; David M. Lovinger, a citizen of United States, residing at 2607 Sunset Place, Nashville, TN 37212; and Peter 1o Martin, a citizen of United States, residing at 3825 Richland Avenue, Nashville, TN 37205; have invented a new and useful "Substituted Dicinnamoylquinides and Their Use in Augmentation of Adenosine Function."
FIELD OF THE INVENTION
The present invention relates generally to the field of use of alkyl, alkoxyl, halogenyl, or hydroxy substituted dicinnamoylquinides. Specifically, the present invention relates to the use of the above-mentioned compounds to treat diseases or conditions that improve from either an acute or chronic increase in adenosine levels.

Inventor: de Paulis et ccl.
Atty. Docket: N-7337 BACKGROUND OF THE INVENTION AND PRIOR ART
Naturally occurring 4-hydroxycinnamoyl mono- and di-esters of quinic acid gamma-lactone are found in roasted coffee (Hucke and Maier, Z Lebensm s Unters Forsch 180, 479-484 (1985), Scholz and Maier, Z Lebensm Unters Forsch 190, 132-134 (1990)), but not in tea or any other caffeine containing beverages. Such cinnamoylquinides have been claimed to be useful for removing wrinkles in skin (US 5589505). Certain cinnamoylquinides have been claimed also to have anti-morphine activity (WIPO 8601508). Particularly, 3-or 4-(3-methoxy-4-hydroxycinnamoyl-1,5-quinide has been claimed to block the binding of tritiated naloxone, a mu opioid receptor antagonist in brain (Wynne et al. Clin Exp Pharmacol Physiol 14, 785-790 (1987)). 1-Vinyloxyformyl-3,4-di(3,4-dimethoxycinnamoyl)-1,5-quinide has been described as an intermediate in the preparation of dicinnamoylquinic acids having aldose reductase Is inhibiting activity and being useful in the treatment of diabetes (JP 04 01184).
Improved methods for the preparation of methoxy- and hydroxy-substituted 3,4-dicinnamoyl-1,5-quinides have been described (US 5395950 and US
5401858). We have discovered a novel use of dicinnamoylquinides as a method of raising adenosine levels in brain and peripheral tissue. Adenosine is a Inventor: de Paulis et al.
Atty. Docket: N-7337 neuromodulator known to produce profound effects on blood flow, neurotransmission, cellular functions, and metabolism. Intracellular levels of adenosine are disclosed to be maintained by an active transport of adenosine across the cell membrane by means of a carrier-mediated, saturable nucleoside transporter, consisting of a 50 kDa protein in the form of a dimer (Thorn and Jarvis, Gen Pharmacol 27, 613-620 (1996)). This transporter protein is widely distributed in thalamic, cortical, and particularly in striatal neurons in the human brain (Glass et al. Brain Res 710, 79-91 (1996)), where it regulates adenosine-dopamine interactions (Dunwiddie and Masino, Ann Rev Neurosci 24, 31-55 (2001)). Of all mammals studied, the human transporter is disclosed to be one of the most sensitive to adenosine (Hammond N-S Arch Pharmacol 361, 373-382 (2000)), resulting in an extremely short half-life of adenosine in blood, i.e. less than 1 s. Inhibition of the adenosine transporter is disclosed to prevent the intracellular metabolism of adenosine and prolongs the presence of high levels of adenosine. This increased level of adenosine has been claimed to have a prophylactic effect on pancreatitis (LJS 5866574). The increased level of adenosine in brain and periphery causes stimulation of adenosine receptor subtypes, similar to the effects seen from unselective adenosine receptor agonists. Stimulation of adenosine A2A receptors has been claimed to promote Inventor: de Paulis et al.
Atty. Docket: N-7337 cell migration, thereby increase wound healing (L1S 6020321). Activation of the adenosine Azs receptor was found to increase vascular endothelial growth factor production, resulting in angiogenic neovascularization (Grant et al. Circ Res 85, 699-706 (1999)). Recent studies have demonstrated that agonists for the s adenosine AzA and As receptors have antiinflammatory properties (Sullivan and Linden, Drug Dev Res 45, 103-112 (1998), Fishman et al. J Cell Physiol 183, 393-398 (2000)). Agonists at the adenosine A1 and A,3 receptors have shown cardioprotective activity in man (Baraldi et al. Med Res Rev 20, 103-128 (2000)), increased cellular availability of adenosine causes reduce the formation to of tumor necrosis factor-alpha in the damaged heart (LTS 5998386), and conjugate compounds of potent adenosine Ai and A3 receptor agonists have shown full cardioprotection in a myocyte model of ischemia (Jacobson et al. J
Biol Chem 275, 30272-30279 (2000)). Methods of using potent adenosine uptake inhibitors to limit reperfusion tissue damage has been reported (US 5840896).
Is Increased adenosine levels has been claimed to enhance the contractile performance of the heart (US 5629298) and prevent cardiac arr hythmias (US
5998387). It is anticipated that increased adenosine levels as a result of administration of the dicinnamoylquinides of Formula 1 also produce the above mentioned beneficial health effects.

Inventor: de Paulis et al.
Atty. Docket: N-7337 SUMMARY OF THE INVENTION
The present invention provides, in part, methods of using 3,4-disubstituted cinnamoyl esters of quinic acid 1,5-lactone, exemplified by Formula l, having no substituent, or a halogen atom or a hydroxyl, alkyl or alkoxyl group in either of the aromatic 3-, 4- and 5-positions to inhibit the human adenosine transporter. By inhibiting the adenosine transporter, the metabolism of intracellular adenosine is prevented and the resulting presence of high levels of extracellular adenosine is prolonged.
1o Methods are provided for using compounds corresponding to Formula 1 (Fig. 1) that block the normal operation of the adenosine transporter, and preferably, result in a higher level of extra-cellular adenosine.
The present invention also provides a method of producing compounds corresponding to Formula 1. Formula 1 is shown below:
is Inventor: de Paulis et al.
Atty. Docket: N-7337 The present invention also provides a method of delivering the compounds corresponding to Formula 1 in order to maintain a systemic level at effective concentrations.
No aspect or embodiment of the present invention is bound by theory or mechanism. Various features and advantages of the invention will be apparent from the following detailed description and from the claims.
Io BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is Formula 1.

R6 'Rs Inventor: de Paulis et al.
Atty. Docket: N-7337 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Thus, although there have been described particular embodiments of the present invention of new and useful Substituted Dicinnamoylquinides and their use in Augmentation of Adenosine Function, it is not intended that such references be construed as limitations upon the scope of this invention except as set forth in the following claims.
3,4-disubstituted cinnamoyl esters of quinic acid 1,5-lactone, exemplified by Formula 1 (Fig. 1), having no substituent, a halogen atom or a hydroxyl, alkyl or alkoxyl group in either of the aromatic 3-, 4- and 5-Io positions inhibit the human adenosine transporter. Interruption of normal adenosine transporter function leads to increased levels of extra-cellular adenosine by preventing adenosine elimination. The elevated adenosine levels stimulate adenosine receptor subtypes, which are known to have several positive health effects, including angiogenic neovascularization, antiinflamation, and cardioprotection. The present invention is not bound by mechanism or theory.
As used herein, "Formula 1" means any chemical composition described by any text and/or figure referring to Formula 1.
_7_ Inventor: de Paulis et al.
Atty. Docket: N-7337 As used herein, "an inappropriate extra-cellular adenosine level"
means an adenosine level present when a subject has a disease or condition that improves, or a symptom of which improves, from either an acute or a chronic increase in adenosine levels.
As used herein, "adenosine transporter" means human equilibrative sensitive adenosine transporter.
As used herein, "substituted dicinnamoylquinides" means 3,4-di(3- or 4-mono-substituted, 3,4- or 3,5-di-substituted, or 3,4,5-tri-substituted)-cinnamoyl-1,5-quinides.
to As used herein, "pharmaceutically" or "pharmacologically acceptable"
means the use of molecular entities or compositions that generally do not produce adverse or allergic reactions when appropriately administered to an animal, or human.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain preferred methods and materials are described below. All publications, patent applications, patents, and other _g_ Inventor: de Paulis et al.
Atty. Docket: N-7337 references mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present document, including definitions, will control.
Unless otherwise indicated, materials, methods, and examples described herein are illustrative only and not intended to be limiting.
The present invention discloses a method of using certain chemical compounds to inhibit the adenosine transporter. Compounds of the invention are illustrated by Formula 1, where Rt-R~ are aromatic substituents each and independently comprised of either a hydrogen or halogen atom, straight or branched C1-Cs alkyl, C1-Cs alkoxy, or a hydroxy group. The synthesis of Io these compounds can be accomplished by the method of Wynne et al. (WO
01508, 1986) as described by Huynh-Ba (US 5395959, 1995, US 5401858, 1995). Briefly, it consists of condensing an excess of the appropriately protected substituted cinnamoyl acid chloride with 1-(2,2,2-trichloroethoxyformyl)-1,5-quinide, prepared by lactonization of quinic acid in 1s acetone with p-toluenesulfonic acid, followed by condensation of the remaining free hydroxyl group with 2,2,2-trichloroethyl chloroformate to give 1-O-(2,2,2-trichloroethoxy-formyl)-3,4-O-isopropylidene-1,5-quinide and hydrolysis of the isopropylidene group in 90% aqueous trichloroacetic acid.
Removal of the 2,2,2-trichloroethoxyformyl group and phenolic protection Inventor: de Paulis et nl.
Atty. Docket: N-7337 groups, if present, gives the final product, which can be purified by fractional crystallization from organic solvents, such as ethylacetate, chloroform, or isopropylether. The ability of the compounds of this invention to inhibit the adenosine transporter is evaluated in binding experiments.
The present invention also provides a method of treating an inappropriate extra-cellular adenosine level in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula 1, wherein Rs, R~, and R5 are the same or different and each independently represent substituents selected from the group 1o consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, and an alkoxyl group; wherein the adenosine transporter is partially or completely inhibited by the treatment. The present invention discloses a method of treating an inappropriate extra-cellular adenosine level wherein the inappropriate extra-cellular adenosine level. In certain embodiments, the inappropriate level is associated with cardiovascular disorders, stroke, heart attack, or depression.
The present invention further provides methods to inhibit the adenosine transporter, the method comprising of administering a composition comprising Formula 1, wherein said administration results in an inhibition of Inventor: de Paulis et al.
Atty. Docket: N-7337 the adenosine transporter. In particular embodiments, the adenosine transporter is the human equilibrative sensitive adenosine transporter.
Particularly preferred compounds of the present invention are compounds of Formula 1 where both aromatic cinnamoyl substituents are comprised of 4-chloro, 4-hydroxy, or 4-methoxy groups, or where the aromatic cinnamoyl substituents are 3,4-dichloro, 3,4-dihydroxy, or 3,4-dimethoxy groups.
The compounds represented by Formula 1 are introduced using any suitable method. A "suitable method" of introduction is one that places a Formula 1 compound in a position to inhibit, either partially or completely, 1o the adenosine transporter. In some preferred embodiments, the administering step of a method of treating an inappropriate extra-cellular adenosine level in a subject in need thereof is via oral, topical, subcutaneous, intramuscular, intravenous or patenteral routes. In some preferred embodiments, the administering step comprises administering the compound I5 more than once. For example, injection, oral, and inhalation methods may be employed, with one of ordinary skill in the art being able to determine an appropriate method of introduction for a given circumstance.
In some preferred embodiments, injection will be used. This injection may be intravenous, intraperitoneal, intramuscular, subcutaneous, Inventor: de Paulis et al.
Atty. Docket: N-7337 intratumoral, intrapleural, or of any other appropriate form. The form of the injectable compositions is typically a liquid or suspension. Additionally, these preparations also may be emulsified. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. In certain embodiments, a beverage or food serves as the excipient. For example, in fortified beverages and food including, but not limited to: aqueous carriers include water, coffee, tea, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and Io nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well known parameters typically related to storage or comfort during injection.
One aspect of the present invention is the use of Formula 1 compounds in pharmaceutical compositions.
In certain embodiments, the present invention provides a method for treating a human suffering from an inappropriate extra-cellular adenosine level, which comprises administration of a therapeutically effective amount of a compound of Formula 1, wherein R1 - Rs are the same or different and each Inventor: de Paulis et al.
Atty. Docket: N-7337 independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, or an alkoxyl group; wherein the adenosine transporter is partially or completely inhibited by the treatment. The above-mentioned method is also a method for the treatment of depression associated within appropriate adenosine levels. In other embodiments, the present invention provides a method for the treatment of cardiovascular disorders associated with inappropriate adenosine levels. In still other embodiments, the present invention provides a method for the treatment of a heart attack associated with inappropriate adenosine levels. In still further Io embodiments, the method for treating a human suffering from an inappropriate extra-cellular adenosine level, is also a method for the treatment of stroke associated with inappropriate adenosine levels.
In certain embodiments, a disease condition is not diagnosed or deemed unrelated to adenosine levels; however the subject is still in need of I5 treatment to increase said levels. The need for increasing adenosine levels is generally determined by a physician or attending healthcare professional (including a veternarian) and includes, without limit, working with subjects and/or healthcare providers to identify standardized methods of testing for said compositions.

Inventor: de Paulis et al.
Atty. Docket: N-7337 In certain other aspects of the present invention there are provided therapeutic kits comprising in suitable container and a pharmaceutical formulation of a Formula 1 compound. Such a kit may further comprise a pharmaceutical formulation of a Formula 1 compound.
s In some embodiments of the present invention, the discovery that Formula 1 compounds are able to inhibit the adenosine transporter will be used in combination with other therapies that also increase adenosine function, such as adenosine receptor agonists. These other therapies are distinguishable from the present invention due to their interaction with the 1o adenosine receptors, rather than the adenosine transporter. These other therapies may be known at the time of this application, or may become apparent after the date of this application.
In other embodiments, the Formula 1 compounds will be administered in a pharmaceutical composition in therapeutically effective amounts. Those t5 of ordinary skill in the art will readily be able to prepare Formula 1 compounds, as described herein, and to inhibit the adenosine transporter based upon the data detailed herein. It is contemplated that many routes of administration may be utilized in conjunction with the Formula 1 compounds of the invention, such as intravenous injection or oral consumption.

Inventor: de Paulis et al.
Atty. Docket: N-7337 Methods for this use of Formula 1 compounds are described herein. In certain embodiments, substituted dicinnamoylquinides are ingested as food additives. In other embodiments, the present invention provides a compound of Formula 1 fortified food product, comprising a food product and a therapeutically effective amount of a compound of Formula l, to provide partial inhibition of an adenosine transporter. In still other embodiments, the above-mentioned Formula 1 fortified food product will provide complete inhibition of the adenosine transporter. For example, coffee and other drinks and foods may be fortified with compounds corresponding to Formula 1.
1o In certain embodiments of the invention, Formula l, and related, compounds are ingested in conjunction with the consumption of coffee or more preferred coffee fortified therewith. Oral formulations, in addition to containing the Formula 1 compounds, may include other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The compositions may take the form of solutions, suspensions, tablets, pills, gels, capsules, sustained release formulations or powders. When the route is topical, the form may include a cream, ointment, salve, spray, or other carrier. The quantity to be administered depends on several factors, for Inventor: de Paulis et al.
Ariy. Docket: N-7337 example, the type of subject being treated and the mass of the subject.
Formula 1 compounds will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. As used herein, "therapeutically effective" means an amount that inhibits, completely or partially, the adenosine transporter so that the level of adenosine in the blood and/or brain increases. In certain embodiments, the present invention providing the method of treating an inappropriate extra-cellular adenosine level in a subject in need thereof will comprise administering to the subject a therapeutically effective amount, wherein the Io therapeutically effective amount is sufficient to raise adenosine levels in blood and brain, of a compound of Formula 1 or a pharmaceutically acceptable formulation thereof. In other embodiments, the therapeutically effective amount of the Formula 1 compound is less than 100 milligrams per kilogram of body weight of the subject. In other embodiments, the therapeutically effective amount of the Formula 1 compound is more than 99 milligrams per kilogram of body weight of the subject. For example, a therapeutically effective amount of a Formula 1 compound results in an increased adenosine level in the blood and/or brain. The formulations are easily administered in a variety of dosage forms, such as the type of Inventor: de Paulis et al.
Atty. Docket: N-7337 injectable solutions described above, with even drug release capsules and the like being employable.
All references described in this patent application are hereby incorporated herein by reference, in their entirety. Also incorporated in this s specification are the exhibits filed herewith. The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way.
Io EXAMPLE 1 3, 4-Diy4-chlorocinnamoyl)-1.5-quinide.
Five g (27 mmol) of 4-chlorocinnamic acid (Aldrich) is dissolved in 50 mL toluene, followed by 5 mL (68 mmol) of thionyl chloride and 0.3 mL
dimethylformamide as catalyst. The mixture is heated to 60 ~C for 2 h. The Is solvent is evaporated and the residue is used direct in the next step. 4-Chorocinnamoyl chloride is dissolved in 25 mL of CH2Clz and added dropwise to a mixture of 3.5 g (10 mmol) of 1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide and 2 g (25 mmol) of pyridine in 50 mL of CH2Clz at 0 ~C (ice - EtOH).
After 16 h at 20 ~C the solvent is removed and the residue is extracted with 2 Inventor: de Paulis et al.
Atty. Docket: N-7337 x 200 mL ethyl acetate. Washing with 2 x 50 mL of 1 N HC1 to remove the pyridine, then with 2 x 50 mL of 2% NaHCOs to remove excess acid, followed by 100 mL of water, drying (Na2S04), evaporation of the solvent, and crystallization from 100 mL of ethyl acetate gave 5.3 g of 3,4-di(4-chlorocinnamoyl)-1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide. Yield 79%.
1H-NMR: 8 ppm 7.68 (d, J--16 Hz, 9'-CH=), 7.60, (d, J=16 Hz, 9"-CH=), 7.47 (d, J--9 Hz, 2H, 2',6'-Ar), 7.40 (d, J 9 Hz, 2H, 3',5'-Ar), 7.38 (d, J--9 Hz, 2H, 2",6"-Ar), '7.31 (d, J=9 Hz, 2H, 3",5"-Ar), 6.51 (d, J--16 Hz, 8'-CH=), 6.30, (d, J--16 Hz, 8"-CH=), 5.72 (t, 4-CHeq), 5.36 (ddd, 5-CHeq), 5.04 (dd, 3-CHaX), 4.86 Io (d, J--12 Hz, 1-OCOOCHCCIs), 4.75 (d, J--12 Hz, 1-OCOOCHCCIs), 3.23 (m, 6-CHaX), 2.72 (d, 6-CHeq), 2.58 (m, 2-CHeq), 2.47 (t, 2-CHaX).
The protected quinide is dissolved in 100 mL of THF, addition of 1 g (15 mmol) of zink powder (65 mesh) and 50 mL acetic acid at 20 °C.
Stirring for 3 h, evaporation of the solvents, extraction with 2 x 200 mL ethyl acetate, washing with 50 mL 1 N NaOH, then 50 mL water, drying (Na2S04) and evaporation of the solvent gave 3,4-di(4-chlorocinnamoyl)-1,5-quinide.
Recrystallization from chloroform gave 2.9 g of pure product. Mp 172 °C.
Yield 78%. tH-NMR: 8 ppm 7.68 (d, J 16 Hz, 9'-CH=), 7.60, (d, J=16 Hz, 9"-CH=), 7.47 (d, J--9 Hz, 2H, 2',6'-Ar), 7.40 (d, J=9 Hz, 2H, 3',5'-Ar), 7.38 (d, J=9 -1 s-Inventor: de Paulis et al.
Atty. Docket: N-7337 Hz, 2H, 2",6"-Ar), '7.31 (d, J=9 Hz, 2H, 3",5"-Ar), 6.51 (d, J=16 Hz, 8'-CH=), 6.30, (d, J 16 Hz, 8"-CH=), 5.08 (m, 4-CHeq), 4.87 (dd, 5-CHeq), 4.41(q, 3-CHaX), 2.39 (m, 6-CHaX), 2.69 (d, 6-CHeq), 2.2.28(m, 2-CHeq), 2.21 (q, 2-CHaY).
s EXAMPLE 2 3, 4-Diferuloyl-1, 5-quircide.
Six g (16 mmol) of 4-O-(2,2,2-trichloroethoxycarboxyl)feruloyl chloride is dissolved in 25 mL of CH2C12 and added dropwise to a mixture of 2 g (5.7 mmol) of 1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide and 2 g (25 mmol) of Io pyridine in 25 mL of CH2C12 at 0 °C (ice - EtOH). After 16 h at 20 ~C the solvent is removed and the residue is extracted with 300 mL ethyl acetate.
Washing with 50 mL of 1 N HCl to remove the pyridine, then with 50 mL of 2% NaHCOs to remove excess acid, followed by 50 mL of water, drying (Na2SO.t), evaporation of the solvent, and crystallization from 100 mL of ethyl Is acetate gave 5 g of 3,4-di[4-O-(2,2,2-trichloroethoxycarboxyl)feruloyl]-1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide. (Yield 83%). Mp 232-234 ~C. ~H-NMR: b ppm 7.69 (d, J=16 Hz, 9'-CH=), 7.61, (d, J--16 Hz, 9"-CH=), 7.20 (m, 6'-CH + 6"-CH), 7.12 (m, 2'-CH + 2"-CH), 7.00 (m, 5'-CH + 5"-CH), 6.47 (d, 8'-CH=), 6.30, (d, 8"-CH=), 5.74 (t, 4-CHeq), 5.38 (m, 5-CHeq), 5.03 (t, 3-CH~"), Inventor: de Paulis et al.
Atty. Docket: N-7337 4.88 (s, 4'-CHzOC00), 4.86 (s, 4"-CH20C00), 4.79 (dd, 1-CH20C00), 3.87 (s, 3'-OCHs), 3.78 (s, 3"-OCHs), 3.21 (d, 6-CHaX), 2.75 (m, 6-CHeq), 2.56 (m, 2-CHeq), 2.52 (t, 2-CHaX).
Deprotection of all trichloroethoxycarboxyl groups is accomplished by dissolving 5 g (4.7 mmol) of 3,4-di[4-O-(2,2,2-trichloroethoxy-carboxyl)feruloyl]-1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide in 50 mL of THF, addition of 1 g (15 mmol) of zink powder (65 mesh) and 50 mL acetic acid at 20 °C. Stirring for 3 h, evaporation of the solvents, extraction with 2 x 75 mL ethyl acetate, washing with 2 x 25 mL 2% NaHCOs, then 50 mL water, to drying (NazS04) and evaporation of the solvent gives a non-crystalline residue. It is dissolved in 15 mL of ethyl acetate and 15 mL of isopropyl ether is added. Filtration gave 2.1 g (85%) of 3,4-diferuloyl-1,5-quinide. Mp 142-~C. Rotation [a]z~D + 196° (c 0.88, DMSO). 1H-NMR: 8 ppm 7.67 (d, J=15.9 Hz, 9'-CH=), 7.55, (d, J 15.8 Hz, 9"-CH=), 7.11 (dd, 6'-CH), 7.09 (dd, 6"-CH), 7.01 I5 (m, 2'-CH + 2"-CH), 6.95 (d, 5'-CH), 6.83 (d, 5"-CH), 6.36 (d, J=15.9 Hz, 8'-CH=), 6.19, (d, J=15.8 Hz, 8"-CH=), 5.69 (t, 4-CHeq), 5.29 (m, 5-CHeq), 4.95 (t, 3-CHaX), 3.93 (s, 3'-OCHs), 3.81 (s, 3"-OCHs), 2.65 (d, 6-CH~X), 2.54 (m, 6-CHeq), 2.41 (m, 2-CHeQ), 2.32 (t, 2-CHaX).
isC-NMR: 8 ppm 177 (7-C=O), 166 (7'-C=O), 165 (7"-C=O), 149 (4'-Inventor: de Paulis et al.
Atty. Docket: N-7337 COH), 148 (4"-COH), 147 (3'-COCHs), 147 (3"-COCHs), 147 (9'-CH=), 146 (9"-CH=), 129 (1'-C), 129 (1"-C), 126 (6'-CH), 125 (6"-CH), 123 (8'-CH=), 123 (8"-CH=), 115 (5'-CH), 115 (5"-CH), 114 (2'-CH), 114 (2"-CH), '74 (4-CH), 72 (5-CH), 68 (1-OH), 66 (3-CH), 38 (6-CH2), 37 (2-CH2).
s 3,4-di(3,4-dichlorocinnamoyl)-1,5-quinide is prepared as described in example 1, starting with 3,4-dichlorocinnamic acid.
1 o EXAMPLE 4 3,4-dicoumaroyl-1,5-quinide is prepared as described in example 2, starting with 4-hydroxycinnamic acid.

is 3,4-dicaffeoyl-1,5-quinide is prepared as described in example 2, except that the starting material is 3,4-dihydroxycinnamic acid and the phenolic protecting groups are methoxyformyl groups and the deprotection reagent is lithium chloride in refluxing pyridine.

Inventor: de Paulis et al.
Atty. Docket: N-7337 Preparation of intermediates 3.4-Isopropylidene-l, 5-guinade.
A suspension of 15 g (78 mmol) of quinic acid (Aldrich) and 0.5 g of p-toluenesulfonic acid in 300 mL of acetone are heated to refluxing temperature for 20 h with the solvent passing through a Soxhlet extractor supplied with g of molecular sieve (4 A), that previously have been activated under vacuum for 2 h at 140 °C. The clear solution is cooled to 5 ~C, 4 g (48 mmol) of NaHCOs is added, and the mixture is stirred for 1 h. Filtration and 1o evaporation of the solvent, followed by crystallization by dissolving the residue in 150 mL of CH2C12 and addition of 150 mL hexane gave 16 g of 3,4-Isopropylidene-1,5-quinide (yield 96%). Mp 140-142 ~C. tH-NMR: 8 ppm 4.72 (m, 5- CHeq), 4.50 (m, 4-CHeq), 4.31 (m, 3-CHaX)> 2.90 (b, 1-COH), 2.78 (d, 6-CHaX), 2.34 (m, 6-CHeq + 2-CHeq), 2.17 (dd, 2-CHaX), 1.52 (s, CCH3), 1.33 (s, Is CCHs).
1-O-(2, 2, 2-Trichloroethoxycarbox~)-3, 4-O-isopropylidene-1, 5-guinide.
Sixteen g (75 mmol) of of 3,4-Isopropylidene-1,5-quinide is dissolved in Inventor: de Paulis et al.
Atty. Docket: N-7337 150 mL of CHZC12 and 14 mL (173 mmol) of anhydrous pyridine is added, followed by dropwise addition of a solution of 17 g (81 mmol) 2,2,2-trichloroethyl chloroformate (Aldrich) in 25 mL of CHZCl2 at 0 °C.
After stirring at room temperature for 2 h, 200 mL of CHZCIz is added and the mixture is washed with 2 x 75 mL 1 N HCI, then with 50 mL water. Drying with anhydrous NazS04, evaporation of the solvent to 100 mL, then adding 400 mL of EtOH gave 20 g of 1-O-(2,2,2-Trichloroethoxycarboxyl)-3,4-O-isopropylidene-1,5-quinide_(yield 68%). Mp 164-166 °C. tH-NMR: 8 ppm 4.84 (t, 5- CHeq), 4.80 (dd, OCH2CCls), 4.56 (dd, 4-CHeq), 4.34 (d, 3-CHaX), 3.06 (d, 6-CHax), 2.66 (m, 6-CHeQ), 2.57 dd, 2-CHeq), 2.40 (dd, 2-CHa,;), 1.53 (s, CCHs), 1.34 (s, CCH3).
1-O-~2, 2, 2-Trichloroethoxycarbox,~ )-1, 5-c~uirtide.
Three mL of water is added to 27 g (165 mmol) of trichloroacetic acid and stirred with warming until clear solution, then 20 g (51 mmol) of 1-O-(2,2,2-Trichloroethoxycarboxyl)-3,4-O-isopropylidene-1,5-quinide is added in portions at 20 °C. Stirring for 4 h, then 200 mL of ice-water and 400 mL of ethyl acetate are added, followed by slowly addition of a solution of 15 g (178 mmol) of NaHCOs in 400 mL water. The organic layer is separated and Inventor: de Paulis et u!.
Atty. Docket: N-7337 washed with 50 mL of 2% NaHCOs, then with 50 mL water. Drying and evaporation of the solvent, addition of 150 mL of 70 °C toluene, cooling over night, and filtration of the crystals gives 12 g (67%) of 1-O-(2,2,2-trichloroethoxycarboxyl)-1,5-quinide. Mp 130-131 °C. tH-NMR: 8 ppm 4.93 (t, s 5- CHeq), 4.77 (dd, OCH2CCls), 4.18 (t, 4-CHeq), 4.02 (t, 3-CHaX), 3.04 (d, CHaX), 2.68 (m, 6-CHeq), 2.36 (m, 2-CH~q), 2.17 (t, 2-CHa,).
4-O-(2,2,2-Trichloroethox~carbox~)ferulic acid.
Ten g (51 mmol) of 3-methoxy-4-hydroxycinnamic acid (Aldrich) is Io dissolved in 140 mL of 1 N NaOH followed by drop-wise addition of 13 g (61 mmol) of 2,2,2-trichloroethyl chloroformate at 0 °C. After 20 min the mixture is neutralized with 2 N HCl and the precipitation is collected by filtration.
Recrystallization from 300 mL 50% of aqueous EtOH gave 16 g of 4-(2,2,2-trichloroethylcarbonato)-3-methoxycinnamic acid (yield 85%). Mp xxx ~C. 1H-Is NMR: 8 ppm 7.75 (d, J--15.9 Hz, 3'-CH=), 7.18 (m, 3H, ArH), 6.42 (d, ~l=15.8 Hz, 2'-CH=), 4.89 (s, OCH2CCls), 3.90 (s, OCHs).
4-O-(2,2,2-Trichloroethoxycarbox~)feruloyl chloride.

Inventor: de Paulis et al.
Atty. Docket: N-7337 Seven g (19 mmol) of 4-(2,2,2-trichloroethylcarbonato)-3-methoxycinnamic acid is dissolved in 75 mL of CH2C12, 5 g (24 mmol) of PC15 was added at 20 °C, and the mixture is heated to 35 °C for 20 min.
Evaporation of the solvent under vacuum and addition of 200 mL of hexane s gave 6 g of 4-O-(2,2,2-trichloroethoxycarboxyl)feruloyl chloride (yield 81%).
Mp xxx °C. 1H-NMR: 8 ppm 7.79 (d, ~I--15.5 Hz, 3'-CH=), 7.18 (m, 3H, ArH), 6.61 (d, J--15.5 Hz, 2'-CH=), 4.89 (s, OCH2CCls), 3.91 (s, OCH3).

to Evaluation of adenosine transport inhibition.
Inhibition of [3H]adenosine transport in homogenates of U-937 cell culture, expressing the human (es) transporter (American Tissue Culture Compository) is performed according to Gu et al. J Neurochem 67, 972-977 (1996). Rate of transport is determined in HEPES buffer at 3'7 °C for 30 min is (with choline replacing sodium). Full displacement curves for the adenosine transporter are obtained by using 8 concentrations of each compound, increasing by a factor of 2 and ranging from 0.25 - 32 ~M. Nonspecific transport is defined with 1 ~,M NBTI ((S~-(4-nitrobenzyl)-6-thioinosine). Cell cultures are grown in high glucose Dulbecco's modified Eagle's medium Inventor: de Paulis et al.
Atty. Docket: N-7337 supplemented with NaHCOs (40 mM), 10% fetal bovine serum (Gibco), 400 mg/L geneticin, and 0.1 mM hypoxanthine. The cells are cultured at 37 ~C
under a COz/air (5/95, v/v) in tissue culture flasks (75 cmz, Falcon). When reaching confluency (70-80%), cells are trypsinized and harvested by centrifugation. Harvested cells are re-suspended in 50 mM TRIS buffer at 4 °C (0.1 mg/mL), the cell membranes are disrupted by Polytron homogenization (12,500 rpm for 25 s), and the suspension is centrifuged at 30,000 x g for 60 min. The supernatent is discarded and remaining the pellet is resuspended in 50 mM TRIS buffer to 0.2 - 0.5 mg protein/mL and frozen at - 80 °C until used. Protein content is measured by the Lowry method using bovine serum albumin as the standard. The transport of 10 ~M [3H]adenosine is blocked by a typical agent of this invention described in example 2 with an inhibitory constant K; of 1.2 ~tM.
I s EXAMPLE 8 Evaluation of adenosine transporter binding affinity.
Inhibition of [3H]NBTI binding in homogenates of U-937 cell culture, expressing the human es transporter is performed according to Marangos et 2o al. J Neurochem 39, 184-191 (1982). The cell membranes in final Inventor: de Paulis et al.
Atty. Docket: N-7337 concentration of 0.03 mg protein/mL are incubated with [3H]NBTI at 2-4 nM
concentration in 50 mM TRIS buffer (pH 7.5) at 4 °C for 1.5 h in a total volume of 1.0 mL. Nonspecific binding is defined with 10 E.tM NBTI. Each determination is carried out in triplicate. Bound and free [3H]NBTI are separated by vacuum filtration through fiberglass filters (Schleicher &
Schuell, Keene, NH) that are presoaked with 0.3% polyethylenimine for 10 min, using a Brandel M-24R cell harvester. The filters are washed three times for 10 s with ice-cold 50 mM TRIS buffer and placed in 10 mL
scintillation fluid (Cytoscint, ICN). Beta spectrometry is performed using a to Beckman L5801 instrument at 47% counting efficiency. ICSO values and Hill slopes (nH) are calculated from log-logit analysis of competition binding data.
K; values of the competing ligand are calculated from ICso values using the Cheng - Prusoff equation, K; = ICsol(1 + LlKv), where L is the concentration of the [3H]NBTI and KD = 0.32 nM is the equilibrium dissociation constant obtained from Scatchard analysis of the binding. Data are expressed as mean ~ standard error of the mean (SEM).

Inventor: de Paulis et al.
Atty. Docket: N-7337 Displacement of [3H]NBTI binding in U-937 cell homogenates expressing the human adenosine transporter by substituted dicinnamoyl-1,5-quinides of formula 1.
Active ingredient Substituents Rl R2 R3 Affinity (Ki ~tM) Hill slope s 3,4-di(4-chlorocinnamoyl)-1,5-quinide H Cl H 1.3 ~ 0.0 1.12 ~ 0.00 3,4-dicaffeoyl-1,5-quinide OH OH H 2.4 ~ 0.7 1.00 ~ 0.03 3,4-diferuloyl-1,5-quinide OCH3 OH H 0.96 ~ 0.13 0.94 ~ 0.02 3,4-dicoumaroyl-1,5-quinide H OH H 0.96 ~ 0.07 1.22 ~ 0.11 Substituents (R4-R6) of the second cinnamoyl group are identical to those of the first.
A Hill slope close to unity indicates binding to a single site.

Pharmaceutical formulations Capsules. 200 g of the active ingredient (compound corresponding to is Formula 1), 1 g magnesium stearate, 5 g sodium laurylsulfate, 50 g lactose, 50 g starch, and 1 g colloidal silicon dioxide are mixed thoroughly and the mixture is filled into 1000 gelatin capsules each containing 200 mg of the active ingredient (compound corresponding to Formula 1).

Inventor: de Paulis et al.
Atty. Docket: N-7337 Oral SOIZbtL072. 20 g of the active ingredient (compound corresponding to Formula 1) is dissolved by stirring in 10 L warm water kept at 50-70 oC, containing 5 g methyl 4-hydroxybenzoate, 10 g 2,3-dihydroxybutanedioic acid, g 1,2,3-propanetriol, and 500 g sucrose. After cooling to room temperature 5 the mixture is flavored and colored with suitable additives to produce 50 doses of a beverage containing 400 mg active ingredient per 200 mL.

Claims (17)

1. A method of treating an inappropriate extra-cellular adenosine level in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a compound of Formula 1 or a pharmaceutically acceptable form thereof, wherein R1, R2, R3, R4, R5, and R6 are the same or different and each independently represents a substituent selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, and an alkoxyl group; wherein the adenosine transporter is partially or completely inhibited by the treatment.

2. The method of claim 1, wherein the inappropriate extra-cellular adenosine level is in cardiovascular disorders.

3. The method of claim 1, wherein the inappropriate extra-cellular adenosine level is associated with a stroke.

4. The method of claim 1, wherein the inappropriate extra-cellular adenosine level is in a heart attack.

5. The method of claim 1, wherein the inappropriate extra-cellular adenosine level is depression.

6. The method of claim 1, wherein the administering step is via oral, inhalation, topical, subcutaneous, intramuscular, intravenous, or patenteral routes.

7. The method of claim 1, wherein the therapeutically effective amount is sufficient to raise adenosine levels in blood and brain.

8. The method of claim 1, wherein the administering step further comprises administering the compound more than once.

9. The method of claim 1, wherein the therapeutically effective amount of the compound is less than 100 milligrams per kilogram of body weight of the subject.

10. The method of claim 1, wherein the therapeutically effective amount of the compound is more than 99 milligrams per kilogram of body weight of the subject.

11. A method for treating a human suffering from an inappropriate extra-cellular adenosine level, which comprises administration of a therapeutically effective amount of a compound of Formula 1, or a pharmaceutically acceptable form thereof, wherein R1 - R6 are the same or different and each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, or an alkoxyl group; wherein the adenosine transporter is partially or completely inhibited by the treatment.

12. The method of claim 11, wherein the method is for the treatment of depression associated with inappropriate adenosine levels.

13. The method of claim 11, wherein the method is for the treatment of cardiovascular disorders associated with inappropriate adenosine levels.

14. The method of claim 11, wherein the method is for the treatment of heart attack associated with inappropriate adenosine levels.

15. The method of claim 11, wherein the method is for the treatment of stroke associated with inappropriate adenosine levels.

16. A compound of Formula 1 fortified food product, comprising:
a food product; and a therapeutically effective amount of a compound of Formula 1 to provide partial inhibition of an adenosine transporter.

17. The food product of claim 16, wherein the therapeutically effective amount of a compound of Formula 1 provides complete inhibition of the adenosine transporter.